| Abstract
| - The design, synthesis, and characterization of a folded high-affinity metal-binding peptide isdescribed. Based on the previously described folded peptide NTH-18, in which an α-helix was constrainedthrough two disulfide bonds to a C-terminal extension of noncanonical secondary structure, a peptide (1)was designed to contain two histidine residues in positions 3 and 7. Air oxidation of 1 led to the formationof peptide 2, which contained two intramolecular disulfide bonds. The presence of the two histidinessignificantly destabilized the α-helical structure of 2 when compared to NTH-18. However, CD spectroscopyrevealed that the addition of certain transition metal ions allowed the reformation of a stable α-helix. CD,NMR, and EPR spectroscopy as well as MALDI-TOF mass spectrometry indicated that 2 bound to Cu2+ toform a 1:1 complex via the imidazoles of the two histidine side chains. A glycine displacement assay revealeda dissociation constant for this complex of 5 nM at pH 8, which is the lowest reported value for a designedCu2+-binding peptide. This peptide displayed more than 100-fold selectivity for Cu2+ over Zn2+, Ni2+, andCo2+. The 1.05 Å crystal structure of the Cu(II)-complex of 2 revealed a square-pyramidal coordinationgeometry and confirmed that 2 bound to copper in an α-helical conformation via its two histidine side chains.The high affinity metal binding of peptide 2 demonstrates that metals can be used for the selective nucleationof α-helices.
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