Project 1/Computational Core

计算核心研究计划已经过修订，以解决具体问题和关注点 提出的审查，并强调这一核心的原则重点。的首要目标 计算核心是1）开发一套计算工具和协议，以促进分析和 EPR光谱数据的解释，包括从DEER实验获得的距离测量， 双自旋标记的蛋白质，和2）为个人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平衡MD模拟， CDB 3，以探索P327 R点突变体引发的可能的构象变化，并初步 结果在项目2研究计划中进行了描述。项目3的基本计算支持包括 常规2D-NMR实验和顺磁共振的常规结构精修计算 增强NMR实验，以及MD模拟，以探索自旋标签的构象趋势 淀粉样β肽中引入的。这种构象分析对于解决距离问题非常重要。 在EPR实验和顺磁共振实验中， 共振增强NMR研究。 开发实用的计算工具和协议，以促进EPR数据分析取决于 关键是项目1中获得的数据，需要几个谨慎的步骤。首先，必须确定， 我们可以使用传统的平衡MD模拟来描述自旋标记侧链动力学和蛋白质 骨架动力学，再加上布朗动力学计算模型的全球蛋白质翻滚， 计算EPR谱直接为单一标记的蛋白质。正如评论者所指出的，以前发表的尝试 利用这类战略并不完全令人满意或令人信服。然而，这些前 研究是基于相当有限的MD模拟，并可能遭受一些其他问题，我们 在下面的研究计划中详细说明。重要的是要建立一个模拟策略， 用于计算EPR谱，以建立我们可以捕获的重要特征和行为， 自旋标记的蛋白质对于不同样品产生独特的EPR谱（例如，清晰，鲜明 与更宽、更复杂的信号相比，完全移动的自旋标记的典型光谱信号 代表部分固定的自旋标记）。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，我们可以计算EPR谱更准确和可靠的比已经 此前报道。但仍需改进，我们对目前基于MD的 EPR谱模拟强调了当前方法中可能存在的不足，并讨论了 具体的策略和测试来解决这些问题。只有在我们令人信服地证明我们 可以直接用MD/布朗动力学模拟协议计算EPR谱， 认真解决EPR DEER实验中获得的自旋标记对距离的计算，或继续 开发更简单的计算策略，不需要多次冗长的MD模拟， 明确的溶剂来估计这些距离。自旋标记分子动力学模拟的可靠性受到许多问题的影响 对距离，包括项目1的评审员提出的几个距离（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 下面的计划，解决这些问题和其他重要因素，以及战略，以实现改善 EPR谱计算和DEER距离估计提出了一个新的具体目标1的上下文中。

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.