Variants of β2-microglobulin cleaved at lysine-58 retain the main conformational features of the...

Variants of b2-microglobulin cleaved at lysine-58 retainthe main conformational features of the native proteinbut are more conformationally heterogeneous andunstable at physiological temperatureMaria C. Mimmi1, Thomas J. D. Jørgensen2, Fabio Pettirossi1, Alessandra Corazza1, Paolo Viglino1,Gennaro Esposito1, Ersilia De Lorenzi3, Sofia Giorgetti4, Mette Pries5, Dorthe B. Corlin6,Mogens H. Nissen5 and Niels H. H. Heegaard6

1 Dipartimento di Scienze e Tecnologie Biomediche and MATI Centre of Excellence, University of Udine, Italy

2 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

3 Department of Pharmaceutical Chemistry, School of Pharmacy, University of Pavia, Italy

4 Department of Biochemistry, School of Pharmacy, University of Pavia, and Biotechnology Laboratories, IRCCS Policlinico San Matteo,

Pavia, Italy

5 Institute of Medical Anatomy, University of Copenhagen, Denmark

6 Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark

Keywords

amyloidosis; cleaved b2-microglobulin;

human b2-microglobulin; NMR; protein

conformation

Correspondence

N. Heegaard, Department of

Autoimmunology, Statens Serum Institut

81 ⁄ 536, Artillerivej 5, DK-2300 Copenhagen

S, Denmark

Fax: +45 32683876

Tel: +45 32683378

E-mail: [email protected]

(Received 31 January 2006, accepted 31

March 2006)

doi:10.1111/j.1742-4658.2006.05254.x

Cleavage of the small amyloidogenic protein b2-microglobulin after lysine-

58 renders it more prone to unfolding and aggregation. This is important

for dialysis-related b2-microglobulin amyloidosis, since elevated levels of

cleaved b2-microglobulin may be found in the circulation of dialysis

patients. However, the solution structures of these cleaved b2-microglobulin

variants have not yet been assessed using single-residue techniques. We

here use such methods to examine b2-microglobulin cleaved after lysine-58

and the further processed variant (found in vivo) from which lysine-58 is

removed. We find that the solution stability of both variants, especially of

b2-microglobulin from which lysine-58 is removed, is much reduced com-

pared to wild-type b2-microglobulin and is strongly dependent on tem-

perature and protein concentration. 1H-NMR spectroscopy and amide

hydrogen (1H ⁄ 2H) exchange monitored by MS show that the overall three-

dimensional structure of the variants is similar to that of wild-type

b2-microglobulin at subphysiological temperatures. However, deviations do

occur, especially in the arrangement of the B, D and E b-strands close to

the D–E loop cleavage site at lysine-58, and the experiments suggest con-

formational heterogeneity of the two variants. Two-dimensional NMR

spectroscopy indicates that this heterogeneity involves an equilibrium

between the native-like fold and at least one conformational intermediate

resembling intermediates found in other structurally altered b2-microglo-

bulin molecules. This is the first single-residue resolution study of a specific

b2-microglobulin variant that has been found circulating in dialysis

patients. The instability and conformational heterogeneity of this variant

suggest its involvement in b2-microglobulin amyloidogenicity in vivo.

Abbreviations

b2m, b2-microglobulin; CE, capillary eletrophoresis; cK58-b2m, b2-microglobulin cleaved after lysine-58; dK58-b2m, b2-microglobulin with

lysine-58 deleted; DRA, dialysis-related amyloidosis; DN3-b2m, b2-microglobulin devoid of N-terminal tripeptide; FID, free induction decay.

FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2461

The conformational behavior of b2-microglobulin (b2m)

is of interest because this molecule is involved in dialy-

sis-related amyloidosis (DRA) [1,2]. This condition,

somehow induced by long-standing dialysis or renal

insufficiency, is characterized by fibrillation and precipi-

tation of b2m in osteoarticular tissues. Under normal

conditions, b2m is a soluble plasma protein and also

part of the MHC class I complexes on the surface of

nucleated cells. It has become clear that this compact,

seven b-stranded protein is conformationally unstable

after cleavages and truncations, and that even intact

b2m may, to a minor extent, adopt an alternative con-

formation at physiological pH [3]. Amyloid fibril forma-

tion from b2m in vitro requires nonphysiological

conditions with respect to pH and ionic strength, the

presence of divalent metal ions, or some of the trunca-

tions ⁄deletions that have been reported to be present in

b2m extracted from amyloid lesions [4–6]. The study of

the behavior of b2m and b2m variants is relevant not

only for DRA, but also for understanding common

pathways of fibril formation in amyloidotic conditions

such as Alzheimer’s disease, transthyretin amyloidoses,

immunoglobulin fragment amyloidosis, or some of the

many other types of amyloidoses [7].

We have previously characterized two b2m variants,

the first obtained by cleavage after Lys58 (cK58-b2m),

and the second by further deletion of the same residue

(dK58-b2m) (Fig. 1). It was shown that the concerted

action of activated complement C1s and carboxypepti-

dase B cleaves b2m after Lys58, leading to cK58-b2m,

and removes the same residue to generate dK58-b2m[8]. This limited proteolysis attacking a susceptible

peptide bond residing in the loop between b-strands D

and E of b2m (Fig. 1) increases the conformational

heterogeneity of the cleaved b2m compared with the

wild-type (wt) molecule [9,10]. The dK58-b2m variant

may occur in vivo and has been reported to be gener-

ated in sera from patients with inflammation patho-

logies, cancer, and renal insufficiency [11–13].

Additionally, we recently showed, using dK58-b2m-

specific antibodies, that dK58-b2m circulates in the

blood of many dialysis patients [14].

The conformations of cK58-b2m and dK58-b2mhave not previously been probed at the single amino

acid level and correlated with the solution stability of

these molecules. We therefore here explore the struc-

tural features and stability of the Lys58-cleaved

b2m variants compared with those of wt b2m by a

Lys58

wt-β2m

cK58-β2m

dK58-β2m

A B C

a

b

1S S

99

581S

S59

1S

S9959

c

99

57Lys58

+

Fig. 1. Structures of b2-microglobulin (b2m) and b2m variants. (A) View of the 20 best-fitting solution structures of wild-type b2m based on

NMR restraints and tethered molecular dynamics. For the sake of simplicity, only the backbone is drawn, apart from the side chain of

Lys58, which is highlighted. Designation of b-strands A–G is indicated. The local trace thickness corresponds to the spatial spreading over

the best overlap of the structural family ensemble. Only the first members of the solution structure families were considered. Drawn with

MOLMOL [34]. (B) NMR-based solution structure of monomeric b2m (pdb entry: 1JNJ) in a ribbon drawing. The Lys58 residue (in red) and the

Cys25 and Cys80 residues (yellow) connected by a disulfide bridge are shown in the backbone trace. Drawn with WEBLABVIEWERPRO 3.7.

