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

Sabah Auhim, Husam, Grigorenko, Bella L, Harris, Tessa, Aksakal, Ozan, Polyakov, Igor, Berry, Colin ORCID: https://orcid.org/0000-0002-9943-548X, dos Passos Gomes, Gabriel, Alabugin, Igor, Rizkallah, Pierre, Nemukhin, Alexander and Jones, D. Dafydd ORCID: https://orcid.org/0000-0001-7709-3995 2021. Stalling chromophore synthesis of the fluorescent protein Venus reveals the molecular basis of the final oxidation step. Chemical Science 12 (22) , pp. 7735-7745. 10.1039/D0SC06693A

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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 (I) 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. 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 on, 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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine Biosciences Advanced Research Computing @ Cardiff (ARCCA)
Additional Information:	This article is licensed under a Creative Commons Attribution 3.0 Unported Licence
Publisher:	Royal Society of Chemistry
ISSN:	2041-6520
Date of First Compliant Deposit:	31 March 2021
Date of Acceptance:	26 March 2021
Last Modified:	20 Dec 2023 10:20
URI:	https://orca.cardiff.ac.uk/id/eprint/140220

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