Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and in the dry state

The structure of the central repetitive domain of high molecular weight (HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis. In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type II β-turns at positions predicted from the model studies; type II β-turns occurred at QPGQ and QQGY sequences and type I β-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of β-turns. The type I β-turns are lost when the protein is dried on a solid surface, probably by conversion to type II β-turns. The homogeneous type II β-turn distribution is compatible with the formation of a β-spiral structure, which provides the protein with elastic properties. The β-turns and thus the β-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis

Phosphorylation in the mannitol specific PTS. Aspects of the phosphorylation processes in the mannitol specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) in Escherichia coli.

Studies naar de samenhang tussen struktuur en funktie van enzymen moeten uiteindelijk leiden tot een beter begrip hiervan. Dit proefschrift beschrijft onderzoek dat in het kader van dit soort studies is uitgevoerd, Het richt zich met name op de karakterisering van enzymen die in ons laboratorium gebruikt worden voor het bestuderen van de genoemde struktuur/funktie relaties. Het betreft de enzymen HPr en IIAmtl; hiervan is HPr een natuurlijk voorkomend enzym maar IIAmtl is van oorsprong een domein van een groter eiwit dat specifiek is voor de opname van mannitol (mtl).... zie : Samenvatting

Potential of 13C and 15N Labeling for Studying Protein-Protein Interactions Using Fourier Transform Infrared Spectroscopy

In this study, we examine the interaction between two bacterial proteins, namely HPr and IIAmtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system, using FTIR spectroscopy. In an interaction involving a 1:1 molar ratio of these two proteins, when they are unlabeled, the overlap of absorbance of the amide I band arising from the peptide group vibrations of the two proteins is such that it is not possible to determine the contribution which each protein makes to the absorbance. Uniform 15N labeling has little effect on the frequency of the amide I band although there is a significant shift of the amide II band. However, we show that uniform (90%) 13C labeling produces a large shift of bands associated with the carbonyl moiety, especially the amide I band. This opens up windows in different regions of the infrared spectrum. Thus, when the same mixture of the two bacterial proteins is made where one of the proteins is uniformly 13C-labeled (in our case HPr), the amide I maxima of this protein shifts by ~45 cm-1 toward lower frequency and reveals the previously overlapped amide I band of the unlabeled IIAmtl. This application of 13C labeling shows the potential of studying protein-protein interactions using FTIR spectroscopy. With thoughtful selection of systems and labeling strategies, numerous studies with proteins should be possible. These could include, among others, enzyme-substrate and protein-ligand interactions.

Exchange of Phosphoryl Groups between HPr Molecules of the Phosphoenolpyruvate-Dependent Phosphotransferase System Is an Autocatalytic Process

HPr, a central component of the phosphoenolpyruvate-dependent phosphotransferase system, can exist in Escherichia coli in a phosphorylated (PHPr) and a nonphosphorylated form. We show that, beside the normal transfer of the phosphoryl group from PHPr to enzymes II and III, PHPr can phosphorylate other HPr molecules in an autocatalytic exchange reaction. The reaction is very fast but is inhibited by labeling the protein with Bolton-Hunter reagent. We demonstrate that the exchange reaction can be used to determine the ΔG° of the phosphoryl group of mutant forms of PHPr relative to wild-type PHPr. Two HPr mutants were constructed by site-directed mutagenesis, HPr P11E and HPr E68A. Both show altered phosphoryl group potentials but show no significantly altered KM or Vmax values compared to wild-type HPr, illustrating the sensitivity of the exchange process. The exchange reaction does not occur between HPr from E. coli and HPr from Staphylococcus carnosus.

Effect of Phosphorylation on Hydrogen-Bonding Interactions of the Active Site Histidine of the Phosphocarrier Protein HPr of the Phosphoenolpyruvate-Dependent Phosphotransferase System Determined by 15N NMR Spectroscopy

The phosphocarrier protein HPr of the phosphoenolpyruvate-dependent sugar transport system of Escherichia coli can exist in a phosphorylated and a nonphosphorylated form. During phosphorylation, the phosphoryl group is carried on a histidine residue, His15. The hydrogen-bonding state of this histidine was examined with 15N NMR. For this purpose we selectively enriched the histidine imidazole nitrogens with 15N by supplying an E. coli histidine auxotroph with the amino acid labeled either at the Nδ1 and Nε2 positions or at only the Nδ1 position. 15N NMR spectra of two synthesized model compounds, phosphoimidazole and phosphomethylimidazole, were also recorded. We show that, prior to phosphorylation, the protonated His15 Nε2 is strongly hydrogen bonded, most probably to a carboxylate moiety. The H-bond should strengthen the nucleophilic character of the deprotonated Nδ1, resulting in a good acceptor for the phosphoryl group. The hydrogen bond to the His15 Nδ1 breaks upon phosphorylation of the residue. Implications of the H-bond structure for the mechanism of phosphorylation of HPr are discussed.

Three-dimensional 15N-1H-1H and 15N-13C-1H nuclear-magnetic resonance studies of HPr a central component of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Assignment of backbone resonances

We have performed three-dimensional NMR studies on a central component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli, denoted as HPr. The protein was uniformly enriched with 15N and 13C to overcome spectral overlap. Complete assignments were obtained for the backbone 1H, 15N and 13C resonances, using three-dimensional heteronuclear 1H NOE 1H-15N multiple-quantum coherence spectroscopy (3D-NOESY-HMQC) and three-dimensional heteronuclear total correlation 1H-15N multiple-quantum coherence spectroscopy (3D-TOCSY-HMQC) experiments on 15N-enriched HPr and an additional three-dimensional triple-resonance 1HN-15N-13Cα correlation spectroscopy (HNCA) experiment on 13C,15N-enriched HPr. Many of the sequential backbone 1H assignments, as derived from two-dimensional NMR studies, were corrected. Almost all discrepancies are in the helical regions, leaving the published antiparallel β-sheet topology almost completely intact.