Experimental and computational investigation of early events in protein folding

Knowledge of the early events in protein folding and its characterization is essential for complete comprehension of the protein folding problem: structure, pathways, and mechanisms. The chemical heterogeneity, topological entanglements, and dynamic nature of the unfolded state conformation pose a huge challenge to a accurate description. Recent years have witnessed a boost in the efforts to develop experimental techniques to characterize the unfolded state of protein under folding conditions. This will have immense implications not only in diagnostic and therapeutic strategies for tackling protein misfolding and aggregation based diseases but also in paving way for development of evolutionarily superior biological structures and design of effective drugs against resistant pathogens.;Using an experimental technique that involves monitoring the tryptophan triplet-triplet optical absorption to measure its lifetime against intramolecular Trp/Cys contact quenching, we investigated the unfolded state of two structurally similar but sequentially nonhomologous still well behaved B1 domains of proteins - L and G. Employing the Szabo, Schulten, and Schulten (SSS) theory with a wormlike chain polymer model, we observe that the loss of denaturant yields an unfolded state that is less diffusive and more compact than the fully denatured state. This reflects the complex internal dynamics of the proteins mediated by transient interactions through the chain.;Polyglutamine amino acid motif is implicated in several neurodegenerative diseases, all of which exhibit aggregation in vivo. To understand the mechanism and correlation between the long glutamine stretches and consequent destabilizion of the proteins leading to amyloid fibrilation, we probed the structural properties of polyglutamine polypeptides using Trp/Cys contact quenching. Modeling the length dependence of contact formation rates with a wormlike chain with excluded volume, we find the polyglutamine peptides to be a unusually "stiff" polymer with a persistence length of ∼ 13.0 A. This propensity for extended conformation can explain both the decrease in stability of the host protein and the propensity to form amyloids once unfolded.;A detailed atomic level characterization and the intricate interplay between kinetic and thermodynamic controls at various phases of protein folding can be obtained using accurate molecular modeling and simulations. This complements the experimental techniques by providing insights at higher temporal and structural resolutions, generally inaccessible to experiments. We consolidated this technique with Trp/Cys contact quenching to characterize the unfolded states of protein L. Making quantitative comparisons between experimentally obtained denaturant-induced unfolded ensemble and computationally simulated temperature-induced unfolded ensemble, we observe a low intrachain diffusion rate that decreases with denaturant concentration. This low diffusion can limit the folding speed and its origin can be quantified by close inspection of the simulated trajectories.