SOLID-STATE NMR METHODS FOR STRUCTURAL STUDIES OF PHOSPHOLIPASE C

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 尽管最近通过X射线晶体学和溶液核磁共振光谱法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，结构变化与激活外周膜蛋白的非底物脂质，这是至关重要的酶活性的这些蛋白质，不了解在原子水平上，因为缺乏实验方法来探测特定的蛋白质-脂质相互作用。 在这个试点项目中，我们正在开发新的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，直接探测脂质-蛋白质相互作用。这些实验是在来自苏云金芽孢杆菌的磷脂酰肌醇特异性磷脂酶C（PI-PLC）上测试的。随着新的方法在手，PI-PLC的界面活化的结构基础可以检查。这些努力代表了我们的长期目标，阐明生物医学重要的人类磷脂酶的结构和机制的第一步。

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.