Department of Chemical and Biological Engineering, University of Colorado Boulder, 3415 Colorado Avenue, Boulder, Colorado 80303, United States.
ACS Synth Biol. 2022 Jan 21;11(1):61-68. doi: 10.1021/acssynbio.1c00398. Epub 2021 Dec 13.
Photosensory domains are powerful tools for placing proteins under optical control, but their integration into light-sensitive chimeras is often challenging. Many designs require structural iterations, and direct comparisons of alternative approaches are rare. This study uses protein tyrosine phosphatase 1B (PTP1B), an influential regulatory enzyme, to compare three architectures for controlling PTPs with light: a protein fusion, an insertion chimera, and a split construct. All three designs permitted optical control of PTP1B activity (i.e., kinetic assays of purified enzyme) and in mammalian cells; photoresponses measured under both conditions, while different in magnitude, were linearly correlated. The fusion- and insertion-based architectures exhibited the highest dynamic range and maintained native localization patterns in mammalian cells. A single insertion architecture enabled optical control of both PTP1B and TCPTP, but not SHP2, where the analogous chimera was active but not photoswitchable. Findings suggest that PTPs are highly tolerant of domain insertions and support the use of screens to evaluate different optogenetic designs.
光感结构域是将蛋白质置于光控之下的有力工具，但将其整合到光敏感嵌合体中常常具有挑战性。许多设计需要结构迭代，而且替代方法的直接比较很少见。本研究使用蛋白酪氨酸磷酸酶 1B（PTP1B），一种有影响力的调节酶，比较了三种用光控制 PTP 的结构：蛋白融合、插入嵌合体和分裂构建体。所有三种设计都允许用光控制 PTP1B 的活性（即，对纯化酶进行动力学测定），并且在哺乳动物细胞中都可以进行；在两种条件下测量的光反应虽然幅度不同，但呈线性相关。基于融合和插入的结构表现出最高的动态范围，并在哺乳动物细胞中保持天然的定位模式。单一的插入结构能够对 PTP1B 和 TCPTP 进行光学控制，但对 SHP2 则不行，类似的嵌合体虽然有活性但不能光开关。研究结果表明，PTP 对结构域插入具有高度的耐受性，并支持使用筛选来评估不同的光遗传学设计。
