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Three analog methanobactin (amb7) peptides containing the primary structure acetyl-Leu1-His2-Cys3-Gly4-Ser5-Cys6-Tyr7-Pro8-His9-Cys10-Ser11-Cys12-Met13 but differing by the number of disulfide bridges, have been studied for their reactivity with Fe(III), Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Ag(I), and Pb(II) using electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) and fluorescence quenching. The Pb(II) bound only with the amb7-0 (0 disulfide bonds), Zn(II) only to amb7-0 and amb7-1 (1 disulfide bond), Ni(II) only to amb7-1 and amb7-2 (2 disulfide bonds), and Cu(II) to amb7-2, concomitant with the oxidation of amb7-0 and amb7-1. The reduction of Cu(II) to Cu(I) resulted with 1-4Cu(I) binding to amb7-1 and amb7-0, while Ag(I) also exhibited 1-6 Ag(I) ions binding through a mechanism indicative of labile proton exchange. The binding of the oxidized Co(III/V) and Mn(III/V) were observed at pH 9 and 11, while Fe(III) did not exhibit significant binding. The observed negatively charged complexes were consistent with the deprotonation of the metal sites of Cys, His, carboxyl terminus and indicative of ion-ion interactions with the metal at pH 7, 9, and 11. The positively charged complexes were indicative of ion-dipole metal binding but at pH 9 and pH 11 gave results similar to those of the negative ion analyses. Fluorescence quenching also qualitatively agreed with the ESI-IM-MS results providing evidence that they revealed the solution-phase metal binding behavior of amb7.

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide's mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide's acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Methanobactin (Mb) from Methylosinus trichosporium OB3b is a member of a class of metal binding peptides identified in methanotrophic bacteria. Mb will selectively bind and reduce Cu(II) to Cu(I), and is thought to mediate the acquisition of the copper cofactor for the enzyme methane monooxygenase. These copper chelating properties of Mb make it potentially useful as a chelating agent for treatment of diseases where copper plays a role including Wilson's disease, cancers, and neurodegenerative diseases. Utilizing traveling wave ion mobility-mass spectrometry (TWIMS), the competition for the Mb copper binding site from Ag(I), Pb(II), Co(II), Fe(II), Mn(II), Ni(II), and Zn(II) has been determined by a series of metal ion titrations, pH titrations, and metal ion displacement titrations. The TWIMS analyses allowed for the explicit identification and quantification of all the individual Mb species present during the titrations and measured their collision cross-sections and collision-induced dissociation patterns. The results showed Ag(I) and Ni(II) could irreversibly bind to Mb and not be effectively displaced by Cu(I), whereas Ag(I) could also partially displace Cu(I) from the Mb complex. At pH ≈ 6.5, the Mb binding selectivity follows the order Ag(I)≈Cu(I)>Ni(II)≈Zn(II)>Co(II)>>Mn(II)≈Pb(II)>Fe(II), and at pH 7.5 to 10.4 the order is Ag(I)>Cu(I)>Ni(II)>Co(II)>Zn(II)>Mn(II)≈Pb(II)>Fe(II). Breakdown curves of the disulfide reduced Cu(I) and Ag(I) complexes showed a correlation existed between their relative stability and their compact folded structure indicated by their CCS. Fluorescence spectroscopy, which allowed the determination of the binding constant, compared well with the TWIMS analyses, with the exception of the Ni(II) complex. Graphical abstract ᅟ.