Static & dynamic electrostatic interactions in proteins

开发和扩展蛋白质中静电相互作用的工作，最终目标是设计能够绘制蛋白质图谱并报告蛋白质动力学和静电学的分子探针，特别是：1)设计和合成新的半胱氨酸(Cys)特异性标记，这是一种通用探针，将使我们能够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选择性地引入半胱氨酸残基，表达蛋白质，并用静电探针标记。例子包括作为模型系统的载脂蛋白(ApoMb)、GB1和T4溶菌酶。3)通过研究利用特定位点标记引入的CN伸展的振动跃迁来测量静态蛋白质相互作用。该振动标签被选择为使用VSE来定量。通过突变CN附近的氨基酸残基，观察到的振动结合位移直接探测到局域场。4)确定是否可以将振动斯塔克效应(VSE)光谱和蛋白质标记相结合来绘制蛋白质中的电场。5)使用动态行程移动法测量静电动态相互作用，在该方法中，实时测量新引入的探头的极性、荧光发射态的能量。与非天然氨基酸侧链的类似数据进行平行比较。这项研究包括大肠杆菌突变蛋白的表达和纯化，新型分子探针的合成，蛋白质的标记和鉴定，以及红外光谱和荧光上转换等一系列实验技术。

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.