SUBMILLISECOND PROTEIN-FOLDING KINETICS STUDIED BY ULTRARAPID MIXING

An ultrarapid-mixing continuous-flow method has been developed to stud y submillisecond folding of chemically denatured proteins, Turbulent f low created by pumping solutions through a small gap dilutes the denat urant in tens of microseconds, We have used this method to study cytoc hrome c folding kinetics in the previously inaccessible time range 80 mu s to 3 ms, To eliminate the heme-ligand exchange chemistry that com plicates and slows the folding kinetics by trapping misfolded structur es, measurements were made with the imidazole complex. Fluorescence qu enching due to excitation energy transfer from the tryptophan to the h eme was used to monitor the distance between these groups, The fluores cence decrease is biphasic. There is an unresolved process with tau < 50 mu s, followed by a slower, exponential process with tau = 600 mu 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 concentr ation, followed by stower 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 p rotein folding.