Fast folding of cytochromec

Abstract

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 I8 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: T = 14-26 ms for H33N,H39K iso-2 versus T = 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" s", the results imply that the direct folding pathway of iso-2 involves passage through on the order of lo9 or fewer partially folded conformers.

Equilibrium unfolding of a small low‐potential cytochrome, cytochrome c553 from Desulfovibrio vulgaris

“…Since cyt c 553 has one of the lowest reduction potentials of any cytochrome c protein, reduction is always stabilizing~Schejter, 1996; Winkler et al, 1997!. Therefore, since evolution acts on the weakest link, a common stability is observed for oxidized, and not A B Mines et al, 1996;Pierce & Nall, 1997!. These experiments have supported the hypothesis that non-native heme ligation~mainly non-native histidines, but there also may be contributions from non-native methionines and the N-terminus!~Hammack et al, 1998! slows down cytochrome c folding at neutral pH, and pathways involving misfolded intermediates have been proposed.…”

“…reduced, forms of cytochrome c proteins. So far, there have only been a few cases where the thermodynamics of neutral corridors have been defined~Malcolm et al, 1990;Pielak et al, 1995!. Several kinetics studies aimed at revealing cytochrome c folding mechanisms and time scales have been made with the proteins from horse and yeast~Elöve Mines et al, 1996;Pierce & Nall, 1997!. These experiments have supported the hypothesis that non-native heme ligation~mainly non-native histidines, but there also may be contributions from non-native methionines and the N-terminus!~Hammack et al, 1998! slows down cytochrome c folding at neutral pH, and pathways involving misfolded intermediates have been proposed.…”

Sequence Composition Effects on Denatured State Loop Formation in Iso-1-cytochrome c Variants: Polyalanine versus Polyglycine Inserts

“…[23][24][25] Hence, "loop closure" can be considered a basic step in protein folding reactions, which sets a "speed limit" for folding. 20,26,27 Very primitive loop structures are the basic starting point in residual structure formation. Thus, investigations into loop formation and their development into residual structure are vital.…”

A size dependent folding contour for cytochrome C

“…Cyt-C is a classic example of the combination of both the types of bonding. More importantly, both fast [10,11] and slow kinetics [2] of the folding of this protein has been reported.…”