A model-free analysis of the protein backbone relaxation parameters shows an average increase of generalized order parameters of 0.015 reflecting a small overall reduction in mobility of
Trang 1Protein stabilization by compatible solutes
studied by NMR
Pedro Lamosa1, David L Turner1,2, Rita Ventura1, Christopher Maycock1and Helena Santos1
1 Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Oeiras, Portugal; 2 Department ofChemistry,
University ofSouthampton, UK
Heteronuclear NMR relaxation measurements and
hydro-gen exchange data have been used to characterize protein
dynamics in the presence or absence of stabilizing solutes
from hyperthermophiles Rubredoxin from Desulfovibrio
gigas was selected as a model protein and the effect of
diglycerol phosphate on its dynamic behaviour was studied
The presence of 100 mM diglycerol phosphate induces a
fourfold increase in the half-life for thermal denaturation
of D gigas rubredoxin [Lamosa, P., Burke, A., Peist, R.,
Huber, R., Liu, M.Y., Silva, G., Rodrigues-Pousada, C.,
LeGall, J., Maycock, C & Santos, H (2000) Appl Environ
Microbiol 66, 1974–1979] A model-free analysis of the
protein backbone relaxation parameters shows an average
increase of generalized order parameters of 0.015 reflecting
a small overall reduction in mobility of fast-scale motions Hydrogen exchange data acquired over a temperature span
of 20C yielded thermodynamic parameters for the struc-tural opening reactions that allow for the exchange This shows that the closed form of the protein is stabilized by an additional 1.6 kJÆmol)1in the presence of the solute The results seem to indicate that the stabilizing effect is due mainly to a reduction in mobility of the slower, larger-scale motions within the protein structure with an associated increase in the enthalpy of interactions
Keywords: chemical exchange; compatible solutes; protein dynamics; rubredoxin; thermostability
Protein stability, activity and dynamics are interrelated
issues with great importance not only in physiological
processes but also in protein engineering The evolution of
protein structures towards extreme thermostability was vital
for hyperthermophiles, microorganisms thriving near the
boiling point of water In general, the proteins of these
organisms are intrinsically resistant to heat denaturation
However, hyperthermophiles also possess intracellular
pro-teins that are not particularly stable, implying the existence
of alternative strategies for their stabilization in vivo [1,2]
Hyperthermophiles accumulate high levels of charged
organic osmolytes in response to supra-optimal growth
temperatures, and this observation led to the hypothesis that
these compounds play a role in thermoprotection of
macromolecules in vivo [3,4] This view is supported by
in vitrostudies showing that these osmolytes protect proteins
against heat [1,5–8] Nevertheless, the molecular basis for
this well established stabilization phenomenon remains
elusive
Several possible mechanisms for protein stabilization
by osmolytes have been proposed [9–11] Arakawa and
Timasheff [12,13] proposed a preferential hydration model
to explain protein stabilization by compatible solutes: solute molecules are excluded from the protein surface, thereby making denaturation entropically less favourable In con-formity, exclusion factors have been measured for a variety
of organic solutes and salts [14–16], however, the correlation between exclusion factors and the degree of protection a solute can bestow upon a particular protein is neither unequivocal nor general [17,18] These apparent inconsis-tencies have sometimes been interpreted as being due to specific protein–solute interactions [7,19] In fact, the magnitude of the stabilizing effect depends on the particular solute–protein pair examined [5,8,19]
Another approach, proposed by Bolen and coworkers, describes the stabilizing or destabilizing nature of inter-actions between solutes and exposed groups in the protein structure [20,21] In this proposal, the stabilizing effect is attributed mainly to a large contribution from interactions with exposed backbone groups in a partially unfolded state, with side-chain interactions modulating the specificity of the effect Overall, the interactions should cause a contraction
of the protein structure with a concomitant decrease in internal mobility [21,22] Indeed, the higher thermal stability
of hyperthermophilic proteins has often been correlated with structure rigidification [23,24] Structural data, both from X-ray and NMR, on series of homologous proteins show evidence for stronger local interactions and/or improved packing of the polypeptide chain, which would bring about a higher conformational rigidity [23] More-over, the lower catalytic efficiency observed in hyperther-mophilic enzymes is usually explained by the decreased
Correspondence to H Santos, Instituto de Tecnologia Quı´mica e
Biolo´gica, Apartado 127, 2780-156 Oeiras, Portugal.
