8.1 Methods for studying protein-protein interactions

Protein-protein interactions are crucial for cellular functions. Scientists use various methods to study these interactions, from in vitro techniques like yeast two-hybrid to in vivo approaches like FRET. Each method has unique strengths and limitations in detecting different types of interactions.

Comparing these techniques is essential for choosing the right approach. Factors like sensitivity, specificity, and throughput vary widely. Some methods excel at detecting stable complexes, while others are better suited for capturing transient or weak interactions in living cells.

Experimental Methods for Studying Protein-Protein Interactions

Methods for protein-protein interaction studies

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• In vitro methods
  • Yeast two-hybrid system detects protein interactions through transcriptional activation of reporter genes (GAL4 system)
  • Co-immunoprecipitation isolates protein complexes using specific antibodies (protein A/G beads)
  • Pull-down assays capture protein complexes using affinity tags (GST, His-tag)
  • Far-Western blotting identifies interactions between immobilized and soluble proteins (protein overlay)
• In vivo methods
  • Fluorescence resonance energy transfer measures energy transfer between fluorophores in close proximity (CFP/YFP pair)
  • Bioluminescence resonance energy transfer uses bioluminescent donor and fluorescent acceptor (luciferase/GFP)
  • Proximity ligation assay detects protein interactions through DNA amplification and fluorescence (rolling circle amplification)
• Biochemical methods
  • Cross-linking covalently links interacting proteins using chemical reagents (formaldehyde, DSS)
  • Surface plasmon resonance measures changes in refractive index upon protein binding (Biacore systems)
  • Isothermal titration calorimetry quantifies heat changes during protein-protein interactions (MicroCal instruments)
• High-throughput methods
  • Protein microarrays immobilize proteins on solid surface for parallel interaction screening (antibody arrays)
  • Mass spectrometry-based approaches identify protein complexes and interaction networks (AP-MS, BioID)

Principles of interaction detection methods

• Yeast two-hybrid system
  • Utilizes transcriptional activation of reporter genes in yeast cells
  • Fusion proteins: DNA-binding domain and activation domain
  • Interaction reconstitutes functional transcription factor
  • Advantages: High-throughput screening, in vivo detection
  • Limitations: High false-positive rate, limited to nuclear interactions
• Co-immunoprecipitation
  • Isolates protein complexes using specific antibodies and affinity matrices
  • Maintains native protein conformations and interactions
  • Advantages: Detects native interactions, applicable to various cell types
  • Limitations: Requires high-quality antibodies, may disrupt weak interactions
• FRET
  • Measures energy transfer between donor and acceptor fluorophores in close proximity
  • Distance-dependent phenomenon ($R_0$ typically 1-10 nm)
  • Advantages: Real-time detection, spatial resolution in living cells
  • Limitations: Requires protein tagging, potential interference with interactions
• Cross-linking
  • Covalently links interacting proteins using bifunctional chemical reagents
  • Stabilizes transient or weak interactions for subsequent analysis
  • Advantages: Captures transient interactions, applicable to complex samples
  • Limitations: May introduce artifacts, challenging to interpret results
• Surface plasmon resonance
  • Measures changes in refractive index upon protein binding to immobilized ligand
  • Real-time monitoring of association and dissociation kinetics
  • Advantages: Label-free detection, real-time kinetics measurement
  • Limitations: Requires protein immobilization, potential surface effects
• Protein microarrays
  • Immobilizes proteins on solid surface for high-throughput interaction screening
  • Allows parallel analysis of thousands of potential interactions
  • Advantages: Parallel analysis of multiple interactions, minimal sample consumption
  • Limitations: Protein denaturation, limited to binary interactions

Comparison of interaction detection techniques

• Sensitivity comparison
  • High: SPR detects interactions with $K_D$ in pM range, ITC measures $\mu$cal/s heat changes
  • Moderate: Co-IP detects interactions with $K_D$ in nM range, FRET sensitive to 1-10 nm distances
  • Low: Y2H prone to false positives, protein microarrays limited by protein stability
• Specificity comparison
  • High: Co-IP maintains native interactions, PLA provides single-molecule resolution
  • Moderate: FRET/BRET distance-dependent, cross-linking captures proximal proteins
  • Low: Y2H prone to non-specific activation, protein microarrays affected by non-native conformations
• Throughput comparison
  • High: Y2H screens millions of interactions, protein microarrays analyze thousands of proteins
  • Moderate: Co-IP coupled with MS identifies hundreds of interactions, pull-down assays for multiple baits
  • Low: SPR analyzes few interactions in detail, ITC provides thermodynamic parameters for single interactions
• In vivo vs in vitro methods
  • In vivo: FRET/BRET in living cells, PLA in fixed tissues
  • In vitro: SPR with purified proteins, ITC for isolated complexes

Types of interactions detectable by methods

• Stable interactions
  • Detectable by Co-IP, pull-down assays, and cross-linking
  • Enzyme-inhibitor complexes (trypsin-BPTI), structural protein assemblies (actin-myosin)
• Transient interactions
  • Detectable by FRET, BRET, cross-linking, and SPR
  • Signaling cascades (MAPK pathway), regulatory interactions (p53-MDM2)
• Weak interactions
  • Detectable by SPR, ITC, and cross-linking
  • Low-affinity binding events ($K_D$ > $\mu$M), temporary associations (chaperone-substrate)
• Binary interactions
  • Detectable by Y2H, FRET, BRET, and SPR
  • Ligand-receptor binding (insulin-insulin receptor), protein dimerization (EGFR)
• Multi-protein complexes
  • Detectable by Co-IP and MS-based approaches
  • Transcription factor complexes (RNA polymerase II), proteasomes (26S)
• Post-translational modification-dependent interactions
  • Detectable by Co-IP, PLA, and MS-based approaches
  • Phosphorylation-dependent binding (SH2 domains), ubiquitination-mediated interactions (E3 ligases)