title: Five amino acid residues responsible for the high stability of Hydrogenobacter thermophilus cytochrome c552: reciprocal mutation analysis.
abstract: Five amino acid residues responsible for extreme stability have been identified in cytochrome c(552) (HT c(552)) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c(552), were replaced with the corresponding residues in the homologous but less stable cytochrome c(551) (PA c(551)) from Pseudomonas aeruginosa. The quintuple HT c(552) variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c(551), suggesting that the five residues in HT c(552) necessarily and sufficiently contribute to the overall stability. In the three HT c(552) variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by -13.3 kJ mol(-1), followed by A7F/M13V (-3.3 kJ mol(-1)) and then I78V (-1.5 kJ mol(-1)). The order of destabilization in HT c(552) was the same as that of stabilization in PA c(551) with reverse mutations such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol(-1), respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability.
authors.affiliation: Graduate School of Biosphere Science, Hiroshima University, CREST of Japan Science and Technology Corp., 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Oikawa
authors.forename: Shota
authors.initials: S
authors.lastname: Nakamura
authors.forename: Takafumi
authors.initials: T
authors.lastname: Sonoyama
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ohshima
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Shin-ichi J
authors.initials: SJ
authors.lastname: Takayama
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: Susumu
authors.initials: S
authors.lastname: Uchiyama
authors.forename: Jun
authors.initials: J
authors.lastname: Hasegawa
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sambongi
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004563:Electrochemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation / Q000235:genetics; D019897:Periplasm / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D013696:Temperature; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D003574:Cytochrome c Group; C022302:cytochrome C-552; D019791:Guanidine