Initiation of protein folding by light can dramaticafly improve the time resolution of kinetic studies. Here we present an example ofan 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 doser 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.Protein folding generally occurs in two phases, one rapid and one slow. The rapid phase is the collapse of the unfolded polypeptide into a compact structure and the formation of secondary structure. The slow phase is the subsequent, often multistep rearrangement to the native conformation (1-7). Because of the limited time resolution (milliseconds) of conventional stopped-flow mixing experiments, the complete time course of protein folding has not yet been observed. A dramatic improvement in time resolution would result if protein folding could be initiated by light.Here we present an example of an optically triggered folding reaction. We take advantage of the fact that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide (CO) to the covalently attached heme group in the unfolded state. The folding reaction can thus be triggered by photodissociating the CO complex. In this study, we used time-resolved absorption spectroscopy with nanosecond lasers to monitor binding events at the heme (8-11). Although rebinding of CO prevents the complete formation of the native conformation, the rapid recovery of the sample permits repetitive photolysis and therefore the acquisition of high signal/noise transient spectra for investigating submillisecond events. Simulations of the multiwavelength data with kinetic models were carried out to generate the spectra of intermediates, as well as the rate constants connecting them. We find that before folding begins there is transient binding of both nonnative and native ligands from the unfolded polypeptide chain on a microsecond time scale. MATERIALS AND METHODSTye VI horse heart cytochrome c from Sigma was purified by ion-exchange chromatography using carboxymethyl-cellulose (Whatman) (12), reduced with sodium dithionite anaerobically, and separated anaerobically from the sodium dithionite by gel filtrat...

An ultrarapid-mixing continuous-f low method has been developed to study submillisecond folding of chemically denatured proteins. Turbulent f low created by pumping solutions through a small gap dilutes the denaturant in tens of microseconds. We have used this method to study cytochrome c folding kinetics in the previously inaccessible time range 80 s to 3 ms. To eliminate the hemeligand exchange chemistry that complicates and slows the folding kinetics by trapping misfolded structures, measurements were made with the imidazole complex. Fluorescence quenching due to excitation energy transfer from the tryptophan to the heme was used to monitor the distance between these groups. The f luorescence decrease is biphasic. There is an unresolved process with < 50 s, followed by a slower, exponential process with ‫؍‬ 600 s at the lowest denaturant concentration (0.2 M guanidine hydrochloride). These kinetics are interpreted as a barrier-free, partial collapse to the new equilibrium unfolded state at the lower denaturant concentration, followed by slower crossing of a free energy barrier separating the unfolded and folded states. The results raise several fundamental issues concerning the dynamics of collapse and barrier crossings in protein folding.

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