(C) Schematic drawing of the variants of b2m generated by limited proteolysis of the wild-type molecule. From the single-chain wild type, a

heterodimeric molecule (cK58-b2m), in which the two chains are connected by a disulfide bridge, is generated by cleavage between the Cys

residues. The further trimming (removal of Lys58) of cK58-b2m generates the dK58-b2m variant.

Stability of b2-microglobulin cleaved at Lys58 M. C. Mimmi et al.

2462 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS

combination of NMR spectroscopy, MS, and capillary

electrophoresis (CE).

Results and Discussion

Solution stability of cleaved b2m variants

monitored by 1H-NMR spectroscopy

When b2m is modified by limited proteolysis cleaving

the chain between the Cys25 and Cys80 residues, a

heterodimeric molecule consisting of two chains held

together by a disulfide bridge is generated. This mole-

cule (cK58-b2m) is further processed in vivo to the

dK58-b2m variant, which lacks the K58 residue

exposed in the A-chain of cK58-b2m (Fig. 1) [11]. The

behavior of cK58-b2m and dK58-b2m in solution was

studied by a series of one-dimensional 1H-NMR spec-

tra collected at different conditions of temperature and

protein concentration. The stability of concentrated

solutions (c. 0.3 mm) in the temperature range between

288 and 310 K was first investigated. The one-dimen-

sional 1H spectra of cK58-b2m and dK58-b2m collec-

ted at 288 K (Fig. 2A) exhibit the typical resonance

pattern of the folded protein, with a few resolved

peaks in the aliphatic and aromatic regions. In partic-

ular, the upfield shifts of Val37, Ile35 and Leu23, due

to the proximity of aromatic residues such as Tyr66,

Phe30, Phe70 and Trp95 (Fig. 2B), are diagnostic of

tertiary structure interactions in the hydrophobic core

and represent a signature of the native fold of the b2mmolecule (Fig. 2A, lower panel) [15]. When the tem-

perature is increased in steps of five degrees up to

298 K, the lower solution stability of dK58-b2m com-

pared to cK58-b2m is highlighted. While the latter at

298 K maintains a folded conformation, the variant

devoid of Lys58 is less stable and undergoes slow

unfolding and aggregation over time, as shown in

Fig. 3. The unfolding is evidenced by the progressive

loss of spectral spreading and the simultaneous growth

of some main envelope at the typical frequencies of

unfolded polypeptides (around 1 p.p.m.). The format-

78910 p.p.m. 2 1 0 p.p.m.

cK58 288K

dK58 288K

L23F70

A

V37L23 L23

L23V37

I35

I35

F70

78910 p.p.m.

wild-type 310K

2 1 0 p.p.m.

V37 L23

I35

L23F70

B

Fig. 2. (A) One-dimensional 1H-NMR aliphatic (right) and aromatic

(left) region of b2-microglobulin (b2m) cleaved after Lys58 (cK58-

b2m) and b2m with Lys58 deleted (dK58-b2m), 0.3 mM, at 288 K and

pH 7.4, and of wild-type b2m, 0.7 mM, at 310 K at pH 6.6, observed

at 500 MHz. The upfield shift of Val37, Ile35 and Leu23, which is

diagnostic of tertiary structure interactions in b2m native folding, is

highlighted. (B) Representation of the b2m hydrophobic core and of

the aliphatic residues giving rise to the most upfield-shifted methyls

in the 1H-NMR spectrum. Val37, Ile35 and Leu23 (green) are placed

in the shielding cone of aromatic rings (red). Only the most important

residues are included in the plot. The plot was drawn using WEBLAB-

VIEWERPRO 3.7 (Accelrys Inc., San Diego, CA, USA).

M. C. Mimmi et al. Stability of b2-microglobulin cleaved at Lys58


ion of large aggregates is suggested by the broadening

linewidth and the related decrease of the overall integ-

ral value under equivalent NMR acquisition condi-

tions. Over the )2 ⁄ 12 p.p.m. region, the spectra of

dK58-b2m shown in Fig. 3 exhibit signal losses of

16% and 33%, respectively, corresponding to 10 and

41 h at 298 K. In the absence of overt precipitation,

this suggests the formation of aggregates with substan-

tially larger linewidths. The loss of stability and the

formation of large, soluble aggregates in dK58-b2msolutions at 310 K over time were suggested previously

by CE analyses, and evidenced by size-exclusion

chromatography with light-scattering detection. In

these experiments a well-defined aggregate formation

with an aggregate size of about 50 nm or

5 · 106 gÆmol)1 was noted [10]. No estimate of the

aggregate dimensions by measurement of translational

diffusion coefficients using diffusion-ordered 2D-NMR

spectroscopy experiments [16,17] was possible in the

present study, because the relatively low sample con-

centration (0.3 mm) prevented reliable exponential fit-

ting of the experimental data.

Upon further increase of the temperature to 310 K,

cK58-b2m eventually slowly undergoes the same

unfolding–aggregation process as observed for dK58-

b2m (data not shown). In accordance with earlier

observations using other methods [10], this thermal

transition is irreversible (data not shown).

Fig. 3. One-dimensional 1H-NMR traces of

b2-microglobulin (b2m) cleaved after Lys58

(cK58-b2m) and b2m with Lys58 deleted

(dK58-b2m) at 298 K and pH 7.4. At 298 K,

cK58-b2m exhibits the typical folded protein

spectrum, whereas dK58-b2m undergoes an

unfolding–aggregation process that is monit-

ored at 0, 10 and 41 h from the temperature

setting. The intensity of the upfield-shifted

resonances of Leu23, Ile35 and Val37 gradu-

ally diminishes, while the envelope around

1 p.p.m. increases. Simultaneous changes

are observed in the aromatic region, invol-

ving a loss of signal dispersion. The overall

integral value is reduced after 41 h.



A different behavior is found when obtaining a ser-

ies of one-dimensional 1H spectra at 310 K using more

dilute solutions of cleaved b2m variants (c. 0.05 mm).

In contrast to the results at 0.3 mm, the unfolding–

aggregation process at a concentration of 0.05 mm is

very slow. This is indicated by only a minor decrease

of the diagnostic upfield-shifted peaks of Leu40,

Val37, Ile35 and Leu23, even after 4 days (Fig. 4).

Nevertheless, a slight and continuous modification of

the tertiary structure is evident from the slow overall

drift of the resonance system with a pattern suggesting

loss of conformational homogeneity. After some 60 h,

for both cK58-b2m and dK58-b2m, the presence of

shoulders within the monitored isolated peaks indicates

the presence of two or more conformers in equilibrium

(peak shoulders are indicated by asterisks in Fig. 4).

Further evidence for conformational heterogeneity

comes from several other envelope changes that appear

when the spectra are superimposed (data not shown).

