Influence of inflammation-related changes on
conformational characteristics of HLA-B27 subtypes as
detected by IR spectroscopy
Heinz Fabian
1
, Bernhard Loll
2
, Hans Huser
3
, Dieter Naumann
1
, Barbara Uchanska-Ziegler
3
and
Andreas Ziegler
3
1 Robert Koch-Institut, Berlin, Germany
2 Institut fu
¨
r Chemie und Biochemie, Abteilung Strukturbiochemie, Freie Universita
¨
t Berlin, Germany
3 Institut fu
¨
r Immungenetik, Charite
´
-Universita
¨
tsmedizin Berlin, Freie Universita
¨
t Berlin, Germany

raphy and exist irrespective of the sequence of the bound peptide and its
binding mode. They might thus influence antigenic properties of the respec-
tive HLA-B27 subtype. Furthermore, a decrease in the pH from 7.5 to 5.6
during the analyses had an influence only on HLA-B*2709 complexed with
the unmodified self-peptide, where His116 is not contacted by any peptide
side chain. This permits us to conclude that histidines, and in particular
His116, influence the stability of MHC:peptide complexes. The conditions
prevailing in inflammatory environments in vivo might thus also exert
an impact on selected conformational features of HLA-B27:peptide
complexes.
Structured digital abstract
l
B*27 and VIPR bind by biophysical (View interaction).
Abbreviations
AS, ankylosing spondylitis; H ⁄ D, hydrogen ⁄ deuterium; HC, heavy chain; HLA, human leukocyte antigen; b
2
m, b
2
-microglobulin; MHC,
major histocompatibility complex; pLMP2, (RRRWRRLTV); pVIPR, (RRKWRRWHL); pVIPR-U5, (RRKWURWHL; U = citrulline);
TIS, (RRLPIFSRL).
FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1713
Introduction
Major histocompatibility complex (MHC) class I mole-
cules are cell-surface membrane glycoproteins that con-
sist of a highly polymorphic heavy chain (HC)
noncovalently associated with a light chain, b
2
-micro-
globulin (b

the affected tissues [12], leading to proton concentra-
tions that can be elevated greatly (100- to 200-fold).
The pH decrease is expected to affect primarily histi-
dine residues, due to their sensitivity to relatively small
pH shifts at physiologically relevant values (the pK
a
of
histidine in proteins is  6.6) [13]. It is well established
that protonation of ionizable groups in folded proteins
may contribute to their conformational stability [14].
Among the HLA-B27 subtypes, HLA-B*2705 (in
short, B*2705) is strongly associated with AS, whereas
another, HLA-B*2709 (in short, B*2709) is not [2,3,11].
The proteins encoded by these two alleles differ only by
a micropolymorphism (Asp116 in B*2705 and His116
in B*2709) within the peptide-binding groove formed
by each of the HC [1,3]. Detailed functional, structural
and thermodynamic studies of these very closely related
subtypes have been carried out to shed light on the
molecular mechanisms underlying their differential
association with AS [15–26]. High-resolution crystal
structures of these subtypes complexed with peptides
constituting the HLA-B27 repertoire reveal that some
peptides, such as the self-peptides TIS (RRLPIFSRL)
or pCatA (IRAAPPPLF), are displayed very similarly
by the two HLA-B27 subtypes [21,26], whereas the viral
peptide pLMP2 (RRRWRRLTV) [22] and the self-pep-
tide pVIPR (RRKWRRWHL) [18] exhibit drastically
different conformations. In addition, pVIPR is bound
in a canonical single conformation by B*2709, but in an

noncitrullinated version of the peptide [18]. Specifi-
cally, pVIPR-U5 is displayed by B*2705 in a canonical
conformation (Fig. 1A), but exhibits a noncanonical
binding mode in the B*2709 subtype, where the side
chain of citrulline at peptide position 5 (pU5) is
embedded within the binding groove and forms a
hydrogen bond to His116 of the HC (Fig. 1B). The
comparative IR spectroscopic analyses described here
address the question of whether the previously
observed difference in flexibility between the B*2705
and B*2709 HC is also found when a peptide assumes,
Influence of inflammatory environment on HLA-B27 H. Fabian et al.
1714 FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS
because of citrullination, a noncanonical binding mode
in the B*2709 subtype. Furthermore, we investigated
whether the micropolymorphism that distinguishes the
two HLA-B27 subtypes exerts an influence on the sta-
bility of the complexes when the pH is lowered to a
value representative of an inflammatory environment.
Results
Infrared absorbance spectra of HLA-B27:pVIPR-U5
complexes
The IR spectroscopic behaviour of the B*2709 ⁄
13
C-b
2
m:pVIPR-U5 and B*2705 ⁄
13
C-b
2

