Interactomics: The study of protein-protein interactions (PPIs) and the networks they form within biological systems.

Why PPIs are important:

• Functional Insight: Essential for understanding how proteins function within cells
• Pathology: Gene mutations can disrupt protein interactions — a primary driver of disease
• Drug Discovery: New drug treatments rely heavily on protein function analysis
• Discovery: Unknown proteins can be discovered by identifying their partners in known pathways

Scale of the problem:

• ~2-4 million proteins per cubic micron in cells
• Number of possible interactions is enormous
• PPIs are intrinsic to virtually every cellular process: cell growth, cell cycle, metabolism, signal transduction

Key challenges:

1. Identifying which proteins interact in the crowded intracellular environment
2. Mapping specific residues that participate in interactions

The Bait and Prey model is the fundamental principle underlying all PPI methods:

Bait (X):

• The protein of interest
• Used to "fish" for interacting partners
• Usually tagged or labeled for detection

Prey (Y):

• Proteins that interact with the bait
• Can be known candidates or unknown proteins from a library

Types of interactions:

• Binary: One bait + one prey
• Complex: One bait + multiple preys simultaneously

The fundamental question: "Does X bind with protein Y?"

A. Experimental Methods:

In Vitro Methods: (Purified proteins, controlled lab environment)

• Co-Immunoprecipitation (Co-IP): Antibodies isolate protein complexes
• GST-Pull Down: Tagged proteins capture binding partners
• Protein Arrays: High-throughput screening on solid surface

In Vivo / Cellular Methods: (Living cells)

• Yeast Two-Hybrid (Y2H): Classic genetic screen for binary interactions
• Mammalian Two-Hybrid (M2H): Y2H in mammalian context
• Phage Display: Connects proteins with encoding DNA
• Proximity Labeling: BioID, TurboID, APEX

Imaging & Real-time:

• FRET: Fluorescence Resonance Energy Transfer
• BRET: Bioluminescence Resonance Energy Transfer

B. Computational Methods:

• Genomic data: Phylogenetic profiles, gene fusion, correlated mutations
• Protein structure: Residue frequencies, 3D distance matrices, surface patches

GST-Pull Down: An affinity purification method similar to Co-IP, but uses a recombinant tagged bait protein instead of an antibody.

Key difference from Co-IP:

The GST Fusion System:

• Bait protein fused to GST (glutathione-S-transferase) tag
• Expressed in E. coli
• GST increases solubility (acts as molecular chaperone)
• GST binds strongly to glutathione-agarose beads

Workflow:

1. Express GST-bait fusion in E. coli
2. Immobilize on glutathione beads
3. Incubate with cell extract (prey source)
4. Wash away non-binders
5. Elute with excess glutathione (competes for GST)
6. Analyze by SDS-PAGE + Western blot

Protein Microarrays: Miniaturized bioanalytical devices with arrayed molecules on a surface for high-throughput analysis.

Three main types:

1. Analytical Protein Arrays:
   • Immobilized capture agents (antibodies)
   • Detect proteins in solution (analyte)
   • Used for: Clinical diagnostics, biomarker discovery
2. Functional Protein Arrays:
   • Proteins of interest are immobilized
   • Capture interacting molecules from analyte
   • Used for: Mapping interactome, identifying protein complexes
3. Reverse Phase Protein Arrays (RPPA):
   • Complex sample immobilized on surface
   • Specific probes detect target proteins within sample
   • Used for: Tissue lysate analysis, pathway profiling

General workflow:

1. Array fabrication (design layout, select probes)
2. Substrate selection & deposition (robotic printing)
3. Immobilization (attach capture molecules)
4. Interaction & detection (fluorescence or MS)

Technical challenges:

• Steric Hindrance:
  • Proteins are large and asymmetrical
  • Immobilization can mask active sites
  • Need site-specific orientation for accessibility
• Low Yield:
  • Inefficient covalent attachment
  • Suboptimal surface density
  • Limits dynamic range
• Non-specific Adsorption:
  • Proteins are "sticky"
  • Hydrophobic/electrostatic binding to substrate
  • Causes high background and false positives
• Conformation Fragility & Denaturation:
  • Proteins are thermodynamically unstable (vs. DNA)
  • Sensitive to pH, temperature, dehydration
  • Loss of 3D structure = loss of activity

Artifacts: Dust particles, scratches, bleeding between spots can cause spurious signals.

Phage Display: A technique where peptides/proteins are displayed on bacteriophage surfaces, creating a physical link between phenotype and genotype.

Fundamental principle:

• Foreign DNA fused to phage coat protein gene
• When phage replicates, fusion protein displayed on surface
• DNA encoding it is packaged inside
• Phenotype (displayed protein) linked to genotype (internal DNA)

Is it in vitro or in vivo?

