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J Biol Chem, Vol. 274, Issue 53, 37533-37537, December 31, 1999

Stabilization of Pseudomonas aeruginosa Cytochrome c₅₅₁ by Systematic Amino Acid Substitutions Based on the Structure of Thermophilic Hydrogenobacter thermophilus Cytochrome c₅₅₂^*

Jun Hasegawa^§, Hideto Shimahara^¶, Masayuki Mizutani, Susumu Uchiyama^¶, Hiroyuki Arai^**, Masaharu Ishii, Yuji Kobayashi^¶, Stuart J. Ferguson, Yoshihiro Sambongi^§§, and Yasuo Igarashi

From the  Daiichi Pharmaceutical Co., Ltd., Edogawa-ku, Tokyo 134-8630, the ^¶ Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, the  Department of Biotechnology, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan, and the  Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c₅₅₁ (PA c₅₅₁) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c₅₅₁ with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c₅₅₂ (HT c₅₅₂), which has similar main chain folding to PA c₅₅₁, but is more stable to heat. Thermodynamic properties of PA c₅₅₁ with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c₅₅₁ with HT c₅₅₂. In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c₅₅₂. We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c₅₅₁ can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c₅₅₂. The higher stability of HT c₅₅₂ may in part be attributed to some of these substitutions.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Proteins isolated from thermophilic organisms are usually stable to heat, indicating that these proteins must themselves embody most of the determinants of protein thermostability. Comparative studies of homologous proteins from mesophiles and thermophiles have provided ideas to explain elevated thermostability which include relatively small solvent-exposed surface area (1), increased packing density (2-4) and core hydrophobicity (5, 6), decreased length of surface loops (4), and generations of ion pairs or hydrogen bonds between polar residues (7, 8). Some recent site-directed mutagenesis studies have indicated that significant stabilization occurs in proteins as a result of mutations to reduce the entropy of the unfolded state (9, 10).

Cytochrome c is characterized by covalent attachment of the heme to the polypeptide chain. This protein has proved useful as a model system for studying the relationship between protein structure and stability because (i) primary and three-dimensional structures of cytochromes c from a wide variety of organisms (both mesophiles and thermophiles) are available, and (ii) heterologous expression systems of both prokaryotic and eukaryotic holocytochromes c have been established (11, 12), which facilitate site-directed mutagenesis studies.

Cytochrome c₅₅₂ (HT c₅₅₂)¹ from a thermophilic hydrogen oxidizing bacterium, Hydrogenobacter thermophilus that grows optimally at 70 °C, is an 80-amino acid protein with a heme. HT c₅₅₂ has 56% sequence identity to an 82-amino acid mono-heme cytochrome c₅₅₁ (PA c₅₅₁) from mesophilic Pseudomonas aeruginosa (13), and the main chain foldings of these proteins are almost the same (14). As expected from the optimal growth temperatures of H. thermophilus and P. aeruginosa, HT c₅₅₂ is more stable to heat than PA c₅₅₁ (15). The genes encoding both proteins have been cloned (16, 17) with a view to identifying (by site-directed mutagenesis) amino acid residues that contribute to the higher stability of HT c₅₅₂ compared with PA c₅₅₁. PA c₅₅₁ and HT c₅₅₂ are thus very suitable proteins for identifying substitutions of amino acid residues that endow stability.

Here we report that holo-PA c₅₅₁, which in terms of visible absorption spectra and thermostability is indistinguishable from the native protein, could be expressed in the periplasm of Escherichia coli. Using this expression system, site-directed mutagenesis studies were performed to show that the stability of PA c₅₅₁ could be significantly increased through selected mutations, which had been chosen by molecular modeling with reference to corresponding amino acid residues in HT c₅₅₂. We discuss the structural origins of higher stability of HT c₅₅₂.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strain, Plasmids, and Growth Condition-- The EcoRI-PstI gene fragment CP1, encoding the 22-amino acid signal sequence and 82-amino acid mature protein of PA c₅₅₁ (18), was inserted into the corresponding restriction sites of pKK223-3 (Amersham Pharmacia Biotech) to generate pKPA1. The pKPA1-based plasmids carrying mutated PA c₅₅₁ gene fragments (see below) were transformed by standard methods into E. coli JCB7120 strain in which the expression of c-type cytochromes is unusually high.² The transformed E. coli cells were grown anaerobically in minimal media in the presence of glycerol, nitrite, and fumarate (19) supplemented with casamino acid (2 mg/ml), tryptophan (20 µg/ml), and ampicillin (50 µg/ml) at 37 °C for the production of wild-type and mutant PA c₅₅₁ proteins.

