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Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions.
Cell Chemical Biology ( IF 6.6 ) Pub Date : 2019-08-15 , DOI: 10.1016/j.chembiol.2019.07.012
Hajime Shinoda 1 , Kai Lu 2 , Ryosuke Nakashima 2 , Tetsuichi Wazawa 2 , Kosuke Noguchi 1 , Tomoki Matsuda 3 , Takeharu Nagai 3
Affiliation  

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.

中文翻译:

耐酸可逆转换的绿色荧光蛋白,可在酸性条件下进行超分辨率成像。

可逆转换的荧光蛋白(RSFP)是超分辨率观察活细胞内蛋白质定位和动力学的关键标签。但是,由于大多数RSFP的高荧光pKa(〜5-6),在酸性条件下(pH 4.5-6.0)的使用受到限制。在这里,我们研究了芽孢杆菌的一种新的光致变色机理,芽孢杆菌是一种最近开发的具有耐酸性的绿色荧光蛋白。芽孢杆菌显示出在酸性条件下具有特别高的对比度的负切换,并且其关闭切换是由发色团羟基苯环随质子化的反式-顺式异构化引起的。通过合理的设计和饱和诱变的结合,我们开发了两种具有增强的开关对比度和关断速度的变体,分别命名为rsGamillus-S和rsGamillus-F。rsGamillus变体的荧光强度,关断速度和开关对比度仅受pH值在4.5至7.5之间的轻微影响。利用这些特性,我们成功地在酸性条件下对细胞结构进行了高对比度超分辨率成像。
更新日期:2019-11-09
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