NMR Analysis of Fab/Fc and IgG Glycoproteins

For NMR measurements, Fab/Fc fragments and full-length IgG glycoproteins were dissolved in 0.5 mL of 5 mM sodium phosphate buffer [pH 6.0, containing 50 mM NaCl and 10% (v/v) D₂O] at a protein concentration of 10 mg/mL. Two-dimensional methyl-transverse relaxation optimized spectroscopy(TROSY) spectral data were acquired at 37 °C using an AVANCE 800 spectrometer equipped with a cryogenic probe (Bruker BioSpin, Fällanden, Switzerland). Assignments for the methionyl methyl resonances of Fc were made based on the previously reported backbone assignments [30 (link)] by analyzing nuclear Overhauser effect (NOE) connectivities observed using AVANCE 800 and AVANCEIII 900 spectrometers equipped with cryogenic probes (Bruker BioSpin, Fällanden, Switzerland). Chemical shifts of ¹H were referenced to 4,4-dimethyl-4-silapentane-1-sulfonic acid (0 ppm), and ¹³C chemical shifts were referenced indirectly using the gyromagnetic ratios of ¹³C and ¹H (γ¹³C/γ¹H = 0.25144952). All NMR data were processed using NMR Pipe [31 (link)] and were analyzed using CCPNMR [32 (link)].