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CL6mN: Rationally Designed Optogenetic Photoswitches with Tunable Dissociation Dynamics.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-08-14 , DOI: 10.1021/acssynbio.0c00362
Abhirup Mukherjee 1 , Chaitanya Sudrik 2 , Yuge Hu 1 , Manish Arha 3 , Mark Stathos 4 , Jieung Baek 5 , David V Schaffer 5, 6, 7 , Ravi S Kane 1
Affiliation  

The field of optogenetics uses genetically encoded photoswitches to modulate biological phenomena with high spatiotemporal resolution. We report a set of rationally designed optogenetic photoswitches that use the photolyase homology region of A. thaliana cryptochrome 2 (Cry2PHR) as a building block and exhibit highly efficient and tunable clustering in a blue-light dependent manner. CL6mN (Cry2-mCherry-LRP6c with N mutated PPPAP motifs) proteins were designed by mutating and/or truncating five crucial PPP(S/T)P motifs near the C-terminus of the optogenetic Wnt activator Cry2-mCherry-LRP6c, thus eliminating its Wnt activity. Light-induced CL6mN clusters have significantly greater dissociation half-lives than clusters of wild-type Cry2PHR. Moreover, the dissociation half-lives can be tuned by varying the number of PPPAP motifs, with the half-life increasing as much as 6-fold for a variant with five motifs (CL6m5) relative to Cry2PHR. Finally, we demonstrate the compatibility of CL6mN with previously reported Cry2-based photoswitches by optogenetically activating RhoA in mammalian cells.

中文翻译:


CL6mN:合理设计的光遗传学光电开关,具有可调谐解离动力学。



光遗传学领域使用基因编码的光开关以高时空分辨率调节生物现象。我们报告了一组合理设计的光遗传学光开关,其使用拟南芥隐花色素2(Cry2PHR)的光裂合酶同源区域作为构建块,并以蓝光依赖性方式表现出高效和可调谐的聚类。 CL6mN(带有N突变 PPPAP 基序的 Cry2-mCherry-LRP6c)蛋白是通过突变和/或截短光遗传学 Wnt 激活剂 Cry2-mCherry-LRP6c C 末端附近的 5 个关键 PPP(S/T)P 基序而设计的,从而消除了它的 Wnt 活性。光诱导的 CL6mN 簇比野生型 Cry2PHR 簇具有显着更长的解离半衰期。此外,可以通过改变 PPPAP 基序的数量来调整解离半衰期,相对于 Cry2PHR,具有五个基序的变体 (CL6m5) 的半衰期增加了 6 倍。最后,我们通过光遗传学激活哺乳动物细胞中的 RhoA 证明了 CL6mN 与先前报道的基于 Cry2 的光开关的兼容性。
更新日期:2020-09-20
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