PROTEIN DYNAMICS FROM NMR WITH IMPROVED STOCHASTIC MODELS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。核磁共振是一种阐明蛋白质动力学的有力方法。一个有用的动态探针是15N-1H键载体，它既参与蛋白质的全局定向，也参与局部位点特异性运动。常用的数据分析方法是无模型（model-free， MF），即假定全局和局部运动解耦，简化局部几何特征。我们发现实验15N弛豫数据对模式耦合和局部几何的一般特征很敏感。这些元素是Freed等人的慢弛豫局部结构（SRLS）方法所固有的，我们已经将其应用于蛋白质的核磁共振自旋弛豫。SRLS模型表达了n -氢键（通常以更快的速度松弛）与其直接（内部）蛋白质环境（通常以较慢的速度松弛）之间的动态耦合。这种偶联是通过电位U/kT实现的，它代表了蛋白质在N-H键局部运动位点施加的空间限制。由于动态耦合，更快的内部N-H动态调整自身以适应蛋白质方向的变化。在轴向局部电位的背景下，SRLS已被应用于大量蛋白质，特别是无配体和抑制剂结合的大肠杆菌腺苷酸激酶（AKeco）。这种酶催化ATP + AMP产生2ADP的反应。它由三个结构域组成，其中两个（LID和AMPbd）执行与催化相关的大振幅节段运动。我们对无配体AKeco的研究得出了结构域AMPbd和LID在溶液中的移动速率。我们研究了AKeco与双底物模拟抑制剂AP5A的配合物，得到了类似于催化反应过渡态的动态元素。该方法正在非轴向局部电位的范围内进一步发展，这需要计算交叉谱密度。

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.