MECHANISM OF EARLY STAGE ABETA AMYLOID ...

MECHANISM OF EARLY STAGE ABETA AMYLOID

FORMATION

By

LEI LI

Submitted in partial fulfillment of the requirements for the Degree of Doctor

of Philosophy

Thesis Advisor: Dr. Michael G. Zagorski

Department of Chemistry

CASE WESTERN RESERVE UNIVERSITY

August 2008

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

03/20/2008

Michael Zagorski

Clemens Burda

We hereby approve the thesis/dissertation of

Ph. D.

Lei Li______________________________________________________

candidate for the ________________________________degree *.

Shu Chen

James Burgess

Gheorghe Mateescu

(signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein.

Table of Contents

TABLE OF CONTENTS i

LIST OF TABLES iv

LIST OF FIGURES v

ACKNOWLEDGEMENTS xv

LIST OF ABBREVIATIONS xvi

ABSTRACT xviii

1. INTRODUCTION: ALZHEIMER’S DISEASE AND AMYLOID Aβ

PEPTIDES 1

1.1 Alzheimer’s Disease 2

1.2 Amyloid Aβ Peptides 6

1.2.1 Aβ and Alzheimer's disease 6

1.2.2 Biogenesis of the Aβ peptides 8

1.2.3 Normal roles of the APP and Aβ peptides 10

1.2.4 Aβ neurotoxicity: Aβ oligomers are the primary neurotoxic species

in Alzheimer’s disease 10

1.2.5 Structural Studies of the Aβ peptides 15

1.3 Objectives and Significance 18

2. SYNTHESIS, PURIFICATION AND DISAGGREGATION OF THE

Aβ PEPTIDES 19

2.1 Peptide Synthesis 20

2.2 HPLC Purification of the Aβ Peptides 24

2.3 Aβ peptide Sample Preparation 25

3. THE EFFECT OF PHENYLALANINE-19 SUBSTITUTION BY

NAPHTHYLALANINE ON THE Aβ(1-40) STRUCTURE 27

3.1 Introduction 28

3.2 Results 30

i

3.2.1 Synthesis of naphthylalanine substituted Aβ(1-40) 30

3.2.2 Conformational studies of naphthylalanine substituted Aβ(1-40) 31

3.2.3 Aβ(1-40) aryl-sulfur interaction studies using NMR 36

3.2.4 NMR studies of naphthylalanine substituted Aβ(1-40) 38

3.2.4.1 Proton chemical shift assignments of naphthylalanine

substituted Aβ(1-40) 38

3.2.4.2 NOE patterns of naphthylalanine substituted Aβ(1-40) 60

3.2.4.3 Proton chemical shift analysis 62

3.2.4.4 Proton chemical shift index analysis 66

3.3 Discussion 70

3.3.1 Aryl-sulfur interaction in Aβ 70

3.3.2 Insights from studies of Aβ(1-40) with Phe19/Phe20 substituted by

naphthylalanine 72

3.4 Material and Methods 74

4. STRUCTURAL STUDIES OF Aβ SOLUBLE OLIGOMERS 76

4.1 Introduction 77

4.2 Results 84

4.2.1 Size Exclusion Chromatography of Aβ 84

4.2.1.1 Aβ oligomers (protofibril preparation protocol) 84

4.2.1.2 ADDLs 87

4.2.1.3 Aβ(1-40) 88

4.2.1.4 Re-injection of Aβ oligomers 89

4.2.1.5 Lyophilized Aβ oligomers 91

4.2.2 Aβ Oligomers Aggregation State Analysis by SEC-MALLS 92

4.2.3 Aβ Oligomers Secondary Structure Analysis by CD 92

4.2.4 NMR Studies of Aβ Oligomers 94

4.2.4.1 HSQC of Aβ oligomers (protofibril preparation protocol) 94

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4.2.4.2 HSQC of ADDLs 99

4.2.5 Diffusion Coefficients Measurements of the Aβ Oligomers 101

4.3 Discussion 103


5. SURFACTANT PROPERTIES OF THE Aβ PEPTIDES 114

5.1 Introduction 115

5.2 Results 115

5.2.1 Critical Micelle Concentration (CMC) of the Aβ Peptides 115

5.2.2 Time Dependent Surface Tension of Aβ Solution 116

5.2.3 Surface Tension of Aβ Oligomers Solution 118

5.2.4 Aβ Conformation and Oligomer Formation Below CMC 119

5.2.5 NMR Studies of Aβ Below CMC 122

5.2.5.1 NMR studies of a simple micelle molecule: sodium dodecyl

sulfate 123

5.2.5.2 2D 15N-1H HSQC of Aβ at different concentrations 124

5.2.5.3 1D 1H-NMR of Aβ at different concentrations 127

5.2.5.4 2D 13C-1H HSQC of Aβ at different concentrations 130

5.2.6 Diffusion Coefficients Measurements of Aβ at Different

Concentrations 138

5.3 Discussion 139


6. CONCLUSIONS AND FUTURE STUDIES 147

7. BIBLIOGRAPHY 150

iii

List of Tables

Table 1.1 Biochemical markers of AD. ↑: markers concentration increase in

AD ↓: markers concentration decrease in AD (Flirski & Sobow, 2005) 5

Table 1.2 Common neurodegenerative diseases characterized by deposition of

aggregated proteins (Skovronsky et al., 2006)

7

Table 2.1 A regular peptide synthesis scheme for Aβ(1-42) on 433A peptide

synthesizer. 1 mmole Fmoc-amino acid were used for each residue 21

Table 2.2 Peptide synthesis scheme for Aβ(1-42) in which methionine 35,

alanine 30, alanine 21` and alanine 2 are 13C labeled. 0.1 mmole Fmoc

amino acids were used for these 13C labeled residues. 1 mmole Fmoc

amino acids were used for other residues 23

Table 3.1 Proton chemical shift assignments for Aβ(1-40)-1-naphthylalanine 40




Table 3.5 Proton chemical shift assignments for Aβ(1-40)-Glycine 56

iv

List of Figures

Figure 1.1 Projected numbers of persons in US population with Alzheimer

disease using the 2000 US Census Bureau middle-series estimates of

population growth, bounded by high- and low-series estimates (Hebert et

al., 2003) 3

Figure 1.2 Plaques and tangles in the cerebral cortex in Alzheimer's disease.

Plaques are extracellular deposits of Aβ surrounded by dystrophic

neurites, reactive astrocytes, and microglia, whereas tangles are

intracellular aggregates composed of a hyperphosphorylated form of the

microtubule-associated protein tau. (Goedert et al., 2006) 7

Figure 1.3 Sequence of Aβ(1-40) and Aβ(1-42) 8

Figure 1.4 APP processing and Aβ production. (A) APP can be cleaved

sequentially by β-secretase (BACE) and γ-secretase: a protease complex

containing presenilin (PS) as the putative catalytic component to produce

Aβ. (B) Alternatively, APP can be processed by α-secretase and γ-

secretase to produce P3 (Esler et al., 2001) 9

Figure 1.5 Amyloid cascade hypothesis. Increased aggregation of Aβ can

occur as a result of (a) overproduction of Aβ42 [as in the case of most

amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2

(PS2) gene mutations], (b) expression of mutations in the Aβ domain of

APP that increases its propensity for aggregation, (c) Apolipoprotein E4

(apoE4) expression, and (d) other genetic and environmental factors,

including aging. Aggregated Aβ accumulates in various forms and

locations, some or all of which may result in cellular toxicities mediated

by a variety of mechanisms. Decreased clearance of aggregates and

failures of cellular defenses to toxicity may exacerbate this process. The

toxic effects of amyloid result eventually in neuronal death and

dysfunction, manifesting as dementia. (Skovronsky et al., 2006) 11

Figure 1.6 Structural model for Aβ(1-40) fibrils (a) Ribbon representation of

residues 9-40, viewed down the long axis of the fibril. Each molecule

v

contains two β-strands (red and blue) that form separate parallel β-sheets

in a double-layered cross-β motif. Two such cross-β units comprise the

protofilament, which is then a four-layered structure. (b) Atomic

representation of residues 1-40 with color coding to indicate residues

with hydrophobic (green), polar (magenta), positively charged (blue), and

negatively charged (red) side chains. Backbone C=O and N-H bond

vectors are approximately perpendicular to the page. Contacts between β-

sheet layers are through side chain-side chain interactions. N-Terminal

residues are assigned random conformations to indicated structural

disorder. Contacts between the two cross-β units are assumed to be along

the hydrophobic faces created by side chains of the C-terminal segment.

The core of the protofilament is hydrophobic with the exception of the

oppositely charged side chains of D23 and K28, which form salt bridges

(Petkova, et al., 2002) 17

Figure 2.1 MALDI mass spectrum of synthesized Aβ(1-42). The theoretical

mass for an unlabeled peptide is 4515, and the observed major molecular

ion peak was 4513.3 in the figure 25

Figure 2.2 1D 1H-NMR spectrum of HPLC purified Aβ(1-42) (100 μM, in 10

mM D2O phosphate buffer, pH 7.3, 5 oC) 25

Figure 3.1 Structural model of Aβ(1-40) based on solid state NMR. A possible

stabilizing aryl-sulfur interaction exists between the Met35 ε-SCH3

group and the Phe19 aromatic ring (5-6 Å), which could become

disrupted by methionine oxidation and thus account for the decreased

aggregation rate. 29

Figure 3.2 structures of 1- and 2-naphthylalanine 30

Figure 3.3 Time dependent CD spectra of wild type and naphthylalanine

substituted Aβ(1-40) peptides (50 μM, pH 7.2~7.4, 25oC) 35

Figure 3.4 13C-1H HSQC spectra of Aβ(1-40) (50 μM, pH 7.2, 5oC). Met35

ε-methyl carbon was 13C labeled, and as internal references the methyl

carbons of three Ala residues in Aβ(1-40) were also 13C labeled. Carbon

decoupler is turned off during HSQC measurement, thus each peak split

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into two by 1JC-H coupling. Chemical shifts of Met35 ε-methyl protons

were measured as an average of two splitted peaks 37

Figure 3.5 The expanded NH region of the TOCSY spectra of 50 µM Aβ(1-

40)-1-Naphthylalanine19 (10 mM phosphate buffer, pH 7.3, 5oC). The

relayed connections among the NH, αH, βH, and γH are shown 41

Figure 3.6 Expanded NH-αH region of the NOESY spectrum of 50 µM Aβ (1-

40)-1-Naphthylalanine19 (mixing time 270 ms, 10 mM phosphate buffer,

pH 7.3, 5oC). Several sequential NOEs are connected as follows: Solid

line (——) residue 28-40; dotted line (••••••) residue 17-26; dashed line

(-----) residue 7-12 42

Figure 3.7 Expanded NH-NH region of the NOESY spectrum of 50 µM Aβ (1-


pH 7.3, 5oC). The NH-NH NOEs are connected as follows: Solid line

(——) residue 30-36; dotted line (••••••) residue 17-25 43




Figure 3.9 Expanded NH-αH region of the NOESY spectrum of 50 µM Aβ (1-


pH 7.3, 5oC). Sequential NOEs are connected as follows: Solid line (—

—) residue 28-40; dotted line (••••••) residue 17-26; dashed line (-----)

residue 7-12 46

Figure 3.10 Expanded NH-NH region of the NOESY spectrum of 50 µM Aβ

(1-40)-2-Naphthylalanine19 (mixing time 270 ms, 10 mM phosphate

buffer, pH 7.3, 5oC). The NH-NH NOEs are connected as follows: Solid

line (——) residue 30-40 47




Figure 3.12 Expanded NH-αH region of the NOESY spectrum of 50 µM Aβ


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buffer, pH 7.3, 5oC). Sequential NOEs are connected as follows: Solid

line (——) residue 28-40; dotted line (••••••) residue 17-25; dashed line

(-----) residue 9-12 50










buffer, pH 7.3, 5oC). Sequential NOEs are connected as follows: Solid

line (——) residue 28-40; dotted line (••••••) residue 17-25 54






40)-Glycine21 (10 mM phosphate buffer, pH 7.3, 5oC). The relayed

connections among the NH, αH, βH, and γH are shown 57


(1-40)-Glycine21 (mixing time 270 ms, 10 mM phosphate buffer, pH 7.3,

5oC). Sequential NOEs are connected as follows: Solid line (——)

residue 28-40; dotted line (••••••) residue 17-25 58


(1-40)-Glycine21 (mixing time 270 ms, 10 mM phosphate buffer, pH 7.3,

5oC). The NH-NH NOEs are connected as follows: Solid line (——)

residue 17-20, dotted line (••••••) residue 30-34 59

Figure 3.20 Summary of the inter-residue NOEs among the backbone NH, αH

and βH for naphthylalanine substituted Aβ(1-40). The NOE intensities

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are reflected by the thickness of the lines. When an unambiguous

assignment was not possible due to peak overlap, the NOEs are drawn

with gray boxes. A: Aβ(1-40)-1-naphthylalanine19 B: Aβ(1-40)-2-

naphthylalanine19 C: Aβ(1-40)-1-naphthylalanine20 D: Aβ(1-40)-2-

naphthylalanine20 E: Aβ(1-40)-Glycine 61

Figure 3.21 αH chemical shift differences between Naphthylalanine

substituted Aβ(1-40) and those expected for each amino acid residue in a

random coil conformation (Wishart and Sykes, 1994) are represented as a

function of residue position of Aβ(1-40). Chemical shift of wild type

Aβ(1-40) are from Hou, et al., 2004 63

Figure 3.22 1Hα chemical shift differences (Δδ = δnaphthylalanine – δAβ(1-

40) ) between Aβ(1-40) and Aβ(1-40)-1-naphthylalanine19, Aβ(1-40)-2-

naphthylalanine19, Aβ(1-40)-1-naphthylalanine20, and Aβ(1-40)-2-

naphthylalanine 65

Figure 3.23 The filtered αH chemical shift indices of Aβ(1-40), Aβ(1-40)-

Glycine21 and Aβ(1-40) with phenylalanine-19 (or phenylalanine-20)

substituted by naphthylalanine 68

Figure 3.24 The raw αH chemical shift indices of Aβ(1-40), Aβ(1-40)-

Glycine21 and Aβ(1-40) with phenylalanine-19 (or phenylalanine-20)

substituted by naphthylalanine 69

Figure 4.1 Schematic representation of molecular weight and retention volume

relationship in SEC 78

Figure 4.2 Schematic representation of light scattering 80

Figure 4.3 Schematic diagrams of SEC-MALLS 81

Figure 4.4 Diffusion measurement pulse sequence (Altieri et al., 1995) 82

Figure 4.5 The attenuated signal for CCl3H with the increasing gradient

strength 83

Figure 4.6 The exponential decay curve fittings of the intensity versus the

gradient strength square 84

Figure 4.7 Size exclusion chromatography of Aβ(1-42). Aβ peptide was

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dissolved in 10 mM phosphate buffer at a concentration of 100 μM and

chromatographed on a superdex 75 column at flow rate of 0.5 ml/min. 10

mM phosphate buffer was used as solvent 85

Figure 4.8 Time dependent size exclusion chromatography of Aβ(1-42). A 100

μM Aβ(1-42) solution was prepared and aged at room temperature. At

indicated aging times, 50 μl Aβ(1-42) solution was injected into the size

exclusion column and chromatographed 86

Figure 4.9 Aβ(1-42) oligomer peak retention time gradually decrease during

the peptide aging period. The retention time was referenced as the elution

time corresponding to the maximum peak height. The Aβ(1-42)

monomer peak elutes at 26 minute through the aging period, while the

Aβ(1-42) oligomer elutes at 15.5 minute when peptide solution was

initially prepared and at 14.5 minute after the peptide was aged for 36

hours 87

Figure 4.10 Size exclusion chromatography of ADDLs*. The ADDL oligomer

peaks eluted at 15.8 minute and the ADDL monomer peak eluted at 26

minute. *Those peaks included in the black rectangle are from F12

medium 88

Figure 4.11 Size exclusion chromatography of Aβ(1-40). The Aβ(1-40)

monomer peak eluted at 27 minute. No oligomer peak was found 89

Figure 4.12 Size exclusion chromatography of Aβ(1-42) oligomer. Only one

peak with retention time 14.9 minute that is corresponding to Aβ

oligomer was observed. No monomer peak (expected retention time ~26

minute) showed in the graph 90

Figure 4.13 Size exclusion chromatography of Aβ(1-42) with an overlay of the

actual molecular mass of oligomer calculated by MALLS using an

empirically determined dN/dC of 0.24 92

Figure 4.14 CD spectra of Aβ(1-42) oligomer (red) and monomer (black). A

100 µM Aβ(1-42) was chromatographed by size exclusion column. The

separated Aβ(1-42) oligomer and monomer peak were collected and

subjected to CD experiments immediately. The concentration were 33

x

and 20 μM for monomer and oligomer, respectively, determined by UV

absorbance. 93

Figure 4.15 The 1H-15N HSQC spectra of SEC separated Aβ(1-42) oligomer

(A) and monomer (B) (10 mM phosphate buffer, pH 7.3, 5 oC). Overlay

of the two spectra (C) reveal an extra peak (marked “?”) in Aβ(1-42)

oligomer spectrum 96

Figure 4.16 The 1H-15N HSQC spectra of SEC separated Aβ(1-42) oligomer

(A) and Aβ(1-42) without SEC separation (B). Those residues that have

chemical shift differences were labeled in the overlay of the two spectra

(C) 97

Figure 4.17 The 1H-15N HSQC spectra of Aβ(1-42) oligomer prepared after

3hours aging (A) and 24 hours aging (B) and the overlay of the two

spectra (C) 98

Figure 4.18 The 1H-15N HSQC spectra of Aβ(1-42) ADDL monomer (A) and

Aβ(1-42) monomer prepared using protofibril preparation protocol(B).

Those residues that have chemical shift differences were labeled in the

overlay of the two spectra (C) 100

Figure 4.19 The exponential decay curve fitting of the integrals in the aliphatic

region for Aβ(1-42) oligomer and monomer, respectively. The diffusion

coefficient was calculated from the exponential decay fitting according to

equation (1) in the introduction 101

Figure 4.20 The exponential decay curve fitting of the integrals in the aliphatic

region of Aβ(1-40) monomer 102

Figure 5.1 Effect of Aβ peptide concentrations on the surface tension of water.

The Aβ peptide solution is prepared in 10 mM phosphate buffer and the

pH is 7.3. Measurement is done at room temperature. Each point is the

mean value of three measurements. The standard deviations for the series

of values are included in the figure but are usually smaller than the size

of the symbols 117

Figure 5.2 The time-dependent surface tension measurements of Aβ(1-40) and

Aβ(1-42) (50 μM in 10 mM phosphate buffer, pH 7.3, room

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temperature). The surface tension of Aβ(1-40) solution remains constant,

however for Aβ(1-42), the surface tension increases over time 118

Figure 5.3 Surface tension of SEC separated Aβ(1-42) oligomer and monomer.

50 μM Aβ(1-42) solution (10 mM phosphate buffer, pH 7.3) was

incubated at room temperature for 5 hours and then chromatographed on

a size exclusion column (Superdex 75, 10 mM pH 7.5 phosphate buffer

as solvent, flow rate 0.5 ml/min). The oligomer and monomer peak were

collected and their surface tension were measured immediately.

Concentration of each solution was determined afterwards by UV

absorbance measurement and indicated in the figure 120

Figure 5.4 Time dependent circular dichroism experiments of Aβ(1-42) at 50

μM and 5 μM, respectively. The peptide solution is prepared in 10 mM

phosphate buffer (pH 7.3) and incubated at room temperature. Each

spectrum was taken at indicated incubation times and is the average of

six measurements. For 5 μM Aβ(1-42) CD measurement, due to the high

noise level between 190 and 200 nm, only spectra between 200 nm and

250 nm were shown 123

Figure 5.5 Size exclusion chromatography of 5 μM Aβ(1-42). The peptide

solution was prepared in 10 mM phosphate buffer and the final pH was

adjusted to pH 7.3. The peptide solution was incubated at room

temperature and chromatographed at indicated incubation times. 10 mM

phosphate buffer was used as effluent and the flow rate 0.5 ml/min 123

Figure 5.6 Chemical shift variation of sodium dodecyl sulfate (SDS) over

different concentrations. The SDS solution was prepared by directly

dissolving SDS in D2O. The pH was adjusted when necessary by adding

diluted HCL or NaOH solution and the final pH was 7.3 125

Figure 5.7 The Overlayed 15N-1H HSQC spectra of Aβ(1-42) at 100 μM (red)

and 1 μM (green) in phosphate buffer (10 mM), pH 7.3, 5 oC 127

Figure 5.8 Overlayed 15N-1H HSQC spectra of Aβ(1-40) at 100 μM (red) and 1

μM (green) in phosphate buffer (10 mM), pH 7.3, 5 oC 128

Figure 5.9 1D 1H NMR spectra of non-isotope labeled Aβ(1-42) recorded at

xii

100 μM, 1 μM, and 0 μM, respectively 129

Figure 5.10 The Overlayed 1D 1H-13C spectra of Aβ(1-42) recorded at 100

μM, 1 μM, respectively. The possible new peaks that were shown in 1

μM spectrum are marked by red arrows, and those peaks that became

broader in 1 μM spectrum are marked by blue arrows 131

Figure 5.11 Overlayed 1D 1H-13C spectra of Aβ(1-40) recorded at 100 μM, 1

μM, respectively. The possible new peaks that were shown in 1 μM

spectrum are marked by red arrows, and those peaks that became broader

in 1 μM spectrum are marked by blue arrows 131

Figure 5.12 2D 1H-13C HSQC spectra of Aβ(1-42) (10 mM phosphate buffer,

pH 7.3) recorded at 0.5 μM, 1 μM, and 100 μM, respectively. The Hα/Cα

peaks assignment to each residue are as shown in the square brackets.

Other peaks were only assigned to corresponding residues, the identity of

the peaks (i.e., whether peaks are Hβ/Cβ or Hγ/Cγ) were not listed 134

Figure 5.13 2D 1H-13C HSQC-TOCSY spectra of 100 μM Aβ(1-42) (10 mM

phosphate buffer, pH 7.3). The relayed connections for several residues

in proton dimension (connections among αH, βH, γH) and in carbon

dimension (connections among αC, βC and γC) are shown 135

Figure 5.14 Expanded Hβ/Cβ region of Aβ(1-42) 1H-13C HSQC spectra: above,

100 μM (red) vs 1 μM (green); below, 100 μM (red) vs 0.5 μM (green).

Those peaks that disappeared or whose intensities greatly decreased in 1

μM or 0.5 μM spectrum are marked with blue arrows 136

Figure 5.15 Expanded Hα/Cα region of overlayed 100 μM (red) and 0.5 μM

(green) Aβ(1-42) 1H-13C HSQC spectra. The peaks that disappeared or

whose intensities greatly decreased in 0.5 μM spectrum are marked with

blue arrows 137

Figure 5.16 Expanded Hα/Cα region of overlayed 100 μM (red) and 0.5 μM

(green) Aβ(1-40) 1H-13C HSQC spectra. The peaks that disappeared or

whose intensities greatly decreased in 0.5 μM spectrum are marked with

blue arrows 137

Figure 5.17 Expanded Hβ/Cβ region (partly) of overlayed 100 μM (red) and 0.5

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μM (green) Aβ(1-40) 1H-13C HSQC spectra. The peaks that disappeared

or whose intensities greatly decreased in 0.5 μM spectrum are marked

with blue arrows 138

Figure 5.18 Aβ(1-40) His Hβ/Cβ chemical shift gradually upfield shifted when

peptide concentration decreases from 100 μM to 0.5 μM. (red: 100 μM,

green: 1 μM, blue: 0.5 μM) 138

xiv

Acknowledgements

I would like to take this opportunity to express my gratitude to all those who have

helped me during my PhD studies.

First of all, I would like to sincerely thank my research advisor Dr. Michael

Zagorski for his guidance, patience, understanding and encouragement during my

graduate studies. I would also like to thank all members past and present of Dr. Zagorski

group: Liming, Rekha, Mihaela, John, Cindy, Ed and Megan for their friendly help and

support during these years.

I would like to thank Dr. Xian Mao and Dr. Dale Ray in CCSB for helping me with

all NMR experiments, and Dr. Yufeng Tong in Department of Physiology & Biophysics

for helping me on NMR softwares.

I want to thank Dr. J. Admin Mann in Department of Chemical Engineering for

helping me with the surface tension experiments.

I gratefully acknowledge all my committee members, Dr. James Burgess, Dr.

Clemens Burda, Dr. Gheorghe Mateescu and Dr. Shu G. Chen, for spending their time to

read this thesis and offering me valuable criticism of my research work.

Last but not the least, I would like to thank my parents, my wife and my daughter,

it is your love and support to enable me to finish this work.

xv

List of Abbreviations

1D one-dimensional

2D two-dimensional

δ chemical shift in ppm

Ǻ angstrom (10-10 meters)

Aβ amyloid beta peptide

AD Alzheimer’s disease

ADDL Aβ derived diffusible ligands

APP amyloid precursor protein

CD circular dichroism

CMC critical micellar concentration

CSI chemical shift index

FMOC 9-Fluorenylmethyloxycarbonyl

HSQC heteronuclear single quantum correlation

HFIP hexafluoro-2-propanol

HPLC high pressure liquid chromatograph

Hz hertz

m/z mass/charge

MALDI matrix assisted laser desorption ionization

MALLS multiple angle laser light scattering

MET methionine

NMR nuclear magnetic resonance

NOE nuclear overhause effect

NOESY nuclear overhauser enhancement spectroscopy

PB phosphate buffer

PHE phenylalanine

ppm parts per million

xvi

PF protofibrils

PFG pulsed field gradient

RP-HPLC reverse-phase high pressure liquid chromatography

SDS sodium dodecyl sulfate

SEC size exclusion chromatography

TFA trifluoroacetic acid

TOCSY total correlation soectroscopy

TSP sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4

UV ultraviolet

WT wild type

xvii

Mechanism of Early Stage Abeta Amyloid Formation

Abstract

by

LEI LI

The brains of patients with Alzheimer’s disease (AD) are characterized by an

abundance of amyloid plaques that contain the Aβ peptide. Numerous studies have

established that the soluble oligomers, and not the insoluble amyloid plaques, constitute

the real culprits responsible for the AD associated neuronal death. This thesis undertakes

two projects that focus on discerning the molecular mechanisms of Aβ aggregation into

soluble oligomers.

The first project was designed to test whether a specific hydrophobic side-chain

interaction occurs during the early stages of Aβ aggregation. A working model of the Aβ

amyloid fibril structure (based on solid-state NMR constraints) has the methyl group of

Met35 positioned above the Phe19 aromatic ring (within 5 Å), and we reasoned that

switching the aromatic (single) ring to a more hydrophobic (two) ring naphthyl system

would stabilize this interaction and increase the random coil → β-sheet conversion that

occurs during Aβ aggregation. Four modified Aβ(1-40) peptides were prepared with the

Phe19 or Phe20 rings substituted with either 1- or 2-naphthyl rings. Circular dichroism

revealed that the Phe19 modified peptides underwent more rapid random → β-sheet

conversions. The 1H NMR spectra of the naphthyl peptides were not appreciably

different from the wild-type peptide, and the chemical shift of the Met35 methyl signal

did not change, suggesting that it may not reside above the naphthyl ring. These results

xviii

xix

suggest that the Phe19-Met35 interaction does not occur in the early stages of Aβ

aggregation, and instead is involved in the later stages of association into β-sheet fibrils.

For the second project, the goal was to determine a high-resolution structural model

of the Aβ oligomer. The NMR spectra of monomers and oligomers (separated by size-

exclusion chromatography) were essentially identical and consistent with random

structure. However, the monomer had significantly lower surface tension and was

random structured by circular dichroism, while the oligomer had higher surface tension

and was β-sheet. These results suggest that the early-formed, soluble Aβ aggregates may

associate into micelle-like structures and that the micelles act as reservoirs that eventually

break down and lead to amyloid fibril formation.

1 Introduction: Alzheimer’s Disease and Amyloid Aβ Peptides

1

1.1 Alzheimer's disease

The name “Alzheimer” was coined after a German neurologist, Dr. Alois

Alzheimer, who in 1907 presented findings about a 51-year-old female patient that

showed severe cognitive disorders pertaining to memory, language, and social

interactions (Alzheimer, 1907; Alzheimer et al., 1995). At that time, both scientists and

the non-science community viewed the dementia as a natural progression of age and as a

result, Alzheimer’s disease (AD) was not differentiated from other types of age-induced

dementia or senility. In fact, it took more than 70-years to show that the dementia of the

Alzheimer type is a disease rather than an inevitable consequence of aging (Möller, H &

Graeber, M, 1998; Blennow, K et al., 2006).

At present, AD has become the most common neurodegenerative disease in the

world with more than 25 million patients and is among the ten leading causes of death in

the developed countries (Minino et al., 2002; Goedert, M. & Spillantini, M., 2006). In the

United States alone, 4.5 million people have the disease and the annual costs for caring

AD patients are over $100 billion (Hebert et al., 2003).

AD rarely occurs in people younger than 60 years old and the prevalence is below

1% in individuals aged 60–64 years. However, the disease shows an almost exponential

increase with age. In people aged between 65 and 75 the AD prevalence is more than

10% and in people over age 75 the prevalence is almost 50%. Owing to the increased life

expectancy and changing population demographics (i.e., the aging of baby boomers), AD

are becoming more and more common in the coming decades. The number of patients is

expected to increase to 13.2 million in the United States by 2050 (and 114 million

worldwide) (Figure 1.1) if new preventive or neuroprotective therapies do not emerge.

2

Figure 1.1 Projected numbers of persons in US population with Alzheimer disease using the 2000 US Census Bureau middle-series estimates of population growth, bounded by high- and low-series estimates (Hebert et al., 2003).

AD is a complex disease and the cause of this disease is not yet fully understood.

There probably is not one single cause, but a combination of many risk factors may affect

each person differently. Besides aging (Evans et al., 1989; Harvey el al., 2003), which is

the most obvious risk factor for the disease, epidemiological statistics have suggested

other factors which include decreased reserve capacity of the brain (Mayeux R., 2003;

Mortimer et al., 2003), head injury (Mortimer et al., 1985; Jellinger, K. A., 2004),

vascular disease (Mayeux R., 2003), genetics (Goate et al., 1991; Corder el al., 1993;

Levy-Lahad et al., 1995; Farrer 1997; Gatz et al., 2006), and poor linguistic ability in

early life (Snowdon et al., 1996). On the other hand, there is evidence suggesting that

dietary intake of homocysteine-related vitamins (vitamin B12 and folate), antioxidants

(such as vitamin C and E), unsaturated fatty acids, and moderate alcohol intake

3

(especially wine) could reduce the risk of Alzheimer’s disease (Luchsinger & Mayeux,

2004).

Clinical diagnosis of AD involves several kinds of evaluations such as medical

history, mental status, memory, reasoning, vision-motor coordination, language skills

evaluation, physical examination, brain scanning, laboratory tests, and psychiatric

evaluation. Nevertheless, physicians can only make a diagnosis of AD with sensitivity of

around 81% and specificity of 70% (Knopman et al., 2001). The most reliable diagnosis

of the disease can be only made by examining brain tissue after patients’ death. However,

early detection is critical to optimize AD treatment since current drugs are more effective

only if they are taken in the initial stages of the disease (Cummings, J. L., 2004) and an

early diagnosis is also important in the case of nonpharmacologic interventions since it

will allow to develop a support system and review financial strategies (Aupperle, P. M.,

2006). Great efforts are being made to identify reliable biomarkers for AD that are

suitable for minimal invasive early diagnosis and prognosis. Several candidate

biomarkers are listed in Table 1.1 (Flirski, M. & Sobow, T., 2005). In addition,

neuroimaging techniques including Computed Tomography CT (Nester, P. J., 2004),

Magnetic Resonance Imaging MRI (Laaksoand. et al., 1996; Jagust et al., 2006) and

Positron Emission Tomography PET (Silverman et al., 2001) also play important roles in

developing early diagnosis.

4

Table 1.1 Biochemical markers of AD. ↑: markers concentration increase in AD ↓: markers concentration decrease in AD (Flirski & Sobow, 2005)

Although a variety of drug treatments can delay AD onset or temporarily reduce its

severity, unfortunately, there is currently no cure or effective long-term treatment for the

disease. Acetylcholinesterase inhibitors (AChEIs) are the mainstays for treating AD and

have become part of standard care (Cummings, J. L., 2004). Four AChEI drugs have been

approved by the U.S. Food and Drug Administration (FDA) for the treatment of AD

which include tacrine (Summers, et al., 1986; Knapp et al., 1994), donepezil (Rogers et

al., 1996; Greenberg et al., 2000), rivastigmine (Rosler et al., 1999; Farlow et al., 2000),

and galantamine (Wilcock et al., 2000; Tariot et al., 2000). Memantine (Lipton S. A.,

2004; Reisberg et al., 2003), a uncompetitive NMDA (N-methyl-D-aspartate) antagonist,

was the fifth drug that was approved in January 2004 by the FDA for moderate to severe

AD treatment. Other AD treatments include intake of vitamins (Grundman, M. 2000),

antioxidants (Luchsinger, J. 2003), ginko biloba (Solomon, P. 2002), nonsteroidal anti-

5

inflammatory drugs (NSAIDs, Etminan et al., 2003), and pharmacological management

of neuropsychiatric symptoms (Brodaty et al., 2003). Currently research is being carried

out to develop anti-amyloid therapy for future AD treatments (Selkoe & Schenk, 2003;

Citron, M. 2004; Siemers, et al., 2005).

1.2 Amyloid beta peptide (Aβ)

1.2.1 Aβ and Alzheimer's disease

A common pathological denominator underlying many diverse neurodegenerative

disorders, including AD, is the aggregation and deposition of misfolded proteins. As

shown in Table 1.2, almost every neurodegenerative disease is associated pathologically

with the insidious accumulation of insoluble filamentous aggregates of normally soluble

proteins in the central nervous system (CNS). Because these filamentous aggregates

display the ultrastructural and tinctorial properties of amyloid (i.e., ~10-nm-wide fibrils

with crossed β-pleated sheet structures that stain with Congo red, thioflavin-S,or other

related dyes), these diseases are usually grouped together and called brain amyloidoses.

For AD, the major characteristic lesions are neuritic plaques and neurofibrillary

tangles (Kidd 1963; Kidd 1964; Terry et al., 1964; Figure 1.2). The finding of the

correlation between plaque counts and dementia severity has put great focus on the

involvement of plaques in the pathogenesis of AD (Blessed et al., 1968; Hardy & Selkoe,

2002). Because of insolubility of those plaques, efforts to identify the protein

composition of plaques were unsuccessful until in the mid-1980s when researchers were

able to purify plaque cores and identify the amino acid sequence of Aβ peptides, the

major component of the plaque (Glenner & Wong, 1984; Masters et al., 1985).

6

Table 1.2 Common neurodegenerative diseases characterized by deposition of aggregated proteins (Skovronsky et al., 2006)

Figure 1.2 Plaques and tangles in the cerebral cortex in Alzheimer's disease. Plaques are extracellular deposits of Aβ surrounded by dystrophic neurites, reactive astrocytes, and microglia, whereas tangles are intracellular aggregates composed of a hyperphosphorylated form of the microtubule-associated protein tau. (Goedert et al., 2006)

7

1.2.2 Biogenesis of Aβ

Aβ is approximately 4 kDa and the length varies from 39 to 43 residues, with most

of the heterogeneity at the carboxyl terminus. However, the two predominant species that

exist in the senile plaques are the Aβ(1-40) and Aβ(1-42). The sequences of the two

peptides are shown in Figure 1.3.

Figure 1.3 Sequence of Aβ(1-40) and Aβ(1-42).

Aβ is generated by endoproteolytic processing of the large type I transmembrane

protein, amyloid precursor protein (APP). (Kang et al., 1987). Enzymes called β- and γ-

secretases cleave the APP to form the N- and C- termini, respectively, of the Aβ peptide

(Vassar et al., 1999, Cai et al., 2001; Schroeter et al., 2003; Kopan and Llagan, 2004;

Huppert et al., 2005); `alternatively, non-amyloidogenic cleavage by α- and γ- proteases

produce the p3 peptide (Gu et al., 2001; Weiderman et al., 2002; Zhao et al., 2004; Kakua

el al., 2006. Figure1.4)

The minor differences in the C-terminal cleavage at Val40 or Ala42 have profound

consequence on the propensity of aggregation and fibril formation. Aβ(1-40) is the

predominant species produced during APP processing. However, Aβ(1-42), which

accounts for only about 10% of total secreted Aβ, is more hydrophobic and much more

prone to aggregate than Aβ(1-40) (Hilbich et al., 1991; Burdick et al., 1992; Jarrett et al.,

1993). Studies also indicate that early-onset plaque formation depends on the levels of

8

Aβ(1-42) (Rockenstein et al., 2001) and fibrillar Aβ(1-42) is able to seed fibril formation

of Aβ(1-40) (Harper & Lansbury, 1997). Moreover, inherited mutations in the APP gene

near the β- and γ- secretase cleavage sites all increase Aβ(1-42) production: mutations

near the β-secretase cleavage site augment elevated production of both Aβ(1-40) and

Aβ(1-42) (Citron et al., 1992; Cai et al., 1993) whereas those near the γ- site specifically

increase production of Aβ(1-42) (Suzuki et al., 1994). Further evidence came form the

discovering of mutations within the presenilin 1- and 2- genes which increase the Aβ(1-

42)/Aβ(1-40 ) ratio throughout life and cause early, aggressive forms of AD (Bentahir et

al. 2006; Kumar-Singh et al. 2006). Taken together, Aβ(1-42) appears to be the major

culprit pathogenic species in AD (Haass & Selkoe, 2007; Findeis, 2007).

Figure 1.4 APP processing and Aβ production. (A) APP can be cleaved sequentially by β-secretase (BACE) and γ-secretase: a protease complex containing presenilin (PS) as the putative catalytic component to produce Aβ. (B) Alternatively, APP can be processed by α-secretase and γ-secretase to produce P3 (Esler et al., 2001)

9

1.2.3 The normal roles of APP and Aβ peptides

The normal functions of APP and Aβ are not fully understood. However, their

importance were suggested by experiments with APP knockout mice (Zheng et al., 1995;

Senechal et al., 2008) in which the mice are still viable and fertile, however, the mice do

show differences from normal mice including lower weight, reduced locomotor activity

and eventual reactive gliosis in the brains, and age-dependent deficits in passive

avoidance learning. In addition, the APP intracellular domain (AICD) resulting from γ-

secretase cleavage of APP regulates phosphoinositide-mediated calcium signaling

through a γ-secretase-dependent signaling pathway, suggesting that the intramembranous

proteolysis of APP may play a signaling role during cell differentiation (Leissring et al.,

2002). Recently, full-length APP was found to functions as an important factor for proper

migration of neuronal precursors into the cortical plate during the development of the

mammalian brain (Tracy et al., 2007). Moreover, Aβ(1-40) is suggested to be produced

as a cellular antioxidant (Teng & Tang, 2005). Both Aβ(1-40) and Aβ(1-42) can

modulate potassium channels in neurons and Aβ(1-40) is also suggested to be able to

counteract the effects of secretase inhibitors (Plant et al., 2003).

1.2.4 Aβ neurotoxicity: Aβ oligomers are the primary neurotoxic species in AD

Initially, processing of APP to produce Aβ was thought to be abnormal and is the

first pathological alteration in AD (Müller & Beyreuther, 1989). However, this notion

was dispelled when Aβ was found to be normally secreted by many types of cells

throughout life and to be present in the cerebrospinal fluid and plasma of humans and

lower mammals (Shoji et al., 1992; Haass et al., 1992; Seubert et al., 1992; Busciglio et

10

al., 1993; Ida et al., 1996; Walsh et al., 2000). Thus, the mere presence of Aβ does not

cause neurodegeneration, rather the neuronal injury appears to be the result of the ordered

self-association of Aβ molecules.

The pathogenetic role of Aβ in AD is described by the central hypothesis for the

cause of the disease: the amyloid cascade hypothesis (Hardy & Selkoe, 2002, Figure 1.5).

Figure 1.5 Amyloid cascade hypothesis. Increased aggregation of Aβ can occur as a result of (a) overproduction of Aβ(1-42) [as in the case of most amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2) gene mutations], (b) expression of mutations in the Aβ domain of APP that increases its propensity for aggregation, (c) Apolipoprotein E4 (apoE4) expression, and (d) other genetic and environmental factors, including aging. Aggregated Aβ accumulates in various forms and locations, some or all of which may result in cellular toxicities mediated by a variety of mechanisms. Decreased clearance of aggregates and failures of cellular defenses to toxicity may exacerbate this process. The toxic effects of amyloid result eventually in neuronal death and dysfunction, manifesting as dementia. (Skovronsky et al., 2006)

11

The amyloid cascade hypothesis states that an imbalance between the production

and clearance of Aβ in the brain is the initiating event of the disease, resulting in the

accumulation and aggregation of Aβ, and ultimately leading to neuronal degeneration and

dementia. Support for this hypothesis includes the findings that mutations implicated in

the familial disease are present in the genes for both the substrate (APP) and presenilin

and both the APP and presenilin mutations increase Aβ42 production (Goate et al., 1991;

Scheuner et al., 1996; Wolfe et al., 1999); people with Down’s syndrome, who possess an

extra APP gene, develop Aβ plaques early in life (Citron et al. 1992; Cai et al. 1993;

Suzuki et al. 1994; Bentahir et al. 2006; Kumar-Singh et al. 2006; Pelfrey et al. 1993; Ida

et al. 1996; Walsh et al. 2000), and finding of a duplication of the APP locus in families

with familial Alzheimer’s disease (Rovelet-Lecrux et al. 2006). In addition, evidence

form in-vitro studies using synthetic Aβ also suggested that aggregated Aβ is toxic to

neurons (Pike et al. 1991; Busciglio et al. 1992; Yankner, 1996; Geula et al. 1998; Kayed

et al 2003).

Self association or aggregation of Aβ can form many different kinds of Aβ

aggregates, including small dimers and the large mature insoluble Aβ fibrils as found in

brains. The insoluble Aβ filamentous aggregates has long time been regarded as the

pathological hallmark of AD, and therefore much research has focused on these insoluble

species. However, in recent years, an increasing body of data has suggested that these

insoluble Aβ fibrils may not be the disease causing agent, rather the soluble, pre-fibrillar

Aβ oligomers are indeed the proximate effectors of synapse loss and neuronal injury

(Haass & Selkoe, 2007).

The indication that the insoluble Aβ aggregate might not be the primary disease-

12

causing species came from human AD studies. It has long been recognized that amyloid

plaque numbers do not always correlate well with the severity of dementia, amyloid

plaques are present in brains from individuals without any cognitive dysfunction or loss

(Katzman 1986; Terry et al. 1991; Dickson et al. 1995; Lue et al., 1999) and mouse

models with synaptotoxicity impaired long-term potentiation (LTP) and cognitive

impairment occur before the formation of amyloid plaques (Larson et al., 1999; Mucke et

al., 2000; Billings et al., 2005). All these data demonstrate that neuronal dysfunction and

loss can occur in the absence of the detectable insoluble plaques. In fact these

observations have been frequently cited as a critical flaw in the amyloid cascade

hypothesis.

In contrast, evidence that the soluble Aβ oligomer might be the toxic species in AD

was supported by the strong correlation of soluble Aβ oligomer with the degree of

synapse loss in AD brain and cognitive decline (Lue et al. 1999). The amount of soluble

amyloid could clearly distinguish tissue from individuals without cognitive dysfunction

from AD tissue, despite similar insoluble plaque burden (McLean et al. 1999; Wang et al.

1999). Injection of soluble Aβ oligomers into the rat brain was sufficient to both disrupt

hippocampal LTP (Walsh et al., 2002 (a)& (b)) and cause deficits in learning behavior

(Cleary et al., 2005). Treatment of cortical neurons with Aβ oligomer led to the

downregulation of N-methyl-D-aspartate (NMDA) receptors and a depression in NMDA-

evoked currents (Snyder et al., 2005). Recently, a specific soluble Aß assemblies (Aß*56)

was identified which inversely correlated with spatial memory in AD adult mice, and

injection of the extracted, soluble Aß*56 into the brains of young rats impaired memory,

suggesting that soluble Aß*56 is the principle cause of memory decline (Lesne et al.,

13

2006).

Many types of assembly of Aβ oligomers (synthetic or natural) have been described

in the literature including: protofibrils (PF). (Harper et al., 1997; Hartley et al., 1999;

Walsh et al., 1997; Walsh et al., 1999), Aβ derived diffusible ligands (ADDLs). (Lambert

et al., 1998; Gong et al., 2003), Aβ*56 (Lesne et al., 2006), annular Aβ assemblies

(Lashuel et al., 2002; Bitan et al., 2003), and Aβ dimers and trimers (Podlisny et al.,

1995; Walsh et al., 2000; Walsh et al., 2002). The term “soluble Aβ oligomers” is not

strictly defined. Usually, “soluble Aβ oligomers” can describe any form of Aß that is

soluble in aqueous buffer and remains in solution following high speed centrifugation.

Measurement of the soluble Aβ oligomers has been achieved using assays that cannot

identify the aggregation state of the species detected (Funato et al. 1998; Morishima-

Kawashima and Ihara 1998; Stenh et al. 2005). Thus, although the detailed assembly

states of these Aβ oligomers are unknown, their failure to pellet following

ultracentrifugation indicates that they are not fibrillar in nature.

The pathogenesis of Aβ oligomers in AD is not clear. There is evidence showing

that ADDLs of synthetic human Aβ (Lambert et al., 1998) and soluble, low-number

oligomers of naturally secreted human Aβ (Walsh et al., 2002; Kamenetz et al., 2003) can

inhibit the maintenance of hippocampal LTP; moreover, Aβ oligomers are found to be

able to bind to synaptic plasma membranes and interfere with the complex system of

receptor and/or channel proteins and signalling pathways that are required for synaptic

plasticity (Kamenetz et a., 2003; Cirrito et a., 2005). Nevertheless, identification of Aβ

oligomers as the primary toxic species provides an opening for understanding the basis of

memory loss in AD and prevention of these toxic soluble species is currently being

14

evaluated as a potential therapeutic intervention for the disease (Lleo et al., 2006).

1.2.5 Structural studies of Aβ

Knowledge of self association of Aβ to form soluble oligomers or high order

aggregates can provide invaluable information in understanding the high neurotoxicity of

these species as well as guide with therapeutic designing. However, despite over a decade

of research efforts, the molecular mechanisms of Aβ oligomer formation remains largely

unknown, which is partly due to the lack of high resolution structural data.

