Biomedical subjects

A Messerschmidt

Publications and source records attributed to A Messerschmidt.

At least 19 recordsLinked to original sources

PubMed2006

First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value.

The EC 'Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, approximately 170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

Animals↗

PubMed2006

Eukaryotic expression: developments for structural proteomics.

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

Animals↗

PubMed2004

Brain abscesses after Serratia marcescens infection on a neonatal intensive care unit: differences on serial imaging.

Serratia are known to be a possible cause of severe cerebral infections in neonates. We describe imaging of three premature infants infected with Serratia marcescens. Born in the 31( st), 25( th) and 28( th) weeks of gestation, they presented with signs of septicaemia on postnatal days 9, 24 and 32. Initial sonography showed cysts in the first child, two areas with anechoic centre and echogenic rim in the second, and several echogenic areas in the third. Lesions were seen on CT, of low density in two cases and minimally increased density in the third. MRI in the first patient showed cysts with incomplete contrast enhancement of the lesions, while patient 2 showed five ring-enhancing fluid-containing lesions with thick walls. In the third patient two abscesses with contrast enhancement and several high-signal spots were seen. We discuss the pathophysiology of the lesions and the impact of the various imaging methods.

Brain Abscess↗

PubMed2004

Fetal skeletal deformities - the diagnostic accuracy of prenatal ultrasonography and fetal magnetic resonance imaging.

OBJECTIVE: To evaluate the diagnostic accuracy of prenatal ultrasonography and fetal magnetic resonance imaging (MRI) in fetuses with skeletal deformities (SD). METHODS: Fourteen pregnant women of 21 - 34 weeks of gestation whose fetuses had SD on prenatal ultrasound (seven fetuses with spina bifida, four with complex malformation syndrome, two with scoliosis, and one with chondrodysplasia) were additionally investigated by fetal MRI using a 1.5T superconducting system with T1-and T2-weighted sequences in three section-planes. Main outcome measures were diagnostic accuracy, potential effect on parental counselling and influence on perinatal management of the additional investigation with fetal MRI. RESULTS: In 10 cases ultrasound had a better diagnostic accuracy than MRI concerning the diagnosis of SD. In four cases with spina bifida MRI provided additional information towards preoperative evaluation for neurosurgery. In five cases parental counselling was improved. There was no influence on perinatal management. CONCLUSION: MRI may provide additional information to ultrasound scan in fetuses with spina bifida and consecutive neurosurgery; in fetuses with other skeletal deformities additional information may be expected only in rare cases.

Adult↗

PubMed2003

Progressive posthemorrhagic hydrocephalus leads to changes of amplitude-integrated EEG activity in preterm infants.

AIM: Intraventricular hemorrhage (IVH) is the most common cause of brain lesions in preterm infants. Among infants with IVH about 35% develop posthemorrhagic hydrocephalus (PPH) which may lead to secondary injury. Therapeutic interventions to reduce the increased intracranial pressure are invasive and carry a high risk of complications. Amplitude-integrated EEG (aEEG) allows continuous neurophysiological surveillance and may help in defining the optimal timing for intervention in infants with progressive PHH. In this report we show, for the first time, a change in aEEG activity in two preterm infants with PHH. METHODS: Cerebral activity was continuously monitored by aEEG provided by the Cerebral Function Monitor (Lectromed, UK) in two preterm infants with PPH. RESULTS: With increasing ventricular width, aEEG showed an increased discontinuity without distinguishable sleep-wake cycling in both infants. One infant showed an abrupt onset of a nearly isoelectric pattern without any change in clinical condition. Clinical signs of increased intracranial pressure developed 6-12 h later in both children. In one patient, aEEG activity returned to normal after successful shunting and reduction of intracranial pressure. CONCLUSION: Continuous neurophysiological monitoring by aEEG may be of value in the diagnostic and therapeutic management of preterm infants with progressive PHH.

Cerebral Hemorrhage↗

PubMed2001

Tobacco uroporphyrinogen-III decarboxylase: characterization, crystallization and preliminary X-ray analysis.

