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U Heinemann

Publications and source records attributed to U Heinemann.

At least 235 records · Page 13Linked to original sources
PubMed1993

Molecular and active-site structure of a Bacillus 1,3-1,4-beta-glucanase.

The three-dimensional structure of the hybrid Bacillus 1,3-1,4-beta-glucanase (beta-glucanase; 1,3-1,4-beta-D-glucan 4-glucanohydrolase, lichenase, EC 3.2.1.73) designated H(A16-M) was determined by x-ray crystallography at a resolution of 2.0 A and refined to an R value of 16.4% using stereochemical restraints. The protein molecule consists mainly of two seven-stranded antiparallel beta-pleated sheets arranged atop each other to form a compact, sandwich-like structure. A channel crossing one side of the protein molecule accommodates an inhibitor, 3,4-epoxybutyl beta-D-cellobioside, which binds covalently to the side chain of Glu-105, as seen in a crystal structure analysis at 2.8-A resolution of the protein-inhibitor complex (R = 16.8%). That Glu-105 may be indispensible for enzyme catalysis by H(A16-M) is suggested by site-directed mutagenesis of this residue, which inevitably leads to an inactive enzyme.

Amino Acid Sequence↗
PubMed1993

Role of NMDA receptors and voltage-activated calcium channels in an in vitro model of cerebral ischemia.

In an in vitro model of cerebral ischemia we investigated the functional consequences of repeated hypoxias and the potential protective effect of the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV) and the calcium channel blocker verapamil in preventing the expression of pathophysiological activity. Rat neocortical slices were exposed to nitrogen for 2-13 min and the hypoxia-induced functional modifications were monitored in layer II/III by recording the extracellular DC potential, the extracellular calcium concentration ([Ca2+]o) and the stimulus-evoked synaptic responses. Hypoxia caused a reversible 2.4-24.6 mV negative shift in the extracellular DC potential associated with a [Ca2+]o decrease from 1.2 to 0.2 mM and a complete loss of synaptic responsiveness. Repeating hypoxias induced an increase in the amplitude of this anoxic depolarization (AD) and a significant decrease in the AD onset latency. Synaptic responses partially recovered at 20 and 60 min intervals between subsequent hypoxic periods, indicating that the initial AD did not induce any short-term irreparable functional deficits. Verapamil (50 microM) caused an increase in the AD onset latency. However, in comparison to untreated controls, verapamil induced a reduction of excitatory and inhibitory responses during hypoxia probably by blocking voltage-activated calcium conductances. In addition, verapamil did not have any significant effect on the hypoxia-induced reduction of [Ca2+]o. Bath application of D-APV (30 microM) prevented the significant reduction in the AD onset latency to the second hypoxia, but had no significant effect on the AD amplitude and duration. The hypoxia-induced decrease in [Ca2+]o was not altered after addition of D-APV to the bathing medium. These data indicate that the influx of calcium through voltage-activated calcium channels and the NMDA receptor-gated ionophore does not significantly contribute to the massive depolarization observed under hypoxic conditions.

2-Amino-5-phosphonovalerate↗
PubMed1993

Synthesis of the Bacillus subtilis histone-like DNA-binding protein HBsu in Escherichia coli and secretion into the periplasm.

A synthetic gene encoding the histone-like DNA-binding protein, HBsu, of Bacillus subtilis was cloned in-frame behind the coding region of the OmpA signal peptide of Escherichia coli. The gene encoding the fusion protein is under control of both the lpp promoter and the lac promoter-operator. Upon induction of gene expression, mature HBsu is secreted into the periplasm. The OmpA signal peptide is correctly removed, resulting in the production of authentic-length HBsu protein. The observed in vitro DNA-binding ability is taken as evidence for the correct folding and assembly of homodimeric HBsu protein. A normally intracellular protein can thus be secreted from E. coli in high yield and with full functionality. By analogy, every histone-like protein or mutant forms thereof may be produced heterologously in E. coli and may be purified without being contaminated by the homologous E. coli HU protein.

Bacillus subtilis↗
PubMed1993

Conformation of d(GGGATCCC)2 in crystals and in solution studied by X-ray diffraction, Raman spectroscopy and molecular modelling.

