Developing 2D UV/vis spectroscopy tools to study biomolecular recognition

开发二维紫外/可见光谱工具来研究生物分子识别

• 批准号: 7937895
• 负责人: SHAUL MUKAMEL
• 金额: $ 47.17万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937895
• 关键词: Address; Antibodies; Antigens; Area; Arts; Biological; Biology; Biophysics; Collaborations; Communicable Diseases; Communities; Complex; Data; Detection; Development; Discipline; Electronics; Environment; Evolution; Feedback; Goals; Health; Human; Immune system; Immunology; Infection; Laboratories; Lasers; Lead; Malignant Neoplasms; Measures; Methodology; Methods; Modeling; Motion; Optics; Phase; Physical Chemistry; Physiologic pulse; Preparation; Protein Binding; Protein Conformation; Protein Dynamics; Proteins; Research; Research Institute; Role; Set protein; Signal Transduction; Source; Spectrum Analysis; Structure; System; Techniques; Technology; Testing; Therapeutic; Validation; Work; X-Ray Crystallography; base; biological systems; chromophore; combat; design; drug development; experience; improved; interdisciplinary collaboration; interest; method development; microbial; molecular recognition; nanosecond; novel; novel strategies; pathogen; protein structure; research study; simulation; technology development; theories; tool; tool development; two-dimensional

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-GM-102: Chemist/biologist collaborations facilitating tool development. The objective of the application is to develop a novel tool to study the role of fast protein dynamics in biomolecular recognition based on two- dimensional coherent UV/vis (2DUV/vis) spectroscopy, the theoretical tools to interpret the data, and a biological system with which to develop and validate the technique. Molecular recognition is central to the functions a protein may naturally possess, or those it may be evolved to possess. However, because biomolecular recognition does not generally follow a simple lock-and-key model, it cannot be characterized by structure determination alone. Biological systems generally show a level of induced fit- or conformational selection-like molecular recognition, and thus fluctuations between different protein conformations (i.e. protein dynamics) are critical. Consequently, understanding protein dynamics and how it might be tailored during evolution is fundamental to our understanding of biology. However, unlike technologies to characterize protein structures and relatively slow dynamics, methodologies to identify and characterize fast (pico- to nanosecond) protein motions remain underdeveloped. 2D UV/vis spectroscopy, a state-of-the-art ultrafast laser spectroscopy technique that only very recently has become feasible due to the advent of femtosecond broadband laser sources and phase-sensitive detection techniques for UV/vis signals, provides a multidimensional view of the structure and dynamical fluctuations of a protein. In this application we propose to develop the experimental and theoretical tools required to study protein dynamics using 2DUV/vis spectroscopy. In addition, we propose to develop and validate the experimental and theoretical tools through application to a biological system, evolutionarily related antibody-chromophre complexes, where the important fast motions may be identified and characterized, and for which fast protein dynamics have been predicted to contribute to molecular recognition. As observed during the early development of NMR methods, the application of the proposed 2DUV/vis methods to an important experimental problem should stimulate broader interest and further development, eventually delivering a new and important tool to the scientific community. The specific aims of this application are: Specific Aim 1: Develop 2DUV/vis system capable of experimentally characterizing fast protein dynamics. Specific Aim 2: Develop theoretical methodology to interpret the data and design new experiments. Specific Aim 3: Explore and validate the application of the developed methodologies, along with X-ray crystallography, to the characterization of biological molecular recognition. The development of methodologies to characterize fast protein dynamics should facilitate the study of many proteins that bind natural chromophores. In addition, there is an increasing number of techniques available to incorporate chromophores into proteins, and the proposed technology development will enable their characterization as well. The proposed research will also have important implications for human health because it will elucidate the mechanisms underlying the evolution of antibodies, our primary line of defense against microbial pathogens and cancer. In addition, great effort has recently been devoted to developing antibody-based therapies, and the results of the proposed should help design rational strategies that optimize the therapeutic potential of antibodies. To successfully address this research challenge, expertise in biology, ultrafast laser spectroscopy, physical chemistry, and theory is required, which is provided through a interdisciplinary collaboration between Drs. Mukamel (physical chemistry, nonlinear spectroscopy, and theory, UC Irvine) and Romesberg (immunology and ultrafast laser spectroscopy, The Scripps Research Institute), thus bridging the disconnect between theory, biophysics, and biology that often limits efforts to apply rigorous physical techniques to biologically relevant problems. The Mukamel lab will perform ab initio simulations of the electronic excitations, compute the 2D signals, interpret the signals in terms of protein structure and dynamics, and design novel pulse sequences to be tested experimentally. The Romesberg lab will implement a 2D UV/vis setup capable of measuring 2D electronic correlation spectra in the visible and near UV spectral region, and perform 2D UV/vis experiments on antibody-chromophore complexes of several sets of evolutionary related antibodies. The feedback between experimental and theoretical methodology development and immediate application to a biologically relevant system will significantly accelerate the refinement of 2D UV/vis spectroscopy as a tool to study biomolecular recognition. PUBLIC HEALTH RELEVANCE: Motions on all timescales contribute to biomolecular recognition, but in contrast to relatively slow dynamics, which may be characterized by a variety of well developed techniques, there is a technology gap for characterizing relatively fast dynamics, and thus, their potential contributions remain poorly understood. We propose to develop 2- dimensional UV/vis spectroscopy to study these fast protein motions, the theoretical tools to understand the data, and a biological system, the evolution of antibodies, to evaluate their importance. In addition to facilitating the study of many other protein- chromophore systems, the proposed research will help to understand how antibodies evolve to protect their host from infection and cancer, and help to devise novel approaches to antibody-based drug development.

