Biomolecular NMR Assignments

, Volume 12, Issue 1, pp 69–77 | Cite as

1H, 13C and 15N backbone resonance assignments of the β-lactamase BlaP from Bacillus licheniformis 749/C and two mutational variants

  • David Thorn
  • Jennifer Kay
  • Noureddine Rhazi
  • Mireille Dumoulin
  • Alessandra Corazza
  • Christian Damblon
Article
  • 63 Downloads

Abstract

Class A β-lactamases have been widely used as versatile scaffolds to create hybrid (or chimeric) proteins for a series of applications ranging from basic research to medicine. We have, in particular, used the β-lactamase BlaP from Bacillus licheniformis 749/C (BlaP) as a protein scaffold to create model polyglutamine (polyQ) proteins in order to better understand the mechanism(s) by which an expanded polyQ sequence triggers the formation of amyloid fibrils. The model chimeras were designed by inserting a polyQ sequence of various lengths at two different locations within BlaP (i.e. position 197 or position 216) allowing a detailed comparison of the effects of subtle differences in the environment of the polyQ sequence on its ability to trigger protein aggregation. In order to investigate the effects of the polyQ insertion at both positions on the structure, stability and dynamics of BlaP, a series of NMR experiments including H/D exchange are foreseen. Accordingly, as necessitated by these studies, here we report the NMR assignment of the wild-type BlaP (BlaP-WT) and of the two reference proteins, BlaP197Q0 and BlaP216Q0, wherein a Pro-Gly dipeptide has been introduced at position 197 and 216, respectively; this dipeptide originates from the addition of the Sma1 restriction site at the genetic level to allow further polyQ sequence insertion.

Keywords

BlaP hybrid proteins Polyglutamine model proteins Protein aggregation Polyglutamine diseases Resonance assignment 

Notes

Acknowledgements

The authors thank Stéphane Preumont for the production and purification of the labeled proteins, and Fabrice Bouillenne and Anne-Marie Matton for their assistance with the purification of proteins. They would also like to thank Frank Löhr for conducting the NMR experiments at the Center for Biomolecular Magnetic Resonance at the Goethe University of Frankfurt, funded by the European Union (Bio-NMR, Project No. 261863). This work was supported by Grants from Fonds de la Recherche Fondamentale et Collective (CJ and 2.4581.12F to MD), Fonds de la Recherche Scientifique (FRS-FNRS, 1.C039.09 and MIS-F.4505.11 to MD), Fonds Spéciaux from the University of Liège (11/108 to MD) and the Belgian program of Interuniversity Attraction Poles administered by the Federal Office for Scientific Technical and Cultural Affairs (P7/4444 and 7/05). JK is a Recipient of FRIA fellowship of FRS-FNRS, DT was a Research Fellow from FRS-FNRS and MD is a Research Associate of FRS-FNRS. The funding sources had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • David Thorn
    • 1
    • 4
  • Jennifer Kay
    • 1
  • Noureddine Rhazi
    • 1
    • 5
  • Mireille Dumoulin
    • 1
  • Alessandra Corazza
    • 2
  • Christian Damblon
    • 3
  1. 1.Laboratory of Enzymology and Protein Folding, Center for Protein Engineering, InBiosUniversity of LiègeLiègeBelgium
  2. 2.Department of MedicineUniversity of UdineUdineItaly
  3. 3.Laboratory of Biological Structural Chemistry, Department of ChemistryUniversity of LiègeLiègeBelgium
  4. 4.Research School of ChemistryThe Australian National UniversityActonAustralia
  5. 5.Molecular Biomimetic and Protein Engineering Laboratory, GIGA-ResearchUniversity of LiègeLiègeBelgium

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