Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

Summary

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its ε subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with ε’s inhibitory C-terminal domain.

Abstract

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit ε with the catalytic complex of Escherichia coli ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit ε could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of ε undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of ε's conformations is predominant. The assay measures kinetics of ε's binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound ε to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on ε's conformational changes.

Introduction

Protein-protein interactions are important for many biological processes, and label-free optical methods like Surface Plasmon Resonance (SPR) have been used in vitro to study kinetics of binding and dissociation¹. Most label-free methods immobilize one biomolecule on a sensor surface and use an optical signal to detect a binding partner from solution as it associates with the immobilized biomolecule¹. While SPR is a highly sensitive method, it is prone to interference due to changes in the refractive index of the solution flowing over the sensor². Although not as sensitive as SPR, Bio-layer Interferometry (BLI) is less affecte....

Protocol

1. Programming the Instrument for BLI Assay

Turn on the instrument at least one hour in advance to allow the lamp to warm up; this is necessary to minimize noise and drift in optical signal during the experiment. Set the desired temperature via the instrument tab to prewarm the sample plate holder. Then set up the experimental design in the Data Acquisition software. Select "New Kinetics Experiment" in the Experiment Wizard tab. This presents a tabbed menu with all steps that must be defined.

1.1. Plate Definition

Define the columns to be used on the 96-well sample plate. Assign columns to ....

Results

Real time binding and dissociation BLI kinetics are shown in Figure 3. This experiment was done with assay buffer in association and dissociation. This experiment was started with a 10 min baseline step since the sensors had been prewet only briefly. Next, biotinylated ε was loaded on the sensors. No detectable dissociation of ε occurred throughout all remaining steps as seen from the reference curve (G) which had no binding partner added. A second reference sensor (H) was devoid of immobilized protein a.......

Discussion

Currently available instruments for BLI allow significant throughput and flexibility in assays for biomolecular interactions. Various solution samples are dispensed in wells of a black microtiter plate, and a set of parallel BLI sensors are programmed to move back and forth between columns of wells on the plate. The samples are stirred by orbital shaking throughout the assay. The system used here has 8 sensors and uses a 96-well sample plate, but another system uses 16 sensors and a 384-well sample plate. Thus, interacti.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Octet-RED96	Pall/FortéBio	30-5048
Bovine Serum Albumin	Sigma	A6003-10G	Fatty Acid free
Biosensor/Streptavidin	Pall/FortéBio	18-5019	Tray of 96 sensors
Microtiter plate	Greiner Bio-one	655209	Black, Polypropylene
Data Acquisition software	Pall/FortéBio	Version 6.4	Newer versions available
Data Analysis software	Pall/FortéBio	Version 6.4	Newer versions available