Fax: + 351 21 4428766, Tel.: + 351 21 4469828,
E-mail: santos@itqb.unl.pt
Abbreviations: DGP, diglycerol phosphate; RdDg, Rubredoxin from
Desulfovibrio gigas.
(Received 4 July 2003, revised 22 September 2003,
accepted 2 October 2003)
Trang 2flexibility of the active site, corroborated by the fact that
mutations increasing thermostability while maintaining
low-temperature activity are extremely rare [25] In fact,
this rigidification has been revealed by H–D exchange
experiments [23,26] However, this view was recently
challenged by the observation of relatively fast exchange
rates in the rubredoxin from Pyrococcus furiosus, the most
stable protein known to date [27] In this context, assessing
the changes in the dynamic behaviour of proteins in the
presence of solutes is expected to shed light on the
stabilization phenomenon
Desulfovibrio gigas rubredoxin (RdDg), a small iron–
sulfur protein with a hydrophobic core formed by the side
chains of six invariant residues, a three-stranded b-sheet,
and an exposed hairpin loop, was chosen as a model
protein Its NMR solution structure was recently obtained
[28] Also, RdDg is highly stabilized by diglycerol phosphate
(DGP), a solute accumulated by the hyperthermophilic
archaeon Archaeoglobus fulgidus [7,29] Addition of 100 mM
DGP yields a fourfold increase in the half-life for thermal
denaturation of RdDg, measured by UV–visible
spectros-copy at 90C [7]
We used NMR for these studies because it provides a
wide range of time-scales for the dynamic analyses
Heteronuclear NMR relaxation data, from which
general-ized order parameters can be derived, provides a tool to
probe the dynamic behaviour of proteins in various
conditions [30–32] Hydrogen exchange rates of labile
protons, such as the amide protons of protein backbones,
can also provide dynamic information on longer
time-scales Amide protons that are buried inside the protein
structure and/or involved in hydrogen bonds require a local
structural opening to allow exchange with solvent protons
[33,34] Therefore, the measurement of amide exchange
rates can be used to evaluate the relationship between
stability and the rigidity of several parts of the protein
structure [35,36]
Materials and methods
Rubredoxin production
Plasmid pRPPL1 [7] harbouring the RdDg gene was
digested with NdeI and EcoRI restriction enzymes The
175-bp DNA fragment obtained was purified from an
agarose gel (2%) and inserted into vector pT7-7 [37]
previously digested with the same restriction enzymes The
resulting construct was named pMSPL1 Escherichia coli
strain BL21(DE) was transformed with pMSPL1 and
grown in medium containing: KH2PO4, 4.5 gÆL)1;
K2HPO4, 10.5 gÆL)1; NaCl, 0.5 gÆL)1; Mg2SO4Æ7H2O, 0.5
gÆL)1; FeCl3Æ3H2O 6 mgÆL)1; U15N-(NH4)2SO4, 2 gÆL)1;
glucose, 4 gÆL)1; vitamin solution, 10 mLÆL)1; trace element
solution 10 mLÆL)1and ampicillin 100 mgÆL)1 One litre of
vitamin solution contained 500 mg aminobenzoic acid,
200 mg nicotinic acid, 100 mg pantothenic acid, 500 mg
pyridoxine, 100 mg thiamine, 200 mg thioctic acid, 200 mg
biotin, 100 mg folic acid and 100 mg riboflavin The
trace element solution contains per litre: CaCl2, 1.06 g;
MnSO4Æ5H2O, 50 mg; CuSO4Æ5H2O, 8 mg; ZnSO4Æ7H2O
40 mg; NaMoO4Æ2H2O, 8 mg; CoCl2Æ6H2O, 8 mg; H3BO3,
6 mg
Transformed E coli cells were grown until D¼ 0.3 and RdDg production induced with isopropyl thio-b-D -galacto-side (IPTG; 25 lgÆL)1final concentration) At this time the culture was supplemented with glycerol (4 mLÆL)1) and ZnCl2(5 mgÆL)1final concentration) and incubated for 8 h Purification of the recombinant protein was performed as described previously [7]
A yield of approximately 10 mgÆL)1of the zinc form of RdDg uniformly labelled with15N was obtained
