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Biomedical subjects

B Steipe

Publications and source records attributed to B Steipe.

15 recordsLinked to original sources
PubMed1997

Ta6Br(2+)12, a tool for phase determination of large biological assemblies by X-ray crystallography.

The title compound Ta6Br(2+)12 is of interest for the analysis of biological structures as a heavy-metal derivative with great potential for the structure determination of large protein systems. In macromolecular crystallography the phases of the measured structure factor amplitudes have to be determined. The most widely used method for novel structures is isomorphous replacement by introducing electron-rich compounds into the protein crystals. These compounds produce measurable changes of the diffraction intensities, which allow phase determination. We synthetized the Ta6Br(2+)12 cluster in high yields, crystallized it, and determined its crystal structure by X-ray diffraction analysis at atomic resolution. The cluster is a regular octahedron consisting of six metal atoms with 12 bridging bromine atoms along the 12 edges of the octahedron. The cluster is compact, of approximately spherical shape with about 4.3 A radius and highly symmetrical. One Ta6Br(2+)12 ion adds 856 electrons to a protein, a considerable contribution to the scattering power even of large proteins or multimeric systems. At low resolution all atoms of the cluster scatter in phase and act as a super heavy-atom, which is easy to locate in the difference Patterson map. We investigated its binding sites in the biologically significant high-resolution structures of an antibody V(L) domain, dimethyl sulfoxide reductase, GTP-cyclohydrolase I, and the proteasome. With the randomly oriented cluster, treated as a single site scatterer, phases could be used only up to 6 A resolution. In contrast, when the cluster is correctly oriented, phases calculated from its 18 atom sites can be used to high resolution. We present the atomic structure of the Ta6Br(2+)12, describe a method to determine its localization and orientation in the unit cell of protein crystals of two different proteins, and analyse its phasing power. We show that phases can be calculated to high resolution. The phase error is lower by more than 30 degrees compared to the single site approximation, using a resolution of 2.2 A. Furthermore, Ta6Br(2+)12 has two different strong anomalous scatterers tantalum and bromine to be used for phase determination.

Binding Sites↗
PubMed1997

Beta-turn propensities as paradigms for the analysis of structural motifs to engineer protein stability.

The thermodynamic stability of a protein provides an experimental metric for the relationship of protein sequence and native structure. We have investigated an approach based on an analysis of the structural database for stability engineering of an immunoglobulin variable domain. The most frequently occurring residues in specific positions of beta-turn motifs were predicted to increase the folding stability of mutants that were constructed by site-directed mutagenesis. Even in positions in which different residues are conserved in immunoglobulin sequences, the predictions were confirmed. Frequently, mutants with increased beta-turn propensities display increased folding cooperativities, suggesting pronounced effects on the unfolded state independent of the expected effect on conformational entropy. We conclude that structural motifs with predominantly local interactions can serve as templates with which patterns of sequence preferences can be extracted from the database of protein structures. Such preferences can predict the stability effects of mutations for protein engineering and design.

Amino Acid Sequence↗
PubMed1995

Evidence against specific binding of salicylic acid to plant catalase.

It was demonstrated that salicylic acid (SA) not only binds to catalase from differentiated higher plants and plant cell suspension cultures but also to those of fungi and animals. SA bound specifically to iron-containing enzymes, such as catalase, aconitase, lipoxidase and peroxidase, while not to iron-free plant enzymes. On the grounds of these experiments, the claim is further challenged that SA is a signalling compound and second messenger in plants that activates plant defense-related genes through elevated H2O2 levels by specifically inhibiting catalase activity. SA may just function as a phytoalexin.

Aconitate Hydratase↗
PubMed1995

Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas. Implications for a single-step purification of bispecific antibodies.

Conventional mouse/mouse or rat/rat hybrid-hybridoma supernatants contain up to 10 different IgG molecules consisting of various combinations of heavy and light chains. Hence, the yield of functional bispecific Ab is low, and purification is often complicated, hampering a general preclinical evaluation of, e.g., bispecific Ab-mediated tumor immunotherapy in animal models. In experiments to overcome this drawback we found that fusion of rat with mouse hybridomas opens the possibility of large scale production of bispecific Ab due to the increased incidence of correctly paired Ab and facilitated purification. In essence, rat/mouse quadroma-derived bispecific Ab have the following advantages: 1) enrichment of functional bispecific Ab because of preferential species-restricted heavy/light chain pairing (observed in four of four rat-mouse quadromas) in contrast to the random pairing in conventional mouse/mouse or rat/rat quadromas, and 2) a possible one-step purification of the quadroma supernatant with protein A. This simple chromatography step does not bind unwanted variants with parental rat/rat heavy chain configuration, and the desired rat/mouse bispecific Ab are retained, which can then easily be separated from parental mouse Ab by sequential pH elution.

