1 Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, 3 Daiichi Pharmaceutical Co., Ltd,1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, 4 Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032 and 5 Graduate School of Biosphere Sciences, Hiroshima University, Kagamiyama 1-4-4, Higashi-Hiroshima, Hiroshima 739-8528, Japan
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Abstract |
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Keywords: combined mutant/cytochrome c/differential scanning calorimetry (DSC)/protein stability
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Introduction |
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We have used homologous cytochromes c from mesophilic and thermophilic bacteria as model proteins to investigate the relationship between stability and structure. Cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and cytochrome c-552 (HT c-552) from a thermophile, Hydrogenobacter thermophilus, are 82 and 80 amino acid proteins, respectively (Sanbongi et al., 1989a), each with a covalently attached heme. These proteins exhibit 56% sequence identity and almost the same backbone conformation (Hasegawa et al., 1998
), but HT c-552 has higher stability compared to PA c-551 (Sanbongi et al., 1989b
). We could then identify several amino acid residues responsible for the higher stability of HT c-552 through site-directed mutagenesis studies on the two proteins (Hasegawa et al., 1999
, 2000
).
The two cytochromes c and their variants have now become suitable model proteins for investigating the mechanisms underlying protein stability and also folding (Tomlinson and Ferguson, 2000; Pertinhez, 2001). In this regard, the thermodynamic properties of these proteins should be studied in detail. Here, we measured the thermal stability of PA c-551 variants and HT c-552 by differential scanning calorimetry (DSC) without a denaturant, with which thermodynamic parameters accompanied by unfolding could be derived directly and thus only reflect the effect of temperature. The resulting parameters were also evaluated with regard to the three-dimensional structure information, thus providing the structural basis for side chain effects on protein thermal stability.
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Materials and methods |
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In addition to the PA c-551 variants previously examined (Hasegawa et al., 1999, 2000
), we prepared three new variants having two of the region I, II and III mutations (F7A/V13M, F34Y/E43Y and V78I, respectively) (Figure 1
). The introduced residues were those at the corresponding positions of HT c-552. PA c-551 variants having mutations in each of the regions I, II and III, are denoted here as R(I), R(II) and R(III), respectively. The combined variant, for example, with mutations in both regions I and II, is designated as R(I+II).
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The methods used for the introduction of mutations and protein preparations were as described previously (Hasegawa et al., 1999). Extinction coefficients were obtained by spectrophotometric measurements after reduction by the addition of a sufficient amount of sodium dithionite and by protein assaying involving amino acid analyses. The resulting values,
551 = 25 200 cm-1 M-1 for PA c-551 variants and
552 = 20 400 cm-1M-1 for HT c-552, were routinely used to determine the concentrations of cytochromes c. The far-UV circular dichroism (CD) spectra of PA c-551 variants were measured with an Aviv Model 202 spectropolarimeter (Aviv, Lakewood, NJ) to determine the effects of mutations on their secondary structures.
DSC measurements and data analysis
DSC measurements were carried out in 50 mM sodium acetate buffer, pH 3.6. The protein solutions were dialyzed extensively against the buffer before the measurements. A degassed protein solution (~1 mg ml-1) was loaded into a calorimeter cell and heated from 10 to 115°C at ~35 psi, at the heating rate of 1 K min-1, with a calorimeter, VP-DSC (Microcal Inc., Northampton, MA, USA) (Plotnikov et al., 1997). Thermodynamic parameters were estimated as mentioned in the Appendix.
The hypothetical value of the difference in Gibbs free energy change, (
Ghyp), for combined PA c-551 variants having mutations in more than one region was calculated, e.g. for the R(I+II) variant:
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CD measurement at different temperatures
To ensure that the thermal transition proceeded in a two-state manner, temperature-induced unfolding was monitored with a CD spectrometer (Privalov, 1979). The temperature-dependent CD ellipticity change at 222 nm was followed in a cuvette of 1 mm path-length. From these measurements, thermodynamic parameters including the van't Hoff enthalpy change of the unfolding,
HvH, were obtained using non-linear least-squares fitting with MATHEMATICA 3.0 according to the method previously described (Marky and Breslauer, 1987
). Here, the temperature-dependent
CP values of individual proteins estimated from DSC measurements were used for the van't Hoff analysis.
