2002 Fiscal Year Final Research Report Summary
Elucidation of protein stability modeled by cytochrome c
| Project/Area Number |
13660341
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | HIROSHIMA UNVERSITY (2002)
Osaka University (2001) |
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Principal Investigator |
SAMBONGI Yoshihito Hiroshima University, Graduate School of Biosphere Science, 大学院・生物圏科学研究科, 助教授 (10222027)
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| Co-Investigator(Kenkyū-buntansha) |
UEDA Ikuo Osaka University, Institute of Scientific and Industrial Research Professor, 産業科学研究所, 教授 (60191912)
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| Project Period (FY) |
2001 – 2002
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| Keywords | cytochrome c / thermopile / DSC / redox function / protein stability |
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| Research Abstract |
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 calorimeter (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, Phe-7 to Ala / val-13 to Met; region II, Glu-34 to Try / Phe-43 to Tyr; and region III, Val-78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpy but also entropic contributions, which reflected improved packing of the side chains. On the other hand, the mutated region II made enthalpy contributions to the stability through electrostatic interactions. The differences in the Gibbs free energy changes og unfolding (Δ(ΔG)) for the mutation(s) in each region and their combination(s) showed that the three regions contributed to the overall stability in an additive manner. However, the mutations in regions I+II+III together resulted in compensation of the increased enthalpy change by the entropy change. The values for heat capacity changes (ΔCp) of the PA c-551 variants were larger than or equal to that of the wild -type. In contrast, HT c-552 had a smaller ΔCp value, resulting in higher ΔG values over a wide temperature range (0 to 100℃), 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 fo protein stabilization.
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