Uroporphyrinogen-III decarboxylase from Nicotiana tabacum is a plastidial enzyme involved in the biosynthesis of chlorophyll and haem. Sedimentation equilibrium with protein producing diffracting crystals clearly indicates that the enzyme is a homodimer under similar ionic strength conditions to those found in the chloroplast stroma. Additionally, dynamic light scattering reveals an ionic strength dependence for this oligomerization state. Crystals were obtained in the hexagonal space group P622 with one molecule per asymmetric unit and diffracted to 2.3 A resolution using synchrotron radiation.

Crystallization↗

PubMed2001

Crystal structure and substrate binding modeling of the uroporphyrinogen-III decarboxylase from Nicotiana tabacum. Implications for the catalytic mechanism.

The enzymatic catalysis of many biological processes of life is supported by the presence of cofactors and prosthetic groups originating from the common tetrapyrrole precursor uroporphyrinogen-III. Uroporphyrinogen-III decarboxylase catalyzes its conversion into coproporphyrinogen-III, leading in plants to chlorophyll and heme biosynthesis. Here we report the first crystal structure of a plant (Nicotiana tabacum) uroporphyrinogen-III decarboxylase, together with the molecular modeling of substrate binding in tobacco and human enzymes. Its structural comparison with the homologous human protein reveals a similar catalytic cleft with six invariant polar residues, Arg(32), Arg(36), Asp(82), Ser(214) (Thr in Escherichia coli), Tyr(159), and His(329) (tobacco numbering). The functional relationships obtained from the structural and modeling analyses of both enzymes allowed the proposal for a refined catalytic mechanism. Asp(82) and Tyr(159) seem to be the catalytic functional groups, whereas the other residues may serve in substrate recognition and binding, with Arg(32) steering its insertion. The crystallographic dimer appears to represent the protein dimer under physiological conditions. The dimeric arrangement offers a plausible mechanism at least for the first two (out of four) decarboxylation steps.

Amino Acid Sequence↗

PubMed2001

Crystal structures of cystathionine gamma-synthase inhibitor complexes rationalize the increased affinity of a novel inhibitor.

Cystathionine gamma-synthase catalyzes the committed step of methionine biosynthesis. This pathway is unique to microorganisms and plants, rendering the enzyme an attractive target for the development of antimicrobials and herbicides. We solved the crystal structures of complexes of cystathionine gamma-synthase (CGS) from Nicotiana tabacum with inhibitors of different compound classes. The complex with the substrate analog dl-E-2-amino-5-phosphono-3-pentenoic acid verifies the carboxylate-binding function of Arg423 and identifies the phosphate-binding pocket of the active site. The structure shows the function of Lys165 in specificity determination and suggests a role for the flexible side-chain of Tyr163 in catalysis. The importance of hydrophobic interactions for binding to the active-site center is highlighted by the complex with 3-(phosphonomethyl)pyridine-2-carboxylic acid. The low affinity of this compound is due to the non-optimal arrangement of the functional groups binding to the phosphate and carboxylate-recognition site, respectively. The newly identified inhibitor 5-carboxymethylthio-3-(3'-chlorophenyl)-1,2,4-oxadiazol, in contrast, shows the highest affinity to CGS reported so far. This affinity is due to binding to an additional active-site pocket not used by the physiological substrates. The inhibitor binds to the carboxylate-recognition site, and its tightly bent conformation enables it to occupy the novel binding pocket between Arg423 and Ser388. The described structures suggest improvements for known inhibitors and give guidelines for the development of new lead compounds.

2-Amino-5-phosphonovalerate↗

PubMed2001

Effects of dimerization on protein electron transfer.

