Báo cáo khoa học: Influence of inflammation-related changes on conformational characteristics of HLA-B27 subtypes as detected by IR spectroscopy potx

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

Keywords
ankylosing spondylitis; citrullination;
conformational differences; HLA-B27
subtypes; IR spectroscopy
Correspondence
D. Naumann, Robert Koch-Institut, P 25,
Nordufer 20,
D-13353 Berlin, Germany
Fax: +49 30 1875 42606
Tel: +49 30 1875 42259
E-mail:
(Received 3 December 2010, revised 8
March 2011, accepted 11 March 2011)
doi:10.1111/j.1742-4658.2011.08097.x
Inflammatory processes are accompanied by the post-translational modifi-
cation of certain arginine residues to yield citrulline, and a pH decrease in
the affected tissue, which might influence the protonation of histidine resi-
dues within proteins. We employed isotope-edited IR spectroscopy to
investigate whether conformational features of two human major histocom-
patibility antigen class I subtypes, HLA-B*2705 and HLA-B*2709, are
affected by these changes. Both differ only in residue 116 (Asp vs. His)
within the peptide-binding grooves, but are differentially associated with
inflammatory rheumatic disorders. Our analyses of the two HLA-B27 sub-
types in complex with a modified self-peptide containing a citrulline
RRKWURWHL (U = citrulline) revealed that the heavy chain is more
flexible in the HLA-B*2705 subtype than in the HLA-B*2709 subtype.
Together with our previous studies of HLA-B27 subtypes complexed with
the unmodified self-peptide RRKWRRWHL, these findings support the
existence of subtype-specific conformational features of the heavy chains
under physiological conditions, which are undetectable by X-ray crystallog-

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

2
m), and a peptide derived from intracellular
proteins [1]. Recognition of these peptide-loaded MHC
molecules by cellular ligands on effector cells triggers
immune responses [2]. For human leukocyte antigen
(HLA) class I molecules, peptides derived from self- or
nonself proteins are usually 8–12 amino acids long and
are accommodated in a binding groove of the molecule
by means of HLA allele-characteristic ‘anchor’ amino
acids. This selectivity of MHC molecules towards cer-
tain anchor residues of peptides provides the basis for
HLA subtype-specific immune responses and impacts
on disease associations as described, for example, for
the group of HLA-B27 alleles [3–6].
It is known that the citrullination of proteins, a
post-translational modification, influences immune
responses and inflammatory reactions [7,8]. This modi-
fication involves arginine, which is strongly basic,
whereas the resulting citrulline is a neutral amino acid
[9]. Citrullination is found within synovial tissue from
patients with reactive arthritis, an HLA-B27-associated
disorder [10]. Moreover, fragments derived from citrul-
linated polypeptides are most likely also available for
presentation by MHC antigens in patients with anky-
losing spondylitis (AS), another spondylarthropathy
that is even more strongly associated with HLA-B27
than reactive arthritis [2,11].
Although distinct, both diseases are also character-
ized by inflammatory processes that are, by their very
nature, associated with a decrease in the pH value of

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

exceptional dual conformation by the AS-associated
B*2705 subtype. One of the conformations observed in
B*2705:pVIPR is identical to that seen in B*2709,
whereas in the other, peptide Arg5 (pArg5) forms a salt
bridge to HC residue Asp116, resulting in a noncanoni-
cal binding mode of the ligand. The dual conformation
of this ligand in B*2705 has also been linked to differ-
ential T-cell responses among the two HLA-B27 sub-
types [15,18].
Recently, an isotope-edited IR spectroscopic study
of B*2709:pVIPR and B*2705:pVIPR demonstrated
that the HC is more flexible in the B*2705 subtype
than in the B*2709 subtype at physiological tempera-
tures [27]. Furthermore, similar conformational differ-
ences between the HC of the two subtypes were also
found in complexes with the viral peptide pLMP2 and
the self-peptide TIS [28]. Collectively, these findings
reveal the existence of subtype-specific, but peptide
sequence- and conformation-independent conforma-
tional differences between the two HLA-B27 HC at
physiological temperatures, which to date have not
been detectable using X-ray crystallography.
To approach the situation prevailing in inflamed tis-
sue, we have now extended these IR spectroscopic
studies to a citrullinated self-peptide, pVIPR-U5
(RRKWURWHL; U = citrulline) and performed
experiments at a lower pH value. This peptide is pre-
sented by the two HLA-B27 molecules in binding
modes that differ drastically not only from each other
[29], but also from the conformations exhibited by the

