Key Techniques for Studying Protein-Protein Interactions (PPIs)

What Are Protein-Protein Interactions (PPIs)?

PPIs are physical contacts established between two or more protein molecules. These interactions are fundamental to virtually all cellular processes, including signal transduction, structural organization, enzymatic regulation, and immune responses. PPIs govern the dynamic formation of protein complexes and are essential for the spatial and temporal coordination of biological functions.

PPIs can be transient or stable, strong or weak, and may occur context-dependent. Their investigation requires a multidisciplinary toolbox comprising molecular biology, biochemistry, biophysics, and proteomics. A precise understanding of PPIs reveals basic biological principles and contributes to drug discovery, where PPIs represent both therapeutic targets and biomarkers.

Yeast Two-Hybrid (Y2H) System

Technique and Mechanism

Y2H assay constitutes a genetic reporter system to detect binary PPIs in vivo. The technique exploits the modular architecture of transcription factors. A canonical transcription factor comprises a DNA-binding domain (DBD) and an activation domain (AD). In Y2H, a "bait" protein fuses to the DBD. A "prey" protein fuses with the AD. The DBD and AD come into proximity upon interaction between bait and prey. The reconstituted transcription factor activates reporter gene expression.

The yeast host strain often bears auxotrophic markers corresponding to reporter genes. Growth on selective media lacking histidine or adenine serves as a phenotypic readout. Alternatively, β-galactosidase activity can be assayed quantitatively. The simplicity and genetic tractability of yeast facilitate large-scale screening.

Construct Design and Reporter Activation

Design of fusion constructs underpins successful Y2H screening. Both bait and prey constructs utilize expression vectors harboring yeast-specific promoters such as ADH1 or GAL1. The reading frame alignments must avoid premature stop codons or unintended truncations.

Reporter activation requires balanced expression of bait and prey. Overexpression can induce false positives due to nonspecific aggregation. Conversely, insufficient expression yields false negatives.

Reporter genes often appear in tandem arrays upstream of a minimal promoter. Activation of multiple reporters reduces false positives.

Figure 1. The classical yeast two-hybrid system (Brückner A, et al., 2009).

Advantages and Limitations

Co-Immunoprecipitation (Co-IP)

Co-IP constitutes a cornerstone for validating PPIs in cell lysates or tissue extracts. Co-IP relies upon an antibody specific to a "bait" protein. The antibody binds the bait within a lysate.

Workflow of Co-IP:

• Cell Lysis. Cells or tissues undergo disruption in a lysis buffer. Buffer formulation depends on the bait's subcellular localization and the interaction's strength.
• Antibody Incubation. The lysate is incubated with a primary antibody directed against the bait
• Bead Capture. Protein A/G beads are added. The resin binds the Fc region of the antibody.
• Wash Steps. Multiple washes with lysis buffer mitigate nonspecific binding.
• Elution. Bound proteins elute via buffer containing a low-pH glycine solution or by denaturation with Laemmli buffer.
• Analysis. Eluted proteins are subject to SDS-PAGE separation. Immunoblotting employs separate antibodies to detect prey proteins. Alternatively, in-gel digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) can identify unknown interactors.

Advantages and Limitations

Pull-Down Assays

Affinity Tag-Based Pull-Down Methodology

Pull-down assays represent an in vitro approach to capture PPIs between a recombinant bait and putative prey proteins. The bait typically carries an affinity tag such as glutathione S-transferase (GST), maltose-binding protein (MBP), or a polyhistidine (His) tag. Expression vectors encode the fusion protein. E. coli commonly serves as the expression host. After induction and bacterial lysis, the fusion protein undergoes affinity chromatography.

Once immobilized on resin, the bait incubates with a source of prey proteins. Sources may include cell lysates, purified recombinant proteins, or in vitro-translated products. Incubation under defined buffer conditions allows transient or stable interactions to form. Following gentle washes, bound prey proteins elute alongside the bait. Analysis via SDS-PAGE and immunoblotting or LC-MS/MS identifies interacting partners.

Comparing Pull-Down and Co-IP Techniques

Fluorescence-Based Approaches for Real-Time Interaction Studies

Fluorescence colocalization

Principle

Fluorescence colocalization relies on the overlap of two fluorescent signals. Proteins are tagged with different fluorophores, such as GFP and RFP. When two tagged proteins localize to the same subcellular compartment, their fluorescence signals overlap under a fluorescence microscope.

