Improved Methods: Membrane Protein Crystallization(RMI)

描述（由申请人提供）：通过x射线晶体学确定的整体膜蛋白结构的数量呈指数级增长。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，基本上超出了所有实验室的能力范围，只有规模最大、资金最充足的实验室才能做到。这项提议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。该建议侧重于几个假设的关键测试；并且在开发一种新的统一技术来帮助膜蛋白纯化，结晶和结构测定（也可用于可溶性蛋白）。具体目标包括：(1)通过还原性甲基化或表面诱变对表面残基进行修饰是否提高了“成功”结晶的可能性？(2)仔细表征纯化膜蛋白溶液中存在的洗涤剂和脂质是否有助于获得适合于结构测定的有序晶体？(3)纯化膜蛋白溶液是否有任何性质与形成适合于结构测定的有序晶体相关（甚至预测）？(4)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结构域，开发统一的新型平台技术。该方法的成功开发将使：简单的光学检测蛋白表达、纯化和结晶；快速一步亲和纯化；包含域以促进晶格的形成；包括异常散射体这样相位就很简单了。这些特定目标的靶标是我实验室目前正在研究（或计划研究）的一系列蛋白质，包括大肠杆菌水通道水通道蛋白Z （AqpZ）、大肠杆菌外膜钴胺转运蛋白BtuB、人质膜铜转运蛋白CTR1、人线粒体外周苯二氮卓类受体PBR和结核分枝杆菌膜蛋白。其他计划的靶点将集中在其他具有基础和生物医学意义的人膜蛋白上。

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.