Label-free Quantification of Direct Protein-protein Interactions with Backscattering Interferometry

Background

Molecular binding interactions are based on driving forces set by hydrogen bonding, hydrophobic and electrostatic interactions, and van der Waals and London dispersion forces. These interactions are fundamental in cellular processes and have led to several significant discoveries in the fields of biochemistry, proteomics, chemistry, and biophysics (Archakov et al., 2003). The struggle to find label-free approaches for interaction studies has remained a major goal in the last three decades. Techniques such as NMR studies (Ames et al., 1994), mass spectrometry (Anderson et al., 2014), surface plasmon resonance (Dell’Orco and Koch, 2016), biolayer interferometry (Kumar et al., 2016), and backscattering interferometry (Bornhop et al., 2007 and 2016; Sulmann et al., 2017) are currently among the most applied methods. Backscattering interferometry (BSI) is a relatively new method and is known for its high sensitivity, immobilization-free, and label-free quantification to study protein-protein or protein ligand interactions. BSI has a refractive index (RI) detector, which measures protein-protein interactions and can also be used to study protein-ligand interactions (Bornhop et al., 2016). More recently, BSI has been successfully used in drug discovery studies (Mizuno et al., 2019), as well as protein-small molecule and protein-ion interactions (Kammer et al., 2018). The current detection limit of the BSI signal is ΔRI = 10^–6. ΔRI is defined as the relative difference in refractive index between two samples having nearly identical RI values. For example, comparing RI values of 1.391312 with 1.391318 yields a ΔRI = 6 × 10^-6 (Bornhop et al., 2007).

Recoverin is a neuronal calcium sensor that interacts with G protein-coupled receptor kinase 1 (GRK1, rhodopsin kinase) in a calcium dependent manner. The interplay of recoverin and GRK1 is important for primary visual processes, as it controls the phosphorylation of rhodopsin (Gorodovikova et al., 1994; Klenchin et al., 1995). Phosphorylation of rhodopsin and several feedback loops are important control steps in photoresponse recovery (Koch and Dell’Orco, 2015). Previous work revealed that the N-terminal amphipathic α-helix of GRK1 comprising the first 25 amino acids is essential for the interaction, as it comes into contact with a hydrophobic pocket in recoverin (Figure 1) (Ames et al., 2006; Higgins et al., 2006). Recoverin-GRK1 complex formation is under the control of calcium. At high saturating calcium concentrations, recoverin exposes its covalently attached myristoyl-group, opening the hydrophobic pocket for the N-terminus of GRK1. In this state, GRK1 is inhibited and unable to phosphorylate rhodopsin. This process is reversed when the calcium concentration drops (Komolov et al., 2009; Zernii et al., 2011). More recently,Abbas et al. (2019) reported that the interaction between recoverin and GRK1 involves a network of switching electrostatic interactions in recoverin by using BSI. Thus, Figure 1 represents the optimal structural configuration of the recoverin-GRK1 complex. By employing a combinatorial approach using BSI and various other techniques, we have further investigated the complex formation using wild-type and mutant forms of recoverin. Results from surface plasmon resonance, isothermal titration calorimetry, tryptophan fluorescence spectroscopy, CD spectroscopy, and molecular dynamics simulations were all in agreement with our BSI studies. The calcium-dependent interaction of recoverin with its target region in GRK1 (peptide encompassing 25 amino acids of the N-terminus) is well suited as a benchmark example for establishing BSI in any laboratory.

In this protocol, we employ BSI to study biomolecular interactions between purified recoverin and the N-terminus of GRK1 containing the first 25 amino acids N-terminally tagged with glutathione-S-transferase (GST-NRK25). We provide a step-by-step protocol including the instrument setup, the detection of signals, designing a binding experiment, and data analysis.

Figure 1. Possible interaction between Recoverin-GRK1. Recoverin is represented by red spheres. The blue spheres represent the C-terminal region of recoverin that interacts with GRK1. GRK1 consists of 558 amino acids and the interaction domain (M1-S25), shown as a yellow coloured helix, is present in the hydrophobic pocket of the protein recoverin.