Protein is not an isolated biological molecule, but has a specific three-dimensional structure and interacts with other molecules (proteins, metabolites, lipids and nucleic acids) to perform functions together, such as mediating signal transduction, regulating metabolism, and energy conversion, etc. Therefore, the modularity and spatial organization of proteins are as important as their expression levels.
Proteins form a complex network by interacting with other proteins, and form a highly organized and dynamic cell system. The research of PPI is helpful to our research on diseases and the development of new drugs. In recent years, in order to analyze PPI, many research methods in vivo, in vitro and in silico have been developed, paving the way for a more comprehensive and systematic study of PPI in the future.
Fig 1. The protein-protein complex structure between Xiap-BIR3 (surface) and Caspase (ribbon) (Rosell, M.; Fernández-Recio, J. 2018)
Creative Proteomics combines lots of classic methods with novel technologies to provide customers with comprehensive PPI dynamic monitoring and data analysis.
|Method||Principle and Description||Advantages|
|Co-immunoprecipitation (Co-IP)||Immunoprecipitation of a target protein using a specific antibody to co-enrich interacting partner proteins.||Suitable for stable or transient interactions.|
|Tandem Affinity Purification (TAP)||Target protein is tagged with two different affinity tags for multi-step affinity purification of the protein and its partners.||Enhances specificity and stringency of purification.|
|Bimolecular Fluorescence Complementation (BiFC)||Fluorescent protein is split into non-fluorescent fragments, each fused with different interacting proteins. Interaction results in fluorescence observable under microscopy.||Allows visualization of protein interaction localization.|
|Förster Resonance Energy Transfer (FRET)||Measures the distance between two attached fluorescent molecules on interacting proteins. Energy transfer occurs within a specific range.||Provides information about spatial proximity and dynamics.|
|Proximity-dependent Biotin Identification (BioID)||Fusion of target protein with a promiscuous biotin ligase leads to biotinylation of nearby proteins. Biotinylated proteins are enriched and identified through affinity purification and mass spectrometry.||Reveals dynamic or weak interactions within the cell.|
|Protein Fragment Complementation Assay (PCA)||Involves splitting a reporter protein into two fragments, each fused with interacting proteins. Interaction leads to reconstitution of reporter protein activity.||Quantitatively analyzes the strength of protein interactions.|
|Cross-Linking Mass Spectrometry (XL-MS)||Proteins are cross-linked in living cells, then identified through mass spectrometry to determine interaction partners.||Reveals protein interactions in a native cellular environment.|
|Affinity Purification - Mass Spectrometry (AP-MS)||Co-purifies target protein and its interacting partners for subsequent identification using mass spectrometry.||Reveals protein interactions under physiological conditions.|
|Yeast Two-Hybrid (Y2H) System||Interacting proteins are fused to different domains of a transcription factor. Interaction reconstitutes the transcription factor, leading to reporter gene expression.||Detects protein interactions in vivo.|
|Fluorescence Resonance Energy Transfer (FRET) Microscopy||Combines FRET with microscopy to visualize protein interactions in live cells.||Enables real-time visualization of interactions at the cellular level.|
|Isothermal Titration Calorimetry (ITC)||Measures heat released or absorbed during protein binding, yielding quantitative thermodynamic data.||Provides direct measurement of binding constants and thermodynamics.|
|Proximity-Labeling Assays||Use engineered enzymes to biotinylate or label interacting proteins, enabling enrichment and identification.||Allows analysis of transient or weak interactions in live cells.|
|Fluorescence Recovery After Photobleaching (FRAP)||Monitors the mobility of fluorescently tagged proteins to assess binding and interaction dynamics.||Provides insights into protein interaction kinetics and mobility.|
|Microscale Thermophoresis (MST)||Measures changes in fluorescence signal due to temperature-induced protein movement, indicating interactions.||Requires minimal sample and provides quantitative binding data.|
Explore the physical interactions between proteins in a controlled laboratory environment. In vitro methods allow direct observation and manipulation of protein interactions, providing essential insights into their biochemical properties and functional roles.
