COMPUTATIONAL APPROACHES TO UNDERSTANDING CONFORMATIONAL CHANGES OF THE S6 HELI

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 离子通道是在膜渗透性中起核心作用的膜蛋白。由于分子在细胞膜上的来回传递是通讯的核心，因此一大类离子通道的最终结果是关闭和打开一个孔也就不足为奇了。通道已经进化出特定的结构域，通过结合配体或与跨膜电场相互作用来与其环境相互作用。这些变化与通道的中心孔变构耦合，导致其打开并允许离子流动。据信，传导孔的结构对于所有这些通道是共同的。大量的突变和结构数据暗示孔内衬内螺旋作为一个门，采用不同的构象时，通道打开和关闭。在闭合状态下，通道孔衬内螺旋形成倒置的“圆锥形”，导致在顶端处的螺旋束阻断离子路径。在开放状态下，这些内螺旋被认为是向外张开的，远离中心孔，允许离子通过通道移动的畅通无阻的路径。在这个项目中，我们将专注于电压门控钾通道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。已知电压依赖性通道中的门控对单个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，这种敏感性的门控突变的变化可能会提供有价值的信息通道门控的机制。我们打算看看一个单一的α-螺旋，使孔内衬使用分子动力学（MD）模拟的机械性能。我们专注于残基前的Pro-Val-Pro（PVP）基序和它们的突变，因为它们是螺旋弯曲和通道门控的关键。脯氨酸通过破坏与i-4残基的氢键来引入螺旋柔性。主链氢键的破坏和不寻常二面角的引入通常为螺旋扭结提供了基础。除了平衡模拟，我们还将进行伞形采样，以比较每个突变的弯曲自由能。我们对来自Kv1.2的S6螺旋的初步分析显示，PVP基序之前的二面角与理想相差甚远。在平衡模拟过程中，极端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。我们的自由能分析的初步结果似乎与最近的实验能量转移。为了确认这种一致性，我们必须进行更广泛的MD模拟，特别是对于需要二维反应坐标（例如，两个二面角，例如φ和psi），用于螺旋弯曲的自由能计算。我们最初的模拟集中在显式水中的单螺旋。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体）中的原子数约为32000。基于单个Intel 2.4GHz CPU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）为1200 SU，生产运行（平衡模拟和伞形采样）为28800 SU，即，每个系统的总仿真时间为250 ns。在我们进行这些模拟之后，我们打算申请更大的资助，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，1999，231-239（2002）. [2]D.H. Hackos，T. Chang和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).