Manipulating Single-Molecule Enzyme Conformations and Activities

操纵单分子酶的构象和活性

• 批准号: 8069189
• 负责人: H Peter Lu
• 金额: $ 30.54万
• 依托单位: BOWLING GREEN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-05 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8069189
• 关键词: 6-hydroxymethyl-7,8-dihydropterin; Adverse effects; Anti-Bacterial Agents; Antibiotics; Area; Atomic Force Microscopy; Bacteria; Binding; Biochemical Reaction; Biological Process; Coupled; Cysteine; Development; Diphosphotransferases; Energy Transfer; Enzymes; Fluorescence; Fluorescence Spectroscopy; Fluorescent Probes; Folate; Frequencies; Glass; Goals; Health; Human; Imaging Techniques; Knowledge; Laboratories; Link; Measurement; Mechanics; Methodology; Microscope; Molecular Conformation; Mutation; Pathway interactions; Pharmaceutical Preparations; Protein Conformation; Protein Dynamics; Proteins; Research; Screening procedure; Site-Directed Mutagenesis; Spectrum Analysis; Staging; Structure-Activity Relationship; Sulfhydryl Compounds; Techniques; Technology; Time; Variant; base; drug development; enzyme activity; enzyme structure; flexibility; fluorescence imaging; inhibitor/antagonist; link protein; next generation; novel; novel strategies; programs; protein function; protein structure function; public health relevance; research and development; response; single molecule

DESCRIPTION (provided by applicant): The key goal of our proposed effort is to demonstrate the application of an unprecedented single-molecule spectroscopy technique and capability. This technique will be capable of manipulating and even controlling protein conformations and manipulating the enzyme protein activity by variation of at least 10:1 in real-time. Specifically, we propose to conduct a systematic technical development and demonstration. Our project consists of three primary aims: (1) Demonstrate correlated single-molecule FRET measurements and AFM single-molecule force pulling-holding manipulation (AFM-FRET) of the conformations of a key enzyme protein related to the biosynthetic pathway of most bacteria (the HPPK enzyme), important for antibiotics drug research and developments; (2) demonstrate single-molecule AFM-FRET analysis of loop sensitive HPPK enzymatic dynamics under mechanical force pulling and holding single-molecule enzyme proteins; and (3) demonstrate a 10:1 range of controlled enzyme reactivity for the HPPK protein by using AFM-FRET tip holding and oscillating single-molecule enzyme proteins. The technology and methodology for the real-time manipulation of protein conformations and activities will demonstrate the novel capability of manipulating single-molecule control/holding protein conformations and dynamics. Our project will provide a new stage in characterizing and analyzing the relationship between protein structure and function. This new stage will feature rapid and sensitive analyzes of protein structures and functions. For example, for various conformations and mutations of a specific enzyme protein involving in critical biological functions, we will be able to explore, analyze, and predict the dynamic function-structure relationship. Ultimately, the knowledge obtained from this project about the conformation sensitive HPPK activity and response to inhibitor binding will eventually be helpful for anti-biotic drug developments. PUBLIC HEALTH RELEVANCE: Our proposed advance in single-molecule spectroscopy and manipulation will be critical for the next generation of single-molecule spectroscopy applicable on human health research: not only observing single-molecule protein dynamics but also be able to manipulate the enzyme reactivity and conformations in real time. We will apply this technical approach to the manipulation of the conformations and activity of enzymes of importance to antibiotics drug developments: a key enzyme protein related to the folate biosynthetic pathway of most bacteria, i.e., 6-hydroxymethyl-7,8- dihydropterin pyrophosphokinase (HPPK). Because that the folate biosynthetic pathway is absent from human being and critical for bacteria, it has been a hot research area in developing anti-bacterial drugs to attack the folate pathway without a harmful side-effect to human.

描述（由申请人提供）：我们提出的努力的关键目标是证明使用前所未有的单分子光谱技术和能力。该技术将能够操纵甚至控制蛋白质构象，并通过实时变化至少10：1来操纵酶蛋白活性。具体而言，我们建议进行系统的技术发展和演示。我们的项目由三个主要目的组成：（1）展示了相关的单分子fret测量和AFM单分子拉力拉力拉力持续操纵（AFM-FRET）对与大多数细菌的生物合成途径相关的关键酶蛋白的构象的构象（HPPK enzyzyme），对抗生素和抗生素的重要性（重要）; （2）证明了在机械力拉动和保持单分子酶蛋白下环环敏感HPPK酶动力学的单分子AFM-FRET分析； （3）通过使用AFM-FRET尖端固定和振荡单分子酶蛋白来证明HPPK蛋白的10：1受控酶反应性。实时操纵蛋白质构象和活动的技术和方法将证明操纵单分子控制/保持蛋白质构象和动力学的新功能。我们的项目将为表征和分析蛋白质结构与功能之间的关系提供一个新的阶段。这个新阶段将以蛋白质结构和功能的快速和敏感分析。例如，对于涉及关键生物学功能的特定酶蛋白的各种构象和突变，我们将能够探索，分析和预测动态功能结构关系。最终，从该项目获得的关于构象敏感的HPPK活性和对抑制剂结合的反应的知识最终将有助于抗生素药物的开发。 公共卫生相关性：我们提出的单分子光谱和操纵方面的提议对于适用于人类健康研究的下一代单分子光谱法至关重要：不仅可以观察单分子蛋白动力学，而且能够实时操纵酶的反应性和构象。我们将采用这种技术方法来操纵对抗生素的重要性的构象和活性：一种与大多数细菌的叶酸生物合成途径有关的关键酶蛋白，即6-羟基甲基甲基-7,8-二羟基二羟基二羟基丙糖蛋白pyrophophoppokinase（hhppppk）。因为人类没有叶酸生物合成途径，对细菌至关重要，因此它一直是开发抗细菌药物的热门研究领域，可以攻击叶酸途径而没有对人的有害副作用。