Energetic Basis and Design of Enzyme Function Demonstrated Using GFP, an Excited-State Enzyme

Lin, Chi-Yun; Romei, Matthew G.; Mathews, Irimpan I.; Boxer, Steven G.

doi:10.1021/jacs.1c12305

Title: Energetic Basis and Design of Enzyme Function Demonstrated Using GFP, an Excited-State Enzyme

Journal Article · 23 February 2022 · Journal of the American Chemical Society

DOI: https://doi.org/10.1021/jacs.1c12305 · OSTI ID:1869440

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Pennsylvania State Univ., University Park, PA (United States)
Stanford Univ., CA (United States)
SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)

We report the past decades have witnessed an explosion of de novo protein designs with a remarkable range of scaffolds. It remains challenging, however, to design catalytic functions that are competitive with naturally occurring counterparts as well as biomimetic or nonbiological catalysts. Although directed evolution often offers efficient solutions, the fitness landscape remains opaque. Green fluorescent protein (GFP), which has revolutionized biological imaging and assays, is one of the most redesigned proteins. While not an enzyme in the conventional sense, GFPs feature competing excited-state decay pathways with the same steric and electrostatic origins as conventional ground-state catalysts, and they exert exquisite control over multiple reaction outcomes through the same principles. Thus, GFP is an “excited-state enzyme”. Herein we show that rationally designed mutants and hybrids that contain environmental mutations and substituted chromophores provide the basis for a quantitative model and prediction that describes the influence of sterics and electrostatics on excited-state catalysis of GFPs. As both perturbations can selectively bias photoisomerization pathways, GFPs with fluorescence quantum yields (FQYs) and photoswitching characteristics tailored for specific applications could be predicted and then demonstrated. The underlying energetic landscape, readily accessible via spectroscopy for GFPs, offers an important missing link in the design of protein function that is generalizable to catalyst design.

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Research Organization:: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)

Sponsoring Organization:: National Institutes of Health (NIH); National Science Foundation (NSF); USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: AC02-76SF00515

OSTI ID:: 1869440

Journal Information:: Journal of the American Chemical Society, Journal Name: Journal of the American Chemical Society Journal Issue: 9 Vol. 144; ISSN 0002-7863

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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