Protein aggregation monitored by capillary

electrophoresis

In contrast to wt b2m, which is freely soluble in

physiological buffers up to at least 10 mgÆmL)1

(0.85 mm), the cleaved variants, in particular dK58-

b2m, are prone to aggregation at high protein

concentrations, especially at increased temperatures.

Visible precipitation occurs over time at concentra-

tions higher than 2 mg mL)1 (0.17 mm) for the

dK58 variant; the cK58 variant is more stable. The

aggregation behavior at different concentrations and

temperatures was characterized by CE (Fig. 5). In

these experiments, the changes in the amount of sol-

uble material were followed over time. As shown in

Fig. 5A, a 1 mgÆmL)1 (0.09 mm) dK58-b2m solution

incubated at increasing temperature initially exhibits

a shift in the conformational equilibrium between

the fast (f) and slow (s) species to more of the (s)

species, which is believed to be a partly unfolded

intermediate (as can be seen below). Subsequently, at

higher temperatures, an irreversible loss of soluble

material occurs. In Fig. 5B, an analysis of soluble

material over time at a fixed sample temperature

of 308 K at two different protein concentrations,

0.9 mgÆmL)1 (0.08 mm) and 2.5 mgÆmL)1 (0.22 mm),

clearly show the loss of solubility in the higher-

concentration solutions of both variants, whereas at

lower concentrations both species have constant

peak areas from 0 to 24 h. This dependence of the

Fig. 4. Details of one-dimensional 1H-NMR traces of diluted b2-microglobulin (b2m) with Lys58 deleted (dK58-b2m) and b2m cleaved after

Lys58 (cK58-b2m) solutions (0.05 mM) at 310 K and pH 7.4. At low concentration, the unfolding process is very slow, as indicated by an only

very minor decrease of the intensity of the diagnostic peaks of Leu23, Ile35, Val37 and Leu40, even after some days. The presence of one or

more conformational isomers, indicated by the resonance splitting of some isolated peaks (highlighted by asterisks), is particularly manifest in

the spectra recorded after more than 100 h of incubation at 310 K, but may also be noticed after 60 h and to some degree in the very first

recorded spectra (t ¼ 0 h). The increasing splitting between the peaks assigned to Ile35 Hd1 and Leu23 Hd2, which is especially noticeable in

the left panel, is consistent with a slow, continuous modification of tertiary structure, which takes place at 310 K in the dilute protein solution.



solution stability of cleaved b2m on its concentration

is in agreement with the NMR results presented

above.

MS analysis of global conformation by amide

hydrogen (1H ⁄ 2H) exchange

We have previously shown that native wt b2m and

dK58-b2m undergo transient cooperative unfolding,

evidenced by a correlated isotopic exchange of amide

hydrogens [10]. This type of exchange mechanism

(EX1) leads to the appearance of distinct bimodal

isotopic envelopes in the mass spectra. The lower mass

peak of this envelope represents the population of mol-

ecules that has not yet undergone cooperative unfold-

ing; the higher mass peak represents the population of

molecules that has been in the unfolded state and thus

undergone correlated exchange. To investigate the

structural stability of the folded states of wt b2m,

cK58-b2m and dK58-b2m, the exchange kinetics of the

folded populations were determined at 298 K (Fig. 6).

At this temperature, a gradual mass increase with

exchange time is observed for the lower-mass popula-

tion. This is due to the noncorrelated exchange mech-

anism, which in structural terms can be explained by

small-amplitude fluctuations within the protected core.

The noncorrelated isotopic exchange kinetics shown in

Fig. 7 was determined by the mass difference of the

lower-mass populations relative to the fully deuterated

control. Fig. 7 shows that at the shortest deuteration

period (t ¼ 0.5 min), all three proteins contain the same

number (i.e. 32; this number is also displayed in Fig. 6)

of 1H atoms not yet exchanged for deuterium. This

indicates that an identical number of protecting hydro-

gen bonds exists in the folded states of wt b2m, cK58-

b2m and dK58-b2m. Furthermore, the cleaved variants,

cK58-b2m and dK58-b2m, exhibit very similar noncor-

related exchange kinetics (Fig. 7). This indicates that

the stability of the hydrogen bond network that confers

protection against isotopic exchange is almost identical

for these proteins. However, with prolonged incubation

this network appears to be slightly more stable in wt

b2m than in the cleaved species (Fig. 7).

Thus, in accordance with the NMR results, the glo-

bal conformation of wt b2m appears to be conserved

in the cleaved forms of b2m. Note that in these experi-

ments only the slowly exchanging hydrogens are

monitored. Thus, contributions from amide hydrogens

in loop regions and from new termini generated in the

cleaved variants are not expected to affect the

exchange count.

Two-dimensional NMR characterization

The detailed interpretation of the 1H-NMR spectra of

b2m variants is based on the parent spectra of wt b2mobtained at different temperature and pH values

0 200 400 600 800 1000 1200 1400 16000

20

40

60

80

100

0.22 mM dK58-β2m

0.08 mM dK58-β2m

0.08 mM cK58-β2m

0.21 mM cK58-β2m

Incubation time (min) at 35°C

% [

P/M

]/[P

/M] s

tart

B

8.5 9.0-0.005

0.000

0.005

0.010

0.015

0.020

0.025

Time (min)

A20

0 n

m51.8°C

49.6°C

45.7°C

41.5°C

34.0°C

22.6°C

9.7°Cf s

A

Fig. 5. Capillary electrophoresis separation of b2-microglobulin (b2m)

with Lys58 deleted (dK58-b2m) incubated at different tempera-

tures. (A) Separation profiles to show that sample temperature

(indicated in the figure) influences the ratio between f and s con-

formers of dK58-b2m in CE. All CE experiments were performed

at a constant capillary temperature of 278 K to preserve the dis-

tribution of conformers in the injected samples. Shown are over-

layed electropherograms with time windows showing the s and f

conformer peaks. Samples were 1 mgÆmL)1 dK58-b2m electro-

phoresed at 278 K using 90 lA constant current after injection for

2 s. (B) Aggregation propensity of b2m cleaved after Lys58

(cK58-b2m) and dK58-b2m at different protein concentrations. Sol-

uble material was monitored by CE as a function of incubation

time. Samples of 0.9 mgÆmL)1 (triangles) or 2.5 mgÆmL)1 (circles)

b2m variants (cK58, open symbols; dK58, filled symbols) were

kept at 308 K, and aliquots (2 s injections of high-concentration

samples and 4 s injections of low-concentration samples) were

analyzed by CE performed at constant current of 80 lA, with the

capillary cooling fluid maintained at 278 K. Samples also contained

0.2 mgÆmL)1 of a marker peptide. Shown are the summed peak

areas P (total area of f + s peaks) divided by the marker peak

area M at different time points as a percentage of the initial

value of P ⁄M at the onset of the experiments where the sample

temperature was brought from 278 K to 308 K.



