A minimal peptide scaffold for beta-turn display: Optimizing a strand position in disulfide-cyclized beta-hairpins

Phage display of peptide libraries has become a powerful tool for the evolu tion of novel ligands that bind virtually any protein target. However, the rules governing conformational preferences in natural peptides are poorly u nderstood, and consequently, structure-activity relationships in these mole cules can be difficult to define. In an effort to simplify this process, we have investigated the structural stability of 10-residue, disulfide-constr ained beta -hairpins and assessed their suitability as scaffolds for beta - turn display. Using disulfide formation as a probe, relative free energies of folding were measured for 19 peptides that differ at a one strand positi on. A tryptophan substitution promotes folding to a remarkable degree. NMR analysis confirms that the measured energies correlate well with the degree of beta -hairpin structure in the disulfide-cyclized peptides. Reexaminati on of a subset of the strand substitutions in peptides with different turn sequences reveals linear free energy relationships, indicating that turns a nd strand-strand interactions make independent, additive contributions to h airpin stability. Significantly, the tryptophan strand substitution is high ly stabilizing with all turns tested, and peptides that display model turns or the less stable C'-C " turn of CD4 on this tryptophan "stem" are highly structured beta -hairpins in water. Thus, we have developed a small, struc tured beta -turn scaffold, containing only natural L-amino acids, that may be used to display peptide libraries of limited conformational diversity on phage.