Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle

Hartley, Andrew M., Worthy, Harley L., Reddington, Samuel C., Rizkallah, Pierre J. ORCID: https://orcid.org/0000-0002-9290-0369 and Jones, D. Dafydd ORCID: https://orcid.org/0000-0001-7709-3995 2016. Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle. Chemical Science 7 (10) , pp. 6484-6491. 10.1039/C6SC00944A

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Abstract

Through the genetic incorporation of a single phenyl azide group into superfolder GFP (sfGFP) at residue 148 we provide a molecular description of how this highly versatile chemical handle can be used to positively switch protein function in vitro and in vivo via either photochemistry or bioconjugation. Replacement of H148 with p-azido-L-phenylalanine (azF) blue shifts the major excitation peak ∼90 nm by disrupting the H-bond and proton transfer network that defines the chromophore charged state. Bioorthogonal click modification with a simple dibenzylcyclooctyne or UV irradiation shifts the neutral-anionic chromophore equilibrium, switching fluorescence to the optimal ∼490 nm excitation. Click modification also improved quantum yield over both the unmodified and original protein. Crystal structures of both the click modified and photochemically converted forms show that functional switching is due to local conformational changes that optimise the interaction networks surrounding the chromophore. Crystal structure and mass spectrometry studies of the irradiated protein suggest that the phenyl azide converts to a dehydroazepine and/or an azepinone. Thus, protein embedded phenyl azides can be used beyond simple photocrosslinkers and passive conjugation handles, and mimic many natural post-translational modifications: modulation though changes in interaction networks.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences Medicine
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:	6 July 2016
Date of Acceptance:	28 June 2016
Last Modified:	05 May 2023 07:31
URI:	https://orca.cardiff.ac.uk/id/eprint/92384

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