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


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.


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



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.


  1. Almeida B, Fernandes S, Abreu IA, Macedo-Ribeiro S (2013) Trinucleotide repeats: a structural perspective. Front Neurol. doi: 10.3389/fneur.2013.00076 Google Scholar
  2. Ambler RP, Coulson AF, Frère JM et al (1991) A standard numbering scheme for the class A beta-lactamases. Biochem J 276(Pt 1):269–270. doi: 10.1042/bj2760269 CrossRefGoogle Scholar
  3. Bundi A, Wüthrich K (1977) 1H NMR titration shifts of amide proton in polypeptide chains resonances. FEBS Lett 77(1):11–14. doi: 10.1016/0014-5793(77)80182-8 CrossRefGoogle Scholar
  4. Delaglio F, Grzesiek S, Vuister GW et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293. doi: 10.1007/BF00197809 CrossRefGoogle Scholar
  5. Hands SL, Wyttenbach A (2010) Neurotoxic protein oligomerisation associated with polyglutamine diseases. Acta Neuropathol 120:419–437. doi: 10.1007/s00401-010-0703-0 CrossRefGoogle Scholar
  6. Huynen C, Filée P, Matagne A et al (2013) Class A β-Lactamases as versatile scaffolds to create hybrid enzymes: applications from basic research to medicine. Biomed Res Int. doi: 10.1155/2013/827621 Google Scholar
  7. Huynen C, Willet N, Buell AK et al (2015) Influence of the protein context on the polyglutamine length-dependent elongation of amyloid fibrils. Biochim Biophys Acta - Proteins Proteom 1854(3):239–248. doi: 10.1016/j.bbapap.2014.12.002 CrossRefGoogle Scholar
  8. Robertson AL, Bottomley SP (2010) Towards the treatment of polyglutamine diseases: the modulatory role of protein context. Curr Med Chem 17:3058–3068. doi: 10.2174/092986710791959800 CrossRefGoogle Scholar
  9. Scarafone N, Pain C, Fratamico A et al (2012) Amyloid-like fibril formation by polyq proteins: a critical balance between the polyq length and the constraints imposed by the host protein. PLoS ONE. doi: 10.1371/journal.pone.0031253 Google Scholar
  10. Vandevenne M, Filee P, Scarafone N et al (2007) The Bacillus licheniformis BlaP beta-lactamase as a model protein scaffold to study the insertion of protein fragments. Protein Sci 16:2260–2271. doi: 10.1110/ps.072912407 CrossRefGoogle Scholar
  11. Vranken WF, Boucher W, Stevens TJ et al (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins Struct Funct Genet 59:687–696. doi: 10.1002/prot.20449 CrossRefGoogle Scholar
  12. Wishart DS, Bigam CG, Yao J et al (1995) 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J Biomol NMR 6:135–140. doi: 10.1007/BF00211777 CrossRefGoogle Scholar
  13. Orr HT, Zoghbi HY (2007) Trinucleotide repeat disorders. Annu Rev Neurosci 30:575–621. doi: 10.1146/annurev.neuro.29.051605.113042 CrossRefGoogle Scholar

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

Personalised recommendations