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Biomedical subjects

E Pohl

Publications and source records attributed to E Pohl.

At least 37 records · Page 2Linked to original sources
PubMed1997

The role of structural domains in RIP II toxin model membrane binding.

The interaction of plant toxin ricin and MLI binding subunits to liposomes containing monosialoganglioside (GM1), bearing a terminal galactose residue, has been examined as a possible receptor model. For the first time we demonstrate that ricin B-chain but not ricin provokes liposome aggregation at 10 M% GM1 concentration, whereas in the presence of either ricin A-chain or galactose the aggregation is inhibited. The B-subunit of plant toxin MLI from Viscum album has similar lectin specificity and activity but cannot aggregate GM1 liposomes. The ability of the B-chain to aggregate liposomes adds a new crucial step in the toxin transmembrane penetration mechanism. We demonstrate here possible ricin B-chain interactions with membranes proceeding via two sites, namely (a) a galactose-binding domain and (b) a hydrophobic interchain domain. In close contact with two phospholipid bilayers, ricin B-chain may determine the geometry of the fusion site. These events can provoke A-chain translocation which follows membrane fusion.

Binding Sites↗
PubMed1997

Comparison of high-resolution structures of the diphtheria toxin repressor in complex with cobalt and zinc at the cation-anion binding site.

The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is a divalent-metal activated repressor of chromosomal genes responsible for siderophore-mediated iron-uptake and of a gene on several corynebacteriophages that encodes diphtheria toxin. Even though DtxR is the best characterized iron-dependent repressor to date, numerous key properties of the protein still remain to be explained. One is the role of the cation-anion pair discovered in its first metal-binding site. A second is the reason why zinc exhibits its activating effect only at a concentration 100-fold higher than other divalent cations. In the presently reported 1.85 A resolution Co-DtxR structure at 100K, the sulfate anion in the cation-anion-binding site interacts with three side chains that are all conserved in the entire DtxR family, which points to a possible physiological role of the anion. A comparison of the 1.85 A Cobalt-DtxR structure at 100K and the 2.4 A Zinc-DtxR structure at room temperature revealed no significant differences. Hence, the difference in efficiency of Co2+ and Zn2+ to activate DtxR remains a mystery and might be hidden in the properties of the intriguing second metal-binding site. Our studies do, however, provide a high resolution view of the cationanion-binding site that has most likely evolved to interact not only with a cation but also with the anion in a very precise manner.

Bacterial Proteins↗
PubMed1996

High-resolution structure of the diphtheria toxin repressor complexed with cobalt and manganese reveals an SH3-like third domain and suggests a possible role of phosphate as co-corepressor.

The crystal structure of diphtheria toxin repressor (DtxR) in complex with the corepressor Co2+ has been determined at 2.0 A resolution and in complex with Mn2+ at 2.2 A resolution. The structure of the flexible third domain could be determined at this high resolution. It appears to contain five antiparallel strands exhibiting a fold very similar to the SH3 domain. A superposition of 46 equivalent C alpha atoms of DtxR and alpha-spectrin SH3 resulted in an rms deviation of 3.0 A. The sequence identity is only 7%. This third domain of DtxR appears to have no interactions with the DNA binding domain nor with the metal binding domain of the repressor. Yet, flexibility in the region between the second and the third domain allows in principle significant conformational changes such as might occur upon DNA binding. The two metal binding sites in the second domain have been unraveled in considerable detail. Metal binding site 1 was well occupied in both the cobalt and manganese structures and showed a surprising sulfate ion as ligand. The sulfate was proven beyond doubt by the high peak at its position in a selenate versus sulfate difference Fourier. The presence of the intriguing sulfate ion at such a crucial position near the metal corepressor suggests the possibility that under physiological conditions phosphate may act as a "co-corepressor" for this class of metal-regulated DNA binding proteins in Corynebacteria, Mycobacteria, and related organisms. The second metal binding site is significantly different in these two DtxR structures. In the 2.0 A cobalt structure, the site is not occupied by a metal ion. In the 2.2 A manganese structure the site is well occupied, at approximately the same position as observed previously in cadmium DtxR. The ligands are Glu105, His106, the carbonyl oxygen of Cys102, and a water molecule. The reasons for differential occupancy of this site in different structures are intriguing and require further investigations.

Amino Acid Sequence↗
PubMed1996

Contribution of the intramolecular disulfide bridge to the folding stability of REIv, the variable domain of a human immunoglobulin kappa light chain.

