proteins
STRUCTURE O FUNCTION O BIOINFORMATICS
Pressure-induced changes in the solution
structure of the GB1 domain of protein G
David J. Wilton,1 Richard B. Tunnicliffe,1 Yuji O. Kamatari,2 Kazuyuki Akasaka,2,3
and Mike P. Williamson1*
1 Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank,
Sheffield S10 2TN, United Kingdom
2 SPring-8 Center, RIKEN Harima Institute, 1-1-1 Kouto, Hyogo 679-5148, Japan
3 Department of Biotechnological Science, School of Biology-Oriented Science and Technology, Kinki University,
930 Nishimitani, Kinokawa 649-6493, Japan
ABSTRACT
The solution structure of the GB1
domain of protein G at a pressure
of 2 kbar is presented. The structure
was calculated as a change from
an energy-minimised low-pressure
structure using 1H chemical shifts.
Two separate changes can be characterised: a compression/distortion,
which is linear with pressure; and a
stabilisation of an alternative folded
state. On application of pressure,
linear chemical shift changes reveal
that the backbone structure changes
by about 0.2 Å root mean square,
and is compressed by about 1%
overall. The a-helix compresses, particularly at the C-terminal end, and
moves toward the b-sheet, while the
b-sheet is twisted, with the corners
closest to the a-helix curling up
towards it. The largest changes in
structure are along the second bstrand, which becomes more twisted.
This strand is where the protein
binds to IgG. Curved chemical shift
changes with pressure indicate that
high pressure also populates an alternative structure with a distortion
towards the C-terminal end of the
helix, which is likely to be caused by
insertion of a water molecule.
INTRODUCTION
There has recently been an increased interest in the effects of pressure on proteins, with a number of international conferences on the topic, as well as reviews,
and an issue of a journal dedicated to the subject.1–4 This interest arises partly
from an increased use of pressure in food treatment; partly because pressure can
dissociate amyloids and other aggregated proteins (often reversibly)5; and partly
because pressure provides a novel insight into protein thermodynamics and function. Pressure affects the stability of different states of proteins via their partial
molar volumes. The partial molar volumes of proteins vary substantially with
functional state, such that the native form has the largest partial molar volume
(because of the existence of internal cavities), while high-energy intermediate
states and unfolded states have smaller volumes.6–8 Therefore increased pressure
can increase the populations of these activated and unfolded forms, without
making other large perturbations to the system as would be caused by temperature or denaturants.9 In addition to causing shifts in equilibria between conformational states, pressure also produces compression within the subensemble of
native states. As a result of the thermodynamic identity10
hðdV Þ2 i ¼ jTV bT
(where h(dV)2i is the average squared volume fluctuation, j is the Boltzmann
constant, T the absolute temperature, V the volume, and bT the isothermal compressibility coefficient), compression of a protein under pressure provides information on volume fluctuations, and thereby provides an important link between
structure and function.
There is very little high-resolution information on the response of protein
structure to pressure. There are high-pressure crystal structures for lysozyme,11
myoglobin,12 urate oxidase13 and cowpea mosaic virus,14 and an NMR structure of ubiquitin.15 Recently we introduced a technique for calculating the
Proteins 2008; 71:1432–1440.
C 2007 Wiley-Liss, Inc.
V
Key words: pressure; chemical shift;
protein G; compression; structural
change.
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PROTEINS
The Supplementary Material referred to in this article can be found online at http://www.interscience.wiley.com/jpages/0887-3585/suppmat/
Yuji O. Kamatari’s current address is Riken Genome Science Center, Yokohama 230-0045, Japan.
*Correspondence to: Mike P. Williamson, Department of Molecular Biology and Biotechnology, University of Sheffield,
Firth Court, Western Bank, Sheffield, UK S10 2TN. E-mail: [email protected]
Received 20 June 2007; Revised 13 August 2007; Accepted 20 September 2007
Published online 12 December 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21832
C 2007 WILEY-LISS, INC.
V
High-Pressure Structure of Protein G
change in structure of a protein on going from low to
high pressure based on changes in 1H chemical shifts,
which has been used for calculating the high-pressure
structures of melittin,16 lysozyme17 and bovine pancreatic trypsin inhibitor (BPTI).18 Here we present a calculated high-pressure structure for the GB1 domain of
protein G at a pressure of 2 kbar. The data allow us to
characterise markedly nonuniform compression within
the folded state of protein G (and thus, from the equation above, nonuniform volume fluctuations), and in
addition to make a preliminary characterisation of an alternative state that is stabilised by high pressure. The volume fluctuations are greatest for b-strand 2, which is
also the location of binding to IgG, while the alternative
state is likely to be the first intermediate in pressure
unfolding.
MATERIALS AND METHODS
The GB1 domain of streptococcal protein G was overexpressed in Escherichia coli BL21 (DE3) pLysS as a Histagged protein (with N-terminal sequence MH6AMD
preceding the normal N-terminal sequence TY. . .) in the
pET15b vector using U-13C glucose and 15NH4Cl.
Growth and purification were as described in Tunnicliffe
et al.19 Backbone assignments were made by comparison
with literature values20,21 and confirmed by 15N-separated TOCSY (total correlation spectroscopy) and
NOESY (nuclear Overhauser enhancement spectroscopy).
Sidechain assignments were carried out using 3D HNCA,
HNCACB, CBCA(CO)NH, CCH-TOCSY, and HCCHTOCSY experiments at 500 and 600 MHz on Bruker
DRX-500 and 600 spectrometers, respectively. A complete
set of assignments is deposited in BioMagResBank (accession number 7280).
Chemical shift values at variable pressure were measured using 15N and 13C HSQC spectra at 30 bar (rather
than at 1 bar, to avoid the risk of getting small bubbles
in the solution), and at 0.5, 1, 1.5, and 2.0 kbar. All
measurements were carried out on a Bruker Biospin
DRX-800 operating at 800 MHz for proton, using a
quartz cell connected to a hand pump, as described.22
Data were processed using FELIX (Accelrys, San Diego,
CA), and peaks were picked into a database, which was
converted into text-readable format and analysed using
Excel (Microsoft, Seattle, WA). Chemical shifts were
measured for a total of 298 assigned protons, including
almost all HN and Ha. However, 9 of these are sidechain
amide protons, which are not used in the calculation,
and 144 are comprised of 65 methylene pairs and 7 valine/leucine methyl pairs: these pairs generate only a single summed restraint each.23 Furthermore, a small number of protons could not be followed either due to overlap at some pressures, or to erratic pressure dependence,
with the result that 206 proton restraints were used. A
full list of restraints (i.e., linear chemical shift changes
with pressure) can be found with the PDB entry.
The method used to calculate a high-pressure protein
structure has been described previously, and is based on
the premise (justified in Refs. 17 and 18) that although
1
H chemical shifts themselves are inadequate for structure refinement, the change in chemical shift in response
to a gradual change in structure can be used as a structural restraint.17,18 1H chemical shifts in proteins can be
calculated to a reasonable accuracy (up to about 0.2 ppm
for carbon-attached protons) using simple geometrical
relationships based on ring current shifts, bond magnetic
anisotropies and electric field effects,24,25 with a slightly
different parametrisation being required for amide protons.26 These relationships permit a simple ‘‘chemical
shift energy’’ term to be calculated based on the difference between calculated and observed shift, which by
partial differentiation can be converted to a force to be
applied during a restrained molecular dynamics calculation.27 In outline, a starting structure is refined in a
fairly complete molecular dynamics force field (including
for example a full Lennard-Jones van der Waals term and
an electrostatic term), until it reaches an energy minimum that does not move on further calculation. We
note that the experimental pressures used here are not
large enough to cause any appreciable change in covalent
structures such as bond lengths and van der Waals radii,
implying there is no reason to modify the molecular dynamics force field. The chemical shifts are calculated for
this structure, and applied as restraints to produce a
‘‘low-pressure’’ structure. Then, the change in shift
between low and high pressure is added to the shifts, and
the new shifts are applied as ‘‘high-pressure’’ restraints.
Because the two sets of calculations are identical except
for the experimentally determined shift restraints, a comparison of the low and high-pressure structures permits
high-resolution details of the pressure-induced structural
change to be obtained, even though the chemical shifts
themselves may have relatively limited accuracy.
We started from a high-resolution crystal structure of
protein G, 1pga,28 for which residue 47 was changed
manually from aspartate to alanine to match the experimental sequence. The structure was equilibrated by a
molecular dynamics calculation in X-PLOR and energy
minimised, to generate a starting structure. The chemical
shift values of this starting structure were calculated
(using the PROTON routine in X-PLOR27) and used as
restraints for a low-pressure structure. The value of the
chemical shift energy term kprot was set to 4000 kcal/mol
per ppm2, this large value ensuring that structural
changes were dominated by the chemical shift restraints.
For the high-pressure structures, the chemical shift
restraints were the computed low-pressure shifts plus the
experimentally observed pressure-induced change in shift.
The low and high-pressure structures were both the average of 50 simulations.
PROTEINS
1433
D.J. Wilton et al.
Calculation of low- and high-pressure
structures
The experimental chemical shift restraints are differences between shifts at low pressure and high pressure, and
accordingly the structures calculated are best described as
changes in structure between low- and high-pressure
structures. Calculations were carried out using a molecular dynamics protocol implemented in X-PLOR, starting
from three different low-pressure starting structures, in
order to ascertain the reliability in the structural details
calculated: more details are provided in the Supplementary Material. For each starting structure, chemical shifts
were calculated in X-PLOR and applied as shift restraints
in order to generate reference low-pressure structures.
Then, the experimental changes in shift between low and
high pressure were added to the calculated (low-pressure)
shifts, to obtain chemical shift restraints for the highpressure structures. The molecular dynamics calculations
Figure 1
Diagrammatic 1H,15N HSQC spectrum of protein G with varying pressure. The
circles show results for pressures of 30 bar, 0.5, 1.0, 1.5, and 2.0 kbar from light
grey to black.
Nonlinear shift changes were fitted to a second-order
polynomial. The color coding of nonlinearity in Figure 8
is based on the magnitude of the second-order coefficient.
RESULTS
NMR chemical shift restraints
The GB1 domain of protein G was overexpressed as a
double-labelled His-tagged protein and purified. No sidechain assignments for this protein were available, so the
NMR spectrum was fully assigned (except for aromatic
signals and the N-terminal His tag) using a set of 3D
experiments. NMR spectra were obtained at a range of
pressures ranging from 30 bar to 2 kbar (1 bar 5 105 Pa
5 0.99 atm). Amide proton shifts were obtained from
15
N1H HSQC spectra (Fig. 1), and carbon-bound
shifts from 13C1H HSQC. Most protons displayed a reversible and linear change in chemical shift with pressure,
and for these protons the pressure-dependent shifts were
fitted to a straight line, from which the gradient was
used as the measure of change in chemical shift with
pressure. However, some protons displayed a curved
pressure dependence, as discussed in more detail below.
These shifts were fitted to a second-order polynomial,
and the initial gradient was used as the linear change in
shift with pressure. A total of 206 proton shifts were
used as restraints, described in more detail in Materials
and Methods.
1434
PROTEINS
Figure 2
Cartoon representation of Protein G. The orientation shown is as used for
moments of inertia calculations. The x direction is approximately parallel to the
helix axis, y is perpendicular to the plane of the sheet, and z is perpendicular to
x and y. Selected residues are numbered. b-strand 1 runs from residues 1 to 8,
strand 2 from 13 to 19, strand 3 from 42 to 47, and strand 4 from 50 to 56,
and the a-helix runs from 23 to 37.
