PHYSICAL BIOCHEMISTRY, PROTEINS, & MOLECULAR RECOGNITION

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The long range goal of our research is to understand the relationships between protein sequence and higher order structure and function. In the past five years we examined interactions within a protein; specifically, the interface between the N- and C-terminal helices of the electron trader protein cytochrome c (Cc). In the next five years we wish to use our expertise to move in a new direction and study interactions between proteins as well. Understanding interactions between proteins is the key to understanding signal transduction, a process essential to nearly all biological processes from development to cancer. Understanding interactions within proteins is crucial for interpreting the data from genome projects and designing novel proteins with medically useful functions. Understanding biological electron transfer is important because genetic defects in electron transfer proteins are thought to play a role in aging and in degenerative diseases. Between proteins. Three complexes will be examined: Cc/cytochrome c peroxidase (CCP), Cc/flavo-cytochrome b2, and Cc/cytochrome b5. Currently there are no detailed equilibrium thermodynamic data on any of these complexes, so the proposed research will lead to new and important information about molecular recognition. Equilibrium binding data will be obtained using isothermal titration calorimetry and electron transfer rates will be measured by our collaborators Millett & Durham. Three hypotheses will be tested: 1) hydrophobic contacts control the strength of binding, but ionic interactions control the rate of electron transfer; 2) contacts in the previously proposed structural models of the complexes are important for both binding and electron transfer; 3) a weak Cc binding site on CCP dominates electron transfer. The binding data will also be used to test several recent hypotheses about the role of surface area and solvation in protein complex formation. Within proteins. We will test the widely held belief that molten globules contain few, if any, specific native long range interactions. We have recently shown that the interaction between the N- and C-terminal helices persists in the molten globule. We now propose to map interactions between the heme, the 6Os helix and the C-terminal helix. The equilibrium thermodynamics of denaturation will be measured using differential scanning calorimetry.