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）：过去三十年来，我们的研究计划受到电压通道蛋白在设计中是模块化的基本宗旨的指导，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，并具有其功能多样性。在新的资金期间，三个主要目标的重点是：1。在开放构象中确定通道的晶体结构，试图理解传感器模块的作用。我们将通过补充用于将其结晶的实验条件与旨在解释开放通道结构进行解码的新型开放构造稳定器结晶的实验条件来追求孔模块的结构功能程序。 2。根据传感器孔的固有特性以及由伴侣模块（包括电压传感器或机械应力传感器）所赋予的调节，阐明了电压依赖性的起源。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，我们将尝试结晶旨在揭开开放结构的最有趣的候选人。建立了在脂质双层中重构的纯化机械敏感的压电蛋白的通道特性，这使我们应用了我们的分子解剖策略，以识别孔模块和力传感器模块的结构决定因素。将生成大量的嵌合体，截短的构建体和精心选择的突变体，并沿着先前报道的线进行功能表征。最终的目的是通过附加压电蛋白的力传感器模块来赋予强大的PM支架机械敏。 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。直接的目标需要从选择性，渗透和纯化的全长XV和F其Sensorless-PM的块中进行深入的功能表征，并在脂质双层中重新建立后。考虑到XV可能代表超极化激活的通道，注意力将引导以建立激活的电压依赖性。紧急任务是使用我们成功应用于KVLM的脂质立方相的方法来结晶XV及其PM。我们在此 +30年间隔中开发的程序，以研究结构 - 功能关系，以及新获得的结构确定能力，以使我们长期承诺的现实，创新和生产力延续，以根据电压门控通道蛋白的模块化设计对电压感测的机械理解。