Sample preparation Purified uniformly 15N-labelled RdDg (Zn form) was concentrated and the buffer removed by ultrafiltration using a YM3 membrane (Amicon) Two samples were prepared in 10%2H2O at a final concentration of 4 mM
In one sample, DGP (potassium salt) was added to a final concentration of 100 mM, while in the other sample KCl was added to the same concentration The pH was adjusted
to 6.9 in both samples and an antibiotic cocktail was added with 70 lMampicillin, 50 lMkanamicin, 50 lMrifampicin and 50 lMchloroamphenicol
For the1H–2H exchange experiments RdDg (Zn form) was used at a final concentration of 1 mM KCl or DGP was added to the protein in 2-mL Eppendorf tubes to a final concentration of 100 mM, the pH was adjusted to 6 in the unlabelled samples and to 5 in the15N-labelled RdDg, and the samples were freeze-dried The dried samples were then dissolved in 2H2O, the pH readjusted (if necessary), and placed in the spectrometer at the desired temperature After allowing a period for temperature equilibration, series of 1D
1H (or 2D1H-15N HSQC for the labelled samples) spectra were acquired
NMR spectroscopy Unless otherwise stated all spectra were recorded at 303 K
in a DRX500 Bruker spectrometer equipped with a 5-mm inverse detection probe head with internal B0gradient coils (Bruker, Rheinstetten, Germany) Temperature was con-trolled using a Eurotherm 818 unit with a B-CU 05 cooling unit One-dimensional1H spectra for the exchange experi-ments were acquired with 72 transients, and continuous low-power water saturation during the relaxation delay of 2.0 s A series of1H–15N correlation spectra was acquired
to measure the 15N relaxation constants R1and R2, and heteronuclear1H–15N NOE using the procedures outlined
in Kay et al [38], modified to include a Watergate 3-9-19 water suppression scheme [39] Values of R1and R2were obtained by fitting the intensities (measured as peak-volumes) over time to a single exponential decay NOE enhancements were taken from the mean value of three integrations of peak volumes in spectra recorded with and without proton saturation The 2D15N–1H HSQC spectra were recorded with standard Bruker pulse programs In these experiments 4096 1H· 512 15N data points were collected using a delay of 2.7 ms for evolution of magneti-zation in the INEPT transfer sequence The 3D 15N–1H HSQC-TOCSY spectrum (40961H· 3215N· 641H data points) was recorded using a delay of 2.7 ms evolution of magnetization in the INEPT transfer sequence and a TOCSY mixing time of 80 ms The data were processed
Trang 3with standard BRUKER software (Bruker) Polynomial
baseline corrections were applied in both dimensions of all
2D spectra
Results
1H and15N chemical shifts
The 2D 1H-15N-HSQC spectrum of RdDg was assigned
with the aid of a 3D1H–15N TOCSY-HSQC spectrum and
published proton chemical shift data [28] (Fig 1)
Ambigu-ities in signal assignment due to overlap in the1H dimension
were solved through spin-system analysis of the 3D
TOCSY-HSQC spectrum, but three signals with AMX
type spin-systems could not be assigned unequivocally
The temperature dependence of the1H and15N chemical
shifts was investigated in the presence of 100 mM KCl or
DGP by acquiring a series of1H–15N HSQC spectra over a
temperature span of 50C (from 30 to 80 C) At 30 C, the
addition of DGP had little or no influence on the proton
NH chemical shifts of RdDg In fact, chemical shift
displacement upon solute addition seems random, with
most shift changes within experimental error, an average
value of 0.004 p.p.m., and a maximum value of 0.087 p.p.m
(Phe30) The displacement of15N chemical shifts follows a
similar pattern, with an average value of 0.031 p.p.m and a
maximum value of 0.607 p.p.m (Ala48) These results agree
with previous findings [28], in which DGP addition caused
no visible change in the proton spectrum
