A Single-Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome.
A Single-Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome.
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单位点突变调节橙色荧光蓝藻色素的荧光和发色。
DOI：
10.1002/cbic.202300358
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Zastrow,MelissaL
中科院分区：
文献类型：
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作者：
Janis,MakenaK;Zou,Wenping;Zastrow,MelissaL
蓝细菌色素（CBCR）cGMP特异性磷酸二酯酶、腺苷酸环化酶和FhlA（GAF）结构域结合胆色素辅因子以赋予对各种蓝细菌感光过程重要的感觉波长。许多分离的GAF结构域自催化结合胆色素，包括来自集胞藻属的CBCR Slr 1393的第三GAF结构域。PCC 6803，其结合藻红胆素（PEB）以产生明亮的橙子荧光蛋白。与绿色荧光蛋白相比，更小的尺寸和缺乏荧光所需的氧使Slr 1393 g3成为新的遗传编码荧光工具的有前途的平台。然而，与在大肠杆菌中表达的总Slr 1393 g3相比，Slr 1393 g3显示出约3%的低PEB结合效率（发色团化）。杆菌在这里，我们使用定点诱变和质粒重新设计方法来改善Slr 1393 g3-PEB结合，并证明其作为活细胞中的荧光标记物的实用性。在单个位点Trp 496处的突变将发射调谐超过约30 nm，可能是通过将PEB的自异构化转变为藻胆素（PUB）。 用于调节Slr 1393 g3和PEB合成酶的相对表达的质粒修饰也改善了发色团化，并且从双质粒系统移动到单质粒系统促进了通过位点饱和诱变和序列截短对一系列突变体的探索。总的来说，用组合的序列截短和W 496 H突变将PEB/PUB发色团化提高到总共23%。
Cyanobacteriochrome (CBCR) cGMP‐specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 fromSynechocystissp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3 % compared to total Slr1393g3 expressed inE. coli. Here we used site‐directed mutagenesis and plasmid redesign methods to improve Slr1393g3‐PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23 % with combined sequence truncation and W496H mutation.