Action spectroscopy of the isolated red Kaede fluorescent protein chromophore

Neville J. A. Coughlan, Mark H. Stockett, Christina Kjaer, Eleanor K. Ashworth, Philip C. Bulman Page, Stephen R. Meech, Steen Brøndsted Nielsen, Lluis Blancafort, W. Scott Hopkins, James N. Bull

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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.
Original languageEnglish
Article number124304
JournalThe Journal of Chemical Physics
Issue number12
Early online date23 Sep 2021
Publication statusPublished - 28 Sep 2021

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