Cytochrome c folding triggered by electron transfer.
BACKGROUND
Experimental and theoretical studies of protein folding suggest that the free-energy change associated with the folding process is a primary factor in determining folding rates. We have recently developed a photochemical electron-transfer-triggering method to study protein-folding kinetics over a wide range of folding free energies. Here, we have used this technique to investigate the relationship between folding rate and free-energy change using cytochromes c from horse (h-cyt c) and yeast (y-cyt c), which have similar backbone folds but different amino-acid sequences and, consequently, distinct folding energies.


RESULTS
The folding free energies for oxidized and reduced h-cyt c and y-cyt c are linear functions of the denaturant (guanidine hydrochloride) concentration, but the concentration required to unfold half of the protein is 1.5 M lower for y-cyt c. We measured the folding rates of reduced h-cyt c and y-cyt c over a range of guanidine hydrochloride concentrations at two temperatures. When driving forces are matched at the appropriate denaturant concentrations, the two homologs have comparable folding rates. The activation free energies for folding h-cyt c and y-cyt c are linearly dependent on the folding free energies. The slopes of these lines are similar (approximately 0.4) for the two proteins, suggesting an early transition state along the folding reaction coordinate.


CONCLUSIONS
The free-energy relationships found for h-cyt c and y-cyt c folding kinetics imply that the height of the barrier to folding depends upon the relative stabilities of the unfolded and folded states. The striking correspondence in rate/free-energy profiles for h-cyt c and y-cyt c suggests that, despite low sequence homology, they follow similar folding pathways.
Mines
G A
GA
Beckman Institute, California Institute of Technology, Pasadena, CA 91125 USA. winkler@bilrc.caltech.edu
Pascher
T
T
Lee
S C
SC
Winkler
J R
JR
Gray
H B
HB
D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D004579:Electron Transport; D006736:Horses; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D017510:Protein Folding
D003574:Cytochrome c Group