In order to investigate the relationship between the rate of protein-protein electron transfer and the structure of the association complex, a dimer of the blue copper protein azurin was constructed and its electron exchange properties were determined. For this purpose, a site for covalent cross-linking was engineered by replacing the surface-exposed asparagine 42 with a cysteine. This mutation enabled the formation of disulfide-linked homo-dimers of azurin. Based on NMR line-broadening experiments, the electron self-exchange (e.s.e.) rate constant for this dimer was determined to be 4.2(+/-0.7) x 10(5)M(-1)s(-1), which is a seven-fold decrease relative to wild-type azurin. This difference is ascribed to a less accessible hydrophobic patch in the dimer. To discriminate between intramolecular electron transfer within a dimer and intermolecular electron transfer between two dimers, the e.s.e. rate constant of (Cu-Cu)-N42C dimers was compared with that of (Zn-Cu)- and (Ag-Cu)-N42C dimers. As Zn and Ag are redox inactive, the intramolecular electron transfer reaction in these latter dimers can be eliminated. The e.s.e. rate constants of the three dimers are the same and an upper limit for the intramolecular electron transfer rate of 10 s(-1) could be determined. This rate is compatible with a Cu-Cu distance of 18 A or more, which is larger than the Cu - Cu distance of 15 A observed in the wild-type crystal structure that shows two monomers that face each other with opposing hydrophobic patches. Modelling of the dimer shows that the Cu-Cu distance should be in the range of 17 A < rCu-Cu < 28 A, which is in agreement with the experimental findings. For efficient electron transfer, it appears crucial that the two molecules interact in the proper orientation. Direct cross-linking may disturb the formation of such an optimal electron transfer complex.

Algorithms↗

PubMed2001

Spectroscopic investigation and determination of reactivity and structure of the tetraheme cytochrome c3 from Desulfovibrio desulfuricans Essex 6.

Cytochrome c3, a small (14-kDa) soluble tetraheme protein was isolated from the periplasmic fraction of Desulfovibrio desulfuricans strain Essex 6. Its major physiological function appears to be that of an electron carrier for the periplasmic hydrogenase. It has been also shown to interact with the high-molecular-mass cytochrome complex in the cytoplasmic membrane, which eventually feeds electrons into the membraneous quinone pool, as well as with the membrane-associated dissimilatory sulfite reductase. The EPR spectra show features of four different low-spin Fe(III) hemes. Orthorhombic crystals of cytochrome c3 were obtained and X-ray diffraction data were collected to below 2 A resolution. The structure was solved by molecular replacement using cytochrome c3 from D. desulfuricans ATCC 27774 as a search model.

Amino Acid Sequence↗

PubMed2001

The three-dimensional structure of cystathionine beta-lyase from Arabidopsis and its substrate specificity.

The pyridoxal 5'-phosphate-dependent enzyme cystathionine beta-lyase (CBL) catalyzes the penultimate step in the de novo biosynthesis of Met in microbes and plants. Absence of CBL in higher organisms makes it an important target for the development of antibiotics and herbicides. The three-dimensional structure of cystathionine beta-lyase from Arabidopsis was determined by Patterson search techniques, using the structure of tobacco (Nicotiana tabacum) cystathionine gamma-synthase as starting point. At a resolution of 2.3 A, the model was refined to a final crystallographic R-factor of 24.9%. The overall structure is very similar to other pyridoxal 5'-phosphate-dependent enzymes of the gamma-family. Exchange of a few critical residues within the active site causes the different substrate preferences between Escherichia coli and Arabidopsis CBL. Loss of interactions at the alpha-carboxyl site is the reason for the poorer substrate binding of Arabidopsis CBL. In addition, the binding pocket of Arabidopsis CBL is larger than that of E. coli CBL, explaining the similar binding of L-cystathionine and L-djenkolate in Arabidopsis CBL in contrast to E. coli CBL, where the substrate binding site is optimized for the natural substrate cystathionine.

Amino Acid Sequence↗

PubMed2000

Cytochrome c nitrite reductase from Wolinella succinogenes. Structure at 1.6 A resolution, inhibitor binding, and heme-packing motifs.

