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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.