Electrostatics and Dynamics in Proteins

蛋白质的静电和动力学

• 批准号: 8795189
• 负责人: STEVEN G. BOXER
• 金额: $ 36.56万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-08-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8795189
• 关键词: Active Sites; Address; Affect; Aldehyde Reductase; Amino Acids; Benchmarking; Binding; Biology; Catalysis; Cell physiology; Cells; Charge; Color; Complex; Coupled; Couples; Coupling; Development; Diabetes Mellitus; Dissociation; Drug usage; Electrons; Electrostatics; Enzymes; Fluorescence; Goals; Green Fluorescent Proteins; Health; Human; Hydrogen Bonding; Image; Imaging Device; In Vitro; Individual; Isomerase; Ketosteroids; Kinetics; Life; Light; Malignant Neoplasms; Measurement; Measures; Membrane; Methodology; Methods; Modeling; Nature; Nucleic Acids; Peptide Hydrolases; Peptides; Process; Protein Tyrosine Kinase; Proteins; Protons; Reaction; Relative (related person); Reporter; Research; Resolution; Role; Series; Site; Structure; System; Tail; Techniques; Testing; Thermodynamics; Time; Tyrosine; Variant; Work; advanced system; base; chromophore; electric field; imaging system; in vivo; inhibitor/antagonist; interest; macromolecule; novel; protein folding; protein protein interaction; protein structure function; research study; sensor; simulation; synthetic peptide

DESCRIPTION (provided by applicant): The long-term goals of this project are to develop spectroscopic methods for probing electric fields in proteins and to apply these methods to obtain quantitative information on fields and their effects on function at the active sites of seveal enzymes and green fluorescent proteins (GFPs). Electrostatic interactions impact every aspect of the structure and function of proteins, nucleic acids, and membranes. Variations in the magnitude and direction of electric fields can significantly affect the rates of elementary processes such as electron and proton transfer, where charge moves over a substantial distance. Similarly, the transition states for many enzyme-catalyzed reactions involve a change in the distribution of charge relative to the starting material and/or products, and the selective stabilization of charge-separated transition states is essential for catalysis. The contours of electric fields steer the binding of substrates, inhibitors and allosteric effectors to macromolecules and directly affect binding constants. On a larger scale, electrostatic interactions affect protein folding, macromolecular interactions, and the assembly of subunits into larger structures. The magnitudes of the electric fields in proteins and the variations in thee fields at different sites are predicted to be enormous, but it is a challenge to obtain quantitativ experimental information on either local variations in electric fields in proteins or the time-dependent changes in these fields coupled to functionally relevant changes in charge distribution. The proposed research outlines a series of approaches and targets that can address these core issues. Aim 1 outlines development of methodology for introducing and characterizing vibrational probes for electric fields in proteins. These methods are used to probe fields in several enzymes. The proposed work focuses on a rigorous comparison between measured and calculated fields and incisive studies of the role of electrostatic interactions in catalytic mechanisms. Aim 2 outlines strategies to understand the mechanism(s) of the recently discovered coupling between light-driven structural dynamics of peptide-protein re-assembly or dissociation in split GFP. A wide range of structural and spectroscopic techniques will be deployed to elucidate the mechanism(s) of this coupling. The split semi-synthetic GFP system will also be used to probe the assembly of the ¿-barrel itself using the built-in reporter chromophore, the origin(s) of color tuning, and both ground and excited state proton transfer. These are achieved by introducing unnatural amino acids as probes or perturbations at functionally interesting sites throughout the protein, now enabled by these semi-synthetic systems. Applications of these novel systems for advanced imaging and as modulators of enzyme function that affect cell physiology with light are described.

描述（由适用提供）：该项目的长期目标是开发用于探测蛋白质电场的光谱方法，并应用这些方法以获取有关田间的定量信息及其对半酶和绿色荧光蛋白（GFPS）活性位点的功能的影响。静电相互作用会影响蛋白质，核酸和机制的结构和功能的各个方面。电场的大小和方向的变化可以显着影响基本过程的速率，例如电子和质子转移，在这些过程中，电荷在很大的距离上移动。同样，许多酶催化反应的过渡状态涉及相对于起始材料和/或产物的电荷分布的变化，并且电荷分离过渡状态的选择性稳定对于催化至关重要。电场的轮廓引导底物，抑制剂和变构作用与大分子的结合，并直接影响结合常数。在更大范围内，静电相互作用会影响蛋白质折叠，大分子相互作用以及将亚基组装成较大的结构。预计蛋白质中电场的幅度和不同位点的特性磁场的变化是巨大的，但是获得有关蛋白质中电场的局部变化的定量实验信息或这些领域的时间依赖性变化，以使这些领域的局部变化与充电分布的功能相关变化。拟议的研究概述了可以解决这些核心问题的一系列方法和目标。 AIM 1概述了用于引入和表征蛋白质电场振动问题的方法的发展。这些方法用于在几种酶中探测字段。所提出的工作集中在测量和计算的场​​之间的严格比较以及静电相互作用在催化机制中的作用的敏锐研究。 AIM 2概述了了解肽 - 蛋白质重新组装或分离GFP中最近发现的耦合的策略，以了解最近发现的耦合。将部署多种结构和光谱技术，以阐明这种耦合的机制。分裂的半合成GFP系统还将使用内置的记者发色团，颜色调整的原点以及地面和激发态质子转移来探测``桶式桶本身的组装。这些是通过在整个蛋白质功能有趣的位点引入不自然的氨基酸作为问题或扰动来实现的，现在由这些半合成系统启用。描述了这些新型系统在高级成像中的应用和用光影响细胞生理的酶功能的调节剂。