In the crystal, d(GGGATCCC)2 forms an A-DNA double helix as known from a single crystal X-ray diffraction study. Accordingly, in the Raman spectra of crystals the A-family marker bands at 664, 705, 807 and 1101 cm-1 and the spectral characteristics in the region 1200 to 1500 cm-1 clearly demonstrate the A-form as the dominant conformation. Bands at 691, 850, and 1080 cm-1, however, indicate that a minor fraction of the octamer molecules in the crystal is in an unusual, still not unequivocally identified conformation possibly belonging to the B-family. In solution, the octamer is in B-like conformation as shown by the presence of B-DNA Raman marker bands at 685, 837, 1094 and 1421 cm-1. Molecular modelling techniques lead to three structures with slightly different B-form geometries as the lowest energies models when a sigmoidal dielectric function with the bulk dielectric constant epsilon = 78 and the value q = -0.5e for the effective phosphate charges was used in the calculations. An A-form structure bearing a strong resemblance to the experimentally determined crystal structure becomes the lowest energy model structure when the electrostatic parameters are changed to epsilon = 30 and q = -0.25e, respectively.

Base Sequence↗
PubMed1993

Three-dimensional structure of the ternary complex between ribonuclease T1, guanosine 3',5'-bisphosphate and inorganic phosphate at 0.19 nm resolution.

The ternary complex formed between RNase T1, guanosine 3',5'-bisphosphate (3',5'-pGp) and Pi crystallizes in the cubic space group I23 with a = 8.706(1) nm. In a previous publication [Lenz, A., Heinemann, U., Maslowska, M. & Saenger, W. (1991) Acta Crystallogr. B47, 521-527], the structure of the complex (in which Pi was not located) was described at a resolution of 0.32 nm. This is now extended to 0.19 nm with newly grown, larger crystals. Refinement with restrained least-squares converged at R = 17.8% for 8027 reflections with [Fo[ > or = 1 sigma ([Fo[); the final model comprises 120 water molecules. 3',5'-pGp is bound to RNase T1 in the anti form, with guanine in the specific recognition site; the 3'-phosphate protrudes into the solvent, and the 5'-phosphate hydrogen bonds with Lys41 O and Asn43 N4. A tetrahedral anion assigned as Pi occupies the catalytic site and hydrogen bonds to the side chains of Tyr38, Glu58, Arg77 and His92. The overall polypeptide fold of RNase T1 in the cubic space group does not differ significantly from that in the orthorhombic space group P2(1)2(1)2(1) except for changes < or = 0.2 nm in loop regions 69-72 and 95-98.

Guanosine Diphosphate↗
PubMed1993

Pharmacological and electrographic properties of epileptiform activity induced by elevated K+ and lowered Ca2+ and Mg2+ concentration in rat hippocampal slices.

We studied some of the physiological and pharmacological properties of an in vitro model of epileptic seizures induced by elevation of [K+]0 (to 8 mM and 10 mM) in combination with lowering of [Mg2+]0 (to 1.4 mM and 1.6 mM) and [Ca2+]0 (to 0.7 mM and 1 mM) in rat hippocampal slices. These concentrations correspond to the ionic constitution of the extracellular microenvironment during seizures in vivo. The resulting activity was rather variable in appearance. In area CA3 recurrent discharges were observed which resulted in seizure-like events with either clonic-like or tonic-clonic-like ictaform events in area CA1. With ion-sensitive electrodes, we measured the field potential and the changes in extracellular ion concentrations which accompany this activity. The recurrent discharges in area CA3 were accompanied by small fluctuations in [K+]0 and [Ca2+]0. The grouped clonic-like discharges in area CA1 were associated with moderate increases in [K+]0 and small decreases in [Ca2+]0 in the order of 2 mM and 0.2 mM, respectively. Large, negative field-potential shifts and increases in [K+]0 to 13 mM, as well as decreases in [Ca2+]0 by up to 0.4 mM, accompanied the tonic phase of ictaform events. The ictaform events were not blocked by D-2-aminophosphonovalerate (2-APV) but were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) alone and in combination with 2-APV and ketamine. In order to determine the pharmacological characteristics of the ictaform events we bath-applied most clinically employed anticonvulsants (carbamazepine, phenytoin, valproate, phenobarbital, ethosuximide, trimethadione) and some experimental anticonvulsants (losigamone, vinpocetine, and apovincaminic acid). Carbamazepine, phenytoin, valproate, and phenobarbital were effective at clinically relevant doses. The data suggest that the high-K+ model of epileptiform activity is a good model of focal convulsant activity.

