Modular Design of Voltage-Gated Channel Proteins

描述（由申请人提供）：我们的研究项目在过去的三十年中一直以基本原则为指导，即电压门控通道蛋白在设计上是模块化的，并且组件模块之间的耦合是其对电压的敏感和控制其功能多样性的基础。在新的筹资期内，重点关注三个主要目标：确定通道的晶体结构为开放构象，试图了解传感器模块的作用。我们将继续进行孔模块的结构-功能程序，通过补充用于结晶的实验条件，使用新的开放构象稳定剂，旨在解码开放通道结构。2. 根据无传感器孔的固有特性阐明电压依赖性的起源，以及由包括电压传感器或机械应力传感器在内的伙伴模块授予的调节。在PM结构的关键位置产生的大量突变有利于孔隙的导电状态；因此，我们将尝试结晶出最有趣的候选者，旨在揭示一个开放的结构。在建立了在脂质双层中重组的纯化的机械敏感压电蛋白的通道特性后，我们应用我们的分子解剖策略来确定孔模块和力传感器模块的结构决定因素。大量的嵌合体、截断的结构体和精心挑选的突变体将按照先前报道的思路产生和功能表征。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通道（Xv）的结构和功能表征。一个直接的目标是在脂质双分子层重构后，对纯化的全长Xv及其无传感器pm的选择性、渗透性和阻滞进行深入的功能表征。考虑到Xv可能代表一个超极化激活通道，我们将注意建立激活的电压依赖性。一项紧迫的任务是使用我们成功应用于KvLm的脂质立方相方法结晶Xv及其PM。我们在这30多年的时间里开发了研究结构-功能关系的程序，以及新获得的结构确定能力，这预示着我们长期致力于在电压门控通道蛋白的模块化设计方面对电压传感的机制理解的现实，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.