Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions
Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions
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DOI：
10.1016/j.chembiol.2019.07.012
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发表时间：
2019-10-17
影响因子：
8.6
通讯作者：
Nagai, Takeharu
中科院分区：
文献类型：
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作者：
Shinoda, Hajime;Lu, Kai;Nagai, Takeharu
可逆切换荧光蛋白（rsfp）是超分辨率观察活细胞内蛋白质定位和动态的关键标签。然而，由于大多数rrsfp的高荧光pK(a)（类似于5-6），它们在酸性条件下（pH 4.5-6.0）的使用受到限制。本文研究了一种新型的耐酸绿色荧光蛋白Gamillus的光致变色机制。Gamillus在酸性条件下表现为负开关，对比度特别高，其关闭开关是由伴质子化的发色团羟基苯基环的反式到顺式异构化引起的。通过合理设计和饱和诱变相结合，我们开发了两个具有增强开关对比度和关闭速度的变体，分别命名为rsGamillus-S和rsGamillus-F。rsGamillus突变体的荧光强度、关断速度和开关对比度受pH值在4.5 ~ 7.5之间变化的影响较小。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。
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 pK(a) (similar to 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.