Calculation of the accessible surface area (ASA) for native and unfolded proteins
ASAN (Richards, 1977) for the native PA c-551 wild-type, the R(I+II+III) variant and HT c-552 was calculated using the program MSRoll (Connolly, 1983
), with the probe radius of water (1.4 Å). The Protein Data Bank accession numbers of the three-dimensional structures are 451C, 1DVV and 1AYG for the wild-type PA c-551, its R(I+II+III) variant and HT c-552, respectively. Since we have no information about the structures of the unfolded proteins, the structures were modeled in their extended conformations, with backbone dihedrals set to
= 120° and
= +120° and side chain torsions to 180° (Creamer et al., 1997
), which were generated with the program Insight II (MSI, San Diego, CA) on a Silicon Graphics workstation. These polypeptide structures were then used to calculate the ASAU values by the same method as that used for the native proteins. ASA of the heme group was calculated separately and added to the ASA of the extended chains.
Surface areas were classified into ASApol and ASAnp, which correspond to polar (N, carbonyl O, and OH) and non-polar (aliphatic C, aromatic C, carbonyl C and S) components, respectively (Richards, 1977). Hydration free energy of the native state (
Gh,N) and unfolded state (
Gh,U), hydration enthalpy (
Hh,UN), hydration entropy (
Sh,UN) and heat capacity change (
CP,UN) of unfolding were calculated from the ASA values according to established procedures (Oobatake and Ooi, 1993
).
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Results |
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The UVvisible spectra of the reduced forms of all PA c-551 variants had nearly identical absorption peaks at 417, 520 and 551 nm. The far-UV CD spectra of all the variants were the same as that of the wild-type, having an absorption peak at 222 nm. These results indicate that the mutations did not have significant effects on the main chain folding and/or on the local structure around the heme. These results were consistent with those of NMR structural analysis of the PA c-551 R(I+II+III) variant that included the F7A/V13M/F34Y/E43Y/V78I mutations; the variant exhibited the same main chain folding as the wild-type PA c-551 (Hasegawa et al., 2000).
Conditions for thermal unfolding by DSC
We examined the pH conditions for DSC thermal unfolding experiments without a denaturant, and found that pH 3.6 worked well (e.g. see the wild-type PA c-551 in Figure 2). With this pH, thermal unfolding of the PA c-551 variants and HT c-552 could be observed up to 120°C in a reversible manner (>70%), providing equilibrium thermodynamic parameters. Therefore, this pH was used to measure thermal stability in this study. pHs <3.6 caused protein unfolding even at room temperature. On the other hand, DSC measurements at pHs >3.6 did not provide the heat capacity (CP) of an unfolded protein, because of high exothermic heat due to protein aggregation (e.g. see the wild-type PA c-551 in Figure 2
). This was most obvious with the highest pH tested, 5.0, possibly because this pH value was close to the pI value (5.1) of PA c-551.
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As shown in Figure 3, the slopes of the observed excess molar heat capacity (CP,obs) at a high temperature (~100°C) became smaller or nearly zero, which was due to the contribution of the heat capacity of a protein in the unfolded state (CP,U). These results indicated that polynomial functions, of which coefficients were calculated from constituent amino acids compositions, were appropriate for estimating CP,U. Consequently, the heat capacity change accompanied by the thermal unfolding,
CP, was found to be temperature dependent in the present analysis.
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Through DSC thermal unfolding experiments on the cytochromes c at pH 3.6 (Figure 4), we obtained the thermodynamic parameters listed in Table I
. The unfolding temperature, Tm, is equivalent to the temperature at which
G becomes zero. The
H,
S,
CP and
G values for PA c-551 variants and HT c-552 at the Tm of wild-type PA c-551 (Tm* = 62.6°C) were compared with those for wild-type PA c-551. The differences in stability between the wild-type PA c-551 and its variants or HT c-552 were also estimated as
(
G) =
G(variants)
G(wild-type PA c-551) using the obtained parameters (Table I
).
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All the PA c-551 variants and HT c-552 had increased H values compared with that of the wild-type PA c-551 (Table I
). Furthermore, PA c-551 variants carrying R(I) mutations except R(I+II+III), i.e. the R(I), R(I+II) and R(I+III) variants, and HT c-552 had
S values similar to that of the wild-type PA c-551. As for the other variants, R(II), R(III) and R(II+III), increased
H values were compensated for the increased
S values. Such compensations (entropyenthalpy compensation relationship; Lumry and Rajender, 1970
) were previously observed for several mutated proteins accompanied by some structural changes (Connelly et al., 1991
; Takano et al., 1995
).
