title: 31P solid-state NMR measurements used to detect interactions between NADPH and water and to determine the ionisation state of NADPH in a protein-ligand complex subjected to low-level hydration.
abstract: 31P-NMR spectra of NADPH and NADPH bound to Lactobacillus casei dihydrofolate reductase have been recorded using the techniques of cross-polarization, magic-angle spinning and high-power proton-decoupling on both lyophilized and hydrated samples. Previous studies on the lyophilized complex of L. casei dihydrofolate reductase with NADPH and methotrexate, measuring the isotropic shifts and principal components of the chemical shift tensors, have shown that the 2'-phosphate group of bound NADPH exists as a mixture of the dianionic and monoanionic states [Gerothanassis, I. P, Barrie, P. J., Birdsall, B. & Feeney, J. (1994) Eur J. Biochem. 226, 211-218]. In the present study on hydrated samples, the characterization of the isotropic shift and chemical shift tensors of the 2'-phosphate signal indicates that the 2'-phosphate is almost exclusively in the dianionic state. This is in agreement with earlier 31P-NMR studies in solution [Feeney, J., Birdsall, B., Roberts, G. C. K. & Burgen, A. S. V. (1975) Nature 257, 564-566]. In experiments examining progressively hydrated (6%, 12%, 15%, by mass) samples, the observed signals become increasingly narrower probably because the microenvironments of the 31P nuclei become more homogeneous upon sample hydration. Chemical exchange between mobile water molecules and bound protons close to individual sites on NADPH has been indirectly monitored on a hydrated sample (15% water, by mass) using a pulse sequence proposed by Harbison and coworkers [Harbison, G. S., Roberts, J. E., Herzfeld, J. & Griffin, R. G. (1988) J. Am. Chem. Soc. 110, 7221-7223]. In this experiment, the two diphosphate signals are totally suppressed while the 2'-phosphate phosphorus signal remains: this indicates a significant polarization of the 2'-phosphate nuclei from protons in exchange with those of mobile water molecules.
authors.affiliation: Department of Chemistry, University of Ioannina, Greece.
authors.forename: I P
authors.initials: IP
authors.lastname: Gerothanassis
authors.forename: P J
authors.initials: PJ
authors.lastname: Barrie
authors.forename: B
authors.initials: B
authors.lastname: Birdsall
authors.forename: J
authors.initials: J
authors.lastname: Feeney
mesh_terms: D001665:Binding Sites; D007477:Ions; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009249:NADP / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D007477:Ions; D008024:Ligands; D011506:Proteins; D014867:Water; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase