Institute of Biochemistry, Graz University of Technology, Petersgasse 12/II, 8010 Graz, Austria.
BioTechMed-Graz, Mozartgasse 12/II, 8010 Graz, Austria.
Sci Adv. 2023 Aug 4;9(31):eadh4721. doi: 10.1126/sciadv.adh4721. Epub 2023 Aug 2.
Regulatory proteins play a crucial role in adaptation to environmental cues. Especially for lifestyle transitions, such as cell proliferation or apoptosis, switch-like characteristics are desirable. While nature frequently uses regulatory circuits to amplify or dampen signals, stand-alone protein switches are interesting for applications like biosensors, diagnostic tools, or optogenetics. However, such stand-alone systems frequently feature limited dynamic and operational ranges and suffer from slow response times. Here, we characterize a LOV-activated diguanylate cyclase (LadC) that offers precise temporal and spatial control of enzymatic activity with an exceptionally high dynamic range over four orders of magnitude. To establish this pronounced activation, the enzyme exhibits a two-stage activation process in which its activity is inhibited in the dark by caging its effector domains and stimulated upon illumination by the formation of an extended coiled-coil. These switch-like characteristics of the LadC system can be used to develop new optogenetic tools with tight regulation.
调控蛋白在适应环境信号方面起着至关重要的作用。特别是对于生活方式的转变，如细胞增殖或凋亡，开关样的特性是理想的。虽然自然界经常使用调控回路来放大或抑制信号，但独立的蛋白质开关在生物传感器、诊断工具或光遗传学等应用中很有趣。然而，这种独立的系统通常具有有限的动态和操作范围，并受到缓慢的响应时间的影响。在这里，我们描述了一种 LOV 激活的二鸟苷酸环化酶（LadC），它可以提供精确的时空控制酶活性，具有超过四个数量级的极高动态范围。为了实现这种显著的激活，该酶表现出一个两阶段的激活过程，其活性在黑暗中被其效应结构域的笼状结构抑制，并在光照下通过形成扩展的卷曲螺旋而被刺激。LadC 系统的这些开关样特性可用于开发具有紧密调控的新型光遗传学工具。
