Uncovering the hidden ground state of green fluorescent protein

Abstract

The fluorescence properties of GFP are strongly influenced by the protonation states of its chromophore and nearby amino acid side chains. In the ground state, the GFP chromophore is neutral and absorbs in the near UV. Upon excitation, the chromophore is deprotonated, and the resulting anionic chromophore emits its green fluorescence. So far, only excited-state intermediates have been observed in the GFP photocycle. We have used ultrafast multipulse control spectroscopy to prepare and directly observe GFP's hidden anionic ground-state intermediates as an integral part of the photocycle. Combined with dispersed multichannel detection and advanced global analysis techniques, the existence of two distinct anionic ground-state intermediates, I(1) and I(2), has been unveiled. I(1) and I(2) absorb at 500 and 497 nm, respectively, and interconvert on a picosecond timescale. The I(2) intermediate has a lifetime of 400 ps, corresponding to a proton back-transfer process that regenerates the neutral ground state. Hydrogen/deuterium exchange of the protein leads to a significant increase of the I(1) and I(2) lifetimes, indicating that proton motion underlies their dynamics. We thus have assessed the complete chain of reaction intermediates and associated timescales that constitute the photocycle of GFP. Many elementary processes in biology rely on proton transfers that are limited by slow diffusional events, which seriously precludes their characterization. We have resolved the true reaction rate of a proton transfer in the molecular ground state of GFP, and our results may thus aid in the development of a generic understanding of proton transfer in biology.

Fig. 1.

Absorption and fluorescence spectra and structural features of GFP. (A) Absorption (blue line) and fluorescence (green line) spectrum of GFP. (B) Structural arrangement of chromophore-binding pocket in GFP in the neutral ground-state A. The coordinates were taken from Protein Data Bank entry 1GFL [Yang et al. (3)].

Fig. 2.

Time-resolved absorbance difference spectra recorded in GFP upon excitation at 400 nm at the time delays indicated.

Fig. 3.

Transient absorption traces recorded in GFP upon excitation at 400 nm in H₂O and D₂O, in the presence or absence of a dump pulse at 540 nm. (A–C) Transient absorption trace of GFP at 509 (A), 496 (B), and 492 (C) nm in a H₂O buffer in the absence (blue curve) and presence (red curve) of a dump pulse at 540 nm at 20-ps delay. The black lines denote the result of the global fitting procedure. The time axis is linear up to 30 ps, logarithmic thereafter in A and B, and linear throughout (C). (D–F) Transient absorption trace at 509 (D), 496 (E), and 492 (F) nm with excitation of GFP in a D₂O buffer in the absence (blue curve) and presence (red curve) of a dump pulse at 540 nm at 100-ps delay. The time axis is linear up to 120 ps, logarithmic thereafter in D and E, and linear throughout (F).

Fig. 4.

Time-resolved absorbance difference spectra recorded on GFP at the delays indicated on excitation at 400 nm in the absence (solid line) and presence (dashed line) of a dump pulse at 540 nm, fired 20 ps after initial excitation. The dotted lines denote a double-difference spectrum, in which a fraction of 0.57 of the undumped spectrum is subtracted from the dumped spectrum, and denote the pure difference spectra corresponding to the anionic ground-state I. The time-resolved spectra were taken at delays of 22 (A), 450 (B), and 900 (C) ps.

Fig. 5.

Photocycle scheme of GFP and SADS of the involved GFP transient states. (Upper) Kinetic scheme and potential energy level surfaces representing the photocycle of GFP, to fit the multipulse and traditional transient absorption data of GFP in H₂O and D₂O. The time constants by which the states evolve into one another as estimated from the target analysis have been indicated at the arrows, with the values in parentheses representing those obtained for D₂O. (Lower) SADS of A* (black), I* (green), I₁ (blue), and I₂ (red) species relative to the A state that result from a target analysis of the time-resolved spectra recorded for GFP in H₂O (solid lines) and D₂O (dotted lines).