GATING OF THE LARGE-CONDUCTANCE MECHANOSENSITIVE CHANNEL

机械感觉机制的分子描述是有限的。E.大肠杆菌是第一个显示出通过打开大的水孔来响应膜拉伸的分离分子，目前是最容易获得的模型系统。0.5 kb的基因，纯化的蛋白质在脂质双层中重组时完全起作用，各种突变体，20种天然同源物，最后，一种同源物的晶体结构现在都是可用的。这个基础项目的目的是详细的功能特性的MscL，特别是它的同源物从M。结核分枝杆菌（Tb-MscL）最近通过X射线晶体学解析为3.5埃，其闭合构象。似乎难以使天然开放沟道结晶，因为开放状态的能量比闭合状态高约19 kbT。长期的目标是预测开放构象和理解开放过程。具体目的是：（1）测定Tb-MscL的电生理特性。(2)使用这些结果来设置开放和亚电导状态的分子模型的约束。的约束将获得从电导，筛分测量使用聚合物，离子选择性的substates和估计的变化与开放的张力依赖性的面内通道膨胀。(3)利用共溶剂和定点突变的影响来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)评估不同厚度双层中MscL门控的能量学。(5)评价MscL对构成双层的两个单层中的张力的敏感性，因为通道在整个双层中是不对称的。这将通过评估在传统的“非偶联”和“偶联”双层古细菌双极脂质，并在一个对称的双层修饰的条件下的门控参数。初步的计算机模型的蛋白质三维结构的开放，封闭和中间构象已经开发。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。

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.