The Arc repressor, which is involved in the switch between lysis and lysogeny of Salmonella bacteriophage P22, does not belong to any of the known classes of DNA-binding proteins. Mutagenesis studies show that the DNA-binding region is located in the 15 N-terminal amino-acid residues. We have now determined the three-dimensional structure of the Arc dimer from an extensive set of interproton-distance data obtained from 1H NMR spectroscopy. A priori, intra- and inter-monomer nuclear Overhauser effects (NOEs) cannot be distinguished for a symmetric dimer. But by using the homology with the Escherichia coli Met repressor we could interpret the NOEs unambiguously in an iterative structure refinement procedure. The final structure satisfies a large set of NOE constraints (1,352 for the dimer). It shows a strongly intertwined dimer, in which residues 8-14 of different monomers form an antiparallel beta-sheet. A model for the Arc repressor-operator complex can account for all available biochemical and genetic data. In this model two Arc dimers bind with their beta-sheet regions in successive major grooves on one side of the DNA helix, similar to the Met repressor interaction. Thus, Arc and Met repressors are members of the same family of proteins, which contain an antiparallel beta-sheet as the DNA-binding motif.

Proteins that are destined for export out of the cytoplasm of Escherichia coli cells are synthesized as precursor proteins with N-terminal extensions or signal sequences, which are essential for translocation of the protein across the inner membrane. Signal sequences contain very little primary sequence homology, and therefore recognition of these sequences is thought to involve specific folding. To assess the conformational flexibility of signal sequences, we have studied the signal peptide of PhoE (MKKSTLALVVMGIVASASVQA) by two-dimensional nuclear magnetic resonance and circular dichroism in different membrane mimetic environments. The secondary structure of the PhoE signal peptide was analyzed via interresidue nuclear Overhauser enhancement measurements, chemical shifts of backbone protons, and by measuring amide proton exchange. The membrane mimetic environments studied were trifluoroethanol (TFE) and micelles of sodium dodecyl sulfate (SDS) or dodecylphosphocholine (DPC). In all systems alpha-helix formation was observed. In TFE, the alpha-helix stretches from the positively charged N-terminus to Ser18. In SDS and DPC micelles, the N- and C-terminal alpha-helical half are separated from each other by a kink at the Gly12 position, with the helical content being higher at the N-terminus and lower at the C-terminus. In zwitterionic DPC micelles, the C-terminal region has a less regular or more flexible structure compared to SDS. The insertion of the PhoE signal peptide into the hydrophobic environment of the micelles was demonstrated by the effect of spin-labeled 12-doxylstearate on the line widths of the peptide proton resonances.(ABSTRACT TRUNCATED AT 250 WORDS)

The Arc repressor of Salmonella bacteriophage P22 is a dimeric sequence-specific DNA-binding protein. The solution structure of Arc has been determined from 2D NMR data using an "ensemble" iterative relaxation matrix approach (IRMA) followed by direct NOE refinement with DINOSAUR. A set of 51 structures was generated with distance geometry and further refined with a combination of restrained energy minimization and restrained molecular dynamics in a parallel refinement protocol. Distance constraints were obtained from an extensive set of NOE build-ups in H2O and 2H2O via relaxation matrix calculations from the ensemble of structures. Methyl group rotation, aromatic ring flaps and internal mobility effects (via order parameters obtained from a free molecular dynamics run in water) were included in these calculations. The best structures were finally refined with direct NOE constraints following a slow-cooling simulated annealing protocol. In this final refinement stage, theoretical NOE intensities were directly compared with the experimental data and forces were derived using a simple two-spin approximation for the gradient of the NOE function. Dynamic assignment was applied to the peaks involving unassigned diastereotopic groups. The structure is determined to a precision (r.m.s.d. from the average excluding the ill defined C and N-terminal region) of 0.55 and 1.10 A for backbone and all atoms, respectively. The final structures, with R factor values around 0.35, have good stereochemical qualities, contain an extensive network of hydrogen bonds consistent with the secondary structure elements and structural features in concordance with genetic data. The overall folding of the solution and crystal structures is the same.

