6J1C

Photoswitchable fluorescent protein Gamillus, N150C/T204V double mutant, off-state

6J1C の概要

• エントリーDOI: 10.2210/pdb6j1c/pdb
• 分子名称: Green fluorescent protein, CHLORIDE ION, GLYCEROL, ... (4 entities in total)
• 機能のキーワード: acid-tolerant, photoswitchable, n150c/t204v double mutant, cis-chromophore, fluorescent protein
• 由来する生物種: Olindias
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 30595.76

構造登録者

Nakashima, R.,Shinoda, H.,Matsuda, T.,Nagai, T. (登録日: 2018-12-28, 公開日: 2019-11-06, 最終更新日: 2023-11-22)

主引用文献

Shinoda, H.,Lu, K.,Nakashima, R.,Wazawa, T.,Noguchi, K.,Matsuda, T.,Nagai, T.
Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions.
Cell Chem Biol, 26:1469-1479.e6, 2019

PubMed Abstract: Reversibly switchable fluorescent proteins (RSFPs) are crucial tags for super-resolution observation of protein localization and dynamics inside living cells. However, due to the high fluorescence pK (∼5-6) of most RSFPs, their usage in acidic conditions (pH 4.5-6.0) has been limited. Here, we investigated a new photochromic mechanism in Gamillus, a recently developed green fluorescent protein with acid tolerance. Gamillus exhibits negative switching with especially high contrast in acidic conditions, and its off switching is caused by trans-to-cis isomerization of the chromophore hydroxyphenyl ring that accompanies protonation. Through a combination of rational design and saturation mutagenesis, we developed two variants with enhanced switching contrasts and off-switching speeds, designated rsGamillus-S and rsGamillus-F, respectively. The fluorescence intensity, off-switching speed, and switching contrast of the rsGamillus variants are only slightly affected by changes in pH between 4.5 and 7.5. Exploiting these properties, we succeeded in high-contrast super-resolution imaging of cellular architectures in acidic conditions.

PubMed: 31422907
DOI: 10.1016/j.chembiol.2019.07.012

実験手法

X-RAY DIFFRACTION (2.09 Å)