Taking the green fluorescence out of the protein: dynamics of the isolated GFP chromophore anion

Abstract

The green fluorescent protein (GFP) is employed extensively as a marker in biology and the life sciences as a result of its spectacular fluorescence properties. Here, we employ femtosecond time-resolved photoelectron spectroscopy to investigate the ultrafast excited state dynamics of the isolated GFP chromophore anion. Excited state population is found to decay bi-exponentially, with characteristic lifetimes of 330 fs and 1.4 ps. Distinct photoelectron spectra can be assigned to each of these timescales and point to the presence of a transient intermediate along the decay coordinate. Guided by ab initio calculations, we assign these observations to twisting about the CC -C bridge followed by internal conversion to the anion ground state. The dynamics in vacuo are very similar to those observed in solution, despite the difference in absorption spectra between the two media. This is consistent with the protein environment restricting rotation about the CC -C bond in order to prevent ultrafast internal conversion and preserve the fluorescence.

Time-resolved photoelectron imaging of the isolated deprotonated nucleotides

“…This can be interpreted as a flow of population from the positive region into the negative, which may be observed due to either a change in electronic state, or relaxation within a single electronically excited state. 49 It is important to note that global fitting cannot precisely capture large amplitude motions in the time resolved photoelectron spectrum; if motion of population occurs which does not behave as one state decaying into another, then the fitting algorithm is only able to approximate this motion. 50 In instances where the motion is relatively fast compared to the instrument response function, however, the global fitting approximation is valid.…”

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.…”

Mapping the Ultrafast Dynamics of Adenine onto Its Nucleotide and Oligonucleotides by Time-Resolved Photoelectron Imaging

“…23 Our tentative conclusion that the dynamics do not directly involve the 1 nπ* state is consistent with those reached for solvated deoxyadenosine, 31 and with certain high-level calculations on solvated Ade: 18,19 the biexponential dynamics observed are a consequence of motion away from the Franck-Condon region towards conical intersections followed by internal conversion. We note that such biexponential decay has been observed in time-resolved photoelectron spectroscopy for dynamics that are occurring strictly on a single surface, 30 indicating that such data are not a prerequisite for the decay through multiple excited states. Finally, in our discussion above and in Fig.…”

“…Quantitative insight can be gained by employing a global fitting procedure, 30 whereby the time-resolved photoelectron spectra, S(eKE, t), are fit simultaneously in energy and time by the following equation:…”