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
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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.
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:10.1155/2013/827621Google Scholar
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.2014.12.002CrossRefGoogle Scholar
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.0031253Google Scholar
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.072912407CrossRefGoogle Scholar
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.20449CrossRefGoogle Scholar