Molecular architecture and electrostatic properties of a bacterial porin.

The integral membrane protein porin from Rhodobacter capsulatus consists of three tightly associated 16-stranded beta barrels that give rise to three distinct diffusion channels for small solutes through the outer membrane. The x-ray structure of this porin has revealed details of its shape, the residue distributions within the pore and at the membrane-facing surface, and the location of calcium sites. The electrostatic potential has been calculated and related to function. Moreover, potential calculations were found to predict the Ca2+ sites.

An mRNA degrading complex in Rhodobacter capsulatus.

An RNA degrading, high molecular weight complex was purified from Rhodobacter capsulatus. N-terminal sequencing, glycerol-gradient centrifugation, and immunoaffinity purification as well as functional assays were used to determine the physical and biochemical characteristics of the complex. The complex comprises RNase E and two DEAD-box RNA helicases of 74 and 65 kDa, respectively. Most surprisingly, the transcription termination factor Rho is a major, firmly associated component of the degradosome.

End group analysis of yeast fatty acid synthetase.

The purified yeast fatty acid synthetase complex has been subjected to amino and carboxyl terminal amino acid end group analysis. Amino end groups were studied by Edman degradation and by dansylation of the sodium dodecyl sulfate- or urea-denatured complex. No N-terminal amino acid could be identified by either method. C-terminal amino acids were investigated by tritium labeling and by digestion of the complex with carboxypeptidases A and B. By both methods, the two amino acids valine and lysine were consistently identified as the C-termini of two different polypeptide chains. After separation of the fatty acid synthetase subunits A and B by sodium dodecyl sulfate polyacrylamide gel electrophoresis lysine was identified as the C-terminus of subunit A and valine as that of subunit B. The results are interpreted as evidence that the yeast fatty acid synthetase complex is basically composed of two nonidentical and multifunctional polypeptide chains.

Prediction of the general structure of OmpF and PhoE from the sequence and structure of porin from Rhodobacter capsulatus. Orientation of porin in the membrane.

By comparing the hydrophilicity profiles and sequences of porin from Rhodobacter capsulatus with those of OmpF and PhoE from Escherichia coli, a set of insertions and deletions for alignment of the sequences has been deduced. With this alignment a similar folding of OmpF and PhoE has been predicted as found by X-ray structure analysis of porin from Rhodobacter capsulates. Furthermore, the orientation of the porin trimer in the outer membrane was inferred from topological data on PhoE. According to this result a single channel of approx. 30 A diameter starts at the outer surface. Near the middle of the outer membrane bilayer this channel branches out into three separate channels, each running within a single porin monomer to the periplasmic surface.

Fluorescent glucagon derivatives. I. Synthesis and characterisation of fluorescent glucagon derivatives.

The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC50 values of 6.8.10(-9), 1.7.10(-8), 1.8.10(-8) and 5.4.10(-9) M were measured in rat liver membranes for NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. From the IC50 values Kd values of 2.16.10(-9), 4.10(-9), 2.10(-9) and 1.72.10(-9) M were calculated for the binding of NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-Trp25-glucagon in phosphate-buffered saline (pH 7.4). Difluorescein-glucagon was also prepared by reacting the amino groups of histidine-1 and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiolTrp25-glucagon. Difluorescein-glucagon bound only weakly to glucagon receptors and displayed antagonist properties. The dimer derivative formed from two difluorescein-2-thiolTrp25-glucagon molecules had similar poor binding qualities, whereas the dimer formed from difluorescein-2-thiolTrp25-glucagon and 2-thiolTrp25-glucagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp25-glucagon and difluorescein-glucagon to rat hepatocytes was followed using flow cytometry.

Structure of the membrane channel porin from Rhodopseudomonas blastica at 2.0 A resolution.

The crystal structure of a membrane channel, homotrimeric porin from Rhodopseudomonas blastica has been determined at 2.0 A resolution by multiple isomorphous replacement and structural refinement. The current model has an R-factor of 16.5% and consists of 289 amino acids, 238 water molecules, and 3 detergent molecules per subunit. The partial protein sequence and subsequently the complete DNA sequence were determined. The general architecture is similar to those of the structurally known porins. As a particular feature there are 3 adjacent binding sites for n-alkyl chains at the molecular 3-fold axis. The side chain arrangement in the channel indicates a transverse electric field across each of the 3 pore eyelets, which may explain the discrimination against nonpolar solutes. Moreover, there are 2 significantly ordered girdles of aromatic residues at the nonpolar/polar borderlines of the interface between protein and membrane. Possibly, these residues shield the polypeptide conformation against adverse membrane fluctuations.

