Nanoliter contact angle probes tumor angiogenic ligand-receptor protein interactions.
Any molecular recognition reaction supported by a solid phase drives a specific change of the solid-solution interfacial tension. Sessile contact angle (CA) experiments can be readily used to track this thermodynamic parameter, prompting this well-known technique to be reinvented as an alternative, easy-access and label-free way to probe and study molecular recognition events. Here we deploy this technique, renamed for this application CONAMORE (CONtact Angle MOlecular REcognition), to study the interaction of the tumor-derived pro-angiogenic vascular endothelial growth factor-A (VEGF-A) with the extracellular domain of its receptor VEGFR2. We show that CONAMORE recognizes the high affinity binding of VEGF-A at nanomolar concentrations to surface-immobilized VEGFR2 regardless of the presence of a ten-fold excess of a non-specific interacting protein, and that it further proofs its specificity and reliability on competitive binding experiments involving neutralizing anti-VEGF-A antibodies. Finally, CONAMORE shows the outstanding capability to detect the specific interaction between VEGFR2 and low molecular weight ligands, such as Cyclo-VEGI, a VEGFR2 antagonist cyclo-peptide, that weighs about 2 kDa.
Olivero
Giulio
G
Chemistry for Technologies Laboratory and INSTM, University of Brescia, Via Branze, 38, 25123 Brescia, Italy.
Maiolo
Daniele
D
Leali
Daria
D
Federici
Stefania
S
Depero
Laura E
LE
Presta
Marco
M
Mitola
Stefania
S
Bergese
Paolo
P
D042501:Angiogenic Proteins / Q000378:metabolism*; D016228:Endothelial Growth Factors / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D055767:Immobilized Proteins; D066298:In Vitro Techniques; D008024:Ligands; D010456:Peptides, Cyclic / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D013816:Thermodynamics; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D042501:Angiogenic Proteins; D016228:Endothelial Growth Factors; D055767:Immobilized Proteins; D008024:Ligands; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C467484:VEGFA protein, human; C513782:VEGFR-2-Fc chimeric protein; D042461:Vascular Endothelial Growth Factor A; C478485:cyclo-VEGI