Circular dichroism and fluorescence of a tyrosine side-chain residue monitors the...

www.bba-direct.com

Biochimica et Biophysica Acta 1699 (2004) 77–86

Circular dichroism and fluorescence of a tyrosine side-chain residue

monitors the concentration-dependent equilibrium between U-shaped

and coiled-coil conformations of a peptide derived from the catalytic

core of HIV-1 integrase

Horea Porumba,b,c,*, Loussinee Zargariana,b, Hayate Merada,b, Richard Maround,Olivier Mauffreta,b, Frederic Troalene, Serge Fermandjiana,b

aDepartement de Biologie et Pharmacologie Structurales, UMR 8113 CNRS, Institut Gustave Roussy, 94805 Villejuif, FrancebLaboratoire de Biotechnologie et Pharmacologie Genetique Appliquee, Ecole Normale Superieure de Cachan, 94235 Cachan, France

cUniversite Paris 13, UFR Sante, Medecine, Biologie Humaine, 93017 Bobigny, FrancedDepartement des Sciences de la Vie et de la Terre, Faculte des Sciences, Universite Saint Joseph, CST-Mar Roukos, Beirut, Lebanon

eLaboratoire de Microchimie et d’Immunologie Moleculaire, Departement de Biologie Clinique, Institut Gustave Roussy, 94805 Villejuif, France

Received 17 June 2003; received in revised form 15 January 2004; accepted 16 January 2004

Available online 13 February 2004

Abstract

The peptide denoted K159 (30 residues) derives from the catalytic core (CC) sequence of HIV-1 integrase (IN, residues 147–175). In the

crystal structure of CC, the corresponding segment belongs to the a4 helix (residues 148–168, including residues Glu 152, Lys 156 and Lys

159, crucial for enzyme activity and DNA recognition), a loop (residues 169–171) and a part of the a5 helix (171–175), involved in enzyme

dimerization. We used the fluorescence and the circular dichroism (CD) properties in the near-UV of the aromatic side chain of a tyrosine

residue added at the C-terminal end of K159 in order to analyze the behavior of the concentrated and diluted peptide in aqueous

trifluoroethanol (TFE), in an attempt to connect the information obtainable at high (NMR), medium (CD) and low (fluorescence)

concentrations of the peptide. Altogether, the C-terminal tyrosine residue provided indirect information on the global conformation of K159

and on the local orientation and environment of the residue. The propensity of TFE to stabilize a-helical conformations in peptides was

confirmed in CD and fluorescence experiments at relatively high (20–160 AM) and low (2–16 AM) concentrations, respectively. At

relatively high concentration, stabilization of the peptide into a-helical conformation favored its auto-association likely in parallel coiled-coil

dimers, as pointed out in our previous work [Eur. J. Biochem. 253 (1998) 236]. This was further confirmed by ANS (1-anilinonaphtalene-8-

sulfonic acid) analysis and fluorescence temperature coefficient measurement. With diluted K159, a Stern–Volmer analysis with positively

and negatively charged quenchers indicated that, when the intermolecular interactions were absent, the tyrosine was in a positively charged

environment, as if the peptide folded into a U-shaped conformation similar to that present in the crystal structure of the enzyme.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Amphipathic helix; ANS; Circular dichroism; Coiled-coil; Concentration dependence; Fluorescence enhancement, quenching and temperature

coefficient; Side-chain as chromophore and fluorophore; Stern–Volmer; Trifluoroethanol

1570-9639/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbapap.2004.01.005

Abbreviations: ANS, 1-anilinonaphtalene-8-sulfonic acid; CC, catalytic

core; CD, circular dichroism; IN, integrase; NMR, nuclear magnetic

resonance; TFE, trifluoroethanol

* Corresponding author. Departement de Biologie et Pharmacologie

Structurales (CNRS UMR 8113), PR2, Institut Gustave Roussy, 39, rue

Camille Desmoulins, 94805 Villejuif Cedex, France. Tel.: +33-1-42-11-51-

29; fax: +33-1-42-11-52-76.

E-mail address: [email protected] (H. Porumb).

1. Introduction

Integration of the HIV-1 genome into the host cell

chromosome is mediated by viral integrase (IN) [1–3].

The enzyme is essential for the viral life cycle and has no

cellular counterpart; therefore, it is a potential target for

developing anti HIV drugs [4,5]. IN has three well defined

domains [6–8], the three-dimensional structure of which are

now well known [9–16]. All of them form dimers in

H. Porumb et al. / Biochimica et Biophysica Acta 1699 (2004) 77–8678

solution, although the full enzyme is likely to function at

least as a tetramer [17–20].

Before the crystal structure of the catalytic domain of IN

was solved [21–23], we have reported that the 147–175 IN

segment had a high helix and coiled-coil forming tendency

[24]. The corresponding peptide, called K159 (Fig. 1B),

exhibited some inhibitory properties against IN, and we

deduced that the inhibition resulted from coiled-coil forma-

tion between the peptide and its counterpart in the enzyme

[25]. Antibodies raised against K159 inhibited the binding to

DNA of IN and of catalytic core (CC). Reactions with K159

of peptide fragments permitted us to determine the location of

the epitope between positions 163–175 [26]. Now we know

that K159 includes thea4 helix and a part of thea5 helix (Fig.

1B). The epitope resides in the loop portion (166–170)

joining helices a4 to a5 and involves a part of a recently

identified nuclear localization signal (NLS) that is essential

for virus replication [27]. It is also suspected that helix a4 is

involved in the specific recognition of the viral DNA, as

several of its residues cross-link to DNA [17,28]. Remark-

ably, most of these residues further interact with a potent

integrase inhibitor, the 5CITEP, i.e. 1-(5-chloroindol-3-yl)-

hydroxy-3-(2H-tetrazol-5-yl)-propenone [29,30], confirming

the biological relevance of helix a4 in IN. We have docu-

mented the propensity of (concentrated) K159 for helix and

coiled-coil formation by nuclear magnetic resonance (NMR)

and CD [31]. The region corresponding to the a4 helix in

K159 displays an amphipathic helix sequence, with heptad

repeats (a, b, c, d, e, f, g—with a and d being generally

hydrophobic amino acid residues) prone to form coiled-coils

[32] (Fig. 1C). In contrast to its counterpart in the protein, the

peptide K159 was only weakly structured in pure aqueous

solution. When sufficiently concentrated (millimolar, tens of

micromolar), K159 underwent both helix stabilization and

auto-association upon addition of moderate amounts of the

hydrophobic solvent trifluoroethanol (TFE).