The chemical shifts of amide protons in RdDg present a
small, linear dependence on temperature (up to 80C), both
in the presence of 100 mMKCl and DGP The variation of
chemical shift with temperature seems random with average
slope of )0.0029 ± 0.0028 p.p.m.ÆK)1 throughout the
protein, with the error given as the standard deviation of
the slopes The segment 25–32 in the protein sequence shows
the largest temperature dependences with an average of )0.0066 ± 0.0045 p.p.m.ÆK)1 The addition of DGP does not significantly change this pattern In fact, the difference
in chemical shift temperature dependence with or without DGP is random and within the experimental error Amide
15N chemical shifts display both positive and negative correlations with temperature, which seem unrelated to protein sequence or residue type and present a relatively small range of values (from 0.018 p.p.m.ÆK)1in Val5 to )0.047 p.p.m.ÆK)1in Phe49) In some residues, such as Ile3, Tyr11, Gly23, Lys25, Phe30 or Ser45, the chemical shifts are temperature independent Many of the plots of 15N chemical shift against temperature are nonlinear This also seems unrelated to protein structure or residue nature Upon solute addition, all signals still exhibit little tempera-ture dependence and tend to maintain their positive or negative correlations
Relaxation data and dynamic parameters Relaxation parameters were measured at 30C for 42 of the
4715N amide nuclei present in the protein (Fig 2), and analysed using the program Model-free v.4.01 [31,40] The diffusion tensor (D) and the rotational correlation time (sm) were evaluated prior to analysis The software package
R1R2_DIFFUSION[31,40] was used to translate the centre of mass of the mean structure of the NMR ensemble [28] to the origin of coordinates, and to estimate D from T1/T2ratios Residues that might be undergoing conformational exchange were identified from the condition: (ÆT2æ) T2,n)/ÆT2æ) (ÆT1æ ) T1,n)/ÆT2æ > 1.5 r and excluded [41] Here, T2,nis the T2
of residue n,ÆT2æ is the average T2, and r is the standard deviation of (ÆT2æ) T2,n)/ÆT2æ) (ÆT1æ) T1,n)/ÆT2æ The axially symmetric diffusion model best fitted the experimental data, and the structure was rotated to its principal axis for use in the model-free analysis The parameters, selected by extensive Monte-Carlo simulations
as described by Mandel et al [31], are summarized in Table 1 After model selection, both the correlation time and the axially symmetric diffusion tensor were optimized simultaneously with all other model-free parameters
In the presence of KCl, there were five residues that did not fit any model in the analysis; these are Tyr11, Tyr13, Leu33, Gly43, and Ala44 Five residues also failed to fit any model in the presence of DGP: Thr7, Val8, Ala16, Leu33, and Val41 The rotational correlation time, sm, determined
in the final calculations, was 3.9 ± 0.2 and 4.6 ± 0.4 ns in the presence of 100 mMKCl and DGP, respectively These values for smare in agreement with the observed negative NOE values and the small size of the protein
Effective correlation times (se) in the range of 20–70 ps were found for 13 residues in 100 mMKCl (Fig 3) In the presence of DGP, 10 residues required the determination of
seto fit the model In both cases, most of these residues are located in the hairpin loop region Only two residues (8 and 46) required an Rexterm for adequate fitting in the presence
of KCl, with values ranging from 0.8 to 4 s)1 When DGP was present, six residues needed an Rexterm (residues 24, 31,
32, 44, 49 and 51), but the fitted value is close to zero in all six cases
The values of the generalized order parameter, S2(Fig 3)
do not display any particular trend over the protein
Fig 1.1H–15N HSQC spectrum of15N-labelled RdDg (Zn-form) in the
presence of 100 m KCl at 30 °C.
