Detailed AFM Force Spectroscopy of the Interaction Between CD44–IgG Fusion Protein and Hyaluronan

Abstract

Atomic force microscopy (AFM) force spectroscopy was used to study the single-molecule rupture events of the interaction between hyaluronan (HA) and the binding domain of its cell surface receptor CD44. AFM probes were amino terminated with 3-aminopropyl triethoxy silane (APTES) followed by covalent coupling of protein A, enabling the binding of the CD44–HA-binding domain, as part of a CD44–Fc fusion protein. HA was covalently bound to APTES-coated silicon surfaces. Single-rupture events were recorded at various loading rates revealing an energy barrier: Eb = 24 ± 1 kT and characteristic distance: xβ = 1.3 ± 0.1 nm for this interaction. This quantification will be of interest in applications and research involving the use of the CD44–Fc fusion protein since we observe a weaker interaction between HA and CD44–Fc than what has been reported for the entire native CD44 molecule.