Thermodynamic characterization of variants of mesophilic cytochrome c and its thermophilic counterpart

Susumu Uchiyama1,2, Jun Hasegawa3, Yuko Tanimoto1, Hiroshi Moriguchi1, Masayuki Mizutani4, Yasuo Igarashi4, Yoshihiro Sambongi5,6 and Yuji Kobayashi1,6

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 References
 
Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6. The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains. Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions. The obtained differences in the Gibbs free energy changes of unfolding [{Delta}({Delta}G)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change ({Delta}CP), resulting in higher {Delta}G values over a wide temperature range (0–100°C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552. Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization.

Keywords: combined mutant/cytochrome c/differential scanning calorimetry (DSC)/protein stability


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 References
 
Proteins from thermophilic bacteria usually exhibit enhanced stability against heat or denaturants compared to the homologs from mesophiles (Jaenicke and Bohm, 1998Go; Jaenicke, 2000Go). A comparative study of thermophilic and mesophilic proteins provides several lines of information on protein stability. In particular, investigation of the relationship between three-dimensional structure and thermodynamic parameters accompanied by protein unfolding provides detailed information on factors contributing to the stability.

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., 1989aGo), each with a covalently attached heme. These proteins exhibit 56% sequence identity and almost the same backbone conformation (Hasegawa et al., 1998Go), but HT c-552 has higher stability compared to PA c-551 (Sanbongi et al., 1989bGo). 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., 1999Go, 2000Go).

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, 2000Go; 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 References
 
PA c-551 variants

In addition to the PA c-551 variants previously examined (Hasegawa et al., 1999Go, 2000Go), we prepared three new variants having two of the region I, II and III mutations (F7A/V13M, F34Y/E43Y and V78I, respectively) (Figure 1Go). 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).



View larger version (60K):
[in this window]
[in a new window]
 
Fig. 1. Backbone ribbon representation of wild-type PA c-551 (green), its R(I+II+III) variant (light blue), and HT c-552 (red). The side chains of Ala7, Met13, Tyr34, Tyr43 and Ile78 in the R(I+II+III) variant are shown together with the respective regions (I, II and III). The figure was created with MOLMOL (Koradi et al., 1996Go).

 
Mutagenesis and protein preparation

The methods used for the introduction of mutations and protein preparations were as described previously (Hasegawa et al., 1999Go). 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, {varepsilon}551 = 25 200 cm-1 M-1 for PA c-551 variants and {varepsilon}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., 1997Go). Thermodynamic parameters were estimated as mentioned in the Appendix.

The hypothetical value of the difference in Gibbs free energy change, {Delta}({Delta}Ghyp), for combined PA c-551 variants having mutations in more than one region was calculated, e.g. for the R(I+II) variant:

where {Delta}({Delta}Gn) is the change in {Delta}({Delta}G) caused by mutation(s) in region I or II. As to the {Delta}({Delta}Ghyp) value of the R(I+II+III) variant (the quintuple mutant, Hasegawa et al., 2000Go), four possible values were calculated from the four combinations of {Delta}({Delta}Gn): {Delta}({Delta}GI+II) and {Delta}({Delta}GIII), {Delta}({Delta}GI+III) and {Delta}({Delta}GII), {Delta}({Delta}GII+III) and {Delta}({Delta}GI), and {Delta}({Delta}GI), {Delta}({Delta}GII) and {Delta}({Delta}GIII).

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, 1979Go). 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, {Delta}HvH, were obtained using non-linear least-squares fitting with MATHEMATICA 3.0 according to the method previously described (Marky and Breslauer, 1987Go). Here, the temperature-dependent {Delta}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, 1977Go) 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, 1983Go), 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 {phi} = –120° and {Psi} = +120° and side chain torsions to 180° (Creamer et al., 1997Go), 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, 1977Go). Hydration free energy of the native state ({Delta}Gh,N) and unfolded state ({Delta}Gh,U), hydration enthalpy ({Delta}Hh,UN), hydration entropy ({Delta}Sh,UN) and heat capacity change ({Delta}CP,UN) of unfolding were calculated from the ASA values according to established procedures (Oobatake and Ooi, 1993Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 References
 
Spectrophotometric characteristics of the PA c-551 variants

The UV–visible 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., 2000Go).

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 2Go). 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 2Go). 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.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2. pH dependence of the heat capacity curves for wild-type PA c-551 in the absence of a denaturant. The pHs tested are indicated. The heat capacity of the unfolded states, especially at pH 5.0, could not be obtained.

