Manipulating Single-Molecule Enzyme Conformations and Activities

操纵单分子酶的构象和活性

基本信息

批准号：
8464146
负责人：
H Peter Lu
金额：
$ 29.48万
依托单位：
BOWLING GREEN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
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/8464146
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 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 screening single molecule

项目摘要

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.

描述（由申请人提供）：我们提出的努力的主要目标是展示前所未有的单分子光谱技术和能力的应用。该技术将能够操纵甚至控制蛋白质构象，并以至少 10:1 的变化实时操纵酶蛋白活性。具体来说，我们建议进行系统的技术开发和示范。我们的项目包括三个主要目标：(1) 展示与大多数细菌生物合成途径相关的关键酶蛋白构象的相关单分子 FRET 测量和 AFM 单分子力牵引操作 (AFM-FRET)。 HPPK 酶），对于抗生素药物的研发非常重要； (2) 展示在机械力拉动和保持单分子酶蛋白的情况下环敏感 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，证明 HPPK 蛋白的受控酶反应性范围为 10:1。实时操纵蛋白质构象和活性的技术和方法将展示操纵单分子控制/保持蛋白质构象和动力学的新能力。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。这个新阶段的特点是对蛋白质结构和功能进行快速、灵敏的分析。例如，对于涉及关键生物功能的特定酶蛋白的各种构象和突变，我们将能够探索、分析和预测动态的功能结构关系。最终，从该项目中获得的有关构象敏感的 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。