Structural Analysis of Biological Membrane Proteins

生物膜蛋白的结构分析

基本信息

  • 批准号:
    7049881
  • 负责人:
  • 金额:
    --
  • 依托单位:
  • 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:
  • 项目状态:
    未结题

项目摘要

Recent advances in genome research have provided new insights into the importance of membrane proteins in cellular functions. In eukaryotes such as yeast, over 14% of open reading frames are predicted to be integral membrane proteins with more than three trans-membrane (TM) segments (25% for two or more TM). Membrane proteins participate in many vital cellular functions; the demand for structural knowledge of membrane proteins has increased more than ever in light of the increased number of these proteins for which important functions have been identified. However structural data for membrane proteins at atomic resolution are only being obtained rather slowly (fewer than 50 unique membrane protein structures available in the Protein Data Bank). The picture gets even more depressing for eukaryotic membrane proteins. Excluding those of mitochondrial origin, only a couple of eukaryotic membrane protein structures were determined to date at atomic resolution; there is not a single recombinant eukaryotic membrane protein structure determined crystallographically! my group has been studying the structure and function of a few selected families of membranes proteins: those involved in cellular multidrug resistance such as P-glycoprotein (P-gp) and its homologs, and the respiratory component cytochrome bc1 complexes (bc1) of mitochondria and bacteria. In collaboration with C. A. Yu (OSU), we have have been successful in obtaining bovine mitochondrial bc1 crystals that diffracted X-rays to higher resolution for native, substrate- and various inhibitor-bound bc1. Our work found that the network of aromatic-aromatic interactions is both effective and specific for inhibitor binding to the hydrophobic active sites of bc1, and provided explanations at atomic resolution for bc1 inhibition by various inhibitors. Moreover the refined structures unveiled rich structural information that suggests mechanisms for substrate reduction and protonation at the quinone reduction site of the cyt. b subunit. Currently my group is refining structures of bc1 with various bound inhibitors that are known to induce conformational switch to the iron-sulfur protein (ISP) subunit. It is believed that correlating structural changes to inhibitor binding and to variations in redox potential may hold the key to understanding the relationship between quinol oxidation and the ISP conformational switch and to providing an explanation for the electron bifurcation at the quinol oxidation site. A major focus of our crystallography unit has been on the expression, purification and crystallization of ABCB1 (P-glycoprotein, P-gp) and its prokaryotic and eukaryotic homologs in collaboration with S. Ambudkar (LCB) and M. Gottesman (LCB). Efforts have been made to purify P-gp from different expression systems such as the baculovirus infected insect cells and the P. pastoris yeast expression system. We have also dedicated resources to expressing, refolding and purifying monoclonal antibodies in the hope of facilitating P-gp purification and crystallization. More recently, we initiated purification and crystallization of the P-gp homologs from the gram-positive bacterium L. lactis (LmrA) and from S. cerevisiae (Pdr5p). Both proteins have been purified to homogeneity and crystallization experiments are underway.Technically, to obtain a membrane protein structure, four obstacles must be overcome: (1) to achieve high-level protein expression, (2) to obtain pure and mono-dispersed proteins in large quantity, (3) to grow diffraction quality crystals, and (4) to solve crystallographic phase problems often at relatively low resolutions for membrane proteins. All these difficulties are due to the fact that membrane proteins have large hydrophobic surface. Membrane proteins are difficult to express in large quantities and in active forms, especially for eukaryotic membrane proteins, in commonly available expression systems. Currently, the most used approach for expressing large amounts of active membrane protein is to screen for high-level expression of a large number of homologs, mostly those of prokaryotes. The concept of high-level expression for membrane protein is perhaps one or two orders of magnitude different from that of high expression of soluble proteins. Even for a very high-level expression, the total amount of membrane protein is rarely over 30% of total membrane proteins in cell membrane (2% for P-gp, 5% for LmrA, and 2% for Pdr5p), making purification of large amount of proteins impossible without large-scale fermentation and cell disruption facilities. Furthermore, success in achieving high-level expression in test tubes does not guarantee success when the production is scaled up. Membrane proteins are often associated with each other in a non-specific manner when purified, which are detrimental to successful crystallization. The problem of polydispersity can be eliminated by screen for different detergents and solvent conditions. When purified, membrane proteins exist in solution as protein-detergent complexes; the available hydrophilic surface that is useful for specific crystal contact is limited. It is quite common to screen for over 30,000 conditions before a diffraction quality crystal form can be found. It has been successful in a few cases to artificially increase the hydrophilic surface by attaching conformational sensitive monoclonal antibodies to target membrane proteins. Our group is also working toward improving existing or devise new technologies to facilitate crystallization of membrane proteins.
基因组研究的最新进展为膜蛋白在细胞功能中的重要性提供了新的见解。在诸如酵母等真核生物中,预计超过14%的开放式阅读框架是具有三个以上的跨膜(TM)段的整体膜蛋白(两个或更多TM)。膜蛋白参与许多重要的细胞功能。鉴于已经确定了重要功能的这些蛋白质数量增加,对膜蛋白的结构知识的需求比以往任何时候都增加。但是,在原子分辨率下的膜蛋白的结构数据仅得到很慢(蛋白质数据库中可用的50个独特的膜蛋白结构少于50个独特的膜蛋白结构)。对真核膜蛋白的图像更加令人沮丧。不包括线粒体起源的那些,在原子分辨率下仅确定了几个真核膜蛋白结构。从结晶上确定的单个重组真核膜蛋白结构没有!我的小组一直在研究一些选定的膜蛋白家族的结构和功能:涉及细胞多药耐药性的蛋白质,例如P-糖蛋白(P-GP)及其同源物,以及Mitochothiria和Capteria of Mitochotia and Clogia of Cytochrome BC1复合物(BC1)。与C. yu(OSU)合作,我们已经成功地获得了牛线粒体BC1晶体,该牛对天然,底物和各种抑制剂结合的BC1的X射线衍射为更高分辨率。我们的工作发现,芳族 - 芳香相互作用网络既有效又特异性对于抑制剂与BC1的疏水活性位点结合,并且在原子分辨率时提供了各种抑制剂BC1抑制的解释。此外,精制结构揭示了丰富的结构信息,这表明了Cyt的喹酮还原位点的底物还原和质子化的机制。 B亚基。目前,我的小组正在使用已知可以诱导构象转换为铁硫蛋白(ISP)亚基的各种结合抑制剂的BC1结构。据信,将结构变化与抑制剂结合和氧化还原电位变化相关联可能是理解喹诺氧化与ISP构象转换之间关系的关键,并为奎因氧化位点上的电子分叉化提供解释。我们晶体学单元的主要重点是与ABCB1(P-糖蛋白,P-GP)及其原核和真核同源物的表达,纯化和结晶,并与S. ambudkar(LCB)和M. Gottesman(LCB)合作。已经努力从不同的表达系统(例如杆状病毒感染昆虫细胞和P. p. pastoris酵母表达系统)中纯化P-gp。我们还专门用于表达,重折叠和净化单克隆抗体,以促进P-gp纯化和结晶。最近,我们从革兰氏阳性细菌(LMRA)和酿酒酵母(PDR5P)中启动了P-gp同源物的纯化和结晶。两种蛋白质都已经纯化为均匀性,并且正在进行结晶实验。在技术上,要获得膜蛋白结构,必须克服四个障碍:(1)以实现高级蛋白质表达,(2)获得纯净和单分散的蛋白质,以使其在大量的范围中以相关质量的质量和晶体质量(4)的固定质量(4),(4)蛋白质。所有这些困难都是由于膜蛋白具有较大的疏水表面。在常见的表达系统中,膜蛋白很难以大量和活性形式表达,尤其是对于真核膜蛋白。当前,表达大量活性膜蛋白的最常用方法是筛选大量同源物(主要是原核生物)的高级表达。膜蛋白的高级表达的概念可能是一个或两个数量级,与可溶性蛋白的高表达不同。即使对于非常高级的表达,膜蛋白的总量很少超过细胞膜中总膜蛋白的30%以上(P-gp为2%,LMRA为5%,PDR5P的2%),没有大规模发酵和细胞破坏设施,就可以纯化大量的蛋白质。此外,在测试管中实现高级表达方面的成功并不能保证当生产扩展时成功。纯化时,膜蛋白通常以非特异性方式相互关联,这对成功结晶有害。可以通过筛选来消除多分散性的问题,以解决不同的洗涤剂和溶剂条件。当纯化时,膜蛋白作为蛋白含量复合物存在于溶液中。可用的可用于特定晶体接触的可用亲水表面受到限制。在发现衍射质量晶体形式之前,筛选30,000多个条件是很常见的。在少数情况下,通过将构象敏感的单克隆抗体连接到靶膜蛋白来人为地增加亲水性表面。我们的小组还致力于改善现有或设计新技术以促进膜蛋白的结晶。

