6j1c

Publication Abstract from PubMed

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 ( approximately 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.

Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions.,Shinoda H, Lu K, Nakashima R, Wazawa T, Noguchi K, Matsuda T, Nagai T Cell Chem Biol. 2019 Oct 17;26(10):1469-1479.e6. doi:, 10.1016/j.chembiol.2019.07.012. Epub 2019 Aug 15. PMID:31422907^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.