Structural Basis of Selective Permeability in Aquaporin

我们研究的长期目标是了解水和多元醇通过人水通道蛋白和水甘油通道蛋白选择性渗透的结构基础。 这项拟议的研究将探讨如何选择性渗透性是建立在这些跨膜通道，如何基板渗透实际上发生在化学水平上，以及某些残基如何赋予选择性渗透人类亚型。为了达到这些目的，我们将使用X射线晶体学来确定两种同源细菌异构体与其底物复合的结构。 我们将关联结构和序列，以确定决定选择性渗透性的残基。然后使用诱变将人残基引入细菌模板。 蛋白质表达、纯化和重建将用于产生类人细菌变体，其将被掺入蛋白脂质体中，其中将通过停流测量来检查选择性渗透性。目的（1）确定GlpF-水的结构，一个E.大肠杆菌水甘油孔蛋白与水分子结合。 比较GlpF-水和GlpF-甘油的结构将阐明甘油分子在通过跨膜通道时如何置换水。 目的（2）确定GlpF与核糖醇及其立体异构体木糖醇复合物的结构，阐明GlpF立体选择性的结构基础。Aim（3）将使AqpZ结晶，得到E. coli水通道蛋白的结构。 比较AgpZ和GlpF的结构将揭示决定水相对于甘油的选择性渗透的残基。 目的（4）将研究水和甘油渗透的决定因素如何赋予人体亚型通道选择性渗透性。 人类决定簇将被交换为AqpZ或GlpF。 人决定簇的选择性渗透性将在细菌模板中表征，并通过与同源人同种型的选择性渗透性进行比较来评价。人水通道蛋白和水甘油通道蛋白调节健康和疾病中的水和小的非电解质的渗透平衡。这项拟议研究的完成将为基于结构的药物设计奠定基础，靶向人类神经调节障碍，而一般的研究结果将阐明在化学水平上通过结晶膜通道的选择性渗透机制。

The long-term goal of our research is to understand the structural basis for selective permeation of water and polyols through human aquaporins and aquaglyceroporins. This proposed research will explore how selective permeability is built into these transmembrane channels, how substrate permeation actually occurs at a chemical level and how certain residues confer selective permeability upon human isoforms. Toward these ends, we will use X-ray crystallography to determine structures of two homologous bacterial isoforms in complex with their substrates. We will correlate structure and sequence to identify residues that determine selective permeability. Mutagenesis will then be used to introduce human residues into the bacterial templates. Protein expression, purification and reconstitution will be used to generate human-like bacterial variants, which will be incorporated into proteoliposomes, where selective permeability will be examined by stopped-flow measurements. Aim (l) will determine the structure of GlpF-water, an E. coli aquaglyceroporin bound with water molecules. Comparing structures of GlpF-water and GlpF-glycerol will elucidate how a glycerol molecule displaces water while passing through the transmembrane channel. Aim (2) will determine the structures of GlpF in complex with ribitol and its stereoisomer xylitol to elucidate the structural basis for stereo-selectivity in GlpF. Aim (3) will crystallize AqpZ, an E. coli aquaporin, and determine its structure. Comparing structures of AgpZ and GlpF will reveal residues that determine selective permeation of water over glycerol. Aim (4) will investigate how determinants of water and glycerol permeation confer channel selective permeability upon human isoforms. Human determinants will be swapped into AqpZ or GlpF. The selective permeability of human determinants will be characterized in bacterial templates and evaluated by comparing with that of cognate human isoforms. Human aquaporins and aquaglyceroporins regulate osmotic balance of water and small non-electrolytes in health and disease. Completion of this proposed study will set the stage for structure-based drug design targeting human osmoregulation disorders, while the general findings will elucidate the mechanism of selective permeability through a crystallized membrane channel at a chemical level.