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W Welte

Publications and source records attributed to W Welte.

At least 37 records · Page 2Linked to original sources
PubMed1998

An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.

FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).

Bacterial Outer Membrane Proteins↗
PubMed1998

Crystal structure of the effector-binding domain of the trehalose-repressor of Escherichia coli, a member of the LacI family, in its complexes with inducer trehalose-6-phosphate and noninducer trehalose.

The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose-6-phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 A resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 A resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central beta-sheet sandwiched between alpha-helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose-6-phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose-6-phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source.

Bacterial Proteins↗
PubMed1998

Structure of the sucrose-specific porin ScrY from Salmonella typhimurium and its complex with sucrose.

The X-ray structure of a sucrose-specific porin (ScrY) from Salmonella typhimurium has been determined by multiple isomorphous replacement at 2.4 A resolution both in its uncomplexed form and with bound sucrose. ScrY is a noncrystallographic trimer of identical subunits, each with 413 structurally well-defined amino acids. A monomer is built up of 18 anti-parallel beta-strands surrounding a hydrophilic pore, with a topology closely similar to that of maltoporin. Two non-overlapping sucrose-binding sites were identified in difference Fourier maps. The higher permeability for sucrose of ScrY as compared to maltoporin is mainly accounted for by differences in their pore-lining residues.

Amino Acid Sequence↗
PubMed1998

[Cooperation guidelines--a contribution to the development of process quality in psychiatric community management].

Cooperation in the provision of care to individuals with chronic psychiatric illnesses represents a significant criterion for quality. In the Tuebingen administrative district, guidelines for cooperation in the handover of patients to facilities providing follow-up care and in parallel care of patients have been developed. Elements of the guidelines include handover standards, case conferences and case management. The results of accompanying research are encouraging, while indicating the necessity of continued discussion in the individual facilities and in the relevant authorities: The acceptance of the guidelines is high and they are considered useful in concrete practice, but implementation is sometimes hesitant and the instrument provided for documentation is barely used due to time limitations. Only by means of compulsory basic documentation will it be possible to evaluate the guidelines and secure the criteria for quality on a long-term basis. As with other models, integration of neurologists and psychiatrists in private practice into the cooperation with inpatient and outpatient facilities is a problem that must yet be resolved.

Case Management↗
PubMed1998

[The Community Psychiatry Organization in Baden-Württemberg].

While as a result of the report of the German Federal Government's Inquiry Commission a nationwide programme for further development of the provision of care to the mentally ill was launched, Baden-Wuerttemberg went it's own ways with its "Programme for the Development of Outpatient Psychiatric Care 1982-1986". In 1994 Baden-Wuerttemberg's concept of a "Communal Psychiatric Combine" ("Gemeindepsychiatrischer Verbund", GPV) was worked out; this concept increases in some respects the existing differences from other states. Apart from an improvement in coordination and cooperation, the GPV is intended to secure comprehensive local care provision for the chronically mentally ill. Furthermore, care provision appropriate to requirements should be made possible and quality assurance realised on all levels. In the following, Baden-Wuerttemberg's understanding of a "Communal Psychiatric Combine" is contrasted with the combine concept of the expert commission. The potential effects of the reginal care situation as well as the chances of implementation are discussed with reference to earlier experiences.

Ambulatory Care↗
PubMed1997

The structure of porin from Paracoccus denitrificans at 3.1 A resolution.

The crystal structure of a non-specific porin from Paracoccus denitrificans at 3.1 A resolution has been solved by molecular replacement using the porin from Rhodopseudomonas blastica as the search model. Paracoccus porin is very similar to other non-specific porins of known structure: a trimer of 16 stranded beta-barrels each with a central pore constricted by a long extracellular loop folding back against the barrel wall. The distinctive distribution of charged residues of this non-specific porin contributes to understanding the relation between structure and ion selectivity.

Amino Acid Sequence↗
PubMed1996

Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore.

In vitro incubation of isolated hexokinase isozyme I or isolated dimer of mitochondrial creatine kinase with the outer mitochondrial membrane pore led to high molecular weight complexes of enzyme oligomers. Similar complexes of hexokinase and mitochondrial creatine kinase could be extracted by 0.5% Triton X-100 from homogenates of rat brain. Hexokinase and creatine kinase complexes could be separated by subsequent chromatography on DEAE anion exchanger. The molecular weight, as determined by gel-permeation chromatography, was approximately 400 kDa for both complexes. The Mr suggested tetramers of hexokinase (monomer 100 kDa) and creatine kinase (active enzyme is a dimer of 80 