The proposed research is to establish the structural basis for how basic-aromatic clusters in membrane-associated proteins are able to sequester specific lipids into lateral membrane domains, how they induce membrane curvature, and how they allow specific peptides to cross cell membranes. The proposal is focused primarily on the MARCKS protein, but also investigates the structure and binding of a wide range of additional basic-aromatic clusters in order to establish how membrane binding and penetration depends on protein sequence.
The specific aims of the proposal are to determine 1) the location and conformation of the MARCKS (151-175) effector domain in membrane bilayers, 2) how the protein sequence determines the depth of penetration into bilayers, 3) whether and how basic-aromatic clusters sequester cholesterol, and 4) the role of basic-aromatic clusters in membrane curvature and peptide penetration. The research relies primarily on magic angle spinning NMR measurements of membrane multilayers and solution NMR of membrane bicelles. The NMR approaches are complemented by fluorescence techniques to investigate binding and membrane interactions. ? ? Basic-aromatic clusters may be a common mechanism for sequestering phosphoinositides and cholesterol in lateral domains. As a result, their importance in human health is significant. Understanding how the levels of phosphoinositides are regulated is central to signal transduction pathways which are involved in cell growth and differentiation, while understanding how the distribution of cholesterol is controlled in plasma membranes has relevance for cardiovascular and related diseases. The strategy is to progress from simple domains induced by the MARCKS (151-175) peptide to the more complex caveolar domains. The NMR measurements proposed will provide high resolution detail which is not available from crystallographic methods or more traditional solution NMR methods. ? ?
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