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An ultrarapid-mixing continuous-flow method has been developed to study submillisecond folding of chemically denatured proteins. Turbulent flow 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 [[micro]second] 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 fluorescence decrease is biphasic. There is an unresolved process with [Tau] < 50 [[micro]second], followed by a slower, exponential process with [Tau] = 600 [[micro]second] 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.

A modified lattice model of energy transfer in a mutant strain of the cyanobacterium Synechocystis 6803, labeled TolE4B, was developed by genetic deletion of proteins joining phycobilisomes to thylakoid membranes and by genetic deletion of the CP43 and CP 47 proteins of photosystem II. The photosystem I (PSI) was left as the only chromatophore in the photosynthetic membranes. An intermediate lifetime is not related to the intact membrane preparation, and the charge separation in PSI is irreversible.