Exploring the impact of sequence on the mechanistic properties of voltage-sensing domains

质膜的存在使得细胞能够维持与细胞存活和正常功能相容的特化细胞内环境。 与外界环境的交流需要信号在这一屏障上来回传递，这种传递必须受到严格的调节，以维持细胞的状态。一类完整的膜蛋白已经进化出电压敏感结构域（VSD），因此它们的作用可以通过膜电位的变化来调节。 这些结构域包含四个跨膜区段，通常标记为S1-S4，其含有能够感测跨膜电场变化的带电残基。负电位将传感器“保持”在电荷更接近细胞内空间的配置中，称为“向下状态”，并且更正的电压将传感器向外驱动穿过膜界面进入“向上状态”。VSD高度保守;然而，VSD序列变异导致电压门控钠通道（Nav）的快速门控和电压门控钾通道（Kv）的较慢门控。此外，在过去的一年半里，已经增选VSD来调节其功能的蛋白质的数量和多样性已经增长到包括质子通道和膜结合磷酸酶。 所有这些蛋白质必须对电压做出不同的反应，以确保特异性。实现这一点的两种方法是通过控制VSD从向下状态切换到向上状态的电压范围以及通过调谐这些转变发生的时间尺度。例如，Nav和Kv通道之间开放动力学的巨大差异对于动作电位的产生是必不可少的。鉴于VSD的高度带电性质及其对电场的极端敏感性，这两种机制将使用VSD的分子模型结合连续体静电计算进行探讨。在Grabe博士的NSF博士后研究期间开发的VSD的下状态分子模型将进一步完善，然后这些模型将用于构建开放过渡的门控轨迹。这将通过比较Kv1.2钾通道的下态模型与上态X射线晶体结构来完成。现实的中间体沿着这条途径将创建通过结合增量刚体插值的螺旋之间的端点状态与分子动力学模拟在每个增量。然后，这些结构将用于Grabe等人（2004）开发的泊松-玻尔兹曼计算，以量化门控的静电。这种能量分析将允许确定不同电压传感器激活的电压范围，以及估计激活动力学。这些都是在这类重要的蛋白质中开发电压感知统一观点的重要步骤，这项工作将为细胞如何感知环境提供新的见解。 这一项目将对教育和培训以及新调查员的职业发展产生更广泛的影响。 PI已经招募了一名研究生和一名本科生进入他的实验室，并在预算中要求对这些学生以及另一名本科生提供支持。 PI还将新的课程材料引入研究生和本科生课程，包括为本科生开设的分子建模课程，该课程将他的实验室项目和新的跨学科数学/计算生物学课程中的材料结合起来，该课程将于2007年秋季为研究生和高级本科生提供。

The presence of the plasma membrane makes it possible for cells to maintain a specialized intracellular environment compatible with cell survival and proper function. Communication with the external environment requires signals to be passed back and forth across this barrier, and this transfer must be tightly regulated so as to maintain cellular conditions. One class of integral membrane proteins has evolved voltage-sensing domains (VSDs) so that their action can be regulated by changes in membrane potential. These domains comprise four transmembrane segments, typically labeled S1-S4, which contain charged residues capable of sensing changes in the electric field across the membrane. Negative potentials 'hold' the sensor in a configuration in which the charges are closer to the intracellular space, termed the 'down state', and more positive voltages drive the sensor outward across the membrane interface into an 'up state'. VSDs are highly conserved; however, VSD sequence variations are responsible for fast gating of voltage-gated sodium channels (Nav) and slower gating of voltage-gated potassium channels (Kv). Moreover within the past year and a half, the number and diversity of proteins that have co-opted VSDs to regulate their function has grown to include proton channels and membrane bound phosphatases. All of these proteins must respond to voltage differently to ensure specificity. Two ways of doing this are by controlling the voltage range over which the VSDs switch from the down state to the up state and by tuning the timescale with which these transitions occur. For instance, the large difference in opening kinetics between Nav and Kv channels is essential for the generation of action potentials. Given the highly charged nature of the VSD and its extreme sensitivity to electric fields, both of these mechanisms will be probed using molecular models of the VSD combined with continuum electrostatic calculations. Down state molecular models of the VSD developed during Dr. Grabe's NSF postdoctoral fellowship will be further refined and these models then will be used to construct gating trajectories of the opening transition. This will be done by comparing the down state model with the up state X-ray crystal structure of the Kv1.2 potassium channel. Realistic intermediates along this pathway will be created by combining incremental rigid body interpolation of helices between the end point states with molecular dynamics simulations at each increment. The structures will then be used in Poisson-Boltzmann calculations, as developed in Grabe et al. (2004), to quantify the electrostatics of gating. This energetic analysis will allow determination of the voltage range over which different voltage sensors activate, as well as the estimation of activation kinetics. These are both important steps in developing a unified view of voltage sensing in this important class of proteins, and this work will provide new insights into how cells sense their environments. The broader impacts of this project will be on education and training, as well as the career development of a new investigator. The PI has already recruited a graduate student and an undergraduate student into his lab, and support for these students as well as another undergraduate student is requested in the budget. The PI also has introduced new course material into graduate and undergraduate curricula, including a molecular modeling course for undergraduate students that incorporates material from his lab project and a new cross-disciplinary mathematical/computational biology course for graduate and advanced undergraduate students to be offered Fall 2007.