8AHA

rsEGFP2 photoswitched to its off-state at 100K

8AHA の概要

• エントリーDOI: 10.2210/pdb8aha/pdb
• 分子名称: Green fluorescent protein, SULFATE ION (3 entities in total)
• 機能のキーワード: rsfp, switchable, ptfp, rsegfp2, fluorescent protein
• 由来する生物種: Aequorea victoria
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 29300.71

構造登録者

Mantovanelli, A.,Adam, V. (登録日: 2022-07-21, 公開日: 2023-07-19, 最終更新日: 2024-02-07)

主引用文献

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

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: 37389576
DOI: 10.1021/jacs.3c01500

実験手法

X-RAY DIFFRACTION (2.38 Å)