Probing protein folding pathways by hydrogen-deuterium exchange: A time-resolved study of BS3 β-lactamase as a model system

Understanding how proteins acquire their functional conformation remains a central challenge in structural biology, particularly for multidomain proteins whose folding trajectories involve intermediates and kinetic traps. Exported enzymes translocated through the Sec system provide a relevant model: they must remain unfolded during passage across the membrane and then spontaneously refold once secretion is complete.
Within this framework, the folding of the BS3 class A β-lactamase from Bacillus licheniformis was characterized at high resolution using hydrogen–deuterium exchange, mass spectrometry and NMR. These approaches reveal a sequence of intermediates guided by an early nucleation core formed by the β4–β5 hairpin, followed by the stepwise organization of the domains and a final maturation phase limited by proline isomerizations. A cleavable variant and evolutionary analyses further show that this motif represents a conserved folding nucleus among β-lactamases and PBPs, highlighting that folding pathways are governed by interdependent rather than autonomous structural units.