P-31 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

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dc.contributor.authorGerothanassis, I. P.en
dc.contributor.authorBarrie, P. J.en
dc.contributor.authorBirdsall, B.en
dc.contributor.authorFeeney, J.en
dc.date.accessioned2015-11-24T16:43:47Z
dc.date.available2015-11-24T16:43:47Z
dc.identifier.issn0014-2956-
dc.identifier.urihttps://olympias.lib.uoi.gr/jspui/handle/123456789/8739
dc.rightsDefault Licence-
dc.subjectp-31 solid-state nmri dihydrofolate reductaseen
dc.subjectnadphen
dc.subjectmagnetization transferen
dc.subjecthydrationen
dc.subjectcasei dihydrofolate-reductaseen
dc.subjectnuclear magnetic-resonanceen
dc.subjectlactobacillus-caseien
dc.subjectescherichia-colien
dc.subjectc-13-nmr spectroscopyen
dc.subjectcrystal-structuresen
dc.subjectmagic angleen
dc.subjectbindingen
dc.subjectmethotrexateen
dc.subjectconformationen
dc.titleP-31 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 hydrationen
heal.abstractP-31-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 P-31-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 P-31 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. Sec. 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.en
heal.accesscampus-
heal.fullTextAvailabilityTRUE-
heal.identifier.primaryDOI 10.1111/j.1432-1033.1996.00262.x-
heal.identifier.secondary<Go to ISI>://A1996TQ25600034-
heal.identifier.secondaryhttp://onlinelibrary.wiley.com/store/10.1111/j.1432-1033.1996.00262.x/asset/j.1432-1033.1996.00262.x.pdf?v=1&t=hmn3nk3f&s=6b190f33f25046fccbcaef40d2de77445d4c4b8d-
heal.journalNameEuropean Journal of Biochemistryen
heal.journalTypepeer reviewed-
heal.languageen-
heal.publicationDate1996-
heal.publisherWiley-Blackwellen
heal.recordProviderΠανεπιστήμιο Ιωαννίνων. Σχολή Θετικών Επιστημών. Τμήμα Χημείαςel
heal.typejournalArticle-
heal.type.elΆρθρο Περιοδικούel
heal.type.enJournal articleen

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