Action spectroscopy of the isolated red Kaede fluorescent protein chromophore

Coughlan, Neville J. A., Stockett, Mark H., Kjaer, Christina, Ashworth, Eleanor K., Bulman Page, Philip C., Meech, Stephen R., Nielsen, Steen Brøndsted, Blancafort, Lluis, Hopkins, W. Scott and Bull, James N. (2021) Action spectroscopy of the isolated red Kaede fluorescent protein chromophore. The Journal of Chemical Physics, 155 (12). ISSN 0021-9606

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Abstract

Incorporation of fluorescent proteins into biochemical systems has revolutionized the field of bioimaging. In a bottom-up approach, understanding the photophysics of fluorescent proteins requires detailed investigations of the light-absorbing chromophore, which can be achieved by studying the chromophore in isolation. This paper reports a photodissociation action spectroscopy study on the deprotonated anion of the red Kaede fluorescent protein chromophore, demonstrating that at least three isomers–assigned to deprotomers–are generated in the gas phase. Deprotomer-selected action spectra are recorded over the S1 ← S0 band using an instrument with differential mobility spectrometry coupled with photodissociation spectroscopy. The spectrum for the principal phenoxide deprotomer spans the 480–660 nm range with a maximum response at ≈610 nm. The imidazolate deprotomer has a blue-shifted action spectrum with a maximum response at ≈545 nm. The action spectra are consistent with excited state coupled-cluster calculations of excitation wavelengths for the deprotomers. A third gas-phase species with a distinct action spectrum is tentatively assigned to an imidazole tautomer of the principal phenoxide deprotomer. This study highlights the need for isomer-selective methods when studying the photophysics of biochromophores possessing several deprotonation sites.

Item Type: Article
Additional Information: Acknowledgments: Funding was provided by the Swedish Foundation for International Cooperation in Research and Higher Education (STINT) Grant for Internationalisation program (Grant No. PT2017-7328 to M.H.S. and J.N.B.), a start-up grant at the University of East Anglia (to J.N.B.), a NSERC Discovery Grant, and a Collaborative Research and Development grant (to W.S.H.), and the provision of computational support from Compute Canada. N.J.A.C. acknowledges a Vanier-Banting Postdoctoral Fellowship from the NSERC. W.S.H. acknowledges an Early Researcher Award from the province of Ontario. M.H.S. acknowledges support from the Swedish Research Council (Grant No. 2016-03675) and the Olle Engkvist Foundation (Grant No. 200-575). L.B. thanks the Ministerio de Ciencia, Innovación y Universidades (Spain), Project No. PID-2019-104654GB-I00. S.B.N acknowledges generous support from the Novo Nordisk Foundation (Grant No. NNF20OC0064958). S.R.M. and P.C.B.P. acknowledge funding from the EPSRC (Grant No. EP/H025715/1). Electronic structure calculations were, in part, carried out on the High Performance Computing Cluster supported by the Research and Specialist Computing Support service at the University of East Anglia. Marlyn Mwita is thanked for recording some preliminary DMS ionograms.
Faculty \ School: Faculty of Science > School of Chemistry
Depositing User: LivePure Connector
Date Deposited: 24 Sep 2021 01:05
Last Modified: 18 Oct 2021 01:05
URI: https://ueaeprints.uea.ac.uk/id/eprint/81509
DOI: 10.1063/5.0063258

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