The CP values of the PA c-551 R(I), R(II) and R(III) variants were close to that of the wild-type, and those of the combined PA c-551 variants were larger (Table I
). Among them, the PA c-551 R(I+II+III) variant had the largest
CP value of 1120 cal mol-1 at the Tm of the wild-type. In contrast, the
CP value of HT c-552 was smaller than that of the wild-type PA c-551. In general, a
G function with a small
CP value results in a shallower parabolic curve and vice versa (Myers et al., 1995
). In this regard, the small
CP value observed for HT c-552 contributed to the positive
G over a wide temperature range (Figure 5
). On the other hand, although the PA c-551 variants having larger
CP values had higher Tm values, their
G values became smaller with decreasing temperature below ~25°C (Figure 5
), thus these variants might loose stability at low temperature.
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The mutations introduced were located in three spatially separated regions in PA c-551, and the main chain folding of the R(I+II+III) variant was found to be the same as that of the wild-type (Hasegawa et al., 2000). Thus, we examined whether the mutation(s) in each region contributed to the overall stability in an additive manner by comparing their
(
G) values. The
(
G) values of all combined PA c-551 variants, except for R(I+II+III), coincided well with the hypothetical
(
G) values [
(
Ghyp)], i.e. the sum of
(
G) for the componential variants (Table I
). The observed
(
G) value of the R(I+II+III) variant was slightly larger than the four possible
(
Ghyp) values (Table I
). This was due to the larger
CP value of R(I+II+III) compared to the other PA c-551 variants at pH 3.6, which caused steeper dependence of
G on temperature. However, considering the experimental accuracy of estimating thermodynamic parameters in this study, the small difference between the
(
G) and
(
Ghyp) values of the R(I+II+III) variant was within experimental error. These results together indicated that the R(I), R(II) and R(III) mutations contributed independently to the enhanced thermal stability, and that each mutation did not have a significant effect on the conformations of other mutated regions. Thus, the structures of the mutated regions for all PA c-551 variants may be represented by those of the corresponding regions in the R(I+II+III) variant. The additivity of
(
G) values has been often observed for other mutated proteins having combined substitutions when the introduced mutations are spatially separated from each other (Kimura et al., 1992
; Kuroda and Kim, 2000
).
ASA and hydration free energies (Gh)
As the three-dimensional structures of the wild-type PA c-551, its R(I+II+III) variant, and HT c-552 were available, we could calculate their ASA and Gh values (Table II
). These values were useful for correlating the macroscopic features in terms of stability with the molecular mechanisms in these proteins. Since the three-dimensional structures of the PA c-551 R(I+II+III) variant and HT c-552 were determined by NMR spectroscopy, we estimated the deviation of ASA in the native state, ASAN, using 20 ensembles of the energy minimized structures. As indicated in Table II
, the calculated deviations were small enough, thus providing comparable ASA and
Gh values to those obtained on X-ray crystallography of the wild-type PA c-551.
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Among the three proteins, Gh of unfolding,
Gh,UN, for the wild-type PA c-551 was smallest (216 kcal mol-1, Table II
), indicating the unfavorable hydration of the native state for stability (Ooi and Oobatake, 1988
). While the R(I+II+III) mutations in PA c-551 had a slight effect on total ASA in the unfolded state (ASAU,total), total ASA in the native state (ASAN,total) of the variant was significantly greater (13%) than that of the wild-type (Table II
); this was due to the large increase in polar ASA in the native state (ASAN,pol), which surpassed the small decrease in non-polar ASA in the native state (ASAN,np). Consequently, the total ASA change (
ASAtotal) of the R(I+II+III) variant, composed of a highly reduced polar ASA change (
ASApol) and a slightly larger
ASAnp, was smaller than that of the wild-type PA c-551. The reduced
ASApol value primarily led to a negative
Gh,UN of 154 kcal mol-1 (Table II
). HT c-552 had a reduced
ASAtotal value due not only to the large decrease in
ASApol but also to the decrease in
ASAnp compared to those of the PA c-551 wild-type, resulting in a negative
Gh,UN value of 147 kcal mol-1 (Table II
). As indicated in Table II
, favorable
Gh,UN for enhanced thermal stabilities of the R(I+II+III) variant and HT c-552 are mainly due to the hydration enthalpy (
Hh,UN) of these proteins. These results indicated that the R(I+II+III) variant and HT c-552 were advantageous in terms of hydration free energy, which contributed to the enhanced thermal stability, compared to the wild-type PA c-551.