TFE is commonly used to push peptides to fold into

structures reproducing the ones achieved within a protein or

membrane environment. Employed at moderate concentra-

tions, TFE re-enforces the strength of the intra-helical

CO. . .NH hydrogen bonds [33,34] without affecting the

hydrophobic forces involved in the condensation of helices

in coiled-coil structures. In contrast, further increasing the

TFE concentration in aqueous solution (that is, above 20%)

destabilizes the hydrophobic drive and entails the coiled-coil

dissociation, in spite of the fact that the individual helices

continue to be stabilized under the effect of the solvent [35].

Recently, CD studies on tropoelastin, on the amphipathic

a-helical synthetic linker peptide of the voltage-gated Shaker

potassium channel, on yeast F1F0-ATPase epsilon-subunit,

or on synthetic channel-forming peptides showed an insig-

Fig. 1. (A) Crystal structure of the catalytic core (CC) of integrase (IN),

drawn after [11]. Shown on the left in a different shade of color is the part of

the helix-loop-helix motif containing the sequence that generated K159,

starting from residue 148 (at the upper left extremity of the helix) to residue

175. The amino acid side chains are also represented. Note that the K159

sequence covers entirely the helix a4 (left) and partially the helix a5 (right)

of the enzyme subunit. Apolar residues appear darker. They constitute a

hydrophobic spine, partially oriented towards the core of the enzyme. The

break in the ribbon representation, for residues before 148, is due to the fact

that the crystal structure is loose, badly defined in that region. It may be that

helix a4, taking advantage of its flexible ‘‘hinges’’, is capable of turning its

hydrophobic spine outwards, rendering it capable to interact with an

identical helix of a neighboring subunit by coiled-coil formation, as

predicted by our previous results [25,26]. (B) Peptide K159, in the same

orientation as that of the parent peptide within the catalytic core. Shown in

dark are the positively and negatively charged residues, marked with an

arrow and an asterisk, respectively. The terminal tyrosine is shown in space-

filling representation. Note its positioning in a positively charged

environment. (C) Native sequence and sequence of K159, indicating the

extent of the a4 and a5 helices and of the loop (marked t). Heptad

representation and secondary structure prediction [51] of K159 peptide

within a protein environment (GOR IV analysis; h, helix; c, coil). Note that

residues a and d in each heptad repeat are hydrophobic.

H. Porumb et al. / Biochimica et Biophysica Acta 1699 (2004) 77–86 79

nificant percentage of a-helix in aqueous solutions, in con-

trast to the substantially higher amounts of a-helix foreseen

by protein secondary structure prediction algorithms.

Through the addition of TFE, the amount ofa-helix increased

to the one expected and rendered the peptides ‘‘functional’’

[36–39]. Restricted flexibility and local irregularities have

been shown to persist in the presence of TFE in the secondary

structures of Neu mutants [40], of angiotensin II AT(1A)

receptor [41], of the nuclear pore complex [42], or of mucin 2

glycoprotein [43]. In addition, contrasting effects of low vs.

high concentrations of TFE, such as different association

patterns, aggregation or change in peptide flexibility, had

been reported on the structure of the HIV-1 protein Vpr [44],

of acidic fibroblast growth factor from newt [45], or apoli-

poprotein E [46]. Along the same lines, we shall show in this

paper that 20% TFE tolerates the presence of the loop in the

case of the diluted K159 peptide.

The contribution of aromatic side-chains to the near-UV

CD is often used to probe the orientation and environment

of the aromatic residues of a polypeptide. Although the

aromatic CD bands are by at least one order of magnitude

weaker than those contributing in the far-UV and thus

require larger amounts of protein in order to be measured,

the small number of aromatic chromophores vs. peptide

chromophores offers the advantage that, if the aromatic side-

chain is affected by a highly localized change in environ-

ment, this can be easily detected, whereas a substantial

fraction of the peptide groups ought to be perturbed in order

to provide a measurable effect in the far-UV.

NMR requires samples in the tens-of-millimolar range,

CD in the tens-of-micromolar range and fluorescence may go

down to the tens-of-nanomolar range. By the use of these

techniques, here and in a series of foregoing papers, we

attempted to follow the behavior of the same peptide over

several logs of concentration. In the present work, circular

dichroism (CD) performed in the aromatic UV region (the

tyrosine band at 275 nm) and a number of fluorescence

techniques parallel the previous CD and NMR results related

to the backbone structure of (concentrated) K159 in the

presence of aqueous TFE [25,26,31,35], and developed this

information by looking at the behavior and environment of

the side-chains, especially that of tyrosine. It is first shown

that the tyrosine phenolic ring can be used as an internal

marker to monitor peptide folding and auto-association. The

use of fluorescence methods then permits to infer on the

structure of the dilute, presumably non-associated peptide in

aqueous TFE mixtures, in a concentration domain inaccessi-

ble to CD and NMR.

2. Materials and methods

2.1. Peptides

Peptide K159 (SQGVV ESMNKELKKIIGQVRDQA-

EHLKTAY) was derived from the IN sequence 147–175

(SQGVI ESMNKELKKIIGQVRDNAEHLKTA), by addi-

tion of an amidated tyrosine at the C-terminus, for quantifi-

cation and reporter purposes, and by the conservative

replacement of I-151 with Vand, respectively, of N-168 with

Q, for reinforcing the propensity towards helix formation.

They were synthesized according to the Fmoc procedure on

an Applied Biosystems model 432A automatic solid phase

synthesizer and were purified by reverse-phase HPLC on an

Aquapore column using a linear gradient from 0% to 100%

acetonitrile, 0.1% trifluoroacetic acid in water. The molecular

mass of each peptide was determined by Electrospray Ioni-

zation Mass Spectrometry (ESIMS) on the Platform-quadru-

pole instrument (VG Biotech). Peptide concentrations were

determined by UV absorption using the molar absorption

coefficient at 280 nm equal to 1197 M�1 cm�1.

