Manipulating Single-Molecule Enzyme Conformations and Activities

描述（由申请人提供）：我们提出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的实时变化操纵酶蛋白活性。具体而言，我们建议进行系统的技术开发和演示。我们的项目包括三个主要目标：(1)展示与大多数细菌（HPPK酶）生物合成途径相关的关键酶蛋白（HPPK酶）构象的相关单分子FRET测量和AFM单分子力拉持操作（AFM-FRET），这对抗生素药物的研究和开发很重要；(2)展示单分子AFM-FRET分析环敏感HPPK酶动力学在机械力拉动和保持单分子酶蛋白；(3)通过使用AFM-FRET尖端保持和振荡单分子酶蛋白，证明HPPK蛋白的酶活性控制范围为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。我们的项目将为蛋白质结构与功能关系的表征和分析提供一个新的阶段。这个新阶段将以快速和敏感的蛋白质结构和功能分析为特色。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，分析和预测动态的功能-结构关系。最终，从这个项目中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将最终有助于抗生素药物的开发。

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.