Use of Open Surface Plasmon Resonance (OpenSPR) to Characterize the Binding Affinity of Protein–Protein Interactions

Background

In order to understand complex mechanisms underlying the regulation of various biological processes, it is often essential to characterize protein–protein interactions under many physiological or pathological conditions. For many years, surface plasmon resonance (SPR) has been widely used to analyze all types of interactions between protein–protein or protein and other molecules such as nucleic acids, lipids, and carbohydrates (Willander and Al-Hilli, 2009; Drescher et al., 2018). Traditionally, Biacore SPR uses a continuous film of gold to detect changes in the angle of re-emitted light when the refractive index of a ligand conjugated to the flat gold surface is altered by its interaction with a local analyte (Willander and Al-Hilli, 2009; Drescher et al., 2018). In contrast, the newly developed Nicoya Lifesciences’ OpenSPR employs gold nanoparticles to detect small changes in the absorbance peak wavelength of a conjugated ligand after its binding to an analyte. Nevertheless, both traditional SPR and contemporary OpenSPR can give real-time information about the binding kinetics and affinities of dynamic interactions between various molecules.

During the last few years, we have used Nicoya Lifesciences’ first generation 1-channel OpenSPR to determine the binding affinities between antigens and antibodies (Chen et al., 2020) or cytokines and putative receptors (Qiang et al., 2022; Zhu et al., 2023). Briefly, a ligand protein is immobilized onto nitrilotriacetic acid (NTA)-conjugated gold nanoparticles in a specific orientation, and an analyte solution is subsequently injected at several increasing concentrations into the microflow system. When broadband white light is shone onto the gold nanoparticles, it produces a strong resonance absorbance peak that is specific to the refractive index of a local ligand conjugated to the gold nanoparticles. If an analyte binds the ligand, it will induce a change in the wavelength of the absorbance peak of the ligand, which can be recorded in the sensorgram as an increase in the SPR signal. After a desired association time, a solution without the analyte is injected to dissociate the complex between ligand and analyte. If the analyte dissociates from the ligand, a decrease in SPR signal would be observed. By dividing the dissociation rate (k_off) by the association rate (k_on), the equilibrium dissociation constant (K_D) can be calculated as a measure of the affinity between these specific ligand–analyte interactants. Thus, the improvement in the detection method (from traditional detection of changes in the angle of re-emitted light to the contemporary detection of changes in the wavelength of the absorbance peak) features OpenSPR as a cost-effective and user-friendly technique for in-depth characterization of protein–protein interactions. Here, we describe the detailed method that we used to characterize procathepsin L (pCTS-L) interaction with two putative pattern recognition receptors (TLR4 and RAGE) using Nicoya Lifesciences’ 1st generation OpenSPR instrument with a 1-channel detection (Zhu et al., 2023).