Backbone 1H,13C, and 15N Chemical Shift Assignments for the Toho-1 b-lactamase R274N/R276N double mutant enzyme

A Toho-1 b-lactamase R274N/R276N double mutant

TROSY offset: amide nitrogens; amide protons.

4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) was used as an internal standard

Toho-1 β-lactamase: backbone chemical shift assignments and changes in dynamics upon binding with avibactam.

Toho-1 b-lactamase, protein NMR, backbone chemical shift assignments

25 M MET
26 A ALA
27 N ASN
......
288 H HIS
289 G GLY
290 F PHE

The sequence for Toho-1 b-lactamase starts at residue 25 and ends,
after 263 residues and three gaps (positions 58, 239, and 253),
at residue 290.

Toho-1 is an extended-spectrum beta-lactamase that has acquired efficient hydrolytic activity not only to penicillins but also to cephalosporins including the expanded-spectrum cephalosporins that were developed to be stable in former beta-lactamases.

Toho-1 b-lactamase R274N/R276N double mutant

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Backbone assignments for Toho-1 b-lactamase R274N/R276N double mutant enzyme
(263 amino acids, 28.66 kDa) are reported based on triple resonance
solution-state NMR experiments on a uniformly 2H,13C,15N-labeled sample.

Bruker Avance III spectrometer operating at 16.44 T (700 MHz-1H) with a cryogenically cooled probe, including Z-axis pulsed field gradients.

Aqueous solution of uniformly 2H-13C-15N labeled Toho-1 b-lactamase with a
concentration of approximately 800-850 uM in pH 6.5, 20 mM MES buffer,
containing 1 mM DTT, 1 mM Na2EDTA, 0.05% NaN3, and 10% (v/v) 2H2O.

Toho-1 b-lactamase R274N/R276N double mutant enzyme

The protein concentration of the sample was approximately 800-850 uM
in pH 6.5, 20 mM MES buffer, containing 1 mM DTT, 1 mM Na2EDTA,
0.05% NaN3, and 10% (v/v) 2H2O.

Interface to IPine Webserver for automated backbone chemical shift assignments

Spectra were processed using Bruker TOPSPIN version 3.6.