Project 1/Computational Core

计算核心研究计划已修订，以解决具体问题和关切 由审查员提出，并强调这一核心的原则重点。会议的主要目标是 计算核心是1)开发一套计算工具和协议，以促进分析和 对EPR光谱数据的解释，包括从鹿实验获得的距离测量 双自旋标记蛋白质，以及2)为各个研究项目提供基本的计算支持。 对于项目1，基本的计算支持需要一系列平衡的MD模拟来支持EPR 光谱计算。在项目2中，这种计算支持包括详细的均衡MD模拟 CDB3探索P327R点突变可能引发的构象变化，并初步 研究结果已在上述项目2研究计划中进行了描述。对项目3的基本计算支持包括 常规2D-核磁共振实验和顺磁共振的常规结构精化计算 增强核磁共振实验，以及探索自旋标记构象趋势的MD模拟 在淀粉样β多肽中引入。这种构象分析对于解决距离将是重要的 EPR实验和顺磁实验中自旋标记侧链构象行为的相关性 共振增强核磁共振研究。 促进EPR数据分析的实用计算工具和协议的开发取决于 关键是基于项目1中获得的数据，并且需要几个谨慎的步骤。首先，重要的是要确定 我们可以使用描述自旋标记侧链动力学和蛋白质的传统平衡MD模拟 骨架动力学，再加上模拟全球蛋白质翻滚的布朗动力学计算， 直接计算单标蛋白质的EPR谱。正如评论家指出的那样，以前发表的尝试 利用这种类型的战略并不完全令人满意或令人信服。然而，这些以前的 研究基于相当有限的MD模拟，并可能受到一些其他问题的影响，我们 在下面的研究计划中详细说明。重要的是要确定模拟策略可以是 用于计算EPR谱，以确定我们可以捕捉到 自旋标记的蛋白质，对不同的样品产生独特的EPR谱(例如，尖锐、明显的 完全移动自旋标签的典型光谱信号与更广泛、更复杂的信号 部分固定的自旋标记的代表)。正如项目1研究计划中所讨论的，我们现在有 初步结果表明，我们可以比以往更准确、更可靠地计算EPR谱 之前报道过。在此基础上，对现有的基于MD的数据挖掘技术进行了详细的分析 下面的EPR波谱模拟突出了当前方法中可能存在的不足，并讨论了 解决这些问题的具体策略和测试。只有在我们令人信服地确定我们 可以直接用MD/Brown联合动力学模拟协议计算EPR谱 认真处理EPR鹿实验中获得的自旋标记对距离的计算，或追求 开发更简单的计算策略，不需要多次、冗长的MD模拟 用明确的溶剂来估算这些距离。影响自旋标签分子动力学模拟的几个问题 配对距离，包括由项目1的评审者提出的几个(例如，势函数参数， 静电处理、周期性边界效应等)我们在修订后的研究中提供了初步数据 解决这些问题和其他重要因素的以下计划，以及实现改进的战略 EPR谱计算和鹿距离估计是在一个新的具体目标1的背景下提出的。

The Computational Core research plan has been revised to address specific questions and concerns raised by the reviewers, and to emphasize the principle focus of this core. The primary goals of the Computational Core are 1) to develop a set of computational tools and protocols to facilitate the analysis and interpretation of EPR spectral data, including distance measurements obtained from DEER experiments for doubly spin-labeled proteins, and 2) provide basic computational support for the individual research projects. For Project 1, basic computational support entails a series of equilibrium MD simulations to support EPR spectral calculations. In Project 2, this computational support includes detailed equilibrium MD simulations for CDB3 to explore possible conformational changes triggered by the P327R point mutant, and preliminary results are described above in the Project 2 Research Plan. Basic computational support for Project 3 includes routine structure refinement calculations for conventional 2D-NMR experiments and paramagnetic resonance enhancement NMR experiments, as well as MD simulations to explore conformational trends for spin labels introduced in the amyloid-beta peptides. This conformational analysis will be important to address distance dependencies on spin label side chain conformational behavior in both EPR experiments and paramagnetic resonance enhancement NMR studies. The development of practical computational tools and protocols to facilitate EPR data analysis depends crucially on data obtained in Project 1, and requires several discreet steps. First, it is important to establish that we can use conventional equilibrium MD simulations that describe spin label side chain dynamics and protein backbone dynamics, coupled with Brownian dynamics calculations that model global protein tumbling, to compute EPR spectra directly for singly labeled proteins. As the reviewers noted, previous published attempts to exploit this type of strategy have not been completely satisfactory or convincing. However, these previous studies were based on rather limited MD simulations, and possibly suffered from some other issues that we address in more detail in the Research Plan below. It is essential to establish that a simulation strategy can be used to compute EPR spectra, in order to establish that we can capture the important features and behavior of spin-labeled proteins that give rise to unique EPR spectra for different samples (e.g., the sharp, distinct spectral signal typical of a completely mobile spin label versus the broader, more complex signals representative of partially immobilized spin labels). As discussed in the Project 1 Research Plan, we now have preliminary results that indicate we can compute EPR spectra more accurately and reliably than has been reported previously. There is still need for improvement, and we present detailed analysis of current MD-based EPR spectral simulations below that highlight possible inadequacies in the current methodology, and discuss specific strategies and tests to address these problems. Only after we have established convincingly that we can calculate EPR spectra directly with the combined MD/Brownian dynamics simulation protocol can we address seriously the calculation of spin label pair distances obtained in EPR DEER experiments, or pursue development of simpler computational strategies that do not require multiple, lengthy MD simulations with explicit solvent to estimate these distances. A number of issues impact the reliable MD simulation of spin label pair distances, including several raised by the reviewers for Project 1 (E.g., potential function parameters, electrostatics treatment, periodic boundary effects, etc.) We present preliminary data in the revised Research Plan below that addresses these issues and other important factors, and the strategies to achieve improved EPR spectral calculations and DEER distance estimates are presented in the context of a new Specific Aim 1.