EXTENSION OF SOLUTION NMR TO 50 KDA PROTEINS AND BEYOND

描述（改编自摘要）：

DESCRIPTION (Adapted from abstract): Nuclear magnetic resonance (NMR) spectroscopy continues to be a central technique in the determination of high resolution models of the structure and dynamics of proteins, nucleic acids, and their complexes. Nevertheless, a significant fraction of the proteins that are known through the analysis of the genomic sequence are inaccessible to solution NMR methods. This is because they are too large, either by themselves and because they require association with large assemblies of lipids, and therefore tumble too slowly for optimal NMR performance. This proposal seeks funds to develop a new approach to rendering the NMR relaxation properties of large proteins amenable to the comprehensive and efficient application of modern triple resonance and related NMR techniques. The basic approach is to arrange simply for the protein molecule to tumble as a much smaller protein. The central goal of this proposal is: To develop a reverse micelle system in a low viscosity fluid capable of solvating, in water, proteins as large as 100 kDa. The applicant calculates that solubilization of proteins in reverse micelles dissolved in liquefied butane, propane, or ethane will tumble with sufficiently short correlation times to allow the full battery of existing triple resonance techniques to be applied, even without benefit of deuteration. Modest pressures ranging up to 50 bar will be required to liquefy these alkanes. Optical spectroscopy under pressure will be employed as a rapid assay. A high resolution probe will be used to characterize the NMR properties of the various test proteins. Should this strategy prove successful, it could provide a general, flexible and extremely powerful approach to using high resolution solution NMR techniques to characterize proteins up to 100 kDa in size and may also offer a route to examining membrane associated or integral membrane proteins.