Biomedical subjects

S Steinbacher

Publications and source records attributed to S Steinbacher.

28 records · Page 2Linked to original sources

PubMed1996

Interactions of phage P22 tails with their cellular receptor, Salmonella O-antigen polysaccharide.

Bacteriophage P22 binds to its cell surface receptor, the repetitive O-antigen structure in Salmonella lipopolysaccharide, by its six homotrimeric tailspikes. Receptor binding by soluble tailspikes and the receptor-inactivating endorhamnosidase activity of the tailspike protein were studied using octa- and dodecasaccharides comprising two and three O-antigen repeats of Salmonella enteritidis and Salmonella typhimurium lipopolysaccharides. Wild-type tailspike protein and three mutants (D392N, D395N, and E359Q) with defective endorhamnosidase activity were used. Oligosaccharide binding to all three subunits, measured by a tryptophan fluorescence quench or by fluorescence depolarization of a coumarin label attached to the reducing end of the dodecasaccharide, occurs independently. At 10 degrees C, the binding affinities of all four proteins to oligosaccharides from both bacterial strains are identical within experimental error, and the binding constants for octa- and dodecasaccharides are 1 x 10(6) M(-1) and 2 x 10(6) M(-1), proving that two O-antigen repeats are sufficient for lipopolysaccharide recognition by the tailspike. Equilibration with the oligosaccharides occurs rapidly, but the endorhamnosidase produces only one cleavage every 100 s at 10 degrees C or about 2 min(-1) at the bacterial growth temperature. Thus, movement of virions in the lipopolysaccharide layer before DNA injection may involve the release and rebinding of individual tailspikes rather than hydrolysis of the O-antigen.

Bacteriophage P22↗

PubMed1995

Isolation, crystallization, crystal structure analysis and refinement of allophycocyanin from the cyanobacterium Spirulina platensis at 2.3 A resolution.

The phycobiliprotein allophycocyanin from the cyanobacterium Spirulina platensis has been isolated and crystallized. The crystals belong to space group P6(3)22 with cell constants a = b = 101.9 A, c = 130.6 A, alpha = beta = 90 degrees, gamma = 120 degrees, with one (alpha beta) monomer in the asymmetric unit. The three-dimensional structure of the (alpha beta) monomer was solved by multiple isomorphous replacement. The crystal structure has been refined in a cyclic manner by energy-restrained crystallographic refinement and model building. The conventional crystallographic R-factor of the final model is 19.6% with data from 8.0 to 2.3 A. The molecular structure of the subunits resembles other solved phycobiliprotein structures. In comparison to C-phycocyanin and b-phycoerythrin the major differences arise from deletions and insertions of segments involved in the protein-chromophore interactions. The stereochemistry of the alpha 84 and beta 84 chiral atoms are C(2)-R, C(3)-R and C(31)-R. The configuration (C(4)-Z, C(10)-Z and C(15)-Z) and the conformation (C(5)-anti, C(9)-syn and C(14)-anti) are equal for both chromophores.

Amino Acid Sequence↗

PubMed1995

Mutations that stabilize folding intermediates of phage P22 tailspike protein: folding in vivo and in vitro, stability, and structural context.

The folding of the trimeric phage P22 tailspike protein is affected by single amino acid substitutions designated temperature-sensitive folding (tsf) mutations. Their phenotypes are alleviated by two repeatedly isolated global suppressor (su) mutations (su V331A and su A334V) and by two additional substitutions (su V331G and su A334I), accessible through site-directed mutagenesis. We investigated the influence of the suppressor mutations on tailspike refolding in vitro, on its maturation at high expression levels in vivo, and on the rates of thermal unfolding of the native protein. All su mutations improved the folding efficiency in vitro and in vivo, but the relative effects of substitutions at position 334 were more pronounced in vivo, whereas the 331 substitutions were more effective in vitro. V331G caused the strongest increase in refolding yields of any single mutation, and was as effective as the V331A/A334V double mutation, where the two single mutations exhibited an additive effect. Both V331A and V331G retarded thermal denaturation, while A334V did not affect, and A334I accelerated unfolding. A334I is the first mutation found to affect the folding of the tailspike and the thermal stability of the native protein in opposite directions. The observed effects can be rationalized on the basis of the recently determined crystal structure of an N-terminally shortened tailspike. As the backbone dihedral angles of Val331 (phi = -119 degrees, psi = -142 degrees) are unusual for non-glycine residues, V331G and V331A may remove steric strain and thereby stabilize folding intermediates and the native protein. The beta-branched side-chains of Val and Ile substituted for Ala334 in the interior of the protein may improve a hydrophobic stack of residues in the large parallel beta-helix. This is likely important in loosely structured early folding intermediates, but not in the very rigid native structure, where the side-chain of Ile can hardly be accommodated.