Cytochrome c nitrite reductase catalyzes the 6-electron reduction of nitrite to ammonia. This second part of the respiratory pathway of nitrate ammonification is a key step in the biological nitrogen cycle. The x-ray structure of the enzyme from the epsilon-proteobacterium Wolinella succinogenes has been solved to a resolution of 1.6 A. It is a pentaheme c-type cytochrome whose heme groups are packed in characteristic motifs that also occur in other multiheme cytochromes. Structures of W. succinogenes nitrite reductase have been obtained with water bound to the active site heme iron as well as complexes with two inhibitors, sulfate and azide, whose binding modes and inhibitory functions differ significantly. Cytochrome c nitrite reductase is part of a highly optimized respiratory system found in a wide range of Gram-negative bacteria. It reduces both anionic and neutral substrates at the distal side of a lysine-coordinated high-spin heme group, which is accessible through two different channels, allowing for a guided flow of reaction educt and product. Based on sequence comparison and secondary structure prediction, we have demonstrated that cytochrome c nitrite reductases constitute a protein family of high structural similarity.

Amino Acid Motifs↗

PubMed2000

Crystal structure of plantacyanin, a basic blue cupredoxin from spinach.

The crystal structure of the basic blue protein (plantacyanin) from spinach (SBP) has been solved to a resolution of 2.05 A by molecular replacement using the homologous protein from cucumber (CBP) as a model. Although the sequence identity of 58% between both proteins is only moderate, the three-dimensional structures turned out to be highly similar and the buried residues, which form the hydrophobic core of the protein, are almost completely conserved. However, the redox potentials of both proteins differ by 40 mV, and a comparison of the two structures leads to a single lysine replacing a proline in the cucumber sequence, which causes a shift of the peptide chain and thus a subtle distortion of the copper ligand geometry in respect to CBP. The crystal contained three monomers of SBP in the asymmetric unit which show considerable variations in outer loop regions owing to crystal packing, but not in the regions presumed to be essential for redox partner recognition and redox potential fine tuning of the copper centers. Still, bond length variations at the copper site are at the same scale between the monomers of SBP as they are in respect to CBP, indicating that in the oxidized state the protein does not impose a high conformational strain on the copper.

Amino Acid Sequence↗

PubMed2000

Equipment for the difficult airway in obstetric units in Germany.

STUDY OBJECTIVE: To examine the availability of specialized equipment for the difficult airway management in obstetric units of German departments of anesthesiology. DESIGN AND SETTING: An anonymous questionnaire survey was mailed to the directors of 993 German departments of anesthesiology. Completed replies were grouped by number of deliveries performed each year. MAIN RESULTS: 55.5% of the hospitals responded. Data of 449 answers were evaluated for this investigation. A difficult airway cart was available in 99.3% of the departments. More detailed investigation revealed that different shaped laryngoscope blades (74.9% of the departments), laryngeal masks (91.0%), a fiberoptic bronchoscope (85.9%), and transtracheal puncture devices (59.9%) were available in the majority of the units. However, only a minority of the departments had these devices directly available in their obstetric operating rooms (OR; laryngeal masks 36.2%, fiberoptic bronchoscope 23.9%, transtracheal puncture set 22.0%). Larger units with more than 1,000 deliveries per year provided their equipment more often directly in the obstetric OR or the facility housing the obstetric unit than did smaller units with less than 1,000 deliveries per year (p< 0.001). CONCLUSIONS: The survey of German departments of anesthesia revealed that specialized equipment for the difficult airway management often is not directly available in the obstetric OR. Anesthesiologists must familiarize themselves as to which difficult airway equipment is available in their unit and where it is stored.

Anesthesia Department, Hospital↗

PubMed2000

Small in-frame deletions and missense mutations in CADASIL: 3D models predict misfolding of Notch3 EGF-like repeat domains.

