Fast events in protein folding initiated by nanosecond laser photolysis.
Initiation of protein folding by light can dramatically improve the time resolution of kinetic studies. Here we present an example of an optically triggered folding reaction by using nanosecond photodissociation of the heme-carbon monoxide complex of reduced cytochrome c. The optical trigger is based on the observation that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide to the covalently attached heme group in the unfolded state. Photodissociation of the carbon monoxide thus triggers the folding reaction. We used time-resolved absorption spectroscopy to monitor binding at the heme. Before folding begins we observe transient binding of both nonnative and native ligands from the unfolded polypeptide on a microsecond time scale. Kinetic modeling suggests that the intramolecular binding of methionine-65 and -80 is faster than that of histidine-26 and -33, even though the histidines are closer to the heme. This optical trigger should provide a powerful method for studying chain collapse and secondary structure formation in cytochrome c without any limitations in time resolution.
Jones
C M
CM
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Henry
E R
ER
Hu
Y
Y
Chan
C K
CK
Luck
S D
SD
Bhuyan
A
A
Roder
H
H
Hofrichter
J
J
Eaton
W A
WA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006418:Heme / Q000378:metabolism; D006736:Horses; D007700:Kinetics; D007834:Lasers; D008715:Methionine; D010782:Photolysis; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013053:Spectrophotometry; D013997:Time Factors
D003574:Cytochrome c Group; D006146:Guanidines; D006418:Heme; D002248:Carbon Monoxide; D008715:Methionine; D019791:Guanidine