Issue 30, 2023

Protomers of the green and cyan fluorescent protein chromophores investigated using action spectroscopy

Abstract

The photophysics of biochromophore ions often depends on the isomeric or protomeric distribution, yet this distribution, and the individual isomer contributions to an action spectrum, can be difficult to quantify. Here, we use two separate photodissociation action spectroscopy instruments to record electronic spectra for protonated forms of the green (pHBDI+) and cyan (Cyan+) fluorescent protein chromophores. One instrument allows for cryogenic (T = 40 ± 10 K) cooling of the ions, while the other offers the ability to perform protomer-selective photodissociation spectroscopy. We show that both chromophores are generated as two protomers when using electrospray ionisation, and that the protomers have partially overlapping absorption profiles associated with the S1 ← S0 transition. The action spectra for both species span the 340–460 nm range, although the spectral onset for the pHBDI+ protomer with the proton residing on the carbonyl oxygen is red-shifted by ≈40 nm relative to the lower-energy imine protomer. Similarly, the imine and carbonyl protomers are the lowest energy forms of Cyan+, with the main band for the carbonyl protomer red-shifted by ≈60 nm relative to the lower-energy imine protomer. The present strategy for investigating protomers can be applied to a wide range of other biochromophore ions.

Graphical abstract: Protomers of the green and cyan fluorescent protein chromophores investigated using action spectroscopy

Supplementary files

Article information

Article type
Paper
Submitted
08 6 2023
Accepted
13 7 2023
First published
13 7 2023
This article is Open Access
Creative Commons BY license

Phys. Chem. Chem. Phys., 2023,25, 20405-20413

Protomers of the green and cyan fluorescent protein chromophores investigated using action spectroscopy

E. K. Ashworth, J. Dezalay, C. R. M. Ryan, C. Ieritano, W. S. Hopkins, I. Chambrier, A. N. Cammidge, M. H. Stockett, J. A. Noble and J. N. Bull, Phys. Chem. Chem. Phys., 2023, 25, 20405 DOI: 10.1039/D3CP02661B

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