Structural Analysis of Biological Membrane Proteins

生物膜蛋白的结构分析

基本信息

批准号：
7592662
负责人：
DI S XIA
金额：
$ 95万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7592662
关键词：
ATP phosphohydrolase ATP-Binding Cassette Transporters Area Bacteria Behavior Binding Biochemical Genetics Biological Biological Models Bos taurus Cattle Cell physiology Class Classification Communication Complex Condition Copper Crystallization Cytochrome bc1 Complex Data Databases Deposition Development Drug Delivery Systems Drug Metabolic Detoxication Environment Enzymes Escherichia coli Fostering Goals Hand Homologous Gene Human Hydroquinones Integral Membrane Protein Intramural Research Program Ion Transport Ions Knowledge Laboratories Length Membrane Proteins Membrane Transport Proteins Methodology Methods Mitochondria Modeling Molecular Molecular Conformation Numbers Open Reading Frames P-Glycoprotein P-Glycoproteins Pharmaceutical Preparations Photosynthetic Complexes Preparation Procedures Proteins Protons Qi Quinones Research Resolution Respiratory Chain Risk Roentgen Rays Scheme Site Solutions Structure Synchrotrons System Techniques Testing United States National Institutes of Health Work X-Ray Crystallography Yeasts benzoquinone design genome sequencing improved inhibitor/antagonist interest mutant oxidation photosynthetic bacteria protein expression protein structure research study structural biology

ATP phosphohydrolase ATP-Binding Cassette Transporters Area Bacteria Behavior Binding Biochemical Genetics Biological Biological Models Bos taurus Cattle Cell physiology Class Classification Communication Complex Condition Copper Crystallization Cytochrome bc1 Complex Data Databases Deposition Development Drug Delivery Systems Drug Metabolic Detoxication Environment Enzymes Escherichia coli Fostering Goals Hand Homologous Gene Human Hydroquinones Integral Membrane Protein Intramural Research Program Ion Transport Ions Knowledge Laboratories Length Membrane Proteins Membrane Transport Proteins Methodology Methods Mitochondria Modeling Molecular Molecular Conformation Numbers Open Reading Frames P-Glycoprotein P-Glycoproteins Pharmaceutical Preparations Photosynthetic Complexes Preparation Procedures Proteins Protons Qi Quinones Research Resolution Respiratory Chain Risk Roentgen Rays Scheme Site Solutions Structure Synchrotrons System Techniques Testing United States National Institutes of Health Work X-Ray Crystallography Yeasts benzoquinone design genome sequencing improved inhibitor/antagonist interest mutant oxidation photosynthetic bacteria protein expression protein structure research study structural biology

展开

项目摘要

Over the past few years, we have made significant progress in obtaining better diffracting bovine mitochondrial cytochrome bc1 (cyt bc1, bc1) crystals and in understanding the mechanism of function by analyzing both native- and inhibitor-bound structures. We proposed a scheme for bc1 inhibitor classification and put forward mechanisms for quinone reduction at the Qi site and quinol oxidation at the Qo site. Recently, we have successfully determined the crystal structures of the wild type and mutant bc1 complex from the photosynthetic bacterium R. sphaeroides (Rsbc1) in complex with various inhibitors, demonstrating our ability to reproducibly obtain atomic resolution structural information on the bacterial bc1 in various forms and our perseverance in pursuing difficult projects. This work accomplishes one of our goals in establishing a model system to systematically study the bc1 complex by combining structural, genetic, and biochemical techniques; it marks another milestone in the study of bc1 complex and in the field of membrane protein structural biology. The development of methodology for membrane protein crystallization has been an integral part of our research on structure determinations of P-gp and its homologues. We have explored various expression systems to achieve consistent high-level protein expression. We have developed and refined a multi-parameter kit to screen for conditions for stabilizing P-gp in solution. As a result, we achieved P-gp preparations at high concentration. To achieve conformation uniformity, we tested mutants and Fab-complexed P-gp in crystallization experiments. We designed and tested tens of thousands crystallization conditions and obtained valuable first-hand knowledge of the behavior of P-gp under these conditions. Although we have yet to reach our goal of structure solution of P-gp, the methods developed have been used for expression, purification, and crystallization of P-gp homologues such as the yeast Pdr5p and the bacterial LmrA from L. lactice. These methods have also been successfully applied to the crystallization of a different class of membrane transporters, the P-type ATPases, including the CopB ion transporter from E. coli and proton transporter Pma1p of yeast, both diffracted X-ray at synchrotron. In addition, my unit has developed procedures to model full-length ABC transporter structures.

在过去的几年中，我们在获得更好的衍射牛线粒体细胞色素BC1（CYT BC1，BC1）晶体方面取得了重大进展，并通过分析天然和抑制剂结构结构来理解功能机理。我们提出了一种BC1抑制剂分类的方案，并提出了在Qi位点减少喹酮的机制，并在QO位点提出了奎诺醇氧化。最近，我们成功地确定了来自光合细菌的野生型和突变体BC1复合物的晶体结构。这项工作实现了我们建立模型系统来通过结合结构，遗传和生化技术来系统研究BC1复合物的目标之一。它标志着BC1复合物研究和膜蛋白结构生物学领域的另一个里程碑。膜蛋白结晶方法的发展一直是我们在P-gp及其同源物结构确定的研究的一部分。我们探索了各种表达系统，以实现一致的高级蛋白表达。我们已经开发并完善了一个多参数套件，以筛选稳定溶液中p-gp的条件。结果，我们在高浓度下实现了P-gp制剂。为了达到构象均匀性，我们在结晶实验中测试了突变体和Fab-complex的P-gp。我们设计和测试了数以万计的结晶条件，并在这些条件下对P-gp的行为获得了宝贵的第一手知识。尽管我们尚未达到P-gp结构溶液的目标，但开发的方法已用于表达，纯化和结晶的P-gp同源物，例如酵母PDR5P和L.乳酸的细菌LMRA。这些方法也已成功应用于不同类别的膜转运蛋白的结晶，包括来自大肠杆菌的CopB离子转运蛋白，包括来自大肠杆菌的COPB离子转运蛋白和酵母的Proton Transporter PMA1P，均衍射为X射线，均具有同步子。此外，我的单元还开发了对全长ABC转运蛋白结构进行建模的程序。