The carbohydrate chains of the bronchial-mucus glycoproteins of six cystic fibrosis patients with blood group 0 were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high-performance liquid chromatography. Structural characterization was performed by 500-MHz 'H-NMR spectroscopy in combination with quantitative sugar analysis. The established structures range in size from tetra-up to heptasaccharides. They are all sialyl analogs of neutral oligosaccharides that were characterized previously [Lamblin G., Boersma A., Lhermitte M., Roussel P., Mutsaers J. Human bronchial mucus is secreted as a gelatinous layer on the surface of the tracheobronchial airway epithelium and is continuously moved towards the pharynx where it is swallowed. It acts as a medium for protection and lubrication of the mucosa and for transport of inhaled particles such as dust, pollen, viruses and bacteria to clear the bronchial epithelium.Bronchial mucins, as most mucins [l], contain about 70% carbohydrate which is 0-glyosidically linked to the peptide backbone as neutral and acidic oligosaccharide chains. The acid functions stem from the presence of sialic and/or sulfate residues [2].Any change in the structure of these oligosaccharides may modify the rheological properties of the bronchial mucus and lead to a non-efficient mucociliary clearance and to destruction phenomena found in cystic fibrosis or other bronchial diseases where hypersecretion of mucus is a pre-eminent feature of the pathological process. Increased adherence of inhaled particles may be a concomitant phenomenon.Since it is difficult to obtain the amount of normal human bronchial secretion required for detailed carbohydrate structural analysis [3], we have taken mucins secreted by patients suffering from cystic fibrosis (CF) for our structural studies. In the future, the results of the structural characterization of their carbohydrates will be compared to mucins secreted by patients with other chronic bronchial hypersecretion diseases.Previously, we have isolated and characterized several lowmolecular-weight oligosaccharides that were obtained from the carbohydrate material released from the mucins occurring in the sputum of six C F patients with blood group 0 [4-61. The structures of 20 neutral and 5 sialyl-oligosaccharides were determined by 500-MHz 'H-NMR spectroscopy in conjunction with sugar composition analysis. The present paper deals with the determination of the structure of another 13 sialyloligosaccharides isolated from the same pool of CF bronchial mucins. MATERIALS AND METHODS Isolation and purijkation of human bronchial sialyl-oligosaccharidesMucin glycoproteins were isolated from the sputum of six patients (blood group 0) suffering from cystic fibrosis as described [2]. Briefly, gelatinous bronchial mucus from the sputum was solubilized by the action of 2-mercaptoethanol (1 %, by vol.) and fractionated on an Ecteola-cellulose column with stepwi...

Wild-type Mnt repressor of Salmonella bacteriophage P22 is a tetrameric protein of 82 residues per monomer. A C-terminal deletion mutant of the repressor denoted Mnt (1-76) is a dimer in solution. The structure of this dimer has been determined using NMR. The NMR assignments of the majority of the 1H, 15N, and 13C resonances were obtained using 2D and triple-resonance 3D techniques. Elements of secondary structure were identified on the basis of characteristic sequential and medium range NOEs. For the structure determination more than 1000 NOEs per monomer were obtained, and structures were generated using distance geometry and restrained simulated annealing calculations. The discrimination of intra- vs intermonomer NOEs was based upon the observation of intersubunit NOEs in [15N,13C] double half-filtered NOESY experiments. The N-terminal part of Mnt (residues 1-44), which shows a 40% sequence homology with the Arc repressor, has a similar secondary and tertiary structure. Mnt (1-76) continues with a loop region of irregular structure, a third alpha-helix, and a random coil C-terminal peptide. Analysis of the secondary structure NOEs, the exchange rates, and the backbone chemical shifts suggests that the carboxy-terminal third helix is less stable than the remainder of the protein, but the observation of intersubunit NOEs for this part of the protein enables the positioning of this helix. The rsmd's between the backbone atoms of the N-terminal part of the Mnt repressor (residues 5-43, 5'-43') and the Arc repressor is 1.58 A, and between this region and the corresponding part of the MetJ repressor 1.43 A.