pH dependent chelation study of Zn(II) and Ni(II) by a series of hexapeptides using electrospray ionization – Ion mobility – Mass spectrometry

Formation of Co(II), Ni(II), Zn(II) complexes of alternative metal binding heptapeptides and nitrilotriacetic acid: Discovering new potential affinity tags

“…Nickel(II) is known to be able to anchor to a side group and bind to deprotonated amide groups in square planar geometry as does Cu(II), 26 although this type of binding requires a low spin state for Ni(II). 14,15 The H and N species, that are related to I but have Cys 2 rather than Asp 2 , did not form [amb 5 -4H+Ni(II)] 2and only formed 5% of [amb 5 -3H+Ni(II)] -, which indicates that at pH 7.0 the Cys 2 is not an effective binding site for Ni(II) and it remains primarily protonated. For species H and N, the Cys 7 resides by His 6 a base that could decrease its effective pKa, while Cys 2 resides by an acidic group Asp 1 that could increase its effective pKa.…”

“…Our previous ESI-IM-MS amb analyses, have shown that the negatively-charged complexes are of particular interest because they correlate to the charge states that relate to the protonation state of the Cys, His, or carboxyl termini which are the potential metal ligand sites and they exhibit a distinct pH-dependence. [8][9][10][11][12][13][14][15] In Figure 3 we show the identity and the relative percentage of these negatively charged complexes for amb 5 G to R. Only G primarily formed the [amb 5 -6H+2Ni(II)] 2complex that related to six negatively-charged sites on the amb 5G peptide. Species G, as in the Co(II) analyses, also formed the negatively charged dimer binding 2Ni(II), [diamb 5ox -6H+2Ni(II)] 2-, where the 2Cys oxidized to form a disulfide bond.…”

“…A new Zn(II) trans-Pro complex was also discovered that although higher in free energy than the cis-Pro conformers, exhibited a DFT CCS He in good agreement with the ESI-IM-MS measured CCS He with Zn(II) coordinated via the substituent groups of the His 1 -Cys 2 -Cys 7 and the carboxylate terminus. 13,15 Further studies of the Zn(II) and Ni(II) chelation by a similar series of modified amb 1A-H peptides, 14 and comparison with the amb 5A-F peptides showed that the additional Tyr in the amb 5 structures contributed to their greater selectivity for Zn(II) at pH 7.0. Why the amb 5 peptides had a preference for chelating Zn(II) over Ni(II) at pH 7.0 is further studied here by extending the amb 5 series to include twelve new primary structures to determine whether differences in their primary structure will influence The primary structure of alternative metal binding-5A (amb 5A ) with the color highlighting the regions modified with respect to the listed primary structures of amb 5 peptides A to R. The structure of amb 5P is also shown, which is the 7xHis-tag.…”

“…Previous electrospray -ion mobility -mass spectrometry (ESI-IM-MS) studies of methanobactin 6,7 from Methylosinus trichosporium (mb-OB3b) and the amb peptides whose primary structure replaced the two enethiol oxazolone of mb-OB3b with the 2His-2Cys substituent groups of a zinc finger protein, have shown their potential for applications as affinity tags. [8][9][10][11][12][13][14][15] For example, the ESI-IM-MS analyses of the amb peptides shows that their formation of metal complexes are dependent on pH which is important for controlling the capture and release of tagged proteins in an IMAC column. A major attribute for ESI-IM-MS analyses is that it allows for the pH-dependent behavior of specific m/z conformers to be examined.…”

“…For example, the negative ion analysis of amb 1 , ac-His 1 -Cys 2 -Gly 3 -Pro 4 -His 5 -Cys 6 , from solutions held at pH 7 showed the predominant species is the singly-charged [amb 1 -H] -, but from solution > pH 8, the predominant species becomes the complex in the form of the [amb 1 -3H+Zn] -. 8,14 These two species correlate to solution behavior because at pH 7 the observed [amb 1 -H]relates to the deprotonation of the carboxyl terminus (pK a ~2.0) and the two His imidazoliums (pK a ~6.0), while at pH >8, the addition of deprotonation of the two Cys (pK a ~8.3) allows for the chelation of Zn(II) and the formation of the [amb 1 -3H+Zn]complex. Moreover, the traveling wave ESI-IM-MS measured collision cross section (CCS He ) of the [amb 1 -3H+Zn]complex is consistent with the tetrahedral coordination of Zn(II) via the 2Cys, His 6 and carboxyl terminus, which is also indicative of the metal complex conformation being preserved during the ESI transfer into the gas phase.…”