Quantification of Ligand–Membrane Interactions Using DNP-NMR Relaxometry

The transverse relaxation rates (<i>R</i>₂) for ¹⁹F spins from a small molecule ligand are measured in the presence of phospholipid vesicles with varied compositions and concentrations. The sensitivity of detection is enhanced by hyperpolarization using dissolution dynamic nuclear polarization (D-DNP), enabling measurement of <i>R</i>₂ relaxation rates in single scans. The binding interaction is described as an equilibrium with a defined number of binding sites on the membrane. From the increase of <i>R</i>₂ as a function of lipid concentration, a parameter (<i>f R</i>_2,b)/<i>K</i>_D is calculated, which depends on the fractional number of binding sites per lipid (<i>f</i>), the relaxation rate (<i>R</i>_2,b) of bound ligand, and the dissociation constant (<i>K</i>_D) for each binding site. The relaxation rate of a bound ligand is modeled based on molecular motions, including rigid body tumbling, diffusion in the bilayer, wobble motions, and CF₃ rotation. By estimation of <i>R</i>_2,b from chemical shift anisotropy and dipole-dipole relaxation, the dissociation constant <i>K</i>_D/<i>f</i> is evaluated to greater than 10 mM for the ligand binding to 200 nm vesicles. The resulting values for the binding affinity were not significantly affected by the presence of cholesterol in the bilayer, but were reduced by vesicle aggregation. These data further demonstrate that <i>R</i>₂ relaxation measurements using DNP hyperpolarization provide a means to detect ligand-membrane binding and kinetic parameters. In addition to <i>R</i>₂, a set of other NMR derived parameters that are sensitive to molecular dynamics, such as <i>R</i>_1ρ, diffusion and cross-relaxation, and spectroscopic techniques, such as Laplace NMR, are compatible with the hyperpolarized method. The measurement of membrane-ligand binding can facilitate applications for drug discovery and biomedical studies.