Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase.
Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase.
复制标题
DOI：
10.1038/nchembio.2063
复制
发表时间：
2016-06
影响因子：
14.8
通讯作者：
Tucker CL
中科院分区：
文献类型：
--
作者：
Taslimi A;Zoltowski B;Miranda JG;Pathak GP;Hughes RM;Tucker CL
拟南芥隐色素2 （AtCRY2）是一种光敏光感蛋白，以前被用于通过与伴侣蛋白CIB1的光依赖性结合来控制蛋白质之间的相互作用。虽然现有的CRY2/CIB二聚化体系已广泛应用于光遗传领域，但仍存在一些局限性。在这里，我们着手优化CRY2/CIB系统的功能，以确定更小的CRY2/CIB版本，显示较少的暗相互作用，并在响应光脉冲时保持更长或更短的信号状态。我们描述了维持光依赖相互作用的最小功能CRY2和CIB1结构域和影响AtCRY2光循环动力学的新信号突变。后一项工作暗示了植物CRYs中α13-α14转基序，其中扰动改变了信号状态寿命。利用长寿命的L348F光周期突变体，我们设计了第二代光激活Cre重组酶PA-Cre2.0，其动态范围提高了五倍，允许在单次短暂光脉冲暴露后进行强大的重组。
Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use controling protein-protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2/CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2/CIB system, to identify versions of CRY2/CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates a α13-α14 turn motif within plant CRYs where perturbations alter signaling state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range allowing robust recombination following exposure to a single, brief pulse of light.