LANOSTEROL BIOSYNTHESIS IN THE MEMBRANE ENVIRONMENT

膜环境中的羊毛甾醇生物合成

• 批准号: 7959547
• 负责人: Sharon Rozovsky
• 金额: $ 7.12万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959547
• 关键词: Active Sites; Anabolism; Binding; Biochemical Reaction; Catalysis; Centers of Research Excellence; Cholesterol; Computer Retrieval of Information on Scientific Projects Database; Coupling; Cyclization; Environment; Enzymes; Family; Funding; Future; Goals; Grant; Human; Hydrophobic Surfaces; Institution; Lanosterol; Lanosterol synthase; Lipid Bilayers; Lipids; Mediating; Membrane; Membrane Proteins; Methodology; NMR Spectroscopy; Preparation; Production; Property; Protein Family; Proteins; Protocols documentation; Reaction; Regulation; Research; Research Personnel; Resources; Skeleton; Source; United States National Institutes of Health; Work; analog; member; mutant; reconstitution; solid state nuclear magnetic resonance; steroid hormone

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 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。 我们建议研究整合膜酶 2,3-氧化角鲨烯环化酶 (OSC) 与其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。 为了到达其脂质底物，OSC 与单位膜酶家族的所有成员一样，仅稳定且永久地驻留在双层的一个小叶中。 与该蛋白质家族中的其他酶一样，OSC 使用大的疏水表面来接触脂质双层，并利用扩展的疏水通道在其活性位点和膜之间穿梭其疏水反应物。 拟议工作的重点是建立能够研究酶反应与膜环境特性之间相互关系的方法。我们的长期目标是了解蛋白质构象变化与底物和产物转移至脂质双层并形成脂质双层之间的相关性（由膜介导）。 初步表征将集中于人类 OSC 的表达、纯化和重构，形成具有明确组成和组织的膜。使用这些方案，我们将研究有效底物呈现所需的膜条件以及膜的物理化学性质对催化的影响。 为此，将使用富含 13C 的过渡态类似物，这些类似物将使用过早中止环化反应的 OSC 突变体进行酶法制备。 此外，还将对与 OSC 结合的化合物及其 NMR 特性进行表征，为未来的固态 NMR 光谱研究做好准备。 拟议的研究将为表征膜环境对单位膜酶功能的贡献提供第一个典型步骤。