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Stalling chromophore maturation of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

Husam Sabah Auhim, Bella L. Grigorenko, Tessa Harris, Igor V. Polyakov, Colin Berry, Gabriel dos Passos Gomes, Igor V. Alabugin, Pierre J. Rizkallah, Alexander V. Nemukhin, View ORCID ProfileD. Dafydd Jones
doi: https://doi.org/10.1101/2020.10.13.337386
Husam Sabah Auhim
aSchool of Biosciences, Molecular Biosciences Division, Cardiff University, Cardiff, UK
bDepartment of Biology, College of Science, University of Baghdad, Baghdad, Iraq
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Bella L. Grigorenko
cChemistry Department, Lomonosov Moscow State University, Moscow, Russian Federation
dEmanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russian Federation
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Tessa Harris
aSchool of Biosciences, Molecular Biosciences Division, Cardiff University, Cardiff, UK
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Igor V. Polyakov
cChemistry Department, Lomonosov Moscow State University, Moscow, Russian Federation
dEmanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russian Federation
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Colin Berry
aSchool of Biosciences, Molecular Biosciences Division, Cardiff University, Cardiff, UK
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Gabriel dos Passos Gomes
eDepartment of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON M5S 3H6, Canada
fDepartment of Computer Science, University of Toronto, 214 College St., Toronto, Ontario M5T 3A1, Canada
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Igor V. Alabugin
gDepartment of Chemistry and Biochemistry, Florida State University, Tallahassee, Fl 32306, USA
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  • For correspondence: jonesdd@cardiff.ac.uk anem@lcc.chem.msu.ru RizkallahP@cardiff.ac.uk alabugin@chem.fsu.edu
Pierre J. Rizkallah
hSchool of Medicine, Cardiff University, CF14 4XN, UK
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  • For correspondence: jonesdd@cardiff.ac.uk anem@lcc.chem.msu.ru RizkallahP@cardiff.ac.uk alabugin@chem.fsu.edu
Alexander V. Nemukhin
cChemistry Department, Lomonosov Moscow State University, Moscow, Russian Federation
dEmanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russian Federation
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  • For correspondence: jonesdd@cardiff.ac.uk anem@lcc.chem.msu.ru RizkallahP@cardiff.ac.uk alabugin@chem.fsu.edu
D. Dafydd Jones
aSchool of Biosciences, Molecular Biosciences Division, Cardiff University, Cardiff, UK
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  • ORCID record for D. Dafydd Jones
  • For correspondence: jonesdd@cardiff.ac.uk anem@lcc.chem.msu.ru RizkallahP@cardiff.ac.uk alabugin@chem.fsu.edu
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Abstract

Fluorescent proteins (FPs) have revolutionised the life sciences but the mechanism of chromophore maturation is still not fully understood. Incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; the crystal structure reveals the presence of O2 located above a dehydrated enolate imidazolone (I) ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an “open” conformation, potentially allowing O2 access to the chromophore. Absorption spectroscopy supported by QM/MM simulations suggest that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H2O2 upon irradiation of this intermediate, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation, and the mechanistic insights opens up new approaches for engineering optically controlled fluorescent proteins.

Competing Interest Statement

The authors have declared no competing interest.

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Posted October 14, 2020.
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Stalling chromophore maturation of the fluorescent protein Venus reveals the molecular basis of the final oxidation step
Husam Sabah Auhim, Bella L. Grigorenko, Tessa Harris, Igor V. Polyakov, Colin Berry, Gabriel dos Passos Gomes, Igor V. Alabugin, Pierre J. Rizkallah, Alexander V. Nemukhin, D. Dafydd Jones
bioRxiv 2020.10.13.337386; doi: https://doi.org/10.1101/2020.10.13.337386
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Stalling chromophore maturation of the fluorescent protein Venus reveals the molecular basis of the final oxidation step
Husam Sabah Auhim, Bella L. Grigorenko, Tessa Harris, Igor V. Polyakov, Colin Berry, Gabriel dos Passos Gomes, Igor V. Alabugin, Pierre J. Rizkallah, Alexander V. Nemukhin, D. Dafydd Jones
bioRxiv 2020.10.13.337386; doi: https://doi.org/10.1101/2020.10.13.337386

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