Introduction of Mutations into PA c₅₅₁ Gene-- Two methods for site-directed mutagenesis were used to introduce a series of mutations in PA c₅₅₁. The first method was the PCR overlap extension technique (20, 21) that was used for the mutations of Phe-7 to Ala/Val-13 to Met (F7A/V13M), Phe-34 to Tyr/Gln-37 to Arg/Glu-43 to Tyr (F34Y/Q37R/E43Y), and Val-78 to Ile (V78I). The conditions for the PCR were as follows: incubation at 94 °C for 2 min followed by 28 cycles of 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min, with a final step of 5 min at 72 °C. Mutations of Phe-7 to Ala (F7A), Val-13 to Met (V13M), Phe-34 to Tyr (F34Y), Gln-37 to Arg (Q37R), Glu-43 to Tyr (E43Y), F34Y/Q37R, F34Y/E43Y, and Q37R/E43Y were introduced by the PCR-based kit, Mutan-Super Express Km (Takara Shuzo). All the PCR products were purified and digested with EcoRI and PstI, and then ligated into the corresponding restriction sites of pKK223-3. The DNA sequences of the entire PCR products were confirmed by the dideoxy-chain termination method using ABI Prism model 310 DNA sequencer.

Production of PA c₅₅₁ Proteins-- The transformed E. coli cells harboring the wild-type or mutant PA c₅₅₁ gene were harvested from the anaerobic culture. Periplasmic protein fractions were recovered by cold osmotic shock, and membrane and cytoplasmic fractions were obtained as described previously (11). Cytochromes c were specifically detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by a heme staining procedure (22). The expressed PA c₅₅₁ proteins in the periplasmic fraction were purified by Hi Trap Q or Mono Q column chromatography (Amersham Pharmacia Biotech), both eluted by 10 mM Tris-HCl buffer (pH 8.0) with an NaCl concentration gradient (0-100 mM), followed by a Superdex 75 column equilibrated and eluted with 50 mM ammonium acetate buffer (pH 7.0). Protein concentrations were determined by the Bio-Rad protein assay kit with bovine serum albumin as a standard. The N-terminal amino acid sequence of the purified wild-type PA c₅₅₁ expressed in the E. coli periplasm was determined by automatic sequencer (Applied Biosystems model 470A).

UV-visible and Circular Dichroic (CD) Spectra-- The UV-visible and CD spectra were measured on Hitachi U-3300 and Jasco J-720 machines, respectively. The protein samples were dissolved in water (pH 5.0 adjusted with HCl), the same conditions as used for thermal denaturation experiments.

NMR Measurements-- Uniformly ¹⁵N-labeled PA c₅₅₁ was obtained from an anaerobic culture with (¹⁵NH₄)₂SO₄ (99.3%, Shoko Co., Ltd.) and ¹⁵N-labeled Algal amino acid mixture (98.2%, Shoko Co., Ltd.) used as nitrogen sources. The protein sample (~1 mM) was dissolved in 90% H₂O, 10% D₂O (pH 5.0 adjusted with HCl), and reduced with sodium dithionite. Three types of NMR measurements were carried out at 25 °C with a Varian Unity Inova 600 spectrometer: two-dimensional ¹H-¹⁵N HSQC (23), three-dimensional ¹⁵N-edited nuclear Overhauser effect spectroscopy-HSQC (24) with a mixing time of 100 ms, and three-dimensional ¹⁵N-edited total correlation spectroscopy-HSQC (24) spectra with a mixing time of 40 ms.

Protein Thermostability-- The wild-type and mutant PA c₅₅₁ proteins (10 µg/ml protein concentration in water, pH 5.0 adjusted with HCl) were subjected to the thermal melting profile analysis by monitoring the changes of CD spectra at 222 nm as described previously (15) with slight modifications. The temperature of the protein solution was continuously raised from 25 °C to 100 °C at the rate of 50 °C per 60 min in the absence of guanidine hydrochloride (GdnHCl) and from 15 °C to 90 °C at the same rate in the presence of 1.5 M GdnHCl. The temperature in the cell was controlled using a Jasco PT343 thermoelectric temperature controller.

