Pr André Matagne
Laboratoire d’enzymologie et de repliement des protéines
Centre d’ingénierie des protéines, Département des Sciences de la Vie
Université de Liège, Belgique
Class A β-lactamases, with their complex architecture and significant size (ca. 29 kDa), provide an excellent model for studying key features of protein folding. These enzymes exhibit two particularly interesting structural aspects : the formation of a β-domain in which the β-sheet is assembled from strands contributed by the N- and C-terminal regions of the polypeptide chain, and the distinctive packing of a large Ω-loop on the protein surface. While protein folding studies have traditionally focused on smaller, single-domain proteins with simple folding kinetics, β-lactamases enable the exploration of folding intermediates and alternative pathways in larger systems.
Using Bacillus licheniformis BS3 β-lactamase as a model, we have elucidated the folding process by combining quenched-flow hydrogen/deuterium exchange pulse-labelling with high-resolution methods including 2D-NMR and proteolytic fragmentation mass spectrometry. Together, these approaches track the formation and stabilization of secondary structure elements over time and reveal kinetically populated folding intermediates. BS3 β-lactamase is particularly well suited for such studies due to its high solubility and stability, ease of purification, measurable biological activity, and well-characterized 3D structure at 1.7 Å resolution. The availability of an assigned 1H-15N HSQC NMR spectrum further supports detailed kinetic and structural analyses, providing new insights into folding pathways and intermediate states in larger, multidomain enzymes.