High-Pressure Structure of Protein G
Table I
Changes in Whole Molecule Properties of Protein G Between 30 and 2000 Bar
Property
Mean Connolly volume (1.4 probe)a,b
Mean van der Waals volume (0.0 probe)a,b
Estimated cavity volumec
Mean surface areab,d
Radius of gyration
Moment of inertia xx componente
Moment of inertia yy componente
Moment of inertia zz componente
Structure
Low P
High P
Diff
% Change
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
11338 6
11386 5
11328 7
6088 7
6086 7
6078 5
3060
3094
3060
3345 6
3363 7
3363 8
10.45
10.49
10.45
36798
38014
35974
37710
37107
38805
18282
18178
18043
11290 5
11254 16
11259 12
6074 5
6044 8
6077 7
3034
3027
3000
3374 6
3354 8
3378 11
10.43
10.43
10.43
36967
38221
36281
37144
36613
38221
18207
17956
17910
248 7
2132 18
269 14
214 9
242 9
22 8
226
266
260
29
28.5
14.9
20.02
20.05
20.01
169
207
307
2566
2494
2584
275
2222
2133
20.42
21.16
20.61
20.23
20.69
20.03
20.9
22.1
22.0
0.87
20.25
0.44
20.23
20.52
20.13
0.46
0.54
0.85
21.50
21.33
21.50
20.41
21.22
20.74
Calculated using VOIDOO.31
Mean value of all 50 structures calculated standard deviation.
c
Connolly volume – vdW volume—estimated surface layer volume. The Connolly volume is the volume enclosed by the surface atoms of the protein plus a 1.4-Å thick
layer (due to the 1.4 Å radius probe used). The surface layer volume is estimated from the radius of gyration of the molecule, r. That is, surface layer volume 5
(4/3)p[(r 1 1.4)3 – r3]. Calculations based on grid searches give broadly similar changes.
d
Calculated using CCP4 routine AREAMOL, part of the CCP4 suite.32
e
Calculated using X-PLOR from the average structure of the 50 structures.
a
b
were then repeated, to produce high-pressure structures.
Each calculation was replicated 50 times to generate an
ensemble of 50 structures, which were subsequently averaged and energy minimised to produce a single resultant
calculated structure. The final outcome of the calculations was therefore three pairs of low- and high-pressure
structures, one of which (that most populated in the molecular dynamics calculations) has been deposited with
the Research Collaboratory of Structural Bioinformatics
Protein Data Bank with codes 2j52 (low) and 2j53
(high).29
If the chemical shift restraints are operating correctly,
the ensemble of structures should possess calculated
chemical shift values that match the restraints closely.
The three sets of 50 low-pressure structures have mean
chemical shift errors of 0.001 0.020 ppm, -0.001 0.020 ppm, and 0.000 0.013 ppm, and the corresponding three sets of 50 high-pressure structures have mean
chemical shifts errors of 0.000 0.025 ppm, 20.001 0.024 ppm, and 0.001 0.018 ppm, respectively (compared with an absolute value shift change from low to
high pressure of 0.054 0.048 ppm), indicating that the
chemical shift restraints were effective in driving the
structure calculation. The changes in chemical shift
between 30 bar and 2 kbar are very significantly greater
than the residual shift errors (P < 0.001, t-test).
Comparison of low- and high-pressure
structures
Protein G is composed of a 4-stranded b-sheet, made
up of two b-hairpins separated by an a-helix that lies
across the face of the sheet (Fig. 2). It is a small and
compact structure, containing no cavities large enough to
contain water molecules, a rather unusual occurrence in
protein structures.30 The high-pressure structures differ
from the low-pressure structures by backbone root mean
square distances of 0.162, 0.168, and 0.227 Å, respectively, for the three different starting structures of protein
G used (aligning and comparing all backbone atoms).
The structural change results in a compression of the
order of 1% at 2 kbar (Table I), corresponding to a compressibility coefficient of 5/Mbar. This should be compared with values of 7.5 and 4.7/Mbar measured in lysozyme in solution and in the crystal, respectively,11,17
and 10/Mbar in urate oxidase.13 It is thus in the normal
range though somewhat small, possibly consistent with
its lack of major internal cavities.
PROTEINS
1435
D.J. Wilton et al.
Figure 3
Two views of the aligned low (blue) and high (red) pressure structures of protein
G (structure 2 of the 3 families used). The difference between the high-pressure
structure and the low-pressure structure has been magnified by a factor of 5, for
clarity, by calculating the vectors representing the structural change for each
atom from low to high pressure and multiplying by 5. Similar changes were seen
for the other two structures, justifying the calculational methodology (see text).
