Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.
Keywords: Src; allosteric; biochemistry; cell biology; chemical biology; enzyme; none; optogenetics; protein engineering; protein kinase.
Cells need to sense and respond to their environment. To do this, they have dedicated proteins that interpret outside signals and convert them into appropriate responses that are only active at a specific time and location within the cell. However, in many diseases, including cancer, these signaling proteins are switched on for too long or are active in the wrong place. To better understand why this is the case, researchers manipulate proteins to identify the processes they regulate. One way to do this is to engineer proteins so that they can be controlled by light, turning them either on or off. Ideally, a light-controlled tool can activate proteins at defined times, control proteins in specific locations within the cell and regulate any protein of interest. However, current methods do not combine all of these requirements in one tool, and scientists often have to use different methods, depending on the topic they are researching. Now, Shaaya et al. set out to develop a single tool that combines all required features. The researchers engineered a light-sensitive ‘switch’ that allowed them to activate a specific protein by illuminating it with blue light and to deactivate it by turning the light off. Unlike other methods, the new tool uses a light-sensitive switch that works like a clamp. In the dark, the clamp is open, which ‘stretches’ and distorts the protein, rendering it inactive. In light, however, the clamp closes and the structure of the protein and its activity are restored. Moreover, it can activate proteins multiple times, control proteins in specific locations within the cell and it can be applied to a variety of proteins. This specific design makes it possible to combine multiple features in one tool that will both simplify and broaden its use to investigate specific proteins and signaling pathways in a broad range of diseases.
© 2020, Shaaya et al.