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

m:pVIPR-U5
complexes was initially studied at pH 7.5 after transfer
into D
2
O-buffer (Fig. 2A). The use of
13
C-labelled b
2
m
for reconstitution with separately expressed unlabelled
HC in the presence of the corresponding peptide
greatly reduced overlapping of its amide I band with
that of the HC in the spectroscopic analyses. The spec-
tra of the two HLA-B27:pVIPR-U5 complexes were
very similar to those previously determined for com-
plexes with the unmodified self-peptides pVIPR [27],
TIS [28] or the viral peptide pLMP2 [28], in a given sub-
type. A feature at  1594 cm
)1
due to
13
C-labelled b
2
m
and a dominant broad absorbance corresponding to the
HC centred at  1640 cm
)1
were observed. The under-
lying HC-specific band components between 1620 and
1700 cm

)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

one additional Asp in B*2705 is practically negligible.
This can be expected even more for His, because its
side-chain absorption band around 1600 cm
)1
is very
weak [31]. The remaining IR intensity at  1545 cm
)1
(amide N–H deformation vibration) 1 h after transfer
into D
2
O buffer, together with the presence of a band
at  3286 cm
)1
(N–H stretching vibration), shows that
a number of amide NH groups of the HC in the two
complexes are protected from H ⁄ D exchange. The
band at  3286 cm
)1
(amide A) is the best indicator of
residual nonexchanged N-H groups, owing to the lack
of other protein absorption in the range 3200–
3400 cm
)1
[30]. Unfortunately, the very strong water
absorption band at  3400 cm
)1
(O–H stretching
vibration) prevents one from obtaining the amide A
band of the HLA-B27:peptide complex in H
2

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

O buffer (0% exchange)
and after thermal denaturation of the complex at
90 °C (fully deuterated state) to 100%. This approach
is only an approximation because: (a) the amide II
bands of different conformations of proteins have
different peak maxima, and (b) IR bands due to amino
acid side-chain absorptions (overlapping bands of Glu
and Asp) between 1550 and 1585 cm
)1
may overlap
with residual amide II band features and may change
as a function of conformational changes. Keeping this
in mind, the IR data suggest that  50% of the amide
protons of the B*2709 ⁄
13
C-b
2
m:pVIPR-U5 complex
remained unexchanged 1 h after transfer into D
2
O
buffer.
3400 3300 3200
ΔA × 5
1700 1600 1500 1400
100 mA
20 mA
3286
Wavenumber (cm
–1

)
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

derivative × 100
Δ
2
nd
derivative × 100
Absorbance
A
B
C
D
E
F
Fig. 2. HLA-B27 complexes measured in D
2
O buffer at pH 7.5. (Lower) (A) IR absorbance spectra of B*2709 ⁄
13
C-b
2
m:pVIPR-U5 (black
trace) and of B*2705 ⁄
13
C-b
2
m:pVIPR-U5 (red trace), both measured at 15 °C 1 h after transfer into D
2
O buffer. The spectra of the two sam-
ples were normalized using the tyrosine absorption band at 1514 cm
)1
as an internal intensity standard. (Middle) Differences of IR spectra
of the HLA-B27:peptide complexes, all measured at 15 °C 1 h after transfer into D

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 (black trace) and B*2705 ⁄
13
C-b
2
m:pVIPR-U5 (red trace) at 15 °C. (E) IR difference spectra (B*2709 ⁄
13
C-b
2
m:pVIPR-U5 ) B*2705 ⁄
13
C-b
2
m:pVIPR-U5) of the second derivatives at 15 °C (black trace) and at 90 °C (red trace). (F) IR difference
spectra of the second derivatives of experiments with two independent preparations of each HLA-B27:pVIPR-U5 complex (black trace;
B*2709; red trace, B*2705), demonstrating the high reproducibility of the experimental data.
Influence of inflammatory environment on HLA-B27 H. Fabian et al.
1716 FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS
Detection of HLA-B27 subtype-dependent
conformational properties
The IR difference spectrum obtained by subtracting
the spectrum of B*2705 ⁄
13
C-b
2
m:pVIPR-U5 from that
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