Denaturation with GdnHCl-- The stability against GdnHCl denaturation of wild-type PA c₅₅₁ or three mutants with F7A/V13M, F34Y/E43Y, or V78I substitutions was assayed. The proteins (10 µg/ml) were incubated in the diluted HCl water (pH 5.0) with varying concentrations of GdnHCl at 25 °C for 2 h before the measurement in order to equilibrate the proteins with the denaturant. The CD ellipticity at 222 nm of the protein solutions was measured at 25 °C.

Materials-- Restriction enzymes, Taq polymerase, T4 DNA ligase, and other reagents for DNA handling were purchased from Takara Shuzo or Toyobo. The authentic PA c₅₅₁ protein from P. aeruginosa and GdnHCl were purchased from Sigma. All other chemicals used were of the highest grade commercially available.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of PA c₅₅₁ in the Periplasm of E. coli-- The wild-type PA c₅₅₁ protein expressed in E. coli JCB7120 strain was fully recovered in the cold osmotic shock fluid containing the periplasmic protein fraction, but not in the membrane and cytoplasmic fractions. The expressed protein had covalently attached heme as judged by heme staining following separation by SDS-polyacrylamide gel electrophoresis (data not shown). The production yield of holo-PA c₅₅₁ in E. coli reached 8 mg/1 liter of culture (equal to 30% of the total periplasmic protein) calculated by using a millimolar coefficient for the  band at 551 nm (21 mM¹ cm¹). All the mutant proteins tested in this study could be obtained in the E. coli periplasm at almost the same yield as the wild-type.

The N-terminal amino acid sequence of the wild-type PA c₅₅₁ protein expressed in the E. coli periplasm was determined as Glu-Asp-Pro-Glu-Val-Leu-Phe-Lys-Asn-Lys-Gly, which was identical to that of the authentic protein purified from the native organism.

Spectroscopy-- The UV-visible (400-600 nm) spectrum of dithionite-reduced wild-type PA c₅₅₁ protein expressed in E. coli showed absorption maxima at 417, 521, and 551 nm, which are characteristic features of authentic PA c₅₅₁ protein (Fig. 1A). The far-ultraviolet CD (200-250 nm) spectrum of the air-oxidized form of the wild-type was also the same as that of the authentic protein, having an absorption peak at 222 nm (Fig. 1B). The same properties in UV-visible and CD spectra were obtained from all the mutant PA c₅₅₁ proteins used in this study (data not shown). Furthermore, the ¹H-¹⁵N HSQC spectrum of the dithionite-reduced wild-type protein was essentially the same as that of authentic protein (Fig. 2) (25). These results together suggested that the wild-type PA c₅₅₁ expressed in E. coli folded correctly and the mutant proteins did not markedly differ in terms of the three-dimensional structure.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   UV-visible and CD spectra. UV-visible (A) and CD (B) spectra of wild-type PA c551 from E. coli (------) and P. aeruginosa (- - -) at 25 °C, pH 5.0 are shown.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   1H-15N HSQC spectrum of the reduced form of the PA c551 from E. coli. The NMR data were processed using the software nmrPipe and nmrDraw (38), and the analysis was assisted by the software Pipp (39). Backbone signal assignment on 1H-15N HSQC spectrum was achieved based on the sequence-specific NOE connectivity (40). The cross-peaks are labeled according to the sequential assignment.

Assay Condition for Thermostability-- The wild-type PA c₅₅₁ from the native organism and E. coli both had the same cooperative melting transition with a T_m value of 50.4 °C in the presence of 1.5 M GdnHCl at pH 5.0 (Fig. 3A). The T_m values of all the mutants could be determined in the presence of the same concentration of the denaturant (Fig. 3A and Table I). Therefore, we carried out the thermal denaturation assays under these conditions throughout the present study. By contrast, in the absence of GdnHCl, the T_m values of the mutant proteins could not be determined because they did not reach a completely denatured state even at 100 °C (Fig. 3B).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Melting profiles of wild-type and mutant PA c551 proteins. Profiles for the wild-type PA c551 from E. coli (), and P. aeruginosa (), the mutants F7A/V13M (×), F34Y/E43Y(), and V78I () are shown in the presence (A) and in the absence (B) of 1.5 M GdnHCl.