|Method||Principle and Description||Advantages|
|Co-immunoprecipitation (Co-IP)||Antibodies pull down one protein and its partners from a mixture for detection.||- Detects endogenous interactions
- Provides information about interacting partners
- Relatively simple
|Pull-Down Assays||Immobilized protein captures binding partners from a mixture.||- Mimics physiological conditions
- Detects weak interactions
|Surface Plasmon Resonance (SPR)||Real-time binding kinetics measured as proteins interact on a sensor chip.||- Label-free detection - Real-time kinetics
- Quantitative affinity determination
|Isothermal Titration Calorimetry (ITC)||Measures heat changes during binding for affinity determination.||- Direct measurement of binding energetics
|Fluorescence Resonance Energy Transfer (FRET)||Interaction detected by energy transfer between labeled proteins.||- Provides spatial and temporal information
- Real-time monitoring
- No need for extensive purification
|Biolayer Interferometry (BLI)||Interference patterns monitored as proteins bind to a biosensor tip.||- Label-free detection
- Real-time kinetics
- Minimal sample requirements
|Yeast Two-Hybrid (Y2H)||Reconstituted transcription factor activity used to detect interactions in yeast cells.||- Suitable for large-scale studies
- Detects protein-protein interactions in a cellular context
|Split-GFP||Green Fluorescent Protein fragments reconstituted upon interaction.||- Direct visualization of interactions
- Simple and visual assay
|Microscale Thermophoresis (MST)||Changes in protein migration due to binding in a temperature gradient used for affinity determination.||- Label-free detection
- Can be used for small molecules and protein-DNA interactions
|Covalent Crosslinking||Chemical crosslinking captures interaction partners for analysis.||- Stabilizes transient interactions
- Preserves weak or labile interactions
|Protein Arrays||Immobilized proteins on a surface detect interactions with target proteins.||- High-throughput screening
- Simultaneous detection of multiple interactions
Harness the power of computational techniques to predict, model, and understand protein interactions in a virtual environment. In silico analysis complements experimental approaches by offering a diverse range of methods to explore complex interaction networks and unravel their underlying mechanisms.
|Molecular Docking||Predicting the 3D structure of protein complexes by exploring possible orientations and conformations of interacting proteins.|
|Molecular Dynamics Simulations||Simulating dynamic behavior of protein complexes to study flexibility, stability, and conformational changes over time.|
|Sequence-Based Prediction Methods||Predicting interactions based on sequence information, including similarity, co-evolution, and domain-domain interactions.|
|Machine Learning and Data Mining||Analyzing large datasets with machine learning algorithms to predict and classify protein-protein interactions based on various features.|
|Evolutionary Analysis||Using comparative genomics and phylogenetic analysis to reveal conserved interacting protein pairs across species.|
|Binding Site Prediction||Predicting protein-protein binding sites using structural or sequence information.|
|Free Energy Calculations||Estimating binding affinity to predict interaction strength between proteins.|
|Hydrogen Bond Analysis||Identifying and analyzing hydrogen bonds in protein-protein interfaces.|
|Energy Minimization||Optimizing protein complex structures for stability.|
|Normal Mode Analysis||Studying conformational changes using vibrational modes.|
Biomedical Research: Uncover crucial protein interactions underlying diseases, aiding in drug target identification and therapeutic development.
Drug Discovery: Validate potential drug candidates by studying their interactions with target proteins, enhancing the efficiency of drug development.
Molecular Pathways: Map intricate signaling pathways, revealing how proteins collaborate to regulate essential cellular processes.
Functional Analysis: Understand protein functions within complex networks, shedding light on biological mechanisms.
Structural Biology: Gain insights into protein complex structures and dynamics, guiding structural biology studies.
Systems Biology: Integrate interaction data to construct comprehensive models of cellular systems and their behavior.
Biotechnology: Optimize enzyme-protein interactions for industrial applications, from biofuel production to bioremediation.
Bioinformatics: Analyze interaction networks computationally, extracting valuable knowledge from complex data.