[15,18] and requires the checking and redetermination

of most of the resonance assignments of the molecule

under investigation according to the standard meth-

odology, i.e. going through scalar and dipolar connec-

tivity patterns for each amino acid residue [19]. This

work could be almost entirely completed for cK58-

b2m, but only partially for dK58-b2m. The difficulty

with both variants, particularly dK58-b2m, is due to

their thermal lability (unfolding and aggregation). This

prevented the use of optimal temperatures (e.g. 310 K)

to improve data quality with concentrated samples

(e.g. 0.5 mm). Increasing the temperature up to 320 K,

whenever possible, generally improves the NMR data

quality for 10–15 kDa proteins by reducing linewidths

and thus favoring spectral analysis. As a compromise

in the present study, the two-dimensional TOCSY

and NOESY spectra of cK58-b2m were obtained at

298 K, whereas the best results with dK58-b2m were

generated at 310 K by working with a very dilute

sample (0.05 mm).

The assignment lists (supplemental Tables 1 and 2)

indicate an overall conservation of the resonance fre-

quencies with respect to the corresponding wt values

and thus confirm the retention of the main features of

the native structure in both variants. The backbone Ha

chemical shifts of cK58-b2m and dK58-b2m (wherever

assignments were available) were compared to the

corresponding values of the wt protein, as shown in

Fig. 8. As expected, the largest deviations of Ha chem-

ical shifts of cK58-b2m are found in proximity to the

cleavage site, more specifically in fragments 56–58 and

59–64, i.e. at the opened loop D–E, and at the start of

strand E [15]. Interestingly, similar deviations are also

found in fragment 26–35, i.e. at the end of strand B

and at loop B–C, which faces the D–E region. Accord-

ing to the well-established correlation between Ha

chemical shifts and secondary structure in polypeptides

[20], the shifts of cK58-b2m Ha resonances compared

to wt b2m reflect changes in the backbone arrangement

within the D–E as well as in the B–C loop. Thus, two

1930 1950 1970 1990

m/z

11600 11700 11800 11900

Da

Rel

ativ

e A

bund

ance

Min

0

0.5dK58, 9+

30

80

160

100% D

oxox

54 Da

dK58

oxox

32 Da3255

cK58 cK58, 9+

Fig. 6. Global amide hydrogen (1H ⁄ 2H)

exchange analysis of the folded conforma-

tions of b2-microglobulin (b2m) cleaved after

Lys58 (cK58-b2m) and b2m with Lys58 dele-

ted (dK58-b2m) at 298 K in deuterated

NaCl ⁄ Pi. The proteins were incubated

pairwise in deuterated NaCl ⁄ Pi buffer. After

various periods of deuteration, isotopic

exchange was quenched by acidification.

Subsequently, the samples were desalted at

quench conditions and analyzed by ESI-MS.

Shown are the ESI mass spectra of a mix-

ture of cK58-b2m and dK58-b2m obtained

after various deuteration periods (given in

minutes in the figure) at 298 K. Left panel:

deconvoluted ESI mass spectra. Right

panel: ESI mass spectra of the m ⁄ z region

with the [M + 9H]9+ ions. The spectra

obtained at t ¼ 0 min (i.e. lowest traces)

were obtained from 1H2O. Ox, Met99-

oxidized species.



opposite changes of secondary structure are found at

the end of strand D and the beginning of strand E, i.e.

a further loss in D and a stabilization in E of the local

b-structure geometry. Compared with wt b2m, the

cK58-b2m molecule is thus most conformationally

different in the cleavage site region (D–E loop), with

additional involvement of the adjacent residues of

strands D and E, and the facing residues of loop B–C.

Unfortunately, this analysis could not be extended to

dK58-b2m, because of the ambiguous assignment of

residues from these regions of the molecule.

Conformational heterogeneity of cK58-b2m and

dK58-b2m

The whole NMR dataset for cK58-b2m and dK58-

b2m revealed the occurrence of at least two different

conformers for each molecule. These conformers were

undergoing slow exchange on the chemical shift time-

scale. Examination of the two-dimensional maps

obtained with concentrated cK58-b2m at 298 K

showed a generalized resonance doubling at the loca-

tions and to the extent reported in Fig. 9. The features

of the pattern of the second conformer resemble the

features of a minor monomeric intermediate occurring

along the b2m-refolding pathway that was named I2and initially identified by Chiti et al. in wt b2m [3].

The I2 conformer was subsequently also detected in

real-time NMR experiments [21]. Further analysis of

other amyloidogenic b2m variants, and in particular of

the species devoid of the N-terminal tripeptide,

DN3-b2m, has shown that the I2 conformer is in equi-

librium with the fully folded species [21,22]. This indi-

cates that it can be precisely identified through NMR

characterization. In the case of DN3-b2m, the observa-

tion of resonance doubling for the side chain signals of

residues Val9, Ser11, Leu23, Val37 and Ala79, which

are all close to one or more aromatic residues in the

cluster of Tyr26, Tyr66, Phe70, Tyr78 and Trp95 [15],

strongly suggested that I2 corresponds to a slightly

destabilized fold that has the overall conformation of

wt b2m, but exhibits a looser packing of its hydropho-

bic core. This interpretation was recently challenged by

Kameda et al. [23], who reported evidence in favor of

a slow trans–cis isomerization of Pro32 during refold-

ing of b2m. Whatever the origin of the conformational

equilibrium that gives rise to the slow refolding step of

b2m, the proposed correspondence of the second form

observed in the cK58-b2m spectra with the I2 con-

former identified in DN3-b2m is based on the similar-

ity of the resonance doubling patterns of the two

variants. This analogy is visualized in Fig. 10, where

details of NOESY spectra are shown. In spite of the

different conditions of temperature and pH, the close

similarity of the patterns is readily appreciated. The

excellent resolution of the resonances in Fig. 10 could

not be exploited for quantitation of the relative con-

centrations of the two forms because, in general,

NOESY cross-peak amplitudes are determined by the

actual motional characteristics of the connected nuc-

lear pairs, and thus may differ between distinct con-

formers [24]. Many other resonance doublings were

observed (the most relevant are reported in Fig. 9), all

consistent with the expected pattern of the I2 interme-

diate that was unambiguously recognized in previous

studies of other b2m variants [21,22]. The best estimate

of the equilibrium populations of the fully folded and

I2 forms for cK58-b2m at 298 K was obtained by

using, for each conformer, the pair of TOCSY connec-

tivities assigned to Val37 Hc1–Hc2. Taking into

account the partial overlap of the specific cross-peaks,

the resulting relative amount of I2 at 298 K was

19 ± 9% of the total protein. The occurrence of an I2intermediate in equilibrium with the main species was

also deduced from the dK58-b2m NMR spectra,

although the lower resolution made it necessary to rely

more on peak shape distortion than on actual peak

separation (Figs 4 and 11B).

