Fast folding of cytochrome c.
Native iso-2 cytochrome c contains two residues (His 18, Met 80) coordinated to the covalently attached heme. On unfolding of iso-2, the His 18 ligand remains coordinated to the heme iron, whereas Met 80 is displaced by a non-native heme ligand, His 33 or His 39. To test whether non-native His-heme ligation slows folding, we have constructed a double mutant protein in which the non-native ligands are replaced by asparagine and lysine, respectively (H33N,H39K iso-2). The double mutant protein, which cannot form non-native histidine-heme coordinate bonds, folds significantly faster than normal iso-2 cytochrome c: gamma = 14-26 ms for H33N,H39K iso-2 versus gamma = 200-1,100 ms for iso-2. These results with iso-2 cytochrome c strongly support the hypothesis that non-native His-heme ligation results in a kinetic barrier to fast folding of cytochrome c. Assuming that the maximum rate of a conformational search is about 10(11) s-1, the results imply that the direct folding pathway of iso-2 involves passage through on the order of 10(9) or fewer partially folded conformers.
Pierce
M M
MM
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Nall
B T
BT
D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D006418:Heme / Q000737:chemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding
D003574:Cytochrome c Group; D006418:Heme