This project contains an integrated research and educational plan involving the mentoring, education, and training of undergraduate and graduate students in biophysical research. The goal of the project is to understand the molecular complexity of cytochrome c (cyt c) folding, providing data vital for characterization of energy landscapes for cyt c. The measurements will test two fundamental hypotheses relevant to understanding protein folding: 1) The extent and distribution of helical propensities of the amino acid sequences play important roles in determining the stability, population, and heterogeneity of the common cyt c folding intermediate; 2) Cyt c molecules primarily sample interchanging extended and compact conformations on the folding energy landscape, the relative populations of which depend on denaturation conditions. The experimental approach will be to employ optical studies of folding on the single-molecule (SM) level, which will allow analysis of conformations and dynamics of individual members of an ensemble of polypeptides. Proposed studies will involve both time-integrated and time resolved SM fluorescence resonance energy transfer measurements, whereby mutations to residues on the protein surface will allow for access to dynamics of protein structure. Cyt c offers a number of key advantages: 1) The covalently attached heme group provides an intrinsic acceptor in energy transfer experiments, thus simplifying sample preparation and minimizing perturbation of protein folding by derivatization. 2) Cyts c are highly soluble, stable, and robust proteins. 3) Cyts c fold simply (i.e. approximated by a two-state model), which is important, as complexity will arise as members of the ensemble are probed. 4) Cyt c folding is well characterized on the macroscopic level, but not yet characterized on the single-molecule level.
