RRC ID 58007
Author Shinoda H, Lu K, Nakashima R, Wazawa T, Noguchi K, Matsuda T, Nagai T.
Title Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions.
Journal 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.
Volume 26(10)
Pages 1469-1479.e6
Published 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
Times Cited 1
Resource
Human and Animal Cells COS-7(RCB0539) HeLa