NMR-based Binding Affinity Determination

All NMR spectra were acquired on a Bruker DRX 600 MHz spectrometer equipped with a ¹H, ¹⁵N, ¹³C, TXI cryoprobe and SampleJet auto-sampler at 298 K (25 °C). Experiments were performed using 50 μM [U-¹⁵N]-CXCL12WT with compound concentrations ranging from 0 μM to 1600 μM prepared as described above and monitored using ¹H-¹⁵N SOFAST-Heteronuclear multiple quantum coherence (HMQC) experiments and chemical shift assignments were acquired from previously published sources.^{36 (link)} Spectra were processed using in-house scripts and chemical shift tracking was performed using a combination of TitrView and CARA software.^{22 (link)} The combined ¹H/¹⁵N chemical shift perturbations were calculated as ((5Δδ_H)² + (Δδ_NH)²)^0.5, where Δδ_H and Δδ_NH represent the respective amide proton and nitrogen chemical shifts. Equilibrium dissociation constants (K_d) were determined using non-linear fitting of the calculated ¹H/¹⁵N chemical shift perturbations as a function of compound concentration to a single-site quadratic equation (protein concentration was held constant at 50 μM).^{37 (link)} For each compound, the residues with the largest chemical shift perturbations were fitted individually and the resulting K_d values and their respective errors were averaged to produce the reported affinity and standard deviation.