STRUCTURE/FUNCTION OF MEMBRANE PROTEIN CHANNELS

这个建议的目的是了解与膜蛋白通道的运输特性有关的双层结构细节。 具体而言，我们希望了解的结构/功能的关系，哺乳动物水通道AQP1和AQP4和孔形成炭疽毒素保护性抗原的高分辨率电子晶体学。我们的具体目标如下。 AIM岛在结构水平上检查汞化合物对AQP1中水转运的抑制作用。 我们将应用电子衍射来定量汞结合后AQP1的三维结构变化。 这将使我们能够了解AQP1功能的基础上，其调制的药理学抑制剂。 AIM II. 检查AQP1中参与选择性水转运的残基的结构/功能作用。 我们将研究极性和带电氨基酸的功能作用，在假定的跨膜区域的水通道蛋白1使用相结合的功能和结构的方法。 氨基酸取代，导致功能改变，而没有结构干扰，将使我们能够确定水运输的关键残基。 AIM III. AQP4水通道的结构研究 AQP4主要在脑内表达，具有最高的渗透水通透性。 我们将结晶水通道蛋白4的脂质双层，并使用它作为一个模型，以了解和确定在结构水平的因素负责的水通道蛋白介导的溶质转运的多样性。 AIM IV.炭疽毒素保护性抗原的结构研究。 保护性抗原（PA63）七聚体的可溶性和膜整合复合物的结构与和不结合致死因子（LF）将使用单粒子图像分析和锥形倾斜重建研究。 这将使我们能够了解LF的结合，并测试所提出的PA63膜嵌入结构域的孔蛋白样模型。 AQP1和同源水通道的选择性表达被认为参与液体吸收和/或分泌，使它们成为重要的药理学靶点。 已显示成孔炭疽毒素具有跨双层递送大分子的潜力。 因此，对这些系统的结构研究将可能对基于结构的药物设计产生影响。

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.