Two conformers of the b2m variants cleaved at

Lys58 were also detected by CE as previously reported

[9] and are shown in Figs 5A and 11A. The precise nat-

ure of the slow conformer peak (labelled ‘s’ in Fig. 5A

and 11A) could not be unequivocally determined in

these experiments. The two populations observed in CE

0

5

10

15

20

25

30

35

0.1 1 10 100 1000

Exchange time[min]

dK58-2mcK58-2mwt-2m

No.

of p

rotiu

m a

tom

s

Fig. 7. Noncorrelated exchange kinetics of the folded conformations

of b2-microglobulin (b2m) cleaved after Lys58 (cK58-b2m) (triangles),

b2m with Lys58 deleted (dK58-b2m) (crosses), and wild-type (wt)

b2m (circles). Shown are mass shifts (expressed as loss of protected

protiated residues to adjust for differences in chain lengths) at 298 K

as a function of time incubated in deuterated NaCl ⁄ Pi.



separations of samples kept at 298 K gave, for the

slow-migrating conformer, concentrations of 38% ±

2% for cK58-b2m and 30% ± 4% for dK58-b2m(triplicate experiments ± SD), relative to the total

peak area, independently of the total b2m concentra-

tions used (examples are shown in Fig. 11A). CE sepa-

rations are accomplished at low temperature in

10–12 min. Thus, solution states are sampled under

dynamic conditions where the conformers are being

separated from each other, whereas NMR spectra

record steady-state solution distributions. Such differ-

ences in experimental conditions may explain the differ-

ences in the relative concentration estimates for the two

conformers. However, both the NMR and CE approa-

dK58-β2m compared to β2m-wt

V9

Y10

S11

A15

N17

G18

G18 K

19S

20

F22

L23

Y26

F30

D34

I35

E36

V37

D38

L40

K41

N42

G43

E44

R45

64I

E47

L65

Y66

Y67

T68

E69

07

FT

71P

72T

73

D76

E77 Y

78A

79C

80 R81

V82

N83

V85

T86

L87

P90

K91

V93

K94

W95

R97

D98

M99

-0,08

-0,06

-0,04

-0,02

0

0,02

0,04

0,06

0,08

0,1

0,12

residue

∆δΗ

α

cK58-β2m compared to β2m-wt

1IQ

2R

3T

4P

5 K6

I7Q

8 V9 Y10

S11

R12

H13

P14

A15

E16

N17

G18 G

18K

19S

201

2N

22

FL2

3N

24C

25Y

26V

27S

28G

29 G29

F30

H31

P32

S33

D34

I35

E36

V37 D38 L3

9L4

0K

41N

42G

43G

43 E44 R

45 I46

E47

K48

V49

E50

H51

S52

D53

S55

F56

S57

D59

W60

S61

Y63

L64

L65

Y66

Y67

T68

E69

F70

T71

T73

E74

K75

D76

E77 Y

78A

79C

80R

81V

82N

83H

84V

85T

86 87L

S88

Q89

P90

K91

I92 V93

K94

W95

D

96R

97D

98M

99

-0,3

-0,25

-0,2

-0,15

-0,1

-0,05

0

0,05

0,1

0,15

0,2

residue

∆δΗ

α

Fig. 8. Two-dimensional NMR study of b2-microglobulin (b2m) cleaved after Lys58 (cK58-b2m) and b2m with Lys58 deleted (dK58-b2m)

at pH 7.4. The assigned backbone Ha chemical shifts of 0.3 mM cK58-b2m at 298 K, and of 0.05 mM dK58-b2m at 310 K, are compared

with the corresponding values of the wild-type (wt) species obtained at 310 K and pH 6.6. The DdHa values (p.p.m.) are reported as

(Dvariant ) Dwt). Residue labels are omitted in regions where the resonance assignment was ambiguous.



ches strongly support the notion of conformational het-

erogeneity of the cK58-b2m and dK58-b2m variants.

Conclusions

Although cK58-b2m is unlikely to have a long lifetime

in vivo, where the exposed Lys58 is rapidly cleaved off

by endogeneous carboxypeptidase B activity [25], this

variant was included in our study because it is more sta-

ble in solution than dK58-b2m and thus more accessible

to analysis. It has very similar characteristics in all the

MS and CE analyses. However, we found for both b2mvariants that the protein concentrations required for

high-resolution NMR spectroscopy were detrimental to

their stability in solution. The two cleaved b2m species

have a pronounced propensity to undergo temperature-

dependent unfolding and aggregation. In addition, the

data show the occurrence of conformational heterogen-

eity in cK58-b2m and dK58-b2m solutions, which is

consistent with their thermal lability. Despite these diffi-

culties, detailed characterization of the conformational

states of the cK58-b2m and dK58-b2m variants has

now been accomplished, and has made it possible, by

reference to the NMR pattern of the DN3 variant of

b2m, to identify a minor conformational species that

also exists in the conformational equilibrium of the

cleaved b2m variants. This conformer is a monomeric

intermediate (I2) occurring on the b2m-refolding path-

way. These findings are consistent with the existence, in

addition to the folded conformation, of a less abundant

form with amyloidogenic features, which has also been

suggested by CE experiments [9,10,22].

V37

Hg1

*

V37

Hg2

*

Y66

Hd*

Y66

He*

F70

Ha

F70

Hd*

F70

HN

F70

Hz

Y78

Ha

Y78

Hd*

Y78

HN

A79

Ha

A79

Hb*

A79

HN

W95

Hd1 W

95 H

e1

W95

Hz2

-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

0.08

0.1

H assignment

∆δH

(p

.p.m

.)

Fig. 9. Resonance doublings in b2-micro-

globulin (b2m) cleaved after Lys58 (cK58-b2-

m) indicating conformational heterogeneity.

The proton chemical shifts of the alternative

conformer (I2) of cK58-b2m are compared

with the corresponding values of the nat-

ively folded form of cK58-b2m in the graph.

The two conformers are recognized in

TOCSY and NOESY maps, obtained at

298 K and pH 7.4. The DdH values (p.p.m.)

are reported as (dI2 ) dN), where N stands

for the natively folded form. Only the most

relevant deviations are shown. Resonance

doubling observed elsewhere was less

pronounced in terms of Dd.

p.p.m.

10.410.610.811.0 p.p.m.

7.0

7.2

7.4

7.6

7.8

8.0

p.p.m.

10.410.610.811.0 p.p.m.

7.0

7.2

7.4

7.6

7.8

8.0

W95 N W95 I2

W95 N

W95 N

W95 N W95 I2 W95 I2

W95 I2

cK58 288K, pH 7.4 ∆N3 310K, pH 6.6

Fig. 10. Details of two-dimensional NMR

NOESY maps of b2-microglobulin (b2m)

cleaved after Lys58 (cK58-b2m) (left) and

b2m devoid of the N-terminal tripeptide

(DN3-b2m) (right), recorded at 500 and

800 MHz, respectively. The intraresidue

connectivities He1–Hd1 (top) and He1–Hf2

(bottom) of Trp95 are indicated for the nat-

ively folded form (N) and for the I2 form, in

equilibrium under the chosen experimental

conditions.