Porin from the halophilic species Ectothiorhodospira vacuolata: cloning, structure of the gene and comparison with other porins.

The gene coding for the anion-specific porin of the halophilic eubacterium Ectothiorhodospira (Ect.) vacuolata was cloned and sequenced, the first such gene so analyzed from a purple sulfur bacterium. It encodes a precursor protein consisting of 374 amino acid (aa)-residues including a signal peptide of 22-aa residues. Comparison with aa sequences of porins from several other members of the Proteobacteria revealed little homology. Only two regions showed local homology with the previously sequenced porins of Neisseria species, Comamonas acidovorans, Bordetella pertussis, Alcaligenes eutrophus, and Burkholderia cepacia. Genomic Southern blot hybridization studies were carried out with a probe derived from the 5' end of the gene coding for the porin of Ect. vacuolata. Two related species, Ect. haloalkaliphila and Ect. shaposhnikovii, exhibited a clear signal, while the extremely halophilic bacterium Halorhodospira (Hlr.) halophila (formerly Ect. halophila) did not show any cross-hybridization even at low stringency. This result is in good accordance with a recently proposed reassignment within the family Ectothiorhodospiraceae, which included the separation of the extremely halophilic species into the new genus Halorhodospira.

Identification and solubilization of a signal peptidase from the phototrophic bacterium Rhodobacter capsulatus.

In Gram-negative bacteria, exported proteins are synthesized with an amino-terminal signal sequence which is cleaved off by the signal peptidase during, or shortly after the translocation process. Here, we report the identification and solubilization of a signal peptidase from the phototrophic bacterium Rhodobacter capsulatus which cleaves homologous and heterologous precursor proteins at the authentic cleavage site. This signal peptidase is the first identified component of the R. capsulatus protein export machinery.

The complete primary structure of GTP:AMP phosphotransferase from beef heart mitochondria.

To complete the amino acid sequence of GTP:AMP phosphotransferase (MgGTP + AMP in equilibrium with MgGDP + ADP) from beef heart mitochondria it was necessary to sequence an intermediate region of about 33 residues after position 102 [(1984) Eur. J. Biochem. 143, 331-339] and find a suitable overlap with the rest of the protein. The required peptides were obtained by cleaving the enzyme with endoproteinase Lys-C. One peptide, covering the region from residue 79 to 144, was sequenced up to residue 124. Another peptide, extending from residue 79 to 169, was subcleaved with Staphylococcus aureus V8 protease and provided the fragment from residue 99 to 139 which was sequenced. Several other peptides from endoproteinase Lys-C cleavage were used to check large sections of the previously published sequence work. The complete sequence contains 225 amino acids and has an Mr of 25 469.

A protein with sequence identity to Skp (FirA) supports protein translocation into plasma membrane vesicles of Escherichia coli.

We have purified to homogeneity a 15 kDa-protein from a ribosomal salt extract of Escherichia coli that compensates in vitro a defect of SecA but not of SecB. Removal of this protein from a cell-free transcription/translation system impairs translocation into plasma membrane vesicles of the precursors of LamB and to a lesser degree also of OmpA. These results suggest a role of the 15 kDa-protein in bacterial protein export. The NH2-terminal 35 amino acids were found to be identical to those of the skp (firA) gene product, to which several putative functions have previously been attributed.

The structure of porin from Rhodobacter capsulatus at 1.8 A resolution.

The structure of the porin from Rhodobacter capsulatus was determined at a resolution of 1.8 A. The analysis started from a closely related crystal structure that had been solved at a medium resolution of 3 A using multiple isomorphous replacement and solvent flattening. The new structure contains the complete sequence of 301 amino acid residues. Refinement of the model is under way; the present R-factor is 22% with good geometry. Except for the lengths of several loops, the resulting chain fold corresponds to the medium resolution model. The membrane channel is lined by a large number of ionogenic side chains with characteristic segregation of differently charged groups.

Isolation and complete amino acid sequence of the beta- and alpha-polypeptides from the peripheral light-harvesting pigment-protein complex II of Rhodobacter sulfidophilus.

