Time-resolved laser spectroscopic studies of rate-limiting events in protein folding and binding

Proteins fold from simple poly-amino acid chains to compact three-dimensional structures capable of performing diverse functions in our cells. This disorder to order transition is both swift (∼microseconds) and specific. While energy landscape theory and kinetic theories of diffusion have enriched our understanding of the mechanism by which proteins fold, we are still searching for the answers to key questions. What is the limiting speed for the folding process? What events drive the folding process at these limiting speeds?;Protein folding begins with the formation of a contact between any two points of the chain diffusing towards each other. The rate of this event sets an upper limit to the overall folding rate. We use laser-triggered nanosecond-resolved multi-wavelength transient absorbance spectroscopy to study contact formation in simple amino acid chains in aqueous solvents. Studying variation of diffusion rates with solvent viscosity and temperature identifies the events limiting the folding rate. Small fast folding proteins are model systems to conduct such experiments, as their folding mimics the initial events in the protein folding process. Our laser-induced temperature-jump nanosecond-resolved fluorescence studies of the tryptophan zipper folding investigates the events that limit beta-hairpin formation in tryptophan zipper at various temperatures and solvent viscosities. We observe a fast (∼100 ns) relaxation and a slower (∼mus) 12 relaxation for the tryptophan zipper in our kinetic studies of TZ2 folding with varying time dependence at different temperatures and viscosities. The presence of more than one relaxation confirms the existence of multiple folding pathways for TZ2. They also open up the possibility of exploring these pathways in different regimes of solvent viscosity.;We study the folding kinetics of the natively unfolded IA3 peptide coupled to its binding to the YPrA enzyme. This interaction results in specific and potent inhibition of YPrA by IA3. Our laser-triggered temperature jump studies enable a better understanding of the mechanism by which IA3 inhibits YPrA. The different folding kinetics of IA3 in the absence and presence of YPrA suggests a mechanism where IA3 first binds to YPrA, and then uses YPrA as a template to stabilize its folded state.