The main objective of this proposal is to develop solid-state NMR techniques that are capable of describing the structures and dynamics of immobile and non-crystalline supramolecular assemblies of membrane proteins that have been largely inaccessible to the traditional experimental methods of structural biology, x-ray crystallography and solution NMR spectroscopy. The key problem hampering widespread use of solid-state NMR spectroscopy for structure determination of membrane-bound peptides and proteins is the lack of a comprehensive resonance assignment protocol. To overcome this problem, multinuclear, multidimensional methods are proposed. Several biologically active membrane-bound polypeptides will be used in the development of methods proposed in this application. Since spin interaction tensors are important in the conformation and dynamics studies of uniaxially oriented membrane-associated polypeptides, simple methods are proposed to study chemical shift anisotropy and dipolar coupling tensors associated with nuclei (~sN and 13C) on the backbone of the peptide of interest rather than using the data from a model peptide in order to probe the global as well as local dynamics of the peptide incorporated in lipid bilayers. Two educational initiatives are proposed to develop Biophysics and Chemical Biology courses that reflect the expansion of this science into the interface between Biology and Physics.
General Significance: More than half of all proteins are strongly associated with cell membranes. Membrane-associated proteins are responsible for many of the properties and functions of biological systems. In order to obtain a detailed understanding of how membrane proteins carry out their functions and how these functions can be altered for biological or biotechnological purposes, it is necessary to determine their three-dimensional structures. Determining the structures of membrane proteins is one of the most important challenges in science at the present time. In this research project, new solid-state NMR techniques will be developed and applied to study several biologically active membrane-bound peptides. The methods and goals of this research will demonstrate the power of solid-state NMR methods applied to systems of fundamental interest, and will also provide a mad map for other investigations of complex membrane proteins using solid-state NMR. Two educational initiatives are proposed to develop Biophysics and Chemical Biology courses that reflect the expansion of this science into the interface between Biology and Physics.