The peripheral light-harvesting bacteriochlorophyll-carotenoid-protein complex B800-850 (LHII) has been isolated from membranes of semi-aerobic dark-grown cells of Rhodobacter sulfidophilus strain W4. A reversed-phase HPLC system resolved one beta- and one alpha-polypeptide in the ratio 1:1. The material obtained was of high purity and suitable for direct microsequence analysis. The primary structures of the beta- and alpha-polypeptides have been determined. The beta-polypeptide consists of 51 amino acid residues, yielding a molecular mass of 5512 Da and having 64.7% hydrophobicity. The alpha-polypeptide consists of 52 amino acid residues, with a calculated molecular mass of 5661 Da and 75% hydrophobicity. The significance of uncommon structure motives with respect to the unusual spectroscopic characteristics of this light-harvesting complex is discussed.

Muscle and liver pyruvate kinases are closely related: amino acid sequence comparisons.

Previous evidence has shown that the M1 and L pyruvate kinase isozymes differ markedly in kinetic and immunological properties, amino acid compositions and peptide maps. However, the amino acid sequence results we present here for the N-terminal region and for a region of the C domain show that the M1 and L isozymes are very similar. The variable length of the N-terminal sequences also explains the difference in regulation by phosphorylation between the M1 and L isozymes. The M1 isozyme lacks the serine residue that has been shown to be phosphorylated in the L isozyme.

Testosterone-induced diminution of two peptides in spleen cells from testosterone-immunosuppressed B10 mice.

High-performance liquid chromatography (HPLC) is used to detect testosterone (T)-sensitive peptides in spleen cells isolated from female C57BL/10 mice immunosuppressed against Plasmodium chabaudi malaria by T treatment. Two peaks with retention times of about 25 min and 34 min, respectively, were identified to be diminished by about 52% and 47%, respectively, in spleen cells from T-treated mice compared to those from untreated mice. Amino acid sequencing revealed that the 24 min peak consisted of the dipeptide Met-Phe and the 34 min peak contained a degradative fragment of the alpha-chain of hemoglobin. Our data suggest that the immunosuppressive T treatment of B10 mice induces a perturbation of erythrophagocytosis in spleens.

Staphylococcal neutral phosphatase. A highly cationic molecule with binding properties for immunoglobulin.

Staphylococcal neutral phosphatase (NPtase) was purified from two Staphylococcus aureus strains by sequential high salt extraction, ultracentrifugation and ion exchange chromatography. The enzyme showed maximum phosphatase activity at neutral pH, appeared as two bands in SDS-PAGE (31 and 32 kDa), and the isoelectric point was > 10. No close similarity between NPtase and other known bacterial proteins in respect of their N-terminal amino acid sequences was found. Purified NPtase bound rat and human polyclonal IgG [intact and F(ab')2 fragments], IgM, IgA, intact myeloma immunoglobulins, myeloma light chains, gamma heavy chain and, with a much lower affinity, Fc fragments. Furthermore, NPtase can bind serum albumin. Heparin, a highly negatively charged molecule, significantly inhibited NPtase binding to immunoglobulins and HSA, but did not inhibit the binding of specific antibodies to NPtase; this indicates that charge interactions are important. The newly characterized staphylococcal phosphatase with binding properties for immunoglobulin is an interesting bacterial protein that could be involved in post-infectious sequelae.

A peptidoglycan binding domain in the porin-associated protein (PAP) of Rhodospirillum rubrum FR1.

The porin-associated protein of Rhodospirillum rubrum FR1 was found to contain a peptidoglycan binding motif. A partial fragment of 179 amino acids, obtained by cleavage of PAP with trypsin, Asp-N protease, and CNBr, was sequenced. Substantial sequence homology was found of the C-terminal part (residues 126-179) of porin-associated protein with OmpA, the peptidoglycan-associated lipoprotein of several bacteria, protein F of Pseudomonas aeruginosa, and PIII of Neisseria gonorrhoeae, the latter being also a porin-associated protein. The 179 amino acid fragment comprised about 67% of the mass spectrometrically determined total mass of PAP of 27850 Da.

Molecular mimicry by Mycoplasma pneumoniae to evade the induction of adherence inhibiting antibodies.

Specific regions of adherence binding sites and epitopes of the P1 adhesin of Mycoplasma pneumoniae were synthesised as octapeptides and used as targets in a modified enzyme-linked immunosorbent assay. Acute phase and convalescent sera from 10 patients with M. pneumoniae infection were tested for antibody reactivity to these octapeptides. In convalescent sera, antibody activities were directed against octapeptides of the epitope regions, whereas no antibody activity was found in acute or convalescent sera to octapeptides of adherence-mediating binding sites could be explained partially from the results of cross-reactivity experiments with adherence-inhibiting anti-P1 adhesin monoclonal antibodies (MAbs). Two of these MAbs showed cross-reactions with intracellular antigens of eukaryotic cell lines in immunofluorescence microscopy experiments. The cross-reacting antigens were isolated and characterised as glyceraldehyde-3-phosphate dehydrogenase and 2-phospho-D-glycerate hydrolyase. Antigenic mimicry of eukaryotic structures by functional sites of the P1 adhesin of M. pneumoniae may influence the pathogenesis of M. pneumoniae infection.

