Investigation of the Selected Transition Metal Binding Characteristics of Methanobactin from methylosinus Trichosporium Ob3b

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Date of Award

Spring 2017

Abstract

The methanobactin (Mb) from Methylosinus trichosporium OB3b (1154.26 Da) 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 they are thought to mediate the acquisition of the copper cofactor for the enzyme methane monooxygenase. Mb will also bind other metal ions and mediate their solubilization and transportation in situ. The structure of Mb consists of nine amino acids and two modified regions containing enethiol-oxazolone rings which are the bidentate binding sites for Cu(I). However, the binding characteristics for Ag(I), Pb(II), Co(II), Fe(III), Mn(II), Ni(II), and Zn(II) have not been determined and the goal of this study is to compare the binding of these metals to the [Mb+Cu(I)- nH](n-1)− complex using traveling wave ion mobility mass spectrometry (TWIMS). The arrival time distribution (ATD) for each metal complex and free Mb was used to quantify the apparent binding affinity and measure their collision cross section at various pH points. In addition, collision-induced dissociation of the disulfide reduced [Mb+M], was used to locate the binding sites of the individual metal(I or II) ions (M), and the normalized collision energy, breakdown curve was measured to determine the relative stability of the [red-Mb+M] complexes. Selected mass spectrometry titration studies were repeated using fluorescence spectroscopy to compare gas-phase and solution-phase techniques.

Advisor

Laurence A Angel

Subject Categories

Chemistry | Physical Sciences and Mathematics

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