Effect of the Tyr5 and His6 substituent groups on the zinc affinities and conformational structures of the acetyl-His1-Cys2-Gly3-Pro4-X5-X6-Cys7 heptapeptides

Abstract

In this study, we investigate the influence of the Tyr₅ and His₆ substituent groups on the zinc-binding affinities and conformational properties of a series of acetylated heptapeptides, acetyl-His₁-Cys₂-Gly₃-Pro₄-X₅-X₆-Cys₇ focusing on the impacts where X₅-X₆ are either Tyr₅-Gly₆, Tyr₅-His₆, Gly₅-Gly₆, or Gly₅-His₆. Utilizing traveling-wave ion mobility-mass spectrometry and molecular modeling techniques we analyze the zinc binding interactions and peptide coordination behavior. The zinc binding peptides (ZBPs) relative zinc affinities were measured across pH 5 to pH 10 by monitoring the solution-phase formation of the [ZBP+Zn(II)]⁻ complex by utilizing native MS in negative ion mode to preserve the solution-phase binding of Zn(II) to the peptides. Furthermore, their relative gas-phase Zn(II) affinities were measured using competitive threshold collision-induced dissociation (TCID) of the [ZBP+Zn(II)+NTA]⁻ complex, by modeling the two competing dissociation channels: [ZBP+Zn(II)]⁻ + NTA or [Zn(II)+NTA]⁻ + ZBP, where NTA is nitrilotriacetic acid. Our examinations also tested whether there was an effect of the formation of the [ZBP+Zn(II)+NTA]⁻ complexes from solutions at different pHs, before they are electrosprayed into the gas-phase for the TCID analyses. Both solution- and gas-phase measurements predicted the heptapeptide with the Gly₅-His₆ residues had the greatest zinc affinity and that the presence of Tyr₅ and His₆ altered the zinc affinity and induced distinct conformational changes due to changes in the coordination of the zinc. This research enhances our understanding of zinc-peptide interactions, with implications for the design of peptide-based metalloproteins, which may guide the design of novel ZBPs for therapeutic, biotechnological or environmental remediation applications.