Structural Analysis of Biological Membrane Proteins

我的团队一直致力于两种膜转运系统的功能机制：质子转运cyt bc1复合物和多药ABC转运体。就我们对其功能机制的理解而言，这两个项目目前处于不同的阶段。对于bc1配合物，我们对其偶联机制已经有了很好的了解。我们已经确定了牛线粒体（Mtbc1）和光合细菌球形红杆菌（Rsbc1）中载脂蛋白和抑制剂结合形式的bc1复合物的晶体结构。我们已经确定了对耦合至关重要的关键结构元素。我们提出了一个表面亲和调制铁硫蛋白（ISP）运动控制假说来解释bc1中的分叉电子转移。对于多药ABC转运体，特别是人p -糖蛋白（hP-gp），其在许多真核系统中的过表达，纯化和与单克隆抗体（mAb）形成复合物的结构解决方案正在取得进展。在过去的几年里，我们通过分析bc1的天然结合结构和抑制物结合结构，在原子分辨率上理解bc1的功能机制方面取得了重大进展。我们提出了bc1抑制剂的分类方案，并提出了QN位点醌还原和QP位点醌氧化的机制。最重要的是，我们已经获得了实验证据来支持我们的表面调制构象开关模型，该模型用于喹啉氧化位点的电子分岔，这是bc1配合物中高质子易位效率的关键。最近，我们成功地测定了sphaeroides光合细菌（R. sphaeroides, Rsbc1）野生型和突变型bc1配合物与多种抑制剂的晶体结构，证明了我们有能力重复地获得不同形式细菌bc1的原子分辨率结构信息，以及我们坚持不懈地追求困难的项目。本研究通过结构、遗传和生化技术的结合，实现了建立一个系统研究bc1复合体的模型系统的目标之一；这是bc1复合体研究和膜蛋白结构生物学领域的又一里程碑。膜蛋白表达、纯化和结晶方法的发展是我们对P-gp及其同源物结构测定研究的一个组成部分。为此，我们一直在探索各种表达系统，以实现少数膜蛋白一致的高水平蛋白表达；其中包括酵母系统，如酿酒葡萄球菌和帕斯德酵母表达系统，细菌系统，如大肠杆菌和乳杆菌表达系统，以及光合细菌球形酵母。我们已经扩展使用Blue-Native技术来检测膜蛋白制剂的单分散性。我们还开发和完善了一个多参数试剂盒筛选条件稳定P-gp在溶液中。我们已经实现了一些完整膜蛋白的高水平表达。除细菌bc1复合体外，还大量纯化了人p -糖蛋白、细菌ABC转运蛋白LmrA和细菌CopB。为了获得单分散的蛋白质样品，我们一直在使用自己开发的Blue-Native技术筛选各种洗涤剂，这是非常成功的。对于构象柔性膜蛋白，如ABC转运蛋白，我们在结晶实验中测试了突变体和fab复合物P-gp。虽然我们还没有达到这些膜蛋白结构解的目标，但这里开发的方法将对其他膜蛋白有用。我的团队一直致力于两种膜转运系统的功能机制：质子转运cyt bc1复合物和多药ABC转运体。就我们对其功能机制的理解而言，这两个项目目前处于不同的阶段。对于bc1配合物，我们对其偶联机制已经有了很好的了解。我们已经确定了牛线粒体（Mtbc1）和光合细菌球形红杆菌（Rsbc1）中载脂蛋白和抑制剂结合形式的bc1复合物的晶体结构。我们已经确定了对耦合至关重要的关键结构元素。我们提出了一个表面亲和调制铁硫蛋白（ISP）运动控制假说来解释bc1中的分叉电子转移。对于多药ABC转运体，特别是人p -糖蛋白（hP-gp），其在许多真核系统中的过表达，纯化和与单克隆抗体（mAb）形成复合物的结构解决方案正在取得进展。在过去的几年里，我们通过分析bc1的天然结合结构和抑制物结合结构，在原子分辨率上理解bc1的功能机制方面取得了重大进展。我们提出了bc1抑制剂的分类方案，并提出了QN位点醌还原和QP位点醌氧化的机制。最重要的是，我们已经获得了实验证据来支持我们的表面调制构象开关模型，该模型用于喹啉氧化位点的电子分岔，这是bc1配合物中高质子易位效率的关键。最近，我们成功地测定了sphaeroides光合细菌（R. sphaeroides, Rsbc1）野生型和突变型bc1配合物与多种抑制剂的晶体结构，证明了我们有能力重复地获得不同形式细菌bc1的原子分辨率结构信息，以及我们坚持不懈地追求困难的项目。本研究通过结构、遗传和生化技术的结合，实现了建立一个系统研究bc1复合体的模型系统的目标之一；这是bc1复合体研究和膜蛋白结构生物学领域的又一里程碑。膜蛋白表达、纯化和结晶方法的发展是我们对P-gp及其同源物结构测定研究的一个组成部分。为此，我们一直在探索各种表达系统，以实现少数膜蛋白一致的高水平蛋白表达；其中包括酵母系统，如酿酒葡萄球菌和帕斯德酵母表达系统，细菌系统，如大肠杆菌和乳杆菌表达系统，以及光合细菌球形酵母。我们已经扩展使用Blue-Native技术来检测膜蛋白制剂的单分散性。我们还开发和完善了一个多参数试剂盒筛选条件稳定P-gp在溶液中。我们已经实现了一些完整膜蛋白的高水平表达。除细菌bc1复合体外，还大量纯化了人p -糖蛋白、细菌ABC转运蛋白LmrA和细菌CopB。为了获得单分散的蛋白质样品，我们一直在使用自己开发的Blue-Native技术筛选各种洗涤剂，这是非常成功的。对于构象柔性膜蛋白，如ABC转运蛋白，我们在结晶实验中测试了突变体和fab复合物P-gp。尽管[摘要被截断为7800个字符]

My group has been working on the mechanisms of function of two membrane transporter systems: proton transporting cyt bc1 complexes and multidrug ABC transporters. These two projects are currently at different stages with respect to our understanding of their mechanisms of function. For the bc1 complex, we have a very good understanding of its mechanism of coupling. We have determined the crystal structures of bc1 complexes from the bovine mitochondria (Mtbc1) and from the photosynthetic bacterium Rhodobacter sphaeroides (Rsbc1) in both apo and inhibitor-bound forms. We have identified critical structural elements that are essential for the coupling. We have proposed a surface-affinity modulated iron-sulfur protein (ISP) motion control hypothesis to explain the bifurcated electron transfer in bc1. For the multidrug ABC transporters, in particular human P-glycoprotein (hP-gp), advances are being made towards its structure solution with respect to its over-expression in a number of eukaryotic systems, its purification and complex formation with monoclonal antibodies (mAb). Over the past few years, we have made significant progress in understanding the mechanism of function of the bc1 at atomic resolution 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 QN site and quinol oxidation at the QP site. Most importantly, we have obtained experimental evidence to support our surface modulated conformation switch model for the electron bifurcation at the quinol oxidation site, which is the key to the high proton translocation efficiency in the bc1 complex. 