 
Fitting the results to CP curves

As shown in Figure 3Go, 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, {Delta}CP, was found to be temperature dependent in the present analysis.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3. Heat capacity curve and fitting function of the PA c-551 R(I+II+III) variant at pH 3.6 (50 mM sodium acetate). The observed heat capacity curve is indicated by a line. {circ}, the non-linear least-squares fitting results and `baseline' (CP,base). The dashed lines represent the heat capacities of the native (CP,N) and unfolded (CP,U) states.

 
The experimental data plots of CP,obs could be well fitted to equation (1) in the Appendix, suggesting that the thermal unfolding could be treated as a two-state model. Thus, thermodynamic parameters [free energy change ({Delta}G), enthalpy change ({Delta}H), entropy change ({Delta}S) and {Delta}CP] were obtained as a function of temperature (see Appendix). For example, CP,obs and the fitting function of the PA c-551 R(I+II+III) variant are shown in Figure 3Go. Also, the ratios of the van't Hoff enthalpy ({Delta}HvH) to the calorimetric enthalpy ({Delta}Hcal) changes were nearly unity for all PA c-551 variants (Table IGo), further supporting that the thermal unfolding proceeded in a two-state manner (Privalov, 1979Go).


View this table:
[in this window]
[in a new window]
 
Table I. Thermodynamic parameters obtained by DSC
 
Thermal unfolding observed on DSC

Through DSC thermal unfolding experiments on the cytochromes c at pH 3.6 (Figure 4Go), we obtained the thermodynamic parameters listed in Table IGo. The unfolding temperature, Tm, is equivalent to the temperature at which {Delta}G becomes zero. The {Delta}H, {Delta}S, {Delta}CP and {Delta}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 {Delta}({Delta}G) = {Delta}G(variants) – {Delta}G(wild-type PA c-551) using the obtained parameters (Table IGo).



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 4. Heat capacity curves of wild-type PA c-551, its variants, and HT c-552 at pH 3.6 (50 mM sodium acetate). Wt, wild-type PA c-551; I, PA c-551 R(I) variant; II, R(II) variant; III, R(III) variant; I+II, R(I+II) variant; I+III, R(I+III) variant; II+III, R(II+III) variant; I+II+III, R(I+II+III) variant.

 
All PA c-551 variants had higher Tm values compared to that of the wild-type, and positive {Delta}({Delta}G) values (Table IGo), indicating that the thermal stability was enhanced by the mutations. The PA c-551 R(I) variant had a larger {Delta}({Delta}G) and a higher Tm compared to those of R(II) and R(III). The Tm and {Delta}({Delta}G) values of the combined variants, R(I+II), R(I+III), R(II+III) and R(I+II+III), were larger than those of the variants having the individual mutations. Among the variants, R(I+II+III) exhibited the highest Tm of 82.4°C and the largest {Delta}({Delta}G) of 4.55 kcal mol-1. HT c-552 was more stable than the R(I+II+III) variant, having a Tm of 94.0°C and a {Delta}({Delta}G) of 6.64 kcal mol-1.

All the PA c-551 variants and HT c-552 had increased {Delta}H values compared with that of the wild-type PA c-551 (Table IGo). 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 {Delta}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 {Delta}H values were compensated for the increased {Delta}S values. Such compensations (entropy–enthalpy compensation relationship; Lumry and Rajender, 1970Go) were previously observed for several mutated proteins accompanied by some structural changes (Connelly et al., 1991Go; Takano et al., 1995Go).

The {Delta}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 IGo). Among them, the PA c-551 R(I+II+III) variant had the largest {Delta}CP value of 1120 cal mol-1 at the Tm of the wild-type. In contrast, the {Delta}CP value of HT c-552 was smaller than that of the wild-type PA c-551. In general, a {Delta}G function with a small {Delta}CP value results in a shallower parabolic curve and vice versa (Myers et al., 1995Go). In this regard, the small {Delta}CP value observed for HT c-552 contributed to the positive {Delta}G over a wide temperature range (Figure 5Go). On the other hand, although the PA c-551 variants having larger {Delta}CP values had higher Tm values, their {Delta}G values became smaller with decreasing temperature below ~25°C (Figure 5Go), thus these variants might loose stability at low temperature.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5. {Delta}G curves of wild-type PA c-551, its variants, and HT c-552. The notations are the same as in Figure 3Go. Tm* is the Tm of wild-type PA c-551.

