Analysis of methanobactin from Methylosinus trichosporium OB3b via ion mobility mass spectrometry.

Methanobactins (mbs) are Low molecular mass copper binding chromopeptides analogous to pyoverdin class iron-binding siderophores. Mb produced by Methylosinus trichosporium OB3b (mb-oB3b) has been used as a model molecuLe for methanobactin although the amino acid sequence of mb-OB3b differs significantly from other characterized mbs. In particular, there is the presence of a pair of cystine residues which are absent in other characterized mbs. The role of the Cys3-Cys6 in copper binding, Cu(ll) reduction and its role on the mb-OB3b structure remains in debate. Here, we use a single-step dithiothreitol treatment as an effective method in reducing the disulfide bond allowing in-depth ion mobility-mass spectrometry (IM-MS) analysis. The IM-MS results show mb-oB3b exists in the gas-phase as three different negatively-charged states and exists in multiple conformational states, when introduced via electrospray ionization from aqueous solution near physiological pH. The disulfide bond serves a structural role and is not involved in the Cu(I/ll) binding capability of mb-OB3b, with the binding of a second Cu(I/ll) related to a further deprotonation of mb-OB3b. Overall, these findings are in good correlation with expected solution-phase behavior of mb-OB3b. The results suggest IM-MS is an effective tool for better understanding the complex nature of this intriguing peptide.