The unfolding processes that are observed by NMR

with temperature increase may be driven by the seeding

probability, which increases with protein concentration

and brings about increased recruitment of monomers

or small oligomers onto the surface of soluble large

aggregates. The occurrence of large, well-defined aggre-

gates has been recently demonstrated by size exclusion

chromatography of dK58-b2m incubated at 310 K [10].

The NMR data clearly indicate that the overall fold-

ing pattern of the cleaved b2m variants is very similar

to that of the wt protein. In fact, distinct differences in

the conformation of the variants are confined to the

cleavage site region (the D–E loop) with additional

involvement of the adjacent residues of strands D and

E and the facing residues of loop B–C. Accordingly,

the noncorrelated amide hydrogen (1H ⁄ 2H) exchange

experiments indicate that only slightly increased pro-

tection is conferred by the hydrogen bonds in wt b2m.

The reduced thermostability of b2m cleaved at Lys58

occurs despite an overall native-like folding and stems

from a single cleavage in a rather mobile and exposed

loop region [15,21]. This cleavage is known to be medi-

ated by complement enzymes that may be activated dur-

ing inflammation. To substantiate a relationship between

Fig. 11. Conformational heterogeneity of cleaved b2-microglobulin (b2m) by NMR and CE. (A) CE analysis of b2-microglobulin cleaved after

Lys58 (cK58-b2m) and b2m with Lys58 deleted (dK58-b2m) kept at 298 K and separated at 283 K. Samples (2.5 mgÆmL)1) were injected for

2 s and analyzed at a constant current of 90 lA in 0.1 M phosphate, pH 7.4, with a capillary temperature setting of 283 K. The f and s con-

formers are indicated. (B) Details of two-dimensional NMR TOCSY spectra of dK58-b2m and cK58-b2m obtained at 500 MHz, and b2m

devoid of the N-terminal tripeptide (DN3-b2m) recorded at 800 MHz. The specific intraresidue connectivities Hd*–Hz of Phe70 arising from

the natively folded form (N) and I2 intermediate (I2) are labeled.



the molecular destabilization characterized here and the

formation of amyloid in vivo, these b2m molecular vari-

ants should be present in amyloid lesions from patients.

Conversely, given the propensity of b2m for specific

cleavage, the pheomenon may be part of a physiological

system marking b2m for clearance in the circulation and

possibly failing in amyloidosis. In any case, the results

reported here provide a further basis for understanding

the link between in vivo stability and the amyloidogeni-

city of conformationally unstable b2m variants.

Experimental procedures

Protein purification

b2m cleaved at Lys58 (cK58-b2m) and with an additional

deletion of residue 58 (dK58-b2m) (Fig. 1) were derived

from wt b2m purified from a pool of urine from nephropa-

thy patients, as described previously [12]. cK58-b2m and

dK58-b2m were generated by treating purified wt b2m with

activated complement C1s in the presence or absence of a

carboxypeptidase B inhibitor, as previously described [11].

Molecular masses of the purified proteins were determined

by MS on a Mariner ESI-TOF biospectrometry workstation

(Applied Biosystems, Foster City, CA, USA) and were in

agreement with the theoretical masses of 11729.2 Da (wt

b2m), 11747.2 Da (cK58-b2m), and 11619.0 Da (dK58-

b2m). The purified proteins were kept in NaCl ⁄Pi (137 mm

NaCl ⁄ 2.7 mm KCl ⁄ 1.5 mm KH2PO4 ⁄ 6.5 mm Na2HPO4,

pH 7.4) at )20 �C until used.

Nuclear magnetic resonance (NMR) spectroscopy

1H-NMR spectra of cK58-b2m and dK58-b2m were

obtained at 500.13 MHz with a Bruker Avance spectrometer

on approximately 0.3 mm and 0.05 mm samples dissolved in

NaCl ⁄Pi at pH 7.4. Deuterium oxide (Cambridge Isotope

Laboratories, Andover, MA, USA, 99.9 atom percentage

D) was added (5% by volume) for frequency lock purposes.

The concentrated dK58-b2m sample (0.3 mm, 3.5 mgÆmL)1)

was obtained from a 2 mgÆmL)1 solution by centrifugal ul-

trafiltration in 5 kDa cut-off vials. The cK58-b2m samples

(0.3 mm and 0.05 mm) and the diluted dK58-b2m sample

(0.05 mm) were filtered before transfer into the NMR tube

using 0.22 lm-pore syringe filters (Millipore, Bedford, MA).

The temperature effect on solution stability was monitored

over time by observing the concentrated proteins at 288,

293, 298 and 310 K. To probe for the effect of dilution, a

series of (one-dimensional) NMR experiments was per-

formed with 0.05 mm solutions at 310 K over 1 week. To

assign resonances, two-dimensional TOCSY [26] and NO-

ESY [27] spectra were recorded for both cK58-b2m and

dK58-b2m. Different temperatures between 288 and 310 K

were explored to collect data with the concentrated samples

until a folded protein conformation appeared to be con-

served. Typical two-dimensional acquisition schemes inclu-

ded: solvent suppression by excitation sculpting [28], 1–1.5-s

steady-state recovery time, mixing times of 38–50 ms for

TOCSY and 150 ms for NOESY, and t1 quadrature detec-

tion by the time proportional phase incrementation method

[29]. The spin-lock mixing in the TOCSY experiments was

obtained with an MLEV17 [30] pulse train at cB2 ⁄ 2p ¼ 7–

10 kHz, sandwiched by two purging pulses of 0.75 ms.

Acquisitions were performed over a spectral width of

8012.820 Hz in both dimensions, with matrix sizes of 1024–

2048 points in t2 and 512 points in t1, and 128–256 scans for

each t1 free induction decay (FID) (total maximum experi-

ment duration was 47 h). In an effort to improve the two-

dimensional data quality for dK58-b2m, a diluted sample

(0.05 mm) was examined at 310 K. A NOESY spectrum was

recorded at 800.13 MHz with a cryoprobe-equipped Bruker

DRX spectrometer. The acquisition was performed over a

spectral width of 12 820.513 Hz in both dimensions and

with a mixing time of 150 ms. The total experimental time

was c. 14 h for 2048 points in t2, 256 points in t1, and 128

scans for each t1 FID. The corresponding TOCSY experi-

ment was performed at 500.13 MHz, using a DIPSI-2 iso-

tropic mixing train lasting 28 ms [31], solvent suppression

by excitation sculpting [28], 1 s steady-state recovery time

and t1 quadrature detection by the echo–antiecho method

[29]. The time needed to collect a signal intensity amenable

to analysis was c. 61 h for 1500 points in t2, 256 points in t1,

and 696 scans for each t1 FID. Apodizations by Gaussian

multiplication in t2 and shifted (72�) square sinebell in t1were applied for processing using the Bruker software. In

general, however, data processing and analysis were per-

formed using Felix (Accelrys Inc., San Diego, CA) software

with shifted (60–90�) square sinebell apodization and zero

filling (up to 2048 · 1024–2048 real points). All spectra were

referenced on the Leu23 CdH3 resonance at )0.58 p.p.m.

