2.2 NMR experiments
High-pressure NMR experiments were performed on an AVANCE3-950
(1H, 950.33 MHz) spectrometer (Bruker, Billerica, MA,
USA) with a cryo-probe system using a pressure-resistant NMR cell
(Daedalus Innovations, Aston, PA, USA). For temperature-variable
experiments, the slot NMR tube for high-concentration electrolyte
samples (SHIGEMI, Hachioji, Japan) was used. To measure PRE,1H/15N heteronuclear single-quantum
correlation (HSQC) spectra of the MTSL-bound OspA and its variants were
collected for the oxidized and reduced forms of MTSL at multiple
pressures and temperatures. A 200 complex × 4096 real data matrix was
acquired with spectral widths of 36.0 (15N,F 1) and 12.98 ppm (1H,F 2). NMR data were processed using Topspin
version 3.2 (Bruker, Billerica, MA, USA),
NMRPipe,26 and
NMRViewJ.27 1H chemical
shifts were referenced to the methyl signals of
2,2-dimethyl-2-silapentane-5-sulfonate (DSS), and 15N
chemical shifts were indirectly referenced to DSS (0 ppm for1H).