Net magnetic susceptibility anisotropy of a protein causes its non- random distribution when an isotropic solution is placed in a strong magnetic field. New methods have been developed for measurement of the small dipolar couplings which result from the small degree of alignment with the magnetic field. Even for small diamagnetic proteins the dipolar couplings potentially yield useful constraints for structure calculation and/or validation. Other significant developments, related to macromolecular structure determination by NMR, include the first experimental determination of a fully asymmetric rotational diffusion tensor, carried out for the HIV protease homodimer, and an experimental correlation between hydrogen bond length and the backbone amide deuteron quadrupole coupling. This latter experiment also represents the first time that backbone amide deuteron quadrupole couplings have been measured in a protein. The structure of the HIV-a accessory protein Nef has been determined by multi-dimensional heteronuclear NMR. Structure determination involved a number of non-standard elements, including the use of perdeuteration of non-exchangeable hydrogens, which yielded a protein fold and suggested mutations for reducing protein aggregation. Standard experiments on the less-aggregating form of the protein then yielded a low-resolution three-dimensional structure. Nef binds with submicromolar affinity to Hck SH3, and the interaction surface was determined on the basis of chemical shift perturbation. This interaction represents the first example of an SH3 domain interacting with more than a single (proline-rich) epitope. Similar experiments were used for identification of a specific, albeit low affinity (Kd about 1mM), binding site for a peptide fragment of the cytosolic N- terminal domain of CD4. Binding of Nef to the CD4 peptide and the Hck SH# are weakly cooperative events.
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