2.2. CD spectroscopy

CD spectra were recorded on a Jobin-Yvon CD6 dichro-

graph in 1-mm and 1-cm path length cells for the far-UVand

near-UV domains (around 222 and 275 nm), respectively.

Peptide spectra were obtained at 25 jC in aqueous solutions

of TFE from 0% to 80% (vol./vol.) and were baseline

corrected. Prior to the CD measurement, the samples were

subjected to centrifugation at 10,000� g for 10 min in a

microcentrifuge. UV absorbance at 280 nm was checked

before and after centrifugation, such as to exclude a possible

loss of material through formation of insoluble aggregates.

Here, the (near-UV) molar differential absorptivity De275 isexpressed per peptide molecule (M� 1 cm� 1).

2.3. Fluorescence spectroscopy

Fluorescence spectra were recorded on a Jobin-Yvon

Fluoromax-2 spectrofluorimeter. The intrinsic fluorescence

of K159, as well as that of tyrosine amide, in the concen-

tration range from 3.6 to 65 AM, was measured at 25 jC, in1-cm path length semi-micro cuvettes containing 800 Al ofwater or aqueous TFE, with excitation at 273 nm, with,

respectively, 2- and 5-nm monochromator slit widths. All

measurements were corrected for the inner filter effect by

multiplying with 2.3A/(1� T ), where A and T are the

absorbance and transmittance of the sample, respectively.

The reported (relative) fluorescence intensities of the sam-

ples, proportional to the quantum yields of their tyrosines, in

arbitrary units, were approximated to the ratios between the

maximum heights of the baseline-corrected fluorescence

emission bands and the absorbencies of the same samples

at the maximum of their excitation spectra. The temperature

coefficients (arbitrary units) were obtained by dividing by

10j the variations of the relative fluorescence intensities

measured between 15 and 25 jC.Unless otherwise stated, ANS (1-anilinonaphtalene-8-

sulfonic acid) fluorescence was measured with excitation

at 372 nm and emission at 530 nm [47]. Its concentration

was maintained constant at 50 AM throughout the experi-


ment performed in aqueous 20% TFE. Fluorescence en-

hancement following interaction with K159 (or with tyro-

sine amide) was expressed as Q =F/Fj� 1, where Fj is the

fluorescence of ANS in the absence of added sample.

Fluorescence enhancement at infinite sample concentration

was obtained, after linear extrapolation, from the ordinate of

the double reciprocal plot 1/Q vs. 1/[sample], where the

sample concentrations (K159 or tyrosine amide) ranged

between 2 and 16 AM for the low concentration domain

and between 20 and 160 AM for the domain of high

concentrations, respectively, as described in Fig. 3B.

Quenching by KI (up to 50 mM) and CsCl (up to 150

mM) was performed (as in Ref. [47], for instance, but see

also Refs. [48,49]) with peptide or tyrosine amide at

constant 3.6 or 65 AM concentrations and plotted according

to the Stern–Volmer equation (Fig. 5A):

Fo=F ¼ ð1þ KSV½X �Þ;

where [X] is the quencher concentration, F is the fluores-

cence of the sample, Fo is the fluorescence in the absence of

quencher, and KSV is the Stern–Volmer constant. The

linearity of the plots showed that the precaution of working

at constant ionic strength would have been unnecessary.

Approximate values of the bimolecular collisional rate

constants, kq, were obtained by the ratio between the KSV

values and the fluorescence lifetime of tyrosine, taken to be

roughly equal to 1 ns.

3. Results

3.1. Rationale

The peptide K159 under study, of 30 residues, derives

from the IN sequence corresponding to residues 147–175. In

the CC crystal structure [9–13], the sequence resolves into

an a helix (a4: residues 148–168), a loop (t: 169–175) and a

shorter, second helix (part of a5: 171–175), as shown in Fig.

1A–C. Otherwise, K159, containing two internal substitu-

tions, meant to reinforce the propensity towards helix for-

mation, retains the same distribution of apolar amino acids in

its a4-like domain. Neither the ‘‘classical’’, conservative

substitutions (I!V, both residues possessing h-branchedaliphatic side-chains, and N!Q, both having amide side-

chains), nor the appended tyrosine is thought to substantially

alter the conformational behavior of the original peptide.

K159 was prepared with the additional tyrosine to the C-

terminal: (i) to aid peptide quantification from its UV absor-

bance at 275 nm, (ii) to be used as a side-chain chromophore

for CD studies in the near-UV, and (iii) to serve as an intrinsic

fluorescence reporter. The choice of tyrosine rather than

tryptophane as fluorescence probe, in spite of the former

being less sensitive as a fluorescent reporter, was imposed by

the need to measure the interaction of the peptide with the full

enzyme, whose intrinsic fluorescence (due to the presence of

tryptophanes) was to be monitored. The terminal location of

the fluorescent reporter was dictated by the fact that the

residues internal to the sequence were meant to specifically

recognize the enzyme’s DNA substrate [28].

The amino acids from positions 2 to 29 of K159 can be

organized in the form of four heptad repeats (residues

labeled from a to g, with those in positions a and d tending

to be hydrophobic, Fig. 1C). This arrangement confers to

the helix a substantial amphipathic character that justifies

the adoption of a coiled-coil conformation in which the

hydrophobic side-chains, disposed in apolar strips that run

along the helix axis, come in close interacting contacts [32].

Also note the fact that in a situation where the whole peptide

were helical, the C-terminal tyrosine would belong to this

apolar strip, taking position a in the incomplete heptad. It

could freely participate in the stabilization of a parallel

coiled-coil structure (not shown) by interacting with the

tyrosine of another helix. In this arrangement, the stacked

tyrosine residues might undergo substantial coupling [50]

but, because of their terminal position, they would still be

accessible to the solvent from at least one direction.

