Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. Here, we describe a strategy for designing oligomers containing both α- and β-amino acid residues (“α/β-peptides”) that mimic several peptides derived from the three-helix bundle “Z-domain” scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF₁₆₅-induced proliferation of human umbilical vein endothelial cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Such reagents would be useful for diagnostic and therapeutic applications.

L- to D-residue isomerization is a post-translational modification (PTM) present in neuropeptides, peptide hormones, and peptide toxins from several animals. In most cases, the D-residue is critical for the biological function of the resulting D-amino acid–containing peptide (DAACP). Here, we provide an example in native neuropeptides in which the DAACP and its all-L-amino acid epimer are both active at their newly identified receptor in vitro and at a neuronal target associated with feeding behavior. On the basis of sequence similarity to a known DAACP from cone snail venom, we hypothesized that allatotropin-related peptide (ATRP), a neuropeptide from the neuroscience model organism Aplysia californica, may form multiple diastereomers in the Aplysia central nervous system. We determined that ATRP exists as a D-amino acid–containing peptide (D2-ATRP) and identified a specific G protein–coupled receptor as an ATRP receptor. Interestingly, unlike many previously reported DAACPs and their all-L-residue analogs, both L-ATRP and D2-ATRP were potent agonists of this receptor and active in electrophysiological experiments. Finally, D2-ATRP was much more stable than its all-L-residue counterpart in Aplysia plasma, suggesting that in the case of ATRP, the primary role of the L- to D-residue isomerization may be to protect this peptide from aminopeptidase activity in the extracellular space. Our results indicate that L- to D-residue isomerization can occur even in an all-L-residue peptide with a known biological activity, and that in some cases, this PTM may help modulate peptide signal lifetime in the extracellular space rather than activity at the cognate receptor.