Trang 4sequence except for the small values in residue 2, which
agrees with the expected flexibility of the N-terminal
region of the protein The difference of S2 values in the
presence and absence of solute are shown in Fig 4 Overall,
the average S2values tend to be higher in the presence of
DGP, but the average difference is only 0.015, and there is
no obvious trend towards segmental rigidification of any
part of the sequence Instead, the whole protein (with the
exception of residues 14–18 and 37–45) tends to display
higher S2values in the presence of the solute (Fig 4)
1
H–2H amide exchange
To evaluate the relative mobility and exposure of the several
segments of the protein sequence,1H–2H amide exchange
rates of15N labelled RdDg were measured at 40 C and
pH 5 by recording 2D HSQC spectra in2H2O as a function
of time and fitting the peak volumes to single exponential decays (Fig 5) The exchange rates of several amides were inaccessible under these experimental conditions: 26 resi-dues exchanged so rapidly that the signals were undetectable
at the start of the spectral acquisition, and nine residues gave signals that remained almost constant throughout the experiment, indicating half-lives greater than 250 h The slowly exchanging residues are clustered around the knuckle that contains the metal centre, while the central region of the b-sheet and the base of the hairpin loop display intermediate exchange rates (Fig 6) The most rapidly exchanging residues are positioned in the hairpin loop, the protein termini and the less structured region of residues 34–36 [28], which is in agreement with a possible higher mobility of these regions
The addition of DGP produced a remarkable increase of half-lives in the 17 amide exchange rates that were measured both in its presence and absence at 40C, reflecting the structural stabilization provided by this solute
The EX2 exchange regime has been established in various rubredoxins (as in most globular stable proteins) [27,42] In fact, EX1 reactions are rarely seen in stable proteins, occurring mostly under the conditions used in some protein refolding experiments [43–46] Under the EX2 regime, the exchange rates are described by Eqn (1):
kex¼ Kopkch½Cat ð1Þ where Kop is the equilibrium constant for structural opening reactions that expose the NH group [33] The term kch[Cat] can be calculated from exchange rates in unstructured peptides and used to obtain Kop, and hence
a value of DG for the opening reactions [47] Assu-ming that the slowest exchanging residues (Val5,
Fig 2.15N amide relaxation parameters of
RdDg as a function of residue number in the
presence of 100 m M KCl (A–C), or 100 m M
DGP (D–F) (A,D) Longitudinal relaxation
time; (B,E) transverse relaxation time; (C,F)
heteronuclear NOE.
Table 1 Summary of parameters used to fit T 1 , T 2 and hNOE S2is the
square of the generalized order parameter characterizing the amplitude
of the internal motions; s e is the effective correlation time for the
internal motions; R ex , is the exchange contribution to T 2 , and the
subscripts f and s indicate fast and slow time scales, respectively.
Model
Optimized
parameters
Fitted residues in the presence of
KCl DGP
4 S2, s e and R ex 1 3
s , S 2
f and s e 0 0
Trang 5Cys6, Thr7, Val8, Cys9, Tyr11, Tyr13, Cys39, Val41, and
Cys42), which are all located near the metal centre,
exchange via a single opening reaction, it is possible to
use the measured exchange rates at five temperatures (between 50 and 70C) at pH 6, to obtain the tempera-ture dependence for the DG of the structural opening
Fig 3 Estimated model-free parameters of RdDg as a function of residue number in the presence of 100 m M KCl (A–C), or 100 m M
DGP (D–F) (A,D) Generalized order parameter; (B,E) effective correlation time; (C,F) chemical exchange rate.
Fig 4 Difference between the generalized or-der parameters in the presence of 100 m M KCl
or DGP of RdDg Only residues whose parameters were calculated in both cases with the same (black bars) or with diff erent dynamic models (grey bars) are included.
