A set of HA-detected experiments for measuring scalar and residual dipolar couplings

Abstract

A new set of HCACO based three-dimensional NMR experiments for measuring residual dipolar couplings in proteins is presented. Using spin-state selection and editing in three dimensions, the experiments allow accurate measurement of intraresidual $^{1}D_{\rm C^{\alpha} H^{\alpha}}$, $^{1}D_{\rm C^{\prime}C^\alpha}$ and $^{2} D_{\rm C^{\prime}H^\alpha}$ scalar and residual dipolar couplings of ¹⁵␣N/¹³C labeled proteins in D₂O and dilute liquid crystals with minimal spectral crowding. The presented experiments are especially suitable for small or medium sized proline-rich proteins, or proteins that require high pH solvent conditions, making ¹H^N detected experiments unattractive. In addition, the tetrahedral coordination of C^α is superior to the planar peptide bond for determination of local alignments in partially structured polypeptides. For the efficient use of spectrometer time and to avoid complications arising from the varying magnitude of the alignment tensor during relatively long experiments, the $^{1}D_{\rm C^{\alpha}H^\alpha}$ and $^{2}D_{\rm C^{\prime}H^\alpha}$ couplings can also be measured simultaneously in an E.COSY like manner with high accuracy. The pulse sequences are balanced for cross-correlation effects and minimized for relaxation losses. The pulse sequences are tested with a sample of ¹⁵N/¹³C human ubiquitin. We find internuclear vector directions determined from the dipolar couplings to have an excellent correlation with those of ubiquitin’s refined solution structure.

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