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J. Biol. Chem., Vol. 275, Issue 48, 37824-37828, December 1, 2000

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Selected Mutations in a Mesophilic Cytochrome c Confer the Stability of a Thermophilic Counterpart^*

Jun Hasegawa^§¶, Susumu Uchiyama^¶, Yuko Tanimoto, Masayuki Mizutani^**, Yuji Kobayashi, Yoshihiro Sambongi^§§, and Yasuo Igarashi^**

From  Daiichi Pharmaceutical Co., Ltd., Edogawa-ku, Tokyo 134-8630, Japan, the  Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan, the ^** Department of Biotechnology, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan, and the  Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan

Received for publication, July 5, 2000, and in revised form, July 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Mesophilic cytochrome c₅₅₁ of Pseudomonas aeruginosa (PA c₅₅₁) became as stable as its thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c₅₅₂ (HT c₅₅₂), through only five amino acid substitutions. The five residues, distributed in three spatially separated regions, were selected and mutated with reference to the corresponding residues in HT c₅₅₂ through careful structure comparison. Thermodynamic analysis indicated that the stability of the quintuple mutant of PA c₅₅₁ could be partly attained through an enthalpic factor. The solution structure of the mutant showed that, as in HT c₅₅₂, there were tighter side chain packings in the mutated regions. Furthermore, the mutant had an increased total accessible surface area, resulting in great negative hydration free energy. Our results provide a novel example of protein stabilization in that limited amino acid substitutions can confer the overall stability of a natural highly thermophilic protein upon a mesophilic molecule.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Heat-stable proteins from thermophilic bacteria usually exhibit main chain foldings similar to those of mesophilic counterparts. Mutational studies on mesophilic proteins modeled with respect to thermophilic counterparts have proved that specific side chain interactions in the thermophiles are partially responsible for the higher stability (1-3). Thermodynamic analysis has also indicated that a thermophilic protein can be stabilized through global interaction throughout the molecule (4). It remains enigmatic as to how many amino acid substitutions contribute to the stability of a natural thermophilic protein (5). In some cases, a mesophilic protein only acquires the stability of the thermophilic counterpart after substantial exchanges of a linear sequence; groups of individual mutations are not sufficient (6). Multiple mutations in mesophilic proteins that completely increase the stability to the levels of thermophilic counterparts would provide important information about relationships between local side chain interactions and overall protein stability and demonstrate that the thermophilic character can depend on a limited number of strong noncovalent interactions.

Cytochrome c is a powerful tool for characterizing protein stability because structural information on a variety of cytochromes c is available, and heterologous expression systems for holopoteins have been established (7). Cytochrome c₅₅₁ (PA c₅₅₁)¹ from a mesophile, Pseudomonas aeruginosa, and cytochrome c₅₅₂ (HT c₅₅₂) from a thermophile, Hydrogenobacter thermophilus, are 82- and 80-amino acid proteins, respectively, each with a covalently attached heme. These proteins exhibit 56% sequence identity (8) and almost the same main chain foldings (9), but HT c₅₅₂ exhibits much higher stability compared with PA c₅₅₁ (10). On a structural comparison between HT c₅₅₂ (9) and PA c₅₅₁ (11), we identified three distal regions responsible for the higher stability of the former (9). The single mutation Val-78 to Ile (V78I), and two double mutations Phe-7 to Ala/Val-13 to Met (F7A/V13M) and Phe-34 to Tyr/Glu-43 to Tyr (F34Y/E43Y), chosen with reference to HT c₅₅₂, in these three regions of PA c₅₅₁ have each been shown to increase protein stability (1).