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is a hereditary microangiopathic condition causing stroke in young adults. The responsible gene has recently been identified as the Notch3 gene. Notch3 encodes a large transmembrane receptor with 34 extracellularly localised epidermal growth factor-like (EGF) repeat domains. We screened 71 unrelated CADASIL families for mutations in two exons coding for the first five EGF-like repeats and found mutations in 70% of the families (n = 50). Two types of mutations were identified: 48 families (96%) had missense mutations and two families (4%) had small in-frame deletions. Seven mutations occurred multiple times. All of them are C to T transitions that affect CpG dinucleotides, suggesting that their multiple occurrence is due to the hypermutability of this sequence. All mutations, including the two deletions, result in the gain or loss of a cysteine residue, thus substantiating the pivotal role of an uneven number of cysteine residues within EGF-like repeat domains of Notch3 in the pathogenesis of CADASIL. To study the potential effects of these mutations 3D homology models of the first six EGF domains were generated on the basis of NMR data from human fibrillin-1. These models predict domain misfolding for a subset of mutations.

Amino Acid Sequence↗

PubMed1999

Heterologous expression of the vanadium-containing chloroperoxidase from Curvularia inaequalis in Saccharomyces cerevisiae and site-directed mutagenesis of the active site residues His(496), Lys(353), Arg(360), and Arg(490).

The vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis is heterologously expressed to high levels in the yeast Saccharomyces cerevisiae. Characterization of the recombinant enzyme reveals that this behaves very similar to the native chloroperoxidase. Site-directed mutagenesis is performed on four highly conserved active site residues to examine their role in catalysis. When the vanadate-binding residue His(496) is changed into an alanine, the mutant enzyme loses the ability to bind vanadate covalently resulting in an inactive enzyme. The negative charges on the vanadate oxygens are compensated by hydrogen bonds with the residues Arg(360), Arg(490), and Lys(353). When these residues are changed into alanines the mutant enzymes lose the ability to effectively oxidize chloride but can still function as bromoperoxidases. A general mechanism for haloperoxidase catalysis is proposed that also correlates the kinetic properties of the mutants with the charge and the hydrogen-bonding network in the vanadate-binding site.

Catalytic Domain↗

PubMed1999

The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum reveals its substrate and reaction specificity.

Cystathionine gamma-synthase catalyses the committed step of de novo methionine biosynthesis in micro-organisms and plants, making the enzyme an attractive target for the design of new antibiotics and herbicides. The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum has been solved by Patterson search techniques using the structure of Escherichia coli cystathionine gamma-synthase. The model was refined at 2.9 A resolution to a crystallographic R -factor of 20.1 % (Rfree25.0 %). The physiological substrates of the enzyme, L-homoserine phosphate and L-cysteine, were modelled into the unliganded structure. These complexes support the proposed ping-pong mechanism for catalysis and illustrate the dissimilar substrate specificities of bacterial and plant cystathionine gamma-synthases on a molecular level. The main difference arises from the binding modes of the distal substrate groups (O -acetyl/succinyl versusO -phosphate). Central in fixing the distal phosphate of the plant CGS substrate is an exposed lysine residue that is strictly conserved in plant cystathionine gamma-synthases whereas bacterial enzymes carry a glycine residue at this position. General insight regarding the reaction specificity of transsulphuration enzymes is gained by the comparison to cystathionine beta-lyase from E. coli, indicating the mechanistic importance of a second substrate binding site for L-cysteine which leads to different chemical reaction types.

Amino Acid Sequence↗

PubMed1999

Structure of cytochrome c nitrite reductase.

The enzyme cytochrome c nitrite reductase catalyses the six-electron reduction of nitrite to ammonia as one of the key steps in the biological nitrogen cycle, where it participates in the anaerobic energy metabolism of dissimilatory nitrate ammonification. Here we report on the crystal structure of this enzyme from the microorganism Sulfurospirillum deleyianum, which we solved by multiwavelength anomalous dispersion methods. We propose a reaction scheme for the transformation of nitrite based on structural and spectroscopic information. Cytochrome c nitrite reductase is a functional dimer, with 10 close-packed haem groups of type c and an unusual lysine-coordinated high-spin haem at the active site. By comparing the haem arrangement of this nitrite reductase with that of other multihaem cytochromes, we have been able to identify a family of proteins in which the orientation of haem groups is conserved whereas structure and function are not.

Crystallography, X-Ray↗