Carbonyl reactivity and post-translational modification of C2 domain lysine clusters

Chemical modification of the expressed proteome remains a major knowledge gap in biochemistry, in part due to the extreme heterogeneity of possible modifications. Recent advances in proteomics have revealed an urgent and unmet need to understand the nonenzymatic reactions that occur between lysine sidechains and cellular carbonyl compounds, including their impact on protein function. This process, called protein carbonylation, occurs constantly and becomes elevated during oxidative stress. Because clusters of lysine residues are important for binding signaling molecules such as phosphoinositide lipids in many cellular pathways, their modification can disrupt functions including membrane trafficking, yet their reactivity and impacts are remarkably understudied. C2 domains are a conserved family of membrane binding domains found in over 200 mammalian proteins. Our preliminary data suggest that a conserved phosphoinositide-binding lysine cluster on C2 domains is especially reactive toward carbonyl compounds, and we believe that C2 domain carbonylation is under-reported in the literature due to intramolecular crosslinking of lysine clusters. We will address this knowledge gap by testing the highly novel hypothesis that the structural properties of C2 domain lysine clusters make them especially reactive with cellular carbonyl compounds, forming carbonylated and crosslinked proteins with altered functionality. Here, we will first use biochemical and mass spectrometry methods to identify the structural determinants of reactivity and crosslinking under physiological conditions. Second, we will measure the functional effects of carbonylation on protein-membrane binding and cellular localization using biophysical and molecular biological tools. The project will integrate training opportunities in state-of-the-art research methods for undergraduates, Masters students, and high school teachers, as well as a new proteomics module for an undergraduate/graduate level biochemistry course.