Protein Phosphorylation as a Biophysical Switch: Structural, Dynamic and Thermodynamic Responses to Phosphorylation

蛋白质磷酸化作为生物物理开关：磷酸化的结构、动态和热力学响应

• 批准号: 0212597
• 负责人: Linda Nicholson
• 金额: --
• 依托单位: Cornell University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0212597&HistoricalAwards=false
• 关键词: Protein; Phosphorylation; Biophysical; Switch; Structural

Nature has designed a broad array of protein-based molecular switches that direct and control the flow of information, energy, and molecular cargo through living cells. One of the most important questions regarding functional switching of proteins is how the covalent attachment of a phosphate group so dramatically alters activity. The overall goal of this research is to elucidate biophysical mechanisms by which specific proteins are regulated by phosphorylation. Two model systems will be investigated, moesin and the c-Src SH3 domain. Moesin is a member of the ERM family of proteins that are involved in dynamic interactions with the actin cytoskeleton and the plasma membrane. SH3 domains are small, modular binding domains that mediate protein-protein interactions through binding to proline-rich sequence motifs. The moesin model system employed in this research consists of two interacting domains, N-FERM and C-ERMAD that comprise the N/C complex. Formation of the N/C complex prevents interactions between moesin and its cellular binding partners, and is regulated by phopshorylation of residue T558 in C-ERMAD and by PIP2 binding to N-FERM. This model system represents a "regulatory rheostat" where phosphorylation is coupled with additional regulatory factors to elicit variable activation. In a second model system, the ligand binding function of the c-Src SH3 domain is regulated by phosphorylation of residue Y57. This represents a prototype molecular switch where phosphorylation of a residue in or near the binding surface alters specificity and affinity of a modular binding domain. For each of the two model systems, multidimensional NMR spectroscopy will be used to observe phosphorylation-induced changes in structure, stability and dynamics, and isothermal titration calorimetry (ITC) for quantifying phosphorylation-induced changes in function. The biophysical mechanism by which each protein is regulated will be determined through amide exchange measurements, backbone and side chain dynamics studies, and determination of macroscopic and site-specific pKas. These investigations will provide a structural, dynamic and thermodynamic framework for understanding molecular switching mechanisms that have not yet been elucidated. Reversible protein phosphorylation is a ubiquitous mechanism utilized by all cells to switch protein function on and off. In order to visualize how protein phosphorylation regulates function, the structure, dynamics and thermodynamics of proteins in both phosphorylated and unphosphorylated states must be examined. Although a few detailed mechanisms are now characterized, our understanding of the varied mechanisms by which regulation by phosphorylation is accomplished is far from complete. This project aims to determine the biophysical mechanisms by which two particular proteins are regulated by phosphorylation. Coupling of the thermodynamic and site-specific perspectives obtained from ITC and NMR, respectively, will yield valuable insights into potential origins of binding energy, and the mechanisms used to alter this energy in biological molecular switches. These studies will advance our understanding of how biological processes are regulated by phosphorylation, and will impact important areas of research such as signal transduction, intracellular trafficking, and regulation of membrane structure. The research objectives will be accomplished primarily through the participation of graduate and undergraduate students who will receive education and training in molecular biology, protein chemistry, physical chemistry, computer technology, and multidimensional NMR spectroscopy.

大自然设计了一系列以蛋白质为基础的分子开关，指导和控制信息、能量和分子货物通过活细胞的流动。关于蛋白质功能转换的最重要的问题之一是磷酸基团的共价连接如何如此显著地改变活性。本研究的总体目标是阐明磷酸化调节特定蛋白质的生物物理机制。两个模型系统将被研究，moesin和c-Src SH3结构域。Moesin是ERM蛋白家族的一员，参与肌动蛋白骨架和质膜的动态相互作用。SH3结构域是小的、模块化的结合结构域，通过结合富含脯氨酸的序列基序介导蛋白质相互作用。本研究采用的moesin模型系统由两个相互作用的结构域N- ferm和C- ermad组成N/C复合物。N/C复合物的形成阻止了moesin与其细胞结合伙伴之间的相互作用，并受C- ermad中残基T558的磷酸化和PIP2与N- ferm结合的调节。该模型系统代表了一个“调节变阻器”，其中磷酸化与额外的调节因子耦合以引发可变激活。在第二个模型系统中，c-Src SH3结构域的配体结合功能由残基Y57的磷酸化调节。这代表了一种原型分子开关，其中结合表面或附近残基的磷酸化改变了模块结合域的特异性和亲和力。对于这两种模型体系，将使用多维核磁共振波谱来观察磷酸化诱导的结构、稳定性和动力学变化，并使用等温滴定量热法（ITC）来量化磷酸化诱导的功能变化。每种蛋白调控的生物物理机制将通过酰胺交换测量、主链和侧链动力学研究以及宏观和位点特异性pka的测定来确定。这些研究将为理解尚未阐明的分子开关机制提供一个结构、动力学和热力学框架。可逆蛋白磷酸化是一种普遍存在的机制，所有细胞都利用它来开关蛋白质功能。为了可视化蛋白质磷酸化如何调节功能，必须检查磷酸化和非磷酸化状态下蛋白质的结构，动力学和热力学。虽然一些详细的机制现在被表征，但我们对磷酸化调控的各种机制的理解远未完成。该项目旨在确定两种特定蛋白质被磷酸化调节的生物物理机制。从ITC和NMR分别获得的热力学和位点特异性视角的耦合，将对结合能的潜在来源以及用于改变生物分子开关中这种能量的机制产生有价值的见解。这些研究将促进我们对磷酸化如何调节生物过程的理解，并将影响信号转导、细胞内运输和膜结构调节等重要研究领域。研究目标将主要通过研究生和本科生的参与来完成，他们将接受分子生物学、蛋白质化学、物理化学、计算机技术和多维核磁共振光谱的教育和培训。