2-Amino-5-phosphonovalerate↗
PubMed1993

Influence of hypoxia on excitation and GABAergic inhibition in mature and developing rat neocortex.

To analyze the functional consequences of hypoxia on the efficacy of intracortical inhibitory mechanisms mediated by gamma-aminobutyric acid (GABA), extra- and intracellular recordings were obtained from rat primary somatosensory cortex in vitro. Hypoxia, induced by transient N2 aeration, caused a decrease in stimulus-evoked inhibitory postsynaptic potentials (IPSPs), followed by a pronounced anoxic depolarization. Upon reoxygenation, the fast (f-) and long-latency (l-) IPSP showed a positive shift in the reversal potential by 24.4 and 14.9 mV, respectively. The peak conductance of the f- and l-IPSP was reversibly reduced in the postanoxic period by 72% and 94%, respectively. Extracellular field potential recordings and application of a paired-pulse inhibition protocol confirmed the enhanced sensitivity of inhibitory synaptic transmission for transient oxygen deprivation. Intracellular recordings from morphologically or electrophysiologically identified interneurons did not reveal any enhanced susceptibility for hypoxia as compared to pyramidal cells, suggesting that inhibitory neurons are not selectively impaired in their functional properties. Intracellularly recorded spontaneous IPSPs were transiently augmented in the postanoxic period, indicating that presynaptic GABA release was not suppressed. Developmental studies in adult (older than postnatal day 28), juvenile (P14-18), and young (P5-8) neocortical slices revealed a prominent functional resistance of immature tissue for hypoxia. In comparison with adult cortex, the hypoxia-induced reduction in excitatory and inhibitory synaptic transmission was significantly smaller in immature cortex. Our data indicate a hypoxia-induced distinct reduction of postsynaptic GABAergic mechanisms, leading to the manifestation of intracortical hyperexcitability as a possible functional consequence.

2-Amino-5-phosphonovalerate↗
PubMed1993

Properties of horizontal cells transiently appearing in the rat dentate gyrus during ontogenesis.

In an ontogenetic study, combining morphological analysis and patch clamp recordings, a transiently appearing horizontal cell type was identified in the dentate gyrus. The cells were exclusively located in the outer third of the stratum moleculare. They were present at postnatal day 2 (P2) and could be identified with fluorescent dyes until around P14. The morphology was bipolar, with a putative axonal and a dendritic process stretching out parallel to the pial surface without any preferential direction. Patch clamp studies in the current and voltage clamp mode were performed in hippocampal slices on visually identified horizontal cells, between P4 and P7, which were subsequently stained with lucifer yellow. The cells had a low resting membrane potential, around -55 mV. They were excitable, displaying broad action potentials (duration 3-20 ms) and, unlike mature dentate granule cells, they also expressed a strong delayed inward rectifier with properties reminiscent of the IQ current. Unlike granule cells, no postsynaptic signals could be observed during elevation of [K+]o or electrical stimulation, suggesting that the horizontal cells did not participate in functional hippocampal circuitry. We suggest that these cells represent migrating cells with subsequent differentiation to granule cells or inhibitory interneurons. Alternatively they may be part of the early radial glia or serve as transient target cells for afferent fibres between the entorhinal cortex and the dentate gyrus.

Animals↗
PubMed1993

Conformational stability of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis, and of the four HBsu variants [F29W], [F47W], [F50W] and [F79W].

From denaturation studies with urea a free energy delta GuH2O of unfolding of 49.8 kJ.mol-1 at 25C was calculated for the histone-like DNA-binding protein HBsu from Bacillus subtilis. Unfolding was monitored by circular dichroism measurements observing the changes of the molar mean residue ellipticity [theta] at 222 nm. For the calculation of delta Gu a two-state model of unfolding, i.e. the unfolding of native dimers into unfolded monomers, was applied. The validity of this model in high ionic strength buffer was proven by measurements at different protein concentrations yielding the same delta Gu values. Four HBsu variants, each carrying one single point mutation ([F29W], [F47W], [F50W] and [F79W]) were analysed with respect to their stability against unfolding at increasing temperatures and urea concentrations. The delta Gu values of mutants were calculated using the two-state model and show a reduced stability of the variants [F29W], [F47W], [F50W] and [F79W] in comparison to the wild type HBsu with delta delta Gu values of -9.2 kJ.mol-1, -7.5 kJ.mol-1, -5.9 kJ.mol-1, and -7.5 kJ.mol-1, respectively. Similar delta delta Gu values were obtained for the HBsu mutant proteins by thermal unfolding experiments.