 
Additivity of {Delta}({Delta}G)

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., 2000Go). Thus, we examined whether the mutation(s) in each region contributed to the overall stability in an additive manner by comparing their {Delta}({Delta}G) values. The {Delta}({Delta}G) values of all combined PA c-551 variants, except for R(I+II+III), coincided well with the hypothetical {Delta}({Delta}G) values [{Delta}({Delta}Ghyp)], i.e. the sum of {Delta}({Delta}G) for the componential variants (Table IGo). The observed {Delta}({Delta}G) value of the R(I+II+III) variant was slightly larger than the four possible {Delta}({Delta}Ghyp) values (Table IGo). This was due to the larger {Delta}CP value of R(I+II+III) compared to the other PA c-551 variants at pH 3.6, which caused steeper dependence of {Delta}G on temperature. However, considering the experimental accuracy of estimating thermodynamic parameters in this study, the small difference between the {Delta}({Delta}G) and {Delta}({Delta}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 {Delta}({Delta}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., 1992Go; Kuroda and Kim, 2000Go).

ASA and hydration free energies ({Delta}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 {Delta}Gh values (Table IIGo). 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 IIGo, the calculated deviations were small enough, thus providing comparable ASA and {Delta}Gh values to those obtained on X-ray crystallography of the wild-type PA c-551.


View this table:
[in this window]
[in a new window]
 
Table II. Calculated molecular volume and ASA of wild-type and R(I+II+III) variant PA c-551 and HT c-552
 
It is well known that the {Delta}CP value is proportional to change in non-polar ASA, ({Delta}ASAnp; Spolar et al., 1989Go). The largest {Delta}ASAnp value of the PA c-551 R(I+II+III) variant was consistent with the largest heat capacity change values, {Delta}CP and {Delta}CP,UN, which were derived from both actual calorimetry measurement and the calculation using ASA values (Table IIGo). These results indicate the existence of further burial of non-polar groups in the variant.

Among the three proteins, {Delta}Gh of unfolding, {Delta}Gh,UN, for the wild-type PA c-551 was smallest (–216 kcal mol-1, Table IIGo), indicating the unfavorable hydration of the native state for stability (Ooi and Oobatake, 1988Go). 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 IIGo); 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 ({Delta}ASAtotal) of the R(I+II+III) variant, composed of a highly reduced polar ASA change ({Delta}ASApol) and a slightly larger {Delta}ASAnp, was smaller than that of the wild-type PA c-551. The reduced {Delta}ASApol value primarily led to a negative {Delta}Gh,UN of –154 kcal mol-1 (Table IIGo). HT c-552 had a reduced {Delta}ASAtotal value due not only to the large decrease in {Delta}ASApol but also to the decrease in {Delta}ASAnp compared to those of the PA c-551 wild-type, resulting in a negative {Delta}Gh,UN value of –147 kcal mol-1 (Table IIGo). As indicated in Table IIGo, favorable {Delta}Gh,UN for enhanced thermal stabilities of the R(I+II+III) variant and HT c-552 are mainly due to the hydration enthalpy ({Delta}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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 References
 
The goal of this study was to characterize thermodynamically the side chain interactions of amino acids that contribute to the thermal stability of HT c-552 compared to the less stable homolog PA c-551. Here we combined the mutations in PA c-551 to examine their contributions in greater detail. The thermal stability of the wild-type PA c-551 and its stabilized variants together with HT c-552 was directly measured by DSC.

Previously, we had obtained, through DSC measurement, the {Delta}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 {Delta}CP was constant regardless of the tested temperature (10~115°C) (Hasegawa et al., 2000Go). With the present DSC measurements at pH 3.6 without a denaturant, we fitted CP,obs using the temperature-dependent {Delta}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 {Delta}CP calculation throughout this study was reasonable, as exemplified by the case of the wild-type PA c-551; its {Delta}CP value at the Tm (718 cal mol-1K-1, Table IGo) was close to that estimated from the Tm dependence of {Delta}H(Tm) (720 cal mol-1 K-1; Hasegawa et al., 2000Go). Through this fitting method, we could obtain thermodynamic parameters {Delta}H, {Delta}S, {Delta}CP and {Delta}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 {Delta}Gh,U values for the three proteins were close to each other (Table IIGo), 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 {Delta}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 {Delta}({Delta}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, 1989Go) and methylene (1.2 kcal mol-1; Jackson et al., 1993Go) 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 {delta} 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., 2000Go).

