Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 118, Issue 13, Pages -Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2101618118
Keywords
biosensor; protein stability; protein engineering; deep mutational scanning
Categories
Funding
- National Natural Science Foundation of China [31870054, 31670802, 31661143021]
- Fundamental Research Funds for the Central Universities [22221818014]
- Research Program of State Key Laboratory of Bioreactor Engineering
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The study presents an in vivo stability biosensor for monitoring protein stability and coupling it with fluorescent readouts. The biosensor was shown to be useful in directed evolution to obtain improved variants of proteins, and in deep mutational scanning to reveal informative stability landscapes in proteins.
Protein stability affects the physiological functions of proteins and is also a desirable trait in many protein engineering tasks, yet improving protein stability is challenging because of limitations in methods for directly monitoring protein stability in cells. Here, we report an in vivo stability biosensor wherein a protein of interest (POI) is inserted into a microbial enzyme (CysGA) that catalyzes the formation of endogenous fluorescent compounds, thereby coupling POI stability to simple fluorescence readouts. We demonstrate the utility of the biosensor in directed evolution to obtain stabilized, less aggregation-prone variants of two POIs (including nonamyloidogenic variants of human islet amyloid polypeptide). Beyond engineering applications, we exploited our biosensor in deep mutational scanning for experimental delineation of the stability-related contributions of all residues throughout the catalytic domain of a histone H3K4 methyltransferase, thereby revealing its scientifically informative stability landscape. Thus, our highly accessible method for in vivo monitoring of the stability of diverse proteins will facilitate both basic research and applied protein engineering efforts.
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