Fig 5 Half-life values for the1H–2H amide exchange reaction in RdDg measured at 40 °C
in the presence of DGP (black bars) or KCl (grey bars) at 100 m M The broken bars rep-resent the slowest exchanging residues with half-life values higher than 250 h, which were too long to be determined in the experimental time frame.
Trang 6around the metal centre, that allows the exchange of
these amide residues to take place In these residues,
DG displayed a linear dependence on temperature
Con-sidering the experimental errors and the narrow range of
temperatures investigated, it is reasonable to treatDH as
constant, and use the Gibbs equation to obtain global
enthalpies and entropies for the opening reaction
In the presence of 100 mMKCl the structural opening
reactions presented an averageDG at 60 C of 28.8 ± 0.9 kJÆ
mol)1, from which aDH of 143 ± 23 kJÆmol)1andDS of
0.34 ± 0.07 kJÆK)1Æmol)1can be derived In the presence of
DGP an averageDG of 30.4 ± 0.9 kJmol)1was found, and
aDH of 160 ± 23 kJmol)1andDS of 0.39 ± 0.07 kJÆK)1Æ
mol)1were calculated
Upon solute addition a stabilization of RdDg took place and an average positiveDG of 1.6 kJÆmol)1can be observed for the structural opening reactions that allow amide exchange around the metal centre to take place Gibbs free energy vs temperature plots result in two almost parallel lines in the presence of KCl or DGP (Fig 7), which suggests that, in this case, the enthalpic contribution is more important to the stabilization phenomenon than the entropic term
Discussion
Chemical shifts are sensitive probes of structural changes in the mean position of atoms within a defined structure or protein conformation The presence of DGP caused no significant alteration in1H or15N chemical shifts or in their temperature dependence This means that, throughout the temperature range investigated (from 30 to 80C), solute addition leaves the mean protein structure unchanged A similar lack of change was observed in chymotrypsin inhibitor 2 and in horse heart cytochrome c, where the presence of 2M glycine produced no visible alteration in proton chemical shifts [36] These results support the view that stabilizing compatible solutes exert their effect through changes in solvent structure and/or subtle changes in the dynamic properties of the protein rather than by changing the structure of the protein itself In fact, protein function depends vitally on its structure and dynamics; if a stabilizing solute induced substantial changes, it might hamper enzyme activity, rendering the stabilizing effect useless
The solute DGP is an effective stabilizer of RdDg [7], however, like other solutes, it does not seem to alter protein structure The increased stability of proteins from hyper-thermophiles, in comparison with their mesophilic homo-logues, has frequently been interpreted as a consequence of
a number of mechanisms that improve internal attractive forces and result in increased rigidity of protein structure [48] However, the terms rigidity or flexibility must be regarded with caution, for there is no single measure of flexibility Proteins undergo a wide variety of motions on vastly different time-scales; a protein may be rigid in the nanosecond, and flexible on the millisecond time-scale Bearing this in mind, it is interesting to note that the increased rigidity of RdDg upon addition of DGP is small in the more rapid, low amplitude motions The effect is mostly
in the longer time-scales and in the restriction of wider concerted motions, which encompass the structural reac-tions that allow protected amides to exchange with the solvent In fact, solute addition caused relatively small changes in the relaxation parameters, in particular for T1, which remained practically unaltered T1 relaxation meas-urements carry information about motions with frequencies
of about 108)1012Hz, while T2 and NOE enhancements also depend upon higher amplitude motional regimes in the micro- and millisecond time scale [38] Thus, solute addition appears to have a greater impact on the wider motions, while leaving the small high frequency fluctuations unchanged Recently, however, Wang et al [49] argued that, in the case of ubiquitin, 15N relaxation measurements alone underestimate the variations in backbone dynamics If this proves to be a general effect, and not just a peculiarity of ubiquitin, it could mean that the variations in backbone
Fig 6 Backbone of RdDg showing different time ranges for the
hydrogen exchange rates of amide groups: slow (dark-blue), medium
(mid-blue), and fast (cyan) Cysteine sulphur atoms are depicted in
yellow The slowest exchanging residues, clustered around the metal
centre, are indicated by residue number.
