Photophysics of a Methylated GFP Chromophore Anion in Vacuo

The photophysical properties of the isolated chromophore anion from the green fluorescent protein have been extensively studied over the years to understand the factors influencing transition energies, excited-state lifetimes, and fluorescence. A commonly used model for the protein chromophore is <i>4'-hydroxybenzylidene-2,3-dimethyl-imidazolinone</i> (<i>p</i>-HBDI). In this work, we have spectroscopically characterized a derivative, brMe-<i>p</i>-HBDI, which features methylation on the carbon bridging the phenol and imidazolinone rings. Experiments were conducted on the anionic form in the gas phase and at cryogenic temperatures using the SAPHIRA ion-storage ring and the LUNA2 fluorescence mass spectrometer, both located in Aarhus. Photoinduced action spectra reveal that brMe-<i>p</i>-HBDI^- cooled to about 20-30 K exhibits maximum absorption at 496.0 ± 0.5 nm. Vibrationally resolved bands appear at shorter wavelengths, while a featureless absorption tail extends toward longer wavelengths, up to approximately 520 nm. The methyl substituent induces a clear redshift (75 meV) in absorption as <i>p</i>-HBDI^- absorbs maximally at 481.51 ± 0.15 nm. The excited-state lifetime of brMe-<i>p</i>-HBDI^- is determined to be 51 ± 3 ps following 495 nm photoexcitation and probing at 800 nm, which is significantly shorter than the nanosecond lifetime previously reported for <i>p</i>-HBDI^-. Consistent with this, no fluorescence was detected from brMe-<i>p</i>-HBDI^- at 100 K, in contrast to <i>p</i>-HBDI^- that is strongly fluorescent according to recent work. These findings are corroborated by (time-dependent) density-functional theory calculations: A methyl substituent at the bridge carbon is predicted to cause a redshift of 77 meV, in excellent agreement with the experimental shift. We find that brMe-<i>p</i>-HBDI^- is planar in the ground state (S₀) but undergoes a twist motion in the S₁ state, leading to a lower-energy nonplanar form where the angle between the two rings is 90°. Our work reveals that even a minor alteration in molecular structure can have a significant impact on the photophysics.