posted on 1998-11-11, 00:00authored byMarcel Ottiger, Ad Bax
Weak alignment of solute macromolecules with the magnetic field can be achieved in a dilute,
aqueous liquid crystalline phase of planar phospholipid micelles, consisting of mixtures of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC). Alignment of proteins in such a medium
is sufficiently weak to retain the simplicity of the isotropic solution NMR spectrum but strong enough to
permit accurate measurement of residual one-bond dipolar couplings. Highly accurate one-bond N−HN, Cα−Hα, Cα−C‘, and C‘−N and two-bond C‘−HN dipolar couplings were measured in 13C/15N-enriched ubiquitin.
Together with knowledge of the protein's three-dimensional structure, the dipolar couplings permit calculation
of the relative, vibrationally corrected average bond lengths for these interactions. Assuming a C‘−N bond
length of 1.329 Å (Engh, R. A.; Huber, R. Acta Crystallogr.1992, A47, 392−400), the relative Cα−C‘ distance
of 1.526 Å is found to be in excellent agreement with results from Engh and Huber (1.525 Å). Using a C‘−N
bond length of 1.329 Å as a reference, N−HN (1.041 ± 0.006 Å) and Cα−Hα (1.117 ± 0.007 Å) are considerably
longer than equilibrium or average internuclear distances derived from ab initio calculations, electron diffraction,
neutron diffraction, or microwave spectroscopy. The increase in effective N−HN and Cα−Hα bond lengths is
attributed to a decrease in the corresponding dipolar couplings resulting from fast librations, which must be of
considerably larger amplitude than the Cα−C‘ and C‘−N angular fluctuations. Accurate knowledge of the
relative effective N−HN, Cα−Hα, Cα−C‘, C‘−N, and two-bond C‘−HN effective internuclear distances is
essential for determining the magnitude of the molecular alignment tensor, for using the dipolar couplings in
macromolecular structure determination, and for extracting angular information from recently described cross
correlation experiments.