3.2. Secondary structure predictions

It is intriguing that several consensus secondary structure

prediction algorithms, such as GOR IV [51], indicate a

propensity of the peptide segment 147–175 to adopt a

helical structure over its entire length (Fig. 1C) and make

no prediction on alternative structures adopted by the resi-

dues AEH (169–171, underlined), known to belong to the

‘‘loop’’ of the motif in the native enzyme. No presence of the

loop was inferred from our previous experiments by CD and

NMR with (concentrated) K159 and its derivatives under

various conditions [25,26,31,35]. We shall show below that,

with diluted K159, under specific circumstances, the data are

consistent with the peptide being folded at the level of the

loop into a hooked, U-shaped conformation similar to that

present in the crystal structure of the enzyme. To begin with,

one should note that the peptide is only weakly structured in

aqueous solution at any concentration. The solvent TFE is

used to create an environment similar to that existent within a

protein, suitable for peptide structuring. The effect of TFE on

concentrated (associated) as well as diluted (non-associated)

K159 will be compared. A series of fluorescence and CD

techniques will be employed to document the association

process and to infer on the conformation of the peptide chain

in either monomer or associated situation.

3.3. Exposition of the terminal tyrosine side-chain

chromophore

Based on peptide bond geometry, we have already shown

that upon increasing the TFE concentration, under the

conditions of the NMR experiment (peptide concentrations

in the millimolar range), the helical stabilization and the

auto-association of the helices into bundles evolve in

parallel at least up to 20% TFE [31]. The connectivities,

Fig. 2. (A) CD spectra of tyrosine amide (curve 1) and of K159 (curve 2) in

the aromatic, near-UV region (42 AM, 20% TFE). (B) CD at 275 nm of

concentrated K159 (42 AM) as a function of TFE concentration: Up to a

concentration of 40% TFE, the points were fitted to a sigmoid whose

midpoint is at C0.5 = 15F 0.2%. As revealed by NMR [31], at this high

peptide concentration the chains start as random coils, go progressively

through partially helical, self-associated dimers and, close to 20% TFE,

they end up as a fully helical parallel coiled-coils. CD spectra of K159 in

the far-UV (peptide bond region) are available from work already published

(Ref. [26], see text for details). The data points obtained above 40% TFE

correspond to the domain where the hydrophobic drive begins to diminish.

Equilibrium centrifugation data obtained at high TFE concentrations with a

related peptide show that, although the peptide remains helical, the

oligomers tend to dissociate [35].


the change in the chemical shift index and the NH proton

temperature coefficients obtained by NMR told us that, at

0% TFE, the peptide is mainly in random conformation,

although there are helix nucleation positions budding in its

N-terminal portion. Between 0% and 20% TFE, the average

percent helical content reaches about 4/5 of its maximum

value, which in itself is of about 80%. Within this domain of

TFE concentrations, the a4 portion of K159, which displays

the higher proportion of helical structure, can therefore take

part into helix-to-helix interactions with neighboring mole-

cules. The tyrosine located at the C-terminal extremity of the

peptide belongs to the region that remains for the moment

unstructured. At higher TFE concentrations, however, this

part of the peptide also becomes helical and can participate

to the coiled-coil formation. The latter tendency is now

complicated by the organic solvent that acts to diminish the

hydrophobic drive towards peptide association. In addition

to the NMR work, we have also shown that, in 20% TFE, the

far-UV CD signal (De222) increased with the peptide con-

centration, therefore confirming the fact that the peptide

association (a concentration-dependent process) and the

adoption of the helical structure evolved in parallel. It has

further been shown that the variation of the De220/De208 ratioas a function of the peptide concentration was sigmoidal

(with mid-point at about 20 AM) in the case of K159, but not

in the case of its kinky derivative, called P159, bearing a

central proline; the proline did not affect the overall helicity

of the peptide but prevented it from forming a coiled coil

[25]. There was no sigmoidal variation of the De220/De208ratio in the case of the more helicogenic derivative of K159

(called EAA26), which was shown by analytical ultracen-

trifugation to form tetramers rather than dimers in 20% TFE;

the tetramers would dissociate into dimers and then mono-

mers when the concentration of TFE exceeded 20% [35]. We

shall retain from the above data that 20 AM is the milestone

separating ‘‘concentrated’’ from ‘‘diluted’’ peptide in 20%

TFE. We shall now show that the spectroscopic properties of

the terminal tyrosine of concentrated K159 reflect the above

structuring and association process and that the behavior of

K159 is different when the peptide is diluted.

With K159 at 42 AM, a concentration that we shall call

high in the framework of this study, the CD in the aromatic

region (De275, Fig. 2A) reflects the helical structuring

(folding) process by TFE, as previously revealed by

NMR and CD in the peptide bond region (De222)[25,31]. Indeed, a CD curve of similar shape to that

recorded at 222 nm is obtained from the Lb band of

tyrosine at 275 nm [52] (Fig. 2B). The increase in the

De275 value produced by TFE is compatible with the

variation of De222 and consistent with the fact that the

tyrosine side-chain, initially in the situation of a fairly

rotating chromophore at the C-terminal of the unstructured

peptide monomer, is forced into a rigid orientation upon

the formation of a helix dimer [53]. This would be a proof

only if the effect were absent at low concentration of the

peptide. Only fluorescence, which has access to the low

concentrations forbidden to CD, will be able to confirm the

issue later on in this work.

Actually, during the nucleation-condensation process,

the C-terminal tyrosine, situated at the a position of an

unfinished heptad (Fig. 1), is allowed to interact with the

tyrosine of another helix lying in a parallel orientation, to

form interacting pairs. The resulting motional restriction in

the tyrosine side-chain contributes to the increase of the

phenolic CD signal. The mid-point of the titration at 275

nm is found around 14% TFE, similar to that of the

titration at 222 nm [24]. The drop in the CD signal

observed above 40% TFE may be due to the dissociation

of the dimer, because of the reduction in the hydrophobic

drive, as we have already demonstrated by analytical

centrifugation (sedimentation equilibrium) and glutaralde-

hyde cross-linking with a peptide derived from K159,

called EAA26 [35].

Fig. 3. (A) Emission spectrum of ANS (100 AM, 20% TFE), in the presence

of dilute K159 (curve 1, 5 AM), of tyrosine amide (curve 2, 50 AM), and of

concentrated K159 (curve 3, 50 AM). (B) Extrapolation of raw data (note

that the scale for the diluted K159 was contracted 10-fold). (C) Maximum

enhancement of the fluorescence of ANS (50 AM) by K159 (diluted, at 2–

16 AM, and concentrated, at 20–160 AM) and by tyrosine amide (20–160

AM) in aqueous 20% TFE, confirming the auto-association of the

concentrated peptide. See Materials and methods for further details.


