Interconnections of Protein Motions, Function and Stability in Enzymes and Photosynthetic Reaction Centers

University Of Washington, Seattle WA

Investigators

Abstract

The investigators will use a combination of experimental and theoretical approaches to explore the connections between protein dynamics, function, and thermal stability. Three proteins will be studied: photosynthetic bacterial reaction centers (RCs), an NADH oxidase of thermophilic bacteria (NOX), and human catechol O-methyltransferase (COMT). The theoretical work will include molecular-dynamics simulations of the three systems, and development of a general density-matrix treatment of reaction dynamics. The density-matrix treatment will be used to explore effects of temperature and the excitation energy on the kinetics of photochemical electron transfer in wild-type and mutant RCs. A new version of the density-matrix model will be developed for ground-state reactions, and will be used to examine the importance of vibrational dynamics and temperature dependence of kinetic isotope effects in electron-, hydride- and proton-transfer reactions. The unusual effects of temperature and urea on NOX will be studied by measuring the lifetimes, anisotropy and fluctuations of fluorescence from mutant enzymes with a single tryptophan residue in the active site or at other sites in the protein. The goal of the work on COMT is to understand how replacing valine 108 by methionine 16 A from the enzyme's active site causes the enzyme to fluctuate among a broader distribution of conformational states and destabilizes the protein to heat and chemical denaturants. The investigators will obtain structures of the 108V and 108M variants of COMT by both NMR and x-ray crystallography, and will use fluorescence, circular dichroism, and NMR spectroscopy to study the effects of substrates and temperature on the structures, motions and activities of the proteins in solution. Intellectual merit: The broad goals of this work are to increase understanding of electron-, hydride- and proton-transfer reactions, to dissect the interlocking roles of protein dynamics and stability in enzymatic catalysis, to clarify the differences between homologous enzymes from thermophilic and mesophilic organisms, and to provide missing information on the structure, dynamics and stability of COMT, an enzyme that plays an important role in human cognition. Broader impact: The theoretical developments and experimental results obtained for the three systems studied in this project should be applicable to many other systems. Photosynthetic bacterial RCs provide important models for the electron- and proton-transfer systems of plants and mitochondria. Understanding how RCs capture energy efficiently under a broad range of conditions also could lead to improved artificial systems for capturing solar energy. An understanding of how the 108M/V polymorphism affects the structure and dynamics of COMT could suggest ways to stabilize the 108M variant, possibly leading to new approaches to preventing or remedying the severe disorders that are linked to this enzyme. The project also will contribute to education by training at least two doctoral students in biochemistry, providing independent research projects for one or more undergraduate students each year, and providing material for discussion in the PI's graduate course in optical spectroscopy. The PI teaches basic biochemistry to approximately 300 undergraduates each year and also teaches a graduate course on reading and writing scientific papers. Promising high school students from under-represented minorities sometimes participate in research projects in the lab during the summer.