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

DOI: 10.1039/D0SC06693A DOI Help

Authors: Husam Sabah Auhim (Cardiff University; University of Baghdad) , Bella L. Grigorenko (Lomonosov Moscow State University; Emanuel Institute of Biochemical Physics, Russian Academy of Sciences) , Tessa K. Harris (Cardiff University) , Ozan E. Aksakal (Cardiff University) , Igor V. Polyakov (Lomonosov Moscow State University; Emanuel Institute of Biochemical Physics, Russian Academy of Sciences) , Colin Berry (Cardiff University) , Gabriel Dos Passos Gomes (University of Toronto) , Igor V. Alabugin (Florida State University) , Pierre J. Rizkallah (Cardiff University) , Alexander V. Nemukhin (Lomonosov Moscow State University; Emanuel Institute of Biochemical Physics, Russian Academy of Sciences) , D. Dafydd Jones (Cardiff University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Chemical Science , VOL 67

State: Published (Approved)
Published: March 2021
Diamond Proposal Number(s): 18812

Open Access Open Access

Abstract: Fluorescent proteins (FPs) have revolutionised the life sciences, but the mechanism of chromophore maturation is still not fully understood. Here we show that incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; a crystal structure indicates the presence of O2 located above a dehydrated enolate form of the imidazolone ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an “open” conformation so forming a channel that allows O2 access to the immature chromophore. Absorbance spectroscopy supported by QM/MM simulations suggests 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, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation and the mechanistic insights open up new approaches for engineering optically controlled fluorescent proteins.

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Documents:
d0sc06693a.pdf

Discipline Tags:

Life Sciences & Biotech Structural biology Chemistry Biochemistry

Technical Tags:

Diffraction Macromolecular Crystallography (MX)