3.4. Peptide association

The recognized nucleation-condensation process of K159

was reassessed using the ANS dye (1-anilinonaphtalene-8-

sulfonic acid). The dye usually inserts itself into the apolar

(hydrophobic) interfaces of protein oligomers and, in so

doing, it blue-shifts and enhances its relative fluorescence

intensity [47]. Its use to monitor peptide association is

uncommon and we performed this experiment primarily as

an exercise, which also confirmed the validity of the

‘‘milestone’’ concentration of 20 AM, delimitating the

associated from the non-associated peptide, obtained from

the De220/De208 ratio (see above).

Indeed, as expected, there is a blue shift and an increase

in the intensity of the emission band of ANS in the presence

of concentrated (20–160 AM) K159 in 20% TFE (Fig. 3A

and B). Under these conditions, after extrapolation at

infinite peptide concentration, ANS nearly doubles its

relative fluorescence intensity as compared to free ANS,

reflecting the (already known) fact that the peptide helices

are engaged in inter-molecular associations, likely coiled-

coil structures (Fig. 3C, [24]). In contrast, no environment

suitable for the enhancement of ANS fluorescence exists in

diluted (2–16 AM) K159 in 20% TFE, which presumably

remains mainly in monomer form at low concentration (Fig.

3C). For the sake of completion, we also report data on the

behavior of the tyrosine amide control. It is not known why

ANS fluorescence is moderately high in the presence of the

free amino acid, as if some stacking took place between

ANS and the tyrosine phenyl ring.

3.5. Tyrosine solvation

It is possible to monitor the TFE dependence of the

association of K159 by following the evolution of the

relative fluorescence intensity of tyrosine, which acts as

an intrinsic reporter. This approach is shown in Fig. 4. First,

one notes that the effect of TFE on free tyrosine amide is

biphasic at any sample concentration (Fig. 4A and B). For

the concentrated tyrosine amide, we willingly exaggerated

the asymptotic nature of the two opposing tendencies (at 42

AM, Fig. 4A). Thus, in the domain of low organic solvent

concentrations (from 0 to about 15% TFE), TFE seems to

‘‘remove’’ the water molecules that hydrate the tyrosine

amide and this leads to increased fluorescence intensity.

Above 15% TFE, it is the TFE that ‘‘solvates’’ the tyrosine

amide. Like water, TFE is (an even better) quencher of

tyrosine fluorescence. The 15% TFE concentration is again

a milestone in the solvation, just as it is in the other

properties of this solvent [34]. Diluted (15 AM) tyrosine

amide in TFE shows a bell-shaped biphasic behavior similar

to that of the concentrated one, except that the curve is less

neat due to lower sensitivity at that dilution (Fig. 4B).

At TFE concentrations above 20%, the relative fluores-

cence intensity of the free tyrosine amide is quenched to a

considerable extent, which is not the case with the tyrosine

belonging to the peptide. The effect of TFE on the intrinsic

fluorescence of K159 at 42 AM concentration is sigmoid in

shape, with midpoint at 11%TFE (Fig. 4A). Obviously, as the

peptide evolves from a random coil structure towards a

helical conformation and as it changes its degree of associ-

Fig. 5. Stern–Volmer analysis of K159 (diluted, at 3.6 AM, and

concentrated, at 65 AM) and of tyrosine amide (42 AM) in aqueous 20%

TFE, with KI and CsCl as negatively and positively charged quenchers,

respectively: representation of the raw data (A), and collisional rate

constants (B), suggesting the positioning of the tyrosine fluorophore in a

positively charged environment within diluted K159.

Fig. 4. Effect of TFE on the fluorescence intensity (arbitrary units) of free

tyrosine amide (squares) and of the tyrosine of K159 (triangles) in:

concentrated solutions (42 AM, C0.5 = 11.3F 0.1%) (A), and diluted

solutions (15 AM, C0.5 = 14F 2%) (B). In either case, in the absence of

TFE, the peptide is structured as a random coil. At 20% TFE concentration,

if concentrated, the peptide chains are fully helical, straight, and associated

as parallel coiled-coils. If diluted, they are monomers possibly adopting a

hook-type conformation.


ation (nucleation-condensation process), this offers a differ-

ent environment to the tyrosine reporter. In the fully associ-

ated coiled-coil, the tyrosines are certainly less exposed to the

solvent than are the free tyrosine amide molecules, and this

accounts for their comparatively larger relative fluorescence

intensity. The variation of the relative fluorescence intensity

of K159 at this high concentration is in line with the near-UV

CD data (De275) described above and with the far-UV CD

(De222) and NMR data previously reported [25,31]. Fluores-

cence is therefore capable of monitoring the nucleation-

condensation process of a peptide chain.

There is some difference between the amplitudes of the

sigmoid curves corresponding to concentrated (42 AM) and

diluted K159 (15 AM)—put side by side for instance the

upper plateau levels in Fig. 4A and B. The difference in the

relative fluorescence intensities might be significant in view

of the experiment that follows.

3.6. Tyrosine environment

We performed a Stern–Volmer analysis with positively

and negatively charged quenchers in order to check on the

nature of the environment of tyrosine in the partially

structured, diluted (presumably monomer) peptide as com-

pared to the auto-associated, concentrated K159 (raw data in

Fig. 5A). The results reveal an important difference between

the environments of tyrosine in diluted vs. concentrated

K159 in 20% TFE aqueous solutions (Fig. 5B): (i) In diluted

K159, the negative iodide quencher is preferentially

attracted towards tyrosine, meaning that the reporter fluo-

rophore is in a positively charged environment. This is

confirmed by the fact that the positively charged cesium

ion is repelled by the positive environment of tyrosine in

diluted K159, so that the collision rate is below that of free

thermal motion. (ii) Free tyrosine amide and the tyrosine

from concentrated K159 collide with either KI or CsCl

quenchers at rates compatible with the thermal motion,

meaning that the tyrosine of concentrated K159 is in an

uncharged environment.

The effect of the peptide concentration (in 20% TFE) on

the quantum yield of tyrosine (expressed as relative ‘‘fluo-

rescence intensity’’, in arbitrary units) also confirms the fact


that the tyrosine fluorophore senses the association process.

