Conformational dynamics of metallo-β-lactamase CcrA during catalysis investigated by using DEER spectroscopy. J Biol Inorg Chem 2015 Apr;20(3):585-94
Date
04/02/2015Pubmed ID
25827593Pubmed Central ID
PMC4733638DOI
10.1007/s00775-015-1244-8Scopus ID
2-s2.0-84939954594 (requires institutional sign-in at Scopus site) 17 CitationsAbstract
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 10 ms after mixing, the DEER spectra show distance increases of 7, 10, and 13 Å 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 10 Å 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.
Author List
Aitha M, Moritz L, Sahu ID, Sanyurah O, Roche Z, McCarrick R, Lorigan GA, 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
Bacterial ProteinsCatalysis
Models, Molecular
Molecular Conformation
Molecular Dynamics Simulation
Spectrum Analysis
beta-Lactamases