Protein-protein interactions (PPIs) are the molecular conversations regulating cellular life. At Creative Biolabs, we use phage display technology to both decipher these dialogues and intervene in them. Depending on the experimental design, this versatile platform allows us to identify novel binding partners by screening cDNA libraries against bait proteins, revealing previously unknown networks. Simultaneously, we utilize it to discover specific modulators—such as peptides or antibody fragments—that can inhibit or enhance these interactions. These screened ligands often serve as precursors for therapeutic agents. Through our Discovery of Novel PPIs Using Phage Display services, we enable researchers to systematically map the interactome and accelerate the development of PPI-targeted drugs.
Fig.1 Timeline of PPI research and modulator development.1
A protein-protein interaction is the physical contact established between two or more protein molecules. These contacts result from biochemical events driven by electrostatic forces, hydrogen bonding, and the hydrophobic effect. When proteins interact, they form protein complexes that dictate specific biological functions. In a biological system, proteins rarely function in isolation. They operate within a vast network known as the interactome. Current estimates suggest that the human interactome contains between 130,000 and 650,000 different types of protein interactions. These interactions regulate essential processes, including DNA replication, signal transduction, cellular metabolism, and the cell cycle. The interface of a protein-protein interaction differs significantly from the binding sites of enzymes. Enzyme binding sites are typically deep crevices or pockets suitable for small molecules. In contrast, PPI interfaces are generally large, flat, and featureless, often spanning surface areas of 1,500 to 3,000 square angstroms. This large surface area is one reason why targeting PPIs was historically challenging.
Although the contact interface between proteins is large, the binding energy is not distributed evenly across the surface. Specific residues contribute disproportionately to the free energy of binding. These residues are known as hot spots. A hot spot is typically defined as a residue where a mutation to alanine results in a significant increase in binding free energy (usually greater than 2.0 kcal/mol). Amino acids such as tryptophan, arginine, and tyrosine are frequently found in these hot spot regions. Identifying these regions is crucial for predicting protein interactions and designing modulators. If a small molecule or peptide can effectively bind to these hot spots, it can disrupt or stabilize the entire interaction.
We classify protein interactions based on their stability and the composition of the protein complex in which they occur. Understanding these classifications is crucial for selecting the most suitable protein-protein interaction methods for study.
The proper function of protein-protein interactions is vital for health. Aberrant interactions are often the root cause of diseases.
PPIs regulate the cell cycle and apoptosis. For instance, the interaction between the tumor suppressor protein p53 and the E3 ubiquitin ligase MDM2 is a critical regulatory point. MDM2 binds to p53 and inhibits its transcriptional activity, leading to p53 degradation. In many cancers, this interaction is dysregulated, preventing p53 from performing its tumor-suppressing function.
Cellular transport relies on highly specific interactions. Carrier proteins interact with particular molecules; this characteristic is essential to ensure that the correct substrates are transported across membranes without interference. Similarly, in signal transduction, an interactive protein must distinguish its specific partner from a crowded cellular environment to transmit accurate signals.
Dysfunctional PPIs are associated with cancer, infectious diseases, and neurodegenerative disorders. For example, the Bcl-2 family of proteins regulates apoptosis. The interaction between anti-apoptotic proteins (like Bcl-2) and pro-apoptotic proteins (like Bax) determines cell survival. In cancer cells, Bcl-2 is often overexpressed, sequestering Bax and preventing cancer cell death. In infectious diseases, viruses utilize PPIs to enter host cells. For example, the HIV-1 virus utilizes its surface glycoprotein, gp120, to bind to the CD4 receptor and the CCR5 co-receptor on human T-cells. Blocking this protein-protein interaction prevents viral entry.
At Creative Biolabs, developing reliable assays for protein-protein interactions is the foundation of our research services. We utilize a comprehensive suite of protein-protein interaction techniques. These include many experimental wet-lab assays.
Phage display is one of our core competencies. It is a high-throughput technique used to study PPIs and discover novel ligands. In this process, we genetically fuse a library of DNA sequences to the gene encoding a coat protein of a bacteriophage. The phage then displays the corresponding peptides or protein fragments on its surface. We screen these phage libraries against an immobilized target protein. Phages that display peptides capable of binding to the target are captured, while non-binders are washed away. This enables us to screen billions of potential interactions rapidly. This method is particularly useful for mapping binding epitopes and identifying peptide modulators for targets that are challenging to target with small molecules. For researchers looking to identify new binding partners or map binding epitopes, we recommend utilizing specialized services. Our Novel PPIs Discovery platform is designed to rapidly screen billions of potential interactions, providing high-affinity candidates for further study.
The Yeast Two-Hybrid system is a genetic method used to screen for protein-protein interactions inside living yeast cells. It relies on the reconstitution of a transcription factor. We fuse the target protein to a DNA-binding domain (the bait) and the potential partner to an activation domain (the prey). If the bait and prey proteins interact, they bring the two domains together, activating a reporter gene. This method is effective for large-scale screening of the interactome.
Co-IP is a standard protein-protein interaction assay used to detect interactions in their native physiological state. We use an antibody to precipitate a specific target protein from a cellular lysate. If the target protein is bound to other proteins in the cell, those partners are co-precipitated with it. This technique is excellent for confirming that two proteins interact within the complex cellular environment.
SPR is a biophysical technique that measures the kinetics of an interaction in real-time without the need for labels. It provides quantitative protein-protein interaction data regarding the association rate, the dissociation rate, and the equilibrium dissociation constant (affinity). This data is crucial for optimizing therapeutic candidates.
The ultimate goal of studying protein interactions is often to develop drugs that can modulate them. We can design molecules to either inhibit or stabilize an interaction.
Inhibition is the most common strategy. We aim to block the interaction between two proteins to stop a disease pathway.
Sometimes, a disease is caused because a protein complex is too unstable. In these cases, we use "molecular glues" to stabilize the interaction. The natural product Fusicoccin A stabilizes the interaction between 14-3-3 proteins and their partners. This demonstrates that small molecules can act as glues to enhance protein interactions.
The field of protein-protein interactions is evolving rapidly. The integration of artificial intelligence with large-scale protein-protein interaction data is accelerating the discovery of novel targets. We are observing a shift from basic inhibition to more complex modulation, including stabilization and degradation. As we continue to map the interactome, the demand for high-throughput screening technologies is increasing. Phage display remains a cornerstone of this discovery process, offering a versatile method to probe the vast landscape of protein interactions. If you are engaged in PPI research, we encourage you to utilize our service to efficiently identify high-quality binding candidates. for more service details!
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