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The structural basis for the photochromism in the fluorescent protein Dronpa is poorly understood, because the crystal structures of the bright state of the protein did not provide an answer to the mechanism of the photochromism, and structural determination of the dark state has been elusive. We performed NMR analyses of Dronpa in solution at ambient temperatures to find structural flexibility of the protein in the dark state. Light-induced changes in interactions between the chromophore and β-barrel are responsible for switching between the two states. In the bright state, the apex of the chromophore tethers to the barrel by a hydrogen bond, and an imidazole ring protruding from the barrel stabilizes the plane of the chromophore. These interactions are disrupted by strong illumination with blue light, and the chromophore, together with a part of the β-barrel, becomes flexible, leading to a nonradiative decay process. [ABSTRACT FROM AUTHOR]

The fast and reversible on/off switching of the fluorescence emission of the GFP-like fluorescent protein Dronpa has attracted considerable interest for applications in subdiffraction imaging. In this paper we study the use of a donut-mode beam in combination with two more overlapping laser beams to increase the imaging resolution through selective switching to the nonfluorescent photoswitched state. We devise and run a series of numerical simulations to determine suitable photophysical parameters of prospective, thermally stable photoswitchable molecules, in terms of photoswitching quantum yields, fatigue resistance, and possible presence of transient nonfluorescent states. Many of our findings are applicable to other measurements that make use of donut beams, and these guidelines can be used in the synthesis and screening of novel photoswitchable compounds. We experimentally demonstrate the possibility of obtaining increased resolution by making use of the efficient and thermally stable Dronpa photoswitching, using equipment that is commonly available. [ABSTRACT FROM AUTHOR]