COMPUTATION OF THE TWO-DIMENSIONAL POTENTIAL OF MEAN FORCE SURFACE OF AQUIFEX A

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 腺苷酸激酶(AK)催化一个磷酸基团从ATP转移到AMP，产生两个ADP作为最终产物。此前，克恩的研究小组确定，开放状态和关闭状态之间的构象转变，而不是化学催化，是反应的速度限制步骤。我们这个项目的主要目标是确定两个国家之间的过渡状态结构(S)。该酶由三个结构域组成：核心、ATP和AMP盖。在我们之前的工作中，我们模拟了Aquifex aeolicus Adylate Kinase(AAK)的配体自由形式和抑制剂结合形式。在这两种模拟中，ATP盖的运动与AMP盖的运动无关。因此，需要一个二维势的平均力(PMF)计算来绘制酶的自由能图景，而不是其他基团对AK同系物进行的一维PMF计算。在前面模拟工作的基础上，我们定义了两个反应坐标，即所选残基质心之间的两个几何夹角。我们使用伞形采样算法对两个状态之间的一些网格点的构象进行了采样。我们需要更多的CPU能力来完成对剩余网格点的模拟，并扩展已经采样的网格点上的轨迹。一旦所有这些模拟都完成后，我们将使用加权直方图分析方法(WHAM)来构建2D PMF表面。从二维PMF表面出发，我们将首先计算开态和闭态之间的自由能差。克恩的研究小组已经通过实验确定了这样的数值。计算值和实验值之间的直接比较将被用来证明计算协议的合理性。我们还将准确地指出过渡态结构(S)，以及过渡态结构(S)中存在的关键相互作用，而不是处于开放或关闭状态。然后，我们将预测哪些突变会影响两个状态之间的转移率。然后，这些预测可以通过实验性的突变研究进一步得到证实。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Adenylate kinase (AK) catalyzes the transfer of one phosphate group from ATP to AMP, producing two ADP as end products. Previously, the Kern's group determined that the conformational transition between the open and the closed states, rather than the chemical catalysis, is the rate limiting step of the reaction. Our main goal of this project is to determine the transition state structure(s) between the two states. The enzyme is made up of three domains: the core, the ATP and the AMP lid. In our previous work we have simulated Aquifex aeolicus adenylate kinase (AAK) in its ligand free form and in its inhibitor bound form. In both simulations, the motion of the ATP lid is independent of the motion of the AMP lid. Therefore a two-dimensional (2D) potential of mean force (PMF) computation is necessary to map the free energy landscape of the enzyme, rather than the one dimensional PMF computations carried out by other groups on AK homologues. Based on our previous simulation work, we have defined two geometrical angles between the centers of mass of selected groups of residues to be the two reaction coordinates. We have used the umbrella sampling algorithm to sample the conformations at some of the grid points between the two states. We need more CPU power to finish the simulations on the remaining grid points, and also to extend the trajectories on the grid points that are already sampled. Once all these simulations are completed, we will use weighted histogram analysis method (WHAM) to construct the 2D PMF surface. From the 2D PMF surface, we will first compute the free energy difference between the open and the closed states. Such a value has been experimentally determined by Kern's group. A direct comparison between the computed and the experimental values will be used to justify the computation protocol. We will also pinpoint the transition state structure(s), and the key interactions present in the transition state structure(s) and not in either the open or the closed state. We will then predict the mutations of which residues influence the transition rate between the two states. These predictions can then be further testified by experimental mutagenesis studies.