Journal of Biomolecular NMR

, Volume 65, Issue 1, pp 41–48 | Cite as

Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase

Article

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.

Keywords

Dissolution dynamic nuclear polarization Nuclear magnetic resonance Protein–ligand interaction Drug discovery 

Abbreviations

D-DNP

Dissolution dynamic nuclear polarization

DHF

Dihydrofolic acid

DHFR

Dihydrofolate reductase

INPHARMA

Interligand NOE for pharmacophore mapping

MTX

Methotrexate

NADPH

Dihydronicotinamide adenine dinucleotide phosphate

NOE

Nuclear Overhauser effect

NOESY

Nuclear Overhauser effect spectroscopy

SPINOE

Spin polarization-induced nuclear Overhauser effect

THF

Tetrahydrofolic acid

TMP

Trimethoprim

Supplementary material

10858_2016_37_MOESM1_ESM.pdf (1 mb)
Supplementary material 1 (PDF 1056 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Chemistry DepartmentTexas A&M UniversityCollege StationUSA
  2. 2.Department of Biochemistry and Molecular Biology, College of MedicineUniversity of FloridaGainesvilleUSA

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