In the present study, the five mutations were introduced together into PA c₅₅₁. Thermodynamic analysis showed that the quintuple mutant of PA c₅₅₁ was as stable as HT c₅₅₂. In order to provide a molecular basis for understanding protein stabilization, we have determined the solution structure of the quintuple mutant. We discuss factors contributing to protein stability in conjunction with structural analyses of HT c₅₅₂, and the wild-type and quintuple mutant PA c₅₅₁ proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Protein Preparations-- Mutations were introduced into the PA c₅₅₁ gene with a polymerase chain reaction-based kit, Mutan-Super Express Km (Takara, Kyoto, Japan), as described previously (1). Transformed Escherichia coli JCB7120 cells harboring PA c₅₅₁ genes were harvested from an anaerobic culture, and the proteins used in this study were purified as described previously (1, 8). The concentrations of the purified protein solutions were determined spectrophotometrically using extinction coefficients of ₅₅₁ = 25,200 cm¹ M¹ and ₅₅₂ = 20,400 cm¹ M¹ for PA c₅₅₁ and HT c₅₅₂, respectively. The uniformly ¹⁵N- or ¹³C/¹⁵N-labeled quintuple mutant PA c₅₅₁ protein was obtained from an anaerobic culture with (¹⁵NH₄)₂SO₄ (99.3%), a ¹⁵N-labeled Algal amino acid mixture (98.2%), and a glycerol-¹³C₃ and ¹³C/¹⁵N-labeled Algal amino acid mixture (97.5%) as nitrogen and carbon sources. The labeled compounds were obtained from Shoko Co., Ltd. (Tokyo, Japan).

Guanidine Hydrochloride (GdnHCl) Denaturation-- Proteins (10 µg/ml) were incubated in diluted HCl water (pH 5.0) with various concentrations of GdnHCl at 25 °C for 2 h before measurements in order to equilibrate the proteins with the denaturant. The CD ellipticity at 222 nm of the protein solutions was measured using a 1-cm path length cuvette at 25 °C with a JASCO J-720 CD spectrophotometer with a PT343 thermoelectric temperature controller. The data were fitted by nonlinear least-squares analysis with KaleidaGraph 3.0 (Synergy Software) employing the Marquart-Levenberg algorithm using a linear extrapolation model as described previously (12). C_m was the concentration of GdnHCl at which the free energy change value, G, became 0.

Thermal Denaturation-- The temperature dependence of the CD ellipticity at 222 nm was monitored using a 1-cm path length cuvette with a JASCO J-720 spectrophotometer with a PT343 thermoelectric temperature controller as described previously (1). Protein solutions (~10 µg/ml), pH 5.0, containing 1.5 M GdnHCl were heated from 15 °C to 90 or 100 °C at a heating rate of 1 K/min. Under these conditions, the thermal transition was highly reversible (95%). The van't Hoff enthalpy, H_van, was determined from the CD denaturation curve according to a two-state mechanism with a temperature-independent heat capacity change, C_P (13). The C_P values used were obtained by differential scanning calorimetric (DSC) measurements at pH 5.0 in the presence of 1.5 M GdnHCl for each protein. The slope of the CD base line for HT c₅₅₂ in the denatured state was assumed to be the same as that in the native state.

DSC measurements were carried out with the VP-DSC developed by MicroCal Inc. (14). Degassed protein solutions, with a concentration of ~1 mg/ml, were loaded into the calorimeter cell, and each sample was heated from 10 to 125 °C under approximately 3 atmospheres, with a heating rate of 1 K/min. Thermodynamic parameters, i.e. T_m, H, S, C_P, and G, were estimated from the heat capacity curve using nonlinear least square fitting based on the previously described method (15, 16). The fitting was performed with MATHEMATICA 3.0 (Wolfram Research Inc.) employing the Marquart-Levenberg algorithm.

NMR Measurement-- A protein sample (~1 mM) of the quintuple mutant was dissolved in a 90% H₂O, 10% D₂O or 99.99% D₂O (v/v) solution (pH 5.0 adjusted with HCl), and then reduced with sodium dithionite. All NMR experiments were performed at 25 °C with a Varian UNITYInova 600 spectrometer. Sequential assignments of the backbone resonances of a polypeptide chain were achieved by means of sets of experiments, HNCACB (17), CBCA(CO)NH (18), HNCO (19), (HB)CBCACO(CA)HA (20), and HNHA (21). Protein side chain assignments were made through HCCH-total correlation spectroscopy experiments (22). Stereospecific assignments of the -methyl protons of valines and -methylene protons were made by analyzing HNHB (23), ¹⁵N-edited NOESY-HSQC (24), and DQF-COSY (25) spectra. All proton signals from the heme moiety were assigned according to the procedure of Keller and Wüthrich (26). The signals of carbons attached to heme protons were assigned with a constant time ¹³C-¹H HSQC spectrum (27). All data were processed using the software NMRPipe (28), and the data analysis was assisted by the software PIPP (29). The ¹H, ¹³C, and ¹⁵N resonance assignments of the quintuple mutant have been deposited in the BioMagResBank, under accession number 4578.