Amide hydrogen (1H ⁄ 2H) exchange monitored

by MS

Deuterated NaCl ⁄Pi was prepared by lyophilization of pro-

tiated buffer followed by redissolution in D2O. To achieve

full deuteration, the deuterated buffers were twice lyophi-

lized and redissolved in D2O. Isotopic exchange was initi-

ated by dilution (1 : 50) of the protiated protein solution

with deuterated buffer, resulting in a final protein concen-

tration of 20 lgÆmL)1. Typically, 10 lL of wt b2m, cK58-

b2m or dK58-b2m (c. 1 mgÆmL)1 in protiated NaCl ⁄Pi)

was added to 490 lL of deuterated NaCl ⁄Pi, pH 7.3 (value

uncorrected for isotope effects). The proteins were incu-

bated pairwise at equimolar concentrations at 25 �C in a

thermomixer. At appropriate intervals, 50 lL aliquots were

withdrawn and quenched by adding 2 lL of 2.5% trifluoro-

acetic acid, which lowered the pH to 2.2 (uncorrected

value). The samples were stored in liquid N2 until analysed



by ESI-MS in positive ion mode on a quadrupole time-of-

flight mass spectrometer (Model Q-TOF 1; Micromass,

Manchester, UK). The MS instrument was coupled to rapid

desalting equipment, as described previously [10]. The total

time for desalting and elution was less than 2 min. The sol-

vent precooling coils, injector with loop, valve and micro-

column were immersed in ice–water slurry (0 �C) to

minimize back-exchange with the protiated solvents. The

desalting step mainly removes deuterium exchanged for

labile hydrogens, i.e. hydrogen attached to N, O and S in

the side chains, and not main chain amide hydrogens at aci-

dic pH [32]. Thus, the mass increase observed after deutera-

tion and desalting primarily reflects deuterium incorporated

into the main chain amide groups. Back-exchange control

experiments were performed to determine the inevitable

deuterium loss that occurs during desalting under quench

conditions (pH 2.2 and 0 �C), where the exchange kinetics

of main chain amide hydrogens is very slow. Aliquots of

fully deuterated wt b2m, cK58-b2m and dK58-b2m under

quench conditions (pH 2.2 and 0 �C) were subjected to

rapid desalting and they were observed to contain 88, 87

and 86 ±1 deuterium atoms, respectively. Since wt b2m,

cK58-b2m and dK58-b2m contain a total of 93, 92 and 91

main chain amide hydrogens, respectively, approximately

five deuterium atoms are back-exchanged with hydrogen

atoms under quench conditions.

CE

A Beckman P ⁄ACE 5010 instrument with sample cooling

and UV detection facilities and placed in a cold room was

used. Electrophoresis buffer was 0.1 m phosphate, pH 7.4.

Detection took place at 200 nm and the separation tube was

a 50 lm inner diameter uncoated fused silica capillary of

57 cm total length with 50 cm to the detector window. Sepa-

rations were carried out at 80 or 90 lA constant current cor-

responding to a field strength of about 490 VÆcm)1. The

capillary cooling fluid and the samples were kept at the tem-

peratures indicated. Samples (30 lL, 0.9 mgÆmL)1 (0.08 mm)

or 2.5 mgÆmL)1 (0.22 mm) of cK58-b2m or dK58-b2m, both

with 0.2 mgÆmL)1 marker peptide (M) added), were protec-

ted against evaporation by 15 lL of overlayed light mineral

oil (Sigma M-3516, St Louis, MO, USA) [33]. Injected sam-

ple volumes were approximately 2.3 or 4.5 nL (2 or 4 s pres-

sure injection for 2.5 mgÆmL)1 and 0.9 mgÆmL)1 samples,

respectively). Data were collected and processed with the

beckman system gold software (Beckman, Fullerton, CA,

USA). The capillary was rinsed after electrophoresis for

1 min with each of 0.1 m NaOH and water and then pre-

rinsed for 2 min with electrophoresis buffer.

Acknowledgements

This work was supported by MIUR (COFIN 2003)

and by Sygesikringen ‘danmarks’ forskningsfond,

Apotekerfonden af 1991, The Danish Medical Reseach

Council, Lundbeckfonden, and M. L. Jørgensen og

Gunnar Hansens Fond. CarlsbergFondet is acknow-

ledged for financial support to TJDJ. The advice of

Professor V. Bellotti and the assistance of Dr A.

Makek are gratefully acknowledged. A special acknow-

ledgement is due to CERM, Florence (Italy), for the

use of their 800 MHz NMR facility.

References

1 Gejyo F, Yamada T, Odani S, Nakagawa Y, Arakawa

M, Kunitomoto T, Kataoka H, Suzuki M, Hirasawa Y

& Shirahama T (1985) A new form of amyloid protein

associated with chronic hemodialysis was identified as

beta 2-microglobulin. Biochem Biophys Res Commun

129, 701–706.

2 Gorevic PD, Casey TT, Stone WJ, DiRaimondo CR,

Prelli FC & Frangione B (1985) b2-microglobulin is an

amyloidogenic protein in man. J Clin Invest 76, 2425–

2429.

3 Chiti F, DeLorenzi E, Grossi S, Mangione P, Giorgetti

S, Caccialanza G, Dobson CM, Merlini G, Ramponi G

& Bellotti V (2001) A partially structured species of

b2-microglobulin is significantly populated under

physiological conditions and involved in fibrillogenesis.

J Biol Chem 276, 46714–46721.

4 Stoppini MS, Arcidiaco P, Mangione P, Giorgetti S,

Brancaccio D & Bellotti V (2000) Detection of frag-

ments of beta2-microglobulin in amyloid fibrils. Kidney

Int 57, 349–350.

5 Linke RP, Hampl H, Bartel-Schwarze S & Eulitz M

(1987) Beta 2-microglobulin, different fragments and

polymers thereof in synovial amyloid in long-term

hemodialysis. Biol Chem Hoppe Seyler 368, 137–144.

6 Linke RP, Hampl H, Lobeck H, Ritz E, Bommer J,

Waldherr R & Eulitz M (1989) Lysine-specific cleavage

of beta 2-microglobulin in amyloid deposits associated

with hemodialysis. Kidney Int 36, 675–681.

7 Merlini G & Bellotti V (2003) Molecular mechanisms of

amyloidosis. N Engl J Med 349, 583–596.