50% of nonexchanged amide protons of the
B*2709 ⁄
13
C-b
2
m:pVIPR-U5 sample as reference, the
IR data indicate that the observed differences in flexi-
bility between the two subtypes might be restricted to
only some residues of their HC.
The difference features around 1688 and 1624 cm
)1
are due to spectral characteristics associated with ami-
de I¢ band components attributed to the b sheets of
the HC, suggesting fine differences in the hydrogen-
bonding pattern of the b-type structures of the HC in
the two HLA-B27 samples. By contrast, no positive or
negative features are observed around 1594 cm
)1
(Fig. 2B), indicating identical peak positions of the b-
sheet band of
13
C-labelled b
2
m in the two samples. In
turn, these characteristics suggest a very similar degree
of H ⁄ D exchange of the amide protons of b
2
min
B*2705 ⁄
13

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-

tion, intensity and shape of band components in
greater detail than by making simple comparisons of
the original spectra [31,32]. Differences in peak posi-
tion and ⁄ or intensities of the amide I¢ bands attributed
to the b sheets of the HC are clearly visible by com-
paring the second derivatives of the spectra of the two
HLA-B27:pVIPR-U5 complexes collected at 15 °C and
pH 7.5 (black and red traces in Fig. 2D). Striking is
the difference in peak position of the amide I¢ band
due to the high-frequency b-sheet band component at
1691 ⁄ 93 cm
)1
which gives rise to obvious positive and
negative features by subtracting the second derivative
IR spectrum of B*2705:pVIPR-U5 from that of
B*2709:pVIPR-U5 (Fig. 2E). These differences,
together with the minor spectral differences of the low-
frequency b-sheet component at  1624 cm
)1
, indicate
the presence of fine differences in the relative orienta-
tion of the b strands of the HC in the two HLA-B27
samples at physiological temperatures. The features
between 1635 and 1655 cm
)1
(Fig. 2E) suggest a more
intense and ⁄ or slightly shifted amide I¢ band compo-
nent in the spectrum of B*2709 compared with that of
B*2705. Subtle structural differences between the HC
of the two HLA-B27 subtypes, which cannot be speci-

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

FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS 1717
region, because spectral differences in the amide I¢
band contour due to H ⁄ D exchange and changes in
secondary structure may overlap. The only way to
overcome this complication is to fully exchange the
protein before monitoring conformational changes in
D
2
O medium. Complete H ⁄ D exchange is also achiev-
able in the case of the HLA-B27:peptide complexes by
keeping the sample solutions close to the denaturation
temperature before cooling them to low temperature,
but this is always accompanied by irreversible aggrega-
tion of the sample, thus rendering it impossible to
obtain the IR spectrum of a completely exchanged
native HLA-B27:peptide complex (data not shown, see
also [27]). The interpretation of the IR spectra
obtained in H
2
O medium is not complicated by the
above-mentioned spectral effects due to H ⁄ D
exchange. We therefore also measured the IR spectra
of the native HLA-B27:peptide complexes in H
2
O buf-
fer, despite the fact that it is more difficult to obtain
IR spectra in this solvent than in D
2
O-containing buf-
fer because of the interfering intense water deforma-

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

from analyses of the spectra in D
2
O buffer (Fig. 2),
that fine differences in the relative orientation of the
strands in the b-sheet structures of the HC in the two
HLA-B27 samples do exist at low temperatures. The
components at  1650 cm
)1
(Fig. 3C) suggest a more
intense and⁄ or slightly shifted amide I band in the
spectrum of B*2709 compared with that of B*2705.
Again, the differences between the second derivative
spectra of B*2705:pVIPR-U5 and B*2709:pVIPR-U5
turned out to be very similar to those found for the
two HLA-B27:pVIPR complexes (compare Fig. 3C
with Fig. 3D). This holds true for the different peak
positions of the high-frequency b-sheet band around
1690 cm
)1
and the spectral differences between the fea-
tures at  1650 and  1626 cm
)1
as well. Moreover,
the subtype-dependent spectral differences were much
more pronounced than the spectral differences between
two independent preparations of the corresponding
complexes (Fig. 3E,F), as demonstrated previously for
the corresponding IR spectra in D
2
O buffer (Fig. 2F)

[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

Fig. 3. HLA-B27 complexes measured in H
2
O buffer at pH 7.5.
(Lower) IR spectra (second derivatives) of B*2709 ⁄
13
C-b
2
m and
B*2705 ⁄
13
C-b
2
m (black and red traces in each panel, respectively),
complexed with (A) pVIPR-U5 or (B) pVIPR at 15 °C. (Middle) Differ-
ences between the second derivative IR spectra of the HLA-
B27:peptide complexes all measured at 15 °C. (C) B*2709 ⁄
13
C-b
2
m:pVIPR-U5 ) B*2705 ⁄
13
C-b
2
m:pVIPR-U5 and (D) B*2709 ⁄
13
C-b
2
m:pVIPR ) B*2705 ⁄
13
C-b