)1
had previously been assigned to different
secondary structure elements of the HC. Bands at
 1650 and 1643 cm
)1
are primarily due to helical
structures which change as a consequence of hydro-
gen ⁄ deuterium (H ⁄ D) exchange, whereas a strong band
component at  1624 cm
)1
and weaker band compo-
nents between 1693 and 1681 cm
)1
are due to the
b sheets of the HC [27].
Because 38% and 23% of the protein backbone of
the HC is formed by b sheet and a-helical structures,
respectively, spectral components attributed to these
structures dominate the IR spectrum. The spectral con-
tributions of the pVIPR-U5 nonapeptide are ‘buried’
under those of the 276 HC residues. The same holds
true for the spectral features associated with the
His fi Asp exchange in the HC. The amino acid side
chain of Asp gives rise to a relatively strong absorp-
tion band between 1550 and 1585 cm
)1
, which over-
laps with an absorption band due to Glu of similar
intensity [30,31]. Taking into account the 34 Asp and
Glu residues in B*2709, the spectral contribution of

O buffer,
even when using IR transmission cells of only a few
lm pathlength. Thus, the amount of nonexchanged
Fig. 1. Structure of the pVIPR-U5 peptide in complex with B*2705
and B*2709. The peptide is depicted from the side of the a2 helix
(not shown). The floor of the peptide-binding groove and the
a1 helix are shown in grey ribbon representation, the subtype-spe-
cific residue 116 (Asp116 or His116) is indicated in green. (A) The
pVIPR-U5 peptide is drawn as a purple stick model bound to
B*2705. (B) The pVIPR-U5 peptide is drawn as a yellow stick
model anchored to B*2709 by a hydrogen bond connecting pU5
O7
and His116
NE2
, as indicated by a dashed red line. Oxygen atoms
are shown in red, nitrogen atoms in blue. B*2705 binds the peptide
in canonical conformation, while it is presented by B*2709 in a
noncanonical binding mode.
H. Fabian et al. Influence of inflammatory environment on HLA-B27
FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1715
amide protons for the partially exchanged state of the
complex at the beginning of the experiment in D
2
O
was approximated (Fig. S1) by setting the difference in
peak intensity of amide II at 1550 cm
)1
between the
spectra of the sample in H
2

)
1448
1515
1545
1594
1640
1694
1688
1651
1624
1560
3292 3293
1445
1542
1562
1598
1624
1651
1688
1624
1650
1691
1693
1592
1514
3281
3314
3306
2
nd

2
O-buffer. (B) B*2709 ⁄
13
C-b
2
m:pVIPR-U5 ) B*2705 ⁄
13
C-
b
2
m:pVIPR-U5 and (C) B*2709 ⁄
13
C-b
2
m:pVIPR ) B*2705 ⁄
13
C-b
2
m:pVIPR. The IR data of B*2709 ⁄
13
C-b
2
m:pVIPR and B*2705 ⁄
13
C-
b
2
m:pVIPR are from previous work by our group [27]. Note that the absorbance scale for the difference spectra (B, C) was expanded by a
factor of five compared with the scale of the absorbance spectra (A). (Upper) (D) Second derivatives of the IR spectra of B*2709 ⁄
13

