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

大自然已经设计了一系列基于蛋白质的分子开关，这些开关可以引导和控制信息、能量和分子货物在活细胞中的流动。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着地改变活性。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。两个模型系统将进行调查，膜突蛋白和c-Src SH 3域。膜突蛋白是ERM蛋白家族的成员，其参与与肌动蛋白细胞骨架和质膜的动态相互作用。 SH 3结构域是小的模块化结合结构域，其通过结合至富含脯氨酸的序列基序来介导蛋白质-蛋白质相互作用。 本研究中所采用的膜突蛋白模型系统包括两个相互作用的结构域，N-FERM和C-ERMAD，它们构成N/C复合物。 N/C复合物的形成阻止膜突蛋白与其细胞结合伴侣之间的相互作用，并通过C-ERMAD中残基T558的磷酸化和PIP 2与N-FERM的结合来调节。该模型系统代表了一个“调节变阻器”，其中磷酸化与其他调节因子结合以引起可变激活。 在第二个模型系统中，c-Src SH 3结构域的配体结合功能通过残基Y 57的磷酸化来调节。这代表了一种原型分子开关，其中结合表面中或附近残基的磷酸化改变了模块化结合结构域的特异性和亲和力。对于两个模型系统中的每一个，多维NMR光谱将用于观察磷酸化诱导的结构、稳定性和动力学变化，等温滴定量热法（ITC）用于定量磷酸化诱导的功能变化。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pKas的测定来确定调节每种蛋白质的生物物理机制。 这些调查将提供一个结构，动力学和热力学框架，了解分子开关机制，尚未阐明。可逆的蛋白质磷酸化是一种普遍存在的机制，所有细胞都利用这种机制来打开和关闭蛋白质功能。 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，现在的特点，我们的理解不同的机制，通过磷酸化的调节是完成远未完成。 该项目旨在确定两种特定蛋白质通过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观点的耦合，将产生有价值的见解结合能的潜在来源，以及用于改变生物分子开关中的这种能量的机制。 这些研究将促进我们对磷酸化如何调节生物过程的理解，并将影响重要的研究领域，如信号转导，细胞内运输和膜结构的调节。 研究目标将主要通过研究生和本科生的参与来实现，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NMR光谱学的教育和培训。

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.