Protein Phosphorylation as a Biophysical Switch: NMR Determination of Structural and Dynamic Responses to Phosphorylation

Nicholson 9808727这项研究的目标是确定磷酸化的结构、动态和功能影响，解决通过可逆磷酸化调节蛋白质的生物物理基础。研究了使用磷酸化作为触发机制的三种不同类型的分子开关：(1)pp60c-Src的SH3和SH2结构域，其代表其中相邻多肽片段的磷酸化改变模块结合域的特异性和亲和力的开关，以及(2)SH2结构域内的特定酪氨酸残基(Y213)，其代表其中靠近结合位点的残基的磷酸化可改变模块结合域的特异性的开关，以及(3)淀粉样前体蛋白(APPC)的47个残基的C末端细胞质尾部中的三个残基，它代表着跨膜蛋白胞质尾部特定残基的磷酸化改变了与细胞因子的相互作用的开关，如内吞作用的组成部分或信号机制。多维核磁共振波谱用于结构确定和动态表征，定点突变和重组蛋白的过度表达，以及滴定微量热法作为定量评估配基特异性和亲和力变化的功能分析方法的组合。可逆的蛋白质磷酸化被认为参与了一系列不同的调控过程的控制，对这些过程的进一步了解对生物技术是基本的和潜在的有用的。这一努力为培养核磁共振波谱学的本科生、研究生和博士后研究员奠定了基础。还将开展几项旨在教育普通公众了解分子世界的外展活动。大自然设计了一系列广泛的分子开关，指导和控制通过活细胞的信息、能量和分子货物的流动。可逆的蛋白质磷酸化，即在蛋白质的特定位置附着和移除一个磷酸基团，是一种调控策略，几乎用于控制所有的生物过程。本研究旨在提供参与蛋白质-蛋白质识别的三个模型开关的原子水平描述：第一和第二个探针如何使邻近多肽片段中或靠近结合部位的残基的磷酸化改变模块结合域区分结合伙伴的能力，而第三个探针研究跨膜蛋白胞质尾部特定残基的磷酸化如何改变与细胞伙伴的相互作用。这些研究将提供基本原理，使所设计的蛋白质的功能能够根据需要进行改变，并将具有巨大的生物技术应用价值。在这些研究中，核磁共振光谱被用来阐明磷酸化和非磷酸化蛋白质的结构和动力学，等温滴定量热法被用来定量评估磷酸化对与已知伙伴结合的功能影响。本科生、研究生和博士后研究员将参与其中，并将结合该项目开展外联活动。

Nicholson 9808727The objectives of this study are to determine the structural, dynamic, and functional effects of phosphorylation, addressing the biophysical basis of protein regulation by reversible phosphorylation. Three distinct types of molecular switches that employ phosphorylation as a trigger mechanism are studied: (1) the SH3 and SH2 domains of pp60c-Src, which represents switches in which phosphorylation of an adjacent polypeptide segment alters the specificity and affinity of modular binding domains, and (2) a specific tyrosine residue (Y213) within the SH2 domain which represents switches in which phosphorylation of a residue in close proximity to a binding site may alter the specificity of a modular binding domain, and (3) three residues in the 47-residue C-terminal cytoplasmic tail of the amyloid precursor protein (APPc), which represents switches in which phosphorylation of specific residues in a cytoplasmic tail of a transmembrane protein alters interactions with cellular factors such as components of the endocytosis or signaling machinery. A combination of approaches such as multidimensional nuclear magnetic resonance (NMR) spectroscopy for structure determination and dynamic characterization, site-directed mutagenesis and overexpression of the recombinant proteins, and titration microcalorimetry as a functional assay for quantitative evaluation of changes in ligand specificity and affinity is employed. Reversible protein phosphorylation is known to be involved in the control of a diverse array of regulatory processes, further understanding of which is fundamental and potentially useful to biotechnology. This endeavor provides a foundation for training undergraduate and graduate students and postdoctoral fellows in NMR spectroscopy. Several outreach activities designed to educate the general public about the molecular world also will be undertaken.Nature has designed a broad array of molecular switches that direct and control the flow of information, energy, and molecular cargo through living cells. Reversible protein phosphorylation, or the attachment and removal of a phosphate group at a specific site on a protein, is a regulatory strategy that is used in the control of almost all biological processes. This study aims to provide an atomic level description of three model switches involved in protein-protein recognition: the first and second probe how phosphorylation of residues either in an adjacent polypeptide segment or in close proximity to the binding site alters the ability of a modular binding domain to discriminate between binding partners, while the third investigates how phosphorylation of specific residues in the cytoplasmic tail of a transmembrane protein alters interactions with cellular partners. These studies will provide fundamental principles that will enable the function of designed proteins to be altered as desired, and will have great value for biotechnology applications. For these studies, NMR spectroscopy is applied to elucidate the structure and dynamics of both phosphorylated and unphosphorylated proteins, and isothermal titration calorimetry used to quantitatively evaluate the functional effects of phosphorylation on binding to known partners. Undergraduate and graduate students and postdoctoral fellows are involved, and outreach activities will be undertaken in conjunction with this project.