GATING OF THE LARGE-CONDUCTANCE MECHANOSENSITIVE CHANNEL

机械感觉机制的分子描述是有限的。大肠杆菌大电导的机械敏感通道（MscL）是第一个通过打开大水孔对膜拉伸做出反应的分离分子，目前是最容易获得的模型系统。0.5 kb的基因，在脂质双层中重组后功能完全的纯化蛋白，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已经可用。本基础项目旨在详细描述MscL的功能特征，特别是结核分枝杆菌的同源物（Tb-MscL），最近通过x射线晶体学在其封闭构象中分辨到3.5埃。由于开放态的能量比封闭态高约19 kbT，因此天然开放通道的结晶似乎很困难。提出的工作的长期目标是预测开放构象和了解开放过程。具体目的是：(1)测定Tb-MscL的电生理特性。(2)利用这些结果对开态和亚电导态的分子模型设置约束。这些约束将从电导、使用聚合物的筛分测量、基态的离子选择性以及与开口的张力依赖相关的面内通道膨胀变化的估计中获得。(3)利用共溶剂和定点诱变的作用，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)评价不同厚度双层中MscL门控的能量学。(5)由于通道在双分子层上是不对称的，因此评估构成双分子层的两个单分子层中MscL对张力的敏感性。这将通过评估由古菌双极性脂质制成的传统“不耦合”和“耦合”双层中的门控参数，并在对称双层修饰的条件下完成。初步建立了蛋白质三维结构的开放、封闭和中间构象的计算机模型。测试这些模型的关键预测将澄清不同蛋白质结构域之间的功能作用和关系，以及张力从脂质双分子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.