描述(由申请人提供)：本申请涉及广泛的挑战领域(06)使能技术和具体的挑战主题，06-GM-102：化学家/生物学家合作促进工具开发。该应用的目的是开发一种新的工具来研究基于二维相干UV/Vis(2DUV/Vis)光谱的快速蛋白质动力学在生物分子识别中的作用，解释数据的理论工具，以及用于开发和验证该技术的生物系统。分子识别是蛋白质可能自然拥有的功能的核心，或者是它可能进化而拥有的那些功能的核心。然而，由于生物分子识别一般不遵循简单的锁和钥匙模型，它不能仅通过结构确定来表征。生物系统通常表现出诱导适配或构象选择类分子识别的水平，因此不同蛋白质构象之间的波动(即蛋白质动力学)是至关重要的。因此，了解蛋白质动力学以及它在进化过程中可能是如何定制的，是我们理解生物学的基础。然而，与表征蛋白质结构和相对缓慢的动力学的技术不同，识别和表征快速(皮秒到纳秒)蛋白质运动的方法仍然不发达。2D UV/Vis光谱是一种最先进的超快激光光谱技术，由于飞秒宽带激光光源和用于UV/Vis信号的相敏检测技术的出现，直到最近才变得可行，它提供了蛋白质结构和动态波动的多维视图。在这一应用中，我们建议开发使用2DUV/VIS光谱研究蛋白质动力学所需的实验和理论工具。此外，我们建议开发和验证实验和理论工具，通过应用于生物系统，进化相关的抗体-生色体复合体，其中重要的快速运动可以被识别和表征，并且快速蛋白质动力学被预测有助于分子识别。正如在核磁共振方法的早期发展中所观察到的那样，所提出的2DUV/VIS方法在一个重要的实验问题上的应用应该会引起更广泛的兴趣和进一步的发展，最终为科学界提供一个新的重要工具。这项应用的具体目标是：具体目标1：开发能够实验表征快速蛋白质动力学的2DUV/VIS系统。具体目标2：发展解释数据和设计新实验的理论方法论。具体目标3：探索和验证所开发的方法以及X射线结晶学在表征生物分子识别方面的应用。描述快速蛋白质动力学的方法学的发展应该有助于研究许多与天然发色团结合的蛋白质。此外，有越来越多的技术可用于将生色团结合到蛋白质中，拟议的技术开发也将使其表征成为可能。这项拟议的研究还将对人类健康产生重要影响，因为它将阐明抗体进化的潜在机制，抗体是我们抵御微生物病原体和癌症的主要防线。此外，最近致力于开发基于抗体的疗法，拟议的结果应该有助于设计合理的策略，优化抗体的治疗潜力。为了成功应对这一研究挑战，需要生物学、超快激光光谱学、物理化学和理论方面的专业知识，这是通过Mukamel博士(物理化学、非线性光谱学和理论，加州大学欧文分校)和Romesberg博士(免疫学和超快激光光谱学，斯克里普斯研究所)之间的跨学科合作提供的，从而弥合理论、生物物理学和生物学之间的脱节，这些脱节往往限制了将严格的物理技术应用于生物学相关问题的努力。Mukamel实验室将对电子激发进行从头计算模拟，计算2D信号，根据蛋白质结构和动力学解释信号，并设计新的脉冲序列进行实验测试。Romesberg实验室将实施2D UV/VIS装置，能够测量可见光和近UV光谱区域的2D电子相关光谱，并对几组进化相关抗体的抗体-发色团复合体进行2D UV/VIS实验。实验和理论方法学发展之间的反馈以及对生物相关系统的即时应用将大大加快2D UV/Vis光谱作为研究生物分子识别工具的精细化。 公共卫生相关性：所有时间尺度上的运动都有助于生物分子识别，但与相对较慢的动力学相反，相对较慢的动力学可能以各种成熟的技术为特征，在表征相对较快的动力学方面存在技术空白，因此，它们的潜在贡献仍然知之甚少。我们建议开发二维UV/Vis光谱来研究这些快速的蛋白质运动，这是理解数据的理论工具，以及一个生物系统，即抗体的进化，以评估它们的重要性。除了促进对许多其他蛋白质-发色团系统的研究外，拟议的研究还将有助于了解抗体如何进化以保护其宿主免受感染和癌症，并有助于设计基于抗体的药物开发的新方法。