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

描述(由申请人提供)：这项R21计划的目标是使用一系列新的氮三乙酸(NTA)试剂获得高质量的组氨酸标记(His-Tag)可溶性蛋白质和His-Tag整合膜蛋白(IMP)的一维和二维晶体，NTA试剂以对称引导的方式促进成核，并通过相分离进入富IMP区域来加速结晶。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。在该项目的初始阶段，将使用一维模板来成核可溶蛋白质的结晶，例如His-tag绿色荧光蛋白(His-GFP)。在这些材料上获得的经验将被扩展到开发新的试剂，用于在二维模板基质中结晶完整的膜蛋白-人白三烯C4合成酶。在这种情况下，具有改进的长程有序性的人白三烯C4合成酶晶体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不连续的数量的蛋白质。一旦蛋白质以这种对称引导的方式聚集在一起，相分离的脂类混合物将被用来横向将完整的膜蛋白浓缩到有利于形成二维晶体的富含蛋白质的区域。在这项建议中开发的材料将用于利用低温电子显微镜和X射线结晶学技术筛选具有印迹对称性和长程有序的晶体的外观。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合成酶将作为测试案例，分别确定所建议的材料是否促进可溶性和完整膜蛋白的结晶。在这个项目中开发的试剂将被送到我们合作者的实验室进行进一步的筛选，包括His标签形式的MalFGK2麦芽糖转运蛋白、细胞色素b6f和核糖转运蛋白。这种“高风险-高回报”的建议有可能彻底改变IMP结构生物学领域，并通过催化许多不同类型的有序His-Tag完整膜蛋白的受控成核和生长，从而加快针对这一重要类别蛋白质的药物开发步伐，用于中分辨率电子结晶学和高分辨率X射线结晶学研究。

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.