A Study of the Molecular Evolution and Structural Stability of Cytochrome c by using Its Interspecies Hybrids

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 09680655
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biophysics
• Research Institution: Jichi Medical School
• Principal Investigator: SAIGO Satoshi Jichi Medical School, Dept. of Physics, Associate Professor, 医学部, 助教授 (70049071)
• Project Period (FY): 1997 – 1999
• Keywords: Molecular Evolution / Cytochrome c / Structural Stability / Chimeric Protein / Amino Acid Substitution / キメラ分子

Research Abstract

The amino acid sequences of homologous proteins from evolutionarily distant organisms are different to large extents as a result of the molecular evolution of the proteins. For example, difference of ca.45% in sequence is seen between horse and yeast cytochromes c (cyt c's). The spatial structures of homologous proteins are, however, strikingly similar. This structural conservation can be produced by two alternative mechanisms. In the first mechanism, no structural change is caused by each of the amino acid substitutions that occur in the proteins. In the second mechanism, structural changes induced by amino acid substitutions are compensated by substitutions that follow them. Interspecies hybrid proteins can be structurally unstable if the second mechanism realizes the structural conservation. In the present study, horse-yeast hybrid cyt c was prepared by semisynthesis. 12 amino acid sequence differences are seen between horse and the hybrid cyt c's. The spectroscopic and physicochemical properties of the hybrid were compared with those of its parent molecules. The spectral features of the hybrid cyt c indicated that the hybrid cyt c has the same heme coordination structure as those of the parent molecules. Analysis of the guanidine hydrochloride denaturation curves of the hybrid and its parent cyt c's showed that the stability of the hybrid is intermediate between those of the parent molecules. In addition, the measurement of the imidazole binding constants of these cyt c's revealed that the stability of the closed heme crevice structure of the hybrid cyt c is also intermediate between those of the parent molecules. These results indicate that the 12 amino acid substitutions seen in the hybrid cyt c are accommodated at their spatial positions with only minor perturbation of the structure of horse cyt c. Furthermore, the results suggest that these amino acid substitutions change additively the stabilities of the global and local structures of cyt c.