Bacteriophage P22↗

PubMed1994

Sequence statistics reliably predict stabilizing mutations in a protein domain.

Immunoglobulin variable domains are generally thought of as well conserved platforms providing the base for antigen binding loops of highly varying sequence and structure. However, domain evolution must ensure a balance between optimizing antigen affinity and the requirements of a stable, cooperatively folding domain. Since random mutations can carry a significant penalty for domain stability, constraints are imposed both on the repertoire of germline sequences and on somatic amino acid replacements during affinity maturation. Analyzing these constraints in the conceptual framework of statistical mechanics, we have been able to predict stabilizing mutations in the McPC603 V kappa domain from sequence information alone with better than 60% success rate. The validity of this concept not only has far reaching implications for antibody engineering but may also be generalized to engineer other proteins for higher stability.

Amino Acid Sequence↗

PubMed1994

Crystal structure of P22 tailspike protein: interdigitated subunits in a thermostable trimer.

The tailspike protein (TSP) of Salmonella typhimurium phage P22 is a part of the apparatus by which the phage attaches to the bacterial host and hydrolyzes the O antigen. It has served as a model system for genetic and biochemical analysis of protein folding. The x-ray structure of a shortened TSP (residues 109 to 666) was determined to a 2.0 angstrom resolution. Each subunit of the homotrimer contains a large parallel beta helix. The interdigitation of the polypeptide chains at the carboxyl termini is important to protrimer formation in the folding pathway and to thermostability of the mature protein.

Bacteriophage P22↗

PubMed1992

[Use of collagen in sealing of arterial puncture holes after heart catheterization].

Percutaneously introduced absorbable purified bovine collagen was used to plug the hole at the site of femoral arterial puncture in 50 patients (42 men, 8 women; median age 58 [22-75] after percutaneous coronary angiography (n = 32; heparin dosage 100 IU/kg) or transluminal coronary balloon dilatation (n = 18; heparin dosage 200 U/kg). Local bleeding ceased after compression of 4 min in 48 of the 50 patients. A pressure bandage was needed additionally in the other two. Bed-rest lasted for 1-24 (median 19) hours. A haematoma of more than 6 cm diameter developed in five patients, but required neither transfusion nor surgical intervention. 39 of the 50 patients were re-examined after a median of 39 days: all of them had had a small pain-free swelling for 2-6 weeks over the puncture site. One patient developed a deep-vein thrombosis one week after the procedure, in another fever occurred after the same period, lasting for two days. These early findings are encouraging. The method may significantly shorten the time of arterial compression and bed-rest, as well as reduce the risk of a large haematoma after arterial punctures.

Adult↗

PubMed1992

Collagen application for sealing of arterial puncture sites in comparison to pressure dressing: a randomized trial.

