Mixed monolayers to promote g-protein adsorption: alpha2A-adrenergic receptor-derived peptides coadsorbed with formyl-terminated oligopeptides.
Pure and mixed monolayers of a synthetic peptide, GPR-i3n, derived from the third intracellular loop of the alpha2 adrenergic receptor and a shorter inactive oligopeptide, N-formyl-(Gly)3-(Cys) (called 3GC), were prepared on gold surfaces. The mixing ratio of the GPR-i3n and 3GC was used to control G-protein binding capability. The GPR-i3n peptide is specially designed for bovine G-protein selectivity and has been proven to have high affinity to G-proteins [Vahlberg, C.; Petoral, R. M., Jr.; Lindell, C.; Broo, K.; Uvdal, K. Langmuir 2006, 22 (17), 7260-7264]. Pure 3GC monolayers show very low protein adsorption capability. In this study, 3GC is chosen as a coadsorbent, with the aim to induce molecular conformational changes during monolayer formation to enhance G-protein adsorption. A full characterization of the mixed monolayers was done. The monolayer thickness and the mass-related surface coverage for both GPR-i3n and 3GC were investigated using radio labeling. The GPR-i3n was labeled by 125I-targeting tyrosine, and the activity was measured by using radioimmunoassay (RIA). The formation and chemical composition of GPR-i3n and 3GC monolayers were investigated using X-ray photoelectron spectroscopy, and it is shown that both GPR-i3n and 3GC bind chemically to the gold surface. The interaction between the mixed monolayers and G-proteins was investigated by means of real-time surface plasmon resonance. There is a higher protein binding capacity to the monolayer when the GPR-i3n peptide is intermixed with the 3GC coadsorbent, despite the fact that the 3GC itself has a very low G-protein binding capability. This supports a molecular reorientation at the surface, while 3GC is intermixed with GPR-i3n.
Balau
Luminita Savitchi
LS
Division of Sensor Science and Molecular Physics, Department of Physics, Chemistry and Biology, IFM, Linköping University, SE-581 83 Linköping, Sweden.
Vahlberg
Cecilia
C
Petoral
Rodrigo M
RM
Uvdal
Kajsa
K
D000327:Adsorption; D000818:Animals; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D006801:Humans; D008567:Membranes, Artificial; D009842:Oligopeptides / Q000737:chemistry*; D011485:Protein Binding; D011863:Radioimmunoassay; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry*; D020349:Surface Plasmon Resonance
C547768:ADRA2A protein, human; D008567:Membranes, Artificial; D009842:Oligopeptides; D018341:Receptors, Adrenergic, alpha-2; D006046:Gold; D019204:GTP-Binding Proteins