Studies have shown that the protein secondary structure, β-sheet structure, plays an

important role in AD pathogenesis. In contrast to α-helix structure in which hydrogen

bonding is formed between residues within the same strand, the hydrogen bonding in β-

sheet structures is among strands. This structural feature of the β-sheet allows the

formation of intermolecular β-pleated sheets, which could be stabilized by protein

oligomerization and aggregation (Soto, 1999). Accumulating evidence has established

that during the self association of the Aβ a conformational change occurs and the Aβ

peptide becomes neurotoxic when aggregated as β-sheet structures (Pike et al., 1995;

Seilheimer et al., 1997; Walsh et al., 2002). Tinctorila analysis of the secondary structure

of Aβ within the amyloid deposits revealed β-sheet structure (Glenner et al., 1972;

Glenner, 1980a; Glenner et al., 1980b). Fiber X-ray diffraction analysis of ex-vivo

amyloid preparation showed a silk-like cross β-pleated sheet organization of Aβ fibrils

(Kirschner et al., 1986). In situ FTIR studies of the amyloid plaques of AD brains also

showed that the core of the plaques gives a strong β-sheet signal from surrounding areas

of the grey matter (Choo et al., 1996)

Aβ peptides in solution (before precipitation as amyloid) can adopt random coil, α-

15

helix, or β-sheet in relative ratios that is modulated by extrinsic factors such as the pH,

peptide concentration, ionic strength, metal ions, membrane-like surfaces, solvent

hydrophobicity and temperature (Teplow, 1998; Zagorski et al., 1999). CD spectra of Aβ

in aqueous buffers indicate the presence of extended random coil conformation and β-

sheet. A time dependent conformational change from the random coil to β-sheet usually

occurs in aged solution (Soto et al., 1995a; Soto et al., 1995b). Fibril formation and

precipitation occurs following β-sheet formation, indicating the formation of the β-sheet

structure is directly related to Aβ peptide aggregation (Barrow et al., 1992; Barrow &

Zagorski, 1991).

Studies have also shown that the principal structural motif in amyloid fibrils is the

cross-β sheet structure. (Kirschner et al., 1986; Miyakawa et al., 1986). X-ray diffraction

analysis of Aβ fibrils suggested a model in which a series of “cross-β” pleated sheets are

oriented parallel to the main axis of the fiber, while the direction of the constituent β-

strands is perpendicular to the fibril axis, making the hydrogen bonds between strands

parallel to the fibril axis (Black & Serpeell, 1996; Inouye et al., 1993; Sunde et al., 1997;

Malinchik et al., 1998; Serpell et al., 2000; Serpell & Smith, 2000). However, this model

is not consistent with results from solid state NMR (11–13) which implies an in-register

parallel (Balbach et al., 2002) or antiparalell (Lansbury et al., 1995; Balbach et al., 2000)

β-sheet alignment of peptide chains within the cross-β motif in Aß fibrils.

Shown in Figure 1.6 is a solid state NMR structural model of Aß(1-40) fibrils that

has received great attention recently (Petkova et al., 2002; Petkova et al., 2006). In this

model, Aβ residues 1–8 are conformationally disordered while residues 12 –24 and 30–

40 adopt β-strand conformations with each β-strand segment forming separate parallel β-

16

sheets through intermolecular hydrogen bonding. Residues 25-29 contain a bend of the

peptide backbone that brings the two β-strands in contact through sidechain-sidechain

interactions. The only charged side chains in the core, Asp 23 and Lys 28, form a salt

bridge.

Figure 1.6 Structural model for Aβ(1-40) fibrils (a) Ribbon representation of residues 9-40, viewed down the long axis of the fibril. Each molecule contains two β-strands (red and blue) that form separate parallel β-sheets in a double-layered cross-β motif. Two such cross-β units comprise the protofilament, which is then a four-layered structure. (b) Atomic representation of residues 1-40 with color coding to indicate residues with hydrophobic (green), polar (magenta), positively charged (blue), and negatively charged (red) side chains. Backbone C=O and N-H bond vectors are approximately perpendicular to the page. Contacts between β-sheet layers are through side chain-side chain interactions. N-Terminal residues are assigned random conformations to indicated structural disorder. Contacts between the two cross-β units are assumed to be along the hydrophobic faces created by side chains of the C-terminal segment. The core of the protofilament is hydrophobic with the exception of the oppositely charged side chains of D23 and K28, which form salt bridges (Petkova, et al., 2006)

17

Structural elucidation of soluble Aβ oligomers has also received broad attention.

For example, Aβ PFs (Harper et al., 1997; Hartley et al., 1999) have flexible structures

that can continue to polymerize in vitro to form amyloid fibrils or can depolymerize to

produce lower-order species. Morphology studies of PFs show that they are narrower

than mature amyloid fibrils (~5 nm versus ~10 nm) and can be up to 150 nm in length.

Tinctorial analysis with Congo red or Thioflavin T indicate PFs contain substantial β-

sheet structure. The annular assemblies of synthetic Aβ (Lashuel et al., 2002; Bitan et al.,

2003) are doughnut-like structures, with an outer diameter of 8–12 nm and an inner

diameter of 2.0–2.5 nm, and can be distinguished from PFs by atomic force microscopy

and electron microscopy. The ADDLs (Lambert et al., 1998; Gong et al., 2003) are even

smaller than annular assemblies and show an apparent hexamer periodicity with hexamer,

dodecamer and octadecamer structures observed. However, despite great research efforts,

site-specific atom level resolution structure of Aβ oligomers still remains largely

unknown due to their intrinsic instability and noncrystallinity.

1.3 Objective and significance of the research

In this thesis we have used NMR along with other biophysical techniques to study

the structural properties of Aβ in solution before it begins to aggregate as fibrils. The

results will provide valuable information in understanding the early events associated

with β-amyloidosis and lead to structure-based design of therapeutic reagents for

treatment of AD.

18

2 Synthesis, Purification and Disaggregation of Aβ Peptides

19

In this thesis, uniformly (>95%) 15N-labeled (or uniformly doubly 15N- and 13C-

labeled) Aβ peptides were purchased from Recombinant Peptides as a recombinant fusion

protein in minimal media containing 15NH4Cl as the sole nitrogen source and/or 13C-

labeled glucose (U-13C6) as the sole carbon source. All other Aβ peptides were

synthesized in our lab. In this chapter, the experimental procedures used to synthesize,

purify and disaggregate the Aβ peptides is described.

2.1 Peptide synthesis

Materials: All Fmoc protected amino acids were obtained from a commercial

source (Anaspec) with side chains protected for the following residues: Ser and Tyr (O-t-

butyl); Asn and Gln (C(O)-NH-trityl); Asp and Glu (COO-t-butyl); His (trityl); Lys (Boc-

NH); Arg(pbf). All other reagents including Fmoc-PEG-PS-Ala or Fmoc-PEG-PS-Val

preloaded resin, NMP, DCM, DMF, Piperidine, HATU, DIEA were obtained from

Applied Biosystems.

Methods: Aβ peptides were synthesized using the standard Fmoc chemistry on a

433A peptide synthesizer (Applied Biosystems). In the procedure, 0.15 g Fmoc-PEG-PS-

Ala resin (for synthesis of Aβ(1-42) or Fmoc-PEG-PS-Val resin (for synthesis of Aβ(1-

40) with a substitution level of 0.2 mmol/g and 1 mmole Fmoc-protected amino acid for

each residue was employed. The peptide chains were assembled from the carboxy

terminus to the amino terminus. Each amino acid was linked to the support by a single

coupling cycle. For a regular synthesis, the single coupling cycle is made of seven

consecutive modules including modules A, C, d, B, D, E, and F. The functions of these

modules are described as follows:

Module A: Activation. The Fmoc Amino Acids are activated by transferring 2 g

20

0.45M HATU solution to the amino acid cartridge

Module C: Capping. The un-reacted amino group on the resin was blocked by

reacting with 0.4 M acetic anhydride

Module d: NMP wash from activator

Module B: Deprotection. The growing peptide N-terminus Fmoc group was

removed by reacting with a solution containing 5% DBU + 20% piperidine

Module D: NMP wash

Module E: Transfer. 1 ml 2 M DIEA solution was transferred to the reaction vessel

Module F: Coupling. The peptide amide bond formation reacts in reaction vessel

The removal of the last Fmoc group was performed on line by a final deprotection

cycle which includes modules I, C, d, B, D, and c in which module c is DCM wash of the

resin. A full program scheme for a regular synthesis of Aβ(1-42) using Fmoc-PEG-PS-

Ala resin is listed in Table 2.1 :

Table 2.1 A regular peptide synthesis scheme for Aβ(1-42) on 433A peptide synthesizer. 1 mmole Fmoc-amino acid were used for each residue

21

The above synthesis method can be used when required Fmoc-amino acids are

cheap and plentiful. However, in case that the amino acids is not readily available, for

example, when synthesizing 13C or 15N labeled peptide, instead of using 1 mmole amino

acid for each residue, 0.1mmole isotope labeled Fmoc-amino acids was used, and the

above mentioned single coupling cycle has to be modified. The new coupling cycle is

defined as “0.1 mmol single coupling” and consisted of eight consecutive modules

including modules a, C, d, B, D, e, F and I in which modules a, e and I are described as

follows:

Module a: Activation. The 0.1 mmole Fmoc Amino Acids were activated by

transferring 1 g 0.09 M HATU solution to the amino acid cartridge

Module e: 1 ml 0.4 M DIEA solution was transferred to the reaction vessel

Module I: Vortex 10 minutes for extended coupling

Compared to the regular single coupling cycle, the activation and transfer modules

for 0.1 mmole Fmoc-amino acids are replaced by module a and e and an extended vortex

function (Module I) are added to the cycle to help completeness of coupling.

A full program scheme for a synthesis of Aβ(1-42) in which methionine-35,

alanine-30, alanine-21 and alanine-2 are 13C labeled are listed in Table 2.2. The synthesis

process was monitored by a built-in conductivity detector by measuring the solvent

conductivity during Fmoc deprotection. A successful synthesis was usually completed in

about 40 hours for Aβ(1-42). After the synthesis, the resin was washed three times with

methanol and dried under vacuum for 4 hours.

22

Table 2.2 Peptide synthesis scheme for Aβ(1-42) in which methionine 35, alanine 30, alanine 21` and alanine 2 are 13C labeled. 0.1 mmole Fmoc amino acids were used for these 13C labeled residues. 1 mmole Fmoc amino acids were used for other residues.

The peptide was cleaved by treating the resin with a freshly prepared mixture of

TFA and a scavenger cocktail reagent (86.5% TFA, 5% thioanisole, 5% water, 2.5%

ethane dithiol, 1% triisopropylsilane) with shaking for 4 hours. The amount of cleavage

reagent was approximately 20 ml/g of the peptide resin. Peptide was precipitated into

cool ethyl ether (100 ml ethyl ether/100 mg resin). Ether was removed by centrifugation.

Crude peptide was washed three times with ethyl ether, then dissolved in water (0.1%

TFA may be added to dissolve the peptide) and subsequently lyophilized to dryness.

Crude Aβ(1-42) peptide was analyzed by MALDI-MS (matrix assisted laser desorption

23

ionization mass spectrometry) with α-cyano-4-hydroxycinnamic acid as the matrix. The

theoretical mass for an unlabeled peptide is 4515. A major molecular ion peak 4513.3 is

observed indicating the synthesis was successful.

2.2 HPLC purification of Aβ peptides

The great aggregation propensity of the Aβ peptide makes HPLC purification a

challenging task. With great care in sample preparation and HPLC condition optimizing,

reproducible HPLC purification of Aβ is possible.

10 mg crude Aβ(1-42) peptides was dissolved in 5 ml TFA. The solution was

sonicated for 15 minutes. TFA was evaporated under a flow of dry nitrogen gas.

Afterwards, 0.5 ml HFIP was added to dissolve the peptide and the solution was

sonicated for another 5 minutes. 0.5 ml water was added to the solution and quickly

mixed. 1 ml crude peptide solution was injeted into HPLC column.

A waters model 600E HPLC system equipped with a Vydaq 259-VHP822

preparative column (22 mm i.d. x 250 mm L) containing highly cross-linked polystyrene-

divinylbenzene copolymer beads with 300 Å pores (Separation Group). The column was

heated to 60°C to improve peak resolution. The solvent system consisted of a linear

gradient of 20-80% acetonitrile in water that contained 0.08-0.1% trifluoroacetic acid.

The peptide elutant was monitored by UV at 220 nm and the major fraction

corresponding to peptides was collected.

The purified peptides were characterized by MALDI (Figure 2.1) and 1D 1H NMR

(Figure 2.2) with an estimated purity greater than 90%.

24

Figure 2.1 MALDI mass spectrum of synthesized Aβ(1-42). The theoretical mass for an unlabeled peptide is 4515, and the observed major molecular ion peak was 4513.3 in the Figure

Figure 2.2 1D 1H-NMR spectrum of HPLC purified Aβ(1-42) (100 μM, in 10 mM D2O phosphate buffer, pH 7.3, 5 oC)

2.3 Aβ peptides sample preparation

Biophysical studies of the synthetic Aβ peptides, especially Aβ(1-42) are usually

25

plagued by many difficulties (Teplow 1998). Because Aβ peptides undergo a time- and

concentration-dependent conformational change and aggregation in the acetonitrile-water

mixture during HPLC purification (Shen. et al., 1995), the lyophilized peptides may

already adopt partially aggregated β-sheet structure. Different commercial sources and

product batches make the peptide structure components even more complicated.

Considerable discrepancy existed between different laboratories or even within the same

group because of batch dependent mixture of aggregates and structures. Thus, to prepare

Aβ solutions reproducibly, direct solubilization of Aβ peptides into aqueous media

should always be avoided, and it is imperative to have an aggregate free, monomeric

solution before each experiment.

To disaggregate the peptides, we used a protocol that was developed in the Teplow

group (Fezoui, et al., 2000) which involves predissolution of peptides in dilute base

solution. With great care, this sample preparation provided reproducible results. The

following is a description of this sample preparation method:

Purified peptides were dissolved in freshly prepared 10 mM NaOH solution in a 1:1

ratio (mg:ml) followed by sonication in an water bath for 1 minute. Solution pH 10.5-

11.0 is required. This base treatment will disrupt any preformed aggregates and provide a

monomeric Aβ stock solution. The basic Aβ stock solution was then combined with a

sodium phosphate buffered solution (10 mM, pH 7.1-7.5, pre-cooled to 0-5 oC) in a 1: 3

ratio (ml: ml) and kept cold (0-5 oC) to prevent aggregation. This will give a 50 μM Aβ

solution with pH 7.3-7.5. For NMR experiments, 5% or 95% D2O will be used in place of

H2O.

26

3 The Effect of Phenylalanine-19 Substitution by Naphthylalanine on the Aβ(1-40) Structure

27

3.1 Introduction

Previous work from our lab and others have established that oxidation of the sulfur

atom to sulfoxide in methionine-35 significantly hinders Aβ amyloid fibril formation

(Watson et al., 1998; Palmblad ret al., 2002; Hou et al., 2002). This effect was thought to

be due to the increased polarity imparted by the methionine-35 sulfoxide group at the

hydrophobic C-terminus of the peptide. However, NMR studies performed on Aβ

peptides with methionine-35 reduced or oxidized showed that the molecular mechanism

behind this inhibition effect is more complicated and suggested oxidation may change the

peptide structure, thus, the Aβ peptides with methionine-35 reduced or oxidized may thus

associate differently during the initial stages of aggregation (Hou, L., et al., 2004).

In the Aβ structural model based on solid state NMR (Figure 3.1, Petkova et al.,

2002), the ε-SCH3 group of methionine-35 is proximate to the center of the

phenylalanine-19 aromatic ring. Such a configuration represents a favorable aryl-sulfur

interaction that is commonly found in protein structures. Inspired by this finding, we

hypothesized that the aryl-sulfur interaction between methionine-35 and phenylalanine-

19 is crucial for Aβ aggregation, as it stabilize the formation of a β-strand structure,

oxidation of the methionine-35 sulfur atom could weaken this aryl-sulfur interaction, and

in doing so weaken the β-strand structure and slow down the Aβ aggregation rate.

NMR studies of wild type Aβ peptides did not show evidence to support the aryl-

sulfur interaction between methionine-35 and phenylalanine-19 (Hou, L., et al., 2004).

However, this could be due to the fact that the aryl-sulfur interaction is weak and not

strong enough for detection. It has benn very well known that the strength of aryl-sulfur

interaction greatly depends on distance and angle between sulfur atom and aromatic rings

28

(Reid et al., 1985). Thus, as an effort to investigate whether there is such aryl-sulfur

interaction in Aβ peptides, we synthesized Aβ peptides with phenylalanine-19 substituted

by naphthylalanine, hoping that the additional phenyl ring introduced by naphthylalanine

may strengthen the aryl-sulfur interaction and facilitate its detection by NMR.

Figure 3.1 Structural model of Aβ(1-40) based on solid state NMR (Petkova et al., 2002). We hypothesized an aryl-sulfur interaction exists between the Met35 ε-SCH3 group and the Phe19 aromatic ring (5-6 Å), which could become disrupted by methionine oxidation and thus account for the decreased aggregation rate of oxidized Aβ peptides.

29

3.2 Results

3.2.1 Naphthyl peptides

Aβ(1-40) were synthesized with Phe19 substituted by 1- or 2-naphthylalanine

(Figure 3.2). As control experiments, Aβ(1-40) with Phe20 substituted by 1- and 2-

naphthylalanine were also synthesized. In this chapter, these naphthylalanine containing

Aβ peptides were denoted as follows:

Aβ(1-40)-1-naphthylalanine19: Phe19 substituted by 1-naphthylalanine




Figure 3.2 Structures of 1- and 2-naphthylalanine

In this chapter, when we are doing NMR studies of naphthylalanine substituted Aβ

peptides, the chemical shift of Ala21 was found to be more affected than other residues.

To further investigate the role of Ala21 in Aβ structures, we then synthesized another

Aβ(1-40) mutant, Aβ(1-40) Flemish mutant Aβ(1-40)-Glycine21, in which Ala21 was

substituted by a Glycine, and studied its structure by NMR.

30

3.2.2 CD studies of naphthylalanine substituted Aβ(1-40)

Before conducting NMR experiments, we did the circular dichroism experiments to

compare the conformational and aggregation properties between the wild type and

naphthylalanine substituted Aβ(1-40) peptides. This enabled us to explore quickly the

influence of naphthylalanine substitution on Aβ structure information.

Figure 3.3 presents the time dependent CD spectra of all studied Aβ(1-40) peptides.

Because Aβ structure and aggregation properties are highly sensitive to

environmental conditions (concentration, pH, temperature, etc), to ensure the obtained

CD spectra of each peptide are comparable, an identical sample preparation protocol

(such protocol can be found in Chapter 2 “synthesis and purification”) was used for

making all peptide solutions. The final concentration of each peptide solution was 50 μM

and the pH were between 7.2 and 7.4. The peptides solutions were incubated at room

temperature, and CD spectra were taken at indicated incubation times.

The CD spectra of wild type Aβ(1-40) and the Flemish mutant (Aβ(1-40)-

Glycine21) indicate both peptides adopt predominantly random structures at 0 hour aging,

as represented by the major negative bands between 195 nm and 200 nm. The CD spectra

of the two peptides after 144 hours aging, however, revealed very few changes compared

to those recorded at 0 hour aging, suggesting no structural changes occurred throughout

the incubation period. In fact, no CD spectra change was observed even after extensive

aging (2-3 weeks). The results obtained here are similar to a previous report (Walsh et al.,

2001) in which the aggregation properties of the two peptides were compared using the

method of Thioflavin T binding assay. Their results indicated that the Flemish mutant

have better solubility in water and aggregated even slower than wild type Aβ(1-40).

31

The Aβ(1-40) peptides with Phe19 substituted by naphthylalanine exhibited

distinct CD behavior than the wild type Aβ(1-40).

The Aβ(1-40)-1-Naphthylalanine19 peptides initially adopts the random structure

as indicated by the negative band at 198 nm. As the peptide is aging, the intensity at 198

nm in the CD spectra starts decreasing while the intensity at 217 nm starts increasing.

These CD spectra intensity changes suggest the peptide conformation is changing from

random to β-sheet. An isodichroic point is observed in the CD spectra, indicating only

two conformations (random and β-sheet structure) existed for the peptide during the

whole aging process. The random to β-sheet conversion stopped before the peptide

conformation reached complete β-sheet structure, as the intensity at 195 nm in the CD

spectra, which would be positive for a typical β-sheet structure, remained negative during

the whole incubation period. Very few CD spectra changes were observed after 144 hours

aging.

Aβ(1-40)-2-Naphthylalanine19 has similar CD results to those of Aβ(1-40)-1-

Naphthylalanine19, except that the random to β-sheet structure conversion rate

accelerated. Unlike Aβ(1-40)-1-Naphthylalanine19, of which the absorbance at 195nm

remains negative throughout the aging process, the Aβ(1-40)-2-Naphthylalanine19 has

positive absorbance at 195 nm with negative absorbance at 217 nm after 72 hours aging

indicating the peptides took primarily β-sheet structure. An isodichroic point is also

observed during the conformational conversion process, however, careful inspection

reveals that the CD spectrum taken after 144 hours aging shifted from the isodichroic

point, suggesting that in addition to the random and β-sheet structure there might be other

conformational structure formed after 144 hours aging.

32

The above CD studies clearly established that naphthylalanine substitution at

peptide position 19 significantly change the structure and aggregation property of Aβ(1-

40) peptides. However whether this substitution effect is unanimous or residue specific

has yet to be determined. Thus same experiments were also performed for Aβ(1-40)

peptide analogs in which Phe20 were substituted by naphthylalanine.

No isodichroic point was observed during the aging process of Aβ(1-40)-1-

Naphthylalanine20. The peptide conformation slowly changed from random to β-sheet

during the first 24 hours as indicated by the intensity decrease at 198nm and intensity

increase at 217 nm in the CD spectra. However, after 24 hours aging, this conformational

conversion stopped and the whole CD spectra intensities started decreasing. This

decrease continued until the CD spectra got to the background spectrum (the spectrum

that is recorded only with phosphate buffer). The conformation of the peptide remained

predominantly random during aging process.

Unlike Aβ(1-40)-1-Naphthylalanine20, the Aβ(1-40)-2-Naphthylalanine20 was

stable for the first 24 hours, as the CD spectra recorded at 0 hour and 24 hour aging were

essentially same. However, after 24 hours aging, the CD spectra intensity started

decreasing with a rate that is even faster than that of Aβ(1-40)-1-Naphthylalanine20.

Within 144 hours, the CD spectra intensity dropped to the level of background spectrum.

No isodichroic point or β-sheet structure was observed. The peptide conformation

remained predominantly random during aging process.

Overall, the above CD studies established that naphthylalanine substitution change

the conformational property of Aβ(1-40). However, these changes can not be simply

explained by the increased hydrophobicity of the peptide (as an additional hydrophobic

33

group, the phenyl ring, is introduced into the peptide by naphthylalanine), as different

naphthylalanine molecules (1-, or 2-naphthylalanine) and different substitution position

(19 or 20) resulted in completely different conformation for the peptide: Naphthylalanine

substitution of Phe19 favors β-sheet structure formation of the peptide, while

naphthylalanine substitution of Phe20 does not change the peptide conformation but

accelerates the aggregation in a manner that no typical β-sheet structure can be observed

during aggregation. A possible rationale for these differences is that the aryl-sulfur

interaction only exists bwtenn Met35 and Phe19, but not bwtenn Met35 and Phe20.

Naphthylalanine substitution of Phe19 will enforce the aryl-sulfur interaction which

subsequently stabilize and favor the β-sheet structure formation. And because there is no

such interaction between Met 35 and Phe 20, naphthylalanine substitution at position 20

will not favor the β-sheet structure formation but simply enhance the peptide

hydrophobicity and aggregation, but the peptide conformation will still remain.

34

Figure 3.3 Time dependent CD spectra of wild type and naphthylalanine substituted Aβ(1-40) peptides (50 μM, pH 7.2~7.4, 25oC)

35

3.2.3 Aβ(1-40) aryl-sulfur interaction studies using NMR

The possible aryl-sulfur interaction between Met35 and Phe19 in Aβ(1-40) was

studied by NMR.