C-b
2
m:pVIPR-U5 at 15 °C (Fig. 2B) is
characterized by spectral features in the amide A, ami-
de I¢, and amide II ⁄ II¢ region. The positive IR differ-
ence band at  3292 cm
)1
demonstrates the presence
of less H ⁄ D-exchanged amide groups in the HC of
B*2709 compared with the B*2705 subtype, which is
supported by the broad positive difference feature at
 1560 cm
)1
. Because less H ⁄ D exchange means less
flexibility of the proteins’ core regions to make them
accessible to the solvent, the IR data demonstrate that
the B*2705 HC is more flexible than the B*2709 HC.
The difference feature at  3292 cm
)1
(Fig. 2B)
accounts for only  2% of the total area under the
amide A band of the B*2709 ⁄
13
C-b
2
m:pVIPR-U5 com-
plex (black trace in Fig. 2A) relative to a baseline
between 3180 and 3410 cm
)1
. Taking the estimated

C-b
2
m:pVIPR-U5 when compared with that
in B*2709. Remarkably, the spectral differences
observed herein for the two complexes with pVIPR-U5
(Fig. 2B) revealed difference bands very similar to
those found previously by us in case of the pVIPR-
complexed B*2709 and B*2705 subtypes (Fig. 2C),
including a positive difference feature in the amide A
region and positive and negative features in the
amide I¢⁄II region of the spectrum. Moreover, the
IR difference spectroscopic features (B*2709 ⁄
13
C-
b
2
m:pVIPR-U5 ) B*2705 ⁄
13
C-b
2
m:pVIPR-U5) did not
change appreciably between 15 and 55 °C (data not
shown), indicating that the conformational differences
between the two HLA-B27 complexes persist over this
temperature range, as observed formerly for the two
HLA-B27:pVIPR complexes [27].
To estimate the number and position of individual
components under the broad amide I ⁄ I¢ band con-
tours, we also employed derivative spectroscopy. This
method allows to visualize fine differences in the posi-

fied at present, are also indicated by the amide A band
components at 3306 and 3314 cm
)1
of B*2709 and
B*2705, respectively (Fig. 2D,E).
The high-temperature IR difference spectrum is fea-
tureless (red trace in Fig. 2E), indicating the loss of all
conformational differences between the two HLA-B27
subtypes. Moreover, the subtype-dependent spectral
differences (black trace in Fig. 2E) were much more
pronounced than the spectral differences between two
independent preparations of the corresponding com-
plexes (Fig. 2F). This provides evidence that the
observed spectral differences at low temperatures are
really significant, and demonstrates the high quality of
the experimental data (also see [28]).
Subtype-dependent conformational properties as
deduced from IR spectroscopy in water
IR measurements in D
2
O provide valuable information
on both the structure and flexibility (H ⁄ D exchange)
of a protein. Moreover, the different kinetics of H ⁄ D
exchange may assist in the assignment of absorption
bands arising from different secondary structure classes
[27,30–33]. By contrast, IR experiments in D
2
O can
also complicate the interpretation in the amide I¢
H. Fabian et al. Influence of inflammatory environment on HLA-B27

tion band at around 1640 cm
)1
[30].
For a direct comparison with the measurements in
D
2
O buffer described previously (Fig. 2), the second
derivatives of the absorbance spectra of the four
complexes and their corresponding differences were
calculated. Interestingly, the IR spectra obtained in
H
2
O buffer revealed subtype-specific spectral features
(Fig. 3). More importantly, many of these features in
the amide I region resembled those described previ-
ously for the corresponding IR spectra in D
2
O buffer
(compare Fig. 2D,E with Fig. 3A,C). Differences in
peak position of the amide I bands at 1692 ⁄ 94 cm
)1
assigned to the high-frequency b-sheet components,
which also give rise to clear positive and negative fea-
tures by subtracting the second derivative IR spectrum
of B*2705:pVIPR-U5 from that of B*2709:pVIPR-U5
(Fig. 3C), together with the spectral differences of the
low-frequency b-sheet component at  1625 cm
)1
were
observed. This corroborates the conclusions derived

