The objective of this CAREER project is to develop a detailed model for cytochrome c folding into its native three dimensional structure. The PI's approach is based on developing a probe that is sensitive to the folding process, and may therefore be used for its characterization. First, residue-specific detail of the structural changes that accompany cytochrome c folding will be generated, both under equilibrium and kinetic folding conditions by replacing cytochrome c C-H bonds with C-D bonds. The C-D bonds are non-perturbative and sensitive to their local environment. Most importantly, the C-D bond absorbs infrared light in a region of the spectrum that is completely free of other protein or denaturant absorptions, and may thus be easily observed and characterized. To characterize the folding of cytochrome c, the C-D spectra will be characterized as the protein folds, both under equilibrium conditions and under kinetic conditions. Specific attention will be given to how different parts of the protein interact with each other during the folding process, but also to how the protein interacts with a bound iron atom which is known to be important not only for function, but for folding as well. Computer simulation studies will be pursued to facilitate the understanding of the experimental observations. The combined theoretical and experimental approach should define the cytochrome c folding process with unprecedented clarity.
The techniques developed to characterize cytochrome c folding will be applicable to other proteins, and thus, these studies will have an impact on a broad range of other areas of biophysics. The educational activities in this CAREER project include training students at the interface of Chemistry and biology and expansion of internet based learning tools. This project is jointly funded by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences and the Experimental Physical chemistry Program in the Chemistry Division.
