A biomimetic system was used to study interactions of the α5β1 receptor with its ligand with an atomic force microscope (AFM). Bioartificial membranes, which display peptides that mimic the cell adhesion domain of the extracellular matrix protein fibronectin, are constructed from peptide-amphiphiles. A novel peptide-amphiphile was designed that contains both GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro, the primary recognition site for α5β1) and PHSRN (Pro-His-Ser-Arg-Asn, the synergy binding site for α5β1) sequences in a single peptide formulation, separated by a spacer. The strength of the PHSRN synergistic effect depends on the accessibility of this sequence to α5β1 integrins. The interaction measured with the immobilized α5β1 integrins and GRGDSP peptide-amphiphiles is found to be specifically related to the integrin-peptide binding. It is affected by divalent cations in a way that accurately mimics the adhesion function of the α5β1 receptor. The dissociation of single α5β1-GRGDSP pairs under loading rates of 1-305 nN/s revealed the presence of two activation energy barriers in the unbinding process. The high-strength regime above 59 nN/s maps the inner barrier along the direction of the force. Below 59 nN/s a low strength regime appears with an outer barrier and a much slower transition rate that defines the dissociation rate (off-rate) in the absence of force (koff0=0.015 s-1).
- Kokkoli, E., and Mardilovich, A. "The Use of Atomic Force Microscopy in Characterizing Ligand-Receptor (α5β1 Integrin) Interactions", Applications of Scanned Probe Microscopy to Polymers, 2005, 897: 182-192.
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