Redox Reactivity of Thioredoxin Disulfide Bonds

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Intellectual Merit.While disulfide bonds are ubiquitous redox-active cofactors used in biology for catalytic, structural and signaling roles, a molecular level understanding of the principles that govern disulfide bond reactivity has proven elusive. In this work, direct electrochemistry will be used as a primary tool to characterize the influence of protein sequence and structure upon the redox properties of the thioredoxin (Trx) superfamily of proteins. Trx proteins are found throughout all the kingdoms of life: a paradigm of disulfide-based mechanisms for charge transfer and redox-homeostasis. While Trx proteins engage in diverse functions, and serve as modules that are a part of complex biological functions, there is a knowledge-gap in our understanding of how Trx proteins are tuned to be specifically reactive. Thus, this project will directly test models of how disulfide bonds are used in Biology, a question critical to many areas of biological chemistry, where the disulfide bond redox state is an essential trait to determine reactivity, signaling, and protein folding. A central question addressed in the project is, "How does Nature tune the redox chemistry of a disulfide bond?" In this project, the PI will (1) Assess the natural range reduction potentials found in Trx proteins, (2) Determine the influence of sequence and structure on reduction potentials, and (3) Examine the stability of disulfide-bond:iron-sulfur cluster complexes. The project involves the use of protein electrochemistry due to the highly sensitive, rapid and quantitative nature of the methodology. The results of the project will provide a new detailed understanding of how thioredoxins are used in Nature's diversity to maintain redox homeostasis.Broader ImpactThe most immediate impact will be upon the training of scientists at all levels (undergraduates, graduate students, post-doctoral faculty fellows) to think quantitatively and chemically in the field of redox biochemistry. However, due to the pervasive and central role that disulfide bond redox chemistry, redox homeostasis and oxidative stress play in the biological chemistry of all life, the broader impacts of this work will touch deeply upon the interface of chemistry and biology. Whether in plant biochemistry, bioenergy sciences or microbial physiology - thioredoxins are a paradigm of understanding how disulfide bonds are used to achieve chemical change in Life. Illuminating this process in a fundamental way will translate into new appreciation of fundamental biology.The research efforts of the PI are paired with education activities in the classroom that brings contemporary biological chemistry to the freshman chemistry audience, training of teacher-scholar postdoctoral fellows at Boston University via the Postdoctoral Faculty Fellow Program, and serving as an instructor in an upcoming graduate/postdoctoral training course in Bioinorganic Chemistry (to be held at Penn State University in 2012), which will disseminate the experimental methodologies of protein electrochemistry to a much broader audience.