Molecular Basis for Voltage-Dependent Channel Function

技术：固体核磁共振实验将在取向膜上进行，取向膜上含有修饰的，标记的电压依赖性的革兰西菌素通道，在零电压和非零电压下。待研究的系统将包括仅通过修改其中一个亚基在通道中心附近引入不对称的通道，以及新发现的同二聚体电压依赖性革兰杀虫素通道。改进后的通道在两个或多个导通状态之间表现出非线性行为和电压依赖门控。螺旋亚基由连接每个亚基的1-5个氨基酸的氢键连接在一起，序列的这些区域负责门控。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选择性地用2H或l5N标记。固态核磁共振波谱将用于表征平均取向和动态的特异性标记位点，传感电压变化在脂质双分子层。同型二聚体样品将是有利的，因为它们所有的通道将具有相同的化学成分。这一特性将使非零电位的研究首次成为可能。Lay：该项目旨在定义离子选择膜通道电压依赖性门控的分子机制，使用唯一已知的门控系统，其化学结构在分子水平上被理解。用于研究的模型系统由几种最近发现的抗生素革兰西菌素a的化学修饰衍生物组成。这些衍生物被设计成具有电压依赖特性，这将有助于进一步了解通道门控的基本原理。固体磁共振实验将在零电压和脉冲电场的存在下，对这些设计的gramicidin通道进行支持。这一结果将对理解生物膜中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.