Molecular Basis for Voltage-Dependent Channel Function

KoeppeGreathouse 9816063技术：固态NMR实验将在含有修饰的、标记的电压依赖性短杆菌肽通道的定向膜上在零电压和非零电压下进行。系统的研究将包括通道，其中的不对称性引入附近的通道中心，通过修改只有一个亚基，和一个新发现的homodimeric电压依赖短杆菌肽通道。修饰后的通道在两个或多个导电状态之间表现出非线性行为和电压依赖性门控。螺旋亚基通过连接每个亚基的氨基酸1-5的氢键保持在一起，并且这些序列区域负责门控。每个亚基的前五个残基内的特定主链和侧链基团将通过固相化学合成用2 H或15 N选择性标记。固态NMR光谱将被用来表征的平均取向和动态的具体标记的网站，感测电压的变化，在脂质双层。同二聚体样品将是有利的，因为它们的所有通道将具有相同的化学组成。这一特点将允许在非零电位的研究是可行的第一次。Lay：该项目旨在定义一个分子机制的电压依赖性门控离子选择性膜通道使用唯一已知的门控系统的化学结构是在分子水平上理解。用于研究的模型系统由几种最近发现的抗生素短杆菌肽A的化学修饰衍生物组成。这些衍生物被设计为具有电压依赖性，这将有助于进一步了解通道门控。固态磁共振实验将进行这些设计的短杆菌肽通道内的定向膜支持在零电压，并在脉冲电场的存在下。这些结果对于理解生物膜通道门控的化学和分子基础具有重要意义。

KoeppeGreathouse9816063Technical:Solid-state NMR experiments will be performed on oriented membranes containing modified, labeled voltage-dependent gramicidin channels at both zero and nonzero voltage. Systems for study will include channels in which an asymmetry is introduced near the channel center, by modifying only one of the subunits, and a newly discovered homodimeric voltage-dependent gramicidin channel. The modified channels show nonlinear behavior and voltage-dependent gating among two or more conducting states. The helical subunits are held together by hydrogen bonds that link amino acids 1-5 of each subunit, and these regions of sequence are responsible for the gating. Specific backbone and side chain groups within the first five residues of each subunit will be selectively labeled with 2H or l5N by solid-phase chemical synthesis. Solid-state NMR spectroscopy will be used to characterize the average orientations and dynamics of specifically labeled sites that sense voltage changes in lipid bilayers. The homodimeric samples will be advantageous because all of their channels will have the same chemical composition. This feature will allow studies at nonzero potential to be feasible for the first time.Lay:This project seeks to define a molecular mechanism for the voltage-dependent gating of ion-selective membrane channels using the only known gated system for which the chemical architecture is understood at the molecular level. The model system for study consists of several recently discovered chemically modified derivatives of the antibiotic gramicidin A. These derivatives were designed to have voltage-dependent properties that would help further the basic understanding of channel gating. Solid-state magnetic resonance experiments will be performed on these designed gramicidin channels supported within oriented membranes at zero voltage, and in the presence of a pulsing electric field. The results will be important for understanding the chemical and molecular basis for channel gating in biological membranes.