The mid-point of the dimerization reaction, as witnessed by

the terminal tyrosine, is around 32 AM, which compares

well (and needs not to be identical) to the 20 AM value

obtained from the far-UV CD, the latter reflecting the

behavior of the entire backbone (Fig. 6A and C). A similar

conclusion emerges from the comparison of the temperature

coefficients of the fluorescence intensities of the tyrosines,

which react to the better solvent exposition of the fluoro-

phores in associated K159 as compared to the non-associ-

ated peptide (compare Fig. 6B and D).

It is not known to what extent and under what circum-

stances the vicinity of tyrosine and lysine, in positions i and

i + 4, could lead to some fluorescence quenching. If that

effect were predominant, then the relative fluorescence

intensities of the unstructured peptide, of the (partially

structured) dilute peptide and of the associated concentrated

one might have ranked rather in reverse order than obtained

experimentally (Fig. 6B). We think that as long as the

portion of the a5 helix bearing the terminal tyrosine

persists in both diluted and concentrated K159 in 20%

TFE (as envisaged above), the geometric relationship

between the lysine side-chain and the tyrosine chromophore

would be the same in a straight or in a U-shaped polypep-

tide chain and thus would not influence the rest of the

analysis.

Altogether, the data are compatible with a model where-

by the diluted K159 (in 20% TFE) is monomeric, with the

a-helical structure interrupted after the a4 portion of the

chain. The apolar spine (residues a and d of the heptads) that

extends all along the sequence (Fig. 1A) might be respon-

sible for the hydrophobic drive that induces the bending of

the rest of the chain. This force acts to close an apolar

pocket between the a4 portion and the C-terminal part of the

molecule. Thus, in the diluted K159, the C-terminal tyrosine

has its phenolic side-chain less exposed to the solvent, in an

area surrounded by the positive charges of the chain (Fig.

1B), as suggested by the Stern–Volmer and the temperature

coefficient results (Figs. 5B and 6D). The folded conforma-

tion might resemble that of the structured peptide within the

native protein (Fig. 1A and B).

Fig. 6. (A) Emission spectra (relative fluorescence intensity) in 20% TFE of

diluted (15 AM, curve 1) and concentrated K159 (42 AM, curve 2), and (B)

effect of sample concentration (in 20% TFE) on the fluorescence intensity

(arbitrary units) of the tyrosine of K159 and of free tyrosine amide, showing

the lower exposition to the solvent of the fluorophore in the diluted peptide.

(C) Effect of peptide concentration on the fluorescence intensity of K159 in

20% TFE (C0.5=32.4F0.2 AM), and (D) on the temperature coefficients of

diluted (15 AM) and concentrated (42 AM) K159 and tyrosine amide. In

concentrated solution the entire sequence of K159 adopts an a-helical

conformation and the resulting shafts interact by coiled-coil formation. It is

possible that, in dilute solutions, the C-terminal extremity of K159, bearing

the tyrosine, is non-helical and torn into a hook, thus maintaining the

phenol group ‘‘hidden’’ from the solvent, this accounting for its higher

fluorescence intensity and lower temperature coefficient, in a region

‘‘guarded’’ by the positive charges of the chain, as implied by the Stern–

Volmer experiment.


In spite of the use of TFE, it turns out that the U-shaped

conformation identified by us for the individual peptide is

biologically relevant. Indeed, the antibodies raised against

K159 as well as against truncated portions of this peptide

have enabled the localization of the epitope, not unexpected-

ly, within the ‘‘loop’’ portion of the peptide, a segment that is

endeavored with greater flexibility [26,46]. The same anti-

bodies recognized the native enzyme, thus indirectly demon-

strating that the proposed U-shaped structure of K159 is a

reality.

In conclusion, the known property of moderate concen-

trations of TFE to induce a-helical peptide conformations has

again been confirmed. The present approach, based on the use

of the tyrosine side-chain phenol group as a CD chromophore

and as a fluorophore reporter, confirms that upon addition of

TFE in concentrated peptide solution the entire sequence of

K159 turns into a-helical conformation and that the resulting

shafts interact by coiled-coil formation (nucleation-conden-

sation process). We contributed to demonstrate the potential

of the fluorescence technique to describe the environment of

reporter side-chains and to monitor the evolution of the

helical structuring process in both concentrated and diluted

solutions, the latter domain not being amenable to NMR and

being hardly accessible to CD.

Acknowledgements

We thank J.P. Levillain for skilled assistance in peptide

synthesis. This project was supported by grants from the

following organizations: SIDACTION (to L.Z.) and ANRS

(to H.M.).

References

[1] P.O. Brown, in: J.M. Coffin, S.H. Hugghes, H.E. Varmus (Eds.),

Retroviruses, Cold Spring Harbor Laboratory, Cold Spring Harbor,

NY, 1997, pp. 161–203.

[2] E. Asante-Appiah, A.M. Skalka, Molecular mechanisms in retrovirus

DNA integration, Antivir. Res. 36 (1997) 139–156.

[3] P. Hindmarsh, J. Leis, Retroviral DNA integration, Microbiol. Mol.

Biol. Rev. 63 (1999) 836–843.

[4] M.S. Hansen, S. Carteau, C. Hoffmann, L. Li, F. Bushman, Retroviral

cDNA integration: mechanism, applications and inhibition, Genet.

Eng. (NY) 20 (1998) 41–61.

[5] Y. Pommier, A.A. Pilon, K. Bajaj, A. Mazumder, N. Neamati, HIV-1

integrase as a target for antiviral drugs, Antivir. Chem. Chemother.

8 (1997) 463–483.

[6] A. Engelman, R. Craigie, Identification of conserved amino acid res-

idues critical for human immunodeficiency virus type 1 integrase

function in vitro, J. Virol. 66 (1992) 6361–6369.

[7] A. Engelman, F.D. Bushman, R. Craigie, Identification of discrete

functional domains of HIV-1 integrase and their organization within

an active multimeric complex, EMBO J. 12 (1993) 3269–3275.

[8] D.C. van Gent, C. Vink, A.A. Groeneger, R.H. Plasterk, Complemen-

tation between HIV integrase proteins mutated in different domains,

EMBO J. 12 (1993) 3261–3267.

