Internal Conversion in the Chromophore of the Green Fluorescent Protein:  Temperature Dependence and Isoviscosity Analysis

Abstract

The temperature dependence of internal conversion in model compounds of the chromophore of the green fluorescent protein and one of its mutants has been measured. The strong temperature dependence persists in all charge forms of the model compounds, in all solvents and in a polymer matrix. The ultrafast internal conversion mechanism is thus an intrinsic property of the chromophore skeleton, rather than one of a specific charge or hydrogen-bonded form. An isoviscosity analysis shows that the coordinate which promotes internal conversion is essentially barrierless at room temperature. At reduced temperatures (or high viscosity) there is evidence for the formation of a small barrier. This may reflect a change in the nature of the microscopic solvent dynamics close to the glass transition temperature. In all cases the viscosity dependence of the rate constant for internal conversion is very weak, being approximately proportional to viscosity raised to the power of 0.25 ( 0.06. This suggests weak coupling between the relevant coordinate and macroscopic solvent viscosity. It is suggested that a potential candidate for the coordinate which promotes internal conversion is the volume-conserving "hula twist".

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.…”

A two-state model of twisted intramolecular charge-transfer in monomethine dyes

“…[20][21][22][23][24][25][26][27][28][29][30] Twisting of the methine bonds in the excited state is predicted to lower the potential energy of the excited state, and to lead to charge-localized (for neutral dyes, charge-separated) states from which internal conversion is possible due to a reduced adiabatic energy gap. 31,32 In a) seth.olsen@uq.edu.au.…”

“…Experiments on several common dyes suggest that solvent viscosity and polarity both affect the rate, although the influence of the former dominates the dynamics in solution 21,46 Other experiments suggest that balance of viscosity and polarity effects may depend on the chemical identity of the dye used. 20,25,47 There are not one, but two accessible twisting channels in the excited state for dyes that are near the cyanine (resonant) limit. The ground state of such dyes is characterized by an equivocal superposition of bonding states, while the excited state by a superposition of diradical character for both bonds of the bridge.…”

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.…”

“…13,16 On the basis of this and on quantum chemistry calculations, it has been proposed that p-HBDI relaxes nonradiatively to the ground state via a concerted, volume-preserving, twist motion around both bridging bounds, the so-called "hula-twist". 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.…”