Manipulating Single-Molecule Enzyme Conformations and Activities

描述(由申请人提供)：我们提出的工作的关键目标是展示一种史无前例的单分子光谱学技术和能力的应用。这项技术将能够操纵甚至控制蛋白质构象，并通过至少10：1的实时变化来操纵酶蛋白质的活性。具体地说，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)展示与大多数细菌的生物合成途径有关的关键酶蛋白(HPPK酶)的构象的相关单分子FRET测量和AFM单分子力拉紧操作(AFM-FRET)，该酶对抗生素药物的研究和开发非常重要；(2)展示在机械力拉动和握持单分子酶蛋白下环敏感的HPPK酶动力学的单分子AFM-FRET分析；以及(3)通过使用AFM-FRET握持和振荡单分子酶蛋白来展示HPPK蛋白的10：1可控的酶活性范围。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和功能之间的关系提供一个新的阶段。这一新阶段将以对蛋白质结构和功能的快速和灵敏的分析为特色。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响应的知识最终将有助于抗生物药物的开发。 与公共健康相关：我们在单分子光谱学和操纵方面的拟议进展将对应用于人类健康研究的下一代单分子光谱学至关重要：不仅观察单分子蛋白质的动力学，而且能够实时操纵酶的反应和构象。我们将应用这种技术方法来操纵对抗生素药物开发至关重要的酶的构象和活性：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-羟甲基-7，8-二氢蝶呤焦磷酸激酶(HPPK)。由于叶酸生物合成途径在人类中不存在，对细菌至关重要，因此开发无毒副作用的叶酸生物合成途径的抗菌药物一直是研究的热点。

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.