Detailed Analysis of Kinetic Binding Traces with Distributions of Surface Sites

Abstract

In kinetic surface binding experiments, deviations from the ideal exponential binding traces are commonly observed. Often, these can be attributed to the intrinsic heterogeneity of the binding properties of the immobilized molecules. Surface binding traces can be fitted with a mathematical model consisting of a heterogeneous distribution of surface sites with quasi-continuous affinity constants and kinetic rate constants. It is implemented in the software EVILFIT both for reaction-controlled binding and, if required, with approximate corrections in the onset of mass transport limitation. Statistical tools prevent over-interpretation and highlight the information content of the data. Examples illustrate impact of the choice of surface chemistry on the surface sites, and also factors such as immobilization density and time after immobilization.

Catabolite repression control protein antagonist, a novel player in Pseudomonas aeruginosa carbon catabolite repression control

“…The determined K D2 value (112.310 nM; Figure 4B) is comparable with the dissociation constant revealed in solution by microscale thermophoresis (134.6 nM) (Sonnleitner et al, 2018). The best fitted binding kinetic for the Hfq/amiE 6ARN /Crc complex deviated from the ideal 1:1 kinetic, which is often observed in surface binding experiments and mainly due to mass transport limitations or heterogeneity of the surface/binding sites (Schuck and Zhao, 2010;Zhao and Schuck, 2017). As Crc is immobilized on the surface, it is possible that not all interaction sites were available for multivalent binding of Crc to Hfq/amiE 6ARN (Pei et al, 2019), which might explain the observed heterogeneous binding affinities.…”

Surface Plasmon Resonance Sensors for Concentration and Reaction Kinetic Detections

“…The pre-immobilized receptors can be divided into two portions, free receptors on the sensor chip surface (B) and receptors have already been combined with analytes (AB). The formation rate of AB can be expressed as [25,26].…”

Dual-Valve and Counter-Flow Surface Plasmon Resonance

“…Surface plasmon resonance (SPR) has been demonstrated as a technique of choice for real-time determinations of binding affinity and kinetics between biologically important molecules. − SPR has gained additional attention in the pharmaceutical industry, because it is viable for detecting binding of small molecules to purified proteins or receptors at cell membranes . Besides, SPR detection does not require the addition of an external or exogenous label to small molecules, which could significantly change the binding behavior.…”