Báo cáo khoa học: Variants of b2-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature potx
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Variantsof b
2
-microglobulin cleavedatlysine-58 retain
the mainconformationalfeaturesofthenative protein
but aremoreconformationallyheterogeneous and
unstable atphysiological temperature
Maria C. Mimmi
1
, Thomas J. D. Jørgensen
2
, Fabio Pettirossi
1
, Alessandra Corazza
1
, Paolo Viglino
1
,
Gennaro Esposito
1
, Ersilia De Lorenzi
3
, Sofia Giorgetti
4
, Mette Pries
5
, Dorthe B. Corlin
6
,
Mogens H. Nissen
5
and Niels H. H. Heegaard
6
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 b
2
-microglobulin;
human b
2
-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: nhe@ssi.dk
(Received 31 January 2006, accepted 31
March 2006)
doi:10.1111/j.1742-4658.2006.05254.x
Cleavage ofthe small amyloidogenic protein b
2
-microglobulin after lysine-
58 renders it more prone to unfolding and aggregation. This is important
for dialysis-related b
2
-microglobulin amyloidosis, since elevated levels of
cleaved b
2
-microglobulin may be found in the circulation of dialysis
patients. However, the solution structures of these cleaved b
2
-microglobulin
variants have not yet been assessed using single-residue techniques. We
here use such methods to examine b
2
-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
b
2
-microglobulin from which lysine-58 is removed, is much reduced com-
pared to wild-type b
2
-microglobulin and is strongly dependent on tem-
perature andprotein concentration.
1
H-NMR spectroscopy and amide
hydrogen (
1
H ⁄
2
H) exchange monitored by MS show that the overall three-
dimensional structure ofthevariants is similar to that of wild-type
b
2
-microglobulin at subphysiological temperatures. However, deviations do
occur, especially in the arrangement ofthe B, D and E b-strands close to
the D–E loop cleavage site at lysine-58, andthe experiments suggest con-
formational heterogeneity ofthe two variants. Two-dimensional NMR
spectroscopy indicates that this heterogeneity involves an equilibrium
between the native-like fold andat least one conformational intermediate
resembling intermediates found in other structurally altered b
2
-microglo-
bulin molecules. This is the first single-residue resolution study of a specific
b
2
-microglobulin variant that has been found circulating in dialysis
patients. The instability andconformational heterogeneity of this variant
suggest its involvement in b
2
-microglobulin amyloidogenicity in vivo.
Abbreviations
b2m, b
2
-microglobulin; CE, capillary eletrophoresis; cK58-b2m, b
2
-microglobulin cleaved after lysine-58; dK58-b2m, b
2
-microglobulin with
lysine-58 deleted; DRA, dialysis-related amyloidosis; DN3-b2m, b
2
-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 b
2
-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 proteinand also
part ofthe 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 atphysiological 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 ofthe 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 ofthe 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 ofthecleaved 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-b2m
have not previously been probed atthe single amino
acid level and correlated with the solution stability of
these molecules. We therefore here explore the struc-
tural featuresand stability ofthe Lys58-cleaved
b2m variants compared with those of wt b2m by a
Lys58
wt-β
2
m
cK58-β
2
m
dK58-β
2
m
A
B
C
a
b
1
SS
99
581
S
S
59
1
S
S
9959
c
99
57
Lys
58
+
Fig. 1. Structures of b
2
-microglobulin (b
2
m) and b
2
m variants. (A) View ofthe 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 ofthe structural family ensemble. Only the first members ofthe 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 ofthevariantsof b 2m generated by limited proteolysis ofthe 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 b
2
-microglobulin cleavedat 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 ofcleaved b2m variants
monitored by
1
H-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
1
H-NMR spec-
tra collected at different conditions oftemperature and
protein concentration. The stability of concentrated
solutions (c. 0.3 m m) in thetemperature range between
288 and 310 K was first investigated. The one-dimen-
sional
1
H spectra of cK58-b2m and dK58-b2m collec-
ted at 288 K (Fig. 2A) exhibit the typical resonance
pattern ofthe 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 ofthenative fold ofthe b2m
molecule (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 andthe simultaneous growth
of some main envelope atthe 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
V37
L23 L23
L23
V37
I35
I35
F70
78910 p.p.m.
wild-type 310K
2 1 0 p.p.m.
V37
L23
I35
L23
F70
B
Fig. 2. (A) One-dimensional
1
H-NMR aliphatic (right) and aromatic
(left) region of b
2
-microglobulin (b2m) cleaved after Lys58 (cK58-
b2m) and b2m with Lys58 deleted (dK58-b2m), 0.3 m
M,at288Kand
pH 7.4, andof wild-type b2m, 0.7 m
M, 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 ofthe b2m hydrophobic core and of
the aliphatic residues giving rise to the most upfield-shifted methyls
in the
1
H-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 b
2
-microglobulin cleavedat Lys58
FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2463
ion of large aggregates is suggested by the broadening
linewidth andthe related decrease ofthe 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-b2m
solutions 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 · 10
6
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 ofthe experimental data.
Upon further increase ofthetemperature 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
1
H-NMR traces of
b
2
-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 ofthe 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.
Stability of b
2
-microglobulin cleavedat Lys58 M. C. Mimmi et al.
2464 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS
A different behavior is found when obtaining a ser-
ies of one-dimensional
1
H spectra at 310 K using more
dilute solutions ofcleaved 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 ofthe resonance system with a pattern suggesting
loss ofconformational 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), thecleaved 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 theconformational equilibrium between
the fast (f) and slow (s) species to moreofthe (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
1
H-NMR traces of diluted b
2
-microglobulin (b2m) with Lys58 deleted (dK58-b2m) and b2m cleaved after
Lys58 (cK58-b2m) solutions (0.05 m
M) at 310 K and pH 7.4. At low concentration, the unfolding process is very slow, as indicated by an only
very minor decrease ofthe intensity ofthe 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 H
d1
and Leu23 H
d2
, 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.
M. C. Mimmi et al. Stability of b
2
-microglobulin cleavedat Lys58
FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2465
solution stability ofcleaved b2m on its concentration
is in agreement with the NMR results presented
above.
MS analysis of global conformation by amide
hydrogen (
1
H ⁄
2
H) 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 ofthe 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 atthe 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
1
H 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, thecleaved variants,
cK58-b2m and dK58-b2m, exhibit very similar noncor-
related exchange kinetics (Fig. 7). This indicates that
the stability ofthe 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 thecleaved species (Fig. 7).
Thus, in accordance with the NMR results, the glo-
bal conformation of wt b2m appears to be conserved
in thecleaved 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 variantsare not expected to affect the
exchange count.
Two-dimensional NMR characterization
The detailed interpretation of the
1
H-NMR spectra of
b2m variants is based on the parent spectra of wt b2m
obtained at different temperatureand pH values
0 200 400 600 800 1000 1200 1400 1600
0
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]
start
B
8.5 9.0
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
Time (min)
A
200 nm
51.8°C
49.6°C
45.7°C
41.5°C
34.0°C
22.6°C
9.7°C
f
s
A
Fig. 5. Capillary electrophoresis separation of b
2
-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 temperatureof 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 arethe summed peak
areas P (total area of f + s peaks) divided by the marker peak
area M at different time points as a percentage ofthe initial
value of P ⁄ M atthe onset ofthe experiments where the sample
temperature was brought from 278 K to 308 K.
Stability of b
2
-microglobulin cleavedat Lys58 M. C. Mimmi et al.
2466 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS
[15,18] and requires the checking and redetermination
of most ofthe resonance assignments ofthe 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 thetemperature 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 ofthe resonance fre-
quencies with respect to the corresponding wt values
and thus confirm the retention ofthemainfeatures of
the native structure in both variants. The backbone H
a
chemical shifts of cK58-b2m and dK58-b2m (wherever
assignments were available) were compared to the
corresponding values ofthe wt protein, as shown in
Fig. 8. As expected, the largest deviations of H
a
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. atthe opened loop D–E, andatthe start of
strand E [15]. Interestingly, similar deviations are also
found in fragment 26–35, i.e. atthe end of strand B
and at loop B–C, which faces the D–E region. Accord-
ing to the well-established correlation between H
a
chemical shifts and secondary structure in polypeptides
[20], the shifts of cK58-b2m H
a
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
Relative Abundance
Min
0
0.5
dK58, 9+
30
80
160
100% D
ox
ox
54 Da
dK58
ox
ox
32 Da
3255
cK58
cK58, 9+
Fig. 6. Global amide hydrogen (
1
H ⁄
2
H)
exchange analysis ofthe folded conforma-
tions of b
2
-microglobulin (b2m) cleaved after
Lys58 (cK58-b2m) and b2m with Lys58 dele-
ted (dK58-b2m) at 298 K in deuterated
NaCl ⁄ P
i
. The proteins were incubated
pairwise in deuterated NaCl ⁄ P
i
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 arethe 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 ofthe m ⁄ z region
with the [M + 9H]
9+
ions. The spectra
obtained at t ¼ 0 min (i.e. lowest traces)
were obtained from
1
H
2
O. Ox, Met99-
oxidized species.
M. C. Mimmi et al. Stability of b
2
-microglobulin cleavedat Lys58
FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2467
opposite changes of secondary structure are found at
the end of strand D andthe beginning of strand E, i.e.
a further loss in D and a stabilization in E ofthe 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 ofthe adjacent residues of
strands D and E, andthe facing residues of loop B–C.
Unfortunately, this analysis could not be extended to
dK58-b2m, because ofthe ambiguous assignment of
residues from these regions ofthe molecule.
Conformational heterogeneity of cK58-b2m and
dK58-b2m
The whole NMR dataset for cK58-b2m and dK58-
b2m revealed the occurrence ofat least two different
conformers for each molecule. These conformers were
undergoing slow exchange on the chemical shift time-
scale. Examination ofthe two-dimensional maps
obtained with concentrated cK58-b2m at 298 K
showed a generalized resonance doubling atthe loca-
tions and to the extent reported in Fig. 9. The features
of the pattern ofthe second conformer resemble the
features of a minor monomeric intermediate occurring
along the b2m-refolding pathway that was named I
2
and initially identified by Chiti et al. in wt b2m [3].
The I
2
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 ofthe N-terminal tripeptide,
DN3-b2m, has shown that the I
2
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 I
2
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 ofthe conformational
equilibrium that gives rise to the slow refolding step of
b2m, the proposed correspondence ofthe second form
observed in the cK58-b2m spectra with the I
2
con-
former identified in DN3-b2m is based on the similar-
ity ofthe resonance doubling patterns ofthe two
variants. This analogy is visualized in Fig. 10, where
details of NOESY spectra are shown. In spite of the
different conditions oftemperatureand pH, the close
similarity ofthe patterns is readily appreciated. The
excellent resolution ofthe resonances in Fig. 10 could
not be exploited for quantitation ofthe relative con-
centrations ofthe two forms because, in general,
NOESY cross-peak amplitudes are determined by the
actual motional characteristics ofthe 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 ofthe I
2
interme-
diate that was unambiguously recognized in previous
studies of other b2m variants [21,22]. The best estimate
of the equilibrium populations ofthe fully folded and
I
2
forms for cK58-b2m at 298 K was obtained by
using, for each conformer, the pair of TOCSY connec-
tivities assigned to Val37 H
c1
–H
c2
. Taking into
account the partial overlap ofthe specific cross-peaks,
the resulting relative amount of I
2
at 298 K was
19 ± 9% ofthe total protein. The occurrence of an I
2
intermediate in equilibrium with themain 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 ofthe b2m variantscleaved at
Lys58 were also detected by CE as previously reported
[9] andare shown in Figs 5A and 11A. The precise nat-
ure ofthe 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]
dK58-2m
cK58-2m
wt-2m
No. of protium atoms
Fig. 7. Noncorrelated exchange kinetics ofthe folded conformations
of b
2
-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 ⁄ P
i
.
Stability of b
2
-microglobulin cleavedat Lys58 M. C. Mimmi et al.
2468 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 ofthe 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
K19
S20
F22
L23
Y26
F30
D34
I35
E36
V37
D38
L40
K41
N42
G43
E44
R45
64I
E47
L65
Y66
Y67
T68
E69
07F
T71
P72
T73
D76
E77
Y78
A79
C80
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
∆δΗα
cK58-β2m compared to β2m-wt
1
I
Q2
R3
T4
P5
K6
I7
Q8
V9
Y10
S11
R12
H13
P14
A15
E16
N17
G18
G18
K19
S20
12N
22F
L23
N24
C25
Y26
V27
S28
G29
G29
F30
H31
P32
S33
D34
I35
E36
V37
D38
L39
L40
K41
N42
G43
G43
E44
R45
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
Y78
A79
C80
R81
V82
N83
H84
V85
T86
87
L
S88
Q89
P90
K91
I92
V93
K94
W95
D96
R97
D98
M99
-0,3
-0,25
-0,2
-0,15
-0,1
-0,05
0
0,05
0,1
0,15
0,2
residue
∆δΗα
Fig. 8. Two-dimensional NMR study of b
2
-microglobulin (b2m) cleaved after Lys58 (cK58-b2m) and b2m with Lys58 deleted (dK58-b2m)
at pH 7.4. The assigned backbone H
a
chemical shifts of 0.3 mM cK58-b2m at 298 K, andof 0.05 mM dK58-b2m at 310 K, are compared
with the corresponding values ofthe wild-type (wt) species obtained at 310 K and pH 6.6. The DdH
a
values (p.p.m.) are reported as
(Dvariant ) Dwt). Residue labels are omitted in regions where the resonance assignment was ambiguous.
M. C. Mimmi et al. Stability of b
2
-microglobulin cleavedat Lys58
FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2469
ches strongly support the notion ofconformational het-
erogeneity ofthe 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 b2m
variants that theprotein 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 ofconformational heterogen-
eity in cK58-b2m and dK58-b2m solutions, which is
consistent with their thermal lability. Despite these diffi-
culties, detailed characterization ofthe conformational
states ofthe cK58-b2m and dK58-b2m variants has
now been accomplished, and has made it possible, by
reference to the NMR pattern ofthe DN3 variant of
b2m, to identify a minor conformational species that
also exists in theconformational equilibrium of the
cleaved b2m variants. This conformer is a monomeric
intermediate (I
2
) 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
W95 He1
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 b
2
-micro-
globulin (b2m) cleaved after Lys58 (cK58-b2-
m) indicating conformational heterogeneity.
The proton chemical shifts ofthe alternative
conformer (I
2
) of cK58-b2m are compared
with the corresponding values ofthe 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 (dI
2
) 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 b
2
-microglobulin (b2m)
cleaved after Lys58 (cK58-b2m) (left) and
b2m devoid ofthe N-terminal tripeptide
(DN3-b2m) (right), recorded at 500 and
800 MHz, respectively. The intraresidue
connectivities H
e1
–H
d1
(top) and H
e1
–H
f2
(bottom) of Trp95 are indicated for the nat-
ively folded form (N) and for the I
2
form, in
equilibrium under the chosen experimental
conditions.
Stability of b
2
-microglobulin cleavedat Lys58 M. C. Mimmi et al.
2470 FEBS Journal 273 (2006) 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS
[...]... data clearly indicate that the overall folding pattern ofthecleaved b2m variants is very similar to that ofthe wt protein In fact, distinct differences in the conformation ofthevariantsare confined to the cleavage site region (the D–E loop) with additional involvement ofthe adjacent residues of strands D and E andthe facing residues of loop B–C Accordingly, the noncorrelated amide hydrogen (1H... capillary temperature setting of 283 K The f and s conformers are indicated (B) Details of two-dimensional NMR TOCSY spectra of dK58-b2m and cK58-b2m obtained at 500 MHz, and b2m devoid ofthe 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 unfolding processes that are. ..Stability ofb2-microglobulincleavedat Lys58 M C Mimmi et al Fig 11 Conformational heterogeneity ofcleavedb2-microglobulin (b2m) by NMR and CE (A) CE analysis ofb2-microglobulincleaved 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 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 aggregates has been recently demonstrated by size exclusion chromatography of dK58-b2m incubated at 310 K [10] The NMR data clearly indicate... 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 andthe amyloidogenicity ofconformationallyunstable b2m variants Experimental procedures Protein purification b2m cleavedat Lys58 (cK58-b2m) and with an additional deletion of residue 58 (dK58-b2m) (Fig... 2461–2474 ª 2006 The Authors Journal compilation ª 2006 FEBS 2471 Stability ofb2-microglobulincleavedat Lys58 M C Mimmi et al the molecular destabilization characterized here andthe formation of amyloid in vivo, these b2m molecular variants 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... indicate that only slightly increased protection is conferred by the hydrogen bonds in wt b2m The reduced thermostability of b2m cleavedat 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 mediated by complement enzymes that may be activated during inflammation To substantiate a relationship... lyophilized and redissolved in D2O Isotopic exchange was initiated by dilution (1 : 50) ofthe protiated protein solution with deuterated buffer, resulting in a final protein concentration of 20 lgÆmL)1 Typically, 10 lL of wt b2m, cK58b2m 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 incubated pairwise... conditions (pH 2.2 and 0 °C), where the exchange kinetics ofmain 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... was supported by MIUR (COFIN 2003) and by Sygesikringen ‘danmarks’ forskningsfond, Stability ofb2-microglobulincleavedat Lys58 Apotekerfonden af 1991, The Danish Medical Reseach Council, Lundbeckfonden, and M L Jørgensen og Gunnar Hansens Fond CarlsbergFondet is acknowledged for financial support to TJDJ The advice of Professor V Bellotti andthe assistance of Dr A Makek are gratefully acknowledged . Variants of b
2
-microglobulin cleaved at lysine-58 retain
the main conformational features of the native protein
but are more conformationally heterogeneous. values
and thus confirm the retention of the main features of
the native structure in both variants. The backbone H
a
chemical shifts of cK58-b2m and dK58-b2m