• Production: In vivo (in E. coli)
• Selection: In vitro (on plates/beads)
• Application: In vivo (therapeutic use)
• Acts as a "bridge" technique

Biopanning (Selection Process):

1. Binding: Phage library exposed to immobilized target
2. Wash: Non-binders removed (acid/urea/competing ligand)
3. Amplification: Bound phages re-infect E. coli and multiply
4. Iteration: Repeat 3-4 cycles to enrich strong binders
5. Sequencing: Identify common motifs in winners

Main limitations:

1. Prokaryotic Expression System:
   • No post-translational modifications (no glycosylation, phosphorylation)
   • May not fold mammalian proteins correctly
   • Codon bias issues
2. Size Constraints:
   • Large protein inserts may disrupt folding or phage assembly
3. Selection Bias:
   • Some peptides toxic to bacteria → lost from library
4. Stringency Risks:
   • First wash too harsh → lose high-affinity candidates
5. In Vivo Translation:
   • Peptide that works in lab may fail in living body (pH, interference)
6. Misfolding:
   • Complex proteins may not adopt correct 3D structure on phage surface

Proximity Labeling: An in vivo method where an enzyme fused to bait labels all nearby proteins with biotin.

Core mechanism:

1. Biotinylation: Enzyme activates biotin → reactive species tags neighbors within ~10-20 nm
2. Capture: Biotin-streptavidin affinity captures tagged proteins
3. Identification: MS identifies the "proteomic atlas" of bait's environment

Comparison:

TurboID is now the gold standard: combines non-toxic nature of BioID with speed of APEX.

SRET = Sequential BRET-FRET

An advanced technique to monitor non-binary interactions (three or more proteins forming a complex).

The molecular components:

1. Donor: Protein 1 fused to Renilla luciferase (Rluc)
2. First Acceptor: Protein 2 fused to GFP/YFP
3. Second Acceptor: Protein 3 fused to DsRed

Sequential energy transfer:

1. BRET phase: Rluc → GFP (if proteins 1 & 2 are close)
2. FRET phase: GFP → DsRed (if proteins 2 & 3 are close)
3. Final emission: DsRed emits — confirms all three are together

Key advantage: Positive SRET signal is definitive proof that all three proteins are physically clustered at the same time.

Application: Studying GPCR oligomerization (homo- and hetero-oligomers) in drug discovery.

PCA Principle:

• Reporter protein (e.g., luciferase) split into two inactive fragments
• Fragments fused to bait and prey proteins
• If bait and prey interact → fragments brought together → reporter reconstituted → signal produced

Logic:

• No interaction → fragments separated → no activity
• Interaction → proximity → reassembly → activity restored

NanoBiT (NanoLuc Binary Technology):

• Current gold standard PCA system
• Large BiT (LgBiT): 18 kDa
• Small BiT (SmBiT): 11 amino acids
• Engineered with very weak intrinsic affinity
• Only reassemble when "forced" together by bait-prey interaction

Advantages of NanoBiT:

• High signal-to-noise ratio
• Low background (no spontaneous assembly)
• Works at physiological protein concentrations
• Superior dynamic range vs. FRET

Inteins = INternal proTEINS

Self-splicing protein segments that excise themselves from a precursor protein, leaving the flanking exteins joined together.

Terminology:

• Intein: Gets removed (internal protein)
• Extein: Flanking sequences that remain (external protein)
• N-extein—[INTEIN]—C-extein → N-extein—C-extein + free intein

Mechanism (Protein Splicing):

1. N-S or N-O acyl shift at N-terminus
2. Transesterification
3. Asparagine cyclization releases intein
4. S-N or O-N acyl shift joins exteins with native peptide bond

Applications:

• Self-cleaving affinity tags: Tag-free protein purification (no extra residues!)
• Expressed Protein Ligation: Join two protein fragments with native bond
• Protein cyclization: Create cyclic proteins
• Conditional protein splicing: Control protein activity

Aptamers: Single-stranded oligonucleotides (ssDNA or RNA) that fold into complex 3D structures and bind targets with high affinity.

How they bind:

• Shape complementarity (not base pairing)
• Non-covalent interactions: hydrogen bonding, van der Waals, aromatic stacking
• Often called "chemical antibodies"

SELEX = Systematic Evolution of Ligands by EXponential Enrichment

1. Create library: 10⁹-10¹¹ random sequences
2. Incubation: Expose library to target
3. Counter-selection: Remove cross-reactive sequences (expose to non-targets)
4. Wash & Elute: Remove non-binders, recover high-affinity sequences
5. Amplification: PCR enrichment of winners
6. Iteration: Repeat 8-15 cycles

Applications: Drugs, therapeutics, diagnostics, bio-imaging, food inspection

A. Experimental-based (validation):

• X-ray crystallography
• NMR spectroscopy
• Cryo-EM

B. Computational based on Genomic Data:

• Phylogenetic profiles: Proteins that co-evolve likely interact
• Gene neighborhood: Genes close on chromosome often encode interacting proteins
• Gene fusion: Proteins fused in one organism may interact in another
• Correlated mutations: Co-evolving residues suggest contact

C. Based on Protein Primary Structure:

• Residue frequencies and pairing preferences
• Sequence profile and residue neighbor list

D. Based on Protein Tertiary Structure:

• 3D structural distance matrix
• Surface patches analysis
• Direct electrostatic interactions
• Van der Waals interactions
• Docking simulations