Static & dynamic electrostatic interactions in proteins

静止的

• 批准号: 6626158
• 负责人: ZINAIDA S ROMANOVA
• 金额: $ 4.64万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6626158
• 关键词: Escherichia coli; aminoacid; bacterial proteins; binding sites; biophysics; chemical reaction; chemical synthesis; computer simulation; cysteine; electric field; fluorescence; fluorescent dye /probe; gene expression; gene mutation; ionic bond; lysozyme; molecular biology; molecular dynamics; myoglobin; postdoctoral investigator; protein binding; protein purification; protein structure; proteins; reagent /indicator; site directed mutagenesis; spectrometry; technology /technique development

To develop and extend work on electrostatic interactions in protein with the ultimate goal of designing molecular probes capable of mapping proteins and reporting on protein dynamics and electrostatics Specifically to: 1) design and synthesize new cysteine (Cys)-specific labels, generic probes that will allow us to map static and dynamic electrostatic fields in any protein. 2) Introduce Cys residues at selective sites throughout model proteins, express proteins and label with probes for electrostatic potentials. Examples include apomyoglobin (apoMb), GB1 and T4 lysozyme as model systems. 3) Measure static protein interactions by studying shifts of vibrational transitions of CN stretch that is introduced using site-specific labeling. This vibrational label is chosen to be quantitatively using VSE. By mutating amino acid residues near to the CN, the observe vibrational bind shifts directly probe the local fields. 4) Establish whether vibrational Stark effect (VSE) spectroscopy and protein labeling can be combined to map electric fields in proteins. 5) Measure electrostatic dynamic interactions using the dynamic Strokes shift method in which the energy of a polar, fluorescent emitting state of a newly introduced probe is measured in real time. Make a parallel comparison with similar data on unnatural amino acid side chains. The proposed research includes expression and purification of mutant proteins from E. coli, synthesis of novel molecular probes, protein labeling and characterization, and, finally, an array of experimental techniques such as infrared spectroscopy and fluorescence up- conversion.

开发和扩展蛋白质中静电相互作用的工作，其最终目的是设计能够绘制蛋白质并报告蛋白质动力学和静电的分子探针，专门针对：1）设计和合成新的半胱氨酸（CYS）（CYS） - 特异性探针，该探针将是通用探针，该探针将是通用探针允许我们绘制任何蛋白质中的静态和动态静电场。 2）在整个模型蛋白质中，在选择性位点引入CYS残基，表达蛋白质和标记，并具有探针的静电电势。例子包括Apomyoglobin（Apomb），GB1和T4溶菌酶作为模型系统。 3）通过研究使用位点特异性标记引入的CN拉伸振动转变的变化来测量静态蛋白相互作用。选择此振动标签是使用VSE定量的。通过突变靠近CN的氨基酸残基，观察到振动结合可以直接探测局部场。 4）确定是否可以将振动性鲜明效应（VSE）光谱和蛋白质标记合并到蛋白质中的电场。 5）使用动态笔触移动方法测量静电动态相互作用，在这种动态中，极性的荧光发射状态的新引入探针的能量是实时测量的。与非天然氨基酸侧链的类似数据并行比较。拟议的研究包括来自大肠杆菌的突变蛋白的表达和纯化，新型分子探针的合成，蛋白质的标记和表征，最后是一系列实验技术，例如红外光谱和荧光上升转化。