Several 88‐residue proteins were designed, synthesized and examined as receptor‐adhesive modular proteins (RAMPs). Three covalent and two noncovalent dimers were made from two 44‐residue peptide chains containing three structural modules: RGD‐A23a (ligand‐spacer‐coil) and A9a‐RGD (coil‐spacer‐ligand). The ligand module contained the tripeptide Arg‐Gly‐Asp (RGD). The coil modules A9a and A23a were five‐heptad α‐helices engineered by Hodges and co‐workers [Int. J. Peptide Protein Res. (1992) 40, 171–1791, By circular dichroic spectroscopy, each of these five RAMPs contained an α‐helical coiled coil. The disulfide‐bridged dimer RGD‐A23a/RGD‐A23a and its reduced form (RGD‐A23a)2 had two N‐terminal RGD sites. The disulfide‐bridged dimer A9a‐RGD/A9a‐RGD and its reduced form (A9a‐RGD)2 had two C‐terminal RGD sites. However, the disulfide‐bridged heterodimer RGD‐A23a/A9a‐RGD had one RGD site at each terminus with a 50 Å coiled coil between them. The temperature at the midpoint of unfolding for each of the covalent homodimers RGD‐A23a/RGD‐A23a (67°C) and A9a‐RGD/A9a‐RGD (69°C) was slightly higher than that of the corresponding noncovalent homodimer (RGD‐A23a)2 (62°C) or (A9a‐RGD)2 (68°C) but much lower than that of the covalent heterodimer RGD‐A23a/A9a‐RGD (79° C). The enthalpy and entropy of thermal unfolding were also significantly greater for the heterodimer than for the four homodimers, consistent with the heterodimer having the most stable coiled coil. Although the distance between its RGD sites was at least 50 A greater than that for the homodimers, this heterodimeric RAMP was only as active as the homodimers A9a‐RGD/A9a‐RGD and (A9a‐RGD)2 in inhibiting the adhesion of A2058 melanoma cells to extracellular matrix proteins. Thus the coiled‐coil heterodimeric RAMP RGD‐A23a/A9a‐RGD was unable to bind to two integrin receptors on the same cell at the same time. The Hodges coiled coil A23a/A9a was useful as an oriented pair of coil modules for placing ligand modules at the opposite ends of an elongated RAMP.
