PROTEIN DYNAMICS FROM NMR WITH IMPROVED STOCHASTIC MODELS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 核磁共振是一种强有力的方法来阐明蛋白质动力学。一个有用的动态探针是15 N-1H键载体，它参与蛋白质的全局重定向和局部位点特异性运动。常用的数据分析方法是无模型（MF），其中全局和局部运动被假定为解耦的，并且局部几何特征被简化。我们发现，实验15 N弛豫数据是敏感的模式耦合和局部几何的一般特征。这些元素是Freed等人的缓慢松弛局部结构（SRLS）方法所固有的，我们已经将其应用于蛋白质的NMR自旋弛豫。SRLS模型表达了N-H键（通常以较快的速率RL松弛）与其直接（内部）蛋白质环境（通常以较慢的速率RC松弛）之间的动态耦合。耦合通过电位U/kT实现，U/kT代表蛋白质在N-H键局部运动位点处施加的空间限制。通过动力学耦合，更快的内部N-H动力学根据蛋白质的变化方向重新调整自身。在轴向局部势的背景下，SRLS已被应用于大量的蛋白质，特别是无配体和结合有寡核苷酸的E。大肠杆菌腺苷酸激酶（AKeco）。这种酶催化ATP + AMP反应产生2ADP。它由三个区域组成，其中两个（LID和AMPbd）执行大幅度催化相关的节段运动。 我们对无配体AKeco的研究得出了结构域AMPbd和LID在溶液中移动的速率。我们的研究AKeco与双底物模拟抑制剂AP 5A的复合物产生了类似于催化反应过渡态的动态元素。该方法正在进一步发展的背景下，非轴局部电位，这需要计算的交叉谱密度。

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. NMR is a powerful method for elucidating protein dynamics. A useful dynamic probe is the 15N-1H bond vector, which engages in both the global reorientation of the protein and local site-specific motions. The commonly used data analysis method is model-free (MF), where the global and local motions are assumed decoupled, and features of local geometry are simplified. We found that the experimental 15N relaxation data are sensitive to mode-coupling and general features of local geometry. These elements are inherent to the Slowly Relaxing Local Structure (SRLS) approach of Freed et al., which we have applied to NMR spin relaxation in proteins. The SRLS model expresses the dynamic coupling between the N-H bond, which typically relaxes at a faster rate, RL, and its immediate (internal) protein surroundings, which typically relax at a slower rate, RC. The coupling materializes through a potential, U/kT, which represents the spatial restrictions imposed by the protein at the site of the local motion of the N-H bond. By virtue of dynamical coupling the faster internal N-H dynamics readjusts itself to the changing orientation of the protein. Within the context of axial local potentials SRLS has been applied to a large number of proteins, in particular ligand-free and inhibitor-bound E. coli adenylate kinase (AKeco). This enzyme catalyzes the reaction ATP + AMP produces 2ADP. It is made up of three domains, two of which (LID and AMPbd) execute large amplitude catalysis-related segmental motions. Our study of ligand-free AKeco yielded the rate at which the domains AMPbd and LID move in solution. Our study of the complex of AKeco with the two-substrate-mimic inhibitor AP5A yielded dynamic elements akin to the transition state of the catalytic reaction. The methodology is being further developed within the context of non-axial local potentials, which requires the calculations of the cross spectral densities.