Effect of pH on the stability

In order to evaluate the effect of pH on stability, we compared the values for changes in {Delta}G at 50.4°C (Tm of wild-type PA c-551 at pH 5.0 with 1.5 M GdmCl; Hasegawa et al., 1999Go), {Delta}({Delta}G)*, obtained under pH 3.6 (this study) and pH 5.0 conditions. The {Delta}({Delta}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 {Delta}({Delta}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 {Delta}({Delta}G)* value at pH 3.6 was 1.02 kcal mol-1 smaller than that under the pH 5.0 conditions. Similarly, the {Delta}({Delta}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., 2000Go). 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., 1999Go). 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., 2000Go). 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., 2000Go). 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 4Go); 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 {Delta}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 {Delta}CP value; however, these interactions lead to loss of stability at low temperature, as previously pointed out (Baldwin, 1986Go; Privalov and Makhatadze, 1993Go). 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., 1999Go), 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 {Delta}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.


    Appendix
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 References
 
The observed excess molar heat capacity (CP,obs) was directly used for the calculation of thermodynamic parameters. CP,obs can be deconvoluted into two terms (Sturtevant, 1987Go; Freire, 1994Go; Yu et al., 1999Go):

(1)
where CP,unf is the excess heat capacity attributed to the transition from the native to the unfolded state, and CP,base the baseline composed of the heat capacity of a protein in the native state and that in the unfolded state. CP,unf can be derived from the following relationship (Privalov, 1979Go; Yu et al., 1999Go):

(2)
where ß is a cooperative factor. ß could be set as 1 in the present study because no concentration dependence was observed for CP,obs (data not shown), which meant the absence of association or dissociation during the protein unfolding. A sedimentation equilibrium experiment at 37°C was also carried out with an analytical ultracentrifuge, Optima XL-I (Beckman Coulter, Palo Alto, CA), to confirm that the proteins did not aggregate during the unfolding experiments. {Delta}H(T) is the temperature-dependent enthalpy change of the transition when the heat capacity change accompanied by the unfolding, {Delta}CP, is not equivalent to zero:

(3)
and

(4)
where Tm is the temperature at which {Delta}G becomes zero. The equilibrium constant, KU, can be expressed as:

(5)

hence


(6)


(7)
where {Delta}S(Tm) is the entropy change of the transition at Tm. CP,base consists of the intrinsic heat capacity of a protein in the native state (CP,N) and that in the unfolded state (CP,U):

(8)

CP,N can be treated as a linear function of temperature (Privalov et al., 1989Go; Freire, 1994Go):

(9)
where BN and DN represent the intercept and slope of the CP,N curve, respectively. The CP,U function can be estimated by summing up the heat capacities of all the amino acid residues comprising individual proteins (Privalov and Makhatadze, 1990Go; Freire, 1994Go). Thus, in the present analysis, CP,U was treated as a polynomial function of temperature:

(10)
where the coefficients, DU, EU and FU, were calculated based on the amino acid compositions of the proteins (Freire, 1994Go). Here, the offset value, BU, was treated as a fitting parameter. {Delta}CP is the heat capacity difference between the native and unfolded states of a protein:

(11)

As a result, {Delta}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, {Delta}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 Revenberg–Marquart algorithm. From these calculations, thermodynamic parameters {Delta}G, {Delta}H, {Delta}S and {Delta}CP could be obtained as a function of temperature.


    Notes
 
2 Present address: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Back

6 To whom correspondence should be addressed. E-mail: yujik{at}protein.osaka-u.ac.jp Back


    Acknowledgments
 
We thank Ms Yoshiko Yagi (Institute for Protein Research) for the precise amino acid analyses, and Dr Takeyuki Sugiura (Daiichi Pharmaceutical Co., Ltd) and Ms Yuko Iko (Osaka University) for critical reading of the manuscript. This work was partly supported by a grant from the Japanese Ministry of Education, Science and Culture, and the Senri Life Science Foundation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 References
 
Baldwin,R.L. (1986) Proc. Natl Acad. Sci. USA, 83, 8069–8072.[Abstract]

Beadle,B.M., Baase,W.A., Wilson,D.B., Gilkes,N.R. and Shoichet,B.K. (1999) Biochemistry, 38, 2570–2576.[CrossRef][ISI][Medline]

