Novel Approaches for Investigating Protein-Protein Interactions

Protein-protein interactions are critical regulators of many cellular processes. The dysregulation of these interactions can lead to abnormal states―such as cancer and cardiovascular disease. Traditional methods for studying protein-protein interactions have included co-immunoprecipitation, crosslinking, far-western blot analysis, and various label-free techniques. But what other emerging technologies might be out there? To further explore this question, Biocompare recently interviewed experts from three companies―Sartorius, SomaLogic, and Promega.

Why protein-protein interactions?

Before the 21^st century, research tended to focus on single proteins. However, most proteins must interact with other proteins for proper function. Nebojsa Janjic, Chief Science Officer at SomaLogic, explains, “So much of biology is about interactions between complementary shapes. An analogy with Lego blocks is not bad, except that there are many more shapes in biology.” Protein-protein interactions involve combinations of hydrophobic binding, van der Waals forces, and salt bridges, with binding domains spanning anywhere from a few peptides to hundreds of amino acids.

Expanding upon traditional methods

Co-immunoprecipitation is perhaps the most common technique to investigate protein-protein interactions. It typically involves selecting an antibody that targets a known protein believed to be associated with a larger complex of proteins. Ultimately, proteins and any bound ligands are precipitated. Janjic explains the basic principle of co-immunoprecipitation: “You ask what your antibody is pulling down in addition to protein A. If I see that Protein B is also present, I will infer that protein A binds to protein B.”

Janjic also highlights the emergence of various label-free technologies, which measure binding interactions without altering the binding partners. “A good example would be something like NMR or hydrogen-deuterium exchange. You don’t need to tag the binding partners with fluorophores or radioisotopes that might change them.”

Nilshad Salim, Product Manager at Sartorius, explains how his company is pioneering label-free technologies. He recommends Sartorius’s Octet platform to investigate protein-protein and protein-drug interactions in real-time.

Salim highlights that Sartorius is the only commercial provider of two key label-free techniques: biolayer interferometry (BLI) and surface plasmon resonance (SPR). “With the Octet portfolio, we provide the best option depending on individual customer requirements rather than fitting the customer needs to one technology,” he emphasizes.

Trans associations elucidate novel pathways

Janjic notes that SomaLogic has developed a multiplex protein assay called SomaScan that can measure 7000 proteins simultaneously from a biological sample. Instead of antibodies, the SomaScan uses aptamers, short, single-stranded DNA or RNA molecules that selectively bind to proteins.

Janjic explains how SomaLogic’s technology has enabled a novel strategy for elucidating protein-protein interactions. He notes that a mutation in one gene often affects the expression level of a protein encoded by a different gene—in what is called a trans association. “When you see trans associations, you can infer that these two proteins may be talking to each other. And the simplest explanation is that these two proteins bind together,” he says.

Intriguingly, some proteins are dramatically more trans-associated than others. But what are these proteins?

Janjic highlights how a 2023 Nature Communications paper used SomaScan technology and the investigation of trans associations to demonstrate that SVEP1, a protein associated with vascular disease, is a ligand for an orphaned receptor called PEAR1. “People didn’t know what the ligand for PEAR1 was. It was an orphan receptor. By understanding this trans association, Nathan Stitziel’s group at Washington University proved that these proteins bind together.”

Janjic further emphasizes how the investigation of trans associations is much more convenient than traditional methods involving co-immunoprecipitation. “With immunoprecipitation, you can only deal with one hypothesis at a time,” he notes. “And you have to start with a hypothesis that Protein A binds to Protein B. That is hard work.”

Bioluminescence to investigate live cell interactions

Marie Schwinn, a Senior Research Scientist at Promega, notes, “We are very excited about newly emerging methods that directly measure protein-protein interactions in live cells.”

Schwinn emphasizes that one of the most prominent challenges when studying protein-protein interactions involves collecting data that accurately represent cellular physiology. “Unfortunately, biochemical or lytic assays do not preserve spatiotemporal information critical to understanding a particular signaling pathway,” she notes. Schwinn says that Promega’s NanoBRET and NanoBiT are ideal for looking at protein-protein interactions in live cells.

NanoBRET and NanoBiT―often called proximity-based assays—enable the direct detection of cellular protein-protein interactions in real-time. Central to these two techniques is the light-producing NanoLuc luciferase enzyme. NanoLuc―ideally suited for assays because it is small, bright, and stable—is a 19 kDa luciferase enzyme engineered from a deep-sea luminescent shrimp.

Schwinn explains how NanoBRET (bioluminescence resonance energy transfer) helps measure the binding of one protein (tagged with NanoLuc) to another protein (tagged with HaloTag bound to a fluorescent ligand). When the proteins of interest interact, the NanoLuc enzyme excites the ligand to produce a fluorescent signal. A typical NanoBRET experiment involves expressing the NanoLuc and HaloTag fusions in live cells. Schwinn explains, “Using CRISPR/Cas9, it is possible to integrate reporter tags like NanoBRET directly into the cellular genome so that protein fusions will be expressed from endogenous loci.”

Schwinn points out that, like NanoBRET, NanoBiT (NanoLuc Binary Technology) is also based on an adaptation of NanoLuc. The NanoBiT system comprises two subunits, Large BiT (18 kDa) and Small BiT (an 11 amino acid peptide), that can be expressed as fusions. When two target proteins tagged with these subunits interact, the subunits come together to generate a bright luminescent signal.

Unveiling key cellular pathways

Although co-immunoprecipitation, crosslinking, and far-western blot analysis are still helpful in understanding protein-protein interactions, many state-of-the-art methods are currently being developed by companies like Sartorius, SomaLogic, and Promega. Ultimately, these emerging techniques are less likely to alter binding patterns, require significantly less work, and can even elucidate processes in live cells. With the help of such technologies, scientists are beginning to unravel key cellular processes associated with cardiovascular disease and cancer.