[9] A. Wlodawer, in: K. Maramorosh, F.A. Murphy, A.J. Shatkin (Eds.),

Advances in Virus Research, vol. 52, Academic Press, San Diego,

1999, pp. 335–350.

[10] F. Dyda, A.B. Hickman, T.M. Jenkins, A. Engelman, R. Craigie, D.R.

Davies, Science 266 (1994) 1981–1986.

[11] G. Bujacz, J. Alexandratos, Z.L. Qing, C. Clement-Mella, A. Wlo-

dawer, FEBS Lett. 398 (1996) 175–178.

[12] Y. Goldgur, F. Dyda, A.B. Hickman, T.M. Jenkins, R. Craigie, D.R.

Davies, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 9150–9154.

[13] S. Maignan, J.-P. Guilloteau, Q. Zhou-Liu, C. Clement-Mella, V.

Mikol, J. Mol. Biol. 282 (1998) 359–368.

[14] P.J. Lodi, J.A. Ernst, J. Kuszewski, A.B. Hickman, A. Engelman,

R. Craigie, G.M. Clore, A.M. Gronenborn, Biochemistry 34 (1995)

9826–9833.

[15] A.P. Eijkelenboom, R.A. Lutzke, R. Boelens, R.H. Plasterk, R.

Kaptein, K. Hard, Nat. Struct. Biol. 2 (1995) 807–810.

[16] M.L. Cai, R. Zheng, M. Caffrey, R. Craigie, G.M. Clore, A.M. Gro-

nenborn, Nat. Struct. Biol. 4 (1997) 567–577.

[17] D. Esposito, R. Craigie, in: K. Maramorosh, F.A. Murphy, A.J. Shat-

kin (Eds.), Advances in Virus Research, vol. 52, Academic Press, San

Diego, 1999, pp. 319–333.

[18] T.S. Heuer, P.O. Brown, Photo-cross-linking studies suggest a

model for the architecture of an active human immunodeficiency

virus type-1 integrase –DNA complex, Biochemistry 37 (1998)

6667–6678.

[19] K. Gao, S.L. Buttler, F. Bushman, Human immunodeficiency virus

type 1 integrase: arrangement of protein domains in active cDNA

complexes, EMBO J. 20 (2001) 3565–3576.

[20] A.A. Podtelezhnikov, K. Gao, F.D. Bushman, J.A. McCammon, Mod-

eling HIV-1 integrase complexes based on their hydrodynamic prop-

erties, Biopolymers 68 (2003) 110–120.

[21] J.C. Chen, J. Krucinski, L.J. Miercke, J.S. Finer-Moore, A.H. Tang,

A.D. Leavitt, R.M. Stroud, Crystal structure of the HIV-1 integrase

catalytic core and C-terminal domains: a model for viral DNA bind-

ing, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 8233–8238.

[22] Z. Chen, Y. Yan, S. Munshi, Y. Li, J. Zugay-Murphy, B. Xu, M.

Felock, P. Felock, A. Wolfe, V. Sardana, E.A. Emini, L.C. Hazuda,

L.C. Kuo, X-ray structure of simian immunodeficiency virus integrase

containing the core and C-terminal domain (residues 50–293)—an

initial glance of the viral DNA binding platform, J. Mol. Biol. 296

(2000) 521–533.

[23] J.Y. Wang, H. Ling, W. Yang, R. Craigie, Structure of a two-domain

fragment of HIV-1 integrase: implications for domain organization in

the intact protein, EMBO J. 20 (2001) 7333–7343.

[24] R.G. Maroun, S. Gayet, M.S. Benleulmi, H. Porumb, L. Zargarian, H.

Merad, H. Leh, J.F. Mouscadet, F. Troalen, S. Fermandjian, Peptide

inhibitors of HIV-1 integrase dissociate the enzyme oligomers, Bio-

chemistry 40 (2001) 13840–13848.

[25] F. Sourgen, R.G. Maroun, V. Frere, M. Bouziane, C. Auclair, F.

Troalen, S. Fermandjian, A synthetic peptide from the human immu-

nodeficiency virus type-1 integrase exhibits coiled-coil properties and

interferes with the in vivo integration activity of the enzyme. Corre-

lated biochemical and spectroscopic results, Eur. J. Biochem. 260

(1996) 145–155.

[26] R.G. Maroun, D. Krebs, M. Roshani, H. Porumb, C. Auclair, F.

Troalen, S. Fermandjian, Conformational aspects of HIV-1 integrase

inhibition by a peptide derived from the enzyme central domain and by

antibodies raised against this peptide, Eur. J. Biochem. 260 (1999)

145–155.

[27] M. Bouyac-Bertoia, J.D. Dvorin, R.A. Fouchier, Y. Jenkins, B.E.

Meyer, L.I. Wu, M. Emerman, M.H. Malim, HIV-1 infection requires

a functional integrase NLS, Mol. Cell 7 (2001) 1025–1035.

[28] L. Zargarian, M.S. Benleumi, J.-G. Renisio, H. Merad, R.G. Maroun,

F. Wieber, O. Mauffret, H. Porumb, F. Troalen, S. Fermandjian, Strat-

egy to discriminate between high and low affinity bindings of human

immunodeficiency virus, type 1 integrase to viral DNA, J. Biol.

Chem. 278 (2003) 19966–19973.

[29] Y. Goldgur, R. Craigie, G.H. Cohen, T. Fujiwara, T. Yoshinaga, T.


ermandjian, H. Sugimoto, T. Endo, H. Murai, D.R. Davies, Struc-

ture of the HIV-1 integrase catalytic domain complexed with an

inhibitor: a platform for antiviral drug design, Proc. Natl. Acad.

Sci. U. S. A. 96 (1999) 13040–13043.

[30] D.J. Hazuda, P. Felock, M. Witmer, A. Wolfe, K. Stillmock, J.A.

Grobler, A. Espeseth, L. Gabryelski, W. Schleif, C. Blau, M.D.

Miller, Inhibitors of strand transfer that prevent integration and

inhibit HIV-1 replication in cells, Science 287 (2000) 646–650.

