Background

The Investigation of protein-protein interactions is an important field in life sciences since protein interactions play crucial roles in living organisms. Many biochemical and physiological reactions are under the control of proteins that require physical communication to other proteins within the cell. For example, human cell invasion by SARS-CoV-2 starts with interactions between the viral Spike protein and the human ACE2 receptor (Shang et al., 2020). Determination of the kinetic and affinity parameters of specific protein complexes provides important information about their properties and helps to discover their function and regulatory properties. Furthermore, the determination of the half maximal effective concentration (EC₅₀) of a metabolite, which inhibits or enhances protein complex formation, is crucial for the understanding of regulatory mechanisms and/or the development of drugs to combat infectious diseases.

Several methods are available to study protein interactions in real time, such as biolayer interferometry (BLI), surface plasmon resonance (SPR), or isothermal titration calorimetry (ITC). BLI is an emergent optical analytic technique that provides a good trade-off between sensitivity, cost intensity, reproducibility, and the ability for high-throughput measurements. The physics of BLI is based on the principle of a wavelength shift when white light is reflected from two layers, first from an internal reference layer and second from a protein coated layer (FortéBio, 2005-2006). The wavelength shift is detected by the machine and is recorded over time. The length of the shift is proportional to the bound proteins on the layer and can be used to calculate the binding affinities and kinetics. Compared to ITC, BLI measurement requires lower amounts of the target proteins, which is especially important for proteins that are difficult to express and purify, e.g., membrane proteins. BLI has lower sensitivity compared to SPR methods; however, in terms of equipment maintenance, BLI is advantageous compared to the micro-fluidic SPR systems, which are prone to clogging and require high sample purity, buffer degassing, and constant maintenance of the sensor chips and micro-fluidic devices. In addition, BLI is tolerant to the use of analytes in complex matrices (Shah and Duncan, 2014), enabling high-throughput measurements of crude analyte preparations.

Recently, we employed BLI to characterize the interaction between the P_II signaling proteins and different binding target proteins, including NAD synthetase (NadE), phosphoenolpyruvate carboxylase (PEPC), P_II-interacting regulator of arginine synthesis (PirA), and P_II-interacting regulator of carbon metabolism (PirC) (Santos et al., 2020; Scholl et al., 2020; Bolay et al., 2021; Orthwein et al., 2021). P_II proteins sense the cellular energy state through the competitive binding of ATP and ADP, and sense carbon/nitrogen balance through binding of the citric acid cycle metabolite 2‐oxoglutarate (2-OG) (Forchhammer and Selim, 2020; Selim et al., 2020). Here, we describe a robust, simple, and quick protocol for the detection of protein interactions using a His-tagged bait protein (ligand) and a non-His-tagged prey protein (analyte). The BLI measurements were performed using a FortéBio^® Octet K2 system (Sartorius). The protocol describes the procedure to determine the dissociation constant (K_D) of the protein complex and also the EC₅₀ of small effector molecules (2-OG here), which can promote or disrupt the protein complex. The protocol is exemplified using the P_II protein from the unicellular cyanobacterium Synechocystis sp PCC 6803 for studying P_II-PirC interactions. In cyanobacteria, the interaction between P_II-PirC regulates the central carbon metabolism in response to the intracellular concentrations of the carbon/nitrogen indicator 2-OG (Orthwein et al., 2021).