NEMD STUDY OF A MUTATED ION CHANNEL, ALPHA-HEMOLYSIN

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。 摘要：我们建议使用分子动力学程序NAMD(Kale et.)对突变的α-溶血素(a-HL)离子通道进行电压驱动的非平衡分子动力学(NEMD)模拟。Al，1999)。该通道首先嵌入人造膜，然后在中性pH的1M氯化钠盐溶液中溶解并平衡；然后在整个系统中施加外部电压。对该系统施加了额外的限制，使蛋白质-溶剂界面处的残基可以自由移动，而蛋白质的其余部分保持冻结。这些模拟是基于我们之前对a-HL系统的计算，在PSC的Lemieux星系团上执行(Grant#mcb030021p)，在那里我们产生了从-400 mV到400 mV的I-V曲线，与实验结果很好地一致(Misakian和Kasianowicz，2003)。我们收集了氯离子和钠离子沿中心孔轴的浓度分布和一维平均作用力(PMF)分布，这表明氯离子在孔的狭窄区域，包括孔的最狭窄部分，作为有利于阴离子的弱选择性过滤器而明显优先。此外，结果揭示了沿着离子通道衬里的一个位置(在lys147处)，它可能为定点突变提供一个很好的候选位置，以便将系统的固有属性从阴离子改变为阳离子选择性。我们想通过使用突变的a-HL结构产生200 mV和-200 mV的I-V数据来测试这一点，突变的a-HL结构带有一个、三个和七个核苷酸突变。这些计算包括平衡和I-V计算，将在匹兹堡超级计算中心的Bigben集群上最有效地执行，因为其可扩展的架构和大量可用CPU。每个I-V数据点在Bigben上应该需要大约5000小时的处理器时间，总共需要30,000个服务单元。Kale，L.，R.Skeel，M.Bhandarkar，R.Brunner，A.Gursoy，N.Krawitz，J.Phillips，A.Shinozaki，K.Varadarajan和K.Schulten。1999年。NAMD2：并行分子动力学的更大可扩展性。计算物理学报151：283-312。和J.J.Kasianowicz。2003年。离子通过溶血素通道的静电控制。膜生物学学报，195(3)，137-146。

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. Abstract: We propose to perform voltage-driven non-equilibrium molecular dynamics (NEMD) simulations of a mutated alpha-hemolysin (a-HL) ion channel, using the molecular dynamics program NAMD (Kale et. al, 1999). The channel is first embedded in an artificial membrane, and solvated in 1M NaCl salt solution at neutral pH and equilibrated; then an external voltage potential is applied across the system. Additional constraints are placed on the system so that the residues at the protein-solvent interface are allowed to move freely while the rest of the protein remains frozen. These simulations are based on our previous calculations of the a-HL system, performed on the Lemieux Cluster at PSC (grant # mcb030021p), where we generated an I-V curve from -400mV to +400mV, in good correspondence with experimental results (Misakian and Kasianowicz, 2003). We collected concentration profiles as well as a 1-D potential of mean force (PMF) profile along the central pore axis for both chloride and sodium ions, which revealed a distinct preference for chloride ions in the narrow regions of the pore, including the most constricted section of the pore, which serves as a weak selectivity filter favoring anions. Additionally, the results revealed a site along the lining of the ion channel (at lys147) which might provide a good candidate for site-directed mutagenesis in order to change the permeant properties of the system from anion to cation selective. We would like to test this by generating I-V data at +200mV and -200 mV using mutated a-HL structures with one, three and seven lys147 -> ser point mutations. These calculations, consisting of equilibration and I-V calculations, would be most effectively carried out on the Bigben cluster at Pittsburgh Supercomputing Center, due to its scalable architecture and large number of available CPUs. Each of the I-V data points should require approximately 5000 hours of processor time on Bigben, for a total request of 30,000 service units. Kale, L., R. Skeel, M. Bhandarkar, R. Brunner, A. Gursoy, N. Krawetz, J. Phillips, A. Shinozaki, K. Varadarajan, and K. Schulten. 1999. NAMD2: Greater scalability for parallel molecular dynamics. Journal of Computational Physics 151: 283-312. Misakian M. and J. J. Kasianowicz. 2003. Electrostatic control of ion transport through the -hemolysin channel. Journal of Membrane Biology, 195 (3), 137-146.