Molecular Dynamics Simulations of Protein Unfolding

描述（由申请人提供）：我们的目标是进行真实的分子动力学（MD）模拟，与实验相一致，以原子分辨率表征蛋白质折叠/展开的途径。尽管人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/展开过程的细节知之甚少。展开对包括淀粉样蛋白疾病在内的许多生物过程具有重要意义。对折叠过程的详细了解也有助于改进结构预测算法。为了实现这一目标，最初和更新的建议是对小球状蛋白的展开进行高温MD模拟。这一努力已经成功地建立了方法，设计了识别和测试过渡态模型的方法，开发了用于评估部分未展开和变性蛋白质残留结构的分析工具，并开发了将MD结果与实验进行比较的方法。但是，仍有一些问题需要解决。在蛋白质展开的实验时间尺度和计算上可行的时间尺度之间存在着巨大的差异。因此，过去的研究采用了苛刻的条件来破坏天然状态的稳定，通常是非常高的温度。现在有可能在实验相关温度下运行更长时间的模拟，特别是对于新发现的超快折叠（-1-15 g）和展开（N5-10 ns）蛋白质。因此，本提案关注这些超快速折叠/展开蛋白质，特别是各种3-螺旋束（嵌入同源结构域En-HD、蛋白A和a3D，一种从头设计的蛋白质）和小的2链和3链[b]结构域（trpzip和WW结构域）（Specific Aim 1）。此外，还将研究化学诱导变性（尿素和氯化胍）的模拟以及引入稳定渗透物（三甲胺n -氧化物）的效果。（Specific Aim 2），因为溶剂诱导构象行为的分子基础仍然未知。为了研究简单螺旋结构和[B-]结构折叠的详细决定因素，将对En-HD和WW结构域超家族的自然序列变异进行模拟。（具体目标3）。随着新的并行模拟程序的开发，更大的系统可以被研究，“试管”模拟将与蛋白质的多个拷贝一起进行，以研究相邻分子（拥挤）对折叠/展开过程的影响。由于这个新程序也使得在合理的时间内进行微秒模拟成为可能，因此将尝试通过降低温度或稀释变性剂来重新折叠md变性的超快蛋白质。（具体目标4）。我们的合作者Alan Fersht教授和William降格罗教授正在对这些系统进行同步实验研究。

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.