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Discussion |
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Previously, we had obtained, through DSC measurement, the CP value of the wild-type PA c-551 from the CP,obs curve at pH 5.0 with 1.5 M guanidine hydrochloride (GdmCl) by non-linear least-squares fitting assuming that
CP was constant regardless of the tested temperature (10~115°C) (Hasegawa et al., 2000
). With the present DSC measurements at pH 3.6 without a denaturant, we fitted CP,obs using the temperature-dependent
CP values for the wild-type PA c-551, its stabilized variants and HT c-552, which gave more accurate thermodynamic parameters. The fitting for
CP calculation throughout this study was reasonable, as exemplified by the case of the wild-type PA c-551; its
CP value at the Tm (718 cal mol-1K-1, Table I
) was close to that estimated from the Tm dependence of
H(Tm) (720 cal mol-1 K-1; Hasegawa et al., 2000
). Through this fitting method, we could obtain thermodynamic parameters
H,
S,
CP and
G as a function of temperature.
We also calculated ASAU values using extended structures of the wild-type PA c-551, R(I+II+III) variant and HT c-552 as unfolded proteins. The ASAU and Gh,U values for the three proteins were close to each other (Table II
), suggesting that the three proteins together with other PA c-551 variants should have almost the same unfolded state from a thermodynamic point of view. Therefore, the changes in the thermodynamic parameters observed in this study can presumably be attributed to changes in the native proteins.
Contributions of regions I and III to the enhanced thermal stability
The significantly similar S values of the PA c-551 R(I), R(I+II) and R(I+III) variants to that of the wild-type may be due to release of water molecules from around the non-polar groups in region I, which is accompanied by the rearrangement of non-polar side chains in this region. Thus, the smaller entropic disadvantage caused by the region I residues in HT c-552 enhances the protein thermal stability.
The (
G) value (1.11 kcal mol-1) of the PA c-551 R(III) variant having the V78I substitution is nearly equivalent to those for hydrophobic interactions of methyl (0.8 kcal mol-1; Sandberg and Terwilliger, 1989
) and methylene (1.2 kcal mol-1; Jackson et al., 1993
) groups. These findings indicate that the additional methyl group of the Ile side chain introduced instead of Val fills the cavity that exists in the hydrophobic interior of the protein. Consistently, the three-dimensional structure of the PA c-551 R(I+II+III) variant shows that the
methyl group of Ile78 fits this cavity, as observed for HT c-552, which is seemingly unaffected by the surrounding solution conditions (Hasegawa et al., 2000
).
Effect of pH on the stability
In order to evaluate the effect of pH on stability, we compared the values for changes in G at 50.4°C (Tm of wild-type PA c-551 at pH 5.0 with 1.5 M GdmCl; Hasegawa et al., 1999
),
(
G)*, obtained under pH 3.6 (this study) and pH 5.0 conditions. The
(
G)* values (in kcal mol-1) at pH 3.6 and 5.0, respectively, were: R(I) variant, 1.89 and 1.40; R(II), 1.38 and 2.40; and R(III), 1.25 and 1.00. No significant differences (within 0.5 kcal mol-1) in the
(
G)* values between pH 3.6 and 5.0 were found for the R(I) and R(III) variants, indicating that the stabilizing factors caused by these mutations were not affected by the pH difference. As to the R(II) variant, its
(
G)* value at pH 3.6 was 1.02 kcal mol-1 smaller than that under the pH 5.0 conditions. Similarly, the
(
G)* value of R(I+II+III) at pH 3.6 was 4.54 kcal mol-1, which is 0.92 kcal mol-1 smaller than that under the pH 5.0 conditions (5.46 kcal mol-1, calculated based on previously determined thermodynamic parameters under these conditions; Hasegawa et al., 2000
). Therefore, the mutations in region II had a less stabilizing effect at pH 3.6 than at pH 5.0. These results suggest that the region II mutations caused the significant improvement of electrostatic interaction(s) in PA c-551. One possible electrostatic interaction is that formed by Tyr34 and Arg47, as predicted from our previous molecular modeling (Hasegawa et al., 1999
). However, additional specific hydrogen bond(s) or ion pair formation is not clearly confirmed in the structural model of the PA c-551 R(I+II+III) variant determined by NMR spectroscopy (Hasegawa et al., 2000
). It is sometimes difficult to determine precise side chain conformation of the protein surface by NMR spectroscopy, due to the lack of a sufficient number of NOE constraints.
