Dynamic Stokes shift in green fluorescent protein variants

Abstract

Solvent reorganization around the excited state of a chromophore leads to an emission shift to longer wavelengths during the excited-state lifetime. This solvation response is absent in wild-type green fluorescent protein, and this has been attributed to rigidity in the chromophore's environment necessary to exclude nonradiative transitions to the ground state. The fluorescent protein mPlum was developed via directed evolution by selection for red emission, and we use time-resolved fluorescence to study the dynamic Stokes shift through its evolutionary history. The far-red emission of mPlum is attributed to a picosecond solvation response that is observed at all temperatures above the glass transition. This time-dependent shift in emission is not observed in its evolutionary ancestors, suggesting that selective pressure has produced a chromophore environment that allows solvent reorganization. The evolutionary pathway and structures of related fluorescent proteins suggest the role of a single residue in close proximity to the chromophore as the primary cause of the solvation response.

Fig. 1.

Chromophore characterization. (A) Absorption spectra of isolated immature mPlum with corresponding chromophore chemical structure. (B) Crystal structure of immature mPlum chromophore including neighboring residues E16 and E215 (S. J. Remington and X. Shu, personal communication). The arrow points to the sp³ hybridized carbon of immature mPlum. The dotted line indicates the hydrogen bond between the glutamic acid and carbonyl of residue 65. (C) Same as A for mature mPlum. (D) Same as B for mature mPlum. The arrow points to the sp² hybridized carbon of mature mPlum.

Fig. 2.

Steady-state fluorescence spectra. Normalized steady-state excitation (dotted line) and fluorescence spectra (solid line) of mRFP at pH 7 (red), mRaspberry at pH 7 (green), and mPlum at pH 7 (black) and pH 11 (blue). The excitation wavelength for the emission spectrum and time-resolved experiments of mPlum is 580 nm (540 nm for mRFP and mRaspberry) and is indicated by an arrow. All excitation spectra were obtained at a wavelength of 650 nm.

Fig. 3.

Time-resolved measurements. (A) Normalized time-resolved fluorescence decays for mPlum, pH 7, at 627 nm (blue) and 692 nm (red) as obtained by upconversion spectroscopy with excitation at 580 nm. Excitation occurs at 1 ps on the time axis. (B) Reconstructed time-resolved fluorescence spectra obtained at 1, 20, 50, 100, and 1,000 ps as obtained by upconversion spectroscopy and TCSPC. The solid lines are log normal line-shape fits to the data. (C) Fluorescence emission maximum as a function of time for mPlum at pH 7 and pH 11 as obtained from upconversion spectroscopy and TCSPC as described in the text. (Inset) Detail for the first 0.2 ns. (D) Fluorescence emission maximum as a function of time for mPlum, mRaspberry, and mRFP in pH 7 buffer as obtained from TCSPC. The excitation wavelength for these measurements was 545 nm.

Fig. 4.

Temperature dependents. (A) Normalized steady-state fluorescence for mPlum at pH 7 from 298 K (red) to 140 K (blue). The excitation wavelength was 466 nm in all cases. (B) Fluorescence emission maximum as a function of time for mPlum at pH 7 for temperatures of 298, 270, 230, and 140 K as obtained by TCSPC.