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

might impact on the thermal denaturation behaviour
of the HLA-B27:pVIPR-U5 complexes at physiological
pH. Following our previous approach [27], we
employed the aromatic ring-stretching vibration
of the tyrosine band at 1514 cm
)1
to follow tempera-
ture-induced conformational changes in the HC.
The decrease in absorbance at 1592 cm
)1
with increas-
ing temperature was used to monitor denaturation of
the secondary structure of b
2
m in the complexes.
The various frequency ⁄ temperature plots obtained
by monitoring the tyrosine ring vibration (Fig. 4A)
revealed very similar thermostabilities of the HC in the
two complexes at pH 7.5 (Table 1). The thermal dena-
turation temperatures estimated from the
13
C-labelled
b
2
m band of the two HLA-B27:pVIPR-U5 complexes
were almost identical (Fig. 4C), with a weak tendency
to reach higher T
m
values than those determined for
the HC-specific tyrosine band (Table 1). Moreover, the

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
m:pVIPR in the temperature range from 15 to
Intensity change (1592 cm
–1
)
Peak position (cm
–1
)
10 20 30 40 50 60 70 80 90
10 20 30 40 50 60 70 80 90
10 20 30 40 50 60 70 80 90
Temperature (°C)
10 20 30 40 50 60 70 80 90
Temperature (°C)
1514.8
1514.6
1514.4
1514.2
1514.0
1514.8
1514.6
1514.4
1514.2
1514.0
AB
CD
Fig. 4. Thermostabilities of HLA-B27:peptide complexes at pH 7.5. All measurements were carried out in D
2
O buffer I and monitored by IR
spectroscopy. For each plot, the first data point (at 15 °C) was obtained 1 h after transfer of the corresponding sample into D

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

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).
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 1719
50 °C was observed (Fig. 4B), indicating that the
microenvironment of at least some Tyr residues must
differ between the two subtypes [27]. In addition, the
gain in stability of B*2709:pVIPR-U5 compared with
B*2709:pVIPR (Table 1) is likely to be a consequence
of an additional peptide–HC interaction (a hydrogen
bond connecting pU5
O7
and His116
NE2
) that is present
only in B*2709:pVIPR-U5. This suspected involvement
of His116 in stabilizing the B*2709 subtype complexed
with pVIPR-U5, but not pVIPR, prompted us to study

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

m:pVIPR–U5, B*2709 ⁄
13
C-b
2
m:pVIPR-U5,
B*2709 ⁄
13
C-b
2
m:pVIPR and B*2705 ⁄
13
C-b
2
m:pVIPR 1 h after transfer into D
2
O buffer. Measurements were performed at pH 7.5 or 5.6
(see Materials and Methods for experimental details). For comparison, the transition temperatures as estimated from IR experiments with
free
13
C-labelled b
2
m are also shown. The T
m
values are the average of experiments with two or three independent preparations of each
sample, with standard deviations of 0.5–1 °C.
IR band B*2705:pVIPR-U5 B*2709:pVIPR-U5 B*2705:pVIPR B*2709:pVIPR b
2
mpH
Tyr band 63.0 64.7 65.9 61.0 7.5
b

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
)

10 20 30 40 50 60 70 80 9010 20 30 40 50 60 70 80 90
C
A
D
B
Fig. 5. Thermostabilities of the HLA-B27:peptide complexes at pH 5.6. All measurements were carried out in D
2
O buffer II and monitored
by IR spectroscopy. For each plot, the first data point (at 15 °C) was obtained 1 h after transfer of the corresponding sample into D
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 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

with X-ray crystallographic data
In an attempt to obtain a structure-based interpreta-
tion for the observed subtype-specific IR spectroscopic
findings, we performed a detailed inspection of the
X-ray structures of the two HLA-B27:pVIPR-U5 sub-
types and analysed, in particular, the crystallographic
temperature factors (B factors) of the different struc-
tural domains. Because both HLA-B27:pVIPR-U5
subtypes crystallized isomorphously, we can exclude
the possibility that differences between the two struc-
tures are due to different packing of the protein chains
in crystallo. A comparison of the X-ray structures
revealed, however, that the structures of the HC and
of b
2
m of the two subtypes appear nearly indistin-
guishable (C
a
root mean square deviation £ 0.6 A
˚
)
[29]. An earlier assessment of the B factors for the
different structural units of B*2709:pVIPR and
B*2705:pVIPR revealed that the peptide-binding
groove exhibits the lowest flexibility, whereas major
parts of the a3 domain and of b
2
m are more flexible
[27]. At the same time, this comparison failed to pro-
vide indications for differences between B*2709 and

