Advanced Proximity Labeling Techniques

Proximity labeling techniques represent a cutting-edge approach wherein a decoy protein of interest is fused with an enzyme capable of indiscriminately labeling neighboring proteins. These labeled proteins are referred to as "prey" proteins.

The field of protein labeling has witnessed significant advancements, prominently featuring proximity-tagging enzymes. Among these, Biotin-dependent biotin identification (BioID) utilizing biotin ligase mutants, primarily the biotin ligase (BirA), has been instrumental. BirA labels lysine residues of proximate proteins with biotin tags when biotin is present. Another remarkable method is Biotin labeling, which relies on engineered ascorbate peroxidase (APEX), enabling rapid biotinylation in the presence of hydrogen peroxide, facilitating the study of protein interaction networks in both spatial and temporal dimensions. PUP-IT, a proximity labeling technique reliant on the PUP ligase PafA, and AirID, a novel proximity marker enzyme derived from targeted BirA mutations from metagenomic data, have further enriched this toolkit. Additionally, methods such as RaPID for studying RNA-protein interactions and CRUIS protein proximity tagging, which combines CRISPR and PUP-IT, have expanded the possibilities in this domain.

These advanced techniques offer several advantages over traditional methods. They demand less stringent pull-down conditions, making them more adaptable. Unlike traditional methods, they do not necessitate protein complexes to remain bound during analysis and can withstand more rigorous lysis conditions. This enables protein labeling in cells under normal physiological conditions, reducing the likelihood of mismatching interacting proteins during lysis and affinity purification steps, ultimately minimizing false positive results. These techniques have found successful application across diverse organisms, including mammalian cells, plants, parasites, mucor, mice, yeast, zebrafish, Drosophila, and worms.

BioID

BioID, a leading proximity labeling technique, functions by labeling proteins in close spatial proximity to the target protein with biotin as evidence of protein interactions. This technique involves the construction of a recombinant expression vector containing the target protein and BirA, followed by transfection into cells for expression amplification. Subsequently, biotin is introduced into the cell medium. BirA, with its biotin-activating capabilities, labels all relevant proteins within a 10 nm radius around the target protein with biotin. Enrichment of biotinylated proteins is achieved using streptavidin-coated magnetic beads, followed by mass spectrometry-based identification.

Split-BioID

Split-BioID represents a protein fragment complementation analysis method based on the BirA* enzyme, mutated at position 118 of BirA, enabling spatial and temporal analysis of protein complexes. Split-BioID involves dividing BirA* into two fragments, which are then fused to two interacting proteins. When these two interacting proteins come into contact, BirA* reassembles, restoring its biotinylation activity. Experimental selection of an appropriate splitting site is essential, with one common approach fusing the split BirA* to the binding sites of the FKBP12-rapamycin complex (FRB) and the FK506 binding protein (FKBP). Addition of rapamycin facilitates the interaction between FRB and FKBP, rejoining the two BirA* fragments and confirming the restoration of biotinylation activity.

Contact-ID

Contact-ID, a variant of split-BioID, further refines proximity labeling. It involves dividing BirA* into two fragments, N-G78 (B1) and G79-C (B2), which are fused to specific cellular locations, such as the ER membrane (ERM) and the outer mitochondrial membrane (OMM). Biotinylation activity is contingent upon the formation of mitochondria-associated membranes (MAMs). Biotinylation occurs only when these fragments physically interact and reorganize at membrane contact sites. This method selectively biotinylates proteins localized to the MAM, with subsequent mass spectrometry analysis elucidating the MAM proteome within living cells.

BioID2

BioID2, a smaller biotin ligase R4OG mutant derived from A. aeolicus, lacks the DNA-binding structural domain present in traditional biotin ligases. Its reduced size enhances targeting and localization. BioID2 requires lower biotin concentrations and shorter biotinylation durations compared to BioID, making it suitable for systems with limited biotin replenishment. BioID2's optimal reaction temperature is around 50°C, making it suitable for thermophilic bacteria. For larger target proteins or protein complexes, flexible linker chains can extend BioID2's labeling range.

TurboID & miniTurbo

TurboID and miniTurbo, generated through R118S mutation of BirA, offer higher catalytic efficiency compared to wild-type BirA. They function as proximity markers within minutes of biotin addition. TurboID, 35 kD in size with 15 mutations compared to wild-type BirA, and miniTurbo, 28 kD in size with 13 mutations, differ in biotinylation activity and background labeling, making miniTurbo preferable in experiments requiring precise labeling control. Split-TurboID, akin to split-BioID, divides TurboID into two fragments, ligates them to different subcellular compartments, and achieves biotin- and rapamycin-dependent expression systems for proximity labeling.

AirID

AirID, developed in 2020, harnesses BirA* from a custom library. It exhibits lower proximal biotinylation activity than TurboID but operates efficiently with lower biotin concentrations, even in biotin-free systems. AirID biotinylates adjacent lysine residues without specific sequence preference, enabling accurate detection of drug-mediated protein-protein interaction inhibition.