Helical membrane proteins have been difficult targets for structural characterization but are ∼30% of the typical genome. This dissertation is focused on the extension of solid state nuclear magnetic resonance methodology to be able to routinely obtain structural restraints and calculate high resolution structures for membrane proteins in a bilayer environment. Rv1861, a M. tuberculosis protein involved in the regulation of transglycosylase activity is used as a model system.
One bottleneck for obtaining structure restraints is the preparation of high quality proteoliposome samples. Rv1861 is readily purified in one detergent but the same detergent fails to produce samples of sufficient quality for structural studies. Based on the physical properties of the original detergent, new detergents were selected and a screening process was devised which improves the reconstitution into liposomes and results in high quality samples. To date, a general procedure for optimizing the detergent used in the preparation of proteoliposome samples of helical membrane proteins for solid state nuclear magnetic resonance has yet to be published.
Another bottleneck for obtaining structural restraints is the assignment of resonances. For both oriented sample and magic angle spinning techniques methods are devised that allow certain crucial assignments to be made. Enough assignments are obtained allowing the measurement of a limited set of structural restraints for Rv1861. The restraints obtained from both techniques are used together resulting in a high quality structure using fewer restraints than required using either technique alone. The methods illustrated here for Rv1861 are generally applicable to all membrane proteins and are expected to drastically increase the rate at which atomic resolution structures are obtained for helical membrane proteins in a lipid bilayer environment.
|Commitee:||Alamo, Rufina, Bruschweiler, Rafael P., Fadool, Debra A., Miller, Brian|
|School:||The Florida State University|
|School Location:||United States -- Florida|
|Source:||DAI-B 75/10(E), Dissertation Abstracts International|
|Keywords:||Helix, Lipid bilayer, Membrane protein, Protein structure, Solid state nuclear magnetic resonance, Tuberculosis|
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