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 expression, purification, and crystallization has been an integral part of our research on structure determinations of P-gp and its homologues. To this end, we have been exploring various expression systems to achieve consistent high-level protein expression for a few membrane proteins; those include yeast systems such as S. cerevisiae and P. pastoris expression systems, bacterial systems such as E. coli and L. lactis expression systems, and photosynthetic bacterum R. sphaeroides. We have extended the use of Blue-Native techniques to detecting monodispersity of membrane protein preparations. We have also developed and refined a multi-parameter kit to screen for conditions for stabilizing P-gp in solution. We have achieved high-level expressions for a number of integral membrane proteins. In addition to the bacterial bc1 complex, the human P-glycolprotein, bacterial ABC transporter LmrA, and bacterial CopB were purified in large quantities. To obtain monodispersed protein samples, we have been using the Blue-Native technique developed in house to screen for various detergents, which is very successful. For conformationally flexible membrane proteins such as ABC transporters, we tested mutants and Fab-complexed P-gp in crystallization experiments. Although we have yet to reach our goal of structure solutions of these membrane proteins, the methods developed here will be useful for other membrane proteins.My group has been working on the mechanisms of function of two membrane transporter systems: proton transporting cyt bc1 complexes and multidrug ABC transporters. These two projects are currently at different stages with respect to our understanding of their mechanisms of function. For the bc1 complex, we have a very good understanding of its mechanism of coupling. We have determined the crystal structures of bc1 complexes from the bovine mitochondria (Mtbc1) and from the photosynthetic bacterium Rhodobacter sphaeroides (Rsbc1) in both apo and inhibitor-bound forms. We have identified critical structural elements that are essential for the coupling. We have proposed a surface-affinity modulated iron-sulfur protein (ISP) motion control hypothesis to explain the bifurcated electron transfer in bc1. For the multidrug ABC transporters, in particular human P-glycoprotein (hP-gp), advances are being made towards its structure solution with respect to its over-expression in a number of eukaryotic systems, its purification and complex formation with monoclonal antibodies (mAb). Over the past few years, we have made significant progress in understanding the mechanism of function of the bc1 at atomic resolution 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 QN site and quinol oxidation at the QP site. Most importantly, we have obtained experimental evidence to support our surface modulated conformation switch model for the electron bifurcation at the quinol oxidation site, which is the key to the high proton translocation efficiency in the bc1 complex. 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 expression, purification, and crystallization has been an integral part of our research on structure determinations of P-gp and its homologues. To this end, we have been exploring various expression systems to achieve consistent high-level protein expression for a few membrane proteins; those include yeast systems such as S. cerevisiae and P. pastoris expression systems, bacterial systems such as E. coli and L. lactis expression systems, and photosynthetic bacterum R. sphaeroides. We have extended the use of Blue-Native techniques to detecting monodispersity of membrane protein preparations. We have also developed and refined a multi-parameter kit to screen for conditions for stabilizing P-gp in solution. We have achieved high-level expressions for a number of integral membrane proteins. In addition to the bacterial bc1 complex, the human P-glycolprotein, bacterial ABC transporter LmrA, and bacterial CopB were purified in large quantities. To obtain monodispersed protein samples, we have been using the Blue-Native technique developed in house to screen for various detergents, which is very successful. For conformationally flexible membrane proteins such as ABC transporters, we tested mutants and Fab-complexed P-gp in crystallization experiments. Although [summary truncated at 7800 characters]