Decades of research has resulted in better understanding of the physiological function and molecular properties of proteins. As proteins rarely act alone, a critical step towards unravelling the complex molecular relationships has been examination of protein- protein interactions (PPI). Read our blog below to learn more about PPI and an interesting technique used to study interactions in living cells.
Proteins: An Overview
Proteins are versatile macromolecules known to control the majority of biological processes including morphology, cell growth, motility, gene expression, proliferation, nutrient uptake, intercellular communication and apoptosis.
Since the pioneering work of protein scientists such as Christian Anfinsen*, it has been clear that protein function is dictated primarily by its structure. Proteins are built from a set of twenty amino acids, each of which has a unique side chain. The linkage of these chains to one another confers a certain conformation or three- dimensional shape to the proteins. However, this conformation is far from static and changes with an alteration in the micro- environment or as proteins interact with other proteins or molecules. This dynamism forms an important layer of connectivity between cell components and cell processes essential to maintain life.
Protein- protein interactions (PPI):
Until the late 1990s, protein function analyses mainly focused on single proteins. However, over 80% of proteins interact with other proteins in vivo and form complexes. This interaction can either be ‘stable’ or ‘transient’. Stable interactions involve complex sub-units of proteins that interact for a very long time; e.g. haemoglobin, core RNA polymerase, etc. Transient interactions are temporary when proteins associate and dissociate in the cellular environment, e.g heterotrimeric G protein, lysin, etc.
Protein-protein interactions can have various measurable effects: they can alter the kinetic properties of proteins, inactivate a protein, change the specificity of a protein for its substrate, allow for substrate channelling and also form new binding sites. Disruptions to the normal patterns of PPIs and protein complexes can be causative or indicative of many disease states, making them attractive targets for drug discovery.
There are various in vitro, in vivo and in silico methods to investigate protein–protein interactions. Below we have described one such technique that examines PPI in living cells.
Protein-protein interactions in living cells:
Historically, only solution-based assays were used to screen compounds in PPI, but a more physiological approach is to examine the interaction in living cells. However, there are very few commercially available technologies that can actually do this. As described in our poster, we have successfully used NanoBRET™ System (Promega) to detect protein- protein interactions in living cells. NanoBRET™ is a proximity-based assay that can detect protein interactions by measuring energy transfer from a bioluminescent protein donor to a fluorescent protein acceptor. The use of full-length proteins expressed at low levels enables PPI monitoring and screening studies that reflect true cellular physiology. NanoBRET™ is a reversible assay which makes it possible to study both induction and inhibition of protein interactions. It has been developed for various epigenetic targets, kinases, transcription factors, receptors and important signalling proteins. Follow the link to our poster to learn more about this assay.
Are you looking for a suitable bioassay to detect PPIs? Why not get in touch with our team and discuss your project?