A photoelectron imaging study of the deprotonated GFP chromophore anion and RNA fluorescent tags.

Helen H Fielding,John M Ward,Alice Henley,Joanne L Woodhouse,Helen C Hailes,Anastasia V Bochenkova,Ross Lewin

doi:10.1039/d1cp01901e

Abstract

Green fluorescent protein (GFP), together with its family of variants, is the most widely used fluorescent protein for in vivo imaging. Numerous spectroscopic studies of the isolated GFP chromophore have been aimed at understanding the electronic properties of GFP. Here, we build on earlier work [A. V. Bochenkova, C. Mooney, M. A. Parkes, J. Woodhouse, L. Zhang, R. Lewin, J. M. Ward, H. Hailes, L. H. Andersen and H. H. Fielding, Chem. Sci., 2017, 8, 3154] investigating the impact of fluorine and methoxy substituents that have been employed to tune the electronic structure of the GFP chromophore for use as fluorescent RNA tags. We present photoelectron spectra following photoexcitation over a broad range of wavelengths (364-230 nm) together with photoelectron angular distributions following photoexcitation at 364 nm, which are interpreted with the aid of quantum chemistry calculations. The results support the earlier high-level quantum chemistry calculations that predicted how fluorine and methoxy substituents tune the electronic structure and we find evidence to suggest that the methoxy substituents enhance internal conversion, most likely from the 2ππ* state which has predominantly Feshbach resonance character, to the 1ππ* state.

Highlights

Green fluorescent protein (GFP), together with its family of variants, is the most widely used fluorescent protein for in vivo imaging
We build on this work and present significantly improved quality photoelectron spectra and photoelectron angular distributions (PADs) of the deprotonated anions of p-HBDIÀ, DF-HBDIÀ and DM-HBDIÀ following photodetachment over the wavelength range 364–230 nm
Analysis of the PADs suggest that indirect detachment via the 2pp* state, which has Feshbach resonance character with respect to the D0 continuum, is likely to contribute to the spectra of p-HBDIÀ, DF-HBDIÀ and DM-HBDIÀ following UV photoexcitation in the 364–315 nm range

Summary

Introduction

Green fluorescent protein (GFP), together with its family of variants, is the most widely used fluorescent protein for in vivo imaging. The VDE of DMHBDIÀ was found to be similar to that of p-HBDIÀ, which was rationalised in terms of opposing inductive and mesomeric effects of the methoxy groups In both biomimetic chromophores, the bright excited shape resonance was found to be shifted higher in energy and resonant autodetachment processes evident in the spectra were attributed to np*-D0 and np*-D1n detachment processes. We build on this work and present significantly improved quality photoelectron spectra and photoelectron angular distributions (PADs) of the deprotonated anions of p-HBDIÀ, DF-HBDIÀ and DM-HBDIÀ following photodetachment over the wavelength range 364–230 nm This allows us to establish that resonant detachment from the 2pp* electronically excited state and internal conversion from the 2pp* state to a lower lying electronically excited state, or S0, play a role in the electronic relaxation mechanism following UV photoexcitation

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Conclusion