NOVEL PREPARATION METHOD FOR ALIGNED MEMBRANE PROTEINS

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This research proposal is aimed at one of the most important and difficult problems associated with membrane protein structure determination by NMR of oriented assemblies; namely sample preparation. it has been shown that the quality of the oriented sample has a dramatic effect on the results of the NMR experiment that subsequently affects the ability to use the data for structure determination. Additionally, although NMR is generally considered a non-destructive technique, long experiment times and repetitive radio frequency pulses inevitably lead to increased sample temperature and hence sample degradation. Advances in sample preparation techniques such as the addition of metal ions to bilayer preparations and chelating agents to bicelle assemblies are proposed. Additionally, the possibility of orienting samples by novel preparation methods that are adaptations of Langmuir-Blodgett methodologies will be explored. These amendments to already existing preparative procedures as well as new methods of sample preparation are aimed at improving the overall temperature stability and longevity of the oriented protein/phospholipid assemblies. The overall quality of the samples prepared by these proposed techniques will be assessed by solid-state NMR. The structures of two membrane proteins, the coat protein of fd bacteriophage and Vpu of HIV-l, prepared in these ways, will be investigated by a variety of solid-state NMR techniques.