Relationships of Protein Dynamics to Structure and Stability

这个项目的目的是了解一个小的模型蛋白，链球菌蛋白G的B1域的动力学与结构特征和折叠稳定性的关系。这将通过使用核磁共振松弛数据来比较B1域的不同突变体中的主链NH和侧链甲基的动力学来实现。首先，对于一系列10个单位点突变，突变位点和远程位点的动态将与突变位点的氨基酸的性质(体积、分支和极性)相关。其次，从动力学数据中估计10个突变体的主链和侧链构象熵，并将其与突变体的折叠稳定性相关联。这一部分将包括对突变体折叠和展开形式的动力学研究。第三，通过内部动力学参数的协变分析，研究B1域内相关运动的程度。将获得10个突变体家族的相关数据和在几个温度下测量的B1结构域的动力学数据。第四，将使用双突变实验来研究动态信息从B1域中的突变位点传播到远程位点的机制。最后，将使用两个B1结构域的变体来研究二级结构环境对氨基酸动力学的影响，其中相同的11个氨基酸序列分别存在于α-螺旋结构或β-发夹结构中。蛋白质的结构稳定性、与其他分子的结合以及催化反应的能力等化学性质往往不能从静态蛋白质结构中充分理解，因为这些性质受到蛋白质动力学的影响。这项研究的主要目标是了解蛋白质的动力学与结构特征和折叠稳定性的关系。这项研究将提供有助于我们理解蛋白质稳定性和功能的见解，并可能对设计具有新特性的蛋白质有用。该项目的主要教育目标是培养一名研究生和一名博士后研究助理。该项目的活动范围从定点突变、蛋白质表达和纯化到核磁共振光谱、其他物理生物化学实验和广泛的计算数据分析。该项目将涉及所有这些实践领域的技能和知识的发展，以及蛋白质稳定性和实验设计的理论方面。

The objective of this project is to understand the relationship of dynamics to structural features and fold stability in a small model protein, the B1 domain of Streptococcal protein G. This will be accomplished by using NMR relaxation data to compare the backbone NH and side chain methyl group dynamics in various mutants of the B1 domain. First, for a series of 10 single-site mutants, the dynamics at the mutation site and remote sites will be correlated with the properties (volume, branching, and polarity) of the amino acid at the mutation site. Second, the backbone and side chain conformational entropy of each of the 10 mutants will be estimated from the dynamics data and correlated with the fold stabilities of the mutants. This section will incorporate dynamics studies of the folded as well as unfolded forms of the mutants. Third, the extent of correlated motions in the B1 domain will be investigated by analysis of the covariation of internal dynamics parameters. Correlation data will be obtained both for the family of 10 mutants and for the dynamics of the B1 domain measured at several temperatures. Fourth, the mechanism by which dynamic information is propagated from a mutation site to a remote site in the B1 domain will be investigated using double-mutant experiments. Finally, the influence of secondary structural environment on amino acid dynamics will be investigated using two B1 domain variants, in which the same 11-amino acid sequence exists in alpha-helical or beta-hairpin structures, respectively.The chemical properties of proteins such as structural stability, binding to other molecules, and ability to catalyze reactions can often not be adequately understood from the static protein structure because these properties are influenced by the dynamics of the protein. The broad goal of this study is to understand the relationship of dynamics to structural features and fold stability in proteins. The study will provide insights that will aid in our understanding of protein stability and function and may be useful in the design of proteins with novel properties. The major educational goal of this project is the training of a graduate student and a postdoctoral research associate. Activities in the project range from site-directed mutagenesis and protein expression and purification to NMR spectroscopy, other physical biochemistry experiments and extensive computational data analysis. The project will involve development of skills and knowledge in all of these practical areas as well as in theoretical aspects of protein stability and in experimental design.