BACKGROUND: Immunoglobulin domains contain about 100 amino acid residues folded into two beta-sheets and stabilized in a sandwich by a conserved central disulfide bridge. Whether antibodies actually require disulfide bonds for stability has long been a matter of debate. The contribution made by the central disulfide bridge to the overall folding stability of the immunoglobulin REIv, the variable domain of a human kappa light chain, was investigated by introducing stabilizing amino acid replacements followed by removal of the disulfide bridge via chemical reduction or genetic substitution of the cysteine residues. RESULTS: Nine REIv variants were constructed by methods of protein engineering that have folding stabilities elevated relative wild-type REIv by (up to) 16.0 kJ mol-1. Eight of these variants can be cooperatively refolded after unfolding and chemical reduction of the disulfide bridge-in contrast to wildtype REIv. The stabilizing effect of one of these residue replacements (T39K) was rationalized by determining the structure of the respective REIv variant at 1.7 A. The loss of folding stability caused by reduction of the intramolecular disulfide bond is on average 19 kJ mol-1. Removal of the disulfide bridge by genetic substitution of C23 for valine resulted in a stable immunoglobulin domain in the context of the stabilizing Y32H amino acid exchange; again, REIv-C23V/Y32H has 18 kJ mol-1 less folding stability than REIv-Y32H. The data are consistent with the notion that all variants studied have the same overall three-dimensional structure with the disulfide bridge opened or closed. CONCLUSIONS: A comparison of the magnitude of the stabilizing effect exerted by the disulfide bond and the length of the mainchain loop framed by it suggests lowering of the entropy of the unfolded state as the sole source of the effect. Disulfide bonds are not necessary for proper folding of immunoglobulin variable domains and can be removed, provided the loss of folding stability is at least partly compensated by stabilizing amino acid exchanges.

Disulfides↗
PubMed1995

Structures of quinoxaline antibiotics.

The crystal structures of three quinoxaline antibiotics-echinomycin 2QN, triostin C and the C222(1) form of triostin A--have been determined, and the structure of the P2(1)2(1)2(1) form of triostin A has been re-refined against our previously reported data. The molecular conformations are compared with those deduced from NMR data and those reported for two complexes of triostin A with oligonucleotides. Although the depsipeptide ring conformations are basically similar, the effective twofold molecular symmetry is violated by the folding of one of the quinoxaline chromophores in echinomycin 2QN and by a rotation of one of the ester planes with the formation of an intramolecular hydrogen bond in triostin C. In the oligonucleotide complexes of triostin A the chirality of the disulfide bridge is inverted. The alanine NH groups are involved in intermolecular hydrogen bonds in all four structures, and (except in echinomycin 2QN) the stacking of the chromophores in the crystal emulates the intercalation involved in DNA complex formation. In echinomycin 2QN, the antibiotic molecules are hydrogen bonded to form a helix along the crystallographic 6(5) screw axes, with a channel of disordered solvent running through the middle of the helix. Crystal data: (1), echinomycin 2QN, C53H66N10O12S2.2.5(C3H6O).2.5(H2O), M(r) = 1289.5, hexagonal, P6(5), a = b = 22.196(15), c = 24.64 (2) A, V = 10,513 (13) A3, Z = 6, Dx = 1.222 Mg m-3, lambda (Cu K alpha) = 1.5418 A, mu = 1.275 mm-1, T = 193 K, R = 9.0% for 4828 I > 2 sigma (I) and 11.8% for all 7102 unique reflections; (2), triostin C, C54H70N12O12S2.0.67(CHCl3).0.67(H2O), M(r) = 1234.2, orthorhombic, P2(1)2(1)2(1), a = 16.054 (8), b = 17.128 (9), c = 22.706 (12) A, V = 6244 (6) A3, Z = 4, Dx = 1.313 Mg m-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.239 mm-1, T = 188 K, R = 7.7% for 4678 I > 2 sigma (I) and 14.0% for all 7260 unique reflections; (3), triostin A, C50H62N12O12S2.2(C7H14O2), M(r) = 1347.6, orthorhombic, P2(1)2(1)2(1), a = 20.94 (2), b = 18.53 (2), c = 18.80 (2) A, V = 7292 (13) A3, Z = 4, Dx = 1.228 Mg m-3, lambda (Cu K alpha) = 1.5418 A, mu = 1.245 mm-1, T = 293 K, R = 6.8% for 2116 I > 2 sigma (I) and 9.3% for all 2928 unique reflections; (4), triostin A, C50H62N12O12S2.HCl.2(C3H7NO), M(r) = 1269.9, monoclinic, C222(1), a = 10.622 (10), b = 17.035 (17), c = 35.21 (3) A, V = 6371 (10) A3, Z = 4, Dx = 1.324 Mg m-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.199 mm-1, T = 153 K, R = 7.5% for 2164 I > 2 sigma (I) and 13.2% for all 3402 unique reflections. Extensive use was made of restraints on the geometrical and displacement parameters in the successful anisotropic refinement of these structures against weak data.

