Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm

Abstract

Phototransformable fluorescent proteins are central to several nanoscopy approaches. As yet however, there is no available variant allowing super-resolution imaging in cell compartments that maintain oxidative conditions. Here, we report the rational design of two reversibly switchable fluorescent proteins able to fold and photoswitch in the bacterial periplasm, rsFolder and rsFolder2. rsFolder was designed by hybridisation of Superfolder-GFP with rsEGFP2, and inherited the fast folding properties of the former together with the rapid switching of the latter, but at the cost of a reduced switching contrast. Structural characterisation of the switching mechanisms of rsFolder and rsEGFP2 revealed different scenarios for chromophore cis-trans isomerisation and allowed designing rsFolder2, a variant of rsFolder that exhibits improved switching contrast and is amenable to RESOLFT nanoscopy. The rsFolders can be efficiently expressed in the E. coli periplasm, opening the door to the nanoscale investigation of proteins localised in hitherto non-observable cellular compartments.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy

“…Whereas both hydrozoan and anthozoan RSFPs have the same overall protein fold and chromophore (4-(p-hydroxybenzylidene)−5imidazolinone (p-HBI)), structural features in their chromophore pockets differ. For example, the p-hydroxyphenyl ring of the onstate chromophore is stabilized in rsEGFP2 by three hydrogen bonds 18 and in Dronpa by π-stacking with the side chain of a histidine residue and by two hydrogen bonds 7 . Therefore, it is not clear yet if the photoswitching mechanisms in hydrozoan and anthozoan RSFPs differ in details or not.…”

“…This fast RSFP is extensively used for tagging proteins in mammalian cells for live-cell RESOLFT nanoscopy 16 . Its p-HBI chromophore, formed autocatalytically from the three residues Ala-Tyr-Gly, is carried by an α-helix entrapped within an 11stranded β-barrel 18 (Fig. 1a).…”

NMR reveals light-induced changes in the dynamics of a photoswitchable fluorescent protein

“…So far, the chromophore connecting helical elements, H2 and H3, have found only little attention in the field of GFP engineering. Concerning the helical region H3, we know that L70 is an important residue for the photo-switching properties of rsEGFP2 (24) and rsFolder (26), while S72 (S73 in rsFolder) has been suggested as one of the 'hopping' points of protons when entering from the solvent onto their way to the chromophore (39). In our NMR data, the H3 region of rsFolder shows increased conformational dynamics in the "off" state, as evidenced by line broadening (C71, F72, S73) and faster H/D exchange (L70, C71).…”

“…Such reversibly photoswitchable fluorescent proteins (RSFP) are required for a variety of applications, ranging from super-resolution microscopy (RESOLFT, NL-SIM, pcSOFI, and others) (19)(20)(21), optical lockin detection (OLID) (22), multiplexed imaging (OPIOM) (23), up to the development of highdensity optical storage media (2). rsFolder has been genetically engineered as a chimera between rsEGFP2 (24) and superfolder GFP (25) to facilitate proper folding in highly reducing environments such as the bacterial periplasm (26). The photophysical properties of rsFolder have been characterized by optical spectroscopy, and high resolution crystallographic structures are available for both the "on" and "off" states (26).…”

“…rsFolder has been genetically engineered as a chimera between rsEGFP2 (24) and superfolder GFP (25) to facilitate proper folding in highly reducing environments such as the bacterial periplasm (26). The photophysical properties of rsFolder have been characterized by optical spectroscopy, and high resolution crystallographic structures are available for both the "on" and "off" states (26). These structures reveal the typical 11-stranded β-barrel fold, enclosing an endogenous p-HBI chromophore, formed by maturation of an Ala-Tyr-Gly tripeptide.…”