Membrane protein folding involves oligomerization of independently stable helices into proper tertiary structures. Knowledge on how proteins oligomerize in the membrane is currently limited by the paucity of high-resolution structures of small oligomeric peptides in lipid bilayers, in particular the lack of intermolecular structure restraints. The first major objective of this project is to develop solid-state NMR (SSNMR) techniques that directly determine the oligomeric number of peptides in lipid bilayers and that yield site-specific intermolecular distance constraints. A 1H-driven anisotropic spin diffusion method under magic-angle spinning will be used to determine the oligomeric number of peptides. The time constants of spin diffusion will be analyzed to yield semi-quantitative distances between different molecules in the aggregate. More quantitative distances will be extracted from heteronuclear dipolar couplings, especially the couplings between protons and low-frequency heteronuclear spins such as 13C and 2H. The high gyromagnetic ratio of the proton will extend the distance reach of SSNMR. 1H-2H dipolar recoupling will be specifically explored to enable the study of intermolecular interfaces through methyl-deuterated amino acid sidechains. This 1H-X distance technique will be extended to allow faster spinning speeds and to increase the efficiency of 1H homonuclear decoupling to enable the measurement of longer distances. With the development of these methods, the structure of two homo-oligomeric helical bundles, the transmembrane peptide of the M2 protein (M2-TMP) of the influenza A virus, and the designed coiled-coil peptide GCN4-MS1, will be investigated. Although the oligomeric states of these peptides are known in detergent micelles, they have not been directly determined in lipid bilayers. Moreover, no intermolecular distances and packing information are known. The aggregation states will now be directly measured in lipid bilayers using 19F and 13C spin diffusion. Intermolecular distances will be extracted both from homonuclear spin diffusion curves and from heteronuclear dipolar recoupling experiments. Furthermore, how the aggregation state and the helical bundle diameter are affected by the membrane composition, membrane thickness, and the amino acid sequence, will be studied. The effect of the size and polarity of the sidechain on the stability of the helical bundle will also be examined through sequence mutations.
This project will have wide-ranging impacts on membrane biophysics, providing heretofore unavailable high-resolution structural information on membrane peptide assemblies. It will extend the frontier of NMR structural biology from secondary and tertiary structure determination to quaternary structure determination. It will enhance the training and education of undergraduate students, graduate students, and under-represented groups. The interdisciplinary nature of the research will benefit graduate students and facilitate the recruiting of undergraduate researchers in chemistry, biochemistry, and biophysics. Women students and undergraduate researchers, who have traditionally participated well in the PI's research, will continued to be encouraged to join the effort. Knowledge gained on membrane protein folding through this project will be incorporated into the teaching of undergraduate thermodynamics, on topics such as Gibbs free energy and chemical equilibrium.
