Improved Methods for Membrane Protein Crystallization(RMI)

膜蛋白结晶 (RMI) 的改进方法

• 批准号: 7265251
• 负责人: Michael Wiener
• 金额: $ 27.08万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-23 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7265251
• 关键词: Affinity Chromatography; Area; Benzodiazepine Receptor; Binding; Cell membrane; Cobalamin; Complex; Copper; Crystallization; Crystallography; Detection; Detergents; Development; Disease; Endopeptidases; Escherichia coli; Funding; Goals; Human; Integral Membrane Protein; Laboratories; Lanthanoid Series Elements; Length; Life; Lipids; Measurement; Membrane; Membrane Proteins; Methods; Methylation; Mitochondria; Modification; Mutagenesis; Mycobacterium tuberculosis; Numbers; Optics; Peptide Hydrolases; Peripheral; Phase; Production; Property; Proteins; Range; Refractory; Research Personnel; Risk; Solutions; Structure; Surface; Technology; Testing; Time; Work; base; cost; improved; novel; programs; protein expression; protein purification; protein structure; rhomboid; structural biology; water channel

DESCRIPTION (provided by applicant): The number of integral membrane protein structures determined by x-ray crystallography is growing exponentially. However, the number of structures is approximately equal to that of soluble proteins twenty five years ago. Moreover, determination of membrane protein structures remains, frankly, too difficult and essentially beyond the reach of all but the largest and most exceedingly well-funded laboratories. The overarching Aims of this proposal are to reduce greatly the difficulty, risk and cost of membrane protein structural biology (via x-ray crystallography). This proposal focuses upon the critical testing of several hypotheses; and upon the development of a novel unified technology to aid membrane protein purification, crystallization and structure determination (that can be used on soluble proteins as well). Specific Aims include: (1) Does modification of surface residues by reductive methylation or surface mutagenesis improve the likelihood of 'successful' crystallization?; (2) Is careful characterization of the detergent and lipid present in purified membrane protein solutions of utility for obtaining well-ordered crystals suitable for structure determination?; (3) Are there any properties of purified membrane protein solutions that are correlative (or even predictive) for formation of well-ordered crystals suitable for structure determination?; (4) Develop a unified novel platform technology based upon the inclusion of optically-active lanthanide-containing domains in proteins or protein-protein complexes. Successful development of this approach will enable: simple optical detection of protein expression, purification and crystallization; fast single-step affinity purification; inclusion of domains to facilitate crystal lattice formation; and inclusion of anomalous scatterers so that phasing is straightforward. The targets for these specific Aims are a range of proteins currently under study (or planned for study) in my laboratory, including the E. coli water channel Aquaporin Z (AqpZ), the E. coli outer membrane cobalamin transporter BtuB, the human plasma membrane copper transporter CTR1, the human mitochondrial peripheral benzodiazepine receptor PBR, and Mycobacterium tuberculosis membrane proteins. Other planned targets will focus upon other human membrane proteins of fundamental and biomedical import.

描述(申请人提供)：X射线结晶学确定的完整膜蛋白结构的数量呈指数增长。然而，结构的数量大约相当于25年前的可溶性蛋白质。此外，坦率地说，膜蛋白结构的测定仍然太困难，基本上超出了所有实验室的能力范围，除了最大和资金最充足的实验室。这项建议的主要目的是大大降低膜蛋白结构生物学(通过X射线结晶学)的难度、风险和成本。这项建议侧重于对几个假设的关键检验；以及开发一种新的统一技术来辅助膜蛋白的纯化、结晶和结构确定(该技术也可用于可溶性蛋白)。具体目标包括：(1)通过还原甲基化或表面突变对表面残基进行修饰是否提高了成功结晶的可能性？(2)对纯化的膜蛋白溶液中存在的洗涤剂和脂类进行仔细的表征是否有助于获得适于结构测定的有序晶体？(3)纯化的膜蛋白溶液是否具有与形成适于结构测定的有序晶体相关的(甚至预测的)性质？(4)开发一种基于蛋白质或蛋白质-蛋白质复合体中包含光学活性的含稀土结构域的统一的新型平台技术。这一方法的成功开发将使以下方面成为可能：对蛋白质表达、纯化和结晶的简单光学检测；快速单步亲和纯化；包含结构域以促进晶格形成；以及包含反常散射体，从而使定相变得简单。这些特定目标的靶标是一系列目前正在研究(或计划研究)的蛋白质，包括大肠杆菌水通道Aquaporin Z(AqpZ)、大肠杆菌外膜钴胺转运蛋白BtuB、人膜铜转运蛋白CTR1、人线粒体外周苯二氮卓类受体PBR和结核杆菌膜蛋白。其他计划的目标将集中在其他具有基础和生物医学意义的人类膜蛋白上。