Deactivation Pathways of an Isolated Green Fluorescent Protein Model Chromophore Studied by Electronic Action Spectroscopy

Abstract

The mechanism of fluorescence and fluorescence quenching of the green fluorescent protein (GFP) is not well-understood. To gain insight into the effect of the surrounding protein on the chromophore buried at its center, the intrinsic electronic absorption and deactivation pathways of a gaseous model chromophore, p-hydroxybenzylidene-2,3-dimethylimidazolone (HBDI) were investigated. No fluorescence from photoactivated gaseous HBDI(-) was detected in the range 480-1100 nm, in line with the ultrafast rate of internal conversion of HBDI(-) in solution. Two different gas-phase deactivation pathways were found: photofragmentation and electron photodetachment. Electronic action spectra for each deactivation pathway were constructed by monitoring the disappearance of HBDI(-) and appearance of product ions as a function of excitation wavelength. The action spectra measured for each pathway are distinct, with electron photodetachment being strongly favored at higher photon energies. The combined (total) gas-phase action spectrum has a band origin at 482.5 nm (23340 cm(-1)) and covers a broad spectral range, 390-510 nm. This extended gas-phase action spectrum exhibits vibronic activity that matches well with the results of previous cold condensed-phase experiments and high-level in vacuo computations, with features evident at +550, +1500, and +2800 cm(-1) with respect to the band origin.

Gas-Phase Fluorescence Excitation and Emission Spectroscopy of Three Xanthene Dyes (Rhodamine 575, Rhodamine 590 and Rhodamine 6G) in a Quadrupole Ion Trap Mass Spectrometer

“…Spectra of R575 were also recorded from acidified solutions containing ∼0.5% (v/v) HNO 3 . Excitation spectra were recorded by monitoring emission at 580 nm, and emission spectra were recorded with excitation at 480 nm.…”

“…Recently, our group [1][2][3][4] and several others [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] have presented works focusing upon the development, characterization and applications of instrumentation for measuring laser-induced fluorescence from molecular ions in the gas phase. The majority of these studies have used highly fluorescent molecules such as the xanthene-based rhodamine dyes.…”

“…The instrumentation developed features laser light sources coupled into trapping mass spectrometers that are used to mass-select the ion population of interest and to store the selected ion population for extended periods of time. Several types of mass spectrometers have been employed, including 3-D Paul-type quadrupole ion traps (QIT) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17], Fourier transform ion cyclotron resonance mass spectrometers (FT-ICR-MS) [18][19][20][21][22][23], and linear radiofrequency ion traps (LIT) [24]. Detection of integrated gas-phase fluorescence has been accomplished using high-sensitivity photomultiplier tubes [5-7, 9-11, 13-17, 21-24] or avalanche photodiodes [18,19,25], while dispersed fluorescence has been measured using spectrographs with charge-coupled device (CCD) detectors [1-4, 8, 12, 22].…”

Excited State Dynamics of the Isolated Green Fluorescent Protein Chromophore Anion Following UV Excitation

“…6,7 In GFP, the chromophore is essentially identical to the deprotonated anion of para-hydroxybenzilidene-2,3-dimethylimidazolinone (HBDI -, shown inset in Figure 1f) and has been widely employed as a model to investigate the intrinsic photophysics of the chromophore within the protein. [8][9][10][11][12][13][14][15][16][17][18] In the gas-phase, the S 1 state is wellcharacterised: the S 1 ← S 0 absorption (action) spectrum is similar to that of the protein and its origin is vertically bound relative to the ground state of the neutral (D 0 ). 8,16 The S 1 state decays primarily by internal conversion on a timescale of 1.4 ps; 14 vibrational autodetachment is also an open channel, although this occurs on a 30 ps timescale.…”

The Effect of Conjugation on the Competition between Internal Conversion and Electron Detachment: A Comparison between Green Fluorescent and Red Kaede Protein Chromophores

“…1,4,[7][8][9][10] The chromophore exists in deprotonated anionic (pHBDI -) or neutral forms and excitation of either of these leads to fluorescence from the deprotonated anionic chromophore at around 508 nm, with a quantum yield of Φ = 0.79 [11][12][13][14] (the neutral form deprotonates upon photoexcitation, yielding the anionic form). 11,15 An interesting feature of the isolated GFP chromophore is that it is non-fluorescent, in both solution and gasphases, 12,14,[16][17] as a result of efficient ultrafast non-radiative decay pathways being accessible in the absence of the protein. 4 For multi-labelling experiments, GFP variants with high visual contrast are desirable.…”