Dilution of dipolar interactions in a spin-labeled, multimeric metalloenzyme for DEER studies. J Inorg Biochem 2014 Jul;136:40-6
Date
04/20/2014Pubmed ID
24742748Pubmed Central ID
PMC4733626DOI
10.1016/j.jinorgbio.2014.03.010Scopus ID
2-s2.0-84939966873 (requires institutional sign-in at Scopus site) 9 CitationsAbstract
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.
Author List
Aitha M, Richmond TK, Hu Z, Hetrick A, Reese R, Gunther A, McCarrick R, Bennett B, Crowder MWAuthor
Brian Bennett D.Phil. Professor and Chair in the Physics department at Marquette UniversityMESH terms used to index this publication - Major topics in bold
Amino Acid SubstitutionBacterial Proteins
Catalysis
Catalytic Domain
Electron Spin Resonance Spectroscopy
Kinetics
Molecular Dynamics Simulation
Mutagenesis, Site-Directed
Spin Labels
beta-Lactamases
