Folding mechanism of Pseudomonas aeruginosa cytochrome c(551): Role of electrostatic interactions on the hydrophobic collapse and transition state properties

We report on the folding kinetics of the small 82 residue cytochrome c(551) from Pseudomonas aeruginosa. The presence of two Trp residues (Trp56 and T rp77) allows the monitoring of fluorescence quenching on refolding in two d ifferent regions of the protein. A single His residue (the iron-coordinatin g His16) permits the study of refolding in the absence of miscoordination e vents. After identification of the kinetic traps (Pro isomerization and agg regation of denatured protein), overall refolding kinetics is described by two processes: (i) a burstphase collapse (faster than milliseconds) which w e show to be a global event leading to a state whose compactness depends on the overall net charge; at the isoeletric pH (4.7), it is maximally compac t, while above and below it is more expanded; and (ii) an exponential phase (in the millisecond time range) leading to the native protein via a transi tion state(s) possibly involving the formation of a specific salt bridge be tween Lys10 and Glu70, at the contact between the N and C-terminal helices. Comparison with the widely studied horse cytochrome c allows the discussio n of similarities and differences in the folding of two proteins which have the same "fold" despite a very low degree of sequence homology (<30 %). (C ) 1999 Academic Press.