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

本研究的目的是确定磷酸化的结构、动态和功能影响，通过可逆磷酸化解决蛋白质调节的生物物理基础。研究了三种不同类型的分子开关，它们采用磷酸化作为触发机制：(1) pp60c-Src的SH3和SH2结构域，它代表了相邻多肽段磷酸化改变模块化结合域特异性和亲和力的开关；(2)SH2结构域内的特定酪氨酸残基（Y213），它代表了靠近结合位点的残基磷酸化可能改变模块化结合域特异性的开关。(3)淀粉样蛋白前体蛋白（APPc） 47个残基c端胞质尾部的三个残基，这代表了跨膜蛋白胞质尾部特定残基磷酸化改变与细胞因子（如内吞作用或信号机制的组成部分）相互作用的开关。结合多种方法，如用于结构测定和动态表征的多维核磁共振（NMR）光谱，位点定向诱变和重组蛋白的过表达，以及用于定量评估配体特异性和亲和力变化的滴定微热法等。已知可逆蛋白磷酸化参与多种调控过程的控制，进一步了解其对生物技术的基础和潜在用途。这一努力为培养核磁共振波谱学的本科生、研究生和博士后奠定了基础。还将开展几项旨在教育公众了解分子世界的外展活动。大自然设计了一系列分子开关，指导和控制信息、能量和分子货物通过活细胞的流动。可逆的蛋白质磷酸化，或在蛋白质上特定位点的磷酸基团的附着和去除，是一种用于控制几乎所有生物过程的调节策略。本研究旨在提供涉及蛋白质-蛋白质识别的三种模型开关的原子水平描述：第一和第二项研究探讨了相邻多肽段或结合位点附近残基的磷酸化如何改变模块化结合域区分结合伙伴的能力，而第三项研究探讨了跨膜蛋白细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，使设计的蛋白质的功能能够按需要改变，并将对生物技术应用具有很大的价值。在这些研究中，核磁共振波谱被用于阐明磷酸化和未磷酸化蛋白的结构和动力学，等温滴定量热法被用于定量评估磷酸化对与已知伴侣结合的功能影响。本项目涉及本科生、研究生和博士后，并将结合本项目开展外展活动。

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.