Anti-Bacterial Agents↗
PubMed1995

Ab initio determination of the crystal structure of cytochrome c6 and comparison with plastocyanin.

BACKGROUND: Electron transfer between cytochrome f and photosystem I (PSI) can be accomplished by the heme-containing protein cytochrome c6 or by the copper-containing protein plastocyanin. Higher plants use plastocyanin as the only electron donor to PSI, whereas most green algae and cyanobacteria can use either, with similar kinetics, depending on the copper concentration in the culture medium. RESULTS: We report here the determination of the structure of cytochrome c6 from the green alga Monoraphidium braunii. Synchrotron X-ray data with an effective resolution of 1.2 A and the presence of one iron and three sulfur atoms enabled, possibly for the first time, the determination of an unknown protein structure by ab initio methods. Anisotropic refinement was accompanied by a decrease in the 'free' R value of over 7% the anisotropic motion is concentrated at the termini and between residues 38 and 53. The heme geometry is in very good agreement with a new set of heme distances derived from the structures of small molecules. This is probably the most precise structure of a heme protein to date. CONCLUSIONS: On the basis of this cytochrome c6 structure, we have calculated potential electron transfer pathways and made comparisons with similar analyses for plastocyanin. Electron transfer between the copper redox center of plastocyanin to PSI and from cytochrome f is believed to involve two sites on the protein. In contrast, cytochrome c6 may well use just one electron transfer site, close to the heme unit, in its corresponding reactions with the same two redox partners.

Chlorophyta↗
PubMed1995

Structure of octreotide, a somatostatin analogue.

Octreotide, a synthetic somatostatin analogue, is an octapeptide with one disulfide bridge. Crystals of octreotide are orthorhombic, space group P2(1)2(1)2(1), a = 18.458 (5), b = 30.009 (7), c = 39.705 (27) A, with three molecules of octapeptide, one ordered oxalate dianion and 52 water molecules in the asymmetric unit. Complete protonation of the NH(2) groups (as assumed in the refinement) would require three oxalate dianions in the asymmetric unit for charge neutrality; a chemical analysis indicated that four are present. In either case they are so disordered that they cannot be distinguished from the water molecules. The 18 951 unique reflections (R(sym) = 0.026) used for structure solution and refinement were recorded with the EMBL imaging-plate scanner using synchrotron radiation. The structure was solved by Patterson interpretation, locating the three disulfide bridges, followed by tangent phase expansion and E-Fourier recycling. The anisotropic refinement against all F(2) data between 1.04 and 10.0 A resolution by blocked restrained full-matrix least-squares techniques converged to a conventional R index based on F of 0.084 [I > 2a(I) and 10.0 > d > 1.04 A] and wR2, the weighted R-index on F(2), of 0.246 (for all data). One peptide molecule adopts a flat beta-sheet structure; the other two possess different irregular backbone conformations, but are similar to each other. All three molecules have a distorted type II' beta-turn around the D-Trp-Lys region, but exhibit different side-chain conformations. The crystal structure is stabilized by a network of inter- and intramolecular hydrogen bonds.

Journal Article↗
PubMed1995

Comparison of different X-ray data-collection systems using the crystal structure of octreotide.

The octapeptide octreotide crystallizes with three peptide molecules and about 20% water in the asymmetric unit, and in many ways possesses diffraction properties similar to those of a 'mini-protein' consisting of 24 amino-acid residues. It diffracts to about 1.0 A but data in the range 1.4-1.0 A are weak. It provides a suitable test of different macromolecular X-ray data-collection techniques, especially of their ability to measure weak reflections accurately. In contrast to typical proteins it is possible to perform a full anisotropic refinement, that we believe provides a more objective test of the quality of the data than the internal consistency of equivalent reflections. We have collected a total of six data sets. The X-ray sources included synchrotron radiation, Cu Kalpha rotating anodes and Mo Kalpha sealed tubes; position-sensitive two-dimensional detectors from four manufacturers and a four-circle diffractometer with scintillation counter were employed. Two of the six data sets were collected at low temperature. Reasonable anisotropic refinement was possible with all area-detector data sets, although significant differences in the precision of the final model were observed. In addition we tested the ability of automated Patterson interpretation to solve the structure using the six independent data sets. The structure solution was only successful using the synchrotron or rotating-anode data sets, i.e. for the more intense sources. It appears that for structure solution the maximum resolution of the data is critical, whereas for refinement the accuracy of the data is more important.

