American Chemical Society
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Elucidation of Structural Restraints for Phosphate Residues with Different Hydrogen Bonding and Ionization States

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journal contribution
posted on 2008-11-06, 00:00 authored by J. Gajda, S. Olejniczak, I. Bryndal, M. J. Potrzebowski
Solid state NMR spectroscopy and gauge including atomic orbital (GIAO) theoretical calculations were employed to establish structural restraints (ionization, hydrogen bonding, spatial arrangement) for O-phosphorylated l-threonine derivatives in different ionization states and hydrogen bonding strengths. These structural restraints are invaluable in molecular modeling and docking procedures for biological species containing phosphoryl groups. Both the experimental and the GIAO approach show that 31P δii chemical shift tensor parameters are very sensitive to the ionization state. The negative values found for the skew κ are typical for −2 phosphates. The distinct span Ω values reflect the change of strength of hydrogen bonding. For species in the −1 ionization state, engaged in very strong hydrogen bonds, Ω is smaller than for a phosphate group involved in weak hydrogen bonding. For phosphates in the −2 ionization state, Ω is significantly smaller compared to −1 species, although the κ for −1 samples never reaches negative values. For −1 phosphate residues, in the case when 1H one pulse and/or combined rotation and multiple pulse spectroscopy (CRAMPS) sequences fail and assignment of proton chemical shift is ambiguous, a combination of 1H−13C and 1H−31P 2D heteronuclear correlation (HETCOR) correlations is found to be an excellent tool for the elucidation of 1H isotropic chemical shifts. In addition, a 2D strategy using 1H−1H double quantum filter (DQF) correlations [a back-to-back (BABA) sequence in this work] is useful for analyzing the topology of hydrogen bonding. In the case of a multicenter phosphorus domain, 2D 31P−31P proton driven spin diffusion experiments give information about the spatial arrangement of the phosphate residues.