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

蛋白质磷酸化作为生物物理开关：核磁共振测定磷酸化的结构和动态响应

• 批准号: 9808727
• 负责人: Linda Nicholson
• 金额: $ 27万
• 依托单位: Cornell University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808727&HistoricalAwards=false
• 关键词: Protein; Phosphorylation; Biophysical; Switch; NMR

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.

尼科尔森 9808727本研究的目的是确定磷酸化的结构、动力学和功能效应，阐明可逆磷酸化调节蛋白质的生物物理基础。 研究了三种不同类型的采用磷酸化作为触发机制的分子开关：（1）pp 60 c-Src的SH 3和SH 2结构域，其代表开关，其中相邻多肽片段的磷酸化改变模块结合结构域的特异性和亲和力，和（2）特定的酪氨酸残基（Y213）在SH 2结构域内，其代表开关，其中紧密接近结合位点的残基的磷酸化可以改变结合位点的特异性。模块结合结构域，和（3）淀粉样前体蛋白（APPc）的47个残基C-末端胞质尾中的三个残基，其代表开关，其中跨膜蛋白的胞质尾中的特定残基的磷酸化改变与细胞因子（例如内吞作用或信号传导机制的组分）的相互作用。 采用多种方法的组合，例如用于结构测定和动态表征的多维核磁共振（NMR）光谱法、重组蛋白的定点诱变和过表达，以及作为定量评价配体特异性和亲和力变化的功能测定的滴定微量热法。已知可逆蛋白质磷酸化参与多种调控过程的控制，进一步了解这一点对生物技术是至关重要的，并可能有用。这一奋进为培养NMR光谱学的本科生、研究生和博士后研究员提供了基础。 此外，还将开展一些旨在教育公众了解分子世界的外联活动。大自然设计了一系列广泛的分子开关，这些开关可以指导和控制活细胞中的信息、能量和分子货物的流动。 可逆的蛋白质磷酸化，或在蛋白质上的特定位点处的磷酸基团的附着和去除，是用于控制几乎所有生物过程的调节策略。 本研究的目的是提供一个原子水平的描述三个模型开关参与蛋白质-蛋白质识别：所述第一和第二探针探测相邻多肽区段中或紧邻结合位点的残基的磷酸化如何改变模块结合结构域区分结合配偶体的能力，而第三个研究跨膜蛋白质尾区中特定残基的磷酸化如何改变与细胞伴侣的相互作用。这些研究将提供基本原理，使设计的蛋白质的功能能够根据需要改变，并将对生物技术应用具有巨大价值。 在这些研究中，NMR光谱被应用于阐明磷酸化和未磷酸化蛋白质的结构和动力学，等温滴定量热法用于定量评估磷酸化对与已知伴侣结合的功能影响。本科生、研究生和博士后研究员都参与其中，外联活动将结合这一项目开展。