NMR Structural Analysis of BmrA-NBD

For NMR experiments, samples were concentrated to 250–400 µM before addition of 10 mM ADP and 10% v/v D₂O and 0.15 mM DSS (final concentrations). NMR spectra of isotope labeled BmrA-NBD in 50 mM BisTris pH 7, 50 mM NaCl were recorded at 298 K on Bruker AVANCE 600, 800, and 900 MHz spectrometers equipped with cryogenic triple resonance probes (Bruker GmbH, Karlsruhe, Germany). TROSY-based ¹⁵N-HSQC, HNCA, HNCO, HN(CA)CO, HN(CO)CA, HNCACB and HNCOCACB experiments were recorded using standard pulse sequences (Salzmann et al. 1998 (link)). All spectra were processed using Bruker TOPSPIN 4.0.8 and analyzed using CARA (Keller 2004 ).
The secondary structure of WT BmrA-NBD based on the backbone chemical shift assignment for the ADP-bound state was determined with TALOS-N (Shen and Bax 2013 (link)).