caa a22 4500 605507236 CHVBK 20210128100631.0 cr unu---uuuuu 210128e20150401xx s 000 0 eng 10.1007/s00775-015-1244-8 doi (NATIONALLICENCE)springer-10.1007/s00775-015-1244-8 Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy [Elektronische Daten] [Mahesh Aitha, Lindsay Moritz, Indra Sahu, Omar Sanyurah, Zahilyn Roche, Robert McCarrick, Gary Lorigan, Brian Bennett, Michael Crowder] Previous crystallographic and mutagenesis studies have implicated the role of a position-conserved hairpin loop in the metallo-β-lactamases in substrate binding and catalysis. In an effort to probe the motion of that loop during catalysis, rapid-freeze-quench double electron-electron resonance (RFQ-DEER) spectroscopy was used to interrogate metallo-β-lactamase CcrA, which had a spin label at position 49 on the loop and spin labels (at positions 82, 126, or 233) 20-35 Å away from residue 49, during catalysis. At 10ms after mixing, the DEER spectra show distance increases of 7, 10, and 13Å between the spin label at position 49 and the spin labels at positions 82, 126, and 233, respectively. In contrast to previous hypotheses, these data suggest that the loop moves nearly 10Å away from the metal center during catalysis and that the loop does not clamp down on the substrate during catalysis. This study demonstrates that loop motion during catalysis can be interrogated on the millisecond time scale. SBIC, 2015 Double electron-electron resonance (DEER) nationallicence Metallo-β-lactamase (MβLs) nationallicence Site-directed spin labeling (SDSL) nationallicence Rapid freeze quench (RFQ) nationallicence MTSL nationallicence CcrA* : Spin-labeledsite-directed variant CcrA:C155S nationallicence CcrA*(49) : Spin-labeledsite-directed variant CcrA:C155S/W49C nationallicence CcrA*(49/82) : Spin-labeledsite-directed variant CcrA:C155S/W49C/N82C nationallicence CcrA*(49/126) : Spin-labeledsite-directed variant CcrA:C155S/W49C/D126C nationallicence CcrA*(49/233) : Spin-labeledsite-directed variant CcrA:C155SW49C/E233C nationallicence DEER : Double electron-electron resonance/pulsed electron-electron double resonance nationallicence EPR : Electron paramagnetic (spin) resonance nationallicence MTSL : (S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate) nationallicence MβL : Metallo-β-lactamase nationallicence RFQ : Rapid freeze quench nationallicence Aitha Mahesh Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut Moritz Lindsay Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut Sahu Indra Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut Sanyurah Omar Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut Roche Zahilyn Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut McCarrick Robert Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut Lorigan Gary Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut Bennett Brian Physics Department, Marquette University, 540 N. 15th Street, 53233, Milwaukee, WI, USA aut Crowder Michael Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/3(2015-04-01), 585-594 0949-8257 20:3<585 2015 20 775 https://doi.org/10.1007/s00775-015-1244-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00775-015-1244-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Aitha Mahesh Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut NATIONALLICENCE 700 1- Moritz Lindsay Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut NATIONALLICENCE 700 1- Sahu Indra Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut NATIONALLICENCE 700 1- Sanyurah Omar Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut NATIONALLICENCE 700 1- Roche Zahilyn Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut NATIONALLICENCE 700 1- McCarrick Robert Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut NATIONALLICENCE 700 1- Lorigan Gary Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut NATIONALLICENCE 700 1- Bennett Brian Physics Department, Marquette University, 540 N. 15th Street, 53233, Milwaukee, WI, USA aut NATIONALLICENCE 700 1- Crowder Michael Department of Chemistry and Biochemistry, Miami University, 650 East High Street, 45056, Oxford, OH, USA aut NATIONALLICENCE 773 0- JBIC Journal of Biological Inorganic Chemistry Springer Berlin Heidelberg 20/3(2015-04-01), 585-594 0949-8257 20:3<585 2015 20 775