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.

该项目的长期目标是更好地理解内部动力学，蛋白质结构和功能之间的互连。 将使用NMR弛豫方法研究野生型骨架和侧链原子的内部动力学和25 kDa色氨酸抑制剂（TRPR）蛋白的功能变化突变体的内部动力学。 TRPR是大型细菌蛋白家族的一部分，该蛋白质通过响应细胞需求而与DNA算子序列结合来调节代谢基因的表达。阻遏物对DNA的亲和力由L-Tryptophan（L-TRP）控制，这是一种激活TRPR的变构效应器。认为TRPR蛋白结构的固有灵活性被认为是L-Tryptaphan核心与TRPR的结合的非合同性的起源，以及在色氨酸抑制剂蛋白的温度敏感突变体中观察到的非本地长期效应的起源，与L75F-TRPR相比，L75F-TRPR无法通过小型结构变化来解释。感兴趣的第二个TRPR突变体是A77V，它与L75F一样，在结构上与野生型TRPR相似，并且具有类似于L75F-TRPR的生物物理特征。尽管结构和生物物理特征相似，但两个TRPR突变体产生了非常不同的表型，并且具有非常不同的L-TRP辅因子结合特性。这种生化差异无法通过存在明显的结构变化来解释。 NMR弛豫实验将检查的假设是，A77V和L775F TRPR蛋白的差异L-TRP结合特性的来源源自固有柔韧性和分子迁移率的差异，这些差异无法从检查蛋白质结构的检查中鉴定出来。该项目的目的是提供有关内部运动在调节TRPR-L-TRP-COFACTOR复合体形成和TRPR-DNA识别方面的生化作用的新见解。这项研究将培训跨学科研究的毕业生和本科生，涉及基于NMR的结构生物学和蛋白质化学基础。此外，PI将使用从这项研究获得的知识来开发生物物理学的研究生水平课程，重点是配体结合，蛋白质 - 蛋白质蛋白和蛋白核酸相互作用的热力学，结构稳定性和动态在调节蛋白质功能中的作用，以及多价蛋白质系统的异质化法规。