Fig 7 Temperature dependence of the average free energy of the
opening reactions that determine hydrogen amide exchange, in the
presence of 100 m M KCl (s) and DGP (d).
Trang 7dynamics we have measured may be underestimated, which
in turn could explain why they are so small Nevertheless,
the faster time-scales would still be the least affected by
solute addition, suggesting that the stabilizing effect
oper-ates via a higher restriction of the wider motions
Most dynamic studies reported in the literature were
performed on proteins with about 100 amino acids, and
rotational correlation times of around 6–9 ns [30,38,50–52]
D gigas rubredoxin has only 52 amino acids and the
smaller correlation time (3.9 ns was estimated in 100 mM
KCl), higher average R2values and lower (more negative)
NOE enhancements [38,53] agree well with values from
other studies in view of the smaller size of RdDg Studies in
which the dynamics of a protein are investigated in different
conditions invariably find increases in correlation times
upon ligand binding which are interpreted in the light of the
higher mass or bigger size of the complex [32,52,54] In this
study, the presence of DGP was found to increase the
correlation time by 0.7 ± 0.6 ns It is difficult to interpret
this effect as a mass variation, since binding of the solute
should cause more dramatic changes in the chemical shifts
than we observed Although close to the experimental error,
a slight increase in correlation time would be consistent with
a higher solvent viscosity, brought about by solute addition
The measured generalized order parameters (S2) for
RdDg are all above 0.7 (with the exception of Asp2), which
denotes a relatively rigid protein structure The least ordered
parts of the NMR structure [28] are the N terminus and the
loop region DGP addition leaves the overall picture
unaltered, but, with the exception of segment 37–45, there
is a trend towards generalized rigidification of the protein
(Fig 4) Most residues that require the determination of se
to fit the model, with or without solute, are located in the
loop Similar behaviour has been observed in the loop
regions or turns of E coli topoisomerase I [52] The average
sevalue for these high frequency motions is also left almost
undisturbed by solute addition The Rex term measures
wider motions than S2 or se and reflects sliding or
breathing motions in protein structure [54,55] In the
presence of 100 mM KCl only two residues required this
term for adequate fitting, which implies that these motions
are limited in RdDg The addition of DGP required the
inclusion of Rexterms in six residues, but in all cases the
fitted value is near zero, suggesting that the solute
completely restricts concerted motions of groups on larger
time-scales
The influence of DGP addition on the order parameters
of RdDg is of the same magnitude as the effect of ligand
binding reported in several studies For instance, in
ketosteroid isomerase from Pseudomonas testosteroni the
active site residues show an average increase in order
parameters upon ligand binding of only 0.03, while order
parameters decrease in the rest of the protein [32] In
4-oxalocrotonate tautomerase, binding of a competitive
inhibitor causes almost no change in average order
param-eters [54] Most of the residues in E coli topoisomerase I
suffer a decrease in order parameters upon DNA binding
while a small portion of the protein, directly involved in
binding, experiences an average order parameter increase of
0.04 [52] In these studies, the increased mobility in large
sections of the protein was interpreted as a compensatory
effect for the unfavourable entropy associated with binding
site rigidification [32,54] In this light, the generalized increase in order parameters in RdDg upon addition of DGP can be interpreted as a thermodynamically unfavour-able entropic process This would lead to an increased stability of the native form of the protein, in agreement with the preferential exclusion model [13,15,56], only if the effect
on the denatured form were still more unfavourable However, in rubredoxins, denaturation occurs in an irreversible process with concomitant loss of the metal centre and therefore stability is determined by the rate of unfolding In fact, kinetic stability may be as important, physiologically, as thermodynamic stability, particularly in hyperthermophilic organisms Many denaturation proces-ses are irreversible at high temperature and, in those caproces-ses, a solute that leavesDG for unfolding unchanged but that is able to increase the activation energy of the denaturing reaction would be an effective stabilizer
