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.

机械强度机制的分子描述受到限制。大肠杆菌大型电导率（MSCL）的机械敏感通道，这是第一个孤立的分子通过打开大型水孔来响应膜拉伸的第一个孤立分子，目前是最容易接近的模型系统。当在脂质双层，多种突变体，二十种天然同源物以及最后一个同源物的晶体结构中重新构造时，0.5 kb基因是纯化的蛋白质完全功能。这个基本项目旨在详细的MSCL功能表征，尤其是其来自结核分枝杆菌（TB-MSCL）的同源物（TB-MSCL），该功能最近通过X射线晶体学对其封闭构型中的3.5埃曲线进行了解决。似乎很难使天然开放通道结晶，因为开放状态的能量比封闭状态高约19 kbt。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）使用这些结果为开放态和亚电导状态的分子模型设置约束。这些约束将从电导，使用聚合物进行筛分，取代的离子选择性以及与开口张力依赖性相关的平面内通道膨胀变化的估计。 （3）使用共溶剂和位点的效果，指示诱变来研究分子内相互作用的性质以及特定蛋白基序的作用，这可能决定了MSCL的拉伸敏感性。 （4）评估不同厚度不同的双层中MSCL门控的能量。 （5）评估组成双层的两个单层中的MSCL对张力的敏感性，因为该通道在双层中是不对称的。这将通过评估传统的“未偶联”和“耦合”双层脂质制成的传统“未偶联”和“耦合”双极脂质的门控参数来完成，并在对称双层修饰的条件下进行。已经开发了蛋白质3维结构的初步计算机模型。测试这些模型的临界预测应阐明不同蛋白质结构域之间的功能作用和关系，以及从脂质双层传达张力的机制到通道门控。