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Protein‑protein interaction
1. BiFC (Bimolecular fluorescence complementation)
1.1 Introduction
Bimolecular Fluorescence Complementation (BiFC) is an imaging technique for visualizing protein‑protein interactions in living cells. The principle involves splitting a fluorescent protein (e.g., YFP, Venus, GFP) into N‑terminal and C‑terminal non‑fluorescent fragments, each fused to a candidate interacting protein (Bait and Prey). When Bait and Prey physically interact within the cell, the two fluorescent fragments are brought into close proximity and refold to reconstitute the intact fluorescent protein structure, generating a detectable fluorescent signal. This signal appears exclusively at the subcellular location where the interaction occurs, enabling simultaneous qualitative detection and spatial localization of the interaction. However, due to its irreversibility, BiFC is not suitable for studying dynamic interactions.
1.2 Products
1.3 Applications
| Applications | Description |
|---|---|
| Interactome Mapping | Y2H is the core technology for mapping proteome‑scale binary protein‑protein interaction (PPI) networks. Through array‑based or pooled library screening strategies, it enables systematic identification of direct PPIs across the entire genome, constructing cellular “wiring diagrams”. Integration with next‑generation sequencing (Y2H‑seq/Stitch‑seq) dramatically increases throughput and coverage. |
| Drug Target Screening and Validation | Y2H screens host proteins or endogenous regulators interacting with disease‑associated proteins (e.g., oncogenes, viral proteins) to discover potential drug targets. Reverse Y2H enables screening of small‑molecule inhibitors that disrupt specific PPIs, providing a high‑throughput platform for PPI‑targeted drug development. |
| Host‑Pathogen Interaction Studies | Y2H is one of the most widely used experimental methods for identifying interactions between pathogen proteins and host cellular proteins (accounting for >90% of information in public databases). By screening host cDNA libraries with pathogen proteins as bait, it reveals molecular mechanisms of pathogen invasion, replication, and immune evasion, providing novel targets for antiviral/antimicrobial drug development. |
| Protein Function and Domain Mapping | By constructing a series of deletion mutants or point mutations of bait proteins and performing pairwise Y2H assays with prey proteins, the key domains (e.g., SH3, leucine zipper) and critical amino acid residues mediating interactions can be precisely mapped. Combined with Deep Mutational Scanning (DMS), the effects of mutations on interactions can be systematically assessed at amino‑acid resolution. |
| Signal Transduction Pathway Analysis | Y2H is widely applied to study intracellular signaling pathways. By screening proteins interacting with key signaling factors (transcription factors, receptors, kinases), complex signal transduction processes are revealed. Combined with Yeast Three‑Hybrid (Y3H), ternary complexes or ligand‑dependent interactions (e.g., hormone‑receptor‑co‑receptor) can be studied to dissect hormone signaling and pathway crosstalk. |
1.4 References:
- Fields S, Song O. A novel genetic system to detect protein‑protein interactions. Nature. 1989 Jul 20;340(6230):245‑6. doi: 10.1038/340245a0. PMID: 2547163.
- Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, Lockshon D, Narayan V, Srinivasan M, Pochart P, Qureshi‑Emili A, Li Y, Godwin B, Conover D, Kalbfleisch T, Vijayadamodar G, Yang M, Johnston M, Fields S, Rothberg JM. A comprehensive analysis of protein‑protein interactions in Saccharomyces cerevisiae. Nature. 2000 Feb 10;403(6770):623‑7. doi: 10.1038/35001009. PMID: 10688190.
- Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, Sakaki Y. A comprehensive two‑hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci USA. 2001 Apr 10;98(8):4569‑74. doi: 10.1073/pnas.061034498. Epub 2001 Mar 13. PMID: 11283351; PMCID: PMC31875.
- Yu H, Braun P, Yildirim MA, Lemmens I, Venkatesan K, Sahalie J, Hirozane‑Kishikawa T, Gebreab F, Li N, Simonis N, Hao T, Rual JF, Dricot A, Vazquez A, Murray RR, Simon C, Tardivo L, Tam S, Svrzikapa N, Fan C, de Smet AS, Motyl A, Hudson ME, Park J, Xin X, Cusick ME, Moore T, Boone C, Snyder M, Roth FP, Barabási AL, Tavernier J, Hill DE, Vidal M. High‑quality binary protein interaction map of the yeast interactome network. Science. 2008 Oct 3;322(5898):104‑10. doi: 10.1126/science.1158684. Epub 2008 Aug 21. PMID: 18719252; PMCID: PMC2746753.