Connelly,P., Ghosaini,L., Hu,C.Q., Kitamura,S., Tanaka,A. and Sturtevant,J.M. (1991) Biochemistry, 30, 1887–1890.[ISI][Medline]

Connolly,M.L. (1983) J. Appl. Crystallogr., 16, 548–558.[CrossRef][ISI]

Creamer,T.P., Srinivasan,R. and Rose,G.D. (1997) Biochemistry, 36, 2832–2835.[CrossRef][ISI][Medline]

Freire,E. (1994) Methods Enzymol., 240, 502–529.[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, 9641–9649.[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, 37533–37537.[Abstract/Free Full Text]

Hasegawa,J., Uchiyama,S., Tanimoto,Y., Mizutani,M., Kobayashi,Y., Sambongi,Y. and Igarashi,Y. (2000) J. Biol. Chem., 275, 37824–37828.[Abstract/Free Full Text]

Jackson,S.E., Moracci,M., elMasry,N., Johnson.C.M. and Fersht,A.R. (1993) Biochemistry, 32, 11259–11269.[ISI][Medline]

Jaenicke,R. (2000) J. Biotechnol., 79, 193–203.[CrossRef][ISI][Medline]

Jaenicke,R. and Bohm,G. (1998) Curr. Opin. Struct. Biol., 8, 738–748.[CrossRef][ISI][Medline]

Kimura S., Nakamura,H., Hashimoto,T., Oobatake,M. and Kanaya,S. (1992) J. Biol. Chem., 267, 21535–21542.[Abstract/Free Full Text]

Koradi,R., Billeter,M. and Wüthrich,K. (1996) J. Mol. Graph., 14, 29–32.

Kuroda,Y. and Kim,P.S. (2000) J. Mol. Biol., 298, 493–501.[CrossRef][ISI][Medline]

Lumry,R. and Rajender,S. (1970) Biopolymers, 9, 1125.[ISI][Medline]

Marky,L. and Breslauer,K. (1987) Biopolymers, 26, 1601–1620.[ISI][Medline]

Myers,J.K., Pace,C.N. and Scholtz,J.M. (1995) Protein Sci., 4, 2138–2148.[Abstract/Free Full Text]

Oobatake,M. and Ooi,T. (1993) Prog. Biophys. Mol. Biol., 59, 237–284.[CrossRef][ISI][Medline]

Ooi,T. and Oobatake,M. (1988) J. Biochem., 103, 114–120.[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, 184–186.[CrossRef][ISI][Medline]

Plotnikov,V.V., Brandts,J.M., Lin,L.-N. and Brandts,J.F. (1997) Anal. Biochem., 250, 237–244.[CrossRef][ISI][Medline]

Privalov,P.L. (1979) Adv. Protein Chem., 33, 167–192.[Medline]

Privalov,P.L. and Makhatadze,G.I. (1990) J. Mol. Biol., 213, 385–391[ISI][Medline]

Privalov,P.L. and Makhatadze,G.I. (1993) J. Mol. Biol., 232, 660–679.[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, 737–750.[ISI][Medline]

Richards,F.M. (1977) Annu. Rev. Biophys. Bioeng., 6, 151–176.[CrossRef][ISI][Medline]

Sanbongi,Y., Ishii,M., Igarashi,Y. and Kodama,T. (1989a) J. Bacteriol., 171, 65–69.[ISI][Medline]

Sanbongi,Y., Igarashi,Y. and Kodama,T. (1989b) Biochemistry, 28, 9574–9578.[ISI][Medline]

Sandberg,W.S. and Terwilliger,T.C. (1989) Science, 245, 54–57.[ISI][Medline]

Spolar,R.S., Ha,J.-H. and Record,T.M.,Jr (1989) Proc. Natl Acad. Sci. USA, 86, 8382–8385.[Abstract]

Sturtevant,J.M. (1987) Annu. Rev. Phys. Chem., 38, 463–488.[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, 62–76.[CrossRef][ISI][Medline]

Tomlinson,E.J. and Ferguson,S.J. (2000) Proc. Natl Acad. Sci. USA, 97, 5156–5160.[Abstract/Free Full Text]

Yu,Y.B., Lavigne,P., Kay,C.M., Hodges,R.S. and Privalov,P.L. (1999) J. Phys. Chem., 103, 2270–2278.[ISI]

Received October 2, 2001; revised February 14, 2002; accepted February 27, 2002.