Structure Calculation-- Approximate interproton distances were obtained from simultaneous ¹³C/¹⁵N-edited NOESY-HSQC (30), ¹⁵N-edited NOESY-HSQC, and NOESY (31) spectra. The mixing time was 60 ms for all NOESY experiments. The distance restraints were grouped into four classes: 1.8-2.7, 1.8-3.3, 1.8-5.0, and 1.8-6.0 Å, corresponding to strong, medium, weak, and very weak NOE cross-peak intensities, respectively. The NOEs including backbone amide protons were grouped into the four classes of 1.8-2.9, 1.8-3.5, 1.8-5.0, and 1.8-6.0 Å. The backbone coupling constants, ³J_NH, were measured in a HNHA experiment. The  and -dihedral angle restraints were derived from ³J_NH coupling constants and chemical shift indices. Values of 60 ± 30° and 40 ± 30° were used for the  and -dihedral angles, respectively, for -helical regions. Hydrogen bond restraints were obtained by analyzing the H/D exchange rates and NOE patterns characteristic of -helices. Two distance restraints, r_NH-O (0-2.3 Å) and r_N-O (0-3.3 Å), were used for each hydrogen bond. Structures were calculated using the YASAP protocol (32) within X-PLOR 3.1 (33). The coordinates of the quintuple mutant of PA c₅₅₁ have been deposited in the Protein Data Bank, under accession number 1DVV.

Calculation of the Accessible Surface Area (ASA) and Gibbs Free Energy of Hydration for the Native State (G_hN)-- ASA values were calculated using the program MSRoll (34), with a probe radius of 1.4 Å. The Protein Data Bank accession numbers for the three-dimensional structures of the native proteins were 451C and 1AYG for PA c₅₅₁ and HT c₅₅₂, respectively. Surfaces were classified into polar and nonpolar components by regarding carbon and sulfur atoms as nonpolar and oxygen and nitrogen as polar (35). G_hN was calculated according to the method proposed by Oobatake and Ooi (36).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Stability against GdnHCl Denaturation-- The far-ultraviolet CD spectrum of the quintuple mutant of PA c₅₅₁ (F7A/V13M/F34Y/E43Y/V78I) was nearly identical to that of the wild type (data not shown). A GdnHCl-induced denaturation curve of the quintuple mutant obtained by CD showed that its C_m value (4.39 M) was highly elevated relative to that of the wild type (Fig. 1A and Table I) and essentially the same as that of HT c₅₅₂ (C_m = 4.47 M). Since PA c₅₅₁ and HT c₅₅₂ exhibit a total of 35 amino acid differences, it is noteworthy that only five of these residues are responsible for the enhanced overall stability against chemical denaturation.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Stability of PA c551 and HT c552 measured by CD. A, chemical denaturation curves induced by GdnHCl measured by CD. The data are plotted as a function of the GdnHCl concentration for the wild-type PA c551 (), the quintuple mutant PA c551 (), and HT c552 (). B, thermal denaturation curves observed by CD. The symbols are the same as in A. The solid lines represent the results of nonlinear least-squares best fits based on methods described under "Experimental Procedures."

View this table: [in this window] [in a new window]   Table I Thermodynamic parameters characterizing GdnHCl and thermal denaturations Cm, Hm, and other values were obtained in GdnHCl and thermal denaturation experiments, respectively. H, S, and G are the values at Tm of the wild-type PA c551 (47.3 °C) estimated from DSC measurements. The hypothetical difference in the free energy change (Ghyp) of the quintuple mutant at the Tm of wild-type PA c551 is the sum of the G values of the PA c551 mutants having the F7A/V13M, F34Y/E43Y, and V78I substitutions. Hm is the calorimetric enthalpy change at Tm of each protein. Hvan is the van't Hoff enthalpy derived from CD measurements. Errors are estimated from three independent measurements.

