Protein-protein interactions are the physical contacts between proteins that underlie all cellular processes. Proteins rarely act in isolation but function as components of complex interaction networks, forming transient or stable complexes that carry out biological functions.
Types of Interactions
Protein-protein interactions range from stable, permanent complexes such as the ribosome or proteasome, to transient interactions that form and dissociate in response to signaling events. Interactions can be obligate, where the subunits are unstable in isolation, or non-obligate, where the individual proteins are stable on their own. The interfaces of stable complexes tend to be larger and more hydrophobic than those of transient interactions, which often involve polar and charged residues.
Interaction Surfaces
Protein-protein interfaces typically bury 600 to 2000 square angstroms of solvent-accessible surface area. The interfaces are enriched in hydrophobic residues, which are buried upon complex formation, and often contain a central hydrophobic patch surrounded by polar interactions. Hot spots are residues that contribute disproportionately to binding energy, typically tryptophan, arginine, and tyrosine. These hot spot residues are surrounded by energetically less important residues that exclude water from the interface.
Binding Affinity and Kinetics
The strength of a protein-protein interaction is described by the dissociation constant, which can range from picomolar for high-affinity interactions to micromolar for weak, transient interactions. The Kd is determined by the rates of association and dissociation. Association rates are influenced by electrostatic steering, where complementary charges on the two proteins accelerate their encounter. Dissociation rates determine interaction lifetime, with slow dissociation producing long-lived complexes and rapid dissociation allowing quick signal termination.
Co-Immunoprecipitation
Co-immunoprecipitation is a widely used method for detecting protein-protein interactions. An antibody against a bait protein is used to capture it along with its interacting partners from cell lysates. The precipitated proteins are separated by SDS-PAGE and identified by western blotting or mass spectrometry. Co-IP can detect interactions under near-physiological conditions, but it may identify indirect interactions and may miss weak or transient interactions. Epitope tagging, where a peptide tag such as FLAG or HA is fused to the bait protein, allows capture using well-characterized tag antibodies.
Affinity Purification Coupled to Mass Spectrometry
AP-MS combines affinity purification of a tagged bait protein with mass spectrometric identification of its interaction partners. The tag is expressed as a fusion protein in cells, and the protein complex is purified under gentle conditions using the tag as a handle. The purified complex is digested with trypsin, and the peptides are identified by mass spectrometry. Control purifications from untagged cells distinguish specific interactors from contaminants. Quantitative AP-MS using SILAC or label-free methods provides additional discrimination.
Yeast Two-Hybrid
The yeast two-hybrid system detects binary protein-protein interactions in living yeast cells. The bait protein is fused to the DNA-binding domain of a transcription factor, and the prey protein is fused to the activation domain. If bait and prey interact, the transcription factor is reconstituted, activating reporter gene expression. Y2H is scalable for high-throughput screening, where a bait protein is tested against libraries of prey proteins. However, Y2H detects interactions in the yeast nucleus, which may not reflect the natural environment of the proteins, and has high false-positive and false-negative rates.
Surface Plasmon Resonance
Surface plasmon resonance measures real-time binding between a protein immobilized on a sensor chip and its interacting partner in solution. SPR provides kinetic information, including association and dissociation rate constants, from which the equilibrium dissociation constant is calculated. The technique is label-free and can measure interactions over a wide range of affinities. SPR is widely used in drug discovery to characterize antibody-antigen interactions and to screen for inhibitors of protein-protein interactions.
Förster Resonance Energy Transfer
FRET detects protein-protein interactions in living cells by measuring energy transfer between a donor and acceptor fluorophore attached to two interacting proteins. When the proteins are within 10 nanometers and in the correct orientation, excitation of the donor leads to emission from the acceptor. FRET can be measured by fluorescence microscopy, allowing spatial and temporal analysis of interactions. Variants include FRAP, which measures protein mobility and binding, and BRET, which uses bioluminescent donors to avoid problems with photobleaching.
