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.photochromism ͉ protonation͞deprotonation ͉ fluorescence microscopy P hotoinduced alteration of chemical and physical properties of photochromic molecules is of great interest because of its potential applications for optoelectronic devices, such as optical memory and optical switches (1). Photoinduced switching of fluorescent properties is one of the most attractive concepts for the realization of a nondestructive read-out system (2-4). Apart from this application, the photoswitching behavior of green fluorescent proteins (GFPs) or GFP-like proteins is being recognized as new methodology of optical marking (5, 6). Intracellular dynamics of selected molecules can be followed by activating the fluorescent proteins to their fluorescent state (7-11). Realization of photoswitching at the single-molecule level will open up exciting opportunities in the field of optoelectronics and biological imaging, where it could provide molecular-scale devices as well as detection of fast dynamics of individual proteins in living cells. Reversible photoswitching at the single-molecule level, however, has not yet been well characterized (12-17). Dickson et al. (12) reported reversible photoswitching of a mutant of GFP. Although they demonstrated a few photoswitching events at the single-molecule level, minutes of illumination was required to achieve the switching. Irie and coworkers (13, 14) also reported reversible photoswitching of diarylethene derivatives, which occurred relatively slowly with a response time of seconds. Although the switching can be repeated Ͼ10 4 times at the ensemble level (1), the number of switching events obtained at the single-m...

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa

“…2). The 9-ps component is assigned to the decay of the singlet excited state of the neutral trans chromophore, in agreement with previous fluorescence measurements 9 . Habuchi et al 9 reported a 14-ps time constant for fluorescence measurements of the off state of Dronpa in 1 H 2 O at pH 7.4, with excitation at 390 nm and detection at 440 nm.…”

“…Spectroscopic contrast is primarily developed via protonation/ deprotonation of the phenolic oxygen such as that in GFP. This has been proposed to develop via excited-state proton transfer (ESPT) [9][10][11] (Supplementary Note 1).…”

“…A central question concerns the proposal that photoswitching of the trans off state to the bright cis on state of Dronpa involves ESPT [9][10][11] (see Supplementary Note 1 ). Photoswitching of Dronpa has been observed at the single molecule level 9 , as well as in ensembles with picosecond time resolution 10 .…”

“…Photoswitching of Dronpa has been observed at the single molecule level 9 , as well as in ensembles with picosecond time resolution 10 . Transient absorption measurements in 1 H 2 O revealed a 4-ps component that was assigned to interconversion from the trans-neutral off ('A2') excited state to the anionic I excited state 10 .…”

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

“…Alexa 647, Dronpa, Dreinklang and rsEGFP) 3,[18][19][20] and (ii) irreversibly photoactivatable and photoconvertible (e.g. PA-GFP, PA-mCherry, mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2 and mMaple) [21][22][23][24][25][26][27] .…”