This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
Membrane proteins constitute 30% of the human genome and, therefore, are of tremendous interest for modern structural biology. However, their X-ray high-resolution structures are often difficult to obtain due to the challenges in crystallization posed by the presence of lipids. From this view, solid-state Nuclear Magnetic Resonance (SSNMR) spectroscopy represents a unique alternative method to study membrane proteins in their native lipid environment without the need for crystallization. This is due to the recent advances in sample preparation techniques using magnetically aligned bilayers, which provide both membrane mimetics and full hydration. In addition, SSNMR methods yield directly orientationally dependent observables as input for structure determination at atomic resolution. This project is aimed at answering important biological and biophysical questions by radically improving current SSNMR techniques. The specific aims of this project are: (i) complete three-dimensional structure determination of membrane proteins (ii) creation of new methods for the studies of inter- and intramolecular contacts in membrane proteins; (iii) detection of ligand binding and elucidation of the associated conformational changes; (iv) efficient algorithms for calculating protein structures from angular-dependent NMR restraints; (v) applications to specific proteins and their ligands. The latter will include ion channels and their agonists and antagonists; viral proteins; and larger membrane-bound receptors (adrenergic and olfactory receptors). These goals will be accomplished through the development of new SSNMR experiments on doubly labeled protein samples for both spectral assignment, elucidation of intramolecular interactions, and measurement of orientationally dependent observables such as dipolar couplings and chemical shifts. The final goal is to assign the spectrum and calculate complete three-dimensional protein structure from a single uniformly labeled protein sample solely based on SSNMR data.
This project will involve postdocs, graduate and undergraduate students with different ethnic and geographical backgrounds. The methods to be developed herein are expected to find their use in other applications including solution NMR and NMR of highly ordered inorganic compounds such as zeolites and other nanostructures. The basic results of this research on solid-state NMR methods of protein structure determination in membranes will be included as part of the curriculum for a graduate course on Macromolecular Structure covering modern methods of biological structure determination. The computational algorithms and simulations programs will be made available to interested research groups.
