A General Mechanism of Green-to-Red Photoconversions of GFP

Abstract

Here we dissect the phenomena of oxidative and reductive green-to-red photoconversion of the Green Fluorescent Protein. We characterize distinct orange- and red-emitting forms (λ_abs/λ_em = 490/565 nm; λ_abs/λ_em = 535/600 nm) arising during the Enhanced Green Fluorescent Protein (EGFP) photoconversion under low-oxygen conditions in the presence of reductants. These forms spectroscopically differ from that observed previously in oxidative redding (λ_abs/λ_em = 575/607 nm). We also report on a new green-emitting state (λ_abs/λ_em = 405/525 nm), which is formed upon photoconversion under the low-oxygen conditions. Based on the spectral properties of these forms, their light-independent time evolution, and the high-level computational studies, we provide a structural basis for various photoproducts. Under the low-oxygen conditions, the neutral quinoid-like structure formed via a two-electron oxidation process is found to be a key intermediate and a most likely candidate for the novel green-emitting state of the chromophore. The observed large Stokes shift is traced to the formation of the zwitterionic form of the chromophore in the excited state. Subsequently, this form undergoes two types of cyclization reactions, resulting in the formation of either the orange-emitting state (λ_abs/λ_em = 490/565 nm) or the red-emitting form (λ_abs/λ_em = 535/600 nm). The T65G mutant lacks one of the proposed cyclization pathways and, indeed, the photoconverted T65G EGFP exhibits a single orange-emitting state. In oxidative redding, the red-emitting state resembles the structure of the chromophore from asFP595 (λ_abs/λ_em = 572/595 nm), which is directly formed upon two-electron oxidation and deprotonation bypassing the formation of the quinoid-like structure. Our results disclose a general "oxidative" mechanism of various green-to-red photoconversions of EGFP, providing a link between oxidative redding and the photoconversion under low-oxygen conditions.

Keywords: EGFP; oxidative redding; photoconversion; photoinduced electron transfer; reductive redding.

FIGURE 1

Orange and red-emitting photoproducts of low-oxygen photoconversion of EGFP. The 2 μM solution of EGFP in the presence of sodium dithionite (10 mM) was illuminated for 5 min with blue light (470 nm, 600 mW/cm²). (A) Fluorescence emission spectra (excitation at 520 nm, solid lines): green – before the photoconversion, from purple to yellow – 0, 5, 10, and 15 min after the photoconversion. The blue dashed line – absorbance after the photoconversion. The purple-to-orange dashed lines – excitation spectra (emission at 620 nm), color-coded similarly to the emission spectra. (B) changes in excitation (dashed lines, orange – for emission at 540 nm, red – for emission at 610 nm) and emission (solid black line, excitation at 540 nm) spectra with time; (C) emission spectra (excitation at 520 nm) of irradiated EGFP samples (5 min, 470 nm, 600 mW/cm²). In the absence of reductants (no FAD, green line) no red-emitting forms are visible even in low-oxygen conditions induced by Ar bubbling. Addition of FAD (magenta line, 10 mM EDTA, 5 μM FAD) or sodium dithionite (orange and red lines) without Ar treatment results in similar spectra.

FIGURE 2

Green-emitting forms and the time course of light-independent interconversion of spectral species. (A) Excitation (green dashed line, emission at 530 nm) and emission (green solid line, excitation at 400 nm) spectra of green-emitting form 5 min after photoconversion. Blue dashed line – absorbance after the photoconversion. (B) Time course of light-independent fluorescence change of the forms with emission at 525 nm (excited at 400 nm), 565 nm, and 600 nm (excited at 525 nm) after the photoconversion, pH = 7.4. The lines represent the mean value, error bars – standard deviation (n = 3 independent photoconversions).

FIGURE 3

Stability of the EGFP photoproducts. (A) Representative pH titration curve of the photoproducts in the presence of sodium dithionite; green line – emission at 525 nm, orange line – emission at 565 nm, red line – emission at 600 nm. (B) Representative time course of the light-independent stage of the photoconversion at different pH, showing better stability of the red-emitting form at high pH. (C) SDS-PAGE analysis of the light-irradiated and untreated EGFP samples in the presence of potassium ferricyanide or sodium dithionite.

FIGURE 4

Fluorescence decay kinetics of the EGFP photoproducts. (A) Photoconversion in the presence of potassium ferricyanide; (B,C) photoconversion in the presence of sodium dithionite. Repetition rate of pulsed light sources: 50 MHz (A) and 10 MHz (B,C).

FIGURE 5

Photoconversion of EGFP-T65G in the presence of sodium dithionite. Orange form: excitation (dashed line, emission at 600 nm) and emission (solid line, excitation at 500 nm) spectra immediately after photoconversion). Green form: excitation (dashed line, emission at 600 nm) and emission (solid line, excitation at 400 nm) spectra immediately after photoconversion). The conditions for photoconversion are similar to the ones used for EGFP.

FIGURE 6

Ground-state equilibrium structures of the red-emitting forms. (A) The non-hydrolyzed DsRed-like chromophore. (B) The hydrolyzed asFP595-like chromophore. Only the QM parts are shown for clarity. All distances are shown in Å.

FIGURE 7

Ground-state equilibrium structure of the quinoid-like form. Only the QM part is shown for clarity. All distances are shown in Å.

FIGURE 8

Ground-state equilibrium structures of the cyclic oxidized chromophores. (A) The neutral orange-emitting chromophore. (B) The red-emitting mOrange-like chromophore. Only the QM parts are shown for clarity. All distances are shown in Å.

FIGURE 9

Proposed mechanism of the green-to-red GFP photoconversions. The molecular species correspond to (1) – the native GFP chromophore, (2) – the radical state, (3,4) – the red-emitting forms in oxidative redding, (5) – the green-emitting quinoid-like form detected upon photoconversion under the low-oxygen conditions, (6,7) – the orange and red-emitting photoproducts observed under the low-oxygen conditions.