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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM046732-06
Application #
6127480
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Wehrle, Janna P
Project Start
1996-04-01
Project End
2004-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
6
Fiscal Year
2000
Total Cost
$263,156
Indirect Cost
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Leroy, Emilie; Defour, Jean-Philippe; Sato, Takeshi et al. (2016) His499 Regulates Dimerization and Prevents Oncogenic Activation by Asparagine Mutations of the Human Thrombopoietin Receptor. J Biol Chem 291:2974-87
Matsushita, Chihiro; Tamagaki, Hiroko; Miyazawa, Yudai et al. (2013) Transmembrane helix orientation influences membrane binding of the intracellular juxtamembrane domain in Neu receptor peptides. Proc Natl Acad Sci U S A 110:1646-51
Defour, Jean-Philippe; Itaya, Miki; Gryshkova, Vitalina et al. (2013) Tryptophan at the transmembrane-cytosolic junction modulates thrombopoietin receptor dimerization and activation. Proc Natl Acad Sci U S A 110:2540-5
Itaya, Miki; Brett, Ian C; Smith, Steven O (2012) Synthesis, purification, and characterization of single helix membrane peptides and proteins for NMR spectroscopy. Methods Mol Biol 831:333-57
Staerk, Judith; Defour, Jean-Philippe; Pecquet, Christian et al. (2011) Orientation-specific signalling by thrombopoietin receptor dimers. EMBO J 30:4398-413
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Sato, Takeshi; Tang, Tzu-Chun; Reubins, Gabriella et al. (2009) A helix-to-coil transition at the epsilon-cut site in the transmembrane dimer of the amyloid precursor protein is required for proteolysis. Proc Natl Acad Sci U S A 106:1421-6
Sengupta, Parijat; Bosis, Eran; Nachliel, Esther et al. (2009) EGFR juxtamembrane domain, membranes, and calmodulin: kinetics of their interaction. Biophys J 96:4887-95
Liu, Wei; Fei, Jeffrey Z; Kawakami, Toru et al. (2007) Structural constraints on the transmembrane and juxtamembrane regions of the phospholamban pentamer in membrane bilayers: Gln29 and Leu52. Biochim Biophys Acta 1768:2971-8
Staerk, Judith; Lacout, Catherine; Sato, Takeshi et al. (2006) An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor. Blood 107:1864-71

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