SYNZIP Protein Interaction Toolbox: <i>in Vitro</i> and <i>in Vivo</i> Specifications of Heterospecific Coiled-Coil Interaction Domains

The synthetic biology toolkit contains a growing number of parts for regulating transcription and translation, but very few that can be used to control protein association. Here we report characterization of 22 previously published heterospecific synthetic coiled-coil peptides called SYNZIPs. We present biophysical analysis of the oligomerization states, helix orientations, and affinities of 27 SYNZIP pairs. SYNZIP pairs were also tested for interaction in two cell-based assays. In a yeast two-hybrid screen, >85% of 253 comparable interactions were consistent with prior in vitro measurements made using coiled-coil microarrays. In a yeast-signaling assay controlled by coiled-coil mediated scaffolding, 12 SYNZIP pairs were successfully used to down-regulate the expression of a reporter gene following treatment with α-factor. Characterization of these interaction modules dramatically increases the number of available protein interaction parts for synthetic biology and should facilitate a wide range of molecular engineering applications. Summary characteristics of 27 SYNZIP peptide pairs are reported in specification sheets available in the Supporting Information and at the SYNZIP Web site [http://keatingweb.mit.edu/SYNZIP/].合成生物学工具箱中已收录日益增多的用于调控转录与翻译的元件，但可用于控制蛋白质结合的元件却寥寥无几。本研究针对22种此前已发表的异特异性合成卷曲螺旋肽进行了表征，这类肽被命名为SYNZIP肽（SYNZIPs）。本研究针对27组SYNZIP肽对的寡聚化状态、螺旋取向及结合亲和力开展了生物物理分析。此外，本研究还通过两种细胞实验对SYNZIP肽对的相互作用进行了检测：在酵母双杂交筛选实验中，253组可比对相互作用中有超过85%与此前通过卷曲螺旋微阵列获得的体外检测结果一致；在由卷曲螺旋介导的支架组装所调控的酵母信号通路实验中，12组SYNZIP肽对在经α因子处理后，成功实现了对报告基因表达的下调。对这些相互作用模块的表征，极大扩充了合成生物学领域可获取的蛋白质相互作用元件库，并将助力诸多分子工程应用的开展。27组SYNZIP肽对的概要特性已在补充材料及SYNZIP官方网站[http://keatingweb.mit.edu/SYNZIP/]提供的规格说明书中予以公布。