Kinetic Analysis of a Protein-protein Complex to Determine its Dissociation Constant (KD) and the Effective Concentration (EC50) of an Interplaying Effector Molecule Using Bio-layer Interferometry

Tim Orthwein; Luciano F Huergo; Karl Forchhammer; Khaled A Selim

doi:10.21769/BioProtoc.4152

Kinetic Analysis of a Protein-protein Complex to Determine its Dissociation Constant (K_D) and the Effective Concentration (EC₅₀) of an Interplaying Effector Molecule Using Bio-layer Interferometry

Bio Protoc. 2021 Sep 5;11(17):e4152. doi: 10.21769/BioProtoc.4152.

Authors

Tim Orthwein¹, Luciano F Huergo², Karl Forchhammer¹, Khaled A Selim^{1

3

4}

Affiliations

¹ Organismic Interactions Department, Interfaculty Institute for Microbiology and Infection Medicine, Cluster of Excellence 'Controlling Microbes to Fight Infections', Tübingen University, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
² Setor Litoral, Federal University of Paraná (UFPR), Matinhos, Brazil.
³ Department of Protein Evolution, Max Planck Institute for Developmental Biology, Tübingen, Germany.
⁴ Pharmaceutical and Drug Industries Research Division, National Research Center, Giza, Egypt.

Abstract

Biolayer interferometry (BLI) is an emerging analytical tool that allows the study of protein complexes in real time to determine protein complex kinetic parameters. This article describes a protocol to determine the K_D of a protein complex using a 6×His tagged fusion protein as bait immobilized on the NTA sensor chip of the FortéBio^® Octet K2 System (Sartorius). We also describe how to determine the half maximal effective concentration (EC₅₀, also known as IC₅₀ for inhibiting effectors) of a metabolite. The complete protocol allows the determination of protein complex K_D and small molecular effector EC₅₀ within 8 h, measured in triplicates. Graphic abstract: Principle of the Biolayer interferometry measurement. (Middle, top) Exemplary result of the BLI measurement using Octet^® (Raw Data). Wavelength shift (Δλ) against time. (A) Baseline 1. Baseline measurement. When the sensor is equilibrated in the kinetics buffer. The light is reflected with no difference. (B) Load. The his-tagged proteins (ligand) are loaded onto the sensor surface. The light is reflected with a shift of the wavelength. (C) Baseline 2. The loaded sensor is equilibrated in the kinetics buffer. No further wavelength shift appears. (D) Association. The loaded sensor is dipped into the analyte solution. The analyte binds to the immobilized ligand along with an increased wavelength shift. (E) Dissociation. Afterward, the sensor is dipped again into the kinetics buffer without the analyte. Some analyte molecules dissociate. The wavelength shift decreases. (Subfigures A-E) The left side shows the position of the sensor during the measurement seen in the representative BLI measurement, marked with the figure label. The right side shows the light path in the sensor. Black waves represent the light emitted to the sensor surface. The red waves show the light reflected from the sensor surface back to the detector.

Keywords: EC50; IC50; KD; Binding kinetics; Biolayer interferometry; PII signaling protein; Protein-protein interaction.