title: Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.
abstract: Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases - TEM-1 and PSE-4 - were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two 'parental' β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions.
authors.affiliation: PROTEO, the Québec Network for Research on Protein Structure, Function and Engineering, Université Laval, Laval, Québec, Canada.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Clouthier
authors.forename: Sébastien
authors.initials: S
authors.lastname: Morin
authors.forename: Sophie M C
authors.initials: SM
authors.lastname: Gobeil
authors.forename: Nicolas
authors.initials: N
authors.lastname: Doucet
authors.forename: Jonathan
authors.initials: J
authors.lastname: Blanchet
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Nguyen
authors.forename: Stéphane M
authors.initials: SM
authors.lastname: Gagné
authors.forename: Joelle N
authors.initials: JN
authors.lastname: Pelletier
mesh_terms: D002942:Circular Dichroism; D005470:Fluorometry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D017433:Protein Structure, Secondary; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001618:beta-Lactamases; C034413:beta-lactamase TEM-1