Table I shows the changes in volume, surface area and
the moments of inertia on application of pressure. There
is an overall reduction in volume of around 0.7%, a
highly significant volume change (t-test, P < 0.001). The
change in van der Waals volume is much smaller
(although still significant, and highly significantly different from the change in Connolly volume), at about
20.3%, as would be expected from the small compressibility of atomic radii. Correspondingly, the change in
cavity volume, although difficult to calculate precisely, is
larger than the overall volume change, demonstrating
that, as one might expect, most of the compression
results from a reduction in the size of cavities. Surface
area changes by very little, and the sign of the change is
not consistent for the three different structures: it is very
dependent on the exact sidechain orientation and its
exact value is not a very useful measure. The changes in
moments of inertia show that the compression is nonuniform. Figure 2 shows the orientation of the molecule
1436
PROTEINS
with respect to the moment of inertia axes. For each of
the three structures the yy component decreases the
most, corresponding to the a helix and b sheet moving
closer together. The xx component increases but by less
than the yy decrease. The increase in the xx component
of the moment of inertia is mainly caused by a reorientation of the loops at the ends of the strands. The zz component also decreases, but by much less than the yy for
structures 1 and 3. The change in the zz component is
explained by parts of the b sheet, around the end of
strand 2, moving closer to the a helix. The overall
change in structure is indicated in Figure 3, where it can
be clearly seen that the two secondary structural elements
pack more closely together at high pressure (particularly
towards the C-terminal end of the helix). Thus for example, the mean distance between helix and sheet has compressed by 0.18 0.06 Å in the family 2 structure shown
in Figure 3, and by 0.07 0.03 and 0.05 0.03 Å in
families 1 and 3, respectively. All these changes are significant compressions (P < 0.001, t-test). Also evident from
Figure 3 is that the sheet buckles and wraps itself more
closely around the helix, with the N-terminal ends of
strands 1, 2, and 4, and the whole of strand 3, moving
toward the helix. In general, the parts of the sheet that
are close to the helix move even closer, while those parts
that are distant (such as the C-terminal end of strand 4)
move away. Strand 2 becomes much more twisted, with
both ends moving toward the helix and the center moving away from the helix: for example, the virtual angle
between the Ca atoms of residues 13, 15, and 19
Figure 4
Difference distance matrix for Ca atoms from starting structure 2, that is,
High P
Low P
– Di,j
. Blue regions indicate where the high-pressure structure
Di,j
distances are less than the corresponding distances at low pressure, that is, the
structure is compressed. Conversely orange /red regions indicate expansion. The
contour levels are set at 0.4, 0.3, 0.2, 0.15, 20.1, 20.15, 20.2, and 20.25.
The secondary structure elements are indicated by open (a helix) and solid
(b strand) rectangles.
High-Pressure Structure of Protein G
Figure 5
Changes in hydrogen bond distances, high pressure to low pressure. For the a
helix, the distances shown are the shortest distances, that is, i to i 1 4. For the
b sheet, they are divided into interstrand distances between residues in strands 1
and 2, strands 1 and 4, and strands 3 and 4. Changes shown are for the
average high and low pressure structure calculated from refined structure 2.
decreases from 146.8 0.38 to 145.0 0.48 in family 2,
and 146.1 0.28 to 144.7 0.28 and 143.1 0.28 to
139.4 0.48 in families 1 and 3, respectively (P < 0.001
in all cases). This increased twist is particularly interesting because it is this strand that forms the interface to
IgG by forming an antiparallel pairing of b-strands.33
The least biased way to examine structural change
between two structures is to calculate the difference distance matrix: sets of interatomic distances are calculated
for both structures and the difference is plotted. Figure 4
shows the difference distance matrix for the Ca atoms. It
can be seen that most of the CaCa distances reduce at
high pressure but there are areas where the distances
increase at high pressure, notably most distances from
the residues in the loops at both ends of strand 2, again
highlighting this strand as the site of biggest changes. It
can also be seen that distances increase between residues
44–53 (the 3rd and 4th b strands plus the turn between
them) and residues 19–29 (the region from the end of b
strand 2 to half way along the a helix), which is related
to the twisting of the sheet. This analysis is for structure
2, but results for the other structure are similar.
