There has been an explosion in the number of high resolution structural models of proteins determined by crystallography and nuclear magnetic resonance (NMR). Yet as the number of known structures approaches 50,000 only a few hundred of those are of integral membrane proteins. The near future of structural biology is envisaged to make significant progress towards the determination of a sufficient number of unique protein structures to allow the prediction of any structure based on sequence alone. Unfortunately, this immensely challenging goal is made even more difficult by the significant barrier presented by integral membrane proteins and, perhaps surprisingly, by membrane anchored proteins. This proposal seeks to escape the restraints presented by these two important classes of proteins and set the stage for significant advances in their structural & biophysical characterization by solution NMR methods. Our approach is unusual and still emerging. It is based on our earlier work using reverse micelle encapsulation to defeat the slow tumbling problem presented by large soluble proteins. In that approach, the protein of interested is encapsulated within the protective aqueous core of a reverse micelle particle and the entire assembly is dissolved in a low viscosity fluid such as liquid ethane. In the low viscosity fluid, the reverse micelle particle tumbles faster than the protein dissolved in bulk water. This provides a significant improvement in the NMR relaxation properties governing the efficiency of the modern triple resonance experiments. The method allows high performance NMR spectra to be obtained on soluble proteins as large as 100 kDa without benefit of deuteration or the TROSY effect. Here we propose to adapt this approach to studies of integral and lipid-anchored peripheral membrane proteins. The reverse micelle method will be used structurally characterize the transmembrane segment of the Kcsa potassium channel. This will provide a foundation for the study of the dynamics of the polypeptide chain forming the channel, which are thought to be important to ion selectivity. Studies with Kcsa will set the stage for a test of the generality of the approach to structural characterization of integral membrane proteins. In parallel, we will employ reverse micelle encapsulation to study the role of covalently attached lipids in the anchoring of proteins to the membrane. We will investigate the structure-function relationships of the myristoylated HIV-1 matrix protein in its binding to the phosphoinositde PIP2. This interaction has been proposed to be promising target for pharmaceutical intervention. The classic myristyl-switch protein recoverin will be used to further define the approach. Analogous studies are proposed for the palmitoylated proteins BET3, a component of a membrane targeting complex, and UL11, the tegument protein of the herpes simplex-1 virus. Interactions between proteins and ligands normally embedded in the membrane will also be investigated. These studies should establish the reverse micelle solubilization method as general approach to structural and dynamic studies of integral and lipid-anchored membrane proteins.
Although integral membrane proteins are vital to many fundamental processes in human biology and represent the majority of targets for pharmaceutical intervention in medicine, it remains extremely difficult to characterize them in structural detail. Here we will develop a new method to determine the structure of integral membrane proteins by nuclear magnetic resonance. This new method also provides a unique view of membrane anchored proteins, some of which are potential targets for anti-viral therapies. ? ? ?
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