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NMR characterization of an engineered domain fusion between maltose binding protein and TEM1 β‐lactamase provides insight into its structure and allosteric mechanism
Author(s) -
Wright Chapman M.,
Majumdar Ananya,
Tolman Joel R.,
Ostermeier Marc
Publication year - 2009
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.22657
Subject(s) - maltose binding protein , allosteric regulation , maltose , chemistry , enzyme , biochemistry , heteronuclear single quantum coherence spectroscopy , substrate (aquarium) , conformational change , stereochemistry , nuclear magnetic resonance spectroscopy , biophysics , binding site , fusion protein , crystallography , biology , recombinant dna , ecology , gene
RG13 is a 72 kDa engineered allosteric enzyme comprised of a fusion between maltose binding protein (MBP) and TEM1 β‐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. Proteins 2010. © 2009 Wiley‐Liss, Inc.