NEMD STUDY OF A MUTATED ION CHANNEL, ALPHA-HEMOLYSIN

这个子项目是许多研究子项目中的一个 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， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 摘要：我们建议使用分子动力学程序NAMD（Kale et.等人，1999）。首先将通道嵌入人工膜中，并在中性pH的1 M NaCl盐溶液中溶剂化并平衡;然后在整个系统上施加外部电压电位。对系统施加额外的限制，使得蛋白质-溶剂界面处的残留物可以自由移动，而蛋白质的其余部分保持冷冻。这些模拟是基于我们之前对a-HL系统的计算，在PSC的Lemieux集群上进行的（授权#mcb 030021 p），我们生成了从-400 mV到+400 mV的I-V曲线，与实验结果良好一致（Misakian和Kasianowicz，2003）。我们收集的浓度曲线以及1-D的平均力（PMF）的曲线沿着的中心孔轴的氯离子和钠离子，这揭示了一个明显的偏好氯离子在狭窄的区域的孔，包括最狭窄的部分的孔，作为一个弱的选择性过滤器有利于阴离子。此外，结果揭示了一个位点沿着内衬的离子通道（在lys 147），这可能提供了一个很好的候选人定点诱变，以改变系统的渗透性能从阴离子到阳离子选择性。我们想通过使用具有一个、三个和七个lys 147-> ser点突变的突变的a-HL结构在+200 mV和-200 mV下产生I-V数据来测试这一点。这些计算，包括平衡和I-V计算，将最有效地在匹兹堡超级计算中心的Bigben集群上进行，由于其可扩展的架构和大量的可用CPU。每个I-V数据点在Bigben上需要大约5000小时的处理时间，总共需要30，000个服务单元。凯尔湖，R. Skeel，M.班达尔卡尔河布伦纳，A。Gursoy，N.菲利普斯，A. Shinozaki，K. Varadarajan和K.舒尔滕1999. NAMD 2：并行分子动力学的更大可扩展性。计算物理学杂志151：283-312。三崎湾和J·J·卡西亚诺维奇2003.通过溶血素通道的离子运输的静电控制。Journal of Membrane Biology，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.