Molecular Dynamics Simulations of Protein Unfolding

描述（由申请人提供）：我们的目标是进行逼真的分子动力学（MD）模拟，与实验一致，以原子分辨率表征蛋白质折叠/解折叠的途径。虽然我们对蛋白质的天然折叠状态的结构细节了解很多，但对部分折叠的构象和折叠/解折叠过程的细节知之甚少。解折叠对许多生物学过程具有重要意义，包括淀粉样疾病。折叠过程的详细知识也应该导致改进的结构预测算法。为了实现这一目标，最初和更新的建议是对小球状蛋白质的展开进行高温MD模拟。这一奋进在建立方法、设计识别和测试过渡态模型的方法、开发用于评估部分未折叠和变性蛋白质中残余结构的分析工具以及开发用于将MD结果与实验进行比较的方法方面取得了成功。但是，仍然存在需要解决的问题。蛋白质解折叠的实验时间尺度和计算上可行的时间尺度之间存在巨大差异。因此，过去的研究采用苛刻的条件来破坏天然状态，通常是非常高的温度。现在可以在实验相关温度下运行更长的模拟，特别是对于新发现的超快折叠（~ 1 - 15 gs）和解折叠（N5-10 ns）蛋白质。因此，该提议集中于这些超快折叠/解折叠蛋白，特别是各种3-螺旋束（有爪同源结构域，En-HD;蛋白A;和a3 D，一种从头设计的蛋白）和小的2-和3-链[B]结构（trpzip和WW结构域）（具体目标1）。此外，模拟化学诱导变性（尿素和胍氯化物）和引入稳定的渗透剂（三甲基胺N-氧化物）的影响将进行研究。（具体目标2），因为溶剂诱导的构象行为的分子基础仍然未知。为了研究简单螺旋和[B]-结构的折叠的详细决定因素，将对En-HD和WW结构域超家族内的天然序列变体进行模拟。（具体目标3）。随着一个新的，并行化的模拟程序正在开发中，更大的系统可以进行研究，和“试管”模拟将追求与蛋白质的多个副本，以研究相邻分子（拥挤）的折叠/展开过程的影响。因为这个新程序也使得在合理的时间内进行微秒级模拟成为可能，所以将尝试通过降低温度或稀释变性剂来重折叠MD变性的超快蛋白质。（具体目标4）。我们的合作者正在对这些系统进行同步实验研究：Alan Fersht教授和William DeGrado教授。

DESCRIPTION (provided by applicant): Our goal is to perform realistic molecular dynamics (MD) simulations, in concert with experiment, to characterize the pathway of protein folding/unfolding at atomic resolution. Although much is known of the structural details of the native, folded state of proteins, very little is known about partially folded conformations and the details of the folding/unfolding process. Unfolding has important implications for many biological processes, including amyloid diseases. Detailed knowledge of the folding process should also lead to improved structure prediction algorithms. In pursuit of this goal, the original and renewed proposals were to perform high-temperature MD simulations of the unfolding of small globular proteins. This endeavor has been successful with respect to establishing the methods, devising ways to identify and test models of transition states, developing analysis tools for the evaluation of residual structure in partially unfolded and denatured proteins, and developing approaches for comparison of MD results with experiment. But, there are still issues that need to be addressed. There has been a huge difference between the experimental time scale of protein unfolding and what is computationally feasible. As a result, past studies employed harsh conditions to destabilize the native state, typically very high temperature. Now it is possible to run longer simulations at experimentally relevant temperatures, particularly for newly discovered ultrafast folding (-1-15 gs) and unfolding (N5-10 ns) proteins. Consequently, this proposal focuses on these ultrafast folding/unfolding proteins, in particular various 3-helix bundles (the engrailed homeodomain, En-HD; protein A; and a3D, a de novo designed protein) and small 2- and 3-stranded [B-structures (trpzip and WW domains) (Specific Aim 1). In addition, simulations of chemically induced denaturation (urea and guanidinium chloride) and the effect of introducing stabilizing osmolytes (trimethylamine N-oxide) will be investigated. (Specific Aim 2), as the molecular basis of solvent-induced conformational behavior remains unknown. To investigate the detailed determinants of folding for simple helical and [B- structures, simulations will be performed of natural sequence variants within the En-HD and WW domain superfamilies. (Specific Aim 3). With a new, parallelized simulation program under development, much larger systems can be investigated, and 'test tube' simulations will be pursued with multiple copies of the protein to investigate the effect of neighboring molecules (crowding) on the folding/unfolding process. Because this new program also makes it possible to perform microsecond simulations in reasonable periods of time, refolding of the MD-denatured ultrafast proteins, by lowering the temperature or diluting out the denaturant, will be attempted. (Specific Aim 4). Concurrent experimental studies on these systems are being pursued by our collaborators: Professors Alan Fersht and William DeGrado.