PROTEIN DYNAMICS FROM NMR WITH IMPROVED STOCHASTIC MODELS

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。 核磁共振是阐明蛋白质动力学的一种有效方法。一个有用的动态探针是15N-1H键载体，它参与蛋白质的全局重定向和局部特定位置的运动。常用的数据分析方法是无模型(MF)，假设全局运动和局部运动是解耦的，并简化了局部几何特征。我们发现实验的15N驰豫数据对模式耦合和局域几何的一般特征很敏感。这些元素是Freed等人的缓慢松弛局部结构(SRLS)方法所固有的，我们已经将该方法应用于蛋白质的核磁共振自旋松弛。SRLS模型表达了N-H键和其直接(内部)蛋白质环境之间的动态耦合，N-H键通常以较快的速率RL松弛，而直接(内部)蛋白质环境通常以较慢的速率Rc松弛。这种耦合是通过一个势U/KT来实现的，它代表了蛋白质在N-H键的局部运动位置施加的空间限制。借助于动力学耦合，更快的内部N-H动力学重新调整自己以适应蛋白质不断变化的取向。在轴向局部电位的背景下，SRLS已经被应用于大量的蛋白质，特别是无配体和抑制剂结合的大肠杆菌腺苷酸激酶(AKECO)。该酶催化ATP+AMP反应生成2ADP。它由三个结构域组成，其中两个结构域(LID和AMPbd)执行与催化相关的大幅度节段运动。我们对无配体AKECO的研究得出了AMPbd和LID结构域在溶液中移动的速度。我们对AKECO与双底物模拟抑制剂AP5A的络合物的研究得到了类似于催化反应过渡态的动态元素。在非轴局域势的背景下，该方法正在进一步发展，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.