Identification of surface exposed amino acids of isolated and membrane-bound CF1 by protease accessibility

Abstract

In order to compare surface-exposed amino acids in isolated and membrane-bound CF₁ the technique of limited proteolysis was employed. The cleavage sites of several proteases were identified by sequence analysis of the resulting peptides after isolation by SDS-PAGE. In isolated CF₁ the N-terminal region of the α subunit was found to be highy sensitive to proteases; the accessible peptide bonds included αE17-G18, αR21-E22, αE22-V23, and αK24-V25. Additional protease-attacked bonds in α subunit were αS86-S87, xαE125-S126. and αR127-L128. In the β subunit of isolated CF₁ the bonds βL14-E15 and βV76-A77 were identified as being accessible. All identified protease accessible amino acids are located at the protein surface according to a molecular model of CF₁ computed after the crystal structure of mitochondrial F₁ by S. Engelbrecht (1997). In membrane bound CF₁ the primarily accessible peptide bond of the N-terminal domain of α is αR21-E22. After this bond is cleaved by trypsin, the αK24-V25 becomes accessible to further trypsin attack. Moreover, the peptide bonds αR14O-S141 and αG16O-R161 are cleaved. According to the Engelbrecht model αG16O is almost completely shielded and actually this amino acid was hardly accessible to protease in isolated CF₁. The β subunit in general is much more sensitive to proteolysis in membrane-bound than in solubilized CF₁. In the β subunit of membrane-bound CF₁ a papain-sensitive bond βG102-G103 was identified. The results indicate major structural alterations when CF₁ is extracted from the CF₀CF₁ complex. Thiol modulation, i.e. reduction of the regulatory disulfide bond between γC199 and γC205 of y subunit, enhances the accesibility of a number of peptide bonds, in particular ααG160-R161, to proteolytic attack by papain. In contrast, thylakoid membrane energization results in masking of this peptide bond.