Bren research summary: Heme protein folding and dynamics.

Project 1: Cytochrome c dynamics.

Using a combination of protein engineering, spectroscopy, and electrochemistry (in collaboration with Sean Elliott of Boston University), we are working to elucidate how mobility modulates electron transfer function of cytochromes c, heme proteins that function in energy transduction. On the “distal” side of the heme, we have characterized a novel “fluxional” motion of the heme axial methionine ligand that plays a role in tuning redox potential and protein stability. We have found that mutations of amino acids in contact with the axial methionine modify its orientation and dynamics, providing us with a powerful tool to probe subtle effects of variations in axial ligand orientation on protein stability and function. On the “proximal” side of the heme, we have discovered surprising differences is heme pocket dynamics, in particular in and near the Cys-X-X-Cys-His heme-binding motif characteristic of cytochromes c. Construction of mutants with altered heme pocket dynamics has allowed us to test relationships between dynamics and heme redox potential. These results are expanding our understanding of the impact of polypeptide and heme mobility on cytochrome structure and function, and of the fundamental factors that control redox potential in metalloproteins.

Project 2: Cytochrome c folding.

Protein folding is characterized by a large degree of inherent heterogeneity. The denatured state and folding intermediates display conformational heterogeneity, and the energy landscape model of folding requires that there is a heterogeneous collection of microscopic routes from conformational states within the denatured state, to the native state. The Bren group is working toward characterizing the complex process of protein folding both in terms of conformational and mechanistic complexity. On the protein conformation side, we are utilizing NMR spectroscopy to characterize denatured and partially denatured forms of our model protein, cytochrome c (utilizing both mitochondrial and bacterial species). This will allow us to test hypotheses regarding how native-like structure in the denatured state nucleates folding. To characterize heterogeneity on the folding energy landscape, we are collaborating with U of R colleague Todd Krauss to perform studies of cytochrome c folding on the single-molecule level. Ultimately, our goal is to perform an integrative analysis of heterogeneous protein folding, making use of our ability to engineer cytochromes c to test fundamental hypotheses as to how the polypeptide sequence directs folding.