Workflow

• Construct expression vectors with fluorescently tagged proteins.
• Transfect cells and allow for expression.
• Use confocal or widefield microscopy to capture images.
• Analyze the degree of signal overlap using correlation coefficients.

Bimolecular Fluorescence Complementation (BiFC)

Principle

BiFC detects PPIs through the reconstitution of a fluorescent protein. The fluorophore (e.g., YFP) is split into two non-fluorescent fragments. Each fragment is fused to a candidate protein. Interaction between the two proteins brings the pieces into proximity, restoring fluorescence.

Workflow

• Design fusion constructs with split fluorophore fragments.
• Co-transfect the constructs into cells.
• Monitor fluorescence formation using live-cell imaging.
• Confirm interaction specificity through negative controls.

Fluorescence Resonance Energy Transfer (FRET)

Principle

FRET measures energy transfer between two fluorophores when nearby. One protein is fused to a donor fluorophore, the other to an acceptor. Upon excitation of the donor, energy transfer to the acceptor leads to detectable emission.

Workflow

• Construct donor- and acceptor-tagged protein vectors.
• Co-express them in cells.
• Excite the donor fluorophore using a specific wavelength.
• Detect energy transfer using FRET imaging techniques (e.g., sensitized emission or acceptor photobleaching).
• Quantify FRET efficiency to assess interaction strength.

Bioluminescence Resonance Energy Transfer (BRET)

Principle

BRET is a non-invasive technique based on energy transfer from a luciferase-tagged donor to a fluorescent acceptor. It does not require external excitation, reducing phototoxicity and background.

Workflow

• Clone proteins of interest with luciferase (donor) and fluorescent protein (acceptor) tags.
• Co-transfect the constructs into live cells.
• Add substrate to activate luciferase.
• Measure emitted light at donor and acceptor wavelengths using a luminometer.
• Calculate the BRET ratio to evaluate PPI occurrence.

Figure 2. Fluorescence-based methods used in the study of protein interactions (Ciruela F. 2008).

Cross-Linking Mass Spectrometry (XL-MS) for Structural Interaction Mapping

Workflow from Cross-Linking to MS-Based Identification

• Sample Preparation and Cross-Linking. Purified protein complexes or cell lysates incubate with cross-linking reagent. Quenching agents terminate the reaction.
• Proteolytic Digestion. Cross-linked proteins denature, reduce disulfide bonds, and alkylate cysteine residues. Subsequent digestion with proteases yields peptide fragments.
• Peptide Enrichment. Cross-linked peptides often represent a minority of total peptides. Enrichment strategies include size-exclusion chromatography (SEC) to isolate higher-molecular-weight cross-linked species or strong cation exchange (SCX) to separate charged peptides.
• LC-MS/MS Analysis. Enriched peptides undergo reversed-phase chromatography coupled to high-resolution mass spectrometers. Data-dependent acquisition selects precursor ions for fragmentation via collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD).
• Data Analysis. Specialized software (e.g., xQuest, MeroX, pLink) matches MS/MS spectra to cross-linked peptide pairs. Scoring algorithms evaluate spectral quality and mass accuracy. Identified cross-links provide inter-residue distance constraints.
• Structural Modeling. Integrative modeling platforms (e.g., Integrative Modeling Platform, IMP) incorporate cross-link restraints with crystallographic or cryo-EM structures.

Advantages and Limitations

Biophysical Techniques for Quantifying PPI Kinetics and Affinity

Surface Plasmon Resonance (SPR)

SPR is an optical detection method that measures changes in the refractive index near a sensor surface. When one protein (ligand) is immobilized on a gold-coated sensor chip, the binding of an analyte protein induces a shift in the angle of reflected light, which is directly proportional to the mass change at the interface.

Workflow

• Immobilize the ligand on a carboxymethyl dextran-coated sensor surface.
• Inject analyte at varying concentrations under continuous buffer flow.
• Monitor real-time binding curves (sensorgrams) that reflect association and dissociation kinetics.
• Analyze kinetic constants (ka, kd) and equilibrium dissociation constant (KD) using curve-fitting models.

Biolayer Interferometry (BLI)

BLI detects interference patterns generated by white light reflecting from two surfaces: an internal reference layer and a protein-coated biosensor tip. Binding events lead to a wavelength shift, indicating mass accumulation on the biosensor.

Workflow

• Load ligand onto a disposable biosensor tip with pre-coated reactive surface (e.g., streptavidin).
• Dip the sensor sequentially into analyte solutions of different concentrations.
• Record optical interference patterns in real time.
• Calculate kinetic and equilibrium parameters based on association and dissociation phases.