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

of two minimally distinct HLA-B27 subtypes affected
by citrullination of peptides? and (b) Can the stabil-
ity of HLA-B27:peptide complexes be impaired by
lowering the pH to levels which prevail in inflamed
tissues?
The isotope-edited IR spectroscopic results described
here corroborate the previously observed differential
flexibility of the two HLA-B27 subtypes. To date, we
had analysed only peptides (pVIPR, TIS, pLMP2) that
are bound to B*2709 in the canonical binding mode,
with the middle of the peptide bulging out of the bind-
ing groove [18,21,22,27,28]. This, however, is not the
case with pVIPR-U5, because the pArg5 fi pU5
exchange leads to a reorientation of this peptide in the
binding groove of the B*2709 subtype, accompanied
by the creation of a novel hydrogen-mediated contact
between citrulline and His116 (Fig. 1) [29]. Therefore,
the increased conformational flexibility of the B*2705
HC in comparison to that of B*2709 found also in
case of pVIPR-U5 (see IR results, Fig. 2) must be
regarded as an intrinsic property of the HC of the two
subtypes and not as a peptide sequence- or binding
mode-related characteristic, particularly because this
modified peptide is bound in a single, canonical con-
formation by B*2705. Ultimately, the polymorphic HC
residue 116 must be responsible for the observed reori-
entation of pVIPR-U5 in comparison with the pVIPR
peptide (Fig. 6). We have already proposed a struc-
ture-based explanation to account for effects observed
in conjunction with the Asp116His exchange, suggest-

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-

tures. This might be because of the very limited infor-
mation on protein dynamics that can be obtained at
100 K, or because the X-ray technique is not sensitive
enough to resolve these differences. As discussed pre-
viously [28], it seems plausible to assume that changes
in the location of the bound water molecules near the
helical regions may also impact on the conformation
of the b sheet of the peptide-binding groove, which in
turn may cause the spectral changes described. Subtle
spectral differences associated with b strands in
proteins, which cannot easily be explained by X-ray
crystallography, are not uncommon. For example, we
have shown before that a comparative IR spectro-
scopic analysis of the protein ribonuclease T1 and
some of its variants can also reveal such alterations
[34]. The only difference observed by X-ray crystal-
lography in these structures is a string of water mole-
cules between the a-helix and the major b-sheet that
are distinctly located in the wild-type protein and in
the variants. As pointed out previously [28], it is con-
ceivable that peptides with a C-terminal basic residue
might lead to an altered binding groove flexibility in
the B*2705 subtype because of the formation of salt
bridges to Asp116 within the molecule’s F pocket
[17,35,36]. This indicates that the conclusion reached
with regard to the enhanced flexibility of the B*2705
HC may not be valid for those complexes that dis-
play a peptide with Arg or Lys at the C-terminus
(see also [19,25,37] for further discussions). These
considerations are irrelevant for the B*2709 subtype,

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.

Influence of inflammatory environment on HLA-B27 H. Fabian et al.
1722 FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS
ings provide also information on the peptide–protein
interaction involving His116 that is found only in
B*2709:pVIPR-U5, and on its contribution to the sta-
bility of this complex. The HC of B*2705 and B*2709
contain 10 and 11 histidine residues, respectively.
When complexed with pVIPR and pVIPR-U5, two
(His9 ⁄ His114 in B*2705) or three (His9 ⁄ His114 ⁄ His116
in B*2709) are involved in peptide–HC interactions via
water molecules in the two subtypes, as well as in
direct interactions with the polymorphic residue 116
(Fig. 7). The adopted side-chain rotamer is identical
for each of the two or three histidines, and the interac-
tion between His9 and pArg2 via a conserved water
molecule is preserved in all four complexes also.
Furthermore, His114 is always located at hydrogen-
bonding distance from the polymorphic residue
Asp ⁄ His116, and is therefore important for the proper
coordination of the side-chain function of this
polymorphic residue. Additional indirect contacts are
formed from His114 to the differently bound peptides.
In the structures of B*2705:pVIPR-U5 (Fig. 7A) and
B*2709:pVIPR (Fig. 7D), His114 is connected via a
conserved water molecule to pLys3, reflecting almost
identical peptide-binding modes, as observed in both
structures. By contrast, in the structures of
B*2709:pVIPR-U5 and B*2705:pVIPR (Fig. 7B,C),
His114 is involved in two indirect hydrogen-bond
interactions with two different side-chain functions of

