TEMPERATURE-DEPENDENT CONFORMATIONAL SAMPLING OF NATIVELY UNFOLDED NUCLEOPORINS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Structural studies of membrane proteins are limited by the availability of crystals diffracting to high resolution. Crystallization in lipidic cubic phase (LCP) matrices (or in meso) has proven to yield high quality crystals of challenging membrane proteins, such as human G protein-coupled receptors. Broader applications of in meso techniques require identification of new lipids with specific phase properties capable of stabilizing proteins with large range of sizes and architectures. At the Joint Center for Innovative Membrane Protein Technologies (JCIMPT) we are working on design and synthesis of such lipids. The phase and structural behavior of novel lipid matrices should be thoroughly characterized prior to being used in specific applications. We propose to overcome the obstacles associated with conventional preparation of lipid samples for x-ray studies, by preparing samples in 96-well sandwich plates and measuring them in situ at the BioSAXS beamline. This approach will allow us to screen for effects of detergents, additive lipids, proteins as well as great variety of precipitants on the lipidic matrices in the high-throughput mode at conditions mimicking those encountered during crystallization trials. During the duration of this proposal we anticipate to fully characterize 10-12 most promising novel lipids pre-selected out of a larger pool of synthesized candidates. The obtained results will be indispensable for selection of proper lipids and guiding in meso crystallization experiments.