SIMULATING DYNAMICS AND ORIENTATIONS OF ENGINEERED SIDE CHAINS IN THE RESTRICTI

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 我们要求在TeraGrid资源，特别是Pople上的计算时间和存储空间，以运行广泛的并行分子动力学模拟，以确定限制性内切酶EcoRI的修饰侧链的优先取向。EcoRI对DNA的六个碱基对序列具有极高的结合特异性，其结构来源通过电子自旋共振(ESR)进行了研究。结合位点定向自旋标记(将包含氮氧化物标记的侧链引入蛋白质)，对EcoRI进行距离测量以提供系统的结构约束。为了从ESR测量中更准确地描绘EcoRI的自旋标记位置的结构和动力学图，需要了解平均氮氧基侧链取向。使用蒙特卡罗旋转异构体搜索方法结合全原子分子动力学对氮氧化物侧链进行建模的初步尝试表明，由于采样限制，实验和模拟之间存在差异。这项请求旨在建立一种程序，其中可以使用广泛的MD模拟来再现两个氮氧化物侧链之间的距离，从而能够提取C？-C？距离和分布。模拟还将允许区分氮氧化物侧链动力学和主链动力学在ESR线形和距离分布上的作用。我们将使用两个独立的氮氧化物力场在蛋白质中的六个不同位置进行10个并行模拟。通过对晶体结构进行双重标记，总共将进行60次模拟，每次100 ns，以模拟不同二级结构上氮氧化物的平均取向和动力学，并研究氮氧化物参数对这些可观测值的影响。我们要求的480K CPU小时将提供执行长时间运行所需的计算能力，从而能够对氮氧化物侧链进行足够的采样。通过这种方式，我们将朝着我们的目标努力，开发一种可应用于不同生物系统的MD模拟程序，以可靠地模拟氮氧化物侧链的取向和动力学，以便更好地了解蛋白质或DNA结构和动力学的更多原子细节。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We request computing time and storage space on TeraGrid resources, specifically Pople, to run extensive parallel molecular dynamics simulations to determine the preferred orientations of a modified side chain of the restriction endonuclease EcoRI. EcoRI exhibits an extremely high binding specificity to a six base pair sequence of DNA, the structural origin of which was investigated using Electron Spin Resonance (ESR). In conjunction with site-directed spin labeling, which introduces a side chain containing a nitroxide label into the protein, distance measurements were performed on EcoRI to provide structural constraints of the system. In order to paint a more accurate picture of the structure and dynamics of the spin labeled locations of EcoRI from ESR measurements, knowledge of the average nitroxide side chain orientation is required. Initial attempts to model the nitroxide side chain using Monte Carlo rotamer search methods in combination with all-atom molecular dynamics have revealed discrepancies between experiment and simulation due to sampling restrictions. This request aims to establish a procedure where extensive MD simulations can be used to reproduce the distance between two nitroxide side chains, enabling the extraction of the C?-C? distance and distribution. The simulations will also allow a discrimination of the role of nitroxide side chain dynamics versus backbone dynamics on ESR lineshapes and distance distributions. We will run 10 parallel simulations at six different sites in the protein using two independent nitroxide force fields. By doubly labeling the crystal structure a total of 60 simulations will be run for 100 ns each to simulate the average nitroxide orientation and dynamics at different secondary structures in addition to investigating the effects of nitroxide parameterization on these observables. Our request of 480K CPU hours will provide the computational power needed to perform long runs that enable sufficient sampling of the nitroxide side chain. In this way, we will work towards our goal of developing an MD simulation procedure that can be applied to different biological systems to reliably model nitroxide side chain orientations and dynamics so that more atomistic details of the protein or DNA structure and dynamics can be better understood.