Studies of the interaction of the maltose-binding protein of Escherichia coli, a closed-groove binder, with 4,6-O-ethylidenemalto-oligosaccharides (dp 2-5) and its regioselective labelling with 3-azibutyl 1-thio-alpha-(6-3H)maltoside.

Four malto-oligosaccharides (dp 2-5), each with a 4,6-O-ethylidene group on the glucosyl unit at the non-reducing terminus, were synthesised and used to prove that the maltose-binding protein (MBP) of E. coli is a closed-groove binder. alpha-D-Glucosylation of 3-azibutyl 1-thio-alpha-D-(6-3H)glucopyranoside yielded a 3H-labelled, photolabile 1-thiomaltoside derivative that was used to chemically modify the binding site of MBP. The 3H-labelled peptide containing 83% of the total radioactivity, which was isolated after tryptic cleavage of the modified MBP and sequenced, is part of the closed end of the MBP groove.

Differential chemical modification of substrate binding areas in porcine-pancreatic alpha-amylase by three regioisomeric photolabile ligands.

Three regioisomeric radiolabelled spacer-modified oligosaccharides: methyl 4'-O-[4-S-(3-azi-4-alpha-D-glucopyranosyloxy-1-[3H]butyl)-6- deoxy- 4-thio-alpha-D-xylo-hex-5-enopyranosyl]-alpha-maltoside (12a, G1-G3*), methyl 4-O-[4-S-(3-azi-4-alpha-maltosyloxy-1-[3H]butyl)-6-deoxy-4-t hio- alpha-D-xylo-hex-5-enopyranosyl]-alpha-D-glucopyranoside (15a, G2-G2*) and methyl 4-S-(3-azi-4-alpha-maltotriosyloxy-1-[3H]butyl)-6-deoxy-4-th io-alpha- D-xylo-hex-5-enopyranoside (16a, G3-G1*) were synthesised and used as photoaffinity probes for the chemical modification of porcine-pancreatic alpha-amylase (PPA). Incorporation of covalently attached radioactivity amounted to 25-38% of the stoichiometric value. Tryptic digestion of the three labelled protein preparations PPA-G1-G3*, PPA-G2-G2*, and PPA-G3-G1* and the purification of the labelled peptides by fractional HPLC yielded altogether six pure components. On the basis of the published three-dimensional structure peptides G1-G3-II, G2-G2-II, and G2-G2-III were part of the catalytic site. G1-G3-I and G2-G2-I were part of the surface binding site. The major component derived from PPA, labelled by G3-G1*, corresponded to an area that is neither close to the active site nor to the surface starch-binding domain, which clearly indicates the presence of a third, hitherto undetected, substrate-binding site.

Immuno-gold electron microscopical detection of heat shock protein 60 (hsp60) in mitochondria of rat hepatocytes and myocardiocytes.

We characterize the specificity of a polyclonal antibody against heat shock protein 60 (hsp60) and present an application for ultrastructural localization studies of this protein. The antibody was obtained from an IgG fraction (AB 121) originally raised against the calcium binding protein calsequestrin by immunoabsorption on isolated rat liver hsp60. As shown by partial N-terminal amino acid sequence analysis of immunoprecipitated proteins AB 121 contained reactivities against hsp60, calsequestrin and the glycoprotein fetuin. In rat heart AB 121 recognized calsequestrin and hsp60. In human and rat liver the only reacting protein was hsp60. In rat erythrocytes the antibody bound to 61 kDa and 58 kDa isoforms of fetuin. According to published data no amino acid sequence homologies nor common motifs are found between calsequestrin, hsp60 and fetuin. As the first application the anti-hsp60 antibody was used for immuno-gold electron microscopical localization of hsp60: in myocardiocytes and hepatocytes of the rat strong labelling was obtained exclusively in mitochondria. No extramitochondrial structures were labelled. The specificity of the antibody and its ability to be visualized by immuno-gold electron microscopy offers the possibility to study the expression of this protein in the liver and in other organs. Possible clinical applications of these studies are discussed, since hsp60 could be a target antigen of autoantibodies in diseases such as autoimmune hepatitis, primary sclerosing cholangitis or primary biliary cirrhosis.