| RRC ID |
58007
|
| 著者 |
Shinoda H, Lu K, Nakashima R, Wazawa T, Noguchi K, Matsuda T, Nagai T.
|
| タイトル |
Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions.
|
| ジャーナル |
Cell Chem Biol
|
| 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 pKa (∼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.
|
| 巻・号 |
26(10)
|
| ページ |
1469-1479.e6
|
| 公開日 |
2019-10-17
|
| DOI |
10.1016/j.chembiol.2019.07.012
|
| PII |
S2451-9456(19)30243-0
|
| PMID |
31422907
|
| MeSH |
Crystallography, X-Ray
Green Fluorescent Proteins / chemistry*
HeLa Cells
Humans
Hydrogen-Ion Concentration
Models, Molecular
Photochemical Processes
Tumor Cells, Cultured
|
| IF |
6.762
|
| 引用数 |
1
|
| リソース情報 |
| ヒト・動物細胞 |
COS-7(RCB0539)
HeLa |
| 遺伝子材料 |
rsGamillus-S/pcDNA3 (RDB19867)
rsGamillus-F/pcDNA3 (RDB19868) |
