d-Amino Acid Scan of Two Small Proteins

Abstract

A "D-scan" of two small proteins, the disulfide-rich Ecballium elaterium trypsin inhibitor II (EETI-II) and a minimized Z domain of protein A (Z33), is reported. For each protein, the stereochemistry of one amino acid at a time was inverted to generate a series of diastereomers. In much the same way an alanine scan determines necessary residues for protein function, the D-scan elucidated the critical stereocenters of the 30-residue EETI-II and the 33-residue Z33. The folding properties and activity of each variant were investigated. A total of 24 out of 30 EETI-II D-scan analogues folded to give a three-disulfide product. Of the 24 variants that folded, half were high-affinity trypsin inhibitors, and three were as active as the wild type (WT). Of these 12 active variants, most were substantially less stable to reduction than WT EETI-II (WT first reduction potential -270.0 ± 1.5 mV, WT second reduction potential -307.2 ± 1.1 mV). Similarly, ten Z33 analogues retained high binding affinity to IgG (KD < 250 nM, WT: 24 ± 1 nM) and 12 additional analogues had reduced but appreciable IgG binding affinity (KD between 250 nM and 2.5 μM). As with EETI-II, most Z33 analogues were substantially less stable than the WT (ΔG(H2O, 263 K) = 2.4 ± 1.2 kcal/mol). Collectively, our findings show that the D-scan is powerful new strategy for studying how the stereochemistry of amino acids affects the structure and function of proteins.

Iterative Nonproteinogenic Residue Incorporation Yields α/β‐Peptides with a Helix–Loop–Helix Tertiary Structure and High Affinity for VEGF

“…Identifying replacement sites among the original proteinogenic residues has required us to accommodate not only points of contact with the target surface on VEGF but also intramolecular packing interactions. The difficulty of this engineering challenge is highlighted by a recently reported d ‐residue scan of a 33‐residue peptide derived from the Z‐domain . This fundamental study revealed that substitution of a single l ‐α residue with its enantiomer at the majority of positions caused a profound decline in affinity for the target protein (an IgG fragment).…”

“…Those prior efforts were limited to mimicry of an isolated secondary structure (a single helix), whereas the approach that delivered high‐affinity VEGF‐binding α/β‐peptides had to accommodate a specific tertiary structure. The challenges of implementing backbone modifications in the context of a discrete tertiary structure are highlighted by a recently described d ‐α‐amino acid scan of a disulfide‐crosslinked, two‐helix Z‐domain derivative; single l → d residue replacements at most of the sites led to a dramatic decline in affinity for the target protein (IgG). We speculate that the principles illustrated by the iterative development process documented here might prove to be useful for generating target‐specific α/β‐peptide ligands based on other tertiary structures.…”

Stereochemical inversion as a route to improved biophysical properties of therapeutic peptides exemplified by glucagon

“…Modifications at the first 7 residues of the N terminus (analogs 1-7) had only a modest effect on fibrillation, while those at position 8, 12, 17 and 28 exhibited a 2-9-fold increase in ThT fluorescence relative to that of native glucagon (4). In contrast, stereochemical inversion at positions 9,11,14,16,19,20,21,23 and 27 appeared to suppress fibrillation, as indicated by an absence of measurable ThT fluorescence.…”

“…More recently, a systematic D-amino acid scan was used to identify GLP-2 (glucagon-like peptide-2) analogs with enhanced pharmacokinetics 15 . Consequently, a D-amino acid scan 16,17 conducted alongside the more common alanine scan 18,19 can serve to more fully interrogate the relationship between structure and function in novel peptides and proteins 20,21 .…”

Heterochiral Knottin Protein: Folding and Solution Structure

“…8–10 Recently, d -amino acid scans have revealed that they may be more tolerated in natural proteins. 11 Horne and co-workers have also demonstrated that in addition to d -amino acids, other types of unnatural amino acids, including β -amino acids, can be incorporated into proteins while preserving tertiary folding. 12 …”