Measuring Sub-Microsceond Protein Folding Kinetics with Independent Probes

Determining the rate of forming the truly folded conformation of ultrafast folding proteins has been an important issue for both experiment and simulation. The double-norleucine mutant of the 35-residue villin subdomain has been the focus of recent computer simulations with atomistic molecular dynamics because it is currently the fastest folding protein, with folding times less than 1 microsecond as measured by tryptophan fluorescence in laser temperature jump experiments. However, the molecular dynamics simulations reported folding times much longer than the times for fluorescence changes. To establish the folding time with greater certainty, we have therefore employed an independent method. In this experiment the decay of the tryptophan triplet state measured by triplet-triplet absorption spectroscopy monitors the change in the population of the unfolded state. The experimental rates from the two methods will be critically compared with each other and with the rates from simulations.