title: NMR characterization of an engineered domain fusion between maltose binding protein and TEM1 beta-lactamase provides insight into its structure and allosteric mechanism.
abstract: RG13 is a 72 kDa engineered allosteric enzyme comprised of a fusion between maltose binding protein (MBP) and TEM1 beta-lactamase (BLA) for which maltose is a positive effector of BLA activity. We have used NMR spectroscopy to acquire [(15)N, (1)H]-TROSY-HSQC spectra of RG13 in the presence and absence of maltose. The RG13 chemical shift data was compared to the published chemical shift data of MBP and BLA. The spectra are consistent with the expectation that the individual domain structures of RG13 are substantially conserved from MBP and BLA. Differences in the spectra are consistent with the fusion geometry of MBP and BLA and the maltose-dependent differences in the kinetics of RG13 enzyme activity. In particular, the spectra provide evidence for a maltose-dependent conformational change of a key active site glutamate involved in deacylation of the enzyme-substrate intermediate.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: Chapman M
authors.initials: CM
authors.lastname: Wright
authors.forename: Ananya
authors.initials: A
authors.lastname: Majumdar
authors.forename: Joel R
authors.initials: JR
authors.lastname: Tolman
authors.forename: Marc
authors.initials: M
authors.lastname: Ostermeier
mesh_terms: D000494:Allosteric Regulation; D020134:Catalytic Domain; D002511:Cephalosporins / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D058619:Maltose-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002511:Cephalosporins; D058619:Maltose-Binding Proteins; D010406:Penicillins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases