AURELIA is an advanced program for the computer-aided evaluation of two-, three- and four-dimensional NMR spectra of any type of molecule. It can be used for the analysis of spectra of small molecules as well as for evaluation of complicated spectra of biological macromolecules such as proteins. AURELIA is highly interactive and offers a large number of tools, such as artefact reduction, cluster and multiplet analysis, spin system searches, resonance assignments, automated calculation of volumes in multidimensional spectra, calculation of distances with different approaches, including the full relaxation matrix approach, Bayesian analysis of peak features, correlation of molecular structures with NMR data, comparison of spectra via spectral algebra and pattern match techniques, automated sequential assignments on the basis of triple resonance spectra, and automatic strip calculation. In contrast to most other programs, many tasks are performed automatically.

The solution structure of histidine-containing phosphocarrier protein from Staphylococcus carnosus was determined by two-and three-dimensional nuclear magnetic resonance (NMR) spectroscopy on uniformly 15 N-enriched protein. The main structural element is an antiparallel (3-pleated sheet with four strands A, B, C, and D arranged with the topology A-D-B-C. Strand A comprises residues 2 to 8, strand B residues 32 to 37, strand C reidues 40 to 43, and strand D residues 59 to 66. Three right-handed helices are arranged on top of the (3-pleated sheet. Helix a reaches from residue 16 to 29, helix b from residue 48 to 53, and helix c from residue 72 to 83. Strands B and C of the (3-pleated sheet are connected by a type II' turn. The hydroxyl proton of Ser-31 is exchanging with the solvent so slowly that cross peaks can be detected in two-dimensional NMR spectra based on homonuclear J-couplings. The imidazole ring of the active-center His-15, which is partly charged in the structure determined at pH 7.2, is located above the N-terminal end of helix a, perpendicular to its axis. The N5 ' atom of His-15, accepting the phosphoryl from enzyme 1, is exposed to the solvent.

The phosphocarrier HPr (heat stable protein) of Staphylococcus carnosus was modified by site-directed mutagenesis of the corresponding ptsH gene in order to analyse the importance of amino acids which were supposed to be part of the active centre of the protein. Three residues which are conserved in all HPrs, Arg17, Pro18 and Glu84, were mutated: Arg17 was changed to His (17RH) and Pro18 and Glu84 were changed into Ala (18PA and 84EA). In addition, Leu86 was changed into Ala (86LA) and one mutant protein was missing the last six residues of the HPr (delta 83). The wild type gene and all mutant genes were overexpressed and the gene products purified to homogeneity. Three-dimensional structures of wild type and mutant proteins were monitored by NMR spectroscopy. All five mutant HPrs had native conformations. The ATP-dependent HPr kinase can phosphorylate all HPr derivatives at Ser46. The PTS activity of the amino-terminal HPr mutant proteins 17RH and 18PA was different compared to wild type HPr. In contrast, the carboxy-terminal mutant HPrs possessed a similar enzyme activity to the wild type HPr. The 17RH and 18PA HPrs with substitution near the active centre His15 showed a very slow phosphorylation by enzyme I but the further transfer of the phosphoryl group to enzyme III was also strongly inhibited. The enzyme activity of the HPr 17RH was significantly improved at low pH. NMR pH-titration experiments showed that Arg17 is not responsible for the low pKa of the active centre His15 but this positively charged residue is essential in this position for the HPr activity.

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Intact rabbit myosin and two different C-terminal fragments of rabbit muscle light meromyosin (LMM) expressed in Escherichia coli, LMM-30, and LMM-30C', were studied by 1H NMR spectroscopy. X-ray small-angle scattering shows that at high ionic strength two polypeptide chains of LMM-30 (which consists of the C-terminal 262 amino acids of myosin heavy chain) or LMM-30C' (which corresponds to LMM-30 but lacks the last 17 residues) assemble to form an alpha-helical coiled-coil as it is found also in myosin. The last 12 C-terminal residues of one polypeptide chain of LMM-30 and the last 9 C-terminal residues of the other chain are very mobile. The last 8 residues of the two strands are equivalent from the NMR point of view and unfolded; the valine residues in position 255 in the two strands are not equivalent, suggesting an interaction between the two strands, Ser-252, Arg-253, and Asp-254 are completely immobilized in one of the polypeptide strands and partly mobile in the other. Essentially the same pattern is observed in intact myosin. In spite of the large molecular weights of LMM-30 and LMM-30C', it is possible to resolve almost all aromatic residues and to determine the pK values of all the 4 tyrosine and of 9 (out of 10) histidine residues. The tyrosine residues in the two strands are equivalent in the two polypeptide chains and both have a pK of 10.5. The pK values of the histidine residues vary between 5.7 and 7.0.