[27]. Altogether, the high degree of similarity between
the corresponding IR spectra in D
2
O and in H
2
O
buffer permits us to conclude that the spectroscopic
1700 15001600
1694
1692
1668
1516
1552
1595
1625
1652
Second derivative
Δ2
nd

derivative
Wavenumber (cm
–1
)
F
E
D
C
B
A

2
m:pVIPR. (Upper) IR-difference spec-
tra of the second derivatives of experiments with two independent
preparations of each HLA-B27:peptide complex at 15°C. (E) com-
plexed with pVIPR-U5 (black trace, B*2709; red trace, B*2705) and
(F) complexed with pVIPR (black trace, B*2709; red trace, B*2705).
The spectra of the samples were normalized by use of the tyrosine
absorption band at 1516 cm
)1
as an internal intensity standard.
Influence of inflammatory environment on HLA-B27 H. Fabian et al.
1718 FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS
features in the amide I ⁄ I¢ regions associated with the
polypeptide backbone both indicate subtle subtype-spe-
cific structural differences, rather than being the conse-
quence of minor subtype-specific differences in H ⁄ D
exchange of the amide protons in the corresponding
HLA-B27:peptide complexes 1 h after transfer into
D
2
O buffer. These subtype-specific structural differ-
ences might influence temporary local or global
unfolding of the HC, and thus its flexibility.
pH-dependent thermal stabilities of
peptide-complexed HLA-B27 subtypes
Having established that HLA-B27 subtype-specific, but
peptide sequence-independent, conformational differ-
ences between the two HC do exist in solution, we next
investigated whether the presence of a hydrogen bond
between the pU5 side chain and His116 of the HC

transition temperature of b
2
m in the two complexes
( 64 °C) was very similar to that of free
13
C-labelled
b
2
m( 65 °C) (Table 1).
By contrast to this finding, distinct thermal denatur-
ation temperatures were observed for the two HLA-
B27 subtypes complexed with the unmodified peptide
pVIPR (Fig. 4B,D). The B*2709 ⁄
13
C-b
2
m:pVIPR
complex was less thermostable than the B*2705 ⁄
13
C-b
2
m:pVIPR complex by 4–5 °C. Moreover, a dif-
ference in peak position of the tyrosine band between
the spectra of B*2709 ⁄
13
C-b
2
m:pVIPR and B*2705 ⁄
13
C-b

2
O buffer. The
temperature dependence of the position of the HC-specific tyrosine band at 1514 cm
)1
is shown for (A) B*2709 ⁄
13
C-b
2
m:pVIPR-U5 (d)
and B*2705 ⁄
13
C-b
2
m:pVIPR-U5 (s), as well as for (B) B*2709 ⁄
13
C-b
2
m:pVIPR (d) and B*2705 ⁄
13
C-b
2
m:pVIPR (s). The other panels depict
the temperature dependence of the peak intensity of the b
2
m-specific b-sheet band at 1592 cm
)1
for (C) B*2709 ⁄
13
C-b
2

the effect of lowering the pH such that it approached
that in an inflamed tissue [12].
To this end, we analysed the influence of a pH value of
5.6 on the thermal stability of all four HLA-B27:peptide
complexes (Fig. 5). The data reveal that both
HLA-B27:pVIPR-U5 complexes (Fig. 5A,C) exhibited
comparable and high thermostabilities with T
m
values of
 63 °C at pH 5.6 (Table 1). By contrast, and as
suspected, a strong impact on the thermostability upon
lowering the pH to 5.6 was observed for B*2709:pVIPR
( 10 °C), but not for B*2705:pVIPR (Table 1). The
lack of a comparable pH-induced decrease in thermal
stability in case of B*2709:pVIPR-U5 allows to conclude
that: (a) it is likely that the hydrogen bond between the
Table 1. Determination of the transition temperatures of HLA-B27:peptide complexes. The transition temperatures (T
m
values in °C) were
calculated either from the intensity ⁄ temperature plot of the b-sheet band of b
2
m at 1592 cm
)1
or from the frequency ⁄ temperature changes
of the tyrosine ring vibration of the HC at 1514 cm
)1
of the IR spectra of B*2705 ⁄
13
C-b
2

