ABSTRACT Gram-negative bacteria have become a serious threat to public health because their resistance to ?-lactam antibiotics, the most widely used and successful class of antibiotics worldwide. These pathogensresist multiple ??lactams chiefly through the acquisition of ??lactamase proteins, which hydrolyticallydestroy the drug. Moreover, under drug pressure, the ??lactamases are evolving broader ?-lactamaseactivity. Understanding the mechanisms for these ?gain-of-activity? mutations is crucial for anticipating andcurbing their effects. An intriguing clue has come from clinical isolates of Acinetobacter baumannii, a Gram-negative pathogenand a global clinical scourge. A baumannii deploys a Class D ?-lactamase, OXA-24, to inactivate penicillinsand carbapenems. Recently, clinical isolates of A. baumannii with expanded resistance were traced tosubstitution mutations within flexible segments of OXA-24 associated with substrate recognition. Theseresults raise our overall hypothesis that conformational dynamics can influence the substrate spectrum ofClass-D ?-lactamases, specifically, in the flexible recognition loops at the protein surface. We therefore propose investigating this hypothesis through flexibility-activity studies of OXA-24 andsubstitution mutants already established to cause ?gain-of-activity? phenotypes in the clinic. Ourinvestigations use liquid state NMR to characterize the conformational ensembles of the free enzyme andsubstrate, and acyl-enzyme complex, for WT-OXA-24 and resistant variants.Aim 1. Compare the conformational sampling of apo OXA-24/40 with that of its clinical variants.Aim 2. Define the site-specific changes in ligand conformational flexibility caused by complexformation.Aim 3. Compare the conformational sampling of the OXA-24/40/ligand complexes with those of itsclinical variants. A predictive understanding of how flexible protein regions respond to resistance-expanding mutationsremains an open challenge. Our proposed research answers this challenge via investigations into the role ofprotein flexibility in expanding gram-negative antibiotic resistance. Our results may suggest new strategiesfor improved inhibitors, and new insights into how proteins evolve new functions.