Stability against Thermal Denaturation-- To address the question of how both the quintuple mutant PA c₅₅₁ and HT c₅₅₂ are thermodynamically stabilized, we performed thermal denaturation experiments using CD and DSC. Fig. 1B shows thermal denaturation curves of the quintuple mutant and HT c₅₅₂ together with the wild-type PA c₅₅₁ observed by CD. The H_van values estimated from CD measurements for the three proteins were almost identical to the calorimetric enthalpy change at the denaturation temperature (T_m), H_m, obtained from DSC measurements (see below and Table I), indicating that thermal denaturation of these proteins proceeded in a two-state manner.

Fig. 2A shows the heat capacity curves of the three proteins measured by DSC. From these curves, thermodynamic parameters were obtained as a function of temperature. We further obtained the C_P value of the wild-type PA c₅₅₁ from T_m-dependent H(T_m) measurements at pH 3.6, 3.8, and 4.0 in the absence of GdnHCl (Fig. 2, B and C) (37). The C_P value obtained from the T_m-dependent H value was 781 cal/mol/K, which was close to that obtained on nonlinear fitting of the C_P curve at pH 5.0 in the presence of 1.5 M GdnHCl (720 cal/mol/K; Table I). These results indicate that the C_P values obtained in this study are reliable ones. It has also been reported that the C_P values do not dramatically change in the presence of GdnHCl up to 2.0 M (38).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   DSC analysis of PA c551 and HT c552. A, molar heat capacity curves in the presence of 1.5 M GdnHCl observed by DSC for the wild-type PA c551 (), the quintuple mutant PA c551 (), and HT c552 (). The solid lines represent the results of nonlinear least-squares best fits based on the two-state model. B, heat capacity curves for wild-type PA c551 at pH 3.6 (), 3.8 (), and 4.0 (). C, enthalpy change of denaturation measured at different pH values versus the corresponding denaturation temperature.

The DSC measurements (Fig. 2A) showed that the quintuple mutant had an increased T_m value of 32.9 °C and enhanced thermodynamic stability (G) of 5.86 kcal/mol at the T_m value of wild-type PA c₅₅₁ (Table I). These values were nearly the same as those of HT c₅₅₂. The quintuple mutant exhibited a large increase in H compared with the wild-type PA c₅₅₁ (Table I), suggesting that the mutant was enthalpically stabilized. In contrast, HT c₅₅₂ was stabilized by a small S rather than by an enthalpic factor (Table I). This is obvious from the heat capacity curve (Fig. 2A), since the peak height and area (nearly representing H_m) of HT c₅₅₂ were smaller than those of the quintuple mutant although their T_m values were nearly the same. These results suggest that the enhanced T_m values of HT c₅₅₂ and the quintuple mutant are mainly due to the five residues (Ala-7, Met-13, Tyr-34, Tyr-43, and Ile-78, numbered as in the quintuple mutant of PA c₅₅₁); however, the stabilizing factors differ in the two stable proteins.

Additivity of Thermal Stabilization-- We estimated G values for the reported PA c₅₅₁ mutants (1) having F7A/V13M, F34Y/E43Y, and V78I substitutions, respectively, by DSC measurements (data not shown). The estimated values were as follows: F7A/V13M, 2.39; F34Y/E43Y, 2.52; and V78I, 0.82 kcal/mol. The G value of the quintuple mutant obtained in this study was almost identical to the value for the hypothetical difference in free energy change, G_hyp, i.e. the sum of the G values for the mutant proteins with the F7A/V13M, F34Y/E43Y, and V78I substitutions, respectively (Table I). This indicates that the mutations in each of the three regions contribute in an additive manner to the enhanced overall stability. The three mutated regions in the quintuple mutant do not interact with each other; thus, they may behave independently without nonlocal structural perturbations.