One hundred patients undergoing routine diagnostic or interventional catheterization were randomly assigned to receive either percutaneously applied collagen (group A; n = 50) or conventional pressure dressing (group B; n = 50) for sealing of the femoral artery. Clinical variables were comparable in both groups. The heparin dose was 100 IU/kg in 30 patients and 200 IU/kg in 20 patients of either group. The average compression time was 4.3 min in group A and 42.3 min in group B (p < .001). Bleeding was not observed in group A but was observed in 6/50 patients in group B. The time to ambulation was 6.4 hr (range, 4-12 hr) in group A and 21.6 hr (range, 10-48 hr) in group B (p < .001). Hematomas with a diameter of > 6 cm developed in 4/50 patients in group A and in 11/50 patients in group B (p < .05). Blood-transfusions or surgical interventions were not required and there was no loss of ankle pulses in either group. In conclusion, percutaneously applied collagen reduced compression time and duration of bedrest after diagnostic catheterization and PTCA. Despite earlier ambulation, the incidence of bleeding was lower with collagen than with conventional pressure dressing.

Aged↗

PubMed1992

[Randomized comparison between collagen administration and pressure dressings for occlusion of the arterial puncture after coronary angiography and coronary dilatation].

One hundred patients undergoing routine diagnostic or interventional catheterization were randomly assigned to receive either percutaneously applied collagen (group A; n = 50) or conventional pressure dressing (group B; n = 50) for sealing of the femoral artery. Clinical variables were comparable in both groups. The heparin dose was 100 IU/kg in 30 patients and 200 IU/kg in 20 patients of both groups. The average compression time was 4.3 min in group A and 42.3 min in group B (p < 0.001). In group A, 1/50 patients required pressure dressing compared to 50/50 patients in group B. Bleeding was not observed in group A, but was observed in 6/50 patients in group B. The time to ambulation was 6.4 (range, 4-12) h in group A and 21.6 (range, 10-48) h in group B (p < 0.001). Hematomas with a diameter of > 6 cm developed in 4/50 patients in group A and in 11/50 patients in group B (p < 0.05). Blood-transfusions or surgical interventions were not required and there was no loss of ankle pulses in either group. In conclusion, percutaneously applied collagen reduced compression time and duration of bedrest after diagnostic catheterization and PTCA. Despite earlier ambulation, the incidence of bleeding was lower with collagen than with conventional pressure dressing.

Angioplasty, Balloon, Coronary↗

PubMedDate not supplied

Interaction of Salmonella phage P22 with its O-antigen receptor studied by X-ray crystallography.

The O-antigenic repeating units of the Salmonella cell surface lipopolysaccharides (serotypes A, B and D1) serve as receptors for phage P22. This initial binding step is mediated by the tailspike protein (TSP), which is present in six copies on the base plate of the phage. In addition to the binding activity, TSP also displays a low endoglycolytic activity, cleaving the alpha(1,3)-O-glycosidic bond between rhamnose and galactose of the O-antigenic repeats. The crystal structure of TSP in complex with receptor fragments allowed to identify the receptor binding site for the octasaccharide product of the enzymatic action of TSP on delipidated LPS and the active site consisting of Asp392, Asp395 and Glu359. The structure comprises a large right-handed parallel beta-helix of 13 turns. These fold independently in the trimer, whereas the N-terminus forms a cap-like structure and the C-terminal parts of the three polypeptide strands merge to a single common domain. In addition, TSP has served as model system for the folding of large, multisubunit proteins. Its folding pathway is influenced by a large number of point mutations, classified as lethal, temperature sensitive or general suppressor mutations, which influence the partitioning between aggregation and the productive folding pathway.

Antibody Specificity↗

PubMedDate not supplied

Structure and reaction mechanism of L-arginine:glycine amidinotransferase.

L-Arginine:glycine amidinotransferase (AT) catalyzes the committed step in creatine biosynthesis by formation of guanidinoacetic acid, the direct precursor of creatine. The X-ray structure of the human enzyme shows a novel fold with fivefold pseudosymmetry of beta beta alphabeta-modules. These modules enclose the active site compartment of the basket-like structure. The active site of AT lies at the bottom of a very narrow channel and contains a catalytic triad with the residues Cys-His-Asp. The transamidination reaction follows a ping-pong mechanism and is accompanied by large conformational changes. During catalysis the amidino group is covalently attached to the active site cysteine to give an amidino-cysteine intermediate.

Amidinotransferases↗