Bacillus subtilis↗
PubMed1993

Trp59 to Tyr substitution enhances the catalytic activity of RNase T1 and of the Tyr to Trp variants in positions 24, 42 and 45.

Using point mutated overproducing strains of E. coli, ribonuclease T1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp, Tyr45Trp or Trp59Tyr and the corresponding double substitutions Tyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr. Steady state kinetics of the transesterification reaction for the two dinucleoside monophosphate substrates guanylyl-3',5'-cytidine and guanylyl-3',5'-adenosine indicate that the tryptophan can be introduced in different positions within the ribonuclease T1 molecule without abolishing enzymatic activity. The Trp59Tyr exchange even enhances catalysis of the cleavage reaction (kcat/Km) relative to the wild type enzyme and similar effects are found with single tyrosine to tryptophan substitutions. For the pH dependencies of the guanylyl-3',5'-cytidine transesterification reaction of wild type ribonuclease T1 and of the variants, typically bell-shaped curves are observed with a plateau in the range pH 4.5-7.0. Their shapes and slopes indicate that the enzymes are comparable in their macroscopic pKa values. At pH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-fold higher transesterification activity for guanylyl-3',5'-adenosine and a 2-fold increase for guanylyl-3',5'-cytidine compared to the wild type enzyme, i.e. this variant catalyses the transesterification of the substrate guanylyl-3',5'-adenosine with the same or better efficiency as guanylyl-3',5'-cytidine.

Base Sequence↗
PubMed1992

Role of histidine-40 in ribonuclease T1 catalysis: three-dimensionalstructures of the partially active His40Lys mutant.

Histidine-40 is known to participate in phosphodiester transesterification catalyzed by the enzyme ribonuclease T1. A mutant enzyme with a lysine replacing the histidine-40 (His40Lys RNase T1) retains considerable catalytic activity [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072]. We report on the crystal structures of His40Lys RNase T1 containing a phosphate anion and a guanosine 2'-phosphate inhibitor in the active site, respectively. Similar to previously described structures, the phosphate-containing crystals are of space group P2(1)2(1)2(1), with one molecule per asymmetric unit (a = 48.27 A, b = 46.50 A, c = 41.14 A). The complex with 2'-GMP crystallized in the lower symmetry space group P2(1), with two molecules per asymmetric unit (a = 49.20 A, b = 48.19 A, c = 40.16 A, beta = 90.26). The crystal structures have been solved at 1.8- and 2.0-A resolution yielding R values of 14.5% and 16.0%, respectively. Comparison of these His40Lys structures with the corresponding wild-type structures, containing 2'-GMP [Arni, R., Heinemann, U., Tokuoka, R., & Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368] and vanadate [Kostrewa, D., Hui-Woog Choe, Heinemann, U., & Saenger, W. (1989) Biochemistry 28, 7692-7600] in the active site, respectively, leads to the following conclusions. First, the His40Lys mutation causes no significant changes in the overall structure of RNase T1; second, the Lys40 side chains in the mutant structures occupy roughly the same space as His40 in the corresponding wild-type RNase T1 structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1992

Alterations in medial perforant path and mossy fiber induced field potentials in amygdala and beta-carboline (FG 7142) kindled rats.

We tested in rat hippocampal slices whether kindling causes sprouting of mossy fibers thereby augmenting excitatory coupling and epileptogenicity. Field potentials (fp's) evoked by orthodromic stimulation of the medial perforant path (MPP) and antidromic stimulation of mossy fibers in slices from control, amygdala kindled (AK) and beta-carboline (FG 7142) kindled rats were recorded in the dentate gyrus granule cell layer. Orthodromic paired pulse inhibition was increased in both groups of kindled animals. In contrast, neither in normal medium nor under additional application of the GABAA receptor antagonist bicuculline nor in Mg(2+)-free artificial cerebrospinal fluid (ACSF) there was evidence for an excitatory field EPSP component upon stimulation of mossy fibers in any of the investigated groups. The results indicate that functional mossy fiber sprouting does not occur as an obligatory step in the induction of kindling epilepsy.

