Immobilized peptides as high affinity capture reagents for multimeric proteins and structural studies of cell-targeting peptides

Protein ligands can be employed as capture reagent in the manufacture of protein-detecting microarrays. Since it would be advantageous to detect low protein concentrations from a complex mixture, the capture reagent must bind the target protein with high specificity as well as high affinity. The use of peptides as capture reagents is appealing because they are easily synthesized and modified to allow precise attachment to a solid surface. However, peptides isolated by combinatorial methods usually bind their target protein with high specificity but low affinity. Therefore, increasing the affinity of peptides toward their target protein would be beneficial for microarray technology. This can be accomplished by promoting two points of contact between the target and peptide ligand. To achieve high affinity, bidentate, binding in solution generally requires careful optimization of the linker connecting the two contact elements. However, we propose that by immobilization of two binding elements on a surface, high affinity binding could be achieved without optimized linkers since the surface itself would provide essentially all possible geometric arrangements of the contact elements. Indeed, we show that linear peptides attached to a solid surface will act as tenacious capture reagents for multimeric proteins. In addition, peptides are useful for cell targeting. Several cell-binding peptides have been isolated by screening combinatorial peptide libraries displayed on the surface of filamenteous phage. Johnston and coworkers isolated a phage-displayed peptide that not only bound, but also entered, fibroblast cells through endocytosis triggered by the clustering of cell-surface receptor molecules. Results obtained by Johnston and coworkers suggest that the cell-binding peptides may be native dimers, and therefore aid receptor clustering. To probe this point a structural characterization of the fibroblast binding peptide was performed using circular dichroism (CD), twodimensional nuclear magnetic resonance (2D-NMR), and sedimentation equilibrium. It is shown that the fibroblast binding peptide does form an α-helical dimer.