SIMULATION OF PROTEIN DYNAMICS AND UNFOLDING IN SOLUTION

开发了计算机模拟溶液中蛋白质的方法， 在第一个资助期内测试的系统，将应用于 积极的实验研究。 我们的计算协议使用 周期盒，明确包括溶剂分子，允许完全键合 长度和角度的灵活性，平滑地截断长距离相互作用 并保持总能量。 它给出了行为良好的轨迹， 计算效率高。 我们新获得的跑得更久的能力， 更真实的模拟，将帮助我们形成强大的相互作用， 实验合作者在标准，NIH，麻省理工学院和洛斯阿拉莫斯。 具体目标是： 1. 我们能模拟尿素和三氟乙醇吗？ 我们将从分子上 动力学来模拟这些变性和复性溶液， 不同的浓度和温度， 实验数据，以获得用于展开的现实解决方案， 重折叠模拟。 2. α-螺旋和β-发夹结构是如何展开的？ 多长时间 螺旋展开带吗？什么是螺旋和发夹的成核中心 阵型？β-发夹是如何展开的， 与α-螺旋展开不同吗这些问题将 在持续1到10纳秒（ns）的展开模拟中回答， 在一定温度范围内进行。 通过缓慢降低温度， 也试图重新折叠部分变性的α-螺旋和β-螺旋， 发夹 3. 蛋白质解折叠的最早期是什么？我们认真 选择了一组五种蛋白质，它们是（a）小的，（B）根据 不同的建筑原则，和（c）在实验研究。 短期模拟（<100 ps）将在一系列温度下运行， 密度 水的相互作用、填料接触和填料体积 变化将被分析，以揭示结构是如何被破坏的。 的 还将研究浓尿素和TFE的影响。 4. 解折叠通路如何依赖于蛋白质结构？运行 在高温下的长时间（1-5 ns）模拟应导致 这里研究的每一种小蛋白质都展开了。 的性质 将分析展开过程中的中间体及其形成方式 和比较 5. 中间状态有多移动的？中间态动力学 通过展开这五种蛋白质产生的蛋白质将在室温下运行， 温度 半胱氨酸、疏水残基和电荷之间的距离 将对两人进行监测。 6. 我们能恢复α-乳白蛋白的一部分的天然折叠吗？ 我们将 使用我们的包装方法和模拟来预测稳定突变 对于已知在低温下为熔融球的部分。

Methods for computer simulation of proteins in solution developed and tested during the first funding period will be applied to systems under active experimental investigation. Our calculation protocol uses a periodic box, includes solvent molecules explicitly, allows full bond length and angle flexibility, truncates long-range interactions smoothly and conserves total energy. It gives well-behaved trajectories that can be computed efficiently. Our newly gained ability to run much longer, more realistic simulations, will help us form strong interactions with experimental collaborators at Standard, NIH, MIT and Los Alamos. Specific aims are: 1. Can we simulate urea and trifluoroethanol? We will molecular dynamics to simulate these denaturing and renaturing solutions at different concentrations and temperatures and calibrate them against experimental data to obtain realistic solutions for use in unfolding and refolding simulations. 2. How do alpha-helix and Beta-hairpin structures unfold? How long does helix unfolding take? What is the nucleation center for helix and hairpin formation? How does Beta-hairpin unfolding, with its long-rang hydrogen bonds, differ from alpha-helix unfolding? These questions will be answered in unfolding simulations lasting 1 to 10 nanoseconds (ns) to be done at a range of temperatures. By reducing temperature slowly, we will also attempt to refold partially denatured alpha-helices and Beta- hairpins. 3. What are the earliest events in protein unfolding? We have carefully chosen a set of five proteins that are (a) small, (b) built according to different architectural principles, and (c) under experimental study. Short simulations (<100ps) will be run at a range of temperatures and densities. Water interactions, packing contacts and packing volume changes will be analyzed to reveal how structure is disrupted. The effects of concentrated urea and TFE will also be investigated. 4. How do unfolding pathways depend on protein architecture? Running prolonged (1-5 ns) simulations at elevated temperatures should caused each of the small proteins studied here to unfold. The nature of the intermediates in unfolding and the way they are formed will be analyzed and compared. 5. How mobile are intermediate states? Dynamics of intermediate states generated by unfolding these five proteins will be run at room temperature. Distances between cysteine, hydrophobic residues and charge pairs will be monitored. 6. Can we restore a native fold to a part of alpha-lactalbumin? We will use our packing methods and simulations to predict stabilizing mutations for a part known to be a molten globule at low temperature.