First we tried to measure the chemical shift of Met35 ε-methyl protons in different

naphthylalanine substituted Aβ(1-40) peptides. The rationale here is, if aryl-sulfur

interaction exists between Met35 and Phe19, naphthylalanine substitution of Phe19 will

strengthen this interaction, because there will be more aromatic rings interacting with the

sulfur atom and the chemical environments of the ε-methyl protons will thus change. As a

result, the chemical shift of the ε-methyl protons will also change.

The chemical shift of Met35 ε-methyl protons were measured by 13C-1H HSQC

experiment. 13C-1H HSQC experiment only detects protons that are directly connected to

the 13C atoms. In our experiments, the Met35 ε-methyl carbon was 13C labeled, thus only

the ε-methyl protons can be detected by NMR. By this way the ε-methyl proton chemical

shifts can be precisely determined and any small chemical shift difference can be easily

identified. A typical 13C-1H HSQC of wild type Aβ(1-40) is shown in Figure 3.4.

36

Figure 3.4 13C-1H HSQC spectra of Aβ(1-40) (50 μM, pH 7.2, 5oC). Met35 ε-methyl carbon was 13C labeled, and as internal references the methyl carbons of three Ala residues in Aβ(1-40) were also 13C labeled. Carbon decoupler is turned off during HSQC measurement, thus each peak split into two by 1JC-H coupling. Chemical shifts of Met35 ε-methyl protons were measured as an average of two splitted peaks.

The 13C-1H HSQC measurements were also performed for all other Aβ(1-40)

naphthyl analogues. Surprisingly, all peptides gave exactly same Met35 ε-methyl

chemical shifts, suggesting there may not be aryl-sulfur interaction between Met35 and

Phe19 in Aβ(1-40)!

Our second NMR approach to study the aryl-sulfur interaction between Met35 and

Phe19 is to measure NOE between Met35 and Naphthylalanine19. NOE measures the

distance between two atoms. However, NOE measurement can only be possible when the

distance is shorter than 5Ǻ. Although in the Aβ molecular model (Figure 3.1), the

measured distance between Phe19 aromatic ring and Met35 ε-methyl is within 5-6 Ǻ, no

NOE observation has ever been published between these two residues. The possible

reason could be because, in wild type Aβ(1-40), the distance between Phe19 aromatic

ring and Met35 side chains is actually farther than 5 Ǻ . If the proposed aryl-sulfur

interaction exists, nathyalanine substitution of Phe19 will enforce this interaction and

37

decrease the distance, and consequently, NOE detection between these two groups should

become possible.

Unfortunately, despite great efforts on many trials using different kinds of NOE

measurement methods, no NOE signal is observed between Met35 and

Naphthylalanine19.

Although the above site specific (for Met35 and Phe19) NMR studies do not

support our hypothesis of aryl-sulfur interactions in Aβ peptides, it may indicate the aryl-

sulfur interactions is not in the early stage of Aβ aggregation. In addition, the CD results

(Figure 3.3) did show that naphthylalanine substitution significantly change the

secondary structure and aggregation property of Aβ(1-40). Fully understanding of this

naphthylalanine substitution effect may be helpful to clarify the structure and aggregation

mechanism of wild type Aβ(1-40). Thus, more NMR studies were performed on those

naphthylalanine substituted Aβ(1-40) peptides.

3.2.4 NMR studies of naphthylalanine substituted Aβ(1-40)

3.2.4.1 1H chemical shift assignments of naphthylalanine substituted Aβ(1-40)

Proton chemical shifts assignments of four naphthylalanine substituted Aβ(1-40)

plus the Aβ(1-40) Flemish mutant were achieved using the standard procedures (Case and

Wright, 1993; Wuthrich, 1986).

Shown in fgure 3.5 and 3.6 are the expanded region of the TOCSY and NOESY

spectrum of Aβ(1-40)-1-naphthylalanine19, respectively. Spin system identification was

made by analyzing the scalar coupling patterns observed in the TOCSY spectrum.

Protons were assigned to specific amino acid types. The correlation of the αH, βH, and

38

γH to each NH are shown in the Figure 3.5. Sequential resonance assignments were

completed on the basis of inter-residue connection between back bone protons (NH, αH).

Sequential connectivities of αN(i, i+1), and NN(i, i+1)) are shown in Figure 3.6 and 3.7,

respectively. The complete assignment was listed in table 3.1.

Chemical shift assignments of all other naphthylalanine substituted Aβ(1-40)

peptides were conducted in similar manners The complete assignments were listed in

table 3.2 to 3.5 and the NMR spectra were shown figures 3.8 to 3.19.

39

Table 3.1 Proton chemical shift assignments for Aβ(1-40)-1-naphthylalanine19 a

Residue NH HA HB Others D1 b 4.12 2.68, 2.79 A2 8.15 4.3 1.36 E3 8.59 4.19 1.90, 1.94 γH 2.12, 2.23 F4 8.43 4.55 3 2,6H 7.15 3,5H 7.24 4H 7.30 R5 8.24 4.27 1.62, 1.72 γH 1.49, δH 3.12 H6 8.51 4.5 3.03, 3.10 2H 7.98 4H 7.06 D7 8.45 4.61 2.67 S8 8.53 4.37 3.88, 3.92 G9 8.64 3.88, 3.95 Y10 8.05 4.53 2.94, 3.02 2,6H 7.06 3,5H 6.78 E11 8.51 4.2 1.83, 1.92 γH 2.18 V12 8.21 3.94 1.95 γH 0.79, 0.89 H13 8.35 4.58 3.02 2H 7.94 4H 6.99 H14 8.23 4.55 3.01 2H 7.96 4H 7.00 Q15 8.48 4.25 2.06 γH 2.31 K16 8.48 4.26 1.73, 1.79 γH 1.35, 1.43 δH 1.65 εH 2.93 ζ NH2

b

L17 8.36 4.33 1.64 γH 1.44 δH 0.84, 0.92 V18 8.04 4.05 1.92 γH 0.78, 0.86 F19 8.46 4.72 3.36, 3.48 2H 7.32 3H 7.43 4,6H 7.85 7,8H 7.42 9H 7.58 F20 8.03 4.45 2.83, 3.02 2,6H 7.20, 3,5H 7.30, 4H 7.27 A21 8.27 4.12 1.38 E22 8.49 4.19 1.93, 2.04 γH 2.27 D23 8.51 4.63 2.61, 2.72 V24 8.27 4.12 2.19 γH 0.97 G25 8.67 3.98 S26 8.27 4.45 3.88, 3.91 N27 8.6 4.74 2.79, 2.87 K28 8.47 4.27 1.77, 1.89 γH 1.40, 1.47 δH 1.68 εH 2.99 ζ NH2

b G29 8.54 3.93 A30 8.14 4.3 1.37 I31 8.31 4.16 1.86 γH 1.20, 1.53 δH 0.87, 0.89 I32 8.43 4.16 1.87 γH 1.22, 1.50 δH 0.88, 0.92 G33 8.6 3.91 L34 8.18 4.35 1.62 γH 1.61 δH 0.89, 0.93 M35 8.58 4.54 2.03, 2.07 γH 2.52, 2.59 εH 2.09 V36 8.39 4.13 2.09 γH 0.96 G37 8.73 3.99 G38 8.38 3.95 V39 8.21 4.19 2.1 γH 0.95 V40 7.96 4.06 2.07 γH 0.91

a the data was obtained in 10 mM phosphate buffer, pH 7.3, 5 oC, the chemical shifts are reported in ppm relative to internal TSP b these peaks were unassignable due to overlap or exchange with solvent

40

Figure 3.5 The expanded NH region of the TOCSY spectra of 50 µM Aβ(1-40)-1-Naphthylalanine19 (10

mM phosphate buffer, pH 7.3, 5oC). The relayed connections among the NH, αH, βH, and γH are shown.

41

Figure 3.6 Expanded NH-αH region of the NOESY spectrum of 50 µM Aβ (1-40)-1-Naphthylalanine19

(mixing time 270 ms, 10 mM phosphate buffer, pH 7.3, 5oC). Several sequential NOEs are connected as

follows: Solid line (——) residue 28-40; dotted line (······) residue 17-26; dashed line (-----) residue 7-12.

42

Figure 3.7 Expanded NH-NH region of the NOESY spectrum of 50 µM Aβ (1-40)-1-Naphthylalanine19

(mixing time 270 ms, 10 mM phosphate buffer, pH 7.3, 5oC). The NH-NH NOEs are connected as follows:

Solid line (——) residue 30-36; dotted line (······) residue 17-25.

43


Residue H HA HB Others D1 b 4.14 2.67, 2.81 A2 8.13 4.29 1.36 E3 8.57 4.18 1.91 γH 2.11, 2.22 F4 8.42 4.54 2.99 2,6H 7.14 3,5H 7.23 4H 7.30 R5 8.22 4.26 1.62, 1.72 γH 1.48 δH 3.11 εH 7.39 H6 8.49 4.49 3.02 2H 7.97 4H 7.06 D7 8.45 4.61 2.66 S8 8.52 4.37 3.87, 3.91 G9 8.63 3.87, 3.94 Y10 8.04 4.51 2.93, 3.02 2,6H 7.06 3,5H 6.78 E11 8.5 4.19 1.82, 1.91 γH 2.16, 2.2 V12 8.2 3.93 1.95 γH 0.78, 0.88 H13 8.35 4.58 3.01 2H 7.94 4H 6.99 H14 8.23 4.51 2.99 2H 7.96 4H 7.00 Q15 8.47 4.24 1.97, 2.05 γH 2.30 εH 6.98, 7.65 K16 8.46 4.25 1.72, 1.78 γH 1.36, 1.42 δH 1.64 εH 2.95 ζ NH2

b L17 8.26 4.28 1.5 γH 1.33, δH 0.78, 0.87 V18 8.07 4.04 1.9 γH 0.73, 0.83 F19 8.44 4.7 3.12 2H 7.34 3,5,8H 7.84 6,7H 7.54 9H 7.63 F20 8.26 4.52 2.88, 3.06 2,6H 7.20, 3,5H 7.29, 4H 7.25 A21 8.25 3.8 1.23 E22 8.3 4.16 1.91, 2.02 γH 2.25 D23 8.47 4.64 2.63, 2.74 V24 8.25 4.11 2.19 0.96 G25 8.65 3.97 S26 8.24 4.42 3.87, 3.91 N27 8.57 4.73 2.79, 2.87 δH 7.74 K28 8.46 4.26 1.76, 1.88 γH 1.4, 1.46 δH 1.66 εH 2.98 ζ NH2

b G29 8.52 3.91 A30 8.13 4.3 1.37 I31 8.3 4.15 1.86 γH 1.19, 1.51 δH 0.88 I32 8.41 4.14 1.86 γH 1.21, 1.49 δH 0.86, 0.92 G33 8.58 3.90, 3.94 L34 8.17 4.34 1.6 γH 1.60 δH 0.87, 0.92 M35 8.56 4.52 2.01, 2.06 γH 2.51, 2.58 εH 2.09 V36 8.37 4.11 2.09 γH 0.95 G37 8.72 3.98 G38 8.37 3.94, 4.02 V39 8.2 4.18 2.09 0.94 V40 7.94 4.05 2.06 0.91


44



45


(mixing time 270 ms, 10 mM phosphate buffer, pH 7.3, 5oC). Sequential NOEs are connected as follows:

Solid line (——) residue 28-40; dotted line (······) residue 17-26; dashed line (-----) residue 7-12.

46



Solid line (——) residue 30-40.

47


Residue H HA HB Others D1 b 4.07 2.65, 2.78 A2 8.12 4.29 1.37 E3 8.58 4.19 1.89, 1.94 γH 2.11,2.22 F4 8.39 4.54 3 2,6H 7.15 3,5H 7.24 4H 7.28 R5 8.2 4.28 1.62 1.73 γH 1.49 δH 3.12 εH 7.39 H6 8.47 4.49 3.03, 3.09 2H 7.99 4H 7.05 D7 8.43 4.62 2.66 S8 8.5 4.37 3.88, 3.91 G9 8.62 3.87 3.87, 3.94 Y10 8.04 4.52 2.94, 3.04 2,6H 7.06 3,5H 6.78 E11 8.5 4.2 1.83, 1.92 γH 2.16, 2.21 V12 8.2 3.94 1.96 γH 0.79, 0.88 H13 8.31 4.57 3.01 H 7.89 4H 6.99 H14 8.19 4.51 2.99, 3.06 2H 7.90 4H 7.00 Q15 8.45 4.25 1.98, 2.06 γH 2.32 εH 6.98, 7.65 K16 8.45 4.26 1.80, 1.88 γH 1.39, 1.45 δH 1.66, 1.75 εH2.97 ζ NH2

b L17 8.31 4.32 1.57, 1.63 γH 1.45 δH 0.84, 0.90 V18 8.1 4.01 1.91 γH 0.70, 0.83 F19 8.25 4.55 2.92 2,6H 7.13, 3,5H 7.23 4H 7.28 F20 8.33 4.71 3.39, 3.59 2H 7.32 3H 7.43 4,6H 7.85 7,8H 7.42 9H 7.58 A21 8.12 4.13 1.32 E22 8.38 4.14 1.93, 2.03 γH 2.27, 2.30 D23 8.54 4.63 2.63, 2.74 V24 8.26 4.12 2.17 γH 0.94 G25 8.63 3.97 S26 8.23 4.42 3.87, 3.91 N27 8.56 4.73 2.79, 2.87 δH 7.02, 7.74 K28 8.45 4.26 1.76, 1.88 γH 1.40, 1.45 δH 1.67 εH 2.98 ζ NH2

b G29 8.52 3.93 A30 8.13 4.31 1.37 I31 8.29 4.15 1.86 γH 1.19, 1.52 γCH3 0.88 I32 8.4 4.16 1.86 γH 1.22, 1.51 δ CH3 0.86 γCH30.93 G33 8.58 3.91, 3.95 L34 8.16 4.35 1.61 γH 1.60 δH 0.88, 0.93 M35 8.56 4.53 2.02, 2.07 γH 2.51, 2.59 εH 2.09 V36 8.36 4.13 2.09 γH 0.96 G37 8.71 3.97, 4 G38 8.37 3.94, 4.02 V39 8.19 4.18 2.1 γH 0.88, 0.95 V40 7.94 4.06 2.06 γH 0.90, 0.92


48



49



Solid line (——) residue 28-40; dotted line (······) residue 17-25; dashed line (-----) residue 9-12.

50




51


Residue H HA HB Others D1 b 4.16 2.69, 2.82 A2 8.13 4.3 1.37 E3 8.56 4.19 1.90, 1.93 γH 2.13, 2.23 F4 8.42 4.55 3.01 2,6H 7.14 3,5H 7.23 4H 7.30 R5 8.24 4.27 1.62, 1.73 γH 1.49 δH 3.12 εH 7.39 H6 8.5 4.52 2.95, 3.04 2H 7.97 4H 7.06 D7 8.45 4.62 2.68 S8 8.52 4.38 3.88, 3.92 G9 8.63 3.88, 3.95 Y10 8.05 4.52 2.95, 3.03 2,6H 7.06 3,5H 6.78 E11 8.5 4.2 1.84, 1.92 γH 2.17, 2.22 V12 8.19 3.94 1.95 γH 0.79, 0.89 H13 8.35 4.59 3.02, 3.04 2H 7.94 4H 6.99 H14 8.26 4.54 3, 3.09 2H 7.96 4H 7.00 Q15 8.49 4.26 1.98, 2.07 γH 2.33 εH 6.99, 7.65 K16 8.47 4.27 1.77, 1.89 γH 1.39 1.44 δH 1.67 εH2.95 ζ NH2

b L17 8.32 4.31 1.6 γH 1.44 δH 0.84, 0.92 V18 8.01 3.94 1.68 γH 0.71, 0.83 F19 8.31 4.57 2.93 2,6H 7.21, 3,5H 7.30, 4H 7.26 F20 8.39 4.71 3.12, 3.25 2H 7.34 3,5,8H 7.84 6,7H 7.54 9H 7.63 A21 8.36 4.2 1.37 E22 8.4 3.99 1.80, 1.95 γH 2.20, 2.23 D23 8.44 4.62 2.63, 2.75 V24 8.24 4.13 2.19 γH 0.96 G25 8.64 3.98 3.97 S26 8.24 4.42 3.87, 3.91 N27 8.57 4.74 2.80, 2.87 δH 7.03, 7.73 K28 8.45 4.26 1.77, 1.88 γH 1.41, 1.47 δH 1.67 εH 2.99 ζ NH2

b G29 8.52 3.92 A30 8.13 4.31 1.37 I31 8.29 4.15 1.86 γH 1.19, 1.51 δ CH3 0.88 γCH30.93 I32 8.4 4.16 1.86 γH 1.20, 1.50 δ CH3 0.86 γCH30.92 G33 8.57 3.91, 3.95 L34 8.16 4.35 1.6 γH 1.60 δH 0.88, 0.93 M35 8.56 4.53 2.02, 2.07 γH 2.51, 2.59 εH 2.09 V36 8.36 4.13 2.09 γH 0.96 G37 8.71 3.98, 4 G38 8.37 3.95, 4.03 V39 8.19 4.18 2.09 γH 0.95 V40 7.93 4.06 2.07 γH 0.91


52



53



Solid line (——) residue 28-40; dotted line (······) residue 17-25.

54




55

Table 3.5 Proton chemical shift assignments for Aβ(1-40)-Glycine21 a

Residue H HA HB Others D1 b 4.12 2,67, 2.81 A2 8.14 4.29 1.37 E3 8.57 4.19 1.90, 2.03 γH 2.12, 2.23 F4 8.41 4.55 3.01 2,6H 7.14 3,5H 7.26 4H 7.30 R5 8.22 4.27 1.73 γH 1.49, 1.62 δH 3.13 εH 7.39 H6 8.49 4.6 2.94, 3.02 2H 7.97 4H 7.06 D7 8.44 4.62 2.67 S8 8.51 4.37 3.88, 3.92 G9 8.64 3.87, 3.96 Y10 8.04 4.52 2.94, 3.03 2,6H 7.06 3,5H 6.78 E11 8.5 4.2 1.83, 1.92 γH 2.16, 2.21 V12 8.2 3.94 1.95 γH 0.79, 0.88 H13 8.34 4.59 3.02 2H 7.94 4H 6.99 H14 8.22 4.53 3.01 2H 7.96 4H 7.00 Q15 8.48 4.26 1.98, 2.33 γH 2.31 εH 6.99, 7.67 K16 8.48 4.27 1.82 γH 1.39, 1.45 δH 1.68, 1.75 εH2.97 ζ NH2

b

L17 8.37 4.33 1.61 γH 1.44 δH 0.84, 0.91 V18 8.11 4.03 1.9 γH 0.74, 0.84 F19 8.41 4.61 2.91, 3.01 2,6H 7.15, 3,5H 7.23, 4H 7.27 F20 8.48 4.59 2.97, 3.13 2,6H 7.22, 3,5H 7.31, 4H 7.28 G21 8.07 3.73, 3.82 E22 8.39 4.27 2.06 γH 2.26 D23 8.65 4.65 2.63, 2.75 V24 8.26 4.12 2.18 γH 0.95 G25 8.64 3.97 S26 8.24 4.43 3.87, 3.91 N27 8.58 4.73 2.81, 2.88 δH 7.74 K28 8.46 4.26 1.77, 1.89 γH 1.41, 1.47 δH 1.67 εH 2.99 ζ NH2

b G29 8.52 3.92 A30 8.14 4.3 1.37 I31 8.3 4.15 1.86 γH 1.21, 1.52 δ CH3 0.88 γCH30.93 I32 8.41 4.15 1.87 γH 1.22, 1.51 δ CH3 0.87 γCH30.93 G33 8.59 3.92, 3.95 L34 8.17 4.34 1.61 γH 1.60 δH 0.89, 0.93 M35 8.57 4.53 2.03, 2.07 γH 2.52, 2.59 εH 2.09 V36 8.37 4.12 2.09 γH 0.96 G37 8.72 3.99 G38 8.37 3.95, 4.02 V39 8.2 4.18 2.09 γH 0.95 V40 7.94 4.05 2.07 γH 0.91


56

Figure 3.17 The expanded NH region of the TOCSY spectra of 50 µM Aβ(1-40)-Glycine21 (10 mM

phosphate buffer, pH 7.3, 5oC). The relayed connections among the NH, αH, βH, and γH are shown.

57

Figure 3.18 Expanded NH-αH region of the NOESY spectrum of 50 µM Aβ (1-40)-Glycine21 (mixing time

270 ms, 10 mM phosphate buffer, pH 7.3, 5oC). Sequential NOEs are connected as follows: Solid line (—

—) residue 28-40; dotted line (······) residue 17-25.

58

Figure 3.19 Expanded NH-NH region of the NOESY spectrum of 50 µM Aβ (1-40)-Glycine21 (mixing

time 270 ms, 10 mM phosphate buffer, pH 7.3, 5oC). The NH-NH NOEs are connected as follows: Solid

line (——) residue 17-20, dotted line (······) residue 30-34.

59

3.2.4.2 NOE patterns of naphthylalanine substituted Aβ(1-40)

The conformation of naphthylalanine substituted Aβ(1-40) were analyzed by

inspecting backbone NOE connectivities. Figure 3.20 shows the summary of backbone

NOEs of each peptide.

The structures of peptides in solution obtained by NMR are usually a population-

weighted average over all structures in the conformational ensemble (Merukta et al.,

1993). However, the predominant conformation could be inferred from NMR parameters.

The peptide backbone NOE can be used to predict the backbone dihedral angles in the

allowed region of Ramachandran plot (Dyson and Wright, 1991; Ramachandran et al.,

1966). Strong αN(i, i+1) NOEs in the absence NN(i, i+1) NOEs suggest that the

backbone dihedral angles are predominantly in the β region for extended structure, while

strong NN(I, i+1) NOEs indicates that the backbone φ and ψ angles are in the αR or αL

region for helix struxtures. When both αN(i, i+1) and NN(I, i+1) NOEs are observed for

consecutive residues, the backbone dihedral angles average between α and β regions.

For naphthylalanine substituted Aβ(1-40), strong αN(i, i+1) were observed for most

of the residues, suggesting the conformational ensemble of the peptide includes a

substantial population of extended structure (Dyson et al., 1988a; Dyson et al., 1988b).

Weak to medium NN(i, i+1) were also observed for some residues indicating the

flexibility of peptide backbones. No medium or long range inter-residual NOE was

observed, suggesting that that α-helical conformation is not present in naphthylalanine

substituted Aβ(1-40).

NOE pattern of the Flemish mutant Aβ(1-40)- Glycine21 is similar to those of

naphthylalanine substituted Aβ(1-40), no long range NOE was observed indicating the

60

peptide conformation is alsao predominantly random extended structure in water

solution.

Figure 3.20 Summary of the inter-residue NOEs among the backbone NH, αH and βH for naphthylalanine substituted Aβ(1-40). A: Aβ(1-40)-1-naphthylalanine19 B: Aβ(1-40)-2-naphthylalanine19 C: Aβ(1-40)-1-naphthylalanine20 D: Aβ(1-40)-2-naphthylalanine20 E: Aβ(1-40)-Glycine21. The NOE intensities are reflected by the thickness of the lines. When an unambiguous assignment was not possible due to peak overlap, the NOEs are drawn with gray boxes.

61

3.2.4.3 Proton chemical shift analysis

1H chemical shifts are strongly dependent on the character and nature of protein

secondary structures. In particular, the αH chemical shifts of all 20 amino acids have an

upfield shift (with respect to the random coil value) of -0.38ppm (average) when the

residues are in α-helical conformation and downfield shift of 0.38ppm when in β-sheet

conformation (Wishart & Skyes, 1994, 1995). Thus, studies on αH chemical shifts

deviation from the random coil values could provide invaluable information on the

conformational structure of the protein.