Microscale Thermophoresis (MST)

MST measures the movement of fluorescently labeled molecules in microscopic temperature gradients. Binding events alter the protein complex's hydration shell, size, or charge, thereby affecting thermophoretic mobility.

Workflow

• Fluorescently labels one protein partner (commonly using dyes or fusion tags).
• Titrate increasing concentrations of the unlabeled binding partner.
• Load samples into capillaries and expose them to infrared laser-induced thermal gradients.
• Detect shifts in thermophoresis profiles, which reflect binding interactions.
• Determine KD values from the binding curve.

Proteomics-Based Approaches for PPI Profiling

Tandem Affinity Purification Coupled with MS (TAP-MS)

Tandem Affinity Purification (TAP) followed by mass spectrometry enables systematic identification of protein complexes in vivo. In TAP, a dual-affinity tag, such as Protein A—TEV cleavage site—Calmodulin-binding peptide (CBP), fuses to the bait protein at the chromosomal locus. Expression occurs under endogenous regulatory elements to preserve physiological levels. The two-step purification involves sequential affinity captures:

• First Affinity Step. Cell lysates incubate with IgG resin. Protein A binds IgG. This step captures the bait-protein complex.
• Protease Cleavage. Tobacco etch virus (TEV) protease cleaves between Protein A and CBP, eluting the complex under mild conditions.
• Second Affinity Step. The eluate binds to calmodulin resin in the presence of Ca²⁺. Washing removes nonspecific proteins.
• Final Elution. EGTA chelates Ca²⁺, releasing CBP-tagged complexes.

The purified complexes undergo tryptic digestion and LC-MS/MS analysis. High-confidence interactors emerge by comparing spectral counts to control purifications. TAP-MS captures stable and transient complexes depending on lysis conditions and purification stringency.

Quantitative Proteomics (SILAC, TMT, iTRAQ)

Quantitative proteomics leverages isotopic labeling and isobaric tags to measure relative protein abundances across conditions. Integration with affinity purification refines PPIs discovery by distinguishing specific interactors from the background. Common approaches include:

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC): Cells grow in media containing light or heavy amino acids. After labeling, cells expressing tagged bait or control undergo combined lysis and affinity capture. MS quantifies heavy-to-light peptide ratios, identifying enriched interactors.

Tandem Mass Tags (TMT): Peptides from different samples (e.g., bait and control) are labeled with isobaric TMT reagents. TMT reagents produce identical mass precursors but release reporter ions of distinct m/z upon fragmentation. Quantification relies on the relative intensities of reporter ions. TMT enables multiplexing of up to 16 samples, enhancing throughput.

Isobaric Tags for Relative and Absolute Quantitation (iTRAQ): Functionally similar to TMT, iTRAQ uses amine-reactive isobaric tags. Multiplexing typically encompasses eight samples. Reporter ion intensities reflect relative peptide abundances.

Chemical Proteomics for Covalent and Reversible Interactions

Chemical proteomics applies reactive probes to capture PPIs based on specific chemistries. Two principal strategies exist:

Activity-Based Protein Profiling (ABPP)

Probes containing a reactive warhead and a tag covalently modify the active sites of enzymes. Subsequent affinity enrichment isolates labeled proteins. Although ABPP primarily identifies enzyme activity profiles, modified probes can trap enzyme-substrate complexes, indirectly reporting PPIs.

Affinity-Based Probes for Reversible Interactions

Small-molecule probes bearing a photoactivatable cross-linker and an enrichment handle capture transient interactions. UV irradiation induces covalent cross-links between the probe and proximal proteins. After cell lysis, click chemistry and attach biotin for enrichment. Eluted proteins or peptides undergo LC-MS/MS analysis. This approach identifies ligand interactor networks and can be extended to capture protein-protein interactions when a bifunctional cross-linker bridges two nearby proteins.

References

• Brückner A, et al. Yeast two-hybrid, a powerful tool for systems biology. International journal of molecular sciences, 2009, 10(6): 2763-2788. DOI: 10.3390/ijms10062763
• Ciruela F. Fluorescence-based methods in the study of protein–protein interactions in living cells. Current opinion in biotechnology, 2008, 19(4): 338-343. DOI: 10.1016/j.copbio.2008.06.003
• Rao V S, et al. Protein‐protein interaction detection: methods and analysis. International journal of proteomics, 2014, 2014(1): 147648. DOI: 10.1155/2014/147648