Modular Design of Voltage-Gated Channel Proteins

电压门控通道蛋白的模块化设计

• 批准号: 8862496
• 负责人: MAURICIO S MONTAL
• 金额: $ 34.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-10 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8862496
• 关键词: Address; Adopted; Anions; Architecture; Attention; Back; Biological Sciences; California; Cells; Cellular biology; Characteristics; Charge; Charybdotoxin; Chimera organism; Collection; Coupling; Crystallization; Dependence; Dissection; Doctor of Philosophy; Funding; Goals; Grant; Individual; Knowledge; Length; Lipid Bilayers; Lipids; Listeria monocytogenes; Location; Mechanical Stress; Membrane; Membrane Proteins; Molecular; Molecular Conformation; National Institute of General Medical Sciences; Neurobiology; Phase; Potassium Channel; Procedures; Property; Protein Subunits; Proteins; Public Health; Regulation; Reporting; Research; Resolution; Role; Sequence Analysis; Structure; Structure-Activity Relationship; Surface; Telefacsimile; Thinking; United States National Institutes of Health; Universities; Vision; Voltage-Gated Potassium Channel; base; design; innovation; inorganic phosphate; insight; interest; monolayer; monoolein; mutant; novel; professor; programs; protein folding; public health relevance; reconstitution; scaffold; self assembly; sensor; small molecule; three dimensional structure; voltage; voltage gated channel

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.

描述（由申请人提供）：我们在过去三十年的研究计划一直遵循以下基本原则：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它们对电压的精致敏感性的基础，并控制着它们的功能。多样性。在新的资助期内，三个主要目标集中在： 1. 确定开放构象中通道的晶体结构，试图了解传感器模块的作用。我们将通过用新型开放构象稳定剂补充用于结晶的实验条件来追求孔模块的结构功能程序，旨在解码开放通道结构。 2.根据无传感器孔隙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。 PM结构上关键位置产生的大量突变体有利于孔的导电状态；因此，我们将尝试具体化最有趣的候选者，旨在揭示一个开放的结构。确定了在脂质双层中重构的纯化机械敏感压电蛋白的通道特性后，我们可以应用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。将产生大量嵌合体、截短的构建体和精心选择的突变体，并按照先前报道的路线进行功能表征。最终目标是通过附加压电蛋白的力传感器模块，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。近期目标是在脂质双层中重构后，对纯化的全长 Xv 和 f 其无传感器-PM 的选择性、渗透和阻断进行深入的功能表征。鉴于 Xv 可能代表超极化激活通道，注意力将集中于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 的脂质立方相方法来结晶 Xv 及其 PM。我们在这 30 多年的时间里开发的用于研究结构-功能关系的程序以及新获得的结构测定能力预示着我们对电压传感机械理解的长期承诺的现实、创新和富有成效的延续。电压门控通道蛋白的模块化设计。