Viscosity-Dependent Fluorescence Decay of the GFP Chromophore in Solution Due to Fast Internal Conversion

Abstract

Time-resolved fluorescence measurements at 275 K show that the excited-state lifetime of a model chromophore of the green fluorescent protein (GFP) substantially increases from subpicoseconds in low-viscosity solvents such as ethanol (η = 1.7 cP) to 30 ps in glycerol (η = 9.9 × 103 cP) and reaches 2.1 ns in glycerol glass at 150 K. At high temperatures the similarity of excited-state decay and ground-state recovery kinetics indicates internal conversion being responsible for the short fluorescence lifetimes. Their viscosity dependence reflects on a motion with a considerable amplitude that is damped by viscous drag and outweighs thermal activation as is concluded from measurements at different temperatures. In solution the neutral and the anionic forms of the model chromophore are similarly nonfluorescent in contrast to wild-type GFP and mutants where the deprotonated form is hardly undergoing internal conversion. Thus, the protein selectively restricts motional degrees of freedom of the chromophore in specific protonation states.

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

“…15,16 On the other hand, substantial viscosity dependence was reported in more viscous solvents, such as polyalcohols. 29 Additionally to being ultrafast, the excited-state dynamics of p-HBDI was also found to be biexponential, with time constants that are the same throughout the emission spectrum but with wavelength-dependent relative amplitudes. [14][15][16] This effect has been attributed to the existence of various conformations of the chromophore in the ground state with a similar absorption spectrum.…”

“…[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.…”

“…24,25,[27][28][29][30][31][32] For example, Solntsev et al 32 have compared the ultrafast excitedstate dynamics of p-HBDI and m-HBDI in aqueous solutions and have found that the deactivation by large amplitude motion was slower with the m-HBDI, due possibly to a higher barrier for Z/E isomerization.…”

Improving autofluorescent proteins: Comparative studies of the effective brightness of Green Fluorescent Protein (GFP) mutants

“…While shifts in the spectra are readily observed, the less accessible but equally important fluorescence quantum yields are also influenced by the interaction between chromophore and protein scaffold. Actually, fluorescence quantum yields F Fl of the isolated chromophore are low in highly viscous media like polyvinylalcohol, but can be as high as 80% in wild-type GFP (Kummer et al, 2002;Litvinenko et al, 2003). This impressively demonstrates that the relaxation pathways of the chromophore's excited electronic state and, thus, the apparent brightness of GFP mutants depend on the local protein environment of the chromophore.…”

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

“…These studies yield a more detailed view on the factors determining the photophysics of GFP, in particular the mechanism which enables the chromophore to emit strongly in the protein even though it is non fluorescent in the denatured form and in aqueous solution. (17) The mechanism of the radiationless decay of the chromophore in solution has been considered in detail elsewhere, both experimentally (18)(19)(20)(21)(22)(23)(24) and in theoretical treatments. (25)(26)(27)(28)(29)(30) The results described below will provide new data against which to test theoretical calculations of excited state properties in the protein, examples of which are beginning to appear.…”