GATING OF THE LARGE-CONDUCTANCE MECHANOSENSITIVE CHANNEL

大电导机械敏感通道的门控

• 批准号: 6499440
• 负责人: SERGEI I SUKHAREV
• 金额: $ 24.49万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-29 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6499440
• 关键词: Mycobacterium tuberculosis; bioenergetics; cell morphology; conformation; hydropathy; lipid bilayer membrane; mechanical pressure; membrane model; membrane reconstitution /synthesis; neural conduction; phospholipids; voltage /patch clamp; voltage gated channel

Molecular descriptions of mechanosensory mechanisms are limited. The mechanosensitive channel of the large conductance (MscL) of E. coli, the first isolated molecule shown to respond to membrane stretch by opening a large aqueous pore, currently is the most accessible model system. The 0.5 kb gene, the purified protein completely functional when reconstituted in lipid bilayers, a variety of mutants, twenty natural homologs, and, finally, the crystal structure of one homolog are now available. This basic project is aimed at detailed functional characterization of MscL and particularly its homolog from M. tuberculosis (Tb-MscL) recently resolved by X-ray crystallography to 3.5 Angstroms in its closed conformation. It appears that it will be difficult to crystallize the native open channel since the energy of the open state is about 19 kbT above the closed state. The long-term goal of the proposed work is to predict the open conformation and understand the opening process. The specific aims are: (1)To measure the electrophysiological properties of Tb-MscL. (2) Use these results to set constraints for molecular models of the open and subconductance states. The constraints will be obtained from conductances, sieving measurements using polymers, ionic selectivity of the substates and estimates of the changes of in-plane channel expansion associated with the tension dependence of opening. (3) Using effects of co-solvents and site directed mutagenesis to study the nature of intramolecular interactions and role of specific protein motifs that may determine the stretch-sensitivity of MscL. (4) Evaluate energetics of MscL gating in bilayers of different thickness. (5) Evaluate MscL sensitivity to tension in the two monolayers that compose a bilayer since the channel is asymmetric across the bilayer. This will be done by evaluating the gating parameters in conventional 'uncoupled' and 'coupled' bilayers made of archaeal bipolar lipids, and under conditions of a symmetrical bilayer modification. Preliminary computer models of the proteins 3-dimensional structure in open, closed and intermediate conformations have been developed. Testing the critical predictions of these models should clarify the functional role and relationships between different protein domains, and the mechanism by which tension is conveyed from the lipid bilayer to channel gating.

对机械感觉机制的分子描述是有限的。大肠杆菌大电导机械敏感通道(MSCL)是第一个通过打开一个大的水孔来响应膜拉伸的分离分子，是目前最容易理解的模型系统。0.5kb的基因，纯化的蛋白在脂双层中重组时完全具有功能，各种突变体，20个天然同源物，最后，一个同源物的晶体结构现在是可用的。这一基础项目旨在详细描述MSCL的功能，特别是其来自结核分枝杆菌的同系物(TB-MSCL)，最近通过X射线结晶学将其解析为闭合构象的3.5埃。这似乎很难结晶自然开放的通道，因为开放状态的能量比关闭状态的能量高出约19KBT。这项拟议工作的长期目标是预测开放构象并了解开放过程。具体目的是：(1)检测TB-MSCL的电生理特性。(2)利用这些结果为开态和亚导状态的分子模型设置约束条件。这些约束将通过电导、使用聚合物的筛选测量、亚态的离子选择性以及与张开的张力相关的面内通道扩展变化的估计来获得。(3)利用共溶剂效应和定点突变研究分子内相互作用的性质和可能决定MSCL拉伸敏感性的特定蛋白质基序的作用。(4)对不同厚度双层膜的MSCL浇注进行了能量学评价。(5)评估MSCL对组成双层的两个单分子膜中的张力的敏感性，因为通道在该双层上是不对称的。这将通过评估传统的由古生物双极脂组成的非偶联和偶联双层中的门控参数来完成，并在对称双层修改的条件下进行。初步建立了蛋白质开放构象、闭合构象和中间构象的三维结构的计算机模型。测试这些模型的关键预测应该澄清不同蛋白质结构域之间的功能作用和关系，以及张力从脂质双层传递到通道门控的机制。