2
m-band 63.8 64.4 66.4 62.5 64.8 7.5
Tyr band 63.4 63.2 62.3 52.8 5.6
b
2
m-band 63.4 63.7 63.8 55.9 48.7 5.6
10 20 30 40 50 60 70 80 90
1513.8
1514.0
1514.2
1514.4
1514.6
1514.8
10 20 30 40 50 60 70 80 90
1513.6
1513.8
1514.0
1514.2
1514.4
1514.6
1514.8
Temperature (°C) Temperature (°C)
Intensity change (1594 cm
–1
)
Peak

position (cm
–1
)

m:pVIPR (s). The other panels
depict the temperature dependence of the peak intensity of the b
2
m-specific b-sheet band at 1594 cm
)1
for (C) B*2709 ⁄
13
C-b
2
m:pVIPR-U5
(d) and B*2705 ⁄
13
C-b
2
m:pVIPR-U5 (s), as well as for (D) B*2709 ⁄
13
C-b
2
m:pVIPR (d) and B*2705 ⁄
13
C-b
2
m:pVIPR (s).
Influence of inflammatory environment on HLA-B27 H. Fabian et al.
1720 FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS
pU5 side chain and His116 also exists in solution at
physiological pH; and (b) the specific peptide–protein
interaction involving His116 contributes to the confor-
mational stability of the HLA-B27 complex.
Correlation of the results from IR spectroscopy

B*2705, which might serve to explain the observed dis-
similarities in amide protection between the HC of the
two subtypes. In the case of complexes with the
unmodified pVIPR peptide, this might have been due
to the different resolutions at which the two structures
were solved (B*2705 at 1.47 A
˚
, B*2709 at 2.2 A
˚
) [18].
Such uncertainties do not exist for the HLA-
B27:pVIPR-U5 subtypes, whose structures had been
determined at high and comparable resolutions of
 1.8 A
˚
[29]. An inspection of the binding grooves of
B*2705:pVIPR-U5 and B*2709:pVIPR-U5, colour-
coded according to the binding groove flexibility in the
crystalline state at 100 K, revealed no clear indications
for subtype-specific differences. In summary, neither
the comparison of the crystallographic temperature
factors nor the detailed comparison of structural fea-
tures provide hints which could help to understand the
IR spectroscopic findings observed in solution at phys-
iological temperatures.
Discussion
This study addresses questions that are relevant for
understanding how an inflammatory environment,
such as that observed in reactive arthritis or AS,
might affect MHC molecules: (a) Is the HC flexibility

ing that a repositioning of water molecules is responsi-
ble for the altered flexibility of the two opposing
helical segments of the binding groove [28]. IR spec-
troscopy cannot be used to localize the regions where
the two HC differ, but molecular dynamics simulations
of complexes of HLA-B27 subtypes with pVIPR have
suggested an increased flexibility of two opposing heli-
cal segments (residues 75–60 and 137–150) of the
B*2705 binding groove in comparison with that of
B*2709 [27]. Corresponding MD simulations of the
two HLA-B27 subtypes with the modified peptide
pVIPR-U5 have not been carried out, but the high
degree of similarity of the subtype-specific spectral dif-
ferences for B*2705 ⁄ B*2709 either with pVIPR or
pVIPR-U5 as observed in this study (Figs 2 and 3)
argues for a comparable nature of the underlying
structural differences.
The observed subtype-specific differences in the IR
b-sheet spectral features of the HC could not be
explained on the basis of their X-ray structures,
because all b strands of the B*2705 and B*2709 HC
H. Fabian et al. Influence of inflammatory environment on HLA-B27
FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1721
complexed with pVIPR-U5 overlay perfectly. Moreover,
a comparative analysis of the B factors for the differ-
ent structural domains provided no clear indications
for differences between the HC of the two subtypes,
suggesting that these conformational characteristics
are only detectable in solution and thus inaccessible
through X-ray data collection at cryogenic tempera-

