Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa

Abstract

Reversible photoswitching of individual molecules has been demonstrated for a number of mutants of the green fluorescent protein (GFP). To date, however, a limited number of switching events with slow response to light have been achieved at the single-molecule level. Here, we report reversible photoswitching characteristics observed in individual molecules of Dronpa, a mutant of a GFP-like fluorescent protein that was cloned from a coral Pectiniidae. Ensemble spectroscopy shows that intense irradiation at 488 nm changes Dronpa to a dim protonated form, but even weak irradiation at 405 nm restores it to the bright deprotonated form. Although Dronpa exists in an acid-base equilibrium, only the photoinduced protonated form shows the switching behavior. At the single-molecule level, 488- and 405-nm lights can be used to drive the molecule back and forth between the bright and dim states. Such reversible photoswitching could be repeated >100 times. The response speed to irradiation depends almost linearly on the irradiation power, with the response time being in the order of milliseconds. The perfect reversibility of the Dronpa photoswitching allows us to propose a detailed model, which quantitatively describes interconversion among the various states. The fast response of Dronpa to light holds great promise for following fast diffusion or transport of signaling molecules in live cells.

Fig. 1.

Photoswitching of Dronpa at the ensemble level. (a) Steady-state spectra of Dronpa at pH 7.4; absorption spectrum (blue line), fluorescence spectrum excited at 488 nm (green line) and 390 nm (red line), and excitation spectrum detected at 540 nm (black line). a.u., arbitrary units. (b and c) Fluorescence decays of Dronpa (pH 7.4) excited at 488 nm and detected at 520 nm (b) and excited at 390 nm and detected at 440 nm (c). (d) Time evolution of the absorption spectrum of Dronpa (pH 7.4) on irradiation with a 488-nm laser (P = 10 mW/cm²). (e and f) Time evolution of the peak absorbance of the deprotonated (e) and protonated (f) form on irradiation with a 488-nm laser. The solid lines show the fitting with a first-order kinetic model. (g) Time evolution of the absorption spectrum of the photoswitched Dronpa (488-nm irradiation) on irradiation with a 405-nm laser (P = 1 mW/cm²). (h and i) Time evolution of the peak absorbance of the deprotonated (h) and protonated (i) form on irradiation with a 405-nm laser. The solid lines show the fitting with a first-order kinetic model.

Fig. 2.

Single-molecule fluorescence measurements of Dronpa on 488-nm excitation. (a) Fluorescence intensity trajectory from a single Dronpa molecule in PVA (pH 7.4) on 488-nm excitation at a power of 220 nW. (b and c) Zoom of the bursts in the trajectory. Blue and red lines show the on-time and the medium-long off-time, respectively. (d–g) Frequency histogram of on-time (d), medium-long off-time (e), long off-time (f), and short off-time (g). Solid lines in d–f show a single-exponential fitting. Solid line in g shows a G fit.

Fig. 3.

Time series of single-molecule fluorescence images of Dronpa in PVA (pH 7.4). A and B denote two different molecules. The images were obtained with a 488-nm excitation at a power of 220 nW. The integration time was 1 ms/pixel with the pixel size of 78 × 78 nm. The same area was scanned with a 405-nm laser at a power of 220 nW (1 ms/pixel with the pixel size of 78 × 78 nm) before scanning images 5, 9, 13, and 17 with a 488-nm laser.

Fig. 4.

Photoswitching of Dronpa at the single-molecule level. (a) Fluorescence intensity trajectory from a single Dronpa molecule in PVA (pH 7.4) on two-color excitation at a power of 220 and 37 nW at 488 and 405 nm, respectively. Lower shows the on–off state of the 488-nm (cyan line) and 405-nm (blue line) light. (b and c) Zoom of the bursts in the trajectory. (d–f) Frequency histogram of on-time (d), medium-long off-time (e), and short off-time (f). Solid lines in d and f show a single-exponential fitting. Solid line in f shows a G fit. (g) Fluorescence intensity trajectory from a single Dronpa molecule in PVA (pH 7.4) on two-color excitation at a power of 320 and 37 nW at 488 and 405 nm, respectively. Lower shows the on–off state of the 488-nm (cyan line) and 405-nm (blue line) light.

Fig. 5.

Schematic diagram of the photoswitching of Dronpa. B, deprotonated form; D, an unknown dark state; A₁, the protonated form that originally exists in the sample; A₂, the protonated form that is formed by the photoswitching; and I, a nonfluorescent intermediate. The photoswitching pathway from B to A₂ is shown by blue arrows, and that from A₂ to B is shown by red arrows.