Snapshots of protein folding. A study on the multiple transition state pathway of cytochrome c(551) from Pseudomonas aeruginosa

Cytochrome c(551) (cyt c(551)) from Pseudomonas aeruginosa is a small prote in (82 residues) that folds via a three-state pathway with the accumulation in the microsecond time-range of a compact collapsed intermediate. The pre sence of a single His residue, at position 16, permits the study of the ref olding at pH 7.0 in the absence of miscoordination events. Here, we report on folding kinetics in the millisecond time-range as a function of urea und er different pH conditions. Analysis of this process (over-and-above prolin e cis-trans isomerization) at pH 7.0, suggests the existence of a multiple transition state pathway in which we postulate three transition states. Tak ing advantage of site-directed mutagenesis we propose that the first "unfol ded-like" transition state (t(1)) originates from the electrostatic propert ies of the collapsed state, while the second transition state (t(2)) involv es the interaction between the N and C-terminal helices and is stabilized b y the salt bridge between Lys10 and Glu70 (similar to1 kcal mol(-1)). Our r esults suggest that, contrary to other cytochromes c, the roll-over effect observed for cyt c(551) at low denaturant concentration can be interpreted in terms of a broad energy barrier without population of any intermediates. The third and more "native-like" transition state (M) can be associated wi th the breaking/formation of the Fe3+-Met61 bond. This strong interaction i s stabilized by the hydrogen bond between Trp56 and heme propionate 17 (HP- 17) as suggested by the increase in the unfolding rate at high denaturant c oncentration of the Trp56Phe site-directed mutant. (C) 2001 Academic Press.