FAST EVENTS IN PROTEIN-FOLDING INITIATED BY NANOSECOND LASER PHOTOLYSIS

Initiation of protein folding by light can dramatically improve the ti me resolution of kinetic studies. Here we present an example of an opt ically triggered folding reaction by using nanosecond photodissociatio n of the heme-carbon monoxide complex of reduced cytochrome c. The opt ical trigger is based on the observation that under destabilizing cond itions 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 rea ction. We used time-resolved absorption spectroscopy to monitor bindin g at the heme. Before folding begins we observe transient binding of b oth nonnative and native ligands from the unfolded polypeptide on a mi crosecond time scale. Kinetic modeling suggests that the intramolecula r 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 opti cal trigger should provide a powerful method for studying chain collap se and secondary structure formation in cytochrome c without any limit ations in time resolution.