Structure of the Quintuple Mutant Protein-- We next determined the solution structure of the quintuple mutant PA c₅₅₁ protein using 1545 NOE-based distance restraints (comprising 592 intraresidue and intraheme, 300 sequential, 257 medium range, 396 long range including NOEs between the heme and polypeptide chain), supplemented with 102 dihedral and 46 hydrogen bond restraints (Fig. 3A). The best 20 structures for the quintuple mutant satisfied the experimental constraints with small deviations from the idealized covalent geometry (Table II). The stereochemical quality of the 20 structures was determined using PROCHECK-NMR (39). Ignoring all glycines and prolines, 98.5% of the remaining residues fell into the most favored and additional allowed regions of  and  spaces. The average atomic root mean square (r.m.s.) deviations for heavy atoms of residues 3-80 were 0.40 ± 0.06 Å for the main chain atoms and 0.84 ± 0.05 Å for all atoms. These values indicate that the determined structures were well converged and that the restrained energy-minimized structure could be used as a representative for comparison with those of the wild-type PA c₅₅₁ and HT c₅₅₂. The main chain folding of the quintuple mutant was similar to those of the wild-type PA c₅₅₁ and HT c₅₅₂ (Fig. 3B; backbone r.m.s. deviation values were 0.84 Å for residues 3-80 of the wild-type PA c₅₅₁ and 0.99 Å for residues 3-78 of HT c₅₅₂, respectively). This indicates that the introduction of five mutations into PA c₅₅₁ does not alter the main chain folding.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Structures of the quintuple mutant and wild-type PA c551 proteins and HT c552. A, stereoview of the 20 structures of the quintuple mutant. B, schematic representation of main chain folding of the quintuple mutant (purple) overlaid with those of the wild-type PA c551 (green) and HT c552 (red).

View this table: [in this window] [in a new window]   Table II Statistics of the 20 structures of the quintuple mutant protein SA represents the 20 individual structures calculated with the X-PLOR program. SAr is the refined structure obtained by energy minimization of the mean structure obtained by simple averaging of the coordinates of the SA structures. FNOE and Ftor were calculated using force constants of 50 kcal/mol/Å2 and 200 kcal/mol/rad2, respectively. Fvdw was calculated using a final value of 4 kcal/mol/Å2 with the van der Waals hard sphere radii set to 0.75 times those in the parameter set PARALLHSA supplied with the X-PLOR program.

Structure Comparison between HT c₅₅₂ and the Quintuple Mutant-- The structure of the quintuple mutant showed that the side chains of the introduced Ala-7 and Met-13 filled a small cavity found in the wild-type (Fig. 4A). The F7A/V13M mutations in this region also changed the Ile-18 side chain conformation to the favorable gauche plus form from the wild-type gauche minus one (Fig. 4A). The r.m.s. deviation value for the Ala-7, Met-13, Tyr-27, and Trp-77 heavy chain atoms and the residues 5-20, 25-29, and 75-79 main chain atoms was 1.28 Å when these atoms were superimposed on the corresponding atoms of HT c₅₅₂.

View larger version (69K): [in this window] [in a new window]   Fig. 4.   Comparison of the side chain packing around the mutation sites in the quintuple mutant and the corresponding regions in the wild-type PA c551 and HT c552. Amino acids mentioned throughout are designated with a one-letter code. Residues in HT c552 are shown with the numbering used for those in PA c551. The mutated side chains of the quintuple mutant and the corresponding ones of the wild-type PA c551 and HT c552 are colored purple, green, and red, respectively. A, the hydrophobic region around Phe-7 and Val-13 of the wild-type PA c551 and the corresponding regions in the quintuple mutant and HT c552. B, the loop and half of the third helix region from Phe-34 to Leu-44 of the wild-type PA c551 and the corresponding regions in the quintuple mutant and HT c552. C, the internal hydrophobic region around Val-78 and the heme of the wild type and the corresponding regions in the quintuple mutant and HT c552.