αH chemical shifts of naphthylalanine substituted Aβ(1-40) were compared with

the random coil values (Merutka et al., 1995) and are plotted in Figure 3.21. The

naphthylalanine substituted Aβ(1-40) αH chemical shifts are generally close to the

random coil values. The chemical shift deviations are usually within the -0.2 ~ 0.2ppm

range, indicating the peptide structure are primarily random extended chain. Most of the

residues near N-terminal have negative values, indicating the propensity to form α-

helical structure. However more residues in the hydrophobic C-terminal region have

positive values, suggesting β-sheet structure is more populated in this region which might

be the driving force during Aβ aggregation.

For peptide Aβ(1-40) Flemish mutant (Aβ(1-40)-Glycine21), its αH chemical shifts

deviation from the random coil values are also plotted in Figure 3.21. The chemical shift

deviation is almost identical to that of Aβ(1-40) (the differences between the two plots

are less than ~0.01ppm), indicating the Flemish mutant also takes primarily random

extended structure as wild type Aβ(1-40).

62

Figure 3.21 αH chemical shift differences between Naphthylalanine substituted Aβ(1-40) and those expected for each amino acid residue in a random coil conformation (Wishart and Sykes, 1994) are represented as a function of residue position of Aβ(1-40). Chemical shift of wild type Aβ(1-40) are from Hou et al.,, 2004

63

To examine the effect of naphthylalanine substitution on Aβ(1-40) structure, the αH

chemical shift were also compared between naphthylalanine substituted and wild type

Aβ(1-40) (Figure 3.22).

The αH chemical shift differences between naphthylalanine substituted and wild

type Aβ(1-40) are usually very small (within -0.05 ~ 0.02ppm). The most pronounced αH

chemical shift differences were observed between residues 18-22. The αH chemical shift

at residue 19 (or residue 20) always downfield shifted ~0.15ppm when phenylalanine is

substituted by naphthylalanine indicating the propensity to form β-sheet structure. Ala21

αH shifted upfield in all four naphthylalanine substituted peptides, however, the degree of

this shift varies significantly among the peptides: Aβ(1-40)-2- naphthylalanine19 has the

Ala21 αH upfield shifted more than 0.4ppm, while Ala21 αH in Aβ(1-40)-2-

naphthylalanine20 has very few chemical shift changes (<0.01ppm); in Aβ(1-40)-1-

naphthylalanine19 and Aβ(1-40)-1- naphthylalanine20, Ala21 αH chemical shift change

are almost identical (~0.1ppm). It also should be noted that Val18 (0.1ppm) and Glu22

(0.2ppm) in Aβ(1-40)-2- naphthylalanine20 have significantly larger αH chemical shift

differences than other three naphthylalanine substituted Aβ(1-40) peptides (Val18

0.01~0.02ppm; Glu22 0.01~0.06ppm).

64

Figure 3.22 1Hα chemical shift differences (Δδ = δnaphthylalanine – δAβ(1-40) ) between Aβ(1-40) and Aβ(1-40)-1-naphthylalanine19, Aβ(1-40)-2-naphthylalanine19, Aβ(1-40)-1-naphthylalanine20, and Aβ(1-40)-2-naphthylalanine20.

65

3.2.4.4 Proton chemical shift index analysis

Chemical shift index analysis (CSI, Wishart &Skyes, 1994b; Wishart et al., 1992)

were also used to study the naphthylalanine substitution effect on Aβ(1-40) secondary

structure.

CSI uses chemical shifts of proton (αH) and/or carbon (including αC, βC and

crobonyl C) to predicate and locate protein secondary structure. The program works in a

two stage digital filter process. First, a ternary shift index of -1, 0, and 1 is assigned to all

the identifiable residues on the basis of chemical shift deviation to the random coil

values. Certain ranges of the deviation are set for different nuclei (for example, ± 0.1ppm

for 1H, -0.5~0.8ppm for αC). 0 is assigned for the chemical shift deviation within the

given range and -1 or 1 is assigned for those out of the given range depending on

negative or positive deviations. Accordingly, CSI of -1, 0, and 1 represents α-helix,

random coil, and β-strand respectively. Usually observation of three or more continuous

residues with same CSI values is indicative of a particular secondary structure. In the

second stage, secondary structures are predicted by using edge-detection, pattern

recongnization and digital smoothing on the raw data (from first stage) to produce

“filtered” chemical shift index. Filtered CSI’s are generally easier to read but

occasionally some important information might be missing during the filtering process.

Thus, both filtered and raw αH chemical shift index of the naphthylalanine substituted

Aβ(1-40) are presented in Figure 3.23 and Figure 3.24, respectively.

The majority of the filtered CSI values for all peptides are zero demonstrating they

adopt predominantly random structures. Residues close to the N-terminal show

continuous negative CSI values indicating α-helical like structure in this region, although

66

the NOE and CD data do not support such conclusion. Two peptides (Aβ(1-40)-2-

naphthylalanine19 and Aβ(1-40)-1- naphthylalanine20) also show tendency towards α-

helical structure in the central region (residue 20-23).

More information can be obtained by analyzing raw CSI values. There are more

residues close to C-terminal having positive CSI values, indicating this region favors β-

strand structure formation. Between residue 16-22, naphthylalanine substituted Aβ(1-40)

have more residues with negative CSI values than the wild type Aβ(1-40) and the flemish

mutant suggesting naphthylalanine substitution might favor α-helical formation in this

region.

67

Figure 3.23 The filtered αH chemical shift indices of Aβ(1-40), Aβ(1-40)-Glycine21 and Aβ(1-40) with phenylalanine-19 (or phenylalanine-20) substituted by naphthylalanine

68

Figure 3.24 The raw αH chemical shift indices of Aβ(1-40), Aβ(1-40)-Glycine21 and Aβ(1-40) with phenylalanine-19 (or phenylalanine-20) substituted by naphthylalanine

69

3.3 Discussion

3.3.1 Aryl-sulfur interaction in Aβ

Elucidation of the Aβ peptide structure is of paramount importance in

understanding the molecular basis of Alzheimer’s disease. Unfortunately, despite decades

of research efforts, no high resolution structure has yet been determined. Recently, a

structural model of Aβ(1-40) fibrils based on the studies of solid state NMR and electron

microscopy has been published (Petkova, et al., 2002). In this model, Aβ residues 12-24

and 30-40 adopt β-strand conformations and form parallel β-sheets through

intermolecular hydrogen bonding. Interestingly, although not explicitly described by the

author, in this model the Met35 ε-CH3S group is proximate to the center of the Phe19

aromatic ring (5-6 Å), which represents an aryl-sulfur interaction that might be crucial to

stabilize the β-strand structure.

It has long been proposed that a strong, favorable interaction exists between

aromatic rings and divalent sulfur atoms (Morgan et al., 1978, 1980) because of high

frequency of contacts observed between sulfur-bearing amino acids (cysteine and

methionine) and those that include an aromatic ring (histidine, tryptophan, tyrosine, and

phenylalanine) in protein crystal structures (Reid et al. 1985; Pal and Chakrabarti 1998;

Pal and Chakrabarti, 2001; Samanta et al. 2000) and small molecule crystal structures

(Zauhar et al. 2000). A statistical analysis of interactions collected from structures in the

Brookhaven Protein Data Bank confirmed that such interactions occurred much more

frequently than would be expected under the assumption of random association between

amino acids, suggesting a favorable aryl-sulfur interaction might have a special

significance for stabilizing the folded conformation of proteins. The geometry,

70

magnitude, and nature of the aryl-sulfur interaction are not well understood. However, it

has been proposed that the interaction may arise from hydrogen bonding to the aryl

hydrogens, SH–π interactions, electrostatic interactions, or hydrophobic interactions (Pal

and Chakrabarti, 2001).

The aryl-sulfur interaction has been extensively studied using many different

techniques including circular dichroism (Viguera and Serrano, 1995) quantum mechanic

calculation(Cheney et al., 1989) and NMR (Bodner and Morgan, 1980). NMR studies of

aryl-sulfur interactions using peptide model systems (Tatko and Waters, 2004) have

shown that chemical shift of ε-methyl group in methionine will shift upfield (0.1-0.2

ppm) when the sulfur atom is in proximity to the aromatic rings. In an α-helix model

system, NMR studies also show that strong NOE can be observed between Phe phenyl

protons and the Met ε-methyl groups when the two groups are in proximity (Stapley et

al., 1995).

To study whether the aryl-sulfur interaction existed in Aβ, we did the solution

NMR studies on wild type Aβ(1-40) as well as Aβ(1-40) with Phe19 substituted by

naphthylalanine. However, neither Met ε-methyl proton chemical shift change nor NOE

between Phe19 phenyl protons and the Met35 ε-methyl groups was observed. Although

our current data does not support the presence of aryl-sulfur interaction between Met35

and Phe19, this doesn't necessarily mean aryl-sulfur interaction does not exist. Because

NMR events of the (non-aggregated) monomeric peptide, the NMR results may suggest

that Phe19-Met35 interaction does not occur in the initial stages of aggregation, but in

stead are involved in the later stages of association into β-sheet aggregates. In addition, as

it has been pointed out, both the distance between sulfur atom and aromatic ring and the

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angles of sulfur atom above aromatic plane play important roles in determining the

strength of aryl-sulfur interaction (Reid et al., 1985). Further studies on Aβ(1-40) using

non-aromatic residues or even bigger aromatic rings (Annulene) to substitute Phe19 will

help to elucidate the aryl-aromatic interaction issue.

3.3.2 Insights from studies of Aβ(1-40) with Phe19/Phe20 substituted by

naphthylalanine

It has long been known that Phe19 and Phe20 are essential in Aβ aggregation in

that peptides without these two residues will not form fibrils (Hilbich et al., 1992; Esler,

et al., 1996). Current CD studies show that, while wild type Aβ(1-40) remain

predominantly random structure and soluble during the whole aging process, Aβ(1-40)

with Phe19 substituted by naphthylalanine adopt β-sheet structure, and Aβ(1-40) with

Phe20 substituted by naphthylalanine are primarily random structure but with accelerated

aggregation rate. Thus the CD results suggest the two Phe residues play distinct roles

during Aβ aggregation. As indicated by our aryl-aromatic interaction hypothesis, the aryl-

sulfur interaction only exists bwtenn Met35 and Phe19, but not bwtenn Met35 and Phe20.

Naphthylalanine substitution of Phe19 will enforce the aryl-sulfur interaction which

subsequently stabilize and favor the β-sheet structure formation. However, because there

is no such interaction between Met 35 and Phe 20, naphthylalanine substitution at

position 20 will not favor the β-sheet structure formation but simply enhance the peptide

hydrophobicity and aggregation.

NMR NOE results of naphthylalanine substituted Aβ(1-40) are similar to that of

wild type Aβ(1-40): both αN(i, i+1) and NN(i, i+1)NOEs are observed for most residues

72

in every peptide indicating the flexibility of peptide backbone. Strong αN(i, i+1) NOEs

indicates the conformational ensemble of the peptide includes a substantial amount of

extended chain structure (Dyson et al., 1988a; Dyson et al., 1988b).

αH chemical shift analysis also established that naphthylalanine substituted Aβ(1-

40) have similar random extended structure as wild type Aβ(1-40). However, significant

αH chemical shift deviation from wild type Aβ(1-40) at residue 18-22 were observed

suggesting there may be some local structure differences in this region. αH chemical shift

index (CSI) analysis of Aβ(1-40)-2- naphthylalanine19 and Aβ(1-40)-1-

naphthylalanine20 also show tendency towards α-helical structure in the central region at

residue 20-23. Interestingly, the above region coincides with the well defined central

hydrophobic region (Leu17-Ala21) which plays an important role during the Aβ

aggregation (Hilbich et al., 1992; Esler et al., 1996; Pallitto et al., 1999; Lowe et al.,

2001; Hou et al., 2004). Although at this time, there is insufficient NMR data to support

the presence of stable structures in this region, it is possible the local structural change in

this region induce the conformational and aggregational property difference between wild

type and naphthylalanine substituted Aβ peptides.

The most pronounced αH chemical shift movement was observed at residue Ala21.

To study the role of this residue, we synthesized Aβ(1-40) with Ala21 substituted by

Glycine, which is in fact Aβ Flemish mutant, and studied its structure by CD and NMR.

However, CD results indicate Aβ Flemish mutant has almost identical conformational

and aggregation properties as Aβ(1-40). NMR studies of Aβ Flemish mutant also suggest

the peptides takes primarily random extended structures in the solution. Those results

indicate Ala21 does not play a significant role in Aβ aggregation.

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3.4 Materials and methods

Materials All Aβ peptides were synthesized and purified as described in Chapter 2.

The Aβ sample solutions were prepared following the disaggregation protocol as

described in Chapter 2. Perdeuterated ethylenediamine tetrracetic acid (Na2EDTA-d12),

3-(trimethylsiyl)-propionate-2,2,3,3-d4 (TSP) and D2O were obtained from Isotec Inc or

Cambridge Isotope Inc. Potassium phosphate buffer solutions and sodium azide (NaN3)

were obtained from Sigma. H2O (HPLC grade) was obtained from Fisher.

CD experiments All CD experiments were performed at room temperature with a

Jasco spectropolarimeter (Model J-810). Quartz cells (Hellma, Inc) of 1-mm path length

were used to obtain spectra at 0.2 nm interval from 190 to 250 nm. Spectra resulted from

averaging and smoothing eight accumulative scans to improve the signal to noise ratio,

followed by subtraction of the CD signals of the instrument background. The data were

analyzed with the Jasco J-810 program of the spectropolarimeter.

NMR experiments The Aβ peptide solutions were prepared at concentration of 50-

100 μM. Based treated Aβ peptide was dissolved in 10 mM phosphate buffer (pH 7.1)

containing Na2EDTA-d12 (0.05 mM), NaN3 (0.05 mM), TSP (0.05 mM) and D2O (5%,

v/v). The pH of the solution was measured at room temperature with a special pH

electrode (Microelectrode, Inc) that fits inside the NMR tube. The final pH was between

7.3 and 7.5, and, if needed, was adjusted with dilute TFA (5%, wt) or NaOH (1 mM) in

D2O. No corrections for pH readings were made for isotope effects. The additives

(Na2EDTA-d12, NaN3, and TSP) were served as chelating reagent to remove metal ions,

antibacterial reagent and internal proton chemical shift reference at 0 ppm, respectively.

NMR spectra were obtained at 5 oC on Bruker Avance 600 or 800MHz

74

spectrometers equipped with TXI cryoprobes. Both spectrometers have actively shielded

z-axis gradient units.

The 1D 13C-1H NMR spectra were acquired with presaturation and WATERGATE

(Piotto, et al., 1992) to suppress the H2O signal. The 2D NMR experiments included the

pulse field gradient (pfg) WATERGATE NOESY (Kay, 1995; Piotto et al., 1992) and pfg

DIPSI TOCSY (Bax & Davis, 1985; Shaka et al., 1988). All of the experiments were

performed in the phase sensitive mode with quadrature detection in both demensions

(States et al.,1982). The carrier was placed in the center of the spectrum at the position of

H2O signal. TOCSY mixing time were set at 70 ms and NOESY mixing time were set at

270 ms.

All NMR data were transferred to a local PC computer and processed using the

FELIX program (Version 2000, Accelrys Inc) or NMRPipe.

The 1D spectra had 8000 Hz spectra width and 32000 data points. Before Fourier

transformation, the data were zero filled once to 32000 real points and the multiplied by

Loretzian-to-Gaussian window function. All 2D spectra had the spectra widths of 7000

Hz in both dimensions. The data were acquired with 2048-4096 points for the F2

dimension and 256-512 complexe increments for the F1 dimension, each consisting of

32-64 scans. Before Fourier transformation, the F2 dimension were zero filled once and

multiplied by a Loretzian-to-Gaussian window function, and the F1 dimension were zero

filled once and multiplied by a 80-90o sinebell window function.

75

4 Structural Studies of Aβ Soluble Oligomer

76

4.1 Introduction

As has been described in the introduction, a growing body of evidences indicates

that Aβ toxicity not lies in the insoluble fibrils that accumulate as senile plaques but

rather in the soluble oligomeric intermediates (Lambert et al., 1998; Hardy & Seloke,

2002; Lesne et al., 2006). It is evident that the elucidation of the Aβ oligomer structures

will help to understand the pathogenesis of the disease and may find immediate

application in drug research. However, despite many research efforts (reviewed by

Caughey and Lansbury, 2003), the molecular structure of the Aβ oligomers remains

unknown. In this chapter we used NMR and other techniques trying to exploit the

structural properties of Aβ oligomers.

The definition of Aβ oligomers is not strictly described. One widely accepted

criteria are that Aβ oligomers exhibit bands larger than monomers on SDS-PAGE gel.

Among many kinds of published Aβ oligomers, two types of Aβ oligomers have been

widely described in vitro, these include protofibrils (Hartley et al., 1999; Walsh et al.,

1997) and ADDLs (Aβ-derived diffusible ligands, Lambert et al., 1998; Klein, 2006).

The protofibrils exist both in Aβ(1-40) and Aβ(1-42). They are short, curly fibrils, 6-8 nm

in diameter, 5-160 nm in length and with molecular weight over 100,000 Da. In contrast,

the ADDLs were only found in Aβ(1-42). They are smaller, globular oligomers with

diameter about 5 nm long and molecular weight between 17,000~27,000 Da. For

completeness purposes, we prepared Aβ oligomers using both protofibril and ADDL

preparation protocols and studied their structural properties.

77

Size exclusion chromatography

To separate Aβ oligomers from monomers, a widely used method is by size

exclusion chromatograph (SEC).

Unlike the reversed-phase liquid chromatography where the separation arises from

different interactions of the solutes with the mobile phase and the stationary phase,

separation in SEC arise from differences in molecular size and the ability of different

molecules to penetrate the pores of the stationary phase to different extents. A schematic

representation of the relationship between molecular weight and elution volume on a size

exclusion column is presented in Figure 4.1

Figure 4.1 Schematic representation of molecular weight and retention volume relationship in SEC

Solute retention in terms of retention volume (VR) can be explained in terms of

three variables: the stationary phase volume (VS), mobile phase volume (VM), and the

distribution coefficient (KD). The largest molecules in a sample will be excluded from all

the pores of the stationary phase and elute with a volume V0 referred to as void volume

which must be the volume of liquid in the interstitial space outside the pores. The

78

stationary phase volume (VS) is usually considered to be the volume of liquid in the

support pores (VI), the smallest molecules in a sample will be able to penetrate all the

pores of the stationary phase and will elute with a mobile phase volume (VM) that is the

sum of stationary phase volume (VI) and the void volume (V0). Molecules of intermediate

size will be able to penetrate some but not all of the pores and will elute with a retention

volume (VR) that is between the void volume (V0) and the mobile phase volume (VM).

The retention volume (VR) can be defined as:

VM=V0 + VI

VR=V0 + KDVI

The distribution coefficient (KD) specifies the fraction of stationary phase volume

(VI) accessible to the molecules and should not exceed unity one.

SEC is a very useful chromatographic method in that it can be performed under

native-like, nondenaturing conditions. In fact, SEC has been widely used in the

Alzheimer research (Walsh et al. 1997, 1999, 2005; Hartley et al., 1999; Ye et al.2004;

Demuro et al., 2005; Lense et al., 2006) for oligomer characterization and preparation

and has been thought to be the best non-SDS-based method for doing so (Bitan G., et al,

2005).

Light scattering

Once the Aβ oligomers have been prepared, their molecular weight will be

measured. The most widely used method for doing so is by using SEC molecular weight

standard curve: briefly, the SEC was calibrated using several known molecular weight

standard compounds, usually globular proteins, then a standard molecular weight curve

79

was constructed by regression analysis, and the molecular weight of the analyze will be

inferred from the standard curve. However, assumptions made in this method is that the

analyte are globular proteins and do not interact with the SEC column matrix. Aβ

peptides are not globular proteins, and there are evidence the they interact with the SEC

column matrix (Walsh, et al 1997). Thus the above method is not the best choice for

measuring Aβ oligomer molecular weight. The molecular weight determination of Aβ

oligomers in this chapter were conducted by measuring their light scattering.

Figure 4.2 Schematic representation of light scattering

When a small particle is illuminated by a light source such as a laser, the particle

will scatter the light in all directions (Figure 4.2). The intensity of the scattered light

depends on the polarizability of the particle, and the polarizability depends on the

molecular weight. This property of light scattering makes it a valuable tool for measuring

molecular weight.

Two different light scattering methods can be used to characterize proteins: the

dynamic light scattering and static light scattering.

The dynamic light scattering (DLS), which is also known as “photon correlation

spectroscopy" (PCS) or "quasi-elastic light scattering" (QELS), uses the scattered light to

80

measure the rate of diffusion of the protein particles. This motion data is conventionally

processed to derive a size distribution for the sample.

The static light scattering (SLS), which is also known as "Rayleigh" scattering or

MALLS (multiple angle laser light scattering), provides a direct measure of molecular

weight. It is therefore very useful for determining whether the native state of a protein is

a monomer or a higher oligomer, and for measuring the masses of aggregates or other

non-native species. We are using SLS to measure the molecular weight of Aβ oligomers.

Unlike molecular weights that are estimated from SEC molecular weight standard

curve, the MW obtained from light scattering is not dependent upon either the Stokes

radius of the protein or a calibration curve that depends upon running several standard

proteins. Light scattering (LS) provides the absolute molecular weight (MW) of

macromolecules in solution. And because of this uniqueness, light scattering is generally

best used on-line in conjunction with size-exclusion chromatography (SEC-MALLS), as

shown in the following diagram.

Figure 4.3 Schematic diagrams of SEC-MALLS

81

As the molecular weight measured from light scattering are independent of the

elution volume, this technique can be used with "sticky" proteins that may interact with

the SEC column matrix, such as Aβ peptides, and also with highly elongated proteins

which elute unusually early for their molecular weight. In fact, light scattering has been

widely used to characterize all soluble Aβ species (Witte et al., 2007; Helper et al., 2006;

Lomakin et al., 1996, 1997; Nichos et al., 2002; Pallitto and Murphy, 2001; Shen et al.,

1994; Walsh et al., 1997, 1999).

Pulsed field gradient diffusion measurement

To asses the different molecular state between Aβ oligomers and monomers, pulsed

field gradient NMR diffusion experiments were also performed.

Self-diffusion is the random translational motion of a molecule in solution without

the effect of a concentration gradient. The diffusion is closely related to the molecular

size and the diffusion coefficient (D) can be used to characterize the oligomeric state of a

molecule. The diffusion coefficient can be determined by using a pulsed field gradient

spin-echo NMR experiment. Shown in Figure 4.4 is an improved version of pulsed field

gradient pulse sequence (Altieri et al., 1995) compared to the original spin echo pulse

sequence (Stejskal and Tanner, 1965).

Figure 4.4 Diffusion measurement pulse sequence (Altieri et al., 1995)

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In order to measure the diffusion coefficients, a series of experiments with varying

gradient strength are recorded. The intensity of the signals decrease with increasing

gradient strength (Figure 4.5, PFG NMR spectra of CCl3H, adapted from Price 1997).The

decrease in signal intensity as a function of gradient strength can be described by the

general equation (Stejskal & Tanner, 1965; Tanner, 1970)

I(2τ) = I(0) e(–γ2Dδ2 (Δ-1/3 δ))g2 (1)

where D is the diffusion coefficient, I(2τ) is the attenuated echo amplitude with gradient

strength g and duration δ, Δ is the time interval between two gradient pulse, and γ is the

gyromagnetic ratio of the nuclei atom (for hydrogen, γ is 2.675*104 G-1s-1. A plot of peak

intensity versus the gradient strength square yields an exponential decay curve which is

fitted to obtain the diffusion coefficient (Figure 4.6)

Figure 4.5 The attenuated signal for CCl3H with the increasing gradient strength

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Figure 4.6 The exponential decay curve fittings of the intensity versus the gradient strength square

4.2 Results

4.2.1 Size exclusion chromatography of Aβ peptides

4.2.1.1 Size Exclusion Chromatography of Aβ oligomer prepared using Aβ

protofibril preparation protocol

Aβ profibril and ADDLs (Aβ-derived diffusible ligands) are two widely studied Aβ

oligomers. We tried both methods to prepare Aβ oligomers.