Those in the b strands vary. Hydrogen bonds between
strands 1 and 2 almost all lengthen. This is because of
the strand 2 moving noticeably upwards towards the a
helix whilst strand 1 does not move as much: this movement is the same as that described above as a twisting of
strand 2. In the other two strand interfaces, there is no
consistent pattern of change. For the other two starting
structures the pattern of change is similar. We conclude
that although in general hydrogen bonds compress with
pressure (as one might expect because they are much
weaker than covalent bonds and therefore more compressible), the changes to individual hydrogen bonds are
determined by larger scale structural changes in the protein. In other studies, there has been a general trend
towards shortening of hydrogen bonds at high pressure,
but there have been large variations. For example in the
recent crystal structure of cowpea mosaic virus, the average hydrogen bond length decreased by 0.01 Å/kbar,
while in urate oxidase, hydrogen bonds in subunit interfaces on average increased by 0.02 Å/kbar.13 In our previous studies, we observed average decreases of 0.006 and
0.014 Å/kbar for lysozyme and BPTI, respectively. The
changes seen here therefore fit well within the range of
observed changes.
Changes in amide proton shifts have generally been
explained as arising from changes in hydrogen bond
lengths. However, as shown in Figure 6, there is a quite
Hydrogen bond length changes
Averaging over all the hydrogen bonds in the protein,
hydrogen bonds shorten by 0.022 0.108 Å over 2 kbar
(i.e., 0.01 Å/kbar). Changes in hydrogen bond lengths
within regular secondary structure are shown in Figure 5
(for the structure calculated from starting structure 2).
The hydrogen bonds along the a helix generally shorten
at high pressure, particularly at the C-terminal end.
Figure 6
Relationship between the change in amide proton shift and the change in the
shortest hydrogen bond involving that proton. This is the data for structure
family 1; those for structures 2 and 3 show even less correlation.
PROTEINS
1437
D.J. Wilton et al.
DISCUSSION
Figure 7
1
HN shifts from 15N,1H HSQC spectra from residues that have a noticeable
nonlinear pressure dependence.
This is the third globular protein for which structural
changes with pressure have been reported based on NMR
chemical shift data. In addition, there is one NOEderived NMR structure and four crystal structures at
high pressure. Because of the physical basis for the calculations, the chemical shift method yields the most precise
measurements of the small structural changes that occur
on application of pressure, but all the structures from
both NMR and crystallography are showing common
themes. Pressure produces a compression of the protein
itself, in the range 5–10/Mbar, with changes in atomic
positions close to 0.1 Å/kbar root mean square. This is
an important result, because the most extensive and
accurate measurements of protein compression have been
derived using ultrasound, which however produces values
for the compression of the protein in its solvent shell.
The value derived is thus the sum of the compression of
weak correlation between the change in shift and the
change in the length of the shortest hydrogen bond
involving that proton. As noted previously,17 amide proton chemical shift changes depend both on the distance
and on the angle of the hydrogen bond.
Curved chemical shifts characterise an
alternative structure
As noted above, for most protons in protein G, the
change in chemical shift with pressure was approximately
linear, indicating a linear compression within the native
ensemble. However, there are a significant number with
nonlinear shift changes. Figure 7 shows those amide proton shifts for which there is the most noticeable nonlinear dependence. The locations of these protons are also
shown in the color-coded cartoon structure in Figure 8.
It can be seen that there is a grouping of amide protons
with nonlinear dependence at the C-terminal end of the
a helix, the following loop, the third b strand and the
C-terminus. This is not related to aromatic ring-current
shifts, which bear no resemblance to the locations of curvature. The presence of nonlinear pressure-dependent
shifts therefore implies that an alternative conformation
is significantly populated at high pressure.34 The data
imply that there is such an alternative conformation for
protein G that differs significantly in this region. Pressure
acts to reduce the partial molar volume of proteins, and
in general this is achieved by the insertion of water molecules into cavities.34–36 It is therefore likely that the
same happens here, and that a water molecule is inserted
into the protein somewhere close to Asn35 towards the
C-terminal end of the helix. There is no cavity here in
the crystal structure large enough to accommodate a
water molecule, but calculations on all three families
indicate small cavities in this region.
