Manipulating Single-Molecule Enzyme Conformations and Activities

Bowling Green State University, Bowling Green OH

Investigators

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Abstract

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.