Singlet oxygen photosensitization by EGFP and its chromophore HBDI

Abstract

The photosensitization of reactive oxygen species and, in particular, singlet oxygen by proteins from the green fluorescent protein (GFP) family influences important processes such as photobleaching and genetically targeted chromophore-assisted light inactivation. In this article, we report an investigation of singlet oxygen photoproduction by GFPs using time-resolved detection of the NIR phosphorescence of singlet oxygen at 1275 nm. We have detected singlet oxygen generated by enhanced (E)GFP, and measured a lifetime of 4 micros in deuterated solution. By comparison with the model compound of the EGFP fluorophore 4-hydroxybenzylidene-1,2-dimethylimidazoline (HBDI), our results confirm that the beta-can of EGFP provides shielding of the fluorophore and reduces the production of this reactive oxygen species. In addition, our results yield new information about the triplet state of these proteins. The quantum yield for singlet oxygen photosensitization by the model chromophore HBDI is 0.004.

FIGURE 1

Normalized absorption spectra of the studied compounds: (A) anionic form of HBDI in NaOH 1M (continuous line) and neutral form in phosphate-citrate buffer pH 5.0 (dotted line); (B) EGFP in PBS buffer pH 7.4.

FIGURE 2

¹O₂ signal photosensitized by HBDI in the anionic form. A solution of HBDI in NaOH 1M was irradiated with 20,000,000 laser pulses at 355 nm and the concomitant phosphorescence of ¹O₂ was detected at 1275 nm (100 ns per channel). The signal was fitted by Eq. 1 with lifetimes of 2 μs and 17 μs for the rise and decay, respectively. (Inset) ¹O₂ phosphorescence spectra obtained from the intensity of the signal at different wavelengths.

FIGURE 3

Normalized emission signals of EGFP in D-PBS irradiated at 532 nm and detected at (A) 1275 nm, 40,000,000 laser pulses, 20 ns per channel, fitted parameters: τ₁ = 4.4 μs, τ₂ = 26 μs. (B) 1100 nm, 40,000,000 laser pulses, 20 ns per channel, fitted parameter: τ = 20 μs. (C) Signal resulting from panels A and B, fitted parameters: τ_Δ = 4.5 μs, τ_T = 28 μs. (D) 1275 nm, [NaN₃] = 7 mM, 40,000,000 laser pulses, 20 ns per channel, fitted parameters: τ₁ = 0.2 μs, τ₂ = 23 μs. (E) 1275 nm, oxygen-saturated D-PBS, 30,000,000 laser pulses, 200 ns per channel, fitted parameter: τ = 3–6 μs.