Lerner Andrew M, Yumerefendi Hayretin, Goudy Odessa J, Strahl Brian D, Kuhlman Brian
Department of Biochemistry & Biophysics , University of North Carolina at Chapel Hill , Chapel Hill , North Carolina 27599 , United States.
Oncology Research Unit , Pfizer Worldwide Research and Development , Pearl River , New York 10965 , United States.
ACS Synth Biol. 2018 Dec 21;7(12):2898-2907. doi: 10.1021/acssynbio.8b00368. Epub 2018 Nov 29.
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.
光遗传学技术利用光响应蛋白来研究活细胞和生物体中的动态过程。这些技术通常依靠重新利用天然存在的光敏感蛋白来控制亚细胞定位和活性。我们之前通过将核输入或输出序列基序嵌入燕麦向光素1的光响应LOV2结构域的C末端螺旋中，构建了两种光遗传学系统，即光激活核穿梭系统（LANS）和光诱导核输出系统（LINX），从而实现了靶蛋白在光依赖下进出细胞核的运输。虽然LANS和LINX是有效的工具，但我们推测连接核心PAS结构域和C末端螺旋的LOV2铰链环内的突变会进一步改善这些开关的功能。在这里，我们鉴定出能有利地改变LANS和LINX光开关的动态范围（靶标与非靶标亚细胞积累的比率）的铰链环突变。我们展示了这些新型光遗传学工具在控制基因转录和表观遗传修饰方面的效用，从而为研究界扩展了光遗传学“工具包”。
