Protein structures from NMR data
This thesis describes the use of nuclear magnetic resonance techniques to determine the structures of two proteins in solution, hen egg-white lysozyme and human interleukin-4. Using 2D 1H methods an extensive set of structural data has been collected for hen lysozyme (1158 NOE distance restraints, 68 o and 24 ?1 dihedral angle restraints) and these data have been used in distance geometry-dynamical simulated annealing calculations to determine an ensemble of NMR structures for the protein. The overall Ca RMSD from the average for a set of 16 calculated structures is 1.8 ± 0.2 A but, excluding 14 residues in exposed disordered regions, this value reduces to 1.3 ± 0.2 Å. Regions of secondary structure, and particularly the four a helices, are well defined (Ca RMSD 0.8 ± 0.3 Å for helices). Detailed comparisons of the NMR structures with crystal structures of the protein have shown the close similarity of the main chain fold and the conformation of interior side chains in solution and in crystals. 3Jaß coupling constant measurement have indicated, however, that the conformational mobility of the side chains of many surface residues is significantly more pronounced than an individual crystal structure would suggest. For human interleukin-4, a strategy involving 15N and 13C labelled recombinant protein together with heteronuclear 3D NMR techniques has been employed to determine the structure of the protein. The work has provided the first structure for this protein, a left-handed four helix bundle with an up-up-down-down connectivity. For an ensemble of 10 final calculated NMR structures there is a Ca RMSD from the average of 1.6 ± 0.4 Å, the definition of the helical core of the protein being particularly good (0.8 ± 0.2 Å). There is, however, some disorder in the long overhand loops of the structure; this reflects the unusually high conformational mobility of these regions. Comparison of the interleukin-4 structure with proteins with related folds, particularly members of the haemopoietic cytokine superfamily, suggests that the fold found here for interleukin-4 may be the adopted structure throughout this cytokine superfamily. In a postscript to this thesis the NMR structure of human interleukin-4 is shown to have a very similar conformation to a crystal structure of the protein which has been solved very recently.