Mutational Effects of Protein Structure and Dynamics

本研究的目的是阐明氨基酸突变对T4噬菌体溶菌酶（T4L）活性位点附近波动特性的影响。哈德森博士将专注于最小扰动突变的大小变化，并将从x射线或预测的晶体结构开始进行约束分子动力学计算。研究计划的一个组成部分包括使用广泛的T4L突变结构数据库，以进一步扩展测试结构预测技术的能力。特定的非键相互作用网络在确定色氨酸138附近区域的整体动力学中的重要性，实验工作中的荧光探针，以及活性位点本身的重要性。将分析T4L突变体的单色氨酸和其他单色氨酸蛋白的荧光复合衰减。模型的开发将以动力学模拟的结果为指导。实验和计算的并置将确定蛋白质内部色氨酸大振幅动力学的性质。将扩展先前对T4噬菌体溶菌酶的模拟工作，以包括现实的溶剂环境。最后，将应用自由能摄动方法来确定突变体T4L在几个室温模拟中观察到的一系列常见结构/构象变化的自由能差。这将提供一个焓/熵分解热力学驱动力与增强的波动门控转变。突变蛋白中非键相互作用的性质将与热力学性质和波动相关函数进行比较，以收集一般原理。

The goal of the proposed research is to elucidate the effects of amino acid mutations on the fluctuation properties of T4 phage lysozyme (T4L) proximate to the active site cleft. Dr. Hudson will focus on mutations of minimal perturbation as regards size change, and will perform constrained molecular dynamics calculations beginning with either an x-ray or a predicted crystal structure. A component of the research plan involves using the extensive data base of T4L mutant structures to further extend the ability to test structure predictive techniques. The importance of particular networks of nonbonded interactions in determining the overall dynamics of the region proximate to tryptophan 138, the fluorescence probe in the experimental work, as well as that of the active site itself will be probed. The complex decay of the fluorescence of the single tryptophan of T4L mutant forms and other single tryptophan proteins will be analyzed. The development of models will be guided by the results of dynamics simulations. The juxtaposition of experiments and calculations will establish the nature of large amplitude tryptophan dynamics on the interior of a protein. The previous simulation work on T4 phage lysozyme to include a realistic solvent environment will be extended. Finally free energy perturbation methods will be applied to determine the free energy differences for series of common structural/conformational changes observed in several room temperature simulations of mutant T4L. This will provide an enthalpic/entropic decomposition of the thermodynamic driving forces associated with the enhanced fluctuations gating the transition. The nature of nonbonded interactions in mutant proteins will be compared with the thermodynamic properties and fluctuation correlation functions to glean general principles.