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Histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, when incubated at elevated temperatures forms many species of protein. The two major species are HPr-1 and HPr-2, which have been shown to lack one or two amides, respectively (Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 7023-7033). The formation of HPr-1 and HPr-2 is shown to be pH-dependent and does not occur readily below pH 6. Investigation of the identities and properties of the two residues that deamidate involved creation of site-directed mutants at the 6 glutamine and 2 asparagine residues of HPr; description of their deamidation species by isoelectric focusing; determination of their relative antibody binding properties; assay of their phosphoacceptor and phosphodonor activities; characterization of tryptic and V8-protease peptides; obtaining two-dimensional nuclear magnetic resonance spectra of HPr, HPr-1, and several mutants. It was determined that the sequential deamidation of Asn-38 and Asn-12 yields HPr-1 and HPr-2. Both residues exist as Asn-Gly pairs, and both deamidations probably form isoaspartyl acid. HPr from Bacillus subtilis and Staphylococcus carnosus which also have Asn-Gly at residues 38 and 39 form HPr-1 species presumably by deamidation. HPr from Streptococcus faecalis which does not have Asn-38 does not form a HPr-1 species. The E. coli mutant HPrs, N12D and Q51E, residues that may be involved in the active site, had impaired phosphohydrolysis properties and decreased phosphoenolpyruvate:sugar phosphotransferase system activity.

While having essentially no amino acid sequence homology, the mitochondrial leader sequences of different pre-proteins carry out the same function of targeting the protein to mitochondria. Among the common attributes that have been noted for leader sequences are a net positive charge and the ability to form amphiphilic alpha-helices. The aim of the research described here was to determine the relative importance of these two attributes in the leader sequence of rat liver mitochondrial aldehyde dehydrogenase. Through site-directed mutagenesis, arginine residues were systematically replaced by glutamine. It was found that individual arginines could be replaced without loss of import competence, so the total charge of the leader sequence was not required for the pre-protein to be imported. However, when two arginines were replaced simultaneously, especially Arg3 and Arg10, the pre-protein lost the ability to be imported. This ability was restored by modifying the leader sequence to increase dramatically its helix-forming potential. This leader sequence did not contain a positive-charged side-chain until Arg11. Therefore, it has been shown that positive charge is not required in the first ten residues provided the sequence could form a relatively more stable alpha-helix. The ability to form an amphiphilic alpha-helix remains the essential factor in determining whether or not a leader sequence can carry out its import function.

Histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, when incubated at elevated temperatures forms many species of protein. The two major species are HPr-1 and HPr-2, which have been shown to lack one or two amides, respectively (Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 7023-7033). The formation of HPr-1 and HPr-2 is shown to be pH-dependent and does not occur readily below pH 6. Investigation of the identities and properties of the two residues that deamidate involved creation of site-directed mutants at the 6 glutamine and 2 asparagine residues of HPr; description of their deamidation species by isoelectric focusing; determination of their relative antibody binding properties; assay of their phosphoacceptor and phosphodonor activities; characterization of tryptic and V8-protease peptides; obtaining two-dimensional nuclear magnetic resonance spectra of HPr, HPr-1, and several mutants. It was determined that the sequential deamidation of Asn-38 and Asn-12 yields HPr-1 and HPr-2. Both residues exist as Asn-Gly pairs, and both deamidations probably form isoaspartyl acid. HPr from Bacillus subtilis and Staphylococcus carnosus which also have Asn-Gly at residues 38 and 39 form HPr-1 species presumably by deamidation. HPr from Streptococcus faecalis which does not have Asn-38 does not form a HPr-1 species. The E. coli mutant HPrs, N12D and Q51E, residues that may be involved in the active site, had impaired phosphohydrolysis properties and decreased phosphoenolpyruvate:sugar phosphotransferase system activity.

The basis for the development of a suite of programs that allow the user to determine motional features (the correlation time and the significance of segmental motion) and the optimum conditions for future experiments from a NOESY signal matrix is presented. This automated evaluation of NOESY data serves as the initial step of an iterative conformational analysis which uses the molecular model manipulation capabilities of modern graphics workstations. Incorporated in these programs is NOESYSIM, a calculation subroutine which uses a set of molecular coordinates (and a correlation time estimate) together with user entered experimental parameters (acquisition time, sweep width, mixing time and cycle repetition time) to generate an accurately calculated NOESY signal matrix reflecting those conditions and the specified conformational model. Conformational refinement then consists of iterative comparisons of the experimental signal matrix with a series (or systematically sampled set) of model coordinates corresponding to a dynamics' course, driven-minimization or torsional grid search. These procedures and developments are illustrated with examples including: solution conformations of prostanoids; studies of the folding preferences and media-dependent changes in conformation for peptide hormones; and the structure elucidation of a novel undecapeptide macrolide antibiotic (lysobactin). For larger molecules, even constrained grid searches have too high a dimensionality and one must resort to distance-constraint based minimizations. A novel procedure for deriving more accurate distance constraints (corrected for secondary NOEs) is detailed and a new strategy for conformation elucidation, based on this procedure, is outlined.