8ahb | pdb_00008ahb

rsEGFP2 photoswitched to its off-state at room temperature and back-switched to its on-state at 100K

spinqualitylabelsall models 8ahb, resolution 1.79Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 8ahb is a 1 chain structure with sequence from Aequorea victoria. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.79Å Ligands: PIA, SO4 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function GFP_AEQVI Energy-transfer acceptor. Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. Fluoresces in vivo upon receiving energy from the Ca(2+)-activated photoprotein aequorin. Publication Abstract from PubMed Single-molecule localization microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below the glass-transition temperature hampers efficient cryo-photoswitching. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent proteins at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at approximately 110 K. At this cryogenic temperature, on-off photoswitching involves the formation of two off-states in cis conformation with blue-shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these off-states can be switched back to the fluorescent on-state by 405 nm light, while both of them are sensitive to UV light at 355 nm. Superior recovery to the fluorescent on-state by 355 nm light was confirmed at the single-molecule level. This suggests, as also shown by simulations, that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 and possibly other FPs could improve the effective labeling efficiency achievable with this technique. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins. Photophysical Studies at Cryogenic Temperature Reveal a Novel Photoswitching Mechanism of rsEGFP2.,Mantovanelli AMR, Glushonkov O, Adam V, Wulffele J, Thedie D, Byrdin M, Gregor I, Nevskyi O, Enderlein J, Bourgeois D J Am Chem Soc. 2023 Jul 12;145(27):14636-14646. doi: 10.1021/jacs.3c01500. Epub , 2023 Jun 30. PMID:37389576 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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