In order to understand how membrane proteins express their diverse biological functions, it is essential to determine their structures. However, membrane proteins present extraordinary technical difficulties for the most commonly used x-ray crystallography and NMR spectroscopy methods of structure determination, which were developed with water soluble globular proteins in mind. Alternative methods of protein structure determination are needed to analyze membrane proteins. The success of the research supported by this grant, especially in the development of a spectroscopic strategy invoking the powerful methods of high resolution solid-state NMR spectroscopy, enables us to propose structural studies of membrane proteins relevant to problems of medical urgency, including AIDS and heavy metal poisoning, and scientific importance, including the architecture of membrane proteins, membrane anchoring of proteins, protein processing, and modification of membrane properties by proteins. Although, experimental NMR studies of membrane proteins are near the limit of feasibility with currently available technology, we have demonstrated that by utilizing the most powerful spectroscopic experiments, spectrometers with the highest magnetic fields, and comprehensive isotopic labeling schemes, it is possible to obtain high resolution solid-state NMR and multidimensional solution NMR spectra of membrane proteins suitable for structural analyses. The overall goals of the research are to develop generally applicable NMR methods for determining the structures and describing the dynamics of membrane proteins and to apply these methods to membrane proteins that have interesting biological functions and are experimentally tractable. These methods will be applied to HIV-l Vpu protein, merP and merT proteins of the bacterial mercury detoxification system, and the coat and procoat proteins of filamentous bacteriophages. In addition, these methods will be applied in collaborative studies to the pulmonary surfactant proteins SP-B and SP-C and to subunit c of the F1F0 ATPase. These membrane proteins participate in biological processes not associated with globular proteins, are relatively small with between 35 and 116 residues, and can be prepared by expression in bacterial systems, automated solid-phase peptide synthesis, or both in substantial quantities specifically, selectively, or uniformly labeled with stable isotopes.
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