The two introduced Tyr-34 and Tyr-43 aromatic side chains in the quintuple mutant were adjacent, as found in HT c₅₅₂, and suggested to undergo a hydrophobic interaction and/or interaction with one another (Fig. 4B). The r.m.s. deviation value for the heavy atoms of the introduced Tyr-34 and Tyr-43 and the main chain atoms of residues 34-44 in the quintuple mutant was 0.91 Å when these atoms were superimposed on the corresponding atoms of HT c₅₅₂. Molecular modeling of PA c₅₅₁ with the F34Y mutation predicts that the  oxygen atom of the introduced Tyr-34 forms a hydrogen bond with the guanidyl base of Arg-47; this was also indicated by our previous thermodynamic analysis (1). However, it was not clear from the NMR data whether an extra hydrogen bond exists in the quintuple mutant, because the guanidyl base of Arg-47 in the mutant was not well defined in the NMR structure.

The mutant structure also showed that the introduced Ile-78 filled a cavity around the heme, which was found in the wild-type (Fig. 4C). The r.m.s. deviation value for the Ile-48, Leu-74, Ile-78, and heme heavy atoms and residues 72-80 main chain atoms in the quintuple mutant was 0.91 Å when these atoms were superimposed on the corresponding atoms of HT c₅₅₂.

These comparisons of side chain interactions in the three mutated regions of the three proteins clearly showed that the regions in the quintuple mutant became more like those in HT c₅₅₂.

Difference in Accessible Surface Area-- We further evaluated, using the structural data, the effects of the five mutations in the three regions on the total ASA (accessible surface area). The quintuple mutant and HT c₅₅₂ in their native states had larger ASA values compared with that of the wild-type PA c₅₅₁; this was due to the larger polar ASA (ASA_pol) value in both cases (Table III). Thus, undefined hydrophilic and polar groups may be more exposed to the solvent. Consequently, the quintuple mutant and HT c₅₅₂ in the native states exhibited greater negative G_hN compared with the wild-type PA c₅₅₁. The negative G_hN values for these two proteins may contribute to the enhanced stability, which is consistent with the results of recent statistical analyses of proteins from thermophiles (40).

View this table: [in this window] [in a new window]   Table III Accessible surface area and hydration free energy GhN was calculated at 298 K.

Conclusion-- Our successful design of a mesophilic protein is, to the best of our knowledge, the first example of protein stabilization to the level of a natural thermophilic counterpart by means of limited amino acid substitutions (2, 41, 42). The formation of extra side chain interactions and exposure of hydrophilic and polar groups of the quintuple PA c₅₅₁ mutant caused the overall elevated stability, which was partly reflected by the increased enthalpy change. The stabilizing strategy for the mutant differed from that in the case of the cold shock protein (43), in which the protein stabilization was mainly achieved through improvement of the electrostatic interaction on the molecular surface.

Our way of carefully comparing the structures of thermophilic and mesophilic homologous proteins and combining selected mutations is valuable for elucidating the relationship between local side chain interactions and overall protein stability. Now that this has been achieved for the first time, it will be worthwhile exploring the possibility of altering other proteins, especially those of industrial and medical interest, in the same manner.

	ACKNOWLEDGEMENTS

We thank R. Muhandirum and L. E. Kay for the pulse sequences and S. J. Ferguson for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by a grant 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.

The atomic coordinates and the structure factors (code 1DVV) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^§ To whom correspondence and requests for reprints may be addressed. Tel.: 81-3-3680-0151; Fax: 81-3-5696-8336; E-mail: haseg7li@ daiichipharm.co.jp.

^¶ These authors contributed equally to this work.

^§§ To whom correspondence and reprint requests may be addressed. E-mail: sambongi@sanken.osaka-u.ac.jp.

Published, JBC Papers in Press, July 28, 2000, DOI 10.1074/jbc.M005861200

	ABBREVIATIONS

The abbreviations used are: PA c₅₅₁, ferrocytochrome c₅₅₁ from P. aeruginosa; HT c₅₅₂, ferrocytochrome c₅₅₂ from H. thermophilus; GdnHCl, guanidine hydrochloride; HSQC, heteronuclear single quantum correlation; NOESY, nuclear Overhauser effect spectroscopy; DQF-COSY, double quantum-filtered correlation spectroscopy; DSC, differential scanning calorimetry; ASA, accessible surface area; r.m.s., root mean square.

	REFERENCES

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