Journal Article↗
PubMed1986

Inhibition of immune phagocytosis by human sera with HLA A, B, C and DR but not with DQ or EM type reactivity.

613 sera from pregnant women were investigated for inhibition of immune phagocytosis (IPI) by monocytes exposed to anti-D (Rh)-sensitized human red blood cells. IPI was detected in 42 (26%) of 165 and in 78 (17%) of 448 sera assessed against monocytes from 15 or 5 panel donors, respectively. All IPI-positive sera reacted in an allotypic pattern. Eight IPI-positive sera tested with autologous monocytes were found to be nonreactive. Upon comparative analysis of 448 sera, a significant correlation was found between the specific patterns of IPI and lymphocytotoxicity by HLA antibodies. In addition, 13 sera (4%) were positive for IPI, but not for lymphocytotoxicity. Twenty IPI-positive sera were tested by indirect immunofluorescence against platelets and T lymphocytes and revealed IgG antibodies in 16 and 11 sera, respectively. IPI activity could be removed from 8 out of 10 positive sera by platelet pool absorption. While virtually all sera exhibiting HLA, A, B or C-like activity (cytotoxicity with all cells) or HLA DR-like activity (exclusive cytotoxicity with B lymphocytes and monocytes) were IPI-positive, IPI was infrequently observed with sera containing HLA DQ-like activity (cytotoxicity with B lymphocytes only). IPI was also rarely seen with sera cytotoxic only to monocytes and not at all with sera containing antibodies against endothelial/monocyte antigens. We conclude that IPI is caused by cytotoxic as well as noncytotoxic HLA A, B, C, DR-specific antibodies. This effect may bear significance for the maternal immune response against fetal antigens and may be useful for pretransplant histocompatibility testing.

Adult↗
PubMed1985

The main inconsequences in the present radiological protection concept for the general population.

The present concept of radiation protection emphasizes the protection of man against ionizing radiation from manmade sources. But the overwhelming radiation exposure in a given population results from the natural radiation environment (NRE) and the use of radiation in medicine, while less than 2% of the total dose is due to occupational exposure, fallout, releases from nuclear industries, etc. Nevertheless, ICRP recommendations do not apply to, or include, "normal" NRE exposure, nor do they deal with medical exposure of patients by applying risk/benefit considerations. The increasing problems associated with various types of NRE exposures (recycling of waste materials, energy-efficient dwellings) and the continuously increasing use of ionizing radiation in medical practices (x-ray and nuclear diagnostic examinations) require a revision of the present radiation protection concept. In this paper suggestions are made concerning the applicability of a unified concept for controlling all sources of ionizing radiation.

Background Radiation↗
PubMed1983

Radiation exposure of the respiratory tract and associated carcinogenic risk due to inhaled radon daughters.

The atmospheric content of radon and its decay products contributes significantly to the radiation exposure of man even in the normal environment. In this study, the risk for lung cancer induction associated with normal exposure to natural radionuclides is assessed in Salzburg, Austria. Altogether more than a thousand rooms have been investigated by using combined radon grab-sampling methods together with continuous measurements of radon and daughters at different control stations. Dose calculations were carried out for 729 demoscopically selected test persons, considering individual differences in age, sex and life-style and atmospheric nuclide concentrations at different sites. Using a specially developed age-dependent lung model, the dose frequency distribution of the absorbed dose to the basal cells of the bronchial epithelium could be evaluated for the population of Salzburg. Dose modifications caused by anatomical and physiological variabilities as well as microdosimetric considerations of the stochastic events during energy deposition result in a significantly increased dispersion of the dose histogram. Induction of lung cancer due to inhalation of radon and decay products can be correlated with cumulative exposure based on experience with lung cancer incidence amongst uranium miners. Using the recommended range of lifetime risk values of 20-45 X 10(-5)/WLM (UN77), it is shown for Salzburg that about 15% of the observed number of lung cancer cases may be induced by natural radionuclides.

Austria↗
PubMed1976

Time function of corticosteroid levels in the blood plasma of rats under the influence of 222Rn inhalation.

The time function of cortocosteroid level in plasma of rats under two different 222Rn concentrations was investigated. Both curves show a maximum after 8h. Whereas the higher activity of 222Rn produces a second maximum after 5 days, the lower activity reaches its second maximum not before 9 days. From both time functions, a two-step mechanism in the intracellular control can be concluded.

Adrenal Cortex Hormones↗