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Control of TurboID-dependent biotinylation intensity in proximity ligation screens

ABSTRACT: Proximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase TurboID has broadened the range of potential applications, as this ligase promotes an intense and faster biotinylation, even in subcellular compartments like the endoplasmic reticulum. On the other hand, the uncontrollable high basal biotinylation rates deny the system´s inducibility and are often associated with cellular toxicity precluding its use in proteomics. We report here an improved method for TurboID-dependent biotinylation reactions based on the tight control of free biotin levels. Blockage of free biotin with a commercial biotin scavenger reversed the high basal biotinylation and toxicity of TurboID, as shown by pulse-chase experiments. Accordingly, the biotin-blockage protocol restored the biological activity of a bait protein fused to TurboID in the endoplasmic reticulum and rendered the biotinylation reaction inducible by exogenous biotin. Importantly, the biotin-blockage protocol was more effective than biotin removal with immobilised avidin and did not affect the cellular viability of human monocytes over several days. The method presented should be useful to researchers interested in exploiting the full potential of biotinylation screens with TurboID and other high-activity ligases for challenging proteomics questions.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Thomas Krüger

LAB HEAD: Axel A. Brakhage

PROVIDER: PXD040302 | Pride | 2023-03-29

REPOSITORIES: Pride

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			Action	DRS
	B2-a.raw	Raw
	B2-b.raw	Raw
	B2-c.raw	Raw
	B5-a.raw	Raw
	B5-b.raw	Raw

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MicroID: A Novel Biotin Ligase Enables Rapid Proximity Ligation Proteomics

Project description:Identifying protein-protein interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity ligation method that uses a promiscuous biotin ligase to detect protein-protein interactions in cells in a highly reproducible manner. Recent advancements in proximity ligation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2 to create a smaller, highly active, biotin ligase that we named MicroID. Truncation of the c-terminal of BioID2 and mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller, highly active construct with lower baseline labeling. Several additional point mutations improved the function of our modified MicroID construct compared to BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID is the smallest biotin ligase (180 AA for microID vs. 257 AA for miniTurbo and 338 AA for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurboID. MicroID also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we developed a MicroID mutant that has lower labeling efficiency, but significantly reduced biotin scavenging compared to BioID2. Finally, we demonstrate utility of MicroID in mass spectrometry experiments by localizing MicroID constructs to subcellular organelles and measuring protein-protein interactions.

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Project description:Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-beads digestion in numerous biological applications. However, the popular on-beads digestion method faces major challenges of streptavidin contamination, the lost information of biotinylation sites, and limited sequence coverage of enriched proteins. Here, we explored thiol-cleavable biotin as an alternative approach to elute biotinylated proteins from streptavidin-coated beads for both chemical biotinylation and biotin ligase-based proximity labeling. All possible amino acid sites for biotinylation were thoroughly evaluated besides the primary lysine residue. We found that biotinylation at lysine residues notably reduces the trypsin digestion efficiency which can be mitigated by the thiol-cleavable biotinylation method. We then evaluated the applicability of thiol-cleavable biotin as a substrate for proximity labeling in living cells, where TurboID biotin ligase was engineered onto the mitochondrial inner membrane facing the mitochondrial matrix. As a proof-of-principle study, thiol-cleavable biotin-assisted TurboID proteomics achieved remarkable intra-organelle spatial resolution with significantly enriched proteins localized in the mitochondrial inner membrane and mitochondrial matrix.

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