LANOSTEROL BIOSYNTHESIS IN THE MEMBRANE ENVIRONMENT

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们建议研究膜酶2，3-氧化角鲨烯环化酶（OSC）和它的脂质环境之间的功能耦合。 OSC是胆固醇生物合成和调节的关键酶。 它催化环化反应产生羊毛甾醇，类固醇和激素的核心骨架。 为了达到其生物学底物，OSC -像单位膜酶家族的所有成员一样-稳定且永久地仅存在于双层的一个小叶中。 与该蛋白质家族中的其他酶一样，OSC使用大的疏水表面接触脂质双层，并利用延长的疏水通道在其活性位点和膜之间穿梭其疏水反应物。 拟议的工作的重点是建立的方法，这将使酶促反应和膜环境的性能之间的相互关系的研究。我们的长期目标是了解蛋白质的构象变化与底物和产物转移到脂质双层并形成脂质双层之间的相关性（由膜介导）。 最初的表征将集中在表达，纯化和重组的人OSC成膜的明确定义的组成和组织。使用这些协议，我们将调查所需的有效的基板介绍和膜的物理化学性质对催化的影响的膜条件。 为此，将使用富含13 C的过渡态类似物，其将使用过早中止环化反应的OSC突变体酶促制备。 此外，化合物结合到OSC和它们的NMR特性将在未来的固态NMR光谱学研究的准备。 拟议的研究将提供第一个典型的步骤，在表征膜环境的贡献的功能的单调的膜酶。

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. We propose to study the functional coupling between the integral membrane enzyme 2,3-oxidosqualene cyclase (OSC) and its lipid environment. OSC is a key enzyme in the biosynthesis and regulation of cholesterol. It catalyzes the cyclization reaction producing lanosterol, the core skeleton of steroids and hormones. To reach its lipidic substrate, OSC - like all members of the monotopic membrane enzyme family - stably and permanently resides in one leaflet of the bilayer only. Like the other enzymes in this protein family, OSC uses large hydrophobic surfaces to contact the lipid bilayer and utilizes extended hydrophobic channels to shuttle its hydrophobic reactants between its active site and the membrane. The focus of the proposed work is to establish the methodology that will enable the study of the interrelationship between the enzymatic reaction and the properties of the membrane environment. Our long-term goal is to understand the correlation - as mediated by the membrane - between the conformational changes of the protein and the transfer of the substrate and product to and form the lipid bilayer. Initial characterization will focus on expression, purification and reconstitution of the human OSC into membranes of well-defined composition and organization. Using these protocols we will investigate the membrane conditions required for efficient substrate presentation and the effect of the membrane's physicochemical properties on catalysis. To that end 13C-enriched transition state analogues will be utilized which will be enzymatically prepared using OSC mutants that prematurely abort the cyclization reaction. In addition, the compounds binding to OSC and their NMR properties will be characterized in preparation for future solid-state NMR spectroscopy studies. The proposed studies will provide the first quintessential step in characterizing the contribution of the membrane environment to the function of a monotopic membrane enzyme.