Proteins must fold into specific three-dimensional structures to be functional, in a process dictated by their primary sequence. Current understanding of the mechanisms by which proteins fold is limited by the deceivingly simple picture arising from standard kinetic experiments. In these experiments, folding appears as a two or three-state process because the inter-conversions between the myriad of intermediate structures that determine the mechanism are too transient to be directly detected. In this proposal, a group of new experimental approaches to circumvent these limitation is presented. To facilitate extracting mechanistic information from kinetics observations, a catalog of small proteins with simple structural patterns; i.e., structural archetypes, will be produced. Their folding properties will be investigated by fast-kinetic methods such as the laser-induced temperature-jump technique. In an alternative approach, the existence of the theoretically predicted downhill scenario for folding will be explored experimentally. Identification of downhill folders is important because during downhill folding all intermediate structures are potentially detectable. Additionally, kinetic methods with improved structural and/or time resolution will be developed. A two-dimensional version of the phi-analysis will be implemented to investigate the population dynamics of transition-state ensembles for folding. Time-dependent information on transient intermediates will be obtained for the first time from equilibrium nuclear magnetic resonance hydrogen-exchange experiments by performing them in kinetic coupling mode. The application of these kinetic techniques to study the folding of structural archetypes and downhill folders will provide direct information about the structural rules governing protein folding.
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| Naganathan, Athi N; Doshi, Urmi; Munoz, Victor (2007) Protein folding kinetics: barrier effects in chemical and thermal denaturation experiments. J Am Chem Soc 129:5673-82 |
| Naganathan, Athi N; Doshi, Urmi; Fung, Adam et al. (2006) Dynamics, energetics, and structure in protein folding. Biochemistry 45:8466-75 |
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