To use Aβ protofibril preparation method, monomeric Aβ(1-42) solution was

freshly prepared (see “Materials and methods”) and incubated at room temperature.

Aliquots of samples were injected into size exclusion column at different incubation

times. A typical SEC of Aβ(1-42) using this preparation methods is shown in Figure 4.7:

there are two clearly resolved peaks, the oligomer peak eluted at about 15 minutes with a

retention volume that is very close to the void volume of the column (~7.5 ml), and the

monomer peak eluted at about 26 minutes which is in the limit of the included volume of

the column ( ~ 24ml).

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Figure 4.7 Size exclusion chromatography of Aβ(1-42). Aβ peptide was dissolved in 10 mM phosphate buffer at a concentration of 100 μM and chromatographed on a superdex 75 column at flow rate of 0.5 ml/min. 10 mM phosphate buffer was used as solvent.

The oligomerization process was followed by monitoring time dependent SEC of

Aβ. As shown Figure 4.8: the peptide solution initially consists of mostly monomers.

Accordingly, the monomer peak is the predominant peak in the SEC. As the peptide

solution is aging, the oligomers are forming, and the oligomer peak is becoming stronger

while the monomer peak is becoming weaker. The oligomer peak got to its maximum

height after 24 hours aging. Then the oligomer peak is also starting weakening, indicating

higher order oligomers or fibrils are formed in the solution which may start precipitating

in the solution or be too large to enter the pores of size exclusion column.

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Figure 4.8 Time dependent size exclusion chromatography of Aβ(1-42). A 100 μM Aβ(1-42) solution was prepar nd aged at room temperature. At indicated aging times, 50 μl Aβ(1-42) solution was injected into the siz clusion column and chromatographed.

An interesting result was obtained when analyzing the retention time of the

monomer and oligomer peaks (Figure 4.9): during the oligomerization process, the

retention time of monomer peak remain relatively constant (~26 min), however, the

retention time of the oligomer peak progressively decrease (from 15.5 min at 0 hour

aging to 14.4 min at 36 hours aging). This result indicates that the size of Aβ(1-42)

monomer is constant during the aging period, however, the Aβ(1-42) oligomer size keeps

increasing, indicating the oligomers formed is indeed a distribution of different sizes.

ed ae ex

Figure 4.9 Aβ(1-42) oligomer peak retention time gradually decrease during the peptide aging period. The retention time was referenced as the elution time corresponding to the maximum peak height. The Aβ(1-42)

minute when peptide solution was initially prepared and at 14.5 minute after the peptide was aged for 36 hours.

monomer peak elutes at 26 minute through the aging period, while the Aβ(1-42) oligomer elutes at 15.5

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4.2.1.2 Size exclusion chromatograph of ADDLs

ADDLs were prepared according to the well established protocols (Klein, 2006, a

detailed preparation protocol is described in “Materials and Methods”). A representative

SEC

aks with retention time corresponding to

oligomerr (15.8 min) and monomer (26 min), respectively. However, unlike SEC of Aβ

oligomers prepared using the protofibril preparation protocol, these two peaks observed

for ADDLs do not change over time. In fact, the ADDLs SEC showed very few

differences even after seven days aging compared to the one at 0 hour aging. Thus, the

SEC difference between the two oligomer preparation protocols indicates the ADDLs are

more stable than the Aβ protofibril oligomer.

of ADDLs is shown in Figure 4.10. In this Figure, the peaks in the black rectangle

are from small molecules that were present in the F12 medium (F12 medium is the

solvents that is used to prepare ADDLs. When F12 medium alone is injected into the

column, SEC shows the same peaks as those in the black rectangle confirming that those

peaks are not from Aβ, but from the F12 medium), only the peaks included in the red

rectangle are from the ADDLs.

The SEC of ADDLs is very similar to that using protofibril preparation method

(Figure 4.7) in that it also contains two pe

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Figure 4.10 Size exclusion chromatography of ADDLs (Red). The ADDL oligomer peaks eluted at 15.8 minute and the ADDL monomer peak eluted at 26 minute. Those peaks included in the black rectangle are from F12 medium

4.2.1.3 Size exclusion chromatograph of Aβ(1-40)

We also tried to prepare Aβ(1-40) oligomer by using either Aβ protofibril or

ADDLs preparation protocols. However, depite different sample preparation methods, the

SEC of Aβ(1-40) are always same. A typical Aβ(1-40) SEC is shown in Figure 4.11.

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Figure 4.11 Size exclusion chromatography of Aβ(1-40). The Aβ(1-40) monomer peak eluted at 27 minute. No oligomer peak was found.

Unlike Aβ(1-42), SEC of Aβ(1-40) only show the monomer peak with retention

time of 27 min compared to 26 min for the monomer peak in SEC of Aβ(1-42). No

oligomer peak was observed for Aβ(1-40) even after weeks of aging. These results

indicate that the size of Aβ(1-40) monomer is smaller than that of Aβ(1-42) monomer

and Aβ(1-40) is less likely to aggregate than Aβ(1-42).

As no oligomer can be formed with Aβ(1-40), the Aβ oligomer referred in this

chapter is exclusively prepared by using Aβ(1-42).

4.2.1.4 Stability of Aβ oligomers separated by SEC

It is clear from the above results that Aβ(1-42) solution usually consists of

monomer and oligomers. By using SEC, the Aβ oligomers can be separated from the

monomer and be collected for other studies. However, a potential risk of using such

prepared Aβ oligomers is that, because oligomers is in fact in equilibrium with the

monomer, the separated oligomer will dissociate to produce the monomer to re-establish

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this equilibrium, and as a result, the collected “oligomer” solution will become again a

mixture of oligomer and monomer. In order to keep the collected oligomer solution

“monomer free”, it is imperative to find proper experiment conditions that can prevent or

slowdown the dissociation process from oligomer to monomer.

In our experience, we found by lowering the temperature (< 5oC), the oligomer to

monomer dissociation process can be successfully inhibited and the collected Aβ

oligomer will be stable for enough long time for other studies. To test the stability of

collected oligomers using this method, a SEC experiment was initially performed to

separate Aβ oligomers from monomer. The Aβ oligomers peak was collected into a vial

surrounded by salt/ice bath. After collection is complete, the vial is transferred into a

refrigerator and incubated for 1 hour. After incubation, the Aβ oligomers solution was re-

injected into the size exclusion column and the second SEC esperiment was performed.

The result of second SEC was shown in Figure 4.12.

Figure 4.12 Size exclusion chromatography of Aβ(1-42) oligomer. Only one peak corresponding to Aβ oligomer was observed. No monomer peak (expected retention time ~26 minute) showed in the experiment.

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Only one peak is observed in Figure 4.12 with retention time corresponding to the

Aβ oligomers. This result clearly prove that, under the experiment condition used

(temperature < 5oC), the freshly prepared Aβ oligomers will be monomer free for at least

1 hour.

According to this result, all Aβ oligomers used in this chapter are freshly prepared

by SEC and the sample temperature was kept cold below 5oC. All other experiments

performed using these oligomers are all performed within one hour of oligomers

preparation. By this way, the sample will contain primarily Aβ oligomers and the

potential interference from Aβ monomer is successfully prevented.

4.2.1.5 Size exclusion chromatograph of lyophilized Aβ oligomers

One possible way to keep Aβ oligomers for future use is by lyophilizing the

collected Aβ oligomers solution. However, once these lyophilized Aβ oligomers is re-

dissolved into solution, whether the oligomer structure will change compared to the one

prior to lyophilization, or, whether oligomer dissociated to produce monomer during the

lyophilization process is unknown. To examine the feasibility of using lyophilization to

preserve Aβ oligomers, we prepared Aβ oligomers by SEC and immediately lyophilized

the sample. The lyophilized Aβ oligomers were redissolved in phosphate buffer and

reinjected into the size exclusion column. Unfortunately, SEC of such recovered Aβ

oligomers showed both oligomer and monomer peaks (data not shown). Same experiment

was also done for lyophilized Aβ monomers. Similarly, SEC of redissolved Aβ

monomers also showed both oligomer and monomer peaks. Thus, those results suggested

that during lyophilization, the Aβ peptides will undergo association and/or dissociation

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process and a mixture of oligomer/monomer will be always produced. Thus,

lyophilization is not a good method for preserving Aβ oligomers.

4.2.2 Aβ(1-42) oligomer aggregation state analysis by SEC-MALLS

To asses the aggregation state of Aβ oligomer, we employed laser ligh scattering

coupled with SEC (SEC-MALLS) which allowed absolute mass determination. Shown in

Figure 4.13 is the representative SEC with molecular weigh measured from light

scattering.

The molecular weigh distribution for the oligomer peak is from 1×103 Kda to 6×104

Kda with an average molecular weigh of 5×103 Kda. However, the light scattering data

for the monomer peak is not available which might be due to the extreme small size of

the Aβ monomer within this peak.

Figure 4.13 Size exclusion chromatography of Aβ(1-42) with an overlay of the actual molecular mass of oligomer calculated by MALLS using an empirically determined dN/dC of 0.24

4.2.3 Aβ(1-42) oligomer secondary structure analysis by CD

As described in Chapter 1, Aβ will adopt either random or β sheet structure in

aqueous solution and a conformational conversion from random to β sheet is usually

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observed in when Aβ solution aged. We noticed that in those studies the author did not

address the aggregation state of Aβ, as our current data suggest the Aβ aqueous solution

is in fact composed of monomer and oligomers. By using SEC, we were able to separate

these two species and study their secondary structure independently.

Shown in Figure 4.14 are the CD spectra of Aβ(1-42) monomer and oligomer,

respectively. Obviously, the monomer adopts predominantly random structure as shown

by the major negative bands at 198nm, and the oligomer are mostly β sheet structure

which is characterized by positive and negative bands at 195 nm and 217 nm,

respectively.

Figure 4.14 CD spectra of Aβ(1-42) oligomer (red) and monomer (black).

The result here clearly shows the distinct structure between Aβ monomer and

oligomer. Furthermore, it suggests that, while the randomly structured Aβ species is Aβ

monomer, the β-sheet structure usually observed in the Aβ aggregation process is actually

from Aβ oligomer, and the β-sheet structure is an intermolecular, not intramolecular

interaction within the Aβ oligomers.

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4.2.4 NMR experiments of Aβ oligomers

4.2.4.1 HSQC of Aβ(1-42) oligomer prepared using protofibril protocol

SEC separated Aβ oligomers were collected for NMR analysis. Great care was

taken during experiments to make sure the oligomer structure doesn’t change between

SEC and NMR experiments. A detailed experiments setup protocol is described in

“Material and method” section.

Shown in Figure 4.15a is the 1H-15N HSQC spectrum of Aβ(1-42) oligomers

formed after 3 hours aging. The 1H-15N HSQC experiment, which detects 1H atoms

directly attached to the 15N atoms, is a standard NMR experiment for proteins which can

provide a fingerprint for the protein backbone structure. The narrow chemical shift

dispersion in the 1H dimension (8.1-8.9 ppm) indicates that the Aβ(1-42) oligomers adopt

predominantly random extended chain structure.

To find out the possible structural differences between Aβ(1-42) oligomer and

monomer, 1H-15N HSQC experiment was also applied for Aβ(1-42) monomer (Figure

4.15). Surprisingly, the two NMR spectra for the two Aβ species were almost identical.

The only obvious difference is the appearance of a new peak (chemical shift: 1H

8.51ppm, 15N 120.97 ppm) in the HSQC of oligomer. Unfortunately, current NMR data is

not sufficient to assign this peak to any Aβ(1-42) residue.

As a control experiment, HSQC experiment was also applied for Aβ solution that

contains both Aβ(1-42) monomer and oligomers. Such spectrum is shown in Figure 4.16.

Surprisingly, this spectrum is also very similar to that of the Aβ(1-42) oligomer. Overlay

of the two spectra did reveal some chemical shift difference for residue Arg 5, Ser 8, His

13 and His 14. Although these differences seems small, they might be significant in

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addressing the structural difference between two sample solutions, as one sample solution

contains exclusively Aβ(1-42) oligomers, while the other contains a mixture of Aβ(1-42)

monomer and oligomers.

Time dependent SEC of Aβ(1-42) (Figure 4.7) has indicate that the Aβ oligomer

size and quantity will change upon different incubation times. To examine whether the

incubation time plays a role in Aβ(1-42) oligomer structure, HSQC of Aβ(1-42)

oligomers prepared at different incubation times were also compared. Shown in Figure

4.17 is the HSQC of Aβ(1-42) oligomers prepared after 3 hours and 24 hours aging,

respectively. The two spectra showed very few differences except the signal-to-noise

level of the 24-hour-aging one is slightly lower, indicating their structure is very similar.

In fact, careful analysis revealed that all above 1H-15N HSQC spectra (from Figure 4.15

to Figure 4.17) are consistent with ranom extended chain structure (Hou et al., 2004; Yan

and Wang, 2006).

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Figure 4.15 The 1H-15N HSQC spectra of SEC separated Aβ(1-42) oligomer and monomer A: oligomer B: monomer C: Overlay of the oligomer (red) and monomer (blue) spectra.

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Figure 4.16 The 1H-15N HSQC spectra of Aβ(1-42) (containg both monomer and oligomer) and Aβ(1-42) oligomer. A: Aβ(1-42) Oligomer B: Aβ(1-42) containing both oligomer and monomer C: overlay of A and B. Those residues that show chemical shift differences were labeled with blue arrows

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Figure 4.17 The 1H-15N HSQC spectra of Aβ(1-42) oligomer prepared after different incubation times. A: 3hours aging B: 24 hours aging C: Overlay of A and B

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4.2.4.2 HSQC of Aβ(1-42) ADDLs

15N-1H HSQC experiments were also performed for collected ADDLs monomer

and oligomer peaks. However, the HSQC experiment for ADDLs oligomer peak is not

successful, possibly because the oligomer concentration within this peak is too low. The

HSQC spectrum of the ADDLs monomer peak is shown in Figure 4.18. This spectrum is

compared with the one from monomer peak using protofibril preparation protocol. The

two spectra are still very close to each other, and each spectrum is consistent with ranom

extended chain structure.

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Figure 4.18 The 1H-15N HSQC spectra of Aβ monomers using different preparation protocols A: Aβ monomer from ADDLs preparation protocol B: Aβ monomer from protofibril preparation protocol. C: overlay of A and B. Those residues that show chemical shift differences were labeled.

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4.2.5 Pulsed field gradient diffusion measurements of the Aβ oligomers and

monomer solution prepared by SEC

The reason that the 1H-15N HSQC of Aβ(1-42) oligomer is very similar to that of

the Aβ(1-42) monomer is not clear. A possible explanation is that the observed NMR

signal of the oligomer is not from the oligomer, but from the residual monomers,

although the SEC experiments has indicated that there is no monomer present in the

oligomer solution (Figure 4.12). To unravel this puzzle, we decided to use pulsed field

gradient (PFG) NMR self-diffusion experiments to measure the size of Aβ oligomer and

monomer to firther determine their aggregation state.

The (PFG) diffusion experiments were initially performed on Aβ(1-42) oligomer

solution that is directly collected from SEC. However, due to the strong water signal, the

experiments were not successful. To exchange H2O with D2O, the collected Aβ(1-42)

oligomer solution were lyophilized and redissolved in D2O buffer. The PFG diffusion

experiments were then applied on those D2O dissolved Aβ oligomer solution. Same

methods were also used for (PFG) diffusion experiments of Aβ(1-42) monomer. The

obtained diffusion coefficients for the two solutions were shown in Figure 4.19.

Figure 4.19 The exponential decay curve fitting of the integrals in the aliphatic region for Aβ(1-42) oligomer and monomer, respectively.

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Surprisingly, the diffusion coefficients for Aβ oligomers and monomer are almost

identical. This unexpected result might be due to the facts that lyophilization will change

Aβ structure, which had been discussed in section 4.2.1.5: the lyophilization process

change the Aβ oligomer (or monomer) solution so that the re-dissolved D2O solution of

Aβ oligomer (or monomer) is actually a mixture of oligomer and monomer. As a result,

the measured diffusion coefficients are not of oligomer or monomer alone, but rather an

average of two.

To measure the diffusion coefficient of Aβ(1-42) oligomer alone, different

preparation methods were tested to try to make an Aβ(1-42) oligomer solution in D2O

that is free of monomers. Unfortunately, it is confirmed that, without using SEC (it is

impractical to use D2O for SEC experiment), the Aβ(1-42) solution always constitute of

monomer and oligomer, and the measured diffusion coefficients for those solutions are

always close to those shown in Figure 4.19.

As a control experiment, the PFG diffusion experiment was also performed for

Aβ(1-40). The result was shown in Figure 4.20.

Figure 4.20 The exponential decay curve fitting of the integrals in the aliphatic region of Aβ(1-40) monomer.

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In our experience, Aβ(1-40) never forms oligomer. Thus the measured diffusion

coefficient is exclusively for Aβ(1-40) monomer. Surprisingly, the measured diffusion

coefficient of Aβ(1-40) monomer is almost identical to those obtained for Aβ(1-42),

indicating the size between Aβ(1-40) monomer and, at the very least, Aβ(1-42) monomer

are very similar.

4.3 Discussion

Numerous studies have benn reported recently trying to elucidate the structural

information of soluble Aβ oligomers. NMR measurements (Hou et al., 2004; Tseng et al.,

1999) suggested that Aβ peptides adopt predominantly monomeric random extended

chain structure in aqueous solution. However, gel filtration analysis indicates the smallest

Aβ species is a dimer (Burdick et al, 1992; Soreghan. et al., 1994; Walsh, et al 1997).

Photochemical oxidative cross-linking suggested that monomer, dimer, trimer, and

tetramers co-exist in a rapid equlibrium for Aβ(1-40) (Bitan, et al 2001) whereas the

same method applied to Aβ(1-42) suggests that pentamer or hexamer are preferentially

formed which can further assemble into even higher order oligomers (Bitan, et al., 2003).

The folding thermodynamics studies suggest Aβ(1-40) is predominantly an unstable and

collapsed monomeric species whereas Aβ(1-42) are trimeric or tetrameric (Chen et al.,

2006), however, SEC-MALLS and analytical ultracentrifugation indicate Aβ solution

exist as a binary mixture of a monomeric peptide and high-molecular mass oligomer

(Hepler et al., 2006).

Apparently, the above results are not very well consistent with each other. These

inconsistencies may be due to the different experiment conditions and analytical method

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that were being used. However, the complex nature of Aβ solution itself, which contains

mixture of monomer, dimer, trimer, oligoner and higher ordre aggregates, also confound

these studies.

As an effort to resolve the above discrepancies and get high resolution structural

information of soluble Aβ oligomer, we employed a strategy which utilized the

separation power of SEC: we first use SEC to separate Aβ oligomer and monomer, and

then use NMR and other analytical techniques to study the structure of these two Aβ

species independently. Advantage of this approach is that Aβ solution components are

simplified and possible interferences from other Aβ species are inhibited.

Two well established protocols to prepare Aβ oligomers were used in our

experiments, one is Aβ protofibrils preparation and another is ADDLs preparation. In

either case, the SEC experiments results are consistent with those from other groups (for

SEC of Aβ using protofibril preparation method, see Walsh, et al 1997; for SEC of

ADDL, see Chromy et al., 2003) in that both oilgomer and monomer peak was obtained,

and the retention times for both oligomer and monomer are very close to those published.

However we did not observe SEC oligomer peak for Aβ(1-40) as other groups did. The

possible reason could be due to the fact that different groups handle the peptide

differently, and as a result, the Aβ peptide will aggregate differently (it is well known

Aβ peptide aggregation is very sensitive to the environmental conditions such peptide

pretreatment, aggregation seed, pH, temperature, and agitation), Nevertheless, we

successfully prepared Aβ(1-42) oligomer, and also because Aβ(1-42) is more pathogenic

and more physiologically relevant to the disease, we mainly use Aβ(1-42) oligomer in

our studies.

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Aβ aggregation state within the oligomer peak characterized by SEC-MALLS

suggests a polydisperse mixture of high mass oligomers. This result is close but with

clear difference to a recently published result using same techniques (Hepler et al., 2006).

In that report, the molecular weight distribution of the oligomer peak is from 150 kDa to

103 kDa, however, our result suggests the molecular weigh distribution is from 103 to

6×104 kDa. Different experiment conditions might explain the results difference (UV

absorbance detector is used in our experiment during SEC-MALLS to measure the

concentration of Aβ oligomer passing through the column, while the other group is using

an interferometric refractometer; the temperature in our experiment is kept at 25oC, while

the refractometer and light scattering cells used in the other group is at 35oC), however

more careful work needs to be done to make this issue clarified. Aggregation state

characterization of Aβ monomer peak is not successful in our experiment because no

light scattering signal can be recorded. This is probably because the size of Aβ species

within the monomer peak is too small to be detected (the light scattering intensity is

proportional to the sixth power of the particle’s diameter which means small particles

will be more difficult to detect than large particles). More concentrated Aβ monomer

solution might help to identify aggregation state within this peak.

The time dependent secondary structure conversion from random to β-sheet

structure has been characteristic during Aβ aggregation process (Barrow et al., 1992;

Teplow, 1998; Hou et al., 2002). Our CD result suggested that thos observed β-sheet

structure is actually from Aβ oligomer and the random structure is from monomer. This

conclusion is partly in accordance with Teplow’s report (Walsh et al., 1997; Bitan et al.,

2003), however, according to their method (deconvolution of CD spectra by using

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software CDANAL), the secondary structure content of the Aβ oligomer is not 100% β-

sheet structure, but constitute of 47% β-sheet, 40% random, and 13% α-helical structures,

and the monomer peak is not 100% random, but constitute of 79% random, 18% β-sheet

and 3% α-helical structures. Because the software used in these studies relies on the

structural information from globular proteins which might not be compatible with

“unstructured” proteins like Aβ, and because no helical structure has ever been observed

in our experience, we reasoned more quantitative and reliable techniques may be needed

to clarify the conformational components within the Aβ oligomer and monomer.

Overall, our results suggested that the Aβ(1-42) solution composed of a mixture of

Aβ monomer and oligomer each with distinct molecular weight and secondary structures.

To get atomic level structural information of the Aβ oligomer structure, we used the

technique of solution NMR.

Previous NMR studies of Aβ (Hou et al., 2004) have suggested that Aβ peptides

adopt predominantly monomeric random extended chain structure in aqueous solution.

No Aβ oligomer structural information has ever been reported by NMR. One possible

reason that no oligomer structure observed in these NMR studies might be due to fact

that the Aβ sample used in these studies were always kept cold (temperature < 5oC). Aβ

monomer is very stable at low temperature and formation of oligomer is greatly inhibited.

Thus, either because no Aβ oligomer formed in these NMR solutions or because Aβ

monomer is the predominant species in the sample, the Aβ oligomer detection by NMR is

prevented. To overcome the above problems, we used SEC to separate Aβ oligomer from

monomer and collected the oligomer peak for NMR studies. By this way, NMR will only

detect Aβ oligomer and the interference from Aβ monomer is eliminated.

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One potential problem when doing NMR studies of Aβ oligomer is that during the

NMR experiment, the oligomer may dissociate to produce monomer which will affect the

result. We have realized this risk and designed the NMR experiments protocol (see

“Materials and Methods”) which will ensure the stability of Aβ oligomer solution before

and after the NMR experiments. Our results clearly show that neither Aβ oligomer

structure change nor Aβ monomer production had happened during the NMR

experiments.

