Electron transfer reactions between proteins control the generation, flow and use of energy in biological systems. Central to many of the processes involved in these events are the cytochromes c. A major goal of this proposal is to contribute to the understanding of the solution structure and dynamics of cytochrome c both free and in complex with biologically relevant structures such as protein redox partners and lipid. The studies described here will augment, unify and clarify a wide body of information regarding the function of this essential protein. Studies are proposed to take advantage of our recently determined high resolution models for the structure cytochrome c in its two redox states in order to directly and comprehensively test a variety of theoretical treatments of cytochrome c mediated electron transfer and to also provide a template for the interpretation of emerging studies of the dynamics of this protein. These efforts lead naturally to quantification of the structural and dynamic consequences of mutations of the cytochrome c molecule that affect stability, redox potential and other important physical parameters of the protein. The complex between cytochrome c and cytochrome b5 and the structural changes that occur upon association of cytochrome c with lipid will also be examined. In a major redirection of this grant we have initiated two exciting new projects. One is centered on taking advantage of the ability for facile mutagenesis of R. capsulatus cytochrome c2 in an effort to identify fleetingly populated folding intermediates by pulsed hydrogen exchange labeling. Parallel to these studies will be the determination of the folding pathway of apocytochrome b562 from the unfolded to the molten globule state. These two folding subprojects will hopefully get at the next issue in protein folding: the underlying themes defining folding pathways. The second new project is an effort to push the use of protein design and engineering to construct minimalist proteins which encaspuslate the essential features of various electron transfer proteins. These systems, termed maquettes, offer an simplified environment within which fundamental issues of inter- and intraprotein electron transfer can be probed. All of these studies will bear directly upon the biological function of the cytochrome class of proteins and will contribute to the understanding of the properties governing electron transfer within and between proteins in general. These studies will make extensive use of modern multinuclear and multidimensional NMR spectroscopy, a variety of methods for the determination of the solution structures of proteins to high resolution, and will also employ detailed analysis of NMR relaxation and hydrogen exchange phenomena to quantitate internal motion in these systems.
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