Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.

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 bac...

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Autores principales:	Christopher M Clouthier, Sébastien Morin, Sophie M C Gobeil, Nicolas Doucet, Jonathan Blanchet, Elisabeth Nguyen, Stéphane M Gagné, Joelle N Pelletier
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ca632b9d42fd4332a332b46c4fd95d68
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spelling	oai:doaj.org-article:ca632b9d42fd4332a332b46c4fd95d682021-11-18T08:03:56ZChimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.1932-620310.1371/journal.pone.0052283https://doaj.org/article/ca632b9d42fd4332a332b46c4fd95d682012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23284969/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Enzyme 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.Christopher M ClouthierSébastien MorinSophie M C GobeilNicolas DoucetJonathan BlanchetElisabeth NguyenStéphane M GagnéJoelle N PelletierPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 12, p e52283 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Christopher M Clouthier Sébastien Morin Sophie M C Gobeil Nicolas Doucet Jonathan Blanchet Elisabeth Nguyen Stéphane M Gagné Joelle N Pelletier Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.
description	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.
format	article
author	Christopher M Clouthier Sébastien Morin Sophie M C Gobeil Nicolas Doucet Jonathan Blanchet Elisabeth Nguyen Stéphane M Gagné Joelle N Pelletier
author_facet	Christopher M Clouthier Sébastien Morin Sophie M C Gobeil Nicolas Doucet Jonathan Blanchet Elisabeth Nguyen Stéphane M Gagné Joelle N Pelletier
author_sort	Christopher M Clouthier
title	Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.
title_short	Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.
title_full	Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.
title_fullStr	Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.
title_full_unstemmed	Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.
title_sort	chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/ca632b9d42fd4332a332b46c4fd95d68
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Chimeric β-lactamases: global conservation of parental function and fast time-scale dynamics with increased slow motions.

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