Early events in protein folding investigated through ultrarapid microfluidic mixing

How a protein folds from a random coil to its native state is one of the most fundamental questions in biophysics. By observing the earliest folding events, it may be possible to gain a better understanding of the entire folding trajectory. Until recently, there has been a gap between the longest possible folding simulations and the earliest possible experimental measurement due to an inherent unobservable dead time. We present an ultrarapid microfluidic mixer with no unobservable dead time and a mixing time more than 500 times shorter than the dead time of the previous state of the art instrument.;We investigated the earliest events in the folding of protein L, and found complexity in a process originally thought to be a simple two-state folder. The experiments performed in a UV fluorescence ultrarapid microfluidic mixer uncovered early kinetics including a 4 mus rise and 43 mus decay in overall tryptophan fluorescence that were entirely within the dead time of earlier stopped-flow experiments.;We measured the time evolution of the diffusion-limited rate of unfolded protein L less than 300 mus after being allowed to start to refold. Using this rate along with a new distance probability distribution produced through hundreds of thousands of MD simulations, we found the intramolecular diffusion coefficient decreases by an astounding 550 fold from when the protein is fully denatured to when it is in folding conditions.