However, despite all the efforts, the 1H-15N HSQC spectra of Aβ oligomer came

out to be surprisingly similar to those of Aβ monomer. Although several chemical shift

differences were observed between oligomer and monomer which may indicate some

local structural differences, the narrow chemical shift dispersion demonstrated that both

Aβ species are adopting random extended structure.

This result is out of our expectation because all other techniques we used rather

than NMR clearly shows that Aβ oligomer has distinct structure than Aβ monomer. The

only plausible explanation is that the Aβ oligomer must be adopting a symmetrical

structure in which each Aβ monomer has an identical chemical environmental and gives

out exactly same NMR information as an Aβ monomer. However, what kind of

symmetrical structure the Aβ oligomer is?

It has long been suggested that Aβ peptides have surfactant like properties and will

form micelles when the concentration is above critical micellar concentration (cmc). In

those studies, the Aβ peptides are reported to have critical micelle concentrations (cmc)

of 17~25 μM and the Aβ micelle serve as the “seeds” during the Aβ ggregation to

promote Aβ fibril formation. Experimental data has also confirmed that when Aβ

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concentration is below the cmc, the fibrillogenesis process is greatly delayed.

Clearly, according to Aβ micelle theory, the Aβ concentration will be critical in

controlling the Aβ structure: when the Aβ concentration is below the cmc, the Aβ

molecule will be monomeric; when the concentration is above the cmc, the excess Aβ

molecules will incorporate and form the micelle whereas the Aβ monomer concentration

is kept constant at critical micellar concentration. To our knowledge, all NMR studies

performed on Aβ peptides up to date all use concentrations far above the the published

critical micelles concentrations. As a result, the studied Aβ molecules are actually all

micelles, and no NMR has ever been done for Aβ monomers!

In our current experiments, to prepare enough Aβ oligomer for NMR study, the Aβ

solution is intentionally made at higher concentration (100 μM). The SEC collected Aβ

oligomer or monomer concentration are about 35 μM which is obviously above the

reported cmc. According to Aβ micelle theory, this suggests that our collected Aβ

oligomer or monomer are actually all Aβ micelles and thus explain why the HSQC of

two Aβ species is so similar..

Based on above results, we hypothesized that Aβ oligomer are composed of micelle

molecules. To study further the relationship between Aβ oligomer and Aβ micelles, and

to compare the structural difference between Aβ micelle and Aβ monomer, i.e. the

structural difference when the Aβ concentration is above or below the cmc, we studied

Aβ micelle property using NMR in the next chapter.

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Materials Unlabeled Aβ peptides were synthesized and purified as described in

Chapter 2.

Uniformly (>95%) 15N-labeled peptides (or uniformly doubly 15N- and 13C-labeled

peptides) were purchased from Recombinant Peptides as a recombinant fusion protein in

minimal media containing 15NH4Cl as the sole nitrogen source and 13C-labeled glucose

(U-13C6) as the sole carbon source.

Aβ oligomer preparation Two Aβ oligomer preparation protocols were used in this

chapter, one is for Aβ protofibril preparation and another is for ADDL preparation.

For Aβ protofibril preparation protocol, disaggregated Aβ peptides (see

disaggregation method in Chapter 2) were dissolved in 10 mM phosphate buffer to the

final concentration of100 μM with pH at 7.3. The Aβ solution was incubated at room

temperature. Aliquots of Aβ were injected into size exclusion column after different

aging times. The oligomerization processe was monitored by SEC.

For ADDL preparation protocol, we employed the standard protocol described in

the literature (Klein, 2002). Briefly, 220 μl cold HFIP was added to 1 mg Aβ(1-42)

peptide and incubated at room temperature for 1 hour. Allow HFIP to evaporate over

night. The residual HFIP was removed by a speedvac. Add 45 μl anhydrous DMSO to the

peptide. The peptide solution was diluted by adding 1.96 ml F12 medium without phenol

red (BioSource Inc, custom preparation) to a final concentration of 100 μM. Put the

solution in the refrigerator (5 oC) and incubate for 24 hours. Following incubation,

centrifuge (14,000 g) the peptide solution at 4 oC for 10 minutes. The supernatant is the

ADDLs. The supernatant is collected and subjected for SEC experiment.

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The SEC separated Aβ oligomer was collected for NMR experiments. Because the

Aβ oligomer may dissociate to produce the monomer which will affect the NMR result, a

specific experiment setup protocol was strictly followed during the experiments to make

sure neither oligomer structural change nor Aβ monomer production will happen before

and after the NMR experiments. This protocol is described as follows:

As shown in the above flow chart, a 100 µM Aβ(1-42) solution was prepared and

incubated at room temperature. After indicated time of aging, 1 ml solution was injected

into size exclusion column. The oligomer peak and monomer peak were collected

directly into ice-cold NMR tubes, respectively. Immediately after peak collection, the

NMR tube was inserted into the NMR spectrometer (pre-cooled down to 5oC) and the

HSQC experiment starts right away. The HSQC experiment usually takes less than 40

minutes. After the HSQC experiment, the NMR solution was re-injected into size

exclusion column and see if there is any monomer peak shown. This final step will tell if

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any dissociation (for collected Aβ oligomer solution) or association (for collected Aβ

monomer solution) may occur during the HSQC experiments. During this protocol, low

temperature is the key because under such condition Aβ peptide structure can be

stabilized and molecule association or dissociation process will be inhibited.

The advantage of the above protocol is that it can give us unambiguous information

about the identity of the Aβ molecules being measured during the NMR experiments:

whether these Aβ molecules are from oligomer or monomer can be clearly concluded by

the second SEC experiment conducted after the NMR. Our results have confirmed that no

Aβ molecules association or dissociation process has happened during the NMR

experiments.

Size exclusion chromatograph Size exclusion chromatography was performed by

using a Waters breeze system (Agilent Technologies, Palo Alto, CA, USA) with a

Superdex 75 10/300 GL column (Amersham Biosciences, Uppsala, Sweden). The

column was equilibrated with 10 mM phosphate buffer pH 7.4 at a flow rate of 0.5

ml/min for 120 minutes. The temperature was kept at 25oC. Separation process was

monitored by UV absorbance at 220 nm.

SEC-MALLS experiment SEC-MALLS experiment was performed on an Agilent

(Wilmington, DE) 1100 series HPLC system equipped with a UV absorbance detector

and a Wyatt DAWN EOS multiangle laser light scattering (MALLS) detector. Wyatt

Astra software (version 4.90.07) was used to analyze all light scattering data. The

refractive index increment for monomeric Aβ(1-42) was from literature (Helper, 2006).

SEC separations conditions were identical as described before.

Circular dichroism spectroscopy The CD spectra were obtained at 22 °C using a J-

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810 spectropolarimeter (Jasco) and a 1-mm path length cell (Hellma). For each sample,

five accumulative readings were averaged and acquired with 0.2-nm resolution, a 2-s

response time, and 50 nm/min scan speed. Spectra were obtained from 190 to 250 nm.

NMR experiments All NMR spectra were acquired at 800 MHz using Bruker

Avance-800 spectrometers equipped with cryoprobe. The 1H chemical shifts were

referenced to an internal standard of sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4

(TSP), whereas the 15N chemical shifts were referenced indirectly relative to the internal

standard 2,2-dimethy-2-silapentane-5-sulfonic acid (DSS) using consensus ratios of

0.10132912. The probe temperatures were calibrated using methanol and dimethyl

sulfoxide solutions. For 15N-1H HSQC experiment, pulse sequence “gNhsqc” was used.

The data were acquired with 2048 points for the F2 dimension and 128-256 complexe

increments for the F1 dimension. The NMR data were transferred to a PC computer and

processed using program NMRPIPE. All NMR spectra were analyzed using program

CARA.

The diffusion coefficients (D) were obtained using the PFG-water-sLED pulse

sequence. Data accumulation involved acquiring an array of 15 spectra (32 scans each, 5

s recycle delay) with different gradient strengths (g) varying from 0.3 to 30 G/cm.

The NMR signal intensities are related to the D value according to the following

relationship:

R = exp (–γ2Dδ2 (Δ-1/3 δ))g2

where R is the ratio of intensities for a resonance with the gradient on (I) to that

with the gradient off (I0), γ is the gyromagnetic ratio of 1H (2.675 × 104 G-1 s-1), g and

δ are the magnitude and duration of the gradient pulses, respectively, and Δ is the time

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interval between the gradient pulses. For our studies, the following parameters were used:

δ = 5.5 ms, g = 0.3-30 G/cm, Δ = 220-320 ms, and a longitudinal eddy-current delay of

40 ms. The upfield methyl signals (0.73-1.52 ppm) and downfield aromatic signals (6.74-

8.66 ppm) were integrated to provide the signal intensities (I and I0), and these data were

approximated as single, exponentially decaying curves [plots of R vs (g2)] using

averaged fitting values obtained from the Origin program (Microcal).

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5 Surfactant Properties of Aβ peptides

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5.1 Introduction

In previous chapter, NMR studies on Aβ oligomers suggest that Aβ oligomers have

symmetrical structure. In fact, Aβ has long been proposed to have a symmetrical micelle

like structure. Studies have shown that Aβ peptides are in fact surfactant like molecules

and have critical micelle concentration (cmc). When the Aβ peptide concentration is

above the cmc, the Aβ monomers will self assemble into micelles and undergo a

conformational change which finally becomes the fibril nucleus. Kayed (2002) indicated

that those formed micelles are Aβ oligomers and their structure are common to most

amyloid proteins which may represent the primary toxic species of amyloids. To fully

understand the Aβ soluble oligomers formation and structural information, it is

imperative to learn first the structure of Aβ micelles and the mechanism of their

formation from Aβ monomers. Unfortunately, despite numerous research efforts, the

structure of Aβ micelles and their formation still largely remains unknown. In this

chapter, we used surface tension measurement method and NMR techniques trying to

explore the structural properties of Aβ micelles.

5.2 Results

5.2.1 Critical micellar concentration (cmc) of Aβ

The critical micellar concentration (cmc) of Aβ(1-42) was determined by the

method of surface tension measurement [Philips, J.N., 1955; Maget, D., 1999]. Figure 5.1

shows the surface tension measured at different Aβ peptide concentrations. The surface

tension variation clearly shows two phases: when peptide concentration is zero, the

measured surface tension equals to 73.6 mN/m which equals to surface tension of pure

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water (actually73.6 mN/m is slightly higher than pure water surface tension due to

presence of phosphate in the buffer). As Aβ concentration gradually increases, the surface

tension starts decreasing linearly and quickly. Such surface tension decrease stopped

when peptide concentration reached cmc. Once peptide concentration is above the cmc,

further increase in peptide concentration can no longer has appreciable influence on the

surface tension and the surface tension becomes relatively constant. The cmc was

determined by measuring the intercept of two slopes as shown in Figure 5.1.

The critical micellar concentration of Aβ(1-40) is determined in a similar way.

Surprisingly, the cmc for the two peptides are both about 8 μM.

Figure 5.1 Effect of Aβ peptide concentrations on the surface tension of water. The Aβ peptide solution is prepared in 10 mM phosphate buffer and the pH is 7.3. Measurement is done at room temperature. Each point is the mean value of three measurements. The standard deviations for the series of values are included in the Figure but are usually smaller than the size of the symbols.

5.2.2 Time dependent surface tension measurement of Aβ solutions

It is well known that both Aβ conformational change and oligomer formation are

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time dependent (Barrow, C.J., et al, 1992; Wash D.M., et al., 1997). Similarly, we also

studied the time dependent surface tension change of Aβ solutions. Shown in Figure 5.2

are the time-dependent surface tension measurement of Aβ(1-40) and Aβ(1-42),

respectively.

For a 50 μM Aβ(1-40) solution, no surface tension changes were observed,

suggesting a stable equilibrium between Aβ(1-40) monomer, micelles and water

molecules. This result is consistent with a recent report (Sabate & Esrelrich, 2005) in

which Aβ(1-40) surface tension was measured over 30 minutes and no changes were

observed.

However, the surface tension of the 50 μM Aβ(1-42) shows a distinct variation

versus time of aging. The surface tension change can be observed within 5 minutes of

sample praparation. As shown in Figure 5.2, the Aβ(1-42) surface tension quickly

increased from 52.7mN to 57 mN within 12 hours aging. After 12 hours aging, the

surface tension keeps increasing, however, the increasing rate is much slower compared

to the rate within first 12 hours. After 24 hours aging, the surface tension became

relatively constant. No evident surface tension change was observed in extended aging.

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Figure 5.2 The time-dependent surface tension measurements of Aβ(1-40) and Aβ(1-42) (50 μM in 10 mM phosphate buffer, pH 7.3, room temperature). The surface tension of Aβ(1-40) solution remains constant, however for Aβ(1-42), the surface tension increases over time.

5.2.3 Surface tension measurement of Aβ oligomers and monomer

Shown in Figure 5.3 is a size exclusion chromatography of Aβ(1-42). The oligomer

peak was collected and its surface tension was measured immediately. As a control

experiment, the Aβ(1-42) monomer peak was also collected and its surface tension was

also measured. Peptide concentration within collected peaks was determined by

measuring their UV absorbance at 220 nm(ε220nm=5×104M-1cm-1). The calculated

concentrations for collected Aβ oligomer and monomer were 8 and 5 μM, respectively.

The surface tension of collected monomer is 58.8 mN/m, which is consistent with

the expected value from Figure 5.1 for a 5 μM Aβ solution. Interesting result was

obtained for Aβ(1-42) oligomer. The concentration of Aβ(1-42) oligomer is higher than

monomer, according to Figure 5.1, Aβ(1-42) oligomer surface tension should be lower

than that of Aβ(1-42) monomer. Surprisingly, the measured surface tension of Aβ(1-42)

oligomer is 72.4 mN which is very close to the surface tension of pure water (73 mN),

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Thus, this result indicates that Aβ oligomers (or Aβ micelles) structure is very stable. The

conversion from oligomers (micelles) to monomer is very slow.

Figure 5.3 Surface tension of SEC separated Aβ(1-42) oligomer and monomer. 50 μM Aβ(1-42) solution (10 mM phosphate buffer, pH 7.3) was incubated at room temperature for 5 hours and then chromatographed on a size exclusion column (Superdex 75, 10 mM pH 7.5 phosphate buffer as solvent, flow rate 0.5 ml/min). The oligomer and monomer peak were collected and their surface tension were measured immediately. Concentration of each solution was determined afterwards by UV absorbance measurement and indicated in the Figure.

5.2.4 Aβ(1-42) conformation and oligomer formation when peptide concentration is

below cmc

Establishment of cmc for Aβ indicates that the Aβ structure will be dependent upon

concentrations: when concentration is below cmc, the Aβ will be primarily monomeric

while when the concentration is above cmc, the Aβ will be mostly micellar structured.

Interestingly, Aβ aggregation is also concentration dependent [Barrow, C.J., et al, 1992]

in that Aβ will aggregate faster at higher concentration than lower concentration. It will

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be both important and interesting to find out the connection between the two events, i.e.,

whether the micelles are on-pathway or off-pathway during Aβ aggregation.

Shown in Figure 5.4 are the time dependent CD spectra of Aβ(1-42) at two

different concentrations. For the 50 μM solution, in which Aβ(1-42) are mostly micelle

structured, CD spectra clearly shows a time dependent conformational change: initially,

the peptide adopt predominantly random structures as represented by the negative bands

at 198nm. Within 12 hours, the conformation quickly changed from random to β-sheet

structure, as indicated by the appearance of positive absorbance peak at 198 nm and

negative absorbance peak at 217 nm in the CD spectra. The conversion from random to

β-sheet structure continues until after 48 hours aging when most peptides are adopting the

β-sheet structure. Further aging did not result in any evident CD spectra change, however,

precipitates starts emerging in solution, suggesting Aβ fibrils start forming.

In contrast, for the 5 μM Aβ(1-42) solution in which the Aβ structure are mostly

monomeric, no evident conformational change can be observed. The peptide

conformation remains random even after 144 hours aging.

Aβ oligomer formation was studied in previous chapters by size exclusion

chromatography, however the Aβ peptdie concentration used in these studies are all

between 50 ~ 100 µM, which are well above the peptide cmc. Here, we studied the Aβ

oligomer formation for the Aβ peptide concentration below cmc. Shown in Figure 5.5 is

the time dependent SEC of a 5 μM Aβ(1-42) solution. The SEC at zero hour aging is

similar to those of a 100 μM Aβ(1-42) solution (see Chapter 4), in which both a strong

monomer peak and a weak oligomer peak are observed. However, unlike the 100 μM

Aβ(1-42) SEC in which the oligomer peak increases over time, the oligomer peak in

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SEC of 5 μM Aβ(1-42) solution remain unchanged during the experimental period,

suggesting very few Aβ oligomer formed. In fact, no appreciable SEC change was

observed even after extended aging (more than 2 weeks).

Clearly the above results indicated that Aβ aggregation and oligomer formation are

greatly inhibited when the peptide concentration is below cmc.

Figure 5.4 Time dependent circular dichroism experiments of Aβ(1-42) at 50 μM and 5 μM, respectively. The peptide solution is prepared in 10 mM phosphate buffer (pH 7.3) and incubated at room temperature. Each spectrum was taken at indicated incubation times and is the average of six measurements. For 5 μM Aβ(1-42) CD measurement, due to the high noise level between 190 and 200 nm, only spectra between 200 nm and 250 nm were shown.

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Figure 5.5 Size exclusion chromatography of 5 μM Aβ(1-42). The peptide solution was prepared in 10 mM phosphate buffer and the final pH was adjusted to pH 7.3. The peptide solution was incubated at room temperature and chromatographed at indicated incubation times. 10 mM phosphate buffer was used as effluent and the flow rate 0.5 ml/min

5.2.5 NMR studies of Aβ using concentration below critical micellar concentration

To our knowledge, almost all NMR studies performed for Aβ so far are using

concentrations that are at least several hundred µM or even mM. The surface tension

studies of Aβ have suggested that Aβ molecules are surfactant-like and have critical

micellar concentration which is around at 8 µM. Thus, Aβ used in those aforementioned

NMR studies are not in their native monomeric state, but are in fact mostly micelles.

However, to get the knowledge of structure and mechanism associated with Aβ

aggregation process (monomer → oligomer → fibril), it is imperative to be able to study

Aβ at its earliest stage, i.e., when Aβ is still monomeric. Only NMR studies of Aβ using

concentration below the cmc, the only condition under which the peptide monomeric

state can be ensured, can provide such information. It also should be noted that Aβ

concentration in vivo are in fact extremely low, usually in the low nanomolar or even

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pico molar ranges [Neve, R.L., 1998]. Studies of Aβ at lower concentration will also be

more physiologically relevant. Thus, in the following part of this chapter, we explored

NMR studies on Aβ using concentration below the cmc and compare the results with

those obtained at higher Aβ concentrations.

5.2.5.1 NMR studies of a simple model micellar molecule: sodium dodecyl sulfate

(SDS)

Before starting experiments on Aβ, we did NMR studies using a simple surfactant

model molecule “sodium dodecyl sulfate, SDS” to confirm the NMR character changes at

different surfactant concentrations.

Sodium dodecyl sulfate (SDS)

The cmc of SDS in pure water is around 8 mM. Three different concentrations of

SDS aqueous solution were prepared: 0.1 mM (<< cmc), 10 mM(~ cmc), and 100 mN(>>

cmc), which corresponds to SDS monomer, SDS mixture of monomer and micelles, and

SDS micelles, respectively. 1D 1H-NMR experiments were performed to measure SDS

chemical shift and self-diffusion coefficient at these concentrations.

Shown in Figure 5.6 is SDS 1D 1H-NMR spectra. As expected, when SDS

concentration increases from 0.1 mM to 100 mM, the SDS structures changes from

monomer to micelles, and as a result, the SDS chemical shifts also change: the SDS α

protons gradually upfield shifted about 0.02 ppm from 3.67 to 3.65 ppm, while the β, γ, δ

and methyl protons downfield shifted about 0.02, 0.01, 0.04 and 0.04 ppm, respectively.

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Figure 5.6 Chemical shift variation of sodium dodecyl sulfate (SDS) over different concentrations. The SDS solution was prepared by directly dissolving SDS in D2O. The pH was adjusted when necessary by adding diluted HCL or NaOH solution and the final pH was 7.3.

The self diffusion coefficient of the SDS molecules at different concentrations were

also measured and the calculated self diffusion coefficients were 6.54×10-7, 3.92×10-7,

and 0.91×10-7 cm2/s for 0.1 mM, 8 mM, and 100 mM SDS solution, respectively. Clearly,

the SDS self diffusion coefficient gradually decreases as SDS molecular size increases

from monomer to micelles.

5.2.5.2 15N-1H HSQC of Aβ(1-42) and Aβ(1-40) at different concentrations

The above two simple experiments on SDS clearly show that chemical shift and

self-diffusion coefficient strongly depend on the structural state of the surfactant

molecule and are two very sensitive parameters to characterize micelle molecules. By

measuring chemical shift/self-diffusion coefficient difference at different surfactant

concentrations, we will be able to tell whether the molecule is in the monomeric or

micelles state. And more importantly, by measuring the chemical shift differences, we

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will be able to infer the local structural difference between the monomeric and micellar

state of a micelle molecule.

Shown in Figure 5.7 is the Aβ(1-42) 15N-1H HSQC spectra recorded at 100 μM and

1 μM, respectively. Surprisingly, the two spectra are very similar. The NH chemical shift

differences between the two spectra are usually less than 0.003 ppm and the 15N chemical

shift differences are usually less than 0.1 ppm. Similar results were obtained for Aβ(1-

40): very few chemical shift or line width difference were observed between 100 μM and

1 μM Aβ(1-40) 15N-1H HSQC spectra (Figure 5.8).

15N-1H HSQC experiments only detect peptide backbones (NH and N). The above

results suggest that the Aβ backbone structure may remain largely unchanged during

structural transition from monomer to micelles. On the other hand, the Aβ residue side

chains may play more important roles during the micelle formation. Thus, chemical shifts

measurements of Aβ residue side chains at different concentrations may give more

information about Aβ monomer-micelle structural differences.

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Figure 5.7 The Overlayed 15N-1H HSQC spectra of Aβ(1-42) at 100 μM (red) and 1 μM (green) in phosphate buffer (10 mM), pH 7.3, 5 oC.

Figure 5.8 Overlayed 15N-1H HSQC spectra of Aβ(1-40) at 100 μM (red) and 1 μM (green) in phosphate buffer (10 mM), pH 7.3, 5 oC.

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5.2.5.3 1D 1H-NMR measurement of Aβ at different concentrations

The side chain chemical shift differences between Aβ micelles and monomer were

initially compared by recording 1D 1H-NMR spectra at different concentrations using

non isotope labeled peptides. However, it turns out that there are some difficulties that

prevented use of non isotope labeled Aβ peptides for NMR study of Aβ monomer:

because Aβ cmc is only 8 μM, the concentration of Aβ monomer sample solution has to

be very low (<8 μM), however, under such low Aβ concentrations, the impurities from

other sources (H2O/D2O, glass ware, phosphate buffer, etc) will severely affect the Aβ

NMR spectra and make the spectra almost unusuable. Shown in Figure 5.9 is the 1D 1H-

NMR spectra of non isotope labeled Aβ(1-42) recorded at 100 μM and 2 μM,

respectively. Clearly, the peaks that are labeled by red arrows do not belong to peptide,

but are impurities from solvents or other sources. Our results confirm that those impurity

peaks may be not that apparent when Aβ peptide concentration is higher than 5 μM

(although some risk still exists depending on different batch preparation), however when

the peptide concentration is below 5 μM, the impurity signals became predominant in the

spectra and make it hard or impossible to correctly assign the peptide NMR peaks and to

get accurate chemical shift information. Thus, to remove those impurity peaks, 13C

isotope labeled Aβ peptides were used and 1D 13C-1H HSQC NMR experiments were

applied.

