The ability of two novel ruthenium(II) polypyridyl-Arg-Gly-Asp (RGD) peptide conjugates to act as molecular probes for reporting on the presence and conformation of integrin alpha(IIb)beta(3) in solution and in live cells was described. The compounds are [Ru(bpy)(2)PIC-RGD](2+); bpy-RGD, and [Ru(dpp)(2)PIC-RGD](2+), dpp-RGD, where dpp is 4,7-diphenyl-1,10-phenanthroline, bpy is 2,2'-bipyridine, and PIC is 2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline. Bpy-RGD is hydrophilic, whereas dpp-RGD is comparatively hydrophobic. Both probes exhibited good affinity and high specificity for purified alpha(IIb)beta(3) in solution. Binding of either complex to the resting integrin resulted in an approximately 8-fold increase of emission intensity from the metal center with dissociation constants (K-d) in the micromolar range for each complex. The K-d for each conjugate/alpha(IIb)beta(3) assembly were compared following treatment of the integrin with the activating agents, Mn2+ and dithiothreitol (DTT), which are commonly used to induce active-like conformational changes in the integrin. For bpy-RGD/alpha(IIb)beta(3) K-d showed relatively little variation with integrin activation, presenting the following trend: denatured alpha(IIb)beta(3) > resting alpha(IIb)beta(3) = pretreated DTT = pretreated Mn2+. K-d for dpp-RGD/ alpha(IIb)beta(3) showed greater variation with integrin activation and the following trend was followed: denatured alpha(IIb)beta(3) > resting alpha(IIb)beta(3) > pretreated Mn2+ = pretreated DTT. Time resolved luminescence anisotropy was carried out to obtain the rotational correlation time of bpy-RGD and dpp-RGD bound to resting or nominally activated integrin. The rotational correlation times of bpy-RGD and dpp-RGD, too fast to measure unbound, decreased to 1.50 +/- 0.03 mu s and 2.58 +/- 0.04 mu s, respectively, when the complexes were bound to resting integrin. Addition of Mn2+ to bpy-RGD/alpha(IIb)beta(3) or dpp-RGD/alpha(IIb)beta(3) reduced the rotational correlation time of the ruthenium center to 1.29 +/- 0.03 mu s and to 1.72 +/- 0.03 mu s, respectively. Following treatment, the rotational correlation time decreased to 1.04 +/- 0.01 mu s and 1.29 +/- 0.03 mu s for bpy-RGD/alpha(IIb)beta(3), and dpp-RGD alpha(IIb)beta(3), respectively. The large relative changes in rotational correlation times observed for Mn2+ or DTT activated integrin indicates significant change in protein conformation compared with the resting integrin. The results also indicated that the metal complex itself affects the final conformational and/or aggregation status of the protein obtained. Furthermore, the extent of conformational change was influenced by whether the probe was bound to the integrin before or after activator treatment. Finally, in vitro studies indicated that both probes selectively bind to CHO cells expressing the resting form of alpha(IIb)beta(3). In each case the probe colocalized with alpha(IIb) specific SZ22 antibody.
Overall, this work indicates that bpy-RGD and dpp-RGD may be useful peptide-probes for rapid assessment of integrin structural status and localization in solution and cells.