Thermal stability of naturally-occurring thermophilic HT c-552
The PA c-551 R(I+II+III) variant has been shown to be as stable as naturally occurring thermophilic HT c-552 against GdmCl at pH 5.0 (Hasegawa et al., 2000). From the present thermodynamic results based on DSC measurements, we found that HT c-552 exhibited significantly higher thermal stability than PA c-551 R(I+II+III) at pH 3.6 without a denaturant.
In addition to the advantage of the enthalpic contribution, HT c-552 is entropically stabilized compared to the PA c-551 R(I+II+III) variant. This is apparently confirmed by the observed CP curve (Figure 4); the height of the CP value at the peak temperature of HT c-552 is smaller than that of the PA c-551 R(I+II+III) variant, although the peak temperature of HT c-552 is significantly higher than that of the PA c-551 R(I+II+III) variant. The well balanced enthalpy and entropy in the native state together with the small
CP of HT c-552 leads to the higher thermal stability over a wide temperature range. Also, the higher stability of HT c-552 could be achieved partly through advantageous hydration free energy. As for the R(I+II+III) variant, it gains thermal stability through improved hydrophobic interactions, which is reflected by the increased
CP value; however, these interactions lead to loss of stability at low temperature, as previously pointed out (Baldwin, 1986
; Privalov and Makhatadze, 1993
). From these comparisons between HT c-552 and the R(I+II+III) variant, it is indicated that the former can be stabilized via a `greater maximum stability and flattened' type (Beadle et al., 1999
), but the latter via a `shifted and flattened' type.
The much higher thermal stability of HT c-552 compared to that of the R(I+II+III) variant determined by DSC will provide a further opportunity for investigating the mechanism underlying the protein stability of a natural thermophilic protein. One of the candidates for HT c-552 is favorable molecular mobility, which may increase the entropic contribution to the thermal stability. The higher thermal fluctuation of HT c-552 may also contribute to its observed smaller CP compared to those of the PA c-551 variants, which cannot be accounted for solely by the change in ASA. Work in this direction is underway.
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Appendix |
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![]() | (1) |
![]() | (2) |
![]() | (3) |
![]() | (4) |
![]() | (5) |
hence
![]() | (6) |
![]() | (7) |
![]() | (8) |
CP,N can be treated as a linear function of temperature (Privalov et al., 1989; Freire, 1994
):
![]() | (9) |
![]() | (10) |
![]() | (11) |
As a result, CP values are temperature dependent. Equations (3)(11) were substituted into equation (2), thus CP,obs could be expressed as a function of temperature with the following fitting parameters, Tm,
H(Tm), BN, DN and BU. Then, non-linear least-squares fitting of CP,obs was performed using MATHEMATICA 3.0 (Wolfram Research Inc., Champaign, IL) employing the RevenbergMarquart algorithm. From these calculations, thermodynamic parameters
G,
H,
S and
CP could be obtained as a function of temperature.
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Notes |
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6 To whom correspondence should be addressed. E-mail: yujik{at}protein.osaka-u.ac.jp
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Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beadle,B.M., Baase,W.A., Wilson,D.B., Gilkes,N.R. and Shoichet,B.K. (1999) Biochemistry, 38, 25702576.[CrossRef][ISI][Medline]
Connelly,P., Ghosaini,L., Hu,C.Q., Kitamura,S., Tanaka,A. and Sturtevant,J.M. (1991) Biochemistry, 30, 18871890.[ISI][Medline]
Connolly,M.L. (1983) J. Appl. Crystallogr., 16, 548558.[CrossRef][ISI]
Creamer,T.P., Srinivasan,R. and Rose,G.D. (1997) Biochemistry, 36, 28322835.[CrossRef][ISI][Medline]
Freire,E. (1994) Methods Enzymol., 240, 502529.[ISI][Medline]
Hasegawa J., Yoshida,T., Yamazaki,T., Sambongi,Y., Yu,Y., Igarashi,Y., Kodama,T., Yamazaki,K., Kyogoku,Y. and Kobayashi,Y. (1998) Biochemistry, 37, 96419649.[CrossRef][ISI][Medline]
Hasegawa,J., Shimahara,H., Mizutani,M., Uchiyama,S., Arai,H., Ishii,M., Kobayashi,Y., Ferguson,S.J., Sambongi,Y. and Igarashi,Y. (1999) J. Biol. Chem., 274, 3753337537.