Animals↗
PubMed1995

High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli.

We have utilized a T7 polymerase/promoter system for the high-level incorporation of methionine analogs with suitable labels for structural research (X-ray and NMR studies) on recombinant annexin V produced in Escherichia coli. Here, we describe, to our knowledge, the first biosynthetic high-level substitution of methionine by 2-aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein. The replacement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X-ray structural analysis. Conditions for expression were optimized concerning the frequency of appearance of revertants, high-level replacement and maximal protein yield. For the incorporation of norleucine and ethionine, E. coli B834 (DE3)(hsd metB), which is auxotrophic for methionine, was grown under methionine-limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted. The factor limiting the high-level incorporation of telluromethionine into protein is its sensitivity towards oxidation. To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and resuspended in fresh media without methionine; telluromethionine was added and protein synthesis induced. Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours). Biosynthetic incorporation of heavy atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy-atom derivatives suitable for X-ray structural analysis, supplementing the traditional trial-and-error preparation of heavy-atom derivatives for the method of multiple isomorphous replacement. Furthermore, the successful high-level incorporation of amino acid analogs can provide single-atom mutations for the detailed study of the structure and function of proteins.

Amino Acids↗
PubMed1995

The crystal structure of holo-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima at 2.5 A resolution.

The crystal structure of holo-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophile Thermotoga maritima was determined by Patterson search methods using the known structure of the Bacillus stearothermophilus enzyme. The structure was refined at a resolution of 2.5 A to an R-factor of 16.63% for 26289 reflections between 8.0 A an 2.5 A with F > 2 sigma(F). The crystallographic asymmetric unit contains two monomers related by approximate 2-fold symmetry and a tetramer is built up by crystallographic symmetry. The root-mean-square deviation of Ca positions of glyceraldehyde-3-phosphate dehydrogenase from T. maritima and B. stearothermophilus is 0.83 A in the NAD+ binding domains and smaller close to the cofactor. In contrast, the largest deviations in the catalytic domains are found at residues involved in coordination of sulphate ion SO4 339, which most likely marks the site of the attacking inorganic phosphate ion in catalysis. A large number of extra salt-bridges may be an important factor contributing to the high thermostability of this protein.

Amino Acid Sequence↗
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

Refined crystal structure of a recombinant immunoglobulin domain and a complementarity-determining region 1-grafted mutant.

We report the solution of the crystal structure of a mutant of the immunoglobulin VL domain of the antibody McPC603, in which the complementarity-determining region 1 segment is replaced with that of a different antibody. The wild-type and mutant crystal structures have been refined to a crystallographic R-factor of 14.9% at a nominal resolution of 1.97 A. A detailed description of the structures is given. Crystal packing results in a dimeric association of domains, in a fashion closely resembling that of an Fv fragment. The comparison of this VL domain with the same domain in the Fab fragment of McPC603 shows that the structure of an immunoglobulin VL domain is largely independent of its mode of association, even in places where the inter-subunit contacts are not conserved between VL and VH. In all three complementarity-determining regions we observe conformations that would not have been predicted by the canonical structure hypothesis. Significant differences between the VL domain dimer and the Fab fragment in the third complementarity-determining region show that knowledge of the structure of the dimerization partner and its exact mode of association may be needed to predict the precise conformation of antigen-binding loops.

Amino Acid Sequence↗
PubMed1990

Crystallization and preliminary X-ray studies of the VL domain of the antibody McPC603 produced in Escherichia coli.

The VL domain, obtained from a recombinant Fv fragment of the antibody McPC603 expressed in Escherichia coli, has been crystallized as a dimer from 2 M-(NH4)2SO4 (pH 4.0). The crystals are hexagonal, space group P6(1)22. The cell dimensions are a = b = 86.48 A, c = 76.64 A, with a VL monomer as the asymmetric unit. The crystals diffract to 2.0 A. The structure was solved by Patterson search using the VL domain of the Fab fragment of McPC603 and the VL dimer REI.

