# Quantification of Ligand–Membrane Interactions Using DNP-NMR Relaxometry

## TL;DR

This paper introduces a method using DNP-NMR to measure how small molecules bind to cell membranes, which can help in drug discovery.

## Contribution

The study presents a novel approach to quantify ligand-membrane interactions using DNP-NMR relaxometry.

## Key findings

- The binding affinity of the ligand was not significantly affected by cholesterol in the bilayer.
- Vesicle aggregation reduced the binding affinity of the ligand.
- DNP-NMR relaxometry can detect ligand-membrane binding and kinetic parameters.

## Abstract

The transverse relaxation rates (R
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 R
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 R
2 as a function of lipid concentration,
a parameter (f R
2,b)/K
D is calculated, which depends on the fractional number
of binding sites per lipid (f), the relaxation rate
(R
2,b) of bound ligand, and the dissociation
constant (K
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 R
2,b from chemical shift anisotropy and dipole–dipole
relaxation, the dissociation constant K
D/f 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 R
2 relaxation measurements
using DNP hyperpolarization provide a means to detect ligand–membrane
binding and kinetic parameters. In addition to R
2, a set of other NMR derived parameters that are sensitive
to molecular dynamics, such as R
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.