The study we present involves the use of a biomimetic system that allows us to study specific interactions in the α5β 1 receptor-GRGDSP ligand system with an atomic force microscope (AFM). Bioartificial membranes that mimic the adhesion domain of the extracellular matrix protein fibronectin are constructed from peptide-amphiphiles. A novel peptide-amphiphile is 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 α 5β1) sequences in a single peptide formulation, separated by a spacer. Two different antibodies are used to immobilize and activate isolated α5β1 integrins on the AFM tip. The interaction measured between immobilized α5β1 integrins and peptide-amphiphiles is specific for integrin-peptide binding and is affected by divalent cations in a way that accurately mimics the adhesion function of the α5β1 receptor. The strength of the PHSRN synergistic effect depends on the accessibility of this sequence to α5β1 integrins. An increase in adhesion is observed compared to surfaces displaying only GRGDSP peptides when the new biomimetic peptide-amphiphiles are diluted with lipidated poly(ethylene glycol), which provides more space for the peptide headgroups to bend and expose more of the PHSRN at the interface.