View this table: [in this window] [in a new window]   Table I Parameters characterizing the thermal denaturation of the mutant PA c551 The temperature of the midpoint of the transition (Tm) and the enthalpy change during unfolding at Tm (H) were calculated from curve fitting of the resulting CD values versus the temperature data on the basis of van't Hoff analysis. This curve fitting was achieved using the function of a least-square analysis in the software MATHEMATICA (Wolflam Inc.). The entropy change during unfolding at Tm (S) was calculated using the equation, S = H/Tm. The differences in the free energy changes of unfoldings between the mutant proteins and wild-type at the wild-type Tm (Gm) were calculated using the equation given by Becktel and Schellman (41), Gm = Tm* S (wild-type), where Tm is the difference in Tm values between the mutant and wild-type proteins, and Sm (wild-type) is the entropy change of the wild-type protein at the Tm. The hypothetical Tm value (Tmhyp) was calculated for each mutant protein with multiple mutations assuming that the effect of each amino acid replacement on the protein stability is independent and cumulative.

Substitutions of Phe-7 and Val-13-- In PA c₅₅₁, a small cavity exists around the side chains of Phe-7 and Val-13, which correspond to Ala and Met, respectively, in HT c₅₅₂. The three-dimensional structure of HT c₅₅₂ shows that the side chains of the Ala and Met fill this cavity (14). The double mutation F7A/V13M in PA c₅₅₁ caused increased thermostability compared with the wild-type (T_m: 12.0 °C; Fig. 3A and Table I). Each single mutation, F7A and V13M, enhanced the thermostability essentially in an additive manner, the individual T_m values being 9.5 and 3.2 °C, respectively (Fig. 3A and Table I, see T_m and T_m^hyp values of F7A/V13M mutant).

Substitutions of Phe-34, Gln-37, and Glu-43-- Phe-34 and Glu-43 in PA c₅₅₁ are both substituted by Tyr residues in HT c₅₅₂. The two Tyr aromatic side chains are closely located in the three-dimensional structure of HT c₅₅₂ and suggested to have hydrophobic interaction with one another (14). The T_m values of PA c₅₅₁ with single F34Y and E43Y mutations were increased by 16.0 and 5.1 °C, respectively, compared with the wild-type (Fig. 3A and Table I). The simultaneous mutation (F34Y/E43Y) caused enhanced thermostability, which was contributed by each single mutation in a cumulative manner (T_m: 20.3 °C, T_m^hyp: 21.1 °C, Fig. 3A and Table I).

Although the single Q37R mutation in PA c₅₅₁ reproducibly made a small contribution to the increased stability (T_m value was 4.3 °C, Table I), the T_m values of PA c₅₅₁ protein with F34Y/Q37R, Q37R/E43Y, and F34Y/Q37R/E43Y mutations (T_m: 12.5, 4.3, and 17.5 °C, respectively; Table I) were each significantly lower than those with F34Y, E43Y, and F34Y/E43Y, respectively.

Substitution of Val-78-- The region around Val-78 in PA c₅₅₁, interacting with heme hydrophobically, should become more hydrophobic if Val were substituted by Ile as in HT c₅₅₂; this is because Ile has one additional methyl group in the side chain compared with that of Val. As expected, the V78I mutation in PA c₅₅₁ caused an 8.4 °C elevation of the T_m value compared with that of the wild-type (Fig. 3A and Table I).

Stability against GdnHCl Denaturation-- We also tested whether F7A/V13M, F34Y/E43Y, and V78I mutations in PA c₅₅₁ stabilize the structure against GdnHCl denaturation. Fig. 4 shows GdnHCl-induced denaturation curves and plots of G versus GdnHCl concentration around the midpoint of denaturation (C_m). The denaturation curve of the wild-type showed that 1.5 M GdnHCl did not denature the protein structure. Thus, in the thermal denaturation experiments, the temperature of the protein samples can be ramped from the non-denatured condition in the presence of 1.5 M GdnHCl. The C_m values of the mutant proteins (F7A/V13M, F34Y/E43Y, and V78I) were elevated as compared with that of the wild-type (C_m: 0.52, 0.73, and 0.19 M, respectively; Table II). Among these mutants, F34Y/E43Y was the most stable, followed by the F7A/V13M, and then the V78I protein, as judged by the comparison of C_m and G^H_2O values. This order of the stability was same as that from the heat denaturations of these mutants.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   GdnHCl-induced denaturation of wild-type and mutant PA c551 proteins. A, denaturation curves are shown as a function of GdnHCl concentration for the wild-type PA c551 (), the mutants F7A/V13M (), F34Y/E43Y (), and V78I (). B, the free energy changes of unfolding (G) are shown as a function of the GdnHCl concentration around the midpoint of the transition. Symbols are the same as those in panel A.

