Time-resolved circular dichroism studies of protein folding intermediates of cytochrome c.
The circular dichroism spectra of cytochrome c (cytc) in 4.6 M guanidine hydrochloride (pH 6.5) indicate that the secondary structure in reduced cytc is near-native, whereas in the CO-bound species (COCytc) it is substantially unfolded. Photolysis of COCytc should thus induce large changes in the secondary structure, which can be probed with time-resolved circular dichroism (TRCD) spectroscopy in the far-UV region. Time-resolved absorption (TROA) and TRCD methods were used to study the photolysis reaction of COCytc in efforts to identify structural intermediates in cytc folding on time scales from nanoseconds to seconds. TROA data from the Soret region, similar to previous studies, showed four intermediates with lifetimes of 2, 50, 225, and 880 micros. The 2-micros process is proposed to involve Fe(II)-Met80 coordination. Approximately 7% of the native CD signal was observed in the TRCD signal at 220 nm within 500 ns, with no significant additional secondary structure formation observed. Further folding after 2 micros may be inhibited by ligation of His26/His33 with Fe(II), which is suggested to be associated with the 50-micros phase. The two slowest components, tau = 225 and 880 micros, are attributed to CO rebinding on the basis of mixed-gas experiments. CO rebinding is expected to compete with protein folding and favor the unfolded state. However, when the two CO rebinding lifetimes are extended into milliseconds by reducing the CO concentration, there is still no significant increase in CD signal at 220 nm.
Chen
E
E
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
Wood
M J
MJ
Fink
A L
AL
Kliger
D S
DS
D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry*; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010782:Photolysis; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013696:Temperature
D003574:Cytochrome c Group; D019791:Guanidine