Crystallization of His-tag Proteins on Nanostructured 1D & 2D Template Interface

His 标签蛋白在纳米结构一维上的结晶

• 批准号: 7082489
• 负责人: DAVID H THOMPSON
• 金额: $ 22.27万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7082489
• 关键词: X ray crystallography; chemical binding; cryoelectron microscopy; crystallization; eicosanoid metabolism; green fluorescent proteins; histidine; membrane proteins; nanotechnology; protein purification; transport proteins; water solubility

DESCRIPTION (provided by applicant): The goal of this R21 proposal is to obtain high-quality 1D and 2D crystals of histidine-tagged (His-tag) soluble proteins & His-tag integral membrane proteins (IMP) using a family of new nitrilotriacetic acid (NTA) reagents that promote nucleation in a symmetry-guided manner and accelerate crystallization via phase separation into IMP-rich domains. These studies will serve as a starting point for the long-term goal of generalizing these materials and methods for this challenging class of proteins. One dimensional templates will be used to nucleate the crystallization of soluble proteins such as His-tag green fluorescent protein (His- GFP) in the initial phase of the project. Experience gained with these materials will then be extended to the development of new reagents for the crystallization of the integral membrane protein, human leukotriene C4 synthase, within a two dimensional template matrix. In this case, human leukotriene C4 synthase crystals with improved long-range order will be nucleated in the presence of symmetrical water-soluble nucleating agents that bind a discrete number of proteins in a predetermined geometric arrangement. Once the proteins have been clustered in this symmetry-guided manner, phase-separating lipid mixtures will be used to laterally concentrate the integral membrane protein into protein-rich domains that favor the formation of two dimensional crystals. The materials developed in this proposal will be used to screen for the appearance of crystals with imprinted symmetry and long-range order using cryogenic electron microscopy and x-ray crystallography techniques. Two proteins of known structure, His-GFP at atomic resolution and human leukotriene C4 synthase at medium resolution, will be used as test cases to determine whether the proposed materials promote crystallization of soluble and integral membrane proteins, respectively. The reagents developed in this project will be sent for additional screening with His-tag forms of MalFGK2 maltose transporter, cytochrome b6f, and ribose transporter in our collaborator's laboratories. This 'high risk- high reward' proposal has the potential to revolutionize the field of IMP structural biology, and accelerate the pace of drug development designed to target this important class of proteins, by catalyzing the controlled nucleation and growth of well-ordered His-tag integral membrane proteins of many different types for medium resolution electron crystallography and high resolution x-ray crystallography studies.

描述（由申请人提供）：该R21提案的目标是使用一系列新的硝基三乙酸（NTA）试剂获得组氨酸标记（His-tag）可溶性蛋白和His-tag整体膜蛋白（IMP）的高质量1D和2D晶体，这些试剂以对称引导的方式促进成核，并通过相分离到富含IMP的结构域加速结晶。这些研究将作为推广这些材料和方法用于这类具有挑战性的蛋白质的长期目标的起点。在项目的初始阶段，一维模板将用于可溶性蛋白质的结晶成核，如His标签绿色荧光蛋白（His- GFP）。从这些材料中获得的经验将扩展到在二维模板基质中开发用于整体膜蛋白结晶的新试剂，即人白三烯C4合成酶。在这种情况下，人类白三烯C4合酶晶体具有改进的远程顺序，将在对称的水溶性成核剂的存在下成核，这些成核剂以预定的几何排列结合离散数量的蛋白质。一旦蛋白质以这种对称引导的方式聚集在一起，相分离的脂质混合物将用于将整体膜蛋白横向浓缩到有利于形成二维晶体的富含蛋白质的区域。本提案中开发的材料将用于使用低温电子显微镜和x射线晶体学技术筛选具有印记对称性和长程有序的晶体外观。两种已知结构的蛋白质，原子分辨率的His-GFP和中等分辨率的人白三烯C4合成酶，将被用作测试案例，以确定所提出的材料是否分别促进可溶性和整体膜蛋白的结晶。本项目开发的试剂将在合作实验室进行MalFGK2麦芽糖转运体、细胞色素b6f和核糖转运体的His-tag形式的额外筛选。这种“高风险高回报”的提议有可能彻底改变IMP结构生物学领域，并通过催化许多不同类型的有序his标签积分膜蛋白的可控成核和生长，加速针对这类重要蛋白质的药物开发步伐，用于中分辨率电子晶体学和高分辨率x射线晶体学研究。