600 mhz nmr instrument

NMR spectra were acquired at 298 K with a 600 MHz NMR instrument (Bruker, Billerica MA) equipped with an AVANCE III console and a triple-resonance cryogenic probe. Samples were at pH 7.5 in a 25 mM HEPES buffer containing 100 mM NaCl, 1 mM dithiothreitol, and 1 mM EDTA. The concentration of ¹⁵N labeled RSV N(31-252) was 0.15 mM. Two-dimensional [¹⁵N, ¹H] transverse relaxation optimized spectroscopy (TROSY) experiments were recorded with increasing ligand concentration [37 (link)]. Evolution times were approximately 53 ms in the ¹H dimension and 29 ms in the ¹⁵N dimension. The total acquisition time was 28 min per experiment.
To estimate the dissociation constant (K_d) from NMR experiments, ¹⁵N-RSV N(31-252) was titrated with a range of ligand concentrations. The weighted average of chemical shift changes (Δδ(N, H)_obs) is calculated using the following equation: $\Delta \mathrm{\delta }{\left(\mathrm{N},\mathrm{H}\right)}_{\mathrm{o}\mathrm{b}\mathrm{s}}=\sqrt{{\left(\Delta {\mathrm{\delta }}_{\mathrm{H}\mathrm{N}}\right)}^2+{\left(\Delta {\mathrm{\delta }}_{\mathrm{{\rm N}}}/5\right)}^2}$ where Δδ_HN and Δδ_N are the chemical shift changes of amide protons and nitrogens, respectively. Then Δδ(N, H)_obs data were fitted by non-linear regression globally against the total concentration of the subtrate (L_T) with the equation: $\mathrm{\Delta }\mathrm{\delta }{\left(\mathrm{N},\mathrm{H}\right)}_{\mathrm{o}\mathrm{b}\mathrm{s}}=\mathrm{\Delta }\mathrm{\delta }{\left(\mathrm{N},\mathrm{H}\right)}_{\text{max}}\frac{\left({\mathrm{K}}_{\mathrm{d}}+{\mathrm{L}}_{\mathrm{T}}+{\mathrm{E}}_{\mathrm{T}}\right)‐\sqrt{{\left({\mathrm{K}}_{\mathrm{d}}+{\mathrm{L}}_{\mathrm{T}}+{\mathrm{E}}_{\mathrm{T}}\right)}^2-4\left({\mathrm{L}}_{\mathrm{T}}{\mathrm{E}}_{\mathrm{T}}\right)}}{2{\mathrm{E}}_{\mathrm{T}}}$ where Δδ(N, H)_max is the maximum chemical shift difference, E_T is the total concentration of N(31-352) in the solution, and L_T is the total concentration of ligand.