PROBING WEAKLY POLAR INTERACTIONS IN CYTOCHROME-C

Theoretical, statistical, and model studies suggest that proteins are stabilized by weakly polar attractions between sulfur atoms and proper ly oriented aromatic rings. The two sulfur-containing amino acids, met hionine and cysteine, occur frequently among functional alleles in ran dom mutant libraries of Saccharomyces cerevisiae iso-1-cytochrome c ge nes at positions that form a weakly polar aromatic-aromatic interactio n in the wild-type protein. To determine if a weakly polar sulfur-arom atic interaction replaced the aromatic-aromatic interaction, the struc ture and stability of two variants were examined. Phenylalanine 10, wh ich interacts with tyrosine 97, was replaced by methionine and cystein e. The cysteine was modified to form the methionine and cystine analog , S-methyl cysteine (Cys(SMe)). Proton NMR studies indicate that chang ing Phe 10 to Met or Cys(SMe) affects only local structure and that th e structures of sulfur-containing variants are nearly identical. Analy sis of chemical shifts and nuclear Overhauser effect data indicates th at both sulfur-containing side chains are in position to form a weakly polar interaction with Tyr 97. The F10M and F10C(SMe) variants are 2- 3 kcal mol-1 less stable than iso-1-cytochrome c at 300 K. Comparison of the stabilities of the F10M and F10C(SMe) variants allows evaluatio n of the potential weakly polar interaction between the additional sul fur atom of F10C(SMe) and the aromatic moiety of Tyr 97. The F10C(SMe) ;C102T variant is 0.7 +/- 0.3 kcal mol-1 more stable than the F10M;C10 2T protein. The increased stability is explained by the difference in hydrophobicity of the sulfur-containing side chains. We conclude that any weakly polar interaction between the additional sulfur and the aro matic ring is too weak to detect or is masked by destabilizing contrib utions to the free energy of denaturation.