View this table: [in this window] [in a new window]   Table II Parameters characterizing GdnHCl denaturation The difference in free energy change between the folded and unfolded states (G) was calculated as described by Pace (42). The free energy change in H2O (GH2O) and the dependence of G on the GdnHCl concentration (m) were determined by a least-squares fit of the data from the transition region using the equation: G = GH2O  m[GdnHCl] (42). The midpoint of the GdnHCl denaturation (Cm) was the concentration of GdnHCl at which the G value became 0. The differences in Cm (Cm) and GH2O (GH2O) between the wild-type and mutant proteins were calculated by subtracting the values of the wild-type from those of mutants.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study we developed an expression system for PA c₅₅₁ using E. coli JCB7120 strain as a host organism. The correctly processed wild-type PA c₅₅₁ expressed in the periplasm of E. coli has the same spectral properties in CD, UV-visible, and NMR, and also has the same thermostability as the authentic protein, indicating that the protein is in "native" state. In previous studies, the PA c₅₅₁ gene on extra-chromosomal plasmids could be expressed heterologously in Pseudomonas putida (26) and in the original organism (18). The holo-PA c₅₅₁ formation was also observed in the periplasm of other E. coli strains.³ The expression level in the present E. coli strain was the highest relative to any previous studies that demonstrated heterologous expression of cytochromes c in other E. coli strains (16, 27, 28),⁴ The efficient production level and easy purification procedure from the E. coli periplasm enabled us to obtain ¹⁵N-labeled PA c₅₅₁ for heteronuclear NMR spectroscopy (detailed structural analysis is in progress). Furthermore, this system will facilitate other studies requiring large amounts of the protein sample such as differential scanning calorimetry and x-ray crystallography. The JCB7120 strain was chosen because it expresses higher levels of endogenous c-type cytochromes than other strains (29).² The basis for this is not known, but it is seemingly not a consequence of enhanced expression of c-type cytochrome biogenesis genes (30) because the JCB7120 strain also produces high level of cytoplasmically expressed HT c₅₅₂ (31), a process that is independent of the biogenesis genes.

Although 35 amino acid residues are substituted between HT c₅₅₂ and PA c₅₅₁, we have postulated, from structure comparison between the proteins, that a few amino acid residues in the three regions formed by Phe-7/Val-13, Phe-34/Gln-37/Glu-43, and Val-78 (Fig. 5; see Fig. 7 in Ref. 14 for the detailed structures) are important for stability. Therefore, we systematically introduced the mutations in these regions of PA c₅₅₁ modeled by the corresponding residues in HT c₅₅₂. All the mutations tested in this study resulted in the increased stability, although HT c₅₅₂ was still more stable than these mutants; its T_m and C_m values were 91.8 °C and 4.49 M, respectively, assayed under the same condition as used for the PA c₅₅₁ proteins.⁵ It is notable that the present findings with a mainly  helical protein contrast with those made with the -sheet rubredoxins from Pyrococcus furiosus (thermophile) and Clostridium pasteurianum (mesophile) (32). For these iron-sulfur proteins substantial exchanges of linear sequence, rather than individual mutations, have been shown to be required to enhance thermostability, implying that many small interactions cumulatively contribute to large increases in the stability.

View larger version (48K): [in this window] [in a new window]   Fig. 5.   Schematic view of mutation points in PA c551 protein. The positions of  carbon atoms of the mutated residues are shown by closed circles. Heme iron is indicated as double lined circle. The atomic coordinates for PA c551 were taken from Protein Data Bank (identification code 451C) (43). The figure was prepared by using the program Molscript (44).

The increased stability of the PA c₅₅₁ with F7A, V13M, F7A/V13M, or V78I mutations indicates that the void spaces in PA c₅₅₁ formed by the side chains of the original residues destabilize the protein structure. Therefore, the mutations designed to fill the void space were effective in increasing stability. It has been suggested in other proteins that higher stability can be achieved when Val is substituted by Ile, having one additional methyl group (33, 34) as found in the PA c₅₅₁ V78I mutant. The stabilization by the E43Y mutation in PA c₅₅₁ could also be attributed to tighter hydrophobic packing between the introduced Tyr residue and Phe-34, Ala-40, or Leu-44, among which the latter two residues are conserved in HT c₅₅₂.

