SOLID-STATE NMR METHODS FOR STRUCTURAL STUDIES OF PHOSPHOLIPASE C

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 尽管最近X射线结晶学和溶液核磁共振波谱在膜蛋白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，与非底物脂类激活外周膜蛋白相关的结构变化对这些蛋白质的酶活性至关重要，由于缺乏探索特定蛋白质-脂质相互作用的实验方法，因此在原子水平上还不能理解。 在这个试点项目中，我们将开发基于魔角旋转固态核磁共振的新方法来研究外周膜蛋白的结构和功能。特别是，将建立二维和三维实验，直接探索脂质-蛋白质相互作用。这些实验将在苏云金芽孢杆菌的磷脂酰肌醇特异性磷脂酶C(PI-PLC)上进行测试。随着新方法的问世，PI-PLC界面活化的结构基础将被研究。这些努力代表了我们朝着阐明具有生物医学重要性的人类磷脂酶的结构和机制的长期目标迈出的第一步。

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. 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 of membrane-associated proteins. As the 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 lack of experimental methods to probe specific protein-lipid interactions. In this pilot project, we will develop 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 will be established to probe directly lipid-protein interactions. These experiments will be 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 will 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.