This leads us to consider the possible effects of solute addition on the activation energy of the denaturing process and we look to the results of the amide exchange experi-ments to provide information about structural openings as
an approximation of the transition state in the unfolding process In fact,1H–2H exchange experiments on series of homologous proteins, or in the presence of stabilizing solutes, have shown a strong correlation between stability and exchange rates [23,26,36]
In RdDg the slowest amide proton exchange rates were found in the metal centre and in the b-sheet region, which reflects the rigidity of these regions [27,34,45] Most of the rapidly exchanging amide protons are exposed to the solvent [28] and little can be said about flexibility on the basis of exchange rates alone The most slowly exchanging residues are protected by the protein structure and require a structural opening reaction to exchange This opening of protein structure to the solvent (although transient) has obvious parallels in the process of denatur-ation, and hence probes protein stability [26,44]
In the presence of 100 mMDGP and at 90C, we found
an increase of 6% inDG (1.6 kJÆmol)1) for the structural opening reaction around the metal centre, affecting the slowest exchanging amide groups Although the linearity of the temperature plots points towards a single opening reaction around the metal centre, a superposition of several opening reactions cannot be ruled out In any event, the decrease inDG, the relative importance of the entropic and enthalpic terms, and therefore the general conclusions, would still hold true Although the localized opening required for amide exchange does not lead to loss of the metal centre, this effect may be compared with a change of 4.2 kJÆmol)1required by the Arrhenius equation to explain the fourfold increase in the half-life for thermal denatura-tion [7]
The temperature dependence of the free energy provides information about the stabilizing or destabilizing nature of enthalpic and entropic contributions Assuming thatDH
is constant over the experimental temperature range, the almost parallel linear fits obtained in the presence of DGP
or KCl indicate that the added stability, in this case, is in essence a consequence of an enthalpy increase for the opening reaction, with small contributions from the entropic term This is in agreement with a small protein rigidification in response to solute addition, inferred from
Trang 8the dynamic behaviour of the protein Thus, we envisage an
improvement in favourable interactions brought about by a
slight restriction of the small high-frequency motions, and a
larger reduction in lower-frequency movements such as
sliding or breathing motions
Conclusion
Protein stability is the result of marginal differences between
various large stabilizing and destabilizing interactions, and
is therefore an elusive subject in which small and subtle
changes may result in considerable added stability In fact, if
taken alone, the dynamic data derived from the relaxation
measurements on RdDg do not look very informative,
because the differences are small and seem random
However, they do point towards some rigidification,
particularly with respect to the slower, wider motions,
which is in agreement with the reduction in the slower amide
exchange rates Taking the results of the two sets of
experiments together, and in view of the strong stabilizing
effect the solute confers upon this protein and the lack of
structural alteration, a clearer picture begins to emerge
Thus, despite the uncertainties in the experimental values, it
appears that the stabilizing effect of DGP is essentially
enthalpic (with small contributions from the entropic term),
involving improved internal attractive forces and promoting
a tighter protein structure with restricted large-scale
motions, without significantly altering the smaller, faster
dynamic motional regimes or perturbing the average protein
structure
Acknowledgements
The SON large-scale facility at Utrecht is acknowledged for valuable
support and the acquisition of several spectra This work was supported
by the European Commission, 5th Framework Programme contract
QLK3-CT-2000-00640, Fundac¸a˜o para a Cieˆncia e Tecnologia,
PRAXIS XXI and FEDER, Portugal (POCTI/BME/35131/99, and
PRAXIS/BIO/12082/98).
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Supplementary material
The following material is available from http://blackwell publishing.com/products/journals/suppmat/EJB/EJB3861/ EJB3861sm.htm
Table S1 Relaxation parameters for D gigas rubredoxin at 303.15 K
Table S2 Model-free parameters for D gigas rubredoxin at 303.15 K
Table S3 Logarithm of the exchange protection factors as a function of temperature