Developing Improved Methods for Modeling and Simulating Protein Structures

我们的第一个计算方法子项目涉及使用对称约束， 分子建模和模拟。对于那些蛋白质或蛋白质组装体 由相同的亚基组成，亚基通常具有相同的构象， 与相邻亚基的相同相互作用。我们将这一限制纳入了 二十多年来，我们的离子通道模型取得了相当大的成功。例如我们 钾通道离子选择区的四重对称模型实际上是 与随后确定的钾通道晶体结构相同。然而，在这方面， 直到最近，我们还没有将对称性约束纳入自动算法， 事实证明，这样做实际上提高了结构模型的质量。我们的一 合作者Andriy Anishkin开发了一个程序，使用谐波约束， 在分子动力学模拟中恢复对称性。我们已经测试了它的效用 通过分析四种离子通道的晶体结构进行离子通道同源建模的程序 命名为KcsA、MlotiK、KirBac、NaK的远亲离子通道。这个项目的第一步 利用其他三种通道结构， 作为模板。接下来，对这十二种模型进行了分子动力学模拟，沿着的还有四种模型。 晶体结构，进行了与蛋白质包埋在一个明确的脂质双层 在通道的每一侧和孔内具有水和离子。八纳秒之后 不受限制的模拟，对称性约束。这个过程重复了两次。 每种型号和晶体结构都需要更多的时间。在没有分子的情况下， 动态仿真、无约束仿真和有约束仿真 然后与原始结构进行比较，以确定哪种结构更好。主要发现 尽管无限制的分子动力学模拟并没有提高同源性， 在大多数情况下，对称性限制的实施确实导致了实质性的改善。 最大的改善发生在孔隙内衬段，其中， 相邻的亚基是广泛的。我们建立离子同调模型的主要目的是 通道是了解他们的结构和药理学足够好地利用模型 基于结构的药物设计。因此，值得注意的是， 基本上改进了形成主要药物结合位点的孔区域的模型。 我们的第二个计算方法子项目涉及简化的分子表示， 蛋白质，从而蛋白质和肽的性质可以模拟更长的时间。一 传统的分子动力学模拟的主要局限性在于， 仅持续很短的时间段，通常小于十分之一微秒。这是 比大多数构象变化所需的时间短得多， 其数量级短于组装淀粉样β结构所需的时间。到 为了克服这个限制，我们小组的Sijung Yun博士一直在使用离散分子动力学 这比传统的分子动力学效率高出七个数量级。 他在波士顿大学攻读博士学位期间帮助开发了这个项目。三 为了提高效率，在离散分子动力学中引入了简化 同时仍然保持准确性。首先，相互作用势被简化为离散的 步这极大地简化了计算期间所需的数学和计算次数。 模拟第二，由于水分子的相互作用被有效势取代。 这大大减少了通过消除水分子模拟的原子数量。三是 用四个珠子表示蛋白质中的一个残基，而不是表示所有的原子 的残留物。这也减少了模拟原子的数量。在这里，尹博士 致力于改进参数并测试已知的蛋白质晶体结构 在进行这些模拟时保持不变。他也一直用这种方法来测试 我们小组开发的淀粉样β六聚体模型。淀粉样β六聚体参与了 阿尔茨海默病的神经毒性。

Our first computational methods subproject involves using symmetry constraints in molecular modeling and simulations. For those cases in which proteins or protein assemblies are composed of identical subunits, the subunits usually have identical conformations and identical interactions with neighboring subunits. We have incorporated this constraint into our models of ion channels for over two decades, with considerable success. For example our four-fold symmetric model of the ion selective region of potassium channels was virtually identical to those of subsequently determined potassium channel crystal structures. However, until recently, we have not incorporated symmetry constraints into an automated algorithm, nor demonstrated that doing so actually improved the quality of structural models. One of our collaborators, Andriy Anishkin, has developed a program that uses harmonic restraints to restore symmetry during molecular dynamic simulations. We have tested the utility of this program for homology modeling of ion channels by analyzing crystal structures of four distantly related ion channels named KcsA, MlotiK, KirBac, NaK. The first step of this project was to develop three homology models of each channel using the other three channel structures as templates. Next, molecular dynamics simulations of these twelve models, along with the four crystal structures, were performed with the proteins embedded in an explicit lipid bilayer with water and ions on each side and within the pore of the channels. After eight nanoseconds of unrestrained simulations, symmetry restraints were imposed. This process was repeated two more times for each model and crystal structure. The models developed without molecular dynamic simulations, with unrestrained simulations, and with symmetry-restrained simulations were then compared to the original structures to determine which was better. The major finding was that although unrestrained molecular dynamic simulations did not improve the homology models, imposition of symmetry restraints did lead to substantial improvement in most cases. The greatest improvement occurred for the pore lining segments, where interaction among adjacent subunits is extensive. Our principal purpose for developing homology models of ion channels is to understand their structure and pharmacology well enough to utilize the models in structure-based drug design. Thus, it is noteworthy that the symmetry restraints substantially improve models of the pore region that forms the principal drug binding sites. Our second computational methods subproject involves simplifying molecular representations of proteins so that properties of proteins and peptides can be simulated for much longer times. A major limitation of conventional molecular dynamic simulations is that they can be performed for only short periods of time, typically less than a tenth of a microsecond. This is substantially shorter than the time required for most conformational changes, and many orders of magnitude shorter than the time required for assembling amyloid beta structures. To overcome this limit, Dr. Sijung Yun of our group has been using discrete molecular dynamics that is about seven orders of magnitude more efficient than conventional molecular dynamics. He helped develop this program during his doctoral work at the University of Boston. Three simplifications are introduced in discrete molecular dynamics in order to increase efficiency while still preserving accuracy. First, interaction potentials are simplified into discrete steps. This greatly simplifies the mathematics and number of calculations required during the simulations. Second, interactions due to water molecules are replaced by effective potentials. This greatly reduces the number of atoms simulated by eliminating water molecules. Third, we used four-bead representation of a residue in a protein instead of representing all the atoms of the residue. This also reduces the number of simulated atoms. While here, Dr. Yun has worked to improve the parameters and test how well known protein crystal structures are maintained when subjected to these simulations. He has also been using this approach to test models of amyloid-beta hexamers developed by our group. Amyloid-beta hexamers are involved in neurotoxicity of Alzheimers disease.