Molecular Basis for Transmembrane Conduction & Signaling

由于原子结构是理解机制和设计调制器的关键， 本提案的长期目标是为理解四种功能类别奠定基础， 膜蛋白在这个水平上的分子结构和机制。在水通道蛋白家族（AQPs）中， 具体的目标包括（1）使用四种AQP中的突变确定选择性的基础， 本实验室已经确定：（2）人水通道蛋白的表达和结构测定 AQP 4，对中风中的附带损害的药物设计的影响，以及疟疾寄生虫水通道蛋白 PfAQP，一个新的抗疟疾药物靶点。一种化学“拴系”方法将识别出可以被 用于证明疟疾水通道蛋白作为药物靶点的原理。先在我们的地标建筑上 跨膜氨转运蛋白AmtB的分子结构和作用机制的研究， 使用突变蛋白的结构和功能分析建立功能的原子基础， 基于我们的结构。该家族的第二个目标是确定AmtB调节的基础， GlnK。包括存在于真核生物中的Rh亚家族的相关家族成员的结构将被描述。 寻求。第三类是尿素转运蛋白，我们试图确定尿素通道的结构 从人类病原体幽门螺杆菌，因为这个通道是必不可少的致病性在胃 溃疡第二个靶标是来自铜绿假单胞菌的尿素转运蛋白。该靶标与人尿素同源 转运蛋白，特别是在跨膜区，因此进入这个新的膜 蛋白质折叠最后，我们试图完善表达条件的膜部分，一个功能，但 神经元烟碱乙酰胆碱受体的简化形式通过异源表达 独特的单一亚基，α 7，形成全功能的同源五聚体AcChR通道。 本研究中使用的技术是表达膜蛋白的最前沿方法， 膜蛋白的提取和纯化、结晶和结构测定。结构 将通过突变分析结合结构和功能测量来测试和改进模型 突变蛋白质。天然和突变蛋白的结构和功能分析将导致 可以直接评估和/或确定其作为药物靶点的治疗优势的程序，以及 提供了一个关键，以推动其作为目标的成功实施。虽然今天的所有目标中有40% 药物是膜蛋白，对于任何一个这样的药物靶点，还没有单一的原子结构 膜蛋白这些蛋白质的结构将建立其作用机制，并提供 针对具有特殊治疗优势的药物发现的模板，以及基本要素 来理解它们的选择性。

Since atomic structure is key to understanding mechanisms and to designing modulators, the long- term objective of this proposal is to establish the basis for understanding four functional classes of membrane proteins at this level of molecular structure and mechanism. In the aquaporin family (AQPs) specific goals include (1) determining the basis of selectivity using mutations in four AQPs whose structures have already determined in our laboratory, (2) expression and structure determination of a human aquaporin AQP4, with impact for drug design against collateral damage in stroke, and the malaria parasite aquaporin PfAQP, a new anti-malarial drug-target. A chemical 'tethering' approach will identify compounds that can be used to prove the principle of the malarial aquaporin as a drug target. Building on our landmark first molecular structure and mechanism of a transmembrane Ammonia transporter, AmtB, research is to establish the atomic basis for function using structural and functional analysis of mutant proteins, designed on the basis of our structure. A second goal in this family is determining the basis for AmtB regulation by GlnK. Structures of related family members that include the Rh subfamily present in eukaryotes will be sought. A third class is that of urea transporters where we seek to determine structures of a urea channel from the human pathogen Helicobacter pylorisince this channel is essential for pathogenicity in stomach ulcer. A second target is the urea transporter from P. aeruginosa. This target is homolgous to human urea transporters, particularly in the transmembrane regions, and thus presents an entry into this new membrane protein fold. Lastly we seek to refine conditions for expression in a membrane fraction, of a functional but simplified version of the neuronal nicotinic acetylcholine receptor obtained by heterologous expression of a unique single subunit, a7, that forms fully functional homopentameric AcChR channels. The techniques used in this research are evolving forefront methods for expression of membrane proteins, extraction and purification, crystallization, and structure determination of membrane proteins. Structural models will be tested and refined by mutational analysis coupled to structural and functional measurements on mutant proteins. The structural and functional analyses of native and mutant proteins will lead to procedures that can directly evaluate and/or establish their therapeutic advantage as drug targets, and provide a key to advancing their successful implementation as targets. While 40% of all targets for today's drugs are membrane proteins there is not yet a single atomic structure for any one such drug target membrane protein. The structures of these proteins will establish their mechanism of action and provide templates for drug discovery aimed at those of particular therapeutic advantage, and the essential elements to understand their selectivity.