This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have begun a major new initiative to measure the structural dynamics of the transmembrane helix protein of Glycophorin-A (GpA). This work will be the most ambitious to date application of 2D IR methods. The large, alpha-helical class of membrane proteins includes cell-surface receptors, ion channels, transporters and redox proteins. Many have a single transmembrane (TM) helix that homo-oligomerizes or associates with other TM helices to form bundles. These TM assemblies are of critical importance in a variety of biological situations and also have advantages for the study of folding in membranes. Integral membrane proteins are estimated to account for nearly one-quarter of all coding sequences in higher organisms, and more than half of all commercial drugs target this class of proteins. Their 3D structures have been studied by NMR on proteins anchored in phospholipid bilayers. A main purpose of this initiative is to measure structures of C=O, N-H, C-N, C(alpha)-H, hydrogen and other weak bonding of residues involved at the helix-helix interfaces of trans-membrane (TM) sections of GpA by means of isotopically selective 2D IR spectroscopy. We plan to obtain the frequency fluctuations to establish the motions of neighboring coupled bonds in the interface region of TM helices and discover how the C(alpha)-H bonds stabilize helix-helix interactions in transmembrane proteins. The 2D IR strategies will be designed to expose both the equilibrium dynamics mediated by the lipid fluctuations and the structural arrangements of coupled residues of GpA in terms of their spatial arrangements across the membrane. The purpose here is to better understand the hydrophobic effects, polarity, hydrogen bonding and other weak interactions between buried residues with the objective of determining the mechanisms and structural basis of helix association. The dual frequency 2D IR, will be used to access the C=O---D-C(alpha) interactions in the TM helical dimer of GpA and the dynamics in the hydrophobic interfacial region. Another purpose of this new approach is to elucidate the correlations between the structural fluctuations occurring at different secondary and tertiary spatial locations. The N-H/N-D exchange in transmembrane helices will be examined by means of dual frequency 2D IR.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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Special Emphasis Panel (ZRG1-MEDB (02))
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University of Pennsylvania
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