Probing stability and dynamics of proteins by protease digestion. II: Identification of the initial chymotryptic cleavage sites of homologous cytochromes c.
Three homologous cytochromes c from horse, rabbit and tuna were subjected to chymotryptic digestion and their initial cleavage sites were identified. The sites in oxidized cytochromes c are the COOH-terminal sides of Tyr-48, Phe-46 and Tyr-46 for horse, rabbit and tuna cytochromes c, respectively. The results show that the chymotrypsin attacks a single site in each protein; the sites are located at the almost identical position on the polypeptide chain. Through the time-course studies of digestion, it was found that the three cytochromes c have different chymotrypsin-susceptibility at the initial cleavage site in the order of horse less than rabbit less than tuna. Studies on chymotryptic digestion of tuna cytochrome c in the reduced form revealed that the haem-reduction does not alter the initial cleavage site but increases the resistance to the proteolysis at the site. The uniqueness of the initial cleavage site in each cytochrome c species suggests that the protease susceptibility reflects some overall properties of the protein. At the same time, it was clarified that the initial cleavage site is also affected by a neighboring region by the fact that another potential cleavage site is located near the site in question. In order to elucidate the initial cleavage site, several physical properties of tuna cytochrome c molecule deduced from the X-ray 3D structure, accessible surface area, temperature factor, effective hydrophobicity and electrostatic potential, were compared with the experimental results and it was concluded that these properties given by a residue have no direct relationship with the chymotrypsin susceptibility.
Miki
Y
Y
Department of Physics, Faculty of Science, University of Tokyo, Japan.
Endo
S
S
Giga-Hama
Y
Y
Tanji
M
M
Wada
A
A
D000595:Amino Acid Sequence; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D006736:Horses; D008969:Molecular Sequence Data; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature; D014413:Tuna
D003574:Cytochrome c Group; D002918:Chymotrypsin