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Figure 5.9 1D 1H NMR spectra of non-isotope labeled Aβ(1-42) recorded at 100 μM, 1 μM, and 0 μM, respectively.

Shown in Figure 5.10 is the 1D 13C-1H HSQC spectra of Aβ(1-42) recorded at 100

μM and 1 μM , respectively. The 13C-1H HSQC experiments only detect protons that are

directly attached to 13C atom, so the impurity peaks plus the backbone NHs are

successfully filtered. Only Hα, Hβ and other side chain protons are recorded.

No evident chemical shift differences were observed between Aβ(1-42) 100 μM

and 1 μM spectra. However, some peak patterns obviously changed, for example, the line

width of peaks at 2.47 and 2.82 ppm (Figure 5.10, blue dotted) in 1 μM spectrum became

broader than those in 50 μM spectrum. More significantly, there seems to be more peaks

in 1 μM spectrum than in 50 μM at 3.22, 2.59, 2.18, 1.79 ppm, respectively (Figure 5.9,

red dotted). Although these differences are seemingly small, they may be important in

illustrating the structural differences between Aβ monomer and micelles.

The 1D 13C-1H HSQC experiments were also applied for Aβ(1-40) at 1 μM and 100

μM (Figure 5.11). Similarly, the 1 μM spectrum also shows more peaks (Figure 5.10, red

dotted) than the 50 μM one, and many peaks became broader (Figure 5.10, blue dotted)

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than those in 50 μM spectrum.

The NMR differences observed in 1D spectra between Aβ monomer and micelles

are encouraging. However, because the experimental conditions are inherently different

(peptide concentrations of Aβ monomer are much lower than that of micelles samples),

the signal to noise of the 1 μM Aβ is inevitably lower than that of 100 μM Aβ, so there is

a risk that those differences are simply the result of spectra artifacts. To verify those

spectra differences and assign the possibly new peaks, 2D NMR experiments must be

applied.

Figure 5.10 The Overlayed 1D 1H-13C spectra of Aβ(1-42) recorded at 100 μM, 1 μM, respectively. The possible new peaks that were shown in 1 μM spectrum are marked by red arrows, and those peaks that became broader in 1 μM spectrum are marked by blue arrows.

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Figure 5.11 Overlayed 1D 1H-13C spectra of Aβ(1-40) recorded at 100 μM, 1 μM, respectively. The possible new peaks that were shown in 1 μM spectrum are marked by red arrows, and those peaks that became broader in 1 μM spectrum are marked by blue arrows.

5.2.5.4 2D 13C-1H HSQC experiments of Aβ(1-42) and Aβ(1-40)

Shown in Figure 5.12 are the 2D 13C-1H HSQC spectra of Aβ(1-42) recorded at 0.5

μM, 1 μM and 100 μM, respectively. The Hα/Cα peaks assignment was made by

referencing chemical shift data from Hou (Hou et al., 2003). Other peaks assignment was

completed by analyzing a 2D HSQC-TOSCY spectrum (Figure 5.13).

Careful examinations of Aβ(1-42) 1 μM and 100 μM 2D 13C-1H HSQC spectra

show basically no chemical shift or other peak differences. The additional peaks

observed in 1 μM Aβ(1-42) 1D 13C-1H HSQC spectrum did not show in the 2D spectrum,

indicating those additional peaks may be just spectra artifacts that are due to the low

signal to noise level. An interesting result was observed by careful comparison between

the 1 μM and 100 μM 2D 13C-1H HSQC spectra: some peaks that can be observed in the

100 μM HSQC spectrum disappeared in the 1 μM spectrum. For example, as illustrated

in Figure 5.14, the Hβ/Cβ peaks corresponding to Asp-1 and Asn-27 have comparable

intensity and line width in the 100 μM spectrum, however, in the 1 μM spectrum, the

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Asp-1 Hβ/Cβ peak disappeared while the Asn-27 Hβ/Cβ peak is still observable. Similarly,

the Phe-19 and Lys-16/Lys-28 Hβ/Cβ peaks have comparable intensity and line width in

the 100 μM spectrum, and in the 1 μM spectrum, the Phe-19 Hβ/Cβ peak disappeared

while the Lys-16/Lys-28 Hβ/Cβ peak are still observable. Same results were observed for

other Hβ/Cβ peaks including Asp-7, Tyr-10 and Phe-20. To look further into this

interesting result, we decided to do the same 2D 13C-1H HSQC experiment for Aβ at even

lower concentration.

2D 13C-1H HSQC spectrum recorded for Aβ(1-42) at 0.5 μM was also shown in

Figure 5.12. 0.5 μM is the minimum concentration of Aβ that 2D NMR experiment can

be applied upon in our hands. Still no evident chemical shift differences were observed

when compared with the 1 μM or 100 μM spectra. However, as in 1 μM spectra, peaks

were also found to be missing from the spectra (Figure 5.14). In fact, in addition to Hβ/Cβ

peaks missing, some Hα/Cα peaks are also missing. As shown in Figure 5.15, the Hα/Cα

peaks of Ser-8 and Ser-26 have comparable intensity and line widths in 100 μM spectra,

however, in the 0.5 μM spectrum, the peak of Ser-8 disappeared while the peak of Ser-26

still existed.

The 2D 13C-1H HSQC experiments were also applied to Aβ(1-40) at 0.5, 1, 100

μM, respectively. Similar to results of Aβ(1-42), no additional peaks were observed in

0.5 or 1 μM 2D HSQC spectra in contrast to those found in 1D spectra, suggesting these

additional peaks in 1D spectra might be just spectra artifacts. Careful comparison

between different concentration Aβ(1-40) 2D spectra reveal similar results as those

found for Aβ(1-42): peaks were also missing in 0.5 or 1 μM spectra compared to 100

μM spectrum (Figure 5.16 and 5.17).

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An interesting result was found for Aβ(1-40): as chemical shifts for most atoms did

not change among different concentration, the Hβ proton chemical shifts of three His

gradually downfield shifted from 3.125, 3.068, 3.023 ppm at 100 μM to 3.117, 3.055,

2.997 ppm at 0.5 μM, respectively (Figure 5.18). Although current data is not sufficient

to assign those peaks to the three individual His residues (His-6, His-13 and His-14), the

observed chemical shifts movements (average chemical shift movements > 0.01 ppm)

suggest those His residues may play important roles in Aβ(1-40) monomer to micelle

structural transition.

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Figure 5.12 2D 1H-13C HSQC spectra of Aβ(1-42) (10 mM phosphate buffer, pH 7.3) recorded at 0.5 μM, 1 μM, and 100 μM, respectively. The Hα/Cα peaks assignment to each residue are as shown in the square brackets. Other peaks were only assigned to corresponding residues, the identity of the peaks (i.e., whether peaks are Hβ/Cβ or Hγ/Cγ) were not listed.

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Figure 5.13 2D 1H-13C HSQC-TOCSY spectra of 100 μM Aβ(1-42) (10 mM phosphate buffer, pH 7.3). The relayed connections for several residues in proton dimension (connections among αH, βH, γH) and in carbon dimension (connections among αC, βC and γC) are shown.

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Figure 5.14 Expanded Hβ/Cβ region of Aβ(1-42) 1H-13C HSQC spectra: above, 100 μM (red) vs 1 μM (green); below, 100 μM (red) vs 0.5 μM (green). Those peaks that disappeared or whose intensities greatly decreased in 1 μM or 0.5 μM spectrum are marked with blue arrows.

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Figure 5.15 Expanded Hα/Cα region of overlayed 100 μM (red) and 0.5 μM (green) Aβ(1-42) 1H-13C HSQC spectra. The peaks that disappeared or whose intensities greatly decreased in 0.5 μM spectrum are marked with blue arrows.

Figure 5.16 Expanded Hα/Cα region of overlayed 100 μM (red) and 0.5 μM (green) Aβ(1-40) 1H-13C HSQC spectra. The peaks that disappeared or whose intensities greatly decreased in 0.5 μM spectrum are marked with blue arrows.

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Figure 5.17 Expanded Hβ/Cβ region (partly) of overlayed 100 μM (red) and 0.5 μM (green) Aβ(1-40) 1H-13C HSQC spectra. The peaks that disappeared or whose intensities greatly decreased in 0.5 μM spectrum are marked with blue arrows.

Figure 5.18 Aβ(1-40) His Hβ/Cβ chemical shift gradually upfield shifted when peptide concentration decreases from 100 μM to 0.5 μM. (red: 100 μM, green: 1 μM, blue: 0.5 μM)

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5.2.6 Aβ(1-42) self diffusion coefficients measurements

In addition to chemical shift, another parameter that is usually used to characterize

micelles molecules is self diffusion coefficients. The Aβ(1-42) self diffusion coefficients

experiments were initially carried out by using non isotope labeled peptides and the

measured self diffusion coefficients were 9.9×10-8, 9.4×10-8, 9.5×10-8, 9.6×10-8, 9.4×10-8

cm2/s for Aβ(1-42) concentrations of 5, 10, 20, 50, 100 μM, respectively. Clearly, for

concentrations between 5 and 100 μM, the self diffusion coefficients show very small

difference. To further explore the self diffusion coefficient of Aβ(1-42) monomer, 1 μM

and 0.5 μM Aβ(1-42) solution were prepared and their self diffusion coefficients were

measured. Unfortunately, as has been discussed in 5.2.3, because the peptide

concentration is too low, the impurity signals from other sources severely distorted the

NMR spectra and make it impossible to accurately integer the peptide NMR peaks, self

diffusion coefficients measurement of the 1 μM and 0.5 μM Aβ(1-42) solutions were not

successful.

Efforts has been made trying to measure the self diffusion coefficients at extreme

low concentrations by using 13C-labeled Aβ(1-42) peptides. Unfortunately, the

measurement is still not successful, the possible reason could be partially due to

prolonged pulse sequence time during which too much transverse relaxation had occurred

before it could be detected. Thus, new NMR pulse sequence might be needed in future

work for self diffusion coefficients measurements of very low concentration Aβ

solutions.

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5.3 Discussion

The sequence of Aβ peptides is amphipathic in that the N-terminal residues 1-28

are composed mostly of polar hydrophilic amino acids while the C-terminal residues 29-

42 are composed mostly of non-polar hydrophobic amino acids. However, the

importance of the amphipathic property of Aβ was not fully realized until Soregan and

co-workers showed that Aβ have properties commonly associated with surfactants or

detergents molecules and proposed for the first time that Aβ peptides organized within

the amyloid fibrils in the same fashion as surfactant molecules organized in micelles.

Since then, numerous researches had been done trying to clarify the role of Aβ micelles

in Aβ aggregation. A model of Aβ fibrillogenesis based upon measurement of small

angle neutron scattering (Lomakin, Teplow, 1996) suggested that, under acidic

conditions, there exists a critical peptide concentration above which the Aβ will form

micelles and the Aβ aggregation nucleus will be produced within these micelles which

will finally form fibrils. Other results from fluorescence experiments (Sabate, 2005)

extended the above Aβ fibrillogenesis model to more physiological condition (pH = 7.5)

and suggested that Aβ micelles serves as both nucleation centers and peptide reservoir

and demonstrated that Aβ micelles are located on-pathway of Aβ aggregation.

Immunoreactivity experiments (Kayed, 2002) indicated that soluble Aβ oligomers, or

micelles, are common structures to most amyloid proteins and may represent the primary

toxic species of amyloids.

Our current CD and SEC data support the above reports, as it is shown in our

experiments that Aβ aggregation and oligomer formation are greatly inhibited when the

peptide concentration is below the critical micelle concentration, suggesting micelles

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formation are crucial for Aβ aggregation and oligomer formation.

Aβ form micelles only when concentration is above critical micellar concentration

(cmc). Unfortunately, the measurement of cmc for Aβ has been in great dispute. The

reported Aβ cmc range from 17.6 to 100 μM, whereas in our measurements Aβ cmc is 8

μM. The reason for the cmc discrepancy is not clear, however different sample

preparation protocol and experimental method may account for these differences.

Most of Aβ micelles studies are confined for Aβ(1-40). Aβ(1-42) cmc measurement

was only reported once in Soregan's paper and the measured cmc for Aβ(1-42) was 25

μM which is identical to Aβ(1-40). Our results are in agreement with Soregan's report, as

in our experiments the cmc for Aβ(1-40) and Aβ(1-42) are also very close, although our

measured cmc for both peptides are 8 μM in contrast to their results of 25 μM. However,

although Aβ(1-42) and Aβ(1-40) have similar cmc value, it should not be concluded that

the micelles formation within the two peptides are same, as it is shown in time dependent

surface tension measurement, the two peptides has distinct surface tension behavior: for

Aβ(1-40), its surface tension remains constant during aging period, while the surface

tension of Aβ(1-42) gradually increases over time. These results suggest that Aβ(1-42)

monomer molecules keeps leaving the water/air interface during aging period and

entering the solution to form Aβ oligomers, while Aβ(1-40) has a more stable

equilibrium between monomer, micelles and water molecules. Recall that Aβ(1-42) is

more pathological and more easily to aggregate than Aβ(1-40), the results above suggest

that, although the cmc of Aβ(1-40) and Aβ(1-42) are close, the micelles formation

between the two peptides and their roles in each peptide aggregation process are

different. We propose both Aβ(1-40) and Aβ(1-42) form micelles, however the

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nucleation process within Aβ(1-40) micelles are slow which result in a relatively slow

aggregation process while the nucleation process in Aβ(1-42) micelles are fast and

oligomer formation is immediate from those micelles. A more through kinetic studies in

the future may be needed to confirm the above hypothesis.

Aβ micelles structure is another subject of extensive studies. It has been reported

(Walsh et al., 1997) that Aβ(1-40) micelles consist of between 25 – 50 monomers and

have elongated geometries with radius ~2.4 nm and cylinder length ~11 nm.

Unfortunately, high resolution atomic structure of Aβ micelles has yet to be determined.

Numerous NMR studies had been done on Aβ peptides, however, it is surprising to

note that no NMR study has ever been reported addressing the Aβ micelles properties.

An unfortunate consequence is that all Aβ NMR studies up to date were using peptide

concentrations that were much higher than cmc, suggesting Aβ within those studies are in

fact all micelles. No NMR study has ever been targeting Aβ at its earliest stage: the Aβ

monomers. Thus it is the aim of our NMR study to explore Aβ monomer structure by

using Aβ concentrations below cmc and compare the structural differences between Aβ

monomers and micelles.

To study micelles molecules, the most commonly used NMR methods are to

measure chemical shift or self diffusion coefficients at different concentrations (Odberg,

L., et al.,1972). When the exchange rate between the monomer and micellar states of a

surfactant molecule is fast, the dependence of the chemical shifts on the surfactant

concentration van be treated in terms of a two-sate model:

δ = Pδmon + (1-P) δ mic ( 1 )

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where δmon and δmic are the chemical shifts of monomeric and micellized molecules,

respectively, and P is the fraction of the monomer. For a micelle molecule, the change of

monomer concentration is usually small when the total micelle molecule concentration is

Ctotal above CMC. Therefore, the fraction P of the monomer can be simply calculated as:

P = CMC / Ctotal ( 2 )

Substituting equation (2) into equation (1), we can get the expression for the chemical

shifts above and below the CMC:

δ = ( CMC / Ctotal )δmon + (1- (CMC / Ctotal)) δmic

= δmic + (CMC / Ctotal) ( δmon - δmic ) for Ctotal > CMC (3a)

= δmon for Ctotal ≤ CMC (3b)

Likewise, the self-diffusion coefficient of the micelle molecule can be treated in a similar

way by the two-state model:

D = ( CMC / Ctotal )Dmon + (1- (CMC / Ctotal)) Dmic

= Dmic + (CMC / Ctotal) ( Dmon - Dmic ) for Ctotal > CMC (4a)

= Dmon for Ctotal ≤ CMC (4b)

where Dmon and Dmic are the self-diffusion coefficients of the monomeric and

micellized molecules, respectively. At concentration above CMC, equation 3 and 4

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predict that chemical shift δ and self-diffusion coefficient D depend linearly on the

inverse concentration 1/ Ctotal. At concentration below CMC, only monomers are present,

and the observed chemical shift and self-diffusion coefficient are independent of the

concentration, equal to δmon and Dmon, respectively. Thus, measuring the chemical shift or

self-diffusion coefficient of the micelle molecule at different concentrations will supply

invaluable information for the structural difference between monomeric and micellized

state.

The chemical shift (and self diffusion coefficients) variation over different

surfactant concentrations were corroborated by studying SDS molecules in our

experiments. However, when the same experiments were applied to Aβ peptides, neither

chemical shift nor self diffusion coefficients show evident difference. This result might

suggest the structural differences between Aβ monomer and micelles are very small.

An interesting result was observed when Aβ concentration is below the cmc: some

peaks that can be observed at higher concentrations simply disappeared. NMR peak

disappearance could have many reasons, including resonance overlapping, line

broadening because of intramolecular conformational exchange, or too fast transverse

relaxation due to slow tumbling of large molecules. Although our current results are very

qualitative and only accurate relaxation measurement of those disappeared peaks can

identify whether their dynamic properties will change at different peptide concentrations,

however for Aβ concentration below cmc, the Aβ molecules should have been all

monomeric and their relaxation rate should be slower than that of Aβ micelles. Thus the

peak disappearance might indicate Aβ monomer structure is not simply random, but have

some defined structure.

143

The importance of the three histidine residues in Aβ(1-40) have been illustrated in a

recent paper from our group (Hou, et al, 2003). In that report, the three histidince 2H

chemical shift were found gradually upfield shifted over time and were proposed to form

salt bridge with other residues. Interestingly, current studies show that, for Aβ(1-40), the

histidine Hβ chemical shift also gradually upfield shifted when peptide concentration

decreases from 1000 μM to 0.5 μM. Although current data is not sufficient to identify the

role of histidine residues in Aβ micelles formation, the fact that such histidine chemical

shift movement only occurred for Aβ(1-40), but not for Aβ(1-42), suggests mechanism of

micelles formation between two peptides are different.

144


Materials In this chapter, the Aβ peptides that are used for surface tension and size

exclusion chromatography experiments are unlabelled and those for NMR experiments

are 15N- or 13C isotope labeled. Unlabeled Aβ peptides were synthesized and purified as

described in Chapter 2. Uniformly (>95%) 15N-labeled peptides (or uniformly doubly

15N- and 13C-labeled peptides) were purchased from Recombinant Peptides as a

recombinant fusion protein in minimal media containing 15NH4Cl as the sole nitrogen

source and 13C-labeled glucose (U-13C6) as the sole carbon source.

Surface Tension measurements Surface tension measurements were carried out at

room temperature using a Wilhelmy plate apparatus consisting of a 1-cm platinum plate

suspended by a Cahn 2000 RG Electrobalance and connected to a digital tensiometer

(Nima Tensiometer, Coventry, U. K.). The atmosphere was nearly saturated with water

by inserting beakers with water inside the measuring chamber.

Aβ solutions were prepared by diluting a stock solution (400 μM Aβ in 10 mM

MaOH) with phosphate buffer (10 mM, pH 7.1) to desired concentration. The final pH

was between 7.1~7.4. The surface tension were measured in a Pyrex crystallization dish.

The plate and dish were cleaned with SDS and then flamed to pyrolyze detergent residue

and any dirt or dust particles. The obtained surface tension was an average of six times

measurement. The variation between individual measurements of the same sample was

±0.5 nN/m.

NMR experiments NMR spectra were obtained at 5 oC on Bruker Avance 800 or

900MHz spectrometers equipped with TXI cryoprobes. Both spectrometers have actively

shielded z-axis gradient units.

145

The 1D 13C-1H NMR spectra were acquired with presaturation and WATERGATE

(Piotto, et al., 1992) to suppress the H2O signal. 2D NMR experiments include

heteronuclear single quantum correlation spectra (HSQC) (Bax, et al., 1990) and 2D

HSQC-TOCSY (Sattler, et al., 1995). The 2D 15N-1H HSQC spectra had spectra width

8000 Hz in 1H dimension and 2500 Hz in 15N dimension. The 2D 13C-1H HSQC spectra

had spectra width 8000 Hz in 1H dimension and 13,000 Hz in 13C dimension, and the 2D

HSQC-TOCSY spectra had spectra width 9000 Hz in 1H dimension and 15,000 Hz in the

13C dimension. The NMR data were transferred to a PC computer and processed using

program NMRPipe. Before Fourier transformation, Gaussian and sinebell window

function were applied in F2 and F1 dimension, respectively. The final matrix was

2K×2K. All NMR spectra were analyzed using program CARA.

146

6 Conclusions and Future Studies

147

The focus of this thesis was to elucidate key molecular features of the early steps of

Aβ peptide aggregation. The results from this work could eventually lead to the

development of more specific therapeutic interventions for treatment of patients with AD.

Studies of the Aβ peptides with 1- or 2-naphthylalanine in replacement of Phe19 or

Phe20 suggest that structural features in the early-formed aggregates and the later stage

amyloid fibrils may be different. Thus, the working loop model of the Aβ(1-40) fibril

structure (as derived from solid-state NMR constraints) has the Met35 ε-methyl

positioned above the Phe19 ring (<5 Ǻ), which is within a range where NMR

perturbations should be detected. Our studies of four modified peptides did not show any

significant changes in the NMR spectral data, although enhancement of the random → β-

sheet conversion was seen by circular dichroism. Because the NMR data was consistent

with random structure (just the wild-type Aβ(1-40) peptide), we presume that the

observed rate enhancement must be due to stability provided in the non-NMR detectable

β-sheet structure. Thus, the misfolding steps associated with the early- and later-stages

of Aβ aggregation into oligomers and fibrils, respectively, may involve different types of

structural motifs. To test this hypothesis, additional experiments would include

comparison of the amyloid fibril structures produced by the wild-type and

naphthylalanine peptides; that is, does the Met35 ε-methyl reside above the naphthyl-19

aromatic ring? This would involve close examination of the fibrils by atomic force

microscopy and solid-state NMR, the latter of which would involve the synthesis of

peptides with 13C labels.

The work involved with the characterization of the soluble Aβ oligomers

demonstrated that they do not adopt a single, well defined structure, and instead exist as

distribution of oligomers with micelle-like features. There are several unique features

about these micelle structures; notably, that the critical micelle concentration of the

Aβ(1-40) and Aβ(1-42) are the same, although only the latter peptide forms oligomers

that can be separated by size-exclusion chromatography. Thus, the added hydrophobicity

148

provided by the Ile41-Ala42 segment may explain why the Aβ(1-42) shows greater

neurotoxicity in cell culture. However, numerous questions remain to be addressed, such

as is the toxic species the micelle? Another important discovery was the selective NMR

peak broadening seen below the critical micelle concentration, which suggests that

mechanisms of Aβ aggregation may be concentration dependent and proceed different

above or below the critical micelle concentration. Numerous experiments should be

undertaken to test this hypothesis, including 1H, 13C, 15N-NMR relaxation measurements

and biological neurotoxicity studies in cells, both done at peptide concentrations above

and below the critical micelle concentration. Another important characteristic parameter

that needs to be clarified is the Aβ micelle aggregation number, which is the number of

monomers within the micelles. There have been reports that Aβ(1-40) micelles consist of

between 25 – 50 monomers based on fluorescence measurement (Walsh et al., 1997;

Sabate & Esrelrich, 2005). However, studies from SEC-MALLS indicated the Aβ

micelles aggregation number is much bigger (several hundred to thousand, Hepler et al.,

2006). The reason for these discrepancies is not clear, however different Aβ sample

preparation method between different groups could confound the results. We will

perform both measurement techniques (fluorescence and SEC-MALLS) to determine Aβ

micelles aggregation number by using an identical Aβ sample preparation protocol.

149

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