Probing the Decay Coordinate of the Green Fluorescent Protein:  Arrest of Cis−Trans Isomerization by the Protein Significantly Narrows the Fluorescence Spectra

Abstract

The fluorescence spectra of the wild-type green fluorescence protein (wt-GFP) and the anionic form of p-hydroxybenzylidenedimethylimidazolone (p-HBDI), which models the protein chromophore, were obtained in the 80-300 K temperature range in glycerol/water solvent. The protein spectra have pronounced and well-resolved vibronic structure, at least at lower temperatures. In contrast, the chromophore spectra are very broad and structureless even at the lowest temperatures. Analysis of the spectra shows that the experimentally observed red-shift of the protein spectrum upon heating is apparently caused by quadratic vibronic coupling of the torsional deformation (TD) of the phenyl single bond of the chromophore to the electronic transition. The broad spectra of the chromophore manifest the contribution of different conformations in the glycerol/water solvent. In particular, the lowest-temperature spectrum reflects the distribution over the same TD coordinate in the excited electronic state, which essentially contributes to the asymmetry of the spectrum. Upon heating, motion along this coordinate leads to a configuration from which the radiationless transition takes place. This narrows the distribution along the TD coordinate, causing a more symmetric fluorescence spectrum. We were able to reconstruct the broad, structureless fluorescence spectra of p-HBDI in glycerol/water solutions at various temperatures by convoluting the original wt-GFP spectra with the function describing the distribution of the transition energies of the p-HBDI chromophore. Thus, both the fluorescence broadening and increase in radiationless transition upon removal of the protein chromophore to bulk solvent are consistent with decay by a barrierless TD of the phenyl single bond.

A diabatic three-state representation of photoisomerization in the green fluorescent protein chromophore

“…Accordingly, it is normally considered that the protein preserves the emitting state by restricting the ability of the chromophore to decay via photoisomerization. This idea supported by analysis of the fluorescence structure of protein and chromophore at low temperatures, 11 and predicted emission energies of chromophore models that are relaxed on the excited state under constraint of planarity. 9,23 The physical mechanism by which the protein constrains the chromophore is still not understood.…”

“…4͒ and denatured proteins 10 do not fluoresce under normal conditions. 4,11 Spectral similarity suggests that the emitting state is localized to the chromophore, 4,11 so this question can be further broken down into three subquestions: What is the state of the chromophore in the protein emitting state?…”

Tuning the Excited-State Dynamics of GFP-Inspired Imidazolone Derivatives

“…[6][7][8] Although GFP was discovered more than 20 years ago, the photophysics of p-HBI and of its mutants is still intensively investigated mainly because of the spectacular decrease of its fluorescence quantum yield, Φ fl , and lifetime, τ, taking place when going from the protein (Φ fl ) 0.79, 9 τ ) 3.3 ns) 8,10 to liquid solutions (Φ fl < 10 -3 , 11 τ < 1 ps). [12][13][14] In this respect, the photophysical properties of the synthetic dimethyl derivative of the GFP chromophore (p-HBDI) 13,[15][16][17][18][19][20][21][22][23][24][25][26] and other related model systems 24,25,[27][28][29][30][31][32] have been extensively investigated.…”

Ultrafast electronic and vibrational dynamics of stabilized A state mutants of the green fluorescent protein (GFP): Snipping the proton wire

“…In general vibronic structure in the neutral GFP chromophore is only observed at low temperatures. (37,38) The broad asymmetric spectrum and large Stokes loss all point to a significant difference between absorbing and emitting state geometries for the neutral chromophore in blGFP. However, the spectral profile hardly evolves at all in the 2 -500 ps time window observed.…”