Chromatography, Affinity↗
PubMed1987

In situ studies of distal convoluted tubule in rat. II. K secretion.

Microperfusion and free-flow micropuncture studies were performed in anesthetized rats to compare the rates of K flux in early and late segments of the distal tubule. Early distal segments were located within the initial 40% of distal tubule length, and late distal segments were located within the terminal 35% of the distal tubule. In early distal segments of control rats, significant K secretion was observed at perfusion rates of 6 and 14 nl/min. In high-K rats significant K secretion was only observed at the lower perfusion rate. Late segments of control rats secreted 70.5 +/- 8.2 at the low flow rate and 139 +/- 18.9 pmol X min-1 X mm-1 at the high flow rate. In K-adapted rats, values were 134.4 +/- 30.2 and 178 +/- 29.3 pmol X min-1 X mm-1. During free flow, we observed a K flux in control rats of 11.0 +/- 9.4 pmol X min-1 X mm-1 (NS) in early segments and a K flux of 55 +/- 6.5 pmol X min-1 X mm-1 in late segments. In K-adapted rats, values were 18.3 +/- 6.7 (P less than 0.05) in early and 123 +/- 17.2 pmol X min-1 X mm-1 in late segments. The majority of our data suggests that the distal convoluted tubule secretes K at a low rate that is not influenced by flow rate or a high-K diet.

Animals↗
PubMed1984

Inactivation of atrial natriuretic substance by kallikrein.

To further characterize the properties of the potent natriuretic and diuretic substance that can be extracted from atrial tissue, we investigated its susceptibility to inactivation by kallikrein and other proteolytic enzymes. Extracts of rat atrial tissue (tissue wet wt 100 mg/ml) were incubated with enzymes under standard conditions and tested by injection into nondiuretic anesthetized rats. One hour of incubation at 37 degrees C with pure porcine pancreatic kallikrein at concentrations of 250 micrograms/ml or greater significantly reduced the activity of atrial natriuretic substance. The reduction in activity was dependent on both enzyme concentration and time of incubation. The kallikrein-catalyzed degradation was completely blocked by aprotinin but was only partially retarded by soybean trypsin inhibitor. Trypsin reduced natriuretic and diuretic activity of extracts at concentrations of 400 micrograms/ml or greater, with nearly complete inactivation at a concentration of 1,000 micrograms/ml. Carboxypeptidase B also caused a concentration-dependent inactivation of the natriuretic material. Last, alpha-chymotrypsin (1,000 micrograms/ml) and elastase (1,000 micrograms/ml) were found to destroy the natriuretic activity. In a separate set of experiments natriuretic activity was observed to be retained by a 1,000 mol wt cutoff membrane. Inactivation of the natriuretic peptide by renal kallikrein is a possible mechanism for in vivo regulation of natriuretic activity.

Animals↗
PubMed1982

Micropuncture studies of the renal effects of atrial natriuretic substance.

Micropuncture studies of the renal effects of atrial natriuretic substance. Injection of atrial extract produced by homogenization, boiling and centrifugation of atrial tissue from one heart caused a 10fold increase in urine flow rate and a 30-fold increase in Na excretion. Similarly prepared extracts of ventricle were without effect. To identify the site of action of atrial natriuretic substance, extract was infused intravenously at rates corresponding to 3 or 6 atria per hour. During infusion at a rate of 3 atria per hour mean urine flow increased from 9.5 +/- 2.8 to 17.2 +/- 1.2 microliter/min and Na excretion from 0.14 +/- 0.06 to 1.78 +/- 0.14 mumol/min. Glomerular filtration rate (GFR), single nephron filtration rate (SNGFR) and proximal and loop of Henle fluid absorption did not change significantly. During infusion of 6 atria per hour, paralleling a greater rise in urine flow rate (from 6.4 +/- 2.09 to 40.3 +/- 7.5 microliter/min) and in sodium excretion (from 0.18 +/- 0.0008 to 5.97 +/- 0.93 mumol/min), filtration rate, measured for either the single nephron or the whole kidney, rose. As a consequence of the rise in GFR, delivery of fluid and chloride into the distal tubule increased significantly. These data suggest that to a major extent the natriuresis is caused by transport inhibition along collecting tubules and collecting ducts. In addition, at high doses a rise in filtration rate contributes to the natriuretic effect of atrial extracts.

Animals↗