Elucidating Protein Folding Mechanisms

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DESCRIPTION (provided by applicant): The production of functional proteins requires that polypeptides find a unique conformation in a vast space of incorrect folds. The consequences of failure are severe; misfolded proteins are implicated in a rapidly growing list of debilitating illnesses that includes type II diabetes as well as AIzheimer's and Creutzfeldt-Jakob diseases. Partially folded polypeptides are key intermediates in both the proper assembly of proteins and in the formation of harmful misfolded structures. Measurements of fluorescence-energy-transfer (FET) kinetics can provide nanosecond timescale snapshots of donor-acceptor distance distributions in dye-labeled proteins as they evolve to native states. These experiments can reveal the polypeptide conformations that are present during folding. Experiments have been designed to address four specific aims: (1) elucidation of the relative energetics of rapidly equilibrating compact and extended structures during folding; (2) characterization of the structures and heterogeneity of compact folding intermediates; (3) exploration of the effects of reduced conformational space by forming native and near-native contacts prior to refolding; and (4) determination of the extent of core hydration during heme protein folding. Natural tryptophan fluorophores as well as covalently attached dyes will serve as donors and acceptors in studies of compact-extended conformational equilibria during the folding of cytochromes c and c'. The role of compact and extended polypeptides in the folding of acyl-coenzyme A-binding protein also will be investigated by FET kinetics. The intracellular environment in which proteins normally fold is densely packed with macromolecules: FET kinetics will be used to probe the effects of high macromolecular concentrations on the compact-extended conformational equilibrium during folding of cytochrome c. The conformational heterogeneity of compact folding intermediates will be evaluated using FET kinetics measurements. Native and near-native contacts in unfolded heme proteins will be created by the introduction of His residues in mutants: the effects of these contacts on folding will be examined in Fe(lll) proteins as well as Co(Ill)- and Zn(ll)-substituted derivatives. Solvent deuterium isotope effects on the triplet lifetime of Zn-cytochrome c' will be measured to evaluate changes in solvation of the porphyrin during folding.