Dynamical Studies of Functionally Altered Mutants of the Tryptophan Repressor Protein

色氨酸阻遏蛋白功能改变突变体的动力学研究

• 批准号: 0444056
• 负责人: Valerie Copie
• 金额: $ 40.46万
• 依托单位: Montana State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0444056&HistoricalAwards=false
• 关键词: Dynamical; Studies; Functionally; Altered; Mutants

The long-term goal of this project is to provide a better understanding of the interconnections between internal dynamics, protein structures, and functions. The internal dynamics of backbone and sidechain atoms of wild-type and functionally altered mutants of the 25 kDa tryptophan repressor (TrpR) protein will be studied using NMR relaxation approaches. TrpR is part of a large family of bacterial proteins that regulate the expression of metabolic genes by binding to DNA operator sequences in response to cellular needs. The repressor's affinity for DNA is controlled by L-tryptophan (L-trp), an allosteric effector that activates the TrpR. The intrinsic flexibility of the TrpR protein structure is thought to be at the origin of the non-cooperativity of binding of the L-tryptophan corepressor to TrpR, and of the non-local long-range effects observed in a temperature-sensitive mutant of the tryptophan repressor protein, L75F-TrpR, which cannot be simply explained by small structural changes when compared to the wild-type TrpR. A second TrpR mutant of interest is A77V which, like L75F, is structurally similar to wild type TrpR and possesses biophysical features analogous to those of L75F-TrpR. Despite similar structures and biophysical features, the two TrpR mutants yield very distinct phenotypes and have very different L-trp cofactor binding properties. Such biochemical differences cannot be explained by the presence of distinct structural changes. The hypothesis to be examined with the NMR relaxation experiments is that the source of the differential L-trp binding properties of the A77V and L775F TrpR proteins originate from differences in intrinsic flexibility and molecular mobility which cannot be identified from inspection of the protein structures. The objective of this project is to provide new insights into the biochemical role of internal motions in modulating TrpR-L-trp-cofactor complex formation and TrpR-DNA recognition. This research will train graduate and undergraduate students in cross-disciplinary research involving NMR-based structural biology and fundamentals of protein chemistry. In addition, the PI will use knowledge obtained from this research to develop a graduate level course in biophysics focusing on the thermodynamics of ligand binding, protein-protein and protein-nucleic acid interactions, role of structural stability and dynamics in modulating protein functions, and allosteric regulations of multivalent protein systems.

这个项目的长期目标是更好地了解内部动力学、蛋白质结构和功能之间的相互联系。用核磁共振弛豫方法研究了25 kDa色氨酸抑制蛋白野生型和功能突变突变体的骨架和侧链原子的内部动力学。TrpR是细菌蛋白质大家族的一部分，通过与DNA操纵子序列结合来调节代谢基因的表达，以响应细胞需求。抑制物对脱氧核糖核酸的亲和力受L-色氨酸(L-色氨酸)控制，L-色氨酸是一种激活Trpr的变构效应。Trpr蛋白结构的内在灵活性被认为是L-色氨酸辅阻遏子与Trpr结合的非协作性的根源，以及在色氨酸抑制蛋白的温度敏感突变体L75F-Trpr中观察到的非局部长程效应，这不能简单地用与野生型Trpr相比的微小结构变化来解释。第二个感兴趣的Trpr突变体是A77V，它与L75F一样，在结构上与野生型Trpr相似，具有与L75F-Trpr相似的生物物理特征。尽管两个Trpr突变体具有相似的结构和生物物理特征，但它们产生了非常不同的表型，并具有非常不同的L-Trp辅因子结合特性。这种生化差异不能用明显的结构变化来解释。核磁共振松弛实验需要检验的假设是，A77V和L775F Trpr蛋白与L-Trp结合特性差异的来源是内在柔韧性和分子流动性的差异，而这些差异不能通过检查蛋白质结构来确定。本项目的目的是对体内运动在调节Trpr-L-Trp-辅因子复合体的形成和Trpr-DNA识别中的生化作用提供新的见解。这项研究将对研究生和本科生进行跨学科研究，涉及基于核磁共振的结构生物学和蛋白质化学基础。此外，PI将利用从这项研究中获得的知识，开发一门研究生级别的生物物理学课程，重点是配体结合的热力学、蛋白质与蛋白质和蛋白质与核酸的相互作用、结构稳定性和动力学在调节蛋白质功能中的作用，以及多价蛋白质系统的变构调节。