Modular Design of Voltage-Gated Channel Proteins

描述（由申请人提供）：我们在过去三十年的研究计划一直受到以下基本原则的指导：电压门控通道蛋白在设计上是模块化的，并且组成模块之间的耦合是其对电压的灵敏度的基础，并控制其功能多样性。在新的资助期内，三个主要目标集中在：1。在一个开放的构象，试图了解传感器模块的作用的通道的晶体结构的测定。我们将通过补充用于结晶的实验条件来追求孔模块的结构-功能程序，其具有新颖的开放构象稳定剂，旨在解码开放通道结构。2.根据无传感器孔的固有特性以及包括电压传感器或机械应力传感器的伙伴模块所赋予的调节来阐明电压依赖性的起源。在PM结构的关键位置产生的许多突变体有利于孔的导电状态;因此，我们将尝试结晶最有趣的候选物，旨在揭示开放结构。已经建立了在脂质双层中重构的纯化的机械敏感压电蛋白的通道特性，导致我们应用我们的分子解剖策略来识别孔模块和力传感器模块的结构决定因素。大量的嵌合体，截短的结构，并仔细选择的突变体将产生和功能特性沿着线先前报道。最终目的是通过附加压电蛋白的力传感器模块来赋予坚固的PM支架机械敏感性。3.新发现的电压激活阴离子选择性通道（Xv）的结构和功能表征。一个直接的目标需要一个深入的功能表征方面的选择性，渗透性，和块的纯化全长Xv和f的无传感器PM后，重建在脂质双层。注意力将被引导到建立激活的电压依赖性，因为Xv可能代表超极化激活通道。一个紧迫的任务是结晶Xv和它的PM使用脂质立方相的方法，我们成功地应用于KvLm。我们在这+30年的时间间隔内开发的研究结构-功能关系的程序以及新获得的结构测定能力预示着我们长期致力于从电压门控通道蛋白的模块化设计方面对电压传感的机械理解的现实，创新和富有成效的延续。

DESCRIPTION (provided by applicant): Our research program over the past three decades has been guided by the fundamental tenet that voltage- gated channel proteins are modular in design and that the coupling between the component modules underlies their exquisite sensitivity to voltage and governs their functional diversity. For the new funding period, the thre main goals focus on: 1. The determination of the crystal structure of the channel in an open conformation attempting to understand the role of the sensor module. We will pursue the structure-function program of the pore module by supplementing the experimental conditions used to crystallize it with novel open-conformation stabilizers aiming to decode the open channel structure. 2. The elucidation of the origin of the voltage dependence in terms of the inherent properties of the sensorless pore, and on the regulation conferred by partner modules including a voltage sensor or a mechanical stress sensor. Numerous mutants generated at key locations on the PM structure favor the conductive state of the pore; accordingly, we will attempt to crystallize the most interesting candidates aiming to uncover an open structure. Having established the channel properties of the purified mechanosensitive Piezo proteins reconstituted in lipid bilayers leads us to apply our molecular dissection strategy to identify the structural determinants of the pore module and of the force sensor module. A vast number of chimeras, truncated constructs, and carefully selected mutants will be generated and functionally characterized along the lines previously reported. The ultimate aim is to confer mechanosensitivity to the robust PM scaffold by appending the force sensor module of Piezo proteins. 3. The structural and functional characterization of a newly discovered voltage-activated anion-selective channel (Xv). An immediate goal entails an in-depth functional characterization in terms of selectivity, permeation, and block of the purified full-length Xv and f its sensorless-PM after reconstitution in lipid bilayers. Attention will be directed to establish te voltage-dependence of activation given that Xv may represent a hyperpolarization-activated channel. An urgent task is to crystallize Xv and its PM using the lipid cubic phase approach we successfully applied for KvLm. The procedures we developed over this +30-year interval to investigate structure-function relationships together with newly acquired capability for structure determination augur a realistic, innovative, and productive continuation of our long-term commitment to a mechanistic understanding of voltage sensing in terms of the modular design of voltage-gated channel proteins.