Abstract
The reversibly switchable fluorescent proteins (RSFPs) commonly used for RESOLFT nanoscopy have been developed from fluorescent proteins of the GFP superfamily. These proteins are bright, but exhibit several drawbacks such as relatively large size, oxygen-dependence, sensitivity to low pH, and limited switching speed. Therefore, RSFPs from other origins with improved properties need to be explored. Here, we report the development of two RSFPs based on the LOV domain of the photoreceptor protein YtvA from Bacillus subtilis. LOV domains obtain their fluorescence by association with the abundant cellular cofactor flavin mononucleotide (FMN). Under illumination with blue and ultraviolet light, they undergo a photocycle, making these proteins inherently photoswitchable. Our first improved variant, rsLOV1, can be used for RESOLFT imaging, whereas rsLOV2 proved useful for STED nanoscopy of living cells with a resolution of down to 50 nm. In addition to their smaller size compared to GFP-related proteins (17 kDa instead of 27 kDa) and their usability at low pH, rsLOV1 and rsLOV2 exhibit faster switching kinetics, switching on and off 3 times faster than rsEGFP2, the fastest-switching RSFP reported to date. Therefore, LOV-domain-based RSFPs have potential for applications where the switching speed of GFP-based proteins is limiting.
Highlights
The resolution of conventional fluorescence microscopes is limited by diffraction to λ, with λ denoting the emission wavelength and NA the numerical aperture of the objective lens
All commonly used reversibly switchable fluorescent proteins (RSFPs) are derived from GFP-related proteins and share several features that limit their applicability as fluorescent reporter proteins
Colonies of E. coli cells expressing YtvA-LOV variants were grown at 37 °C and screened for increased off-switching amplitude after repeated RESOLFT-type on/off-switching with an automated microscope
Summary
The resolution of conventional fluorescence microscopes is limited by diffraction to λ , with λ denoting the emission wavelength and NA the numerical aperture of the objective lens. The light intensity required to switch molecules into a dark state can be decreased if the on- and the off-state are long-lived This is the basis for RESOLFT microscopy, which applies the switching of fluorescent proteins between two different conformations, one of which is non-fluorescent[1]. In this study[15], the wild-type LOV domain was used, which has a relatively weak fluorescence and switches back to the fluorescent state under UV light to less than 10% This hampers its use for RESOLFT applications, where multiple switching cycles have to be performed. We aimed at improving the switching and brightness of YtvA-LOV by site-directed and error-prone mutagenesis This resulted in the improved reversibly switchable protein rsLOV1 that exhibits an effective brightness ~10-fold higher compared to the wild-type protein under RESOLFT imaging conditions due to improved on-switching. We generated a second variant rsLOV2 with an even ~2-fold further increased fluorescence that was used for STED imaging
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