1438
PROTEINS
Figure 8
Low-pressure structure of protein G, color coded by nonlinear amide proton
shifts. Residues in blue have virtually linear pressure dependence; the
nonlinearity of the amide proton shift is indicated by colors ranging from green
(small nonlinear term) through yellow and orange to red (highest nonlinear
term).
High-Pressure Structure of Protein G
the protein plus the compression of the hydration layer,
which are difficult to separate. The compressibility of the
hydration layer is expected to be negative,37 but its magnitude is difficult to calculate. The results summarised
here on change in the protein itself (5–10/Mbar) are
comparable in size with the partial specific adiabatic
compressibilities measured by ultrasound (0–14/
Mbar),37,38 which suggests that the compressibility of
the hydration layer has been overestimated previously by
as much as 5–10/Mbar: in other words, the effect of the
hydration layer appears to be less than had been previously thought.
Other details found here also match those seen in previous studies. Compression is far from uniform, with
local regions of expansion. Hydrogen bonds show a small
overall compression, but with wide variations. Helices
tend to show a simple uniform compression, whereas
sheets have a tendency to deform under pressure, but
with little reduction in volume. Of particular interest is
the finding that the region of protein G that shows the
greatest changes in structure at high pressure (i.e., the
most compressible region and therefore the region with
the highest volume fluctuations) also forms the active
site in binding to IgG, implying that the active site has
the greatest volume fluctuations. Thus not only does the
structure of protein G match that of its target, but also
its dynamics are such that it is able to adapt itself to
bind: in other words, evolution seems to have produced
a protein well adapted to binding its target not only
structurally but also dynamically. In agreement with this
conclusion, a recent study of dynamics in protein G
found large-amplitude slow correlated motions in the
same region, which the authors speculated may be functionally important.39 Our result supports that speculation, by showing that the compressibility, and therefore
the volume fluctuations, of protein G are greatest at the
b-strand that forms the recognition interface with IgG;
and that this fluctuation requires concerted changes to
hydrogen bond geometry. This observation therefore provides further support to the suggestion that high pressure
allows characterisation of functionally important mobility
not easily observed by other techniques.40 The additional
energy input into protein G due to a 2 kbar pressure is
pDV 5 4 kJ mol21. This is far too small an energy to
deform covalent bonds or to perturb the natural dynamics of the protein. Pressure therefore provides a very
powerful tool for altering the energy landscape of a protein, without increasing the internal energy.41
This study has also provided a preliminary characterisation of an alternative state populated reversibly at high
pressure, which is not the gradual change characterised
by the linear 1H shift changes, but rather an all-or-nothing step change. The structure is different in the region
of the C-terminal end of the helix, very likely by the
insertion of a water molecule into a cavity, though of
course other structural changes could be responsible.
There is no single large cavity in this region that is
clearly ‘‘waiting’’ for a water molecule, as there was in
the case of lysozyme that was studied previously by us,17
but analysis of the structure and the chemical shift
changes suggests that the experimental observations
would be consistent with insertion of a single water molecule close to the amide proton of Asn35, which would
lead to disruption of the C-terminal end of the helix and
local structural rearrangement. The structural change is
therefore not directly related to the function of the protein, which involves b-strand 2, but is presumably an
early stage in pressure unfolding. Interestingly, Kitahara
et al.15 have characterised a very similar alternative state
in the structurally related protein ubiquitin, involving a
reorientation of the helix and the C-terminal region. By
contrast to protein G, this structural change in ubiquitin
could have functional significance. It therefore appears
that (as in numerous other examples) the unfolding
pathway is more highly conserved than the function.
ACKNOWLEDGMENT
We thank Prof David Baker (University of Washington,
Seattle) for providing the plasmid for protein G.
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