Cryogenic Ion Spectroscopy of the <i>Ortho</i> Green Fluorescent Protein Chromophore Anion

Engineering new fluorescent probes, such as fluorescent proteins, requires a firm understanding of factors governing chromophore photophysics. Here we study <i>o</i>-HBDI^-, a derivative of the green fluorescent protein chromophore anion where the phenolate oxygen is shifted from the <i>para</i> to <i>ortho</i> position. Absorption by cryogenically cooled ions in the gas phase was monitored indirectly via photon emission or electron photodetachment versus excitation wavelength and combined with dispersed fluorescence spectra to determine the Stokes shift. The higher barrier to internal conversion in <i>o</i>-HBDI^- relative to that in <i>p</i>-HBDI^- gives a longer excited-state lifetime and allows fluorescence to persist over a broad range of excitation energies. The 0-0 transition is markedly red-shifted, consistent with time-dependent density functional theory calculations on <i>trans-E/Z</i> isomers. A small Stokes shift suggests that fluorescent <i>trans</i> isomers retain planarity after excitation, as also supported by theory, while nonfluorescent <i>cis</i> isomers undergo twist deformations. Our findings highlight structural factors influencing chromophore fluorescence.