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.

机械感觉机制的分子描述是有限的。大肠杆菌的大电导 (MscL) 机械敏感通道是第一个被证明可以通过打开大水孔来响应膜拉伸的分离分子，目前是最容易访问的模型系统。 0.5 kb 基因、在脂质双层中重建时完全发挥功能的纯化蛋白质、各种突变体、20 种天然同源物，以及最后一种同源物的晶体结构现已可用。该基础项目旨在详细描述 MscL 的功能特征，特别是其来自结核分枝杆菌 (Tb-MscL) 的同源物，最近通过 X 射线晶体学解析其闭合构象为 3.5 埃。由于开放态的能量比封闭态高约 19 kbT，因此似乎很难使天然开放通道结晶。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)测量Tb-MscL的电生理特性。 (2) 使用这些结果为开态和亚电导态的分子模型设置约束。这些约束将通过电导、使用聚合物的筛分测量、亚态的离子选择性以及与开口的张力依赖性相关的面内通道扩张的变化的估计来获得。 (3) 利用共溶剂和定点诱变的作用来研究分子内相互作用的性质以及可能决定MscL拉伸敏感性的特定蛋白质基序的作用。 (4) 评估不同厚度双层中 MscL 门控的能量学。 (5) 评估组成双层的两个单层中 MscL 对张力的敏感性，因为通道在双层上不对称。这将通过评估由古菌双极性脂质制成的传统“非偶联”和“偶联”双层中的门控参数以及在对称双层修饰的条件下来完成。开放、封闭和中间构象的蛋白质三维结构的初步计算机模型已经开发出来。测试这些模型的关键预测应该阐明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。