Tateyama Samu, Kobayashi Itsuki, Hisatomi Osamu
Department of Earth and Space Science, Graduate School of Science , Osaka University , Toyonaka , Osaka 560-0043 , Japan.
Biochemistry. 2018 Nov 27;57(47):6615-6623. doi: 10.1021/acs.biochem.8b00995. Epub 2018 Nov 13.
Photozipper (PZ) is a light-activatable basic leucine zipper (bZIP) protein composed of a bZIP domain and a light-oxygen-voltage-sensing domain of aureochrome-1. Blue light induces dimerization and subsequently increases the affinity of PZ for the target DNA sequence. We prepared site-directed PZ mutants in which Asn131 (N131) in the basic region was substituted with Ala and Gln. N131 mutants showed spectroscopic and dimerization properties almost identical to those of wild-type PZ and an increase in helical content in the presence of the target sequence. Quantitative analyses by an electrophoretic mobility shift assay and quartz crystal microbalance (QCM) measurements demonstrated that the half-maximal effective concentrations of N131 mutants to bind to the target sequence were significantly higher than those of PZ. QCM data also revealed that N131 substitutions accelerated the dissociation without affecting the association, suggesting that a base-specific interaction of N131 occurred after the association between PZ and DNA. Activation of PZ by illumination decreased both the standard errors and the unstable period of QCM data. Optical control of transcription factors will provide new knowledge of the recognition of the target sequence.
光拉链（PZ）是一种光可激活的碱性亮氨酸拉链（bZIP）蛋白，由一个bZIP结构域和金藻色素-1的光-氧-电压传感结构域组成。蓝光诱导二聚化，随后增加PZ对靶DNA序列的亲和力。我们制备了定点PZ突变体，其中碱性区域的Asn131（N131）被Ala和Gln取代。N131突变体表现出与野生型PZ几乎相同的光谱和二聚化特性，并且在存在靶序列的情况下螺旋含量增加。通过电泳迁移率变动分析和石英晶体微天平（QCM）测量进行的定量分析表明，N131突变体与靶序列结合的半数有效浓度显著高于PZ。QCM数据还显示，N131取代加速了解离而不影响结合，这表明N131的碱基特异性相互作用发生在PZ与DNA结合之后。光照激活PZ降低了QCM数据的标准误差和不稳定期。转录因子的光学控制将为靶序列识别提供新知识。