however, because peptides with basic C-termini bind
only very rarely to this subtype in vivo [4,16].
How distinct dynamic characteristics of the two
HLA-B27 subtypes impact on their function is cur-
rently unknown. We have previously argued that the
interaction of these molecules with receptors on effec-
tor cells might be altered in dependence on HC flexibil-
ity [28]. However, in the absence of thorough analyses
of dynamic properties concerning entire assemblies of
MHC class I molecules and receptors on T cells or
natural killer cells, it is currently not clear whether
interactions of the binding partners are indeed influ-
enced. Analysis of a T-cell receptor footprint on an
HLA-A2:peptide complex by NMR spectroscopy [38]
is a first step towards understanding this intricate
issue, although the flexibility of the MHC molecule
was not investigated in this study.
In addition to the general subtype-specific conforma-
tional features discussed above, our experimental find-
Fig. 6. Conserved interactions between the displayed peptide and amino acid residues residing on the a1- or a2 helix. All structures are
superimposed and the view is towards the carboxylate of the C-terminal p9 residue. Only the peptide segments from p5 to p9 are shown,
and side chains are omitted with the exception of pArg5 (pVIPR) and pU5 (pVIPR-U5). HC residues Asp77 and Trp147, which are involved in
conserved interactions with peptide residues, are shown as grey sticks. Hydrogen-bonding interactions of the peptide residues p8 (main
chain carbonyl) to Trp147 (indole NE atom) and of p9 (main chain amide) to Asp77 (carboxylate OD1 atom) are depicted with red dashed
lines. (A) B*2705:pVIPR in canonical conformation (green) and in noncanonical conformation (orange). Only the latter conformation allows
the peptide to anchor to the HC by a salt bridge from pArg5 to Asp116. The conformation of pVIPR in B*2709 (brown) is indistinguishable
from the canonical binding mode of this peptide in B*2705. (B) B*2705 in complex with pVIPR-U5 is shown in purple and B*2709 with
pVIPR-U5 in yellow. In the latter complex, a hydrogen bond is formed between His116 and pU5. The pU5 side chain points to different
directions in the two subtypes. Despite differences in peptide sequences and conformations, the formation of highly conserved hydrogen
bonds from the a1- and a2 helices to the peptide main chain atoms is still permitted in all four structures.

the bound peptide. In addition, the side chains of pU5
and pTrp7 (B*2709:pVIPR-U5) are connected via the
same water molecule to the imidazole function of
His114 (Fig. 7B). Irrespective of the occurrence of the
pVIPR peptide in a dual conformation (B*2705), a
water molecule is establishing an indirect contact with
Fig. 7. Structural features of the pVIPR and pVIPR-U5 peptides bound to B*2705 and B*2709. The view is identical to that in Fig. 1. In all
panels, the subtype-specific residue 116 is indicated in green. The conserved His9 and His114 residues (magenta sticks) are involved in pep-
tide–HC interactions via water molecules that are represented by red spheres. Hydrogen bonds and salt bridges are depicted by dashed red
lines. (A) pVIPR-U5 drawn in purple as presented by B*2705. (B) pVIPR-U5 drawn in yellow as presented by B*2709. (C) The binding of
pVIPR by B*2705 occurs in a dual conformation with roughly equal occupancy. Although one of the binding modes resembles that found in
B*2709 (green, compare Fig. 6A), the other (orange) is distinct from the first between pLys3 and pTrp7. It is characterized by the formation
of a salt bridge between pArg5 and Asp116 and is thus similar to the conformation of pVIPR-U5 when bound to B*2709 (compare B). (D)
pVIPR (brown) is bound by B*2709 in a single conformation, with the middle of the peptide bulging out of the binding groove. Note that
hydrogen bonds involving His9, His114 and His116 (only B*2709) are retained in a nearly identical manner by the two complexes of each
HLA-B27 subtype.
H. Fabian et al. Influence of inflammatory environment on HLA-B27
FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1723
pLys3 in the canonical conformation or with pArg5 in
the noncanonical binding mode. Therefore, histidine
residues within the binding groove of HLA-B27 mole-
cules serve important functions in maintaining a bound
peptide in place.
As indicated previously, it was to be expected that
the distinct water networks within the HLA-B27 bind-
ing groove might be influenced by lowering the pH to
values that predominate in an inflammatory milieu
[12]. Furthermore, the unique direct contact between
pU5 and His116 (B*2709:pVIPR-U5) and the lack of
it (B*2709:pVIPR) must be regarded as particularly

