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: 7831184
• 负责人: SHAUL MUKAMEL
• 金额: $ 47.69万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7831184
• 关键词: Address; Antibodies; Antigens; Area; Arts; Biological; Biology; Biophysics; Classification; 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; public health relevance; 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）使能技术和特定的挑战主题，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快速蛋白质动力学在基于二维相干紫外/维斯（2DUV/维斯）光谱的生物分子识别中的作用，解释数据的理论工具，以及开发和验证该技术的生物系统。分子识别对于蛋白质可能天然拥有的功能或可能进化拥有的功能至关重要。然而，由于生物分子识别通常不遵循简单的锁和钥匙模型，因此它不能仅通过结构测定来表征。生物系统通常表现出一定水平的诱导拟合或构象选择样分子识别，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，理解蛋白质动力学以及它在进化过程中如何被定制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（皮科至纳秒）蛋白质运动的方法仍然不发达。2D UV/维斯光谱是一种最先进的超快激光光谱技术，由于飞秒宽带激光源和UV/维斯信号相敏检测技术的出现，该技术最近才变得可行，提供了蛋白质结构和动力学波动的多维视图。在此应用中，我们建议开发所需的实验和理论工具，研究蛋白质动力学使用2DUV/维斯光谱。此外，我们建议开发和验证的实验和理论工具，通过应用到生物系统，进化相关的抗体-发色团复合物，其中重要的快速运动可以识别和表征，并已预测快速蛋白质动力学有助于分子识别。正如在NMR方法的早期发展过程中所观察到的那样，将所提出的2DUV/维斯方法应用于一个重要的实验问题应该会激发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。本申请的具体目标是：具体目标1：开发能够通过实验表征快速蛋白质动力学的2DUV/维斯系统。具体目标2：开发理论方法来解释数据和设计新的实验。具体目标3：探索和验证所开发的方法学的应用，沿着X射线晶体学，生物分子识别的表征。快速蛋白质动力学特性的方法学的发展应促进许多蛋白质结合天然发色团的研究。此外，有越来越多的技术可用于将发色团纳入蛋白质中，拟议的技术开发也将使其表征成为可能。这项拟议中的研究也将对人类健康产生重要影响，因为它将阐明抗体进化的机制，抗体是我们对抗微生物病原体和癌症的主要防线。此外，最近已经投入了很大的努力来开发基于抗体的疗法，并且所提出的结果应该有助于设计优化抗体的治疗潜力的合理策略。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博士之间的跨学科合作提供的。（物理化学，非线性光谱学和理论，UC Irvine）和Romesberg（免疫学和超快激光光谱学，斯克里普斯研究所），从而弥合了理论，生物物理学，而生物学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，从蛋白质结构和动力学角度解释信号，并设计新的脉冲序列进行实验测试。Romesberg实验室将实施能够测量可见光和近紫外光谱区域的2D电子相关光谱的2D UV/维斯装置，并对几组进化相关抗体的抗体-发色团复合物进行2D UV/维斯实验。实验和理论方法的发展和生物相关系统的直接应用之间的反馈将显着加速细化的二维紫外/维斯光谱作为一种工具，研究生物分子识别。 公共卫生关系：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以通过各种成熟的技术来表征，对于表征相对较快的动力学存在技术差距，因此，它们的潜在贡献仍然知之甚少。我们建议开发二维UV/维斯光谱来研究这些快速蛋白质运动，理解数据的理论工具，以及生物系统，抗体的进化，以评估其重要性。除了促进许多其他蛋白质-发色团系统的研究外，拟议的研究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。