Engineering Improved Photoswitches for the Control of Nucleocytoplasmic Distribution,ACS Synthetic Biology

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Engineering Improved Photoswitches for the Control of Nucleocytoplasmic Distribution
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-11-15 00:00:00 , DOI: 10.1021/acssynbio.8b00368
Andrew M. Lerner ₁ , Hayretin Yumerefendi _{1,

2} , Odessa J. Goudy ₁ , Brian D. Strahl _{1,

3} , Brian Kuhlman _{1,

3}

Affiliation

Optogenetic techniques use light-responsive proteins to study dynamic processes in living cells and organisms. These techniques typically rely on repurposed naturally occurring light-sensitive proteins to control subcellular localization and activity. We previously engineered two optogenetic systems, the light activated nuclear shuttle (LANS) and the light-inducible nuclear exporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus. While LANS and LINX are effective tools, we posited that mutations within the LOV2 hinge-loop, which connects the core PAS domain and the C-terminal helix, would further improve the functionality of these switches. Here, we identify hinge-loop mutations that favorably shift the dynamic range (the ratio of the on- to off-target subcellular accumulation) of the LANS and LINX photoswitches. We demonstrate the utility of these new optogenetic tools to control gene transcription and epigenetic modifications, thereby expanding the optogenetic “tool kit” for the research community.

中文翻译：

工程改良的光电开关，用于控制核质分布

光遗传学技术使用光响应蛋白来研究活细胞和生物体中的动态过程。这些技术通常依赖于重新定向的天然存在的光敏蛋白来控制亚细胞定位和活性。我们之前通过将核导入或导出序列基序嵌入到Avena sativa光响应LOV2结构域的C末端螺旋中，设计了两个光遗传系统，即光激活核穿梭（LANS）和光诱导核输出（LINX）。肌钙蛋白1，因此能够实现光依赖的靶蛋白向细胞核的转运和向细胞核的转运。虽然LANS和LINX是有效的工具，但我们认为，连接核心PAS域和C末端螺旋结构的LOV2铰链环内的突变将进一步改善这些开关的功能。在这里，我们确定铰链环突变有利地转移了LANS和LINX光开关的动态范围（目标外亚细胞积累的比率）。我们证明了这些新的光遗传学工具可用于控制基因转录和表观遗传修饰，从而为研究界扩展了光遗传学“工具套件”。

更新日期：2018-11-15

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