STRUCTURE/FUNCTION OF MEMBRANE PROTEIN CHANNELS

膜蛋白通道的结构/功能

• 批准号: 6519649
• 负责人: ALOK K MITRA
• 金额: $ 10.06万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519649
• 关键词: Bacillus; Xenopus oocyte; bacterial disease; bacterial toxins; biological fluid transport; chemical models; cryoscopy; crystallization; divalent metal; electron crystallography; image processing; lipid bilayer membrane; membrane permeability; mercury; molecular polarity; osmosis; pore forming protein; protein structure function; site directed mutagenesis; structural biology; water channel; water flow

The objective of this proposal is to understand structural details in the bilayer that relate to transport properties of membrane protein channels. Specifically, we wish to understand the structure/function relationships for the mammalian water channels AQP1 and AQP4 and the pore-forming anthrax toxin protective antigen by high-resolution electron crystallography. Our specific aims are listed below. AIM I. Examine at the structural level the inhibition of water transport in AQP1 by mercurial compounds. We will apply electron diffraction to quantitate in 3-dimensions structural changes in AQP1 upon mercurial binding. This will allow us to understand AQP1 function based on its modulation by the pharmacological inhibitor. AIM II. Examine the structural/functional roles of residues involved in the selective water transport in AQP1. We will examine the functional roles of polar and charged amino acids in the putative membrane-spanning region of AQP1 using a combined functional and structural approach. Amino-acid substitutions that lead to altered function without structural perturbation will allow us to identify residues critical for water transport. AIM III. Structural studies on the AQP4 water channel. The AQP4 water channel expressed primarily in brain elicits highest osmotic water permeability. We will crystallize AQP4 in the lipid bilayer and use it as a model to understand and identify at the structural level factors responsible for diversity in solute transport mediated by aquaporins. AIM IV. Structural studies on anthrax toxin protective antigen. The structure of the soluble and membrane-integrated complex of protective antigen (PA63) heptamers with and without bound lethal factor (LF) will be studied using single particle image analysis and conical-tilt reconstructions. This will allow us to understand the binding of LF and test the proposed porin-like model for the membrane-embedded domain of PA63. The selective expression of AQP1 and homologous water channels believed to be involved in fluid absorption and/or secretion makes them an important pharmacological target. The pore-forming anthrax toxin has been shown to have a potential for delivery of macromolecules across the bilayer. Thus structural studies on these systems will potentially have impact on structure-based drug design.

这项提议的目的是了解双层中与膜蛋白通道的运输特性有关的结构细节。具体地说，我们希望通过高分辨电子结晶学来了解哺乳动物水通道AQP1和AQP4与成孔炭疽毒素保护性抗原的结构/功能关系。我们的具体目标如下所示。目的I.从结构水平研究汞化合物对水通道蛋白1水转运的抑制作用。我们将利用电子衍射来定量研究汞结合时AQP1的三维结构变化。这将使我们能够了解AQP1的功能，这是基于药物抑制剂对AQP1的调节。目的II.研究AQP1中参与选择性水运输的残基的结构/功能作用。我们将使用功能和结构相结合的方法研究极性和带电氨基酸在AQP1假定的膜跨越区域中的功能作用。氨基酸取代导致功能改变而不引起结构扰动，这将使我们能够识别对水运输至关重要的残基。目的III.水通道AQP4的结构研究。主要在脑内表达的AQP4水通道具有最高的渗透水通透性。我们将在脂质双层中结晶AQP4，并将其作为一个模型，以在结构水平上理解和识别导致水通道蛋白介导的溶质运输多样性的因素。目的IV.炭疽毒素保护性抗原的结构研究。用单粒子图像分析和锥体倾斜重建方法研究了含有和不含有结合致死因子(LF)的保护性抗原(PA63)七聚体的可溶性和膜结合复合体的结构。这将使我们能够了解LF的结合，并测试所提出的PA63膜嵌入域的孔蛋白样模型。AQP1和被认为参与液体吸收和/或分泌的同源水通道的选择性表达使其成为重要的药理靶点。形成孔洞的炭疽毒素已被证明具有通过双层传递大分子的潜力。因此，对这些体系的结构研究可能会对基于结构的药物设计产生影响。

项目成果

Large-scale structural changes accompany binding of lethal factor to anthrax protective antigen: a cryo-electron microscopic study.

大规模结构变化伴随着致死因子与炭疽保护性抗原的结合：一项冷冻电子显微镜研究。

• DOI: 10.1016/j.str.2004.09.010
• 发表时间: 2004
• 期刊: Structure (London, England : 1993)
• 作者: Ren,Gang;Quispe,Joel;Leppla,StephenH;Mitra,AlokK
• 通讯作者: Mitra,AlokK