Hasegawa,J., Uchiyama,S., Tanimoto,Y., Mizutani,M., Kobayashi,Y., Sambongi,Y. and Igarashi,Y. (2000) J. Biol. Chem., 275, 3782437828.
Jackson,S.E., Moracci,M., elMasry,N., Johnson.C.M. and Fersht,A.R. (1993) Biochemistry, 32, 1125911269.[ISI][Medline]
Jaenicke,R. (2000) J. Biotechnol., 79, 193203.[CrossRef][ISI][Medline]
Jaenicke,R. and Bohm,G. (1998) Curr. Opin. Struct. Biol., 8, 738748.[CrossRef][ISI][Medline]
Kimura S., Nakamura,H., Hashimoto,T., Oobatake,M. and Kanaya,S. (1992) J. Biol. Chem., 267, 2153521542.
Koradi,R., Billeter,M. and Wüthrich,K. (1996) J. Mol. Graph., 14, 2932.
Kuroda,Y. and Kim,P.S. (2000) J. Mol. Biol., 298, 493501.[CrossRef][ISI][Medline]
Lumry,R. and Rajender,S. (1970) Biopolymers, 9, 1125.[ISI][Medline]
Marky,L. and Breslauer,K. (1987) Biopolymers, 26, 16011620.[ISI][Medline]
Myers,J.K., Pace,C.N. and Scholtz,J.M. (1995) Protein Sci., 4, 21382148.
Oobatake,M. and Ooi,T. (1993) Prog. Biophys. Mol. Biol., 59, 237284.[CrossRef][ISI][Medline]
Ooi,T. and Oobatake,M. (1988) J. Biochem., 103, 114120.[Abstract]
Pertinhez,T.A., Bouchard,M., Tomlinson,E.J., Wain,R., Ferguson,S.J., Dobson,C.M. and Smith,L.J. (2001) FEBS Lett., 495, 184186.[CrossRef][ISI][Medline]
Plotnikov,V.V., Brandts,J.M., Lin,L.-N. and Brandts,J.F. (1997) Anal. Biochem., 250, 237244.[CrossRef][ISI][Medline]
Privalov,P.L. (1979) Adv. Protein Chem., 33, 167192.[Medline]
Privalov,P.L. and Makhatadze,G.I. (1990) J. Mol. Biol., 213, 385391[ISI][Medline]
Privalov,P.L. and Makhatadze,G.I. (1993) J. Mol. Biol., 232, 660679.[CrossRef][ISI][Medline]
Privalov,P.L., Tiktopulo,E.I., Venyaminov,S.Y., Griko,Y.V., Makhatadze,G.I. and Khechinashvili,N.N. (1989) J. Mol. Biol., 205, 737750.[ISI][Medline]
Richards,F.M. (1977) Annu. Rev. Biophys. Bioeng., 6, 151176.[CrossRef][ISI][Medline]
Sanbongi,Y., Ishii,M., Igarashi,Y. and Kodama,T. (1989a) J. Bacteriol., 171, 6569.[ISI][Medline]
Sanbongi,Y., Igarashi,Y. and Kodama,T. (1989b) Biochemistry, 28, 95749578.[ISI][Medline]
Sandberg,W.S. and Terwilliger,T.C. (1989) Science, 245, 5457.[ISI][Medline]
Spolar,R.S., Ha,J.-H. and Record,T.M.,Jr (1989) Proc. Natl Acad. Sci. USA, 86, 83828385.[Abstract]
Sturtevant,J.M. (1987) Annu. Rev. Phys. Chem., 38, 463488.[CrossRef][ISI]
Takano,K., Ogasahara,K., Kaneda,H., Yamagata,Y., Fujii,S., Kanaya,E., Kikuchi,M., Oobatake,M. and Yutani,K. (1995) J. Mol. Biol., 254, 6276.[CrossRef][ISI][Medline]
Tomlinson,E.J. and Ferguson,S.J. (2000) Proc. Natl Acad. Sci. USA, 97, 51565160.
Yu,Y.B., Lavigne,P., Kay,C.M., Hodges,R.S. and Privalov,P.L. (1999) J. Phys. Chem., 103, 22702278.[ISI]
Received October 2, 2001; revised February 14, 2002; accepted February 27, 2002.