Amygdala↗
PubMed1992

Determination of DNA-binding parameters for the Bacillus subtilis histone-like HBsu protein through introduction of fluorophores by site-directed mutagenesis of a synthetic gene.

A synthetic gene encoding the histone-like DNA-binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli. Yields of the purified protein are at least 20 mg/l culture medium. The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild-type protein. N-terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E. coli. Site-directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA-binding parameters by fluorescence spectroscopy. According to gel-retardation experiments, the mutant protein [Phe47----Trp]HBsu shows identical DNA binding to wild-type HBsu protein. Analysis of fluorescence binding data reveals that [Phe47----Trp]HBsu binds double-stranded DNA with a dissociation constant in the micromolar range. Computer-assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely. A dimer of [Phe47----Trp]HBsu appears to contact three or four base pairs of DNA. These results are in partial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.

Amino Acid Sequence↗
PubMed1992

RNase T1 mutant Glu46Gln binds the inhibitors 2'GMP and 2'AMP at the 3' subsite.

On the basis of molecular dynamics and free-energy perturbation approaches, the Glu46Gln (E46Q) mutation in the guanine-specific ribonuclease T1 (RNase T1) was predicted to render the enzyme specific for adenine. The E46Q mutant was genetically engineered and characterized biochemically and crystallographically by investigating the structures of its two complexes with 2'AMP and 2'GMP. The ribonuclease E46Q mutant is nearly inactive towards dinucleoside phosphate substrates but shows 17% residual activity towards RNA. It binds 2'AMP and 2'GMP equally well with dissociation constants of 49 microM and 37 microM, in contrast to the wild-type enzyme, which strongly discriminates between these two nucleotides, yielding dissociation constants of 36 microM and 0.6 microM. These data suggest that the E46Q mutant binds the nucleotides not to the specific recognition site but to the subsite at His92. This was confirmed by the crystal structures, which also showed that the Gln46 amide is hydrogen bonded to the Phe100 N and O atoms, and tightly anchored in this position. This interaction may either have locked the guanine recognition site so that 2'AMP and 2'GMP are unable to insert, or the contribution to guanine recognition of Glu46 is so important that the E46Q mutant is unable to function in recognition of either guanine and adenine.

Adenosine Monophosphate↗
PubMed1992

C-C-A-G-G-C-m5C-T-G-G. Helical fine structure, hydration, and comparison with C-C-A-G-G-C-C-T-G-G.

The self-complementary DNA duplex C-C-A-G-G-C-m5C-T-G-G has been refined against 1.75-A x-ray diffraction data to an R value of 17.4%. In the crystal of space group P6, 10-base pair DNA fragments with characteristic sequence-related fine structure stack end to end to form long antiparallel B-type double helices. As shown by a structure analysis at lower resolution (Heinemann, U., and Alings, C. (1991) EMBO J. 10, 35-43), the overall geometry of C-C-A-G-G-C-m5C-T-G-G is similar to that of the unmethylated analog C-C-A-G-G-C-C-T-G-G despite a different crystal environment. The present high resolution structure analysis permits a detailed comparison of the two duplexes and their hydration spheres. Helical parameters are significantly correlated between both molecules, with the exception of the base pair propeller. Sugar pucker and backbone torsion angles alpha, gamma, delta, and chi show similar mean values, but their individual values deviate significantly between duplexes. In contrast, torsion angles beta, epsilon, and zeta change along the strands of both duplexes in much the same way. The effect of single-site methylation on DNA conformation appears to be small and limited to the base pairs directly involved. Methylation tends to push base pairs toward the minor groove of the helix. A regular minor groove hydration pattern involves dual hydrogen bonding of water molecules to O-4' and base atoms of C-C-A-G-G-C-m5C-T-G-G.

Base Sequence↗
PubMed1992

His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study.

In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Monophosphate↗