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

prominent candidates for being affected by pH-induced
changes. The drastic difference ( 10 °C) found in the
thermal stability between complexes of B*2709 with
these two peptides at pH values of 7.5 and 5.6 (Fig. 5
and Table 1) demonstrates that the pU5–His116 con-
tact [29] (a) is most likely present also at physiological
temperature in the noncrystalline state, and (b) stabi-
lizes the B*2709:pVIPR-U5 complex. As expected, the
B*2705 subtype is not affected by the  100-fold
elevated H
+
concentration because of the replacement
of His116 by Asp116 (Fig. 5 and Table 1). Experi-
ments with further peptides and intermediate pH val-
ues will have to be carried out to assess the general
relevance of these findings. Nevertheless, it is already
evident now that moderate changes in pH values
within tissues have the potential to destabilize selected
MHC:peptide complexes by influencing the proton-
ation state of histidine residues, even when they are
deeply embedded within the protein’s interior.
A decreased pH value (5 or lower) is also typical for
endosomal compartments in which the loading of
MHC class II antigens and CD1 molecules (b
2
m-com-
plexed, lipid-binding MHC class I-like proteins) is
accomplished [39,40]. A histidine residue within the
a1-domain of class II antigens (His33) has been sug-
gested to be primarily responsible for facilitating the

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

13
C-labelled b
2
m, E. coli cells were cultured in minimal
medium containing a mixture of trace minerals, ammonium
chloride and uniformly
13
C-labelled glucose (Cambridge
Isotope Labs, Andover, MA, USA), as described previously
[27]. For complex formation, the proteins were solubilized
with urea, and refolded by dilution in the presence of the
peptide [17,43]. Following reconstitution, the entire mixture
was concentrated using Amicon Ultra-15 devices of 10-kDa
molecular weight cutoff (Millipore Corp., Billerica, MA,
USA), and the complexes were isolated by size-exclusion
chromatography in 10 mm sodium phosphate buffer,
pH 7.5, 150 mm NaCl. For the IR measurements, the sam-
ple solutions were exchanged at room temperature with the
corresponding D
2
O buffer (buffer I: 10 mm sodium phos-
phate buffer, pH 7.5, 150 mm NaCl; buffer II: 100 mm
MES pH 5.6) using Vivaspin 500 concentrators (Sartorius
AG, Go
¨
ttingen, Germany) with a membrane of 10- and
5-kDa molecular mass cut-off for the complexes and for
free b
2
m, respectively. This procedure took  40 min for

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

the dry air purging of the spectrometer, the sample in the
cell jacket was mounted on a motor-driven sample shuttle.
This allowed recording of the background immediately
before recording of the sample spectrum without opening
the sample chamber of the spectrometer. Buffer spectra
Influence of inflammatory environment on HLA-B27 H. Fabian et al.
1724 FEBS Journal 278 (2011) 1713–1727 ª 2011 The Authors Journal compilation ª 2011 FEBS
were recorded under identical conditions and subtracted
from the spectra of the proteins in the relevant buffer and
at the relevant temperature. Spectral contributions from
residual water vapour, if present, were eliminated using a
set of water vapour spectra. The final unsmoothed protein
spectra were used for further analysis. Band positions and
band intensities were determined by standard functions of
the Bruker OPUS software, implemented into home-built
macros for data analysis. Second derivatives were obtained
using the Savitzky–Golay algorithm with 13-point smooth-
ing. Intensity ⁄ temperature and frequency ⁄ temperature plots
of the IR data were created using origin software and
analysed by non-linear fitting procedures [27] to estimate
the thermal midpoint of a transition.
Acknowledgements
We are grateful to Dr Michael Beyermann, Leibniz-
Institut fu
¨
r Molekulare Pharmakologie, Berlin,
Germany, for synthesis and purification of the peptide
pVIPR, and to Christina Schnick for excellent techni-
cal assistance. We thank Dr Markus Wahl for continu-
ous encouragement and support. This work was

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.
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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

O buffer (partially H ⁄ D exchanged
state), and in D
2
O buffer after cooling from 90 to
25 °C (fully deuterated state).
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
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 1727