Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions. Issue 10 (17th October 2019)

• Record Type: Journal Article
• Authors: Shinoda, Hajime; Lu, Kai; Nakashima, Ryosuke; Wazawa, Tetsuichi; Noguchi, Kosuke; Matsuda, Tomoki; Nagai, Takeharu
• Is Part Of: Cell chemical biology. Volume 26:Issue 10(2019)
• Page Start: 1469
• Page End: 1479.e6
• Publication Date: 2019-10-17
• Subjects: fluorescent proteins -- reversible photoswitching -- super-resolution fluorescence imaging -- PALM -- trans-cis isomerization -- acid tolerance -- pH; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2019.07.012
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: 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 p K a (∼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. Graphical Abstract: Highlights: rsGamillus-S and rsGamillus-F are acid-tolerant green RSFPs (p K a = 3.6, respectively) Off switching of rsGamilluses is driven by chromophore trans -to- cis isomerization rsGamilluses are applicable to super-resolution imaging in acidic conditions Abstract : Shinoda et al. developed reversibly photoswitchable variants of an acid-tolerant monomeric GFP, Gamillus, named "rsGamillus-S" and "rsGamillus-F". Both rsGamillus proteins exhibit superior acid tolerance (p K a = 3.6) compared with other reported reversibly photoswitchable GFPs. They demonstrated the applicability of rsGamillus proteins for super-resolution imaging in acidic conditions.