Abstract
Proximity-dependent biotinylation techniques have been gaining wide applications in the systematic analysis of protein-protein interactions (PPIs) on a proteome-wide scale in living cells. The engineered biotin ligase TurboID is among the most widely adopted given its enhanced biotinylation efficiency, but it faces the background biotinylation complication that might confound proteomic data interpretation. To address this issue, we report herein a set of split TurboID variants that can be reversibly assembled by using light (designated “OptoID”), which enable optogenetic control of biotinylation based proximity labeling in living cells. OptoID could be further coupled with an engineered monomeric streptavidin that permits real-time monitoring of biotinylation with high temporal precision. These optical actuators and sensors will likely find broad applications in precise proximity proteomics and rapid detection of biotinylation in living cells.
Highlights
Proximity labeling (PL) has been gaining more and more applications to identify protein-protein interactions (PPIs) on a proteome-wide scale under more physiologically relevant conditions within living cells (Qin et al, 2021)
To monitor the biotin labeling process catalyzed by TurboID in living cells, we utilized an engineered monomeric streptavidin tagged with enhanced green fluorescent protein (EGFP). Monomeric Streptavidin (mSA) has been shown to tightly bind biotin with a binding affinity of 2.8 nM in vitro (Lim et al, 2013)
We described a photo-switchable version of split-TurboID, designated OptoID, that can be conditionally assembled by using light
Summary
Proximity labeling (PL) has been gaining more and more applications to identify protein-protein interactions (PPIs) on a proteome-wide scale under more physiologically relevant conditions within living cells (Qin et al, 2021). Promiscuous enzymes, such as proximity-dependent biotin identification (BioID) (Choi-Rhee et al, 2004; Roux et al, 2012) and ascorbic acid peroxidase (APEX) (Rhee et al, 2013; Lam et al, 2015), have been engineered to label endogenous proteins within a few nanometers. The prototypical version of BioID has a low activity
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