项目成果

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DI S XIA其他文献

DI S XIA的其他文献

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{{ truncateString('DI S XIA', 18)}}的其他基金

DETECTION OF THE SUBSTRATE UBIQUINONE/UBIQUINOL IN THE BOVINE MITOCHONDRIAL C
牛线粒体C中底物泛醌/泛醇的检测
  • 批准号:
    7181848
  • 财政年份:
    2005
  • 资助金额:
    --
  • 项目类别:
DETECTION OF THE SUBSTRATE UBIQUINONE/UBIQUINOL IN THE BOVINE MITOCHONDRIAL C
牛线粒体C中底物泛醌/泛醇的检测
  • 批准号:
    7181867
  • 财政年份:
    2005
  • 资助金额:
    --
  • 项目类别:
STRUCTURAL ANALYSIS OF BIOLOGICAL MEMBRANE PROTEINS
生物膜蛋白的结构分析
  • 批准号:
    6289383
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Structural Analysis of Biological Membrane Proteins
生物膜蛋白的结构分析
  • 批准号:
    7291706
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Structural Analysis of Biological Membrane Proteins
生物膜蛋白的结构分析
  • 批准号:
    6559198
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Structural Analysis of Biological Membrane Proteins and
生物膜蛋白的结构分析和
  • 批准号:
    6763516
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Structural Analysis of Biological Membrane Proteins
生物膜蛋白的结构分析
  • 批准号:
    6433116
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Structural Analysis of Biological Membrane Proteins
生物膜蛋白的结构分析
  • 批准号:
    7338455
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Structural Analysis of Biological Membrane Proteins and
生物膜蛋白的结构分析和
  • 批准号:
    6951337
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:
Structural Analysis of Biological Membrane Proteins
生物膜蛋白的结构分析
  • 批准号:
    7732998
  • 财政年份:
  • 资助金额:
    --
  • 项目类别:

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