Characterization of the interaction of multivalent glycosylated ligands with bacterial lectins by biolayer interferometry

Abstract

The study of multivalent carbohydrate–protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. Bio-Layer Interferometry (BLI) is emerging as a tool to monitor such complex interactions. In this study, various glycoclusters and dendrimers were prepared and evaluated as ligands for lectins produced by pathogenic bacteria Pseudomonas aeruginosa (LecA and Lec B) and Burkholderia ambifaria (BambL). Reliable kinetic and thermodynamic parameters could be measured, and immobilization of either lectin or ligands resulted in high quality data. The methods gave results in full agreement with previous ITC experiments, and presented strong advantages since they require less quantity and purity for the biomolecules.

Multivalent glycocyclopeptides: conjugation methods and biological applications

“…Similar observations were reported more recently with the same compounds and lectin by BioLayer Interferometry. 129,130 This study clearly highlights the interest of glycodendrimer microarrays for the screening of multivalent ligands in parallel with lectins using low quantity of materials and rapid experimental procedures.…”

Luminescent Ruthenium(II) Complexes Used for the Detection of 8-Oxoguanine in the Human Telomeric Sequence

“…The association and dissociation constants between the complexes and the targets were measured, which give access to the dissociation equilibrium constant ( K D ). BLI was first implemented to study interactions between large biomolecules, such as protein–membrane interactions and more recently for the interaction of small compounds with G4 DNA. ,, The studies were done under the same saline conditions than luminescence titrations (10 mM HEPES pH 7.4, 100 mM NaCl, and 50 mM KCl). BLI sensorgrams recorded for the interaction of complex 1 with the hTel and hTel-oxoG10 are displayed in Figure A,B, respectively (see the Supporting Information for the other sensorgrams).…”

“…BLI was first implemented to study interactions between large biomolecules, such as protein−membrane interactions and more recently for the interaction of small compounds with G4 DNA. 38,45,46 The studies were done under the same saline conditions than 5).…”

Cell-free expression and characterization of multivalent rhamnose-binding lectins using biolayer interferometry

“…Finally, we demonstrated the utility of this method for estimating binding kinetics and affinity ( Figure 2C ). As previously described for multivalent lectin-ligand systems with weak dissociation, we estimated the kinetic parameters k on , k off , and k D from the linear relationship between k obs , fit by a 1:1 association model, and the lectin concentration, determined by 14 C-Leucine incorporation ( Figure 2C-D, Figure S6 ) (Picault et al 2022). Values of k on were not affected much by the architecture of the substrates, with variations from 3.5 × 10 −5 M -1 s -1 to 1.9 × 10 −5 M -1 s -1 ( Table S3 ).…”

“…Biolayer interferometry (BLI) is a technique that has been recently used to characterize protein binding parameters without a purification step, indicating possible compatibility with CFE reactions (Pogoutse et al 2016). In addition, BLI has been used to study multivalent lectin-carbohydrate interactions in purified systems (Laigre et al 2018; Picault et al 2022). Notably, this technique requires less material than surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC), is label free, and can be run in a plate-based format, making it more compatible with small-scale cell-free expression (Concepcion et al 2009).…”