Cytochrome c(551) (cyt c(551)) from Pseudomonas aeruginosa is a small protein (82 residues) that folds via a three-state pathway with the accumulation in the microsecond time-range of a compact collapsed intermediate. The presence of a single His residue, at position 16, permits the study of the refolding 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 under different pH conditions. Analysis of this process (over-and-above proline cis-trans isomerization) at pH 7.0, suggests the existence of a multiple transition state pathway in which we postulate three transition states. Taking advantage of site-directed mutagenesis we propose that the first "unfolded-like" transition state (t(1)) originates from the electrostatic properties of the collapsed state, while the second transition state (t(2)) involves the interaction between the N and C-terminal helices and is stabilized by the salt bridge between Lys10 and Glu70 ( approximately 1 kcal mol(-1)). Our results 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 with the breaking/formation of the Fe(3+)-Met61 bond. This strong interaction is 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 concentration of the Trp56Phe site-directed mutant. Study holds ProTherm entries: 15966, 15967, 15968, 15969, 15970 Extra Details: stability; folding kinetics; transition states; chevron plot; phi values
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:46 p.m.
|Number of data points||19|
|Proteins||Cytochrome c-551 ; Cytochrome c-551|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence EDPEVLFKNKGCVACHAIDTKMVGPAYKDVAAKFAGQAGAEAELAQRIKNGSQGVWGPIPMPPNAVSDDEAQTLAKWVLSQK|