SOLID-STATE NMR METHODS FOR STRUCTURAL STUDIES OF PHOSPHOLIPASE C

用于磷脂酶 C 结构研究的固态核磁共振方法

• 批准号: 8364946
• 负责人: Tatyana Polenova
• 金额: $ 3.4万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364946
• 关键词: Architecture; Bacillus thuringiensis; Centers of Research Excellence; Crystallization; Funding; Goals; Grant; Hand; Human; Knowledge; Lipids; Magic; Membrane Proteins; Methods; NMR Spectroscopy; National Center for Research Resources; Peripheral; Phosphatidylinositols; Phospholipase; Phospholipase C; Pilot Projects; Principal Investigator; Production; Proteins; Research; Research Infrastructure; Resources; Solutions; Source; Structure; Testing; United States National Institutes of Health; X-Ray Crystallography; base; cost; interfacial; research study; solid state nuclear magnetic resonance; success; two-dimensional

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. Despite the recent successes in structural characterization of membrane proteins by X-ray crystallography and solution NMR spectroscopy, these studies remain a challenge due to the inherent insolubility and difficulties in crystallization of many membrane-associated proteins. As a result, our knowledge about the architecture and function of membrane proteins remains sparse. In particular, structural changes associated with activation of peripheral membrane proteins by non-substrate lipids, which are critical for the enzymatic activity of these proteins, are not understood at the atomic level because of a lack of experimental methods to probe specific protein-lipid interactions. In this pilot project, we are developing new magic angle spinning solid-state NMR based methods to study the structure and function of peripheral membrane proteins. In particular, two- and three-dimensional experiments are established to probe lipid-protein interactions directly. These experiments are tested on a phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis. With the new methods in hand, the structural basis of the interfacial activation of PI-PLC can be examined. These efforts represent the first step toward our long-term goal to elucidate the structure and the mechanism of biomedically important human phospholipases.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 尽管最近通过 X 射线晶体学和溶液核磁共振波谱在膜蛋白的结构表征方面取得了成功，但由于许多膜相关蛋白固有的不溶性和结晶困难，这些研究仍然是一个挑战。因此，我们对膜蛋白的结构和功能的了解仍然很少。特别是，由于缺乏探测特定蛋白质-脂质相互作用的实验方法，与非底物脂质激活外周膜蛋白相关的结构变化（这对于这些蛋白质的酶活性至关重要）在原子水平上尚不清楚。 。 在这个试点项目中，我们正在开发新的基于魔角旋转固态核磁共振的方法来研究外周膜蛋白的结构和功能。特别是，建立了二维和三维实验来直接探测脂质-蛋白质相互作用。这些实验在苏云金芽孢杆菌的磷脂酰肌醇特异性磷脂酶 C (PI-PLC) 上进行了测试。利用现有的新方法，可以检查 PI-PLC 界面激活的结构基础。这些努力代表了我们朝着阐明生物医学上重要的人类磷脂酶的结构和机制的长期目标迈出的第一步。