Abstract
Reliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the binding kinetics of Ni(II) ions to genetically modified flagellin layers, we demonstrate that: (1) Grating-Coupled Interferometry (GCI) is well suited to resolve the binding of ions, even at very low protein immobilization levels; (2) it supplies high quality kinetic data from which the number and strength of available binding sites can be determined, and (3) the rate constants of the binding events can also be obtained with high accuracy. Experiments were performed using a flagellin variant incorporating the C-terminal domain of the nickel-responsive transcription factor NikR. GCI results were compared to affinity data from titration calorimetry. We found that besides the low-affinity binding sites characterized by a micromolar dissociation constant (Kd), tetrameric FliC-NikRC molecules possess high-affinity binding sites with Kd values in the nanomolar range. GCI enabled us to obtain real-time kinetic data for the specific binding of an analyte with molar mass as low as 59 Da, even at signals lower than 1 pg/mm2.
Highlights
Reliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task
While Grating-Coupled Interferometry (GCI) has already shown its potential in the binding kinetics measurement of a small polyphenol molecule, the 458 Da epigallocatechin gallate (EGCG)[17], too, there are no examples in the scientific literature of exploiting its top-level sensitivity below molar mass of 60 Da
We have recently shown that flagellin or flagellin-based fusion proteins rapidly form an oriented, dense and stable monolayer on hydrophobic surfaces, where the hypervariable D3 domain is oriented towards the liquid phase[51,52]
Summary
Reliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. The recently developed grating-coupled interferometry (GCI) is a hybrid phase-shifting Mach–Zehnder interferometer, which can present several advantages over other optical biosensor techniques It employs a simple waveguide-grating structure, moving parts are completely eliminated decreasing noise levels, the incorporated coupling gratings possess high alignment tolerance, and the applied T a2O5 waveguide has high refractive index contrast with a relatively long interaction path with the sample. While GCI has already shown its potential in the binding kinetics measurement of a small polyphenol molecule, the 458 Da epigallocatechin gallate (EGCG)[17], too, there are no examples in the scientific literature of exploiting its top-level sensitivity below molar mass of 60 Da. For demonstrating the performance of the GCI technology to study the binding kinetics of ions, we selected a newly developed genetically modified flagellin variant with Ni-binding ability, which can be potentially applied in further bio- and chemical sensor applications
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