Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase

Abstract

Protein–ligand interaction is often characterized using polarization transfer by the intermolecular nuclear Overhauser effect (NOE). For such NOE experiments, hyperpolarization of nuclear spins presents the opportunity to increase the spin magnetization, which is transferred, by several orders of magnitude. Here, folic acid, a ligand of dihydrofolate reductase (DHFR), was hyperpolarized on 1H spins using dissolution dynamic nuclear polarization (D-DNP). Mixing hyperpolarized ligand with protein resulted in observable increases in protein 1H signal predominantly in the methyl group region of the spectra. Using 13C single quantum selection in a series of one-dimensional spectra, the carbon chemical shift ranges of the corresponding methyl groups can be elucidated. Signals observed in these hyperpolarized spectra could be confirmed using 3D isotope filtered NOESY spectra, although the hyperpolarized spectra were obtained in single scans. By further correlating the signal intensities observed in the D-DNP experiments with the occurrence of short distances in the crystal structure of the protein–ligand complex, the observed methyl proton signals could be matched to the chemical shifts of six amino acids in the active site of DHFR-folic acid binary complex. These data demonstrate that 13C chemical shift selection of protein resonances, combined with the intrinsic selectivity towards magnetization originating from the initially hyperpolarized spins, can be used for site specific characterization of protein–ligand interactions.

Determination of protein–ligand binding modes using fast multi-dimensional NMR with hyperpolarization

“…The information is similar to that from conventional 3D ltered NOESY experiments shown in ref. 23, but is obtained in a fraction of the time with hyperpolarization. In each of the three reconstructed spectra, distinct signal patterns are observed, allowing the determination of the source of polarization for each observable methyl group.…”

“…21 Polarization transferred from a hyperpolarized ligand can also be observed directly as selectively enhanced protein signals, revealing structural information related to the ligand-protein interaction. 22,23 Knowledge of the protein-ligand complex structure provides essential information to guide the ligand optimization process in structure-based drug design (SBDD). Computational docking is a rapid and inexpensive method for predicting the orientation and conformation of the ligand when binding to the target protein.…”

“… 21 Polarization transferred from a hyperpolarized ligand can also be observed directly as selectively enhanced protein signals, revealing structural information related to the ligand–protein interaction. 22 , 23 …”

Applications of dissolution dynamic nuclear polarization in chemistry and biochemistry

“…Site specific characterization of protein–ligand interactions can also be achieved using hyperpolarized ligand, through ligand–protein polarization transfer. For example, enhanced 1 H signal of the protein dihydrofolate reductase in the methyl group spectral region was observed when mixed with the hyperpolarized ligand folic acid . When using isotope labeled protein, 13 C single‐ or multiquantum selection can further be used to acquire NMR spectra, where chemical shift ranges of the heteronucleus can be identified.…”

“…Protein–solvent interactions play an important role in governing the structure of macromolecules. On the basis of intermolecular interactions, such as between proteins and water, spin magnetization of a small molecule is transferred to a target macromolecule through chemical exchange of labile protons, as well as cross relaxation due to nuclear Overhauser effect . NMR‐based measurements of exchange or cross relaxation rates indicate such interactions and thus provide structural information of macromolecules.…”