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Protein, Enzyme, and Biological Water Dynamics: 2D Vibrational Echo Spectroscopy

蛋白质、酶和生物水动力学：二维振动回波光谱

基本信息

批准号：
8136495
负责人：
MICHAEL D FAYER
金额：
$ 30.41万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-04-01 至 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8136495
关键词：
Active Sites Affinity Alanine Azides Behavior Binding Binding Proteins Biological Biological Process Chemicals Complex Coupling Development Encapsulated Environment Enzymes Equilibrium Equus caballus Freedom Gel Gramicidin Guanidines Health Heme Hemeproteins Hydrogen Bonding Integral Membrane Protein Lasers Ligands Location Measurement Mechanics Mediating Membrane Membrane Proteins Methanol Methionine Methodology Methods Micelles Modeling Modification Molecular Probes Nature Oxygen Oxygenases Peroxidases Phospholipids Play Progress Reports Property Protein Dynamics Proteins Protons Recombinants Research Role Site Spectrum Analysis Structural Protein Structure Surface System Techniques Time United States National Institutes of Health Urea Vertebral column Water Work aqueous base biological systems cytochrome c disulfide bond enzyme substrate macromolecule molecular dynamics mutant nanopore neuroglobin nitrophorin novel strategies protein function protein protein interaction protein structure research study surfactant tool water channel

项目摘要

DESCRIPTION (provided by applicant): Research is proposed to study an interrelated set of problems involving the dynamics and dynamics-structure relationships of proteins, enzymes, enzyme-substrate binding, and the nature of biological water. The proposal is organized into two related parts. The first part discusses protein and enzyme dynamics. The second part involves the properties of biological water and its impact on biological systems. The principal experimental tools are ultrafast 2D-IR vibrational echo spectroscopy and other ultrafast IR methods. The vibrational echo experiments are akin to 2D-NMR except that they directly examine the structural/mechanical degrees of freedom of biological systems on time scales not accessible by other methods. The 2D-IR results are analyzed in conjunction with molecular dynamics simulations and other theoretical approaches. Building on our initial successful work in elucidating the relationship between substrate binding and protein dynamics with 2D-IR spectroscopy, novel approaches will be applied to the important question of how protein structural dynamics are modified by binding of exogenous ligands in the active site. CO and azide probes will be introduced selectively within the active site of several peroxidases. The interplay between key structural motifs and protein function will be examined for several systems. Neuroglobin (Ngb) is a heme protein with a single disulfide bond that is implicated in modulating the protein oxygen binding affinity. Structural and dynamic transformations that occur within Ngb will be probed by biochemically and mutagenically disrupting the disulfide bond. The relationship between protein function and structural transformation will also be examined in other systems such as nitrophorins. Recently developed methodology to introduce site-specific probes of protein dynamics selectively within the active site and at specific locations in the protein will be employed. The effects of nanoscopic confinement on protein unfolding will be probed by studying the denaturation of cytochrome c (cyt c) in aqueous and sol-gel nanopore environments. Denaturation studies with guanidine HCl, urea, methanol, and pH as chemical denaturants will probe the dynamical properties of molten globule states. Biological water differs markedly from bulk water behavior because of the effects of nanoscopic confinement and intimate contact to biological macromolecules. Our successful 2D-IR measurements of the dynamics of nanoscopic water will be extended to reverse micelles with phospholipid and non-ionic surfactants. The dynamics of water at protein interfaces will be determined by confining proteins in the reverse micelles and observing the water hydrogen bond dynamics. Water properties at membrane surfaces play an important role in biological processes because of water's interaction with transmembrane proteins and other biomolecules. The dynamics and interactions of water at the surfaces of model phospholipids membranes will be studied using 2D-IR spectroscopy. The dynamics of water in gramicidin, a model for transmembrane proton channel proteins, in multibilayers will be determined via ultrafast IR spectroscopies. PUBLIC HEALTH RELEVANCE The structural dynamics of complex biological molecules, such as proteins and enzymes, determine how they perform their biological functions. This project is using advanced infrared laser techniques to directly examine biomolecular structural dynamics and how biomolecule interactions with the surrounding medium, particularly water in biological environments, influence structural dynamics. The methodology builds on previous successful applications and developments of state-of-the-art ultrafast infrared laser experiments.

描述(由申请人提供)：建议研究一组相互关联的问题，涉及蛋白质、酶、酶-底物结合的动力学和动力学-结构关系，以及生物水的性质。该提案分为两个相关的部分。第一部分讨论蛋白质和酶的动力学。第二部分涉及生物水的性质及其对生物系统的影响。主要的实验工具是超快2D-IR振动回波光谱和其他超快红外方法。振动回波实验类似于2D-核磁共振，不同之处在于它们直接在时间尺度上检查生物系统的结构/机械自由度，而其他方法无法获得。结合分子动力学模拟和其他理论方法对2D-IR结果进行了分析。在我们用2D-IR光谱阐明底物结合和蛋白质动力学之间关系的初步成功工作的基础上，新的方法将被应用于如何通过活性位点上的外源配体结合来改变蛋白质结构动力学这一重要问题。CO和叠氮化物探针将被选择性地引入几种过氧化物酶的活性部位。关键结构基序和蛋白质功能之间的相互作用将在几个系统中进行研究。脑红蛋白(NGB)是一种具有单一二硫键的血红素蛋白，参与调节蛋白质的氧结合亲和力。发生在NGB内的结构和动态变化将通过生物化学和突变破坏二硫键来探测。蛋白质功能和结构转换之间的关系也将在其他系统中进行研究，例如亚硝基。最近开发的方法，以引入有选择地在活性部位内和在蛋白质中的特定位置的蛋白质动力学的位置特定的探针将被使用。通过研究细胞色素c(Cytc)在水溶液和溶胶-凝胶纳米孔环境中的变性，将探讨纳米限制对蛋白质展开的影响。以盐酸胍、尿素、甲醇和pH为化学变性剂的变性研究将探索熔融球状状态的动力学性质。由于纳米尺度的限制和与生物大分子的亲密接触的影响，生物水的行为明显不同于整体水的行为。我们对纳米水动力学的成功2D-IR测量将扩展到磷脂和非离子表面活性剂的反胶束。蛋白质界面的水动力学将通过将蛋白质限制在反胶束中并观察水的氢键动力学来确定。由于水与跨膜蛋白和其他生物分子的相互作用，膜表面的水性质在生物过程中起着重要的作用。将用2D-IR光谱研究水在模型磷脂膜表面的动力学和相互作用。通过超快红外光谱分析，将测定多层膜中跨膜质子通道蛋白模型--甘草素中的水的动态变化。公共卫生相关性复杂生物分子的结构动力学，如蛋白质和酶，决定了它们如何履行其生物功能。该项目使用先进的红外激光技术直接研究生物分子结构动力学，以及生物分子与周围介质，特别是生物环境中的水相互作用如何影响结构动力学。该方法建立在以前最先进的超快红外激光实验的成功应用和发展的基础上。