COMPUTATIONAL APPROACHES TO UNDERSTANDING CONFORMATIONAL CHANGES OF THE S6 HELI

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 离子通道是在膜通透性中起核心作用的膜蛋白。由于分子在膜上来回传递是通讯的中心，因此关闭和打开一个孔是一大类离子通道的最终结果也就不足为奇了。通道通过结合配体或与跨膜的电场相互作用，进化出特定的结构域来与其环境相互作用。这些变化以变构方式耦合到通道的中心孔，使其打开并允许离子流动。传导孔的结构被认为是所有这些通道的共同结构。大量的突变和结构数据表明，孔衬内螺旋是一扇门，当通道打开和关闭时，它采用不同的构象。在闭合状态下，内层螺旋的通道形成一个倒置的“帐篷”，在顶端形成螺旋束，阻断离子通道。在开放状态下，这些内部螺旋被认为是向外扩展，远离中央毛孔，允许离子通过通道畅通无阻地移动。在这个项目中，我们将重点研究电压门控钾通道Kv1.2的孔衬内S6螺旋的构象变化，以及单个氨基酸突变如何影响这些构象。众所周知，电压依赖通道中的门控对单一氨基酸突变非常敏感[1，2]。我们将研究改变通道机制的突变。我们这个项目的动机是，这种对突变变化的门控敏感性可能提供关于通道门控机制的有价值的信息。我们打算使用分子动力学(MD)模拟来研究组成毛孔衬里的单个α-螺旋的机械特性。我们关注Pro-Val-Pro(PVP)基序之前的残基及其突变，因为它们对螺旋弯曲和通道门控至关重要。通过破坏I-4残基的氢键，Pro引入了螺旋柔性。这种主链氢键的破坏和不寻常的二面角的引入往往为螺旋扭曲提供了基础。除了平衡模拟，我们还将进行伞形抽样，以比较每个突变的弯曲自由能。我们对Kv1.2中S6螺旋的初步分析表明，PVP基序之前的二面角非常不理想。在平衡模拟中，极端的二面角松弛到典型的α螺旋值。这一结果表明，前脯氨酸残基的二面角是螺旋弯曲的良好反应坐标。我们关于自由能分析的初步结果似乎与最近的实验能量移动一致。为了确认这种一致性，我们必须进行更广泛的MD模拟，特别是对于伞状采样，在这种情况下，我们需要二维反应坐标(例如，两个二面角，如Phi和psi)来计算螺旋弯曲的自由能。我们最初的模拟集中在显性水中的单个螺旋上。我们将使用NAMD进行所有MD模拟。每个系统(一个野生型和五个突变型)的原子数量约为32000个。基于单个英特尔2.4 GHz处理器上的初始测试运行的估计计算时间如下：初始设置(最小化、加热和平衡)1200SU，生产运行(平衡模拟和伞形抽样)28800 SU，即每个系统总共250 ns的模拟。在我们进行了这些模拟之后，我们打算申请一笔更大的拨款，以将我们的分析扩展到膜中的全通道结构。参考文献[1]O.Yifrach和R.MacKinnon，Cell，第111卷，231-239(2002)。[2]D.H.Hackos，T.Chang，K.J.Swartz，J.Gen.Physiol，第119卷，521-531(2002)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ion channels are membrane proteins that play a central role in membrane permeability. Since passing molecules back and forth across the membrane is central to communication, it is not surprising that closing and opening a pore is the end result of a large class of ion channels. Channels have evolved specific domains to interact with their environment by binding ligands or interacting with the electric field across the membrane. These changes are allosterically coupled to the central pore of the channel causing it to open and allow ion flow. It is believed that the architecture of the conduction pore is common to all of these channels. Extensive mutational and structural data implicate the pore-lining inner helix as a gate that adopts different conformations when channels open and close. In the closed state, the channels pore-lining inner helices form an inverted 'teepee' resulting in a helical bundle at the apex that blocks the ion pathway. In the open state, these inner helices are thought to splay out, away from the central pore, allowing an unobstructed pathway for ions to move through the channel. In this project, we will focus on conformational changes of the pore-lining inner S6 helix of the voltage-gated potassium channel Kv1.2 and how single amino acid mutations affect these conformations. It is known that gating in voltage-dependent channels is very sensitive to a single amino acid mutations[1,2]. We will examine mutations that change the mechanics of the channel. Our motivation for this project is that this sensitivity of gating to mutational changes may provide valuable information about the mechanism of channel gating. We intend to look at the mechanical properties of a single alpha-helix that makes up pore lining using molecular dynamics (MD) simulations. We focus on residues preceding the Pro-Val-Pro (PVP) motif and their mutations because they are critical for helix bending and channel gating. Proline introduces helix flexibility by disrupting hydrogen bonding with the i-4 residue. This disruption of backbone hydrogen bonding and introduction of unusual dihedral angles often provides the basis for helix kinking. In addition to equilibrium simulations, we will also carry out umbrella sampling to compare free energy of bending for each mutation. Our initial analysis of the S6 helix from Kv1.2 revealed that the dihedral angles preceding the PVP motif are extremely far from ideal. During the equilibrium simulations, the extreme dihedral angles relax to typical alpha-helical values. This results suggest that the dihedral angles of pre-proline residues are good reaction coordinates for helix bending. Our preliminary result on free energy analysis seems consistent with recent experimental energy shifts. In order to confirm this consistency we must carry out more extensive MD simulations, especially for the umbrella sampling where we need two-dimensional reaction coordinates (e.g., two dihedral angles such as phi and psi) for free energy calculation of the helix bending. Our initial simulations focus on a single helix in explicit water. We will use NAMD for all MD simulations. The number of atoms in each system (one wild-type and five mutants) is about 32000. The estimated computing time based on initial test runs on a single Intel 2.4GHz CPU is as follows: 1200 SUs for initial setups (minimization, heating, and equilibration) and 28800 SUs for production runs (both equilibrium simulations and the umbrella sampling), i.e., total 250ns simulation for each system. After we carry out these simulations we intend to apply for a larger grant to extend our analyses to full channel structure in a membrane. References [1] O. Yifrach and R. MacKinnon, Cell, Vol 111, 231-239 (2002). [2] D.H. Hackos, T. Chang, and K.J. Swartz, J. Gen. Physiol, Vol. 119, 521-531 (2002).