8 Nissen MH, Johansen B & Bjerrum OJ (1997) A simple

method for the preparation and purification of C1 com-

plement cleaved beta 2-microglobulin from human

serum. J Immunol Methods 205, 29–33.

9 Heegaard NHH, Roepstorff P, Melberg SG & Nissen

MH (2002) Cleaved b2-microglobulin partially attains a

conformation that has amyloidogenic features. J Biol

Chem 277, 11184–11189.

10 Heegaard NHH, Jørgensen TJD, Rozlosnik N, Corlin

DB, Pedersen JS, Tempesta AG, Roepstorff P, Bauer R

& Nissen MH (2005) Unfolding, aggregation, and

seeded amyloid formation of lysine-58-cleaved b2-micro-

globulin. Biochemistry 44, 4397–4407.



11 Nissen MH, Roepstorff P, Thim L, Dunbar B &

Fothergill JE (1990) Limited proteolysis of beta

2-microglobulin at Lys-58 by complement component

C1s. Eur J Biochem 189, 423–429.

12 Nissen MH, Thim L & Christensen M (1987) Purifica-

tion and biochemical characterization of the complete

structure of a proteolytically modified beta-2-micro-

globulin with biological activity. Eur J Biochem 163,

21–28.

13 Plesner T & Wiik A (1979) Demonstration of electro-

phoretic heterogeneity of serum beta 2-microglobulin in

systemic lupus erythematosus and rheumatoid arthritis:

evidence against autoantibodies to beta 2-microglobulin.

Scand J Immunol 9, 247–254.

14 Corlin DB, Sen JW, Ladefoged S, Lund GB, Nissen

MH & Heegaard NH (2005) Quantification of

cleaved {beta}2-microglobulin in serum from patients

undergoing chronic hemodialysis. Clin Chem 51,

1177–1184.

15 Verdone G, Corazza A, Viglino P, Pettirossi F, Giorg-

etti S, Mangione P, Andreola A, Stoppini M, Bellotti V

& Esposito G (2002) The solution structure of human

beta2-microglobulin reveals the prodromes of its amy-

loid transition. Protein Sci 11, 487–499.

16 Morris KF & Johnson CS Jr (1992) Diffusion-ordered

2D NMR spectroscopy. J Am Chem Soc 114, 3139–3141.

17 Stejskal EO & Tanner JE (1965) Spin diffusion measure-

ments: spin echoes in the presence of a time-dependent

field gradient. J Chem Phys 42, 288–292.

18 Okon M, Bray P & Vucelic D (1992) 1H NMR assign-

ments and secondary structure of human beta 2-micro-

globulin in solution. Biochemistry 31, 8906–8915.

19 Wuthrich K (1986) NMR Spectroscopy of Proteins and

Nucleic Acids. Wiley & Sons, New York.

20 Wishart DS & Sykes BD (1994) Chemical shifts as a

tool for structure determination. Methods Enzymol 239,

363–392.

21 Corazza A, Pettirossi F, Viglino P, Verdone G, Garcia

J, Dumy P, Giorgetti S, Mangione P, Raimondi S,

Stoppini M, et al. (2004) Properties of some variants of

human beta2-microglobulin and amyloidogenesis. J Biol

Chem 279, 9176–9189.

22 De Lorenzi E, Grossi S, Massolini G, Giorgetti S, Man-

gione P, Andreola A, Chiti F, Bellotti V & Caccialanza

G (2002) Capillary electrophoresis investigation of a

partially unfolded conformation of beta (2)-microglobu-

lin. Electrophoresis 23, 918–925.

23 Kameda A, Hoshino M, Higurashi T, Takahashi S,

Naiki H & Goto Y (2005) Nuclear magnetic resonance

characterization of the refolding intermediate of beta2-

microglobulin trapped by non-native prolyl peptide

bond. J Mol Biol 348, 383–397.

24 Macura S & Ernst RR (1980) Elucidation of cross

relaxation in liquids by two-dimensional NMR spectro-

scopy. Mol Phys 41, 95–117.

25 Nissen MH (1993) Proteolytic modification of b2-micro-

globulin in human serum. Danish Med Bull 40, 56–64.

26 Braunschweiler L & Ernst RR (1983) Coherence trans-

fer by isotopic mixing: application to proton correlation

spectroscopy. J Magn Reson 53, 521–528.

27 Jeener J, Meier BH, Bachmann P & Ernst RR (1979)

Investigation of exchange processes by two-dimensional

NMR spectroscopy. J Chem Phys 71, 286–292.

28 Hwang TL & Shaka AJ (1995) Water suppression that

works. Excitation sculpting using arbitrry waveforms

and pulsed field gradients. J Magn Reson 112, 275–279.

29 Marion D & Wuthrich K (1983) Application of phase

sensitive two-dimensional correlated spectroscopy

(COSY) for measurements of 1H)1H spin–spin coup-

ling constants in proteins. Biochem Biophys Res Com-

mun 113, 967–974.

30 Bax A & Davis DG (1985) MLEV-17 based two dimen-

sional homonuclear magnetization transfer spectro-

scopy. J Magn Reson 65, 355–360.

31 Shaka AJ, Lee CJ & Pines A (1988) Iterative schemes

for bilinear operators: applications to spin decoupling.

J Magn Reson 77, 274–293.

32 Bai Y, Milne JS, Mayne L & Englander SW (1993)

Primary structure effects on peptide group hydrogen

exchange. Proteins 17, 75–86.

33 Heegaard NHH, Sen JW & Nissen MH (2000) Congo-

philicity (Congo red affinity) of different b2-microglobu-

lin conformations characterized by dye affinity capillary

electrophoresis. J Chromatogr A 894, 319–327.

34 Koradi R, Billeter M & Wuthrich K (1996) MOLMOL:

a program for display and analysis of macromolecular

structures. J Mol Graph 14, 51–32.

Supplementary material

The following supplementary material is available

online:

Table S1. 1H-NMR chemical shift (p.p.m.) of cK58-

b2m in H2O/D2O = 95/5, at 298 K and pH = 7.4.

Hydrogen nuclei are labeled as recommended by

IUPAC-IUBMB-IUPAB.

Table S2. 1H-NMR chemical shift (p.p.m.) of dK58-

b2m in H2O/D2O = 95/5, at 310 K and pH = 7.4.

Hydrogen nuclei are labeled as recommended by

IUPAC-IUBMB-IUPAB. The chemical shift values of

diastereotopic pairs are listed with a separation slash

for resolved resonances, whereas a single value indi-

cates that only one resonance was observed either

because of degeneracy or magnetic equivalence or

because of a single unambiguous assignment.

This material is available as part of the online article

from http://www.blackwell-synergy.com



Variants of β2-microglobulin cleaved at lysine-58 retain the main conformational features of the...

Documents

Transcript of Variants of β2-microglobulin cleaved at lysine-58 retain the main conformational features of the...