8AHB
rsEGFP2 photoswitched to its off-state at room temperature and back-switched to its on-state at 100K
Summary for 8AHB
| Entry DOI | 10.2210/pdb8ahb/pdb |
| Descriptor | Green fluorescent protein, SULFATE ION (3 entities in total) |
| Functional Keywords | rsfp, switchable, ptfp, rsegfp2, fluorescent protein |
| Biological source | Aequorea victoria |
| Total number of polymer chains | 1 |
| Total formula weight | 28916.46 |
| Authors | Mantovanelli, A.,Adam, V. (deposition date: 2022-07-21, release date: 2023-07-19, Last modification date: 2024-02-07) |
| Primary citation | Mantovanelli, A.M.R.,Glushonkov, O.,Adam, V.,Wulffele, J.,Thedie, D.,Byrdin, M.,Gregor, I.,Nevskyi, O.,Enderlein, J.,Bourgeois, D. Photophysical Studies at Cryogenic Temperature Reveal a Novel Photoswitching Mechanism of rsEGFP2. J.Am.Chem.Soc., 145:14636-14646, 2023 Cited by PubMed Abstract: 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 isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at ∼110 K. At this cryogenic temperature, on-off photoswitching involves the formation of two off-states in conformation with blue-shifted absorption relative to that of the 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. PubMed: 37389576DOI: 10.1021/jacs.3c01500 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.79 Å) |
Structure validation
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