Membrane proteins typically contain hydrophobic stretches of roughly 18-25 residues that independently fold into alpha-helical secondary structure and span the lipid bilayer. These proteins may have only a single transmembrane helix or may contain several membrane-spanning helices that pack together to form the core of the protein. The transmembrane helix in single-pass membrane proteins serves to anchor the protein in the membrane and often contains sequence motifs that drive the assembly of a protein complex. The objective of the proposed research is two-fold: to refine methods for structural measurements in membrane bilayers using solid-state NMR approaches and to address basic questions involving how transmembrane helices interact. The three specific aims which form the core of this proposal focus on membrane proteins of biomedical interest. - The E5 papillomavirus protein. E5 is a viral protein that causes cell transformation by serving as a molecular scaffold for dimerization of the PDGF-beta receptor. High resolution solution NMR studies in detergent micelles and magic angle spinning NMR measurements in membrane bilayers are proposed for establishing the structure of the E5 dimer and its interactions with the PDGF-beta receptor. - The neu/erbB-2 receptor. Mutation of a single amino acid in the transmembrane domain of the neu receptor, Val664Glu, causes constitutive activation leading to breast and ovarian cancer. MAS NMR distance measurements are proposed for refining our dimer structure of neu transmembrane helices. - Phospholamban. Phospholamban is a 52-residue integral membrane protein that regulates the calcium ATPase in the sarcoplasmic reticulum of cardiac and smooth muscle. MAS NMR distance measurements are proposed for establishing the detailed packing interactions of the transmembrane and cytosolic helices, and how phospholamban regulates the calcium ATPase.
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