The G_m value for the F34Y mutant is 1.9 kcal/mol, which is comparable to the free energy of the hydrogen bond (2~4 kcal/mol). Consistent with this calculation, three-dimensional molecular modeling of PA c₅₅₁ with the F34Y mutation suggest that the  oxygen atom of the introduced Tyr-34 forms a hydrogen bond with the guanidyl base of Arg-47 in PA c₅₅₁: the distance between these atoms is estimated to be 3.2 Å. However, Lys-45 in HT c₅₅₂ (corresponding to the PA c₅₅₁ Arg-47) does not form a hydrogen bond with the "original" Tyr residue. If Lys-45 could be substituted by Arg in HT c₅₅₂, much higher thermostability should be obtained in the thermophilic protein. The T_m value for the PA c₅₅₁ F34Y mutant (16 °C) is the largest among those of the single amino acid mutants of PA c₅₅₁, and this thermostabilization is one of the most dramatic observed for a single substitution in any protein. It is equivalent to that of a yeast iso-1-cytochrome c mutant, which exhibits the highest elevation of T_m ever observed (35).

The NMR solution structure of HT c₅₅₂ indicates that Arg-35 and the two Tyr residues (corresponding to Gln-37, Phe-34, and Glu-43 in PA c₅₅₁, respectively) form aromatic-amino interactions (14). These interactions have been suggested to cause the higher thermostability of HT c₅₅₂. However, the Q37R mutation negatively affected thermostability of the three PA c₅₅₁ proteins with F34Y, E43Y, or F34Y/E43Y mutations. This observation clearly indicates that the aromatic-amino interaction(s) are formed between the introduced Arg and Tyr residues as in HT c₅₅₂, but these interactions may disturb the hydrogen bond formation between Tyr-34 and Arg-47, and/or the hydrophobic interactions between Tyr-43 and Phe-34, Ala-40, Tyr-41, or Leu-44.

In this study, successful enhancement of protein stability has been achieved by filling small void spaces, increasing hydrophobicity, and generation of a hydrogen bond in the three local regions of PA c₅₅₁. Hydrogenophilus thermoluteolus (formerly Pseudomonas hydrogenothermophila; Ref. 36), which grows optimally at 52 °C, has a homologous cytochrome c₅₅₂ (HP c₅₅₂, having 65% amino acid identity to HT c₅₅₂), although the partial sequence (60 amino acids) is only available. HP c₅₅₂ has been shown to have high thermostability like HT c₅₅₂ (37). It should be noted that, in the HP c₅₅₂ protein, corresponding amino acid residues to PA c₅₅₁ Phe-7, Val-13, and Phe-34 are, respectively, Ala, Met, and Tyr as found in HT c₅₅₂. These findings may support the proposition that the substitutions of Phe to Ala, Val to Met, and Phe to Tyr at these positions play important roles in the protein stability, assuming that HP c₅₅₂ protein has a three-dimensional structure similar to that of HT c₅₅₂ and PA c₅₅₁. Our study strongly indicates that the HT c₅₅₂ can be used as an ideal model protein, it is 26 residues smaller than the yeast iso-1-cytochrome c, for elucidating the roles of amino acid residues in protein stability by mutating the mesophilic homologue, PA c₅₅₁ protein.

	ACKNOWLEDGEMENT

We thank Prof. J. Cole (University of Birmingham) for providing us the E. coli JCB7120 strain and helpful discussion.

	FOOTNOTES

^* This work was supported in part by grants from the Japanese Ministry of Education, Science and Culture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence and requests for reprints may be addressed: Daiichi Pharmaceutical Co. Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan. Fax: 81-3-5696-8336; E-mail: haseg7li@daiichipharm.co.jp.

^** Supported by a fellowship from the Japan Society for the Promotion of Science.

^§§ Supported by a fellowship from the Japan Society for the Promotion of Science and by grants from the Naito Foundation and the Wellcome Trust. To whom correspondence and requests for reprints may be addressed. Present address: Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan; E-mail: sambongi@sanken.osaka-u.ac.jp.

² J. Cole, personal communication.

³ Y. Sambongi and S. J. Ferguson, unpublished results.

⁴ Y. Sambongi, unpublished results.

⁵ J. Hasegawa, unpublished results.

	ABBREVIATIONS

The abbreviations used are: HT c₅₅₂, ferrocytochrome c₅₅₂ from H. thermophilus; GdnHCl, guanidine hydrochloride; PCR, polymerase chain reaction; PA c₅₅₁, ferrocytochrome c₅₅₁ from P. aeruginosa; HP c₅₅₂, ferrocytochrome c₅₅₂ from H. thermoluteolus; CD, circular dichroism; HSQC, heteronuclear single quantum correlation.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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