Our long-term goal is to understand the mechanisms of protein oxidation by distinct reactive oxygen species, and their role in age- and oxidative stress-related changes of intracellular calcium homeostasis in skeletal and cardiac muscle. We shall focus on (i) a detailed investigation of the in vitro and in vivo oxidation by reactive oxygen species of two major proteins involved in cellular calcium regulation: SR Ca-ATPase and calmodulin, and (ii) on the characterization of the molecular mechanism by which reactive oxygen species react with particular protein domains as a function of solvent accessibility, metal-binding propensities, and anchimeric effects exerted by neighboring amino acids. Specifically, we aim to: (1) characterize the molecular products of the in vitro oxidation of Ca-ATPase and calmodulin by hydroxyl radicals, superoxide anion radical, hydroperoxide, single oxygen, peroxyl radicals, oxyl radicals, nitric oxide, nitrogen dioxide and peroxynitrite (oxoperoxonitrate); (2) compare the product patterns of the in vitro oxidation with protein oxidation product patterns obtained in vivo from proteins isolated from rats of various defined ages; (3) characterize the oxidation of the proteins, particularly calmodulin, as a function of solution structure and binding to target proteins; (4) investigate in detail why certain protein domains are more susceptible to oxidation than others. The combination of aims (1) and (2) will yield information on whether and which reactive oxygen species are damaging proteins in the course of aging of an organism. This is an important goal since various reactive oxygen species are implied in the """"""""free radical theory of aging,"""""""" but, based on chemical studies, they are expected to react very differently with target proteins forming different molecular products. The combination of aims (3) and (4) will yield information on how protein sequence and structure contribute to the oxidation sensitivity and product specificity in proteins exposed to oxidative stress. Experimentally, these studies will involve the generation of reactive oxygen species by chemically defined means, and the analysis of protein products by HPLC/MS and NMR. With these studies, we envisage a contribution to the basic understanding of oxidative stress and aging on the molecular level, an approach which should benefit a more rational development of dietary or pharmaceutical therapies of oxidative stress- and/or age-related dysfunctions.
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