Improved Methods: Membrane Protein Crystallization(RMI)

改进方法：膜蛋白结晶（RMI）

• 批准号: 7012589
• 负责人: Michael Wiener
• 金额: $ 28.56万
• 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/7012589
• 关键词: X ray crystallography; crystallization; detergents; membrane proteins; optics; protein purification; protein structure; rare earth element; structural biology; technology /technique development

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（由申请人提供）：通过X射线晶体学确定的完整膜蛋白结构的数量呈指数增长。然而，结构的数量大约等于25年前可溶性蛋白质的数量。此外，坦率地说，膜蛋白结构的测定仍然太困难，除了最大和资金最充足的实验室之外，基本上超出了所有实验室的能力范围。该提案的总体目标是大大降低膜蛋白结构生物学（通过X射线晶体学）的难度，风险和成本。该提案侧重于几个假设的关键测试;以及开发一种新的统一技术，以帮助膜蛋白纯化，结晶和结构测定（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诱变修饰表面残基是否能提高“成功”结晶的可能性？(2)对纯化膜蛋白溶液中存在的去污剂和脂类的仔细鉴定是否对获得适合于结构测定的有序晶体有用？(3)纯化的膜蛋白质溶液是否有与适合结构测定的有序晶体形成相关（甚至是预测）的性质？(4)开发基于在蛋白质或蛋白质-蛋白质复合物中包含光学活性含镧系元素结构域的统一的新平台技术。这种方法的成功开发将实现：蛋白质表达、纯化和结晶的简单光学检测;快速一步亲和纯化;包含结构域以促进晶格形成;以及包含异常散射体，以便定相简单。这些特定目标的目标是我实验室目前正在研究（或计划研究）的一系列蛋白质，包括E。coli水通道蛋白AqpZ（AquaporinZ）、大肠杆菌水通道蛋白AqpZ、大肠杆菌水通道蛋白AqpZ、大肠杆菌水通道蛋白AqpZ。大肠杆菌外膜钴胺素转运蛋白BtuB、人质膜铜转运蛋白CTR 1、人线粒体外周苯二氮受体PBR和结核分枝杆菌膜蛋白。其他计划的目标将集中在其他人类膜蛋白的基本和生物医学进口。