title: Dilution of dipolar interactions in a spin-labeled, multimeric metalloenzyme for DEER studies.
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.
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, OH 45056, United States.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Aitha
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, OH 45056, United States.
authors.forename: Timothy K
authors.initials: TK
authors.lastname: Richmond
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, OH 45056, United States.
authors.forename: Zhenxin
authors.initials: Z
authors.lastname: Hu
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, OH 45056, United States.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Hetrick
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, OH 45056, United States.
authors.forename: Raquel
authors.initials: R
authors.lastname: Reese
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, OH 45056, United States.
authors.forename: Althea
authors.initials: A
authors.lastname: Gunther
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, OH 45056, United States.
authors.forename: Robert
authors.initials: R
authors.lastname: McCarrick
authors.affiliation: Department of Biophysics, National Biomedical EPR Center, Medical College of Wisconsin, Milwaukee, WI 53226, United States.
authors.forename: Brian
authors.initials: B
authors.lastname: Bennett
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, 160 Hughes Hall, Oxford, OH 45056, United States. Electronic address: crowdemw@miamioh.edu.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Crowder
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D013113:Spin Labels; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D013113:Spin Labels; D001618:beta-Lactamases
keywords: Double electron electron resonance (DEER); MTSL; Metallo-β-lactamase (MβL); Site-directed spin labeling (SDSL)
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