[31] D. Krebs, R.G. Maroun, F. Sourgen, F. Troalen, D. Davoust, S. Fer-

mandjian, Helical and coiled-coil forming properties of peptides de-

rived from and inhibiting human immunodeficiency virus type 1

integrase assessed by 1H-NMR. Use of NH temperature coefficients

to probe coiled-coil structures, Eur. J. Biochem. 253 (1998) 236–244.

[32] A. Lupas, Prediction and analysis of coiled coil structures, Methods

Enzymol. 266 (1996) 513–525.

[33] P. Luo, R.L. Baldwin, Mechanism of helix induction by trifluoroetha-

nol: a framework for extrapolating the helix-forming properties of

peptides from trifluoroethanol/water mixtures back to water, Bio-

chemistry 36 (1997) 8413–8421.

[34] A. Cammers-Goodwin, T.J. Allen, S.L. Oslick, K.F.McClure, J.H. Lee,

D.S. Kemp, Mechanisms of stabilization of helical conformations of

polypeptides by water containing trifluoroethanol, J. Am. Chem. Soc.

118 (1996) 3082–3090.

[35] R.G. Maroun, D. Krebs, S. El Antri, A. Deroussent, E. Lescot, F.

Troalen, H. Porumb, M.E. Goldberg, S. Fermandjian, Self-association

and domains of interactions of an amphipathic helix peptide inhibitor

of HIV-1 integrase assessed by analytical ultracentrifugation and NMR

experiments in trifluoroethanol/H(2)O mixtures, J. Biol. Chem. 274

(1999) 34174–34185.

[36] L.D. Muiznieks, S.A. Jensen, A.S. Weiss, Structural changes and

facilitated association of tropoelastin, Arch. Biochem. Biophys. 410

(2003) 317–323.

[37] O. Ohlenschlager, H. Hojo, R. Ramachandran, M. Gorlach, P.I. Haris,

Three-dimensional structure of the S4–S5 segment of the Shaker

potassium channel, Biophys. J. 82 (2002) 2995–3002.

[38] C. Aznar-Derunes, C. Manigand, P. Picard, A. Dautant, M. Goetz,

J.M. Schmitter, G. Precigoux, Study of the yeast Saccharomyces

cerevisiae F1F0-ATPase epsilon-subunit, J. Pept. Sci. 8 (2002)

365–372.

[39] J.R. Broughman, L.P. Shank, W. Takeguchi, B.D. Schultz, T. Iwamoto,

K.E. Mitchell, J.M. Tomich, Distinct structural elements that direct

solution aggregation and membrane assembly in the channel-forming

peptide M2GlyR, Biochemistry 41 (2002) 7350–7358.

[40] R.S. Houliston, R.S. Hodges, F.J. Sharom, J.H. Davis, Comparison of

proto-oncogenic and mutant forms of the transmembrane region of the

Neu receptor in TFE, FEBS Lett. 535 (2003) 39–43.

[41] R.K. Salinas, C.S. Shida, T.A. Pertinhez, A. Spisni, C.R. Nakaie, A.C.

Paiva, S. Schreier, Trifluoroethanol and binding to model membranes

stabilize a predicted turn in a peptide corresponding to the first ex-

tracellular loop of the angiotensin II AT(1A) receptor, Biopolymers 65

(2002) 21–31.

[42] Y. Pilpel, O. Bogin, V. Brumfeld, Z. Reich, Polyproline Type II Con-

formation in the C-Terminal Domain of the Nuclear Pore Complex

Protein gp210, Biochemistry 42 (2003) 3519–3526.

[43] K. Uray, M.R. Price, Z. Majer, E. Vass, M. Hollosi, F. Hudecz, Iden-

tification and solution conformation of multiple epitopes recognized

by a MUC2 mucin-specific monoclonal antibody, Arch. Biochem.

Biophys. 410 (2003) 254–260.

[44] N. Morellet, S. Bouaziz, P. Petitjean, B.P. Roques, NMR structure of

the HIV-1 regulatory protein VPR, J. Mol. Biol. 327 (2003) 215–227.

[45] S. Srisailam, T.K. Kumar, D. Rajalingam, K.M. Kathir, H.S. Sheu,

F.J. Jan, P.C. Chao, C. Yu, Amyloid-like fibril formation in an all

beta-barrel protein-partially structured intermediate state(s) is a pre-

cursor for fibril formation, J. Biol. Chem. 278 (2003) 17701–17709.

[46] V. Raussens, C.M. Slupsky, R.O. Ryan, B.D. Sykes, NMR structure

and dynamics of a receptor-active apolipoprotein E peptide, J. Biol.

Chem. 277 (2002) 29172–29180.

[47] R.S. Kiss, C.M. Kay, R.O. Ryan, Amphipatic a-helix bundle organi-

zation of lipid-free chicken apolipoprotein A-I, Biochemistry 38

(1999) 4327–4334.

[48] S.S. Lehrer, Solute perturbation of protein fluorescence. The quench-

ing of the tryptophyl fluorescence of model compounds and of lyso-

zyme by iodide ion, Biochemistry 10 (1971) 3254–3263.

[49] M.R. Eftink, C.A. Ghiron, Fluorescence quenching studies with pro-

teins, Anal. Biochem. 114 (1981) 199–227.

[50] T.M. Cooper, R.W. Woody, The effect of conformation on the CD of

interacting helices: a theoretical study of tropomyosin, Biopolymers

30 (1990) 657–676.

[51] J. Garnier, J.-F. Gibrat, B. Robson, GOR secondary structure predic-

tion method version IV, Methods Enzymol. 266 (1996) 540–553.

[52] M.E. Holtzer, K. Adams, E.G. Lovett, A. Holtzer, Tyrosines in two-

stranded coiled coils are CD active near 280 nm even in the absence

of interhelix tyrosine-tyrosine interactions, Biopolymers 38 (1996)

669–671.

[53] S. ElAntri, P. Bittoun, O. Mauffret, M. Monnot, O. Convert, E. Les-

cot, S. Fermandjian, Effect of distortions in the phosphate backbone

conformation of six related octanucleotide duplexes on CD and 31P

NMR spectra, Biochemistry 32 (1993) 7079–7088.

Circular dichroism and fluorescence of a tyrosine side-chain residue monitors the...

Documents

Transcript of Circular dichroism and fluorescence of a tyrosine side-chain residue monitors the...