Green fluorescent proteins are light-induced electron donors

Abstract

Proteins of the green fluorescent protein (GFP) family are well known owing to their unique biochemistry and extensive use as in vivo markers. We discovered that GFPs of diverse origins can act as light-induced electron donors in photochemical reactions with various electron acceptors, including biologically relevant ones. Moreover, via green-to-red GFP photoconversion, this process can be observed in living cells without additional treatment.

Figure 1

Oxidant-mediated green-to-red photoconversion of EGFP in vitro. (a) Confocal images of benzoquinone-mediated photoconversion of EGFP immobilized on a metal-affinity bead in green (left) and red (center) channels, and their overlay (right). A region on the upper side of the bead was preirradiated with a high-intensity 488-nm laser (1.5 W/cm²). Scale bar, 20 µm. (b) Benzoquinone concentration dependences on green fluorescence decrease (green open squares) and red fluorescence increase (red closed squares) during oxidative redding of the immobilized EGFP. After one activating scan with 488-nm laser, remaining green fluorescence (normalized according to initial value) and originating red fluorescence (normalized according to maximal value) were measured and shown in the graph. Each data point is an average of three independent experiments. Error bars, s.d. (c) Proposed scheme of the oxidative redding. In the first step, an excited green chromophore (Chr_g) donates one electron to an oxidant molecule (A). As a result, a short-lived intermediate (Chr^•+) is formed. If it reacts with an electron acceptor during its lifetime, the final red fluorescent form (Chr_r) is formed; otherwise the intermediate comes into a permanently bleached state (Chr_bl).

Figure 2

Green-to-red photoconversion of GFPs in live cells. (a) Fluorescence microscopy of Phoenix Eco cells transiently expressing EGFP-N1 in green (upper row) and red (center row) channels. Bottom row represents an overlay of the green and red images. Numbers above images designate duration of blue light irradiation (1W/cm²) that induces green-to-red photoconversion. Note high heterogeneity of cells after 30 s of irradiation that results in different colors on the overlaid image. Scale bar, 20 µm. (b) Quantification of fluorescence changes in two selected individual cells—cell 1 (filled squares) and cell 2 (open triangles)—marked in a. (c, d) Redding efficiency normalized according to initial green fluorescence level for designated cell lines transiently expressing EGFP in cytoplasm (c) or mitochondria (d). Maximal (red columns), average (yellow columns) and minimal (green columns) responses are shown for each cell line. (e, f) Green-to-red photoconversion in a live button polyp, Zoanthus sp. (e) The Zoanthus specimen used in this work. (f) Confocal optical section through a tip of the native tentacle in green (left images) and red (center images) channels and their overlay (right images) before (upper row) and after (bottom row) photoconversion. Here, green fluorescence is characteristic of the ectoderm cells, whereas endoderm cells contain symbiotic algae Zooxantella, which resembles red spheres due to chlorophyll fluorescence. Local photoconversion was induced in the region marked by a white square using irradiation with 100% 488 nm laser line (0.15 W/cm²). Scale bar, 50 µm.