peptide exchange that is necessary for the loading of
MHC class II molecules and the initiation of immune
responses [41]. To the best of our knowledge, the
results presented here are the first to provide hints of
the involvement of a naturally occurring MHC poly-
morphism that exerts a pH-dependent effect on the
conformational stability of an MHC molecule,
although the suggested protonation of His116 in
B*2709:pVIPR complexes must be regarded as indirect
and needs to be substantiated by further experiments.
Direct proof for the protonation of individual histidine
residues might be obtained by neutron diffraction tech-
niques, as revealed by the extremely complex picture
of differential protonation for the 38 histidine residues
of the haemoglobin tetramer [42].
Materials and methods
Sample preparation
HPLC-purified, citrulline-modified peptide pVIPR-U5 was
purchased from Alta Bioscience (Birmingham, UK). The
nonapeptide pVIPR was synthesized and purified by
M. Beyermann (Leibniz-Institut fu
¨
r Molekulare Pharmakologie,
Berlin, Germany). The extracellular domains of B*2705 and
B*2709 and
13
C-labelled b
2
m were expressed separately
in Escherichia coli as inclusion bodies [17,43]. To obtain

each sample. The final sample concentrations were between
10 and 20 mgÆmL
)1
before collection of IR data of the
complexes and  2mgÆmL
)1
for b
2
m.
IR spectroscopy
The protein solutions were always freshly prepared and
placed into demountable calcium fluoride IR cells [30] with
an optical pathlength of 50 lm for measurements in D
2
O
buffer or 8 lm for samples in H
2
O buffer. IR spectra were
recorded with IFS-28B and IFS-66 FTIR spectrometers
(Bruker Optics, Ettlingen, Germany) equipped with deuter-
ated triglycine sulfate detectors and continuously purged
with dry air. For each sample, 128 interferograms were
co-added and Fourier-transformed to yield spectra with a
nominal resolution of 4 cm
)1
. The sample temperature was
controlled by means of thermostated cell jackets. Spectra at
discrete temperatures were obtained by heating the protein
solutions from 15 to 90 °C in steps of 2.5 °C. In order to
minimize problems due to baseline drifts or variations in

supported by the Deutsche Forschungsgemeinschaft
(grants Na226 ⁄ 12-3, UC8 ⁄ 1-2, and SFB 449 ⁄ B6), and
a grant from the Robert Koch-Institut, Berlin (to Hans
Huser). Andreas Ziegler also acknowledges support
from the VolkswagenStiftung (grant I ⁄ 79 989)
and Bernhard Loll from the Fonds der Chemischen
Industrie and the Forschungsfo
¨
rderung der Freien
Universita
¨
t Berlin.
References
1 Madden DR (1995) The three-dimensional structure of
peptide–MHC complexes. Annu Rev Immunol 13, 587–
622.
2Lo
´
pez de Castro JA (2007) HLA-B27 and the patho-
genesis of spondyloarthropathies. Immunol Lett 108,
27–33.
3 D’Amato M, Fiorillo MT, Carcassi C, Mathieu A,
Zuccarelli PP, Bitti R, Tosi R & Sorrentino R (1995)
Relevance of residue 116 of HLA-B27 in determining
susceptibility to ankylosing spondylitis. Eur J Immunol
25, 3199–3201.
4Lo
´
pez de Castro JA, Alvarez I, Marcilla M, Paradela
A, Ramos M, Sesma L & Vazquez M (2004) HLA-B27:

12 Stehen KH, Steen AE & Reeh PW (1995) A dominant
role of acidic pH in inflammatory excitation and sensiti-
zation of nociceptors in rat skin, in vitro. J Neurosci 15,
3982–3989.
13 Grimsley G, Scholtz JM & Pace CN (2009) A summary
of the measured pK values of the ionisable groups in
folded proteins. Protein Sci 18, 247–251.
14 Pace CN, Grimsley GR & Scholtz JM (2009) Protein
ionisable groups: pK values and their contribution to
protein stability and solubility. J Biol Chem 284, 13285–
13289.
15 Fiorillo MT, Maragno M, Butler R, Dupuis ML &
Sorrentino R (2000) CD8(+) T-cell autoreactivity to an
HLA-B27-restricted self-epitope correlates with ankylos-
ing spondylitis. J Clin Invest 106, 47–53.
16 Ramos M, Paradela A, Vazquez M, Marina A, Vazquez J
&Lo
´
pez de Castro JA (2002) Differentail association
of HLA-B*2705 and B*2709 to ankylosing spondylitis
correlates with limited peptide subsets but not with
altered cell surface stability. J Biol Chem 277, 28749–
28756.
17 Hu
¨
lsmeyer M, Hillig RC, Volz A, Ru
¨
hl M, Schro
¨
der W,

association. J Biol Chem 279, 652–663.
21 Hu
¨
lsmeyer M, Welfle K, Po
¨
hlmann T, Misselwitz R,
Alexiev U, Welfle H, Saenger W, Uchanska-Ziegler B &
Ziegler A (2005) Thermodynamic and structural equiva-
lence of two HLA-B27 subtypes complexed with a self-
peptide. J Mol Biol 346, 1367–1379.
22 Fiorillo MT, Ru
¨
ckert C, Hu
¨
lsmeyer M, Sorrentino R,
Saenger W, Ziegler A & Uchanska-Ziegler B (2005)
Allele-dependent similarity between viral and self-pep-
tide presentation by HLA-B27 subtypes. J Biol Chem
280, 2962–2971.
23 Ru
¨
ckert C, Fiorillo MT, Loll B, Moretti R, Biesiadka J,
Saenger W, Ziegler A, Sorrentino R & Uchanska-
Ziegler B (2006) Conformational dimorphism of self-
peptides and molecular mimicry in a disease-associated
HLA-B27 subtype. J Biol Chem 281, 2306–2316.
24 Winkler K, Winter A, Ru
¨
ckert C, Uchanska-Ziegler B
& Alexiev U (2007) Natural MHC class I polymor-

Uchanska-Ziegler B (2008) Citrullination-dependent dif-
ferential presentation of a self-peptide by HLA-B27
subtypes. J Biol Chem 283, 27189–27199.
30 Fabian H & Ma
¨
ntele W (2002) Infrared spectroscopy of
proteins. In Handbook of Vibrational Spectroscopy
(Chalmers JM & Griffiths PR eds), pp. 3399–3425. Wi-
ley, Chichester.
31 Barth A & Zscherp C (2002) What vibrations tell us
about proteins. Q Rev Biophys 35, 369–430.
32 Surewicz WK & Mantsch HH (1988) New insight into
protein secondary structure from resolution-enhanced
infrared spectra. Biochim Biophys Acta 952, 115–130.
33 Fabian H, Schultz CP, Naumann D, Landt O, Hahn U
& Saenger W (1993) Secondary structure and tempera-
ture-induced unfolding and refolding of ribonuclease T1
in aqueous solution. J Mol Biol 232, 967–981.
34 Fabian H, Schultz CP, Backmann J, Hahn U, Saenger
W, Mantsch HH & Naumann D (1994) Impact of point
mutations on the structure and thermal stability of ribo-
nuclease T1 in aqueous solution probed by Fourier
transform infrared spectroscopy. Biochemistry
33,
10725–10730.
35 Stewart-Jones GBE, di Gleria K, Kollnberger S,
McMichael AJ, Jones EY & Bowness P (2005) Crystal
structure and KIR3DL1 recognition of three immuno-
dominant viral peptides complexed to HLA-B*2705.
Eur J Immunol 35, 341–351.

as control element for ligand release from HLA-DR
molecules. Proc Natl Acad Sci USA 99, 16946–16950.
42 Kovalevsky AY, Chatake T, Shibayama N, Park SY,
Ishikawa T, Mustyakimov M, Fisher Z, Langan P &
Morimoto Y (2010) Direct determination of proton-
ation states of histidine residues in a 2 A
˚
neutron struc-
ture of deoxy-human normal adult haemoglobin and
implications for the Bohr effect. J Mol Biol 398, 276–
291.
43 Garboczi DN, Hung DT & Wiley DC (1992) HLA-
A2–peptide complexes: refolding and crystallization of
molecules expressed in Escherichia coli and complexed
with single antigenic peptides. Proc Natl Acad Sci USA
89, 3429–3433.
Influence of inflammatory environment on HLA-B27 H. Fabian et al.
1726 FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS
Supporting information
The following supplementary material is available:
Fig. S1. IR absorbance spectra of a B*2709 ⁄
13
C-
b
2
m:pVIPR-U5 complex in H
2
O buffer, 1 h after
transfer into D
2