TEMPERATURE-DEPENDENT CONFORMATIONAL SAMPLING OF NATIVELY UNFOLDED NUCLEOPORINS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 膜蛋白的结构研究受到衍射型高分辨率晶体可用性的限制。在脂质立方相(LCP)基质中(或在介观中)结晶已被证明可以产生高质量的具有挑战性的膜蛋白晶体，如人G蛋白偶联受体。介观技术的广泛应用要求鉴定具有特定相性质的新脂类，能够稳定具有大范围大小和结构的蛋白质。在创新膜蛋白技术联合中心(JCIMPT)，我们正在设计和合成这种脂类。在用于特定应用之前，应对新型脂质基质的相和结构行为进行彻底的表征。我们建议通过在96孔夹层板中制备样品，并在BioSAXS光束线上进行现场测量，来克服用于X射线研究的常规脂质样品制备的障碍。这种方法将使我们能够在模拟结晶试验中遇到的条件下，以高通量模式筛选洗涤剂、添加剂脂类、蛋白质以及各种沉淀剂对脂质基质的影响。在这项提议的持续期间，我们预计将从更大的合成候选者库中预选出10-12种最有希望的新脂质的充分特征。所得结果将对选择合适的脂类和指导介观结晶实验是必不可少的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.