Document Type

Article

Language

eng

Format of Original

7 p.

Publication Date

7-2014

Publisher

Elsevier

Source Publication

Journal of Inorganic Biochemistry

Source ISSN

0162-0134

Original Item ID

doi: 10.1016/j.jinorgbio.2014.03.010

Abstract

The metallo-β-lactamases (MβLs), which require one or two Zn(II) ions in their active sites for activity, hydrolyze the amide bond in β-lactam-containing antibiotics, and render the antibiotics inactive. All known MβLs contain a mobile element near their active sites, and these mobile elements have been implicated in the catalytic mechanisms of these enzymes. However little is known about the dynamics of these elements. In this study, we prepared a site-specific, double spin-labeled analog of homotetrameric MβL L1 with spin labels at positions 163 and 286 and analyzed the sample with DEER (double electron electron resonance) spectroscopy. Four unique distances were observed in the DEER distance distribution, and these distances were assigned to the desired intramolecular dipolar coupling (between spin labels at positions 163 and 286 in one subunit) and to intermolecular dipolar couplings. To rid the spin-labeled analog of L1 of the intermolecular couplings, spin-labeled L1 was “diluted” by unfolding/refolding the spin-labeled enzyme in the presence of excess wild-type L1. DEER spectra of the resulting, spin-diluted enzyme revealed a single distance corresponding to the desire intramolecular dipolar coupling.

Comments

Accepted version. Journal of Inorganic Biochemistry, Vol. 136 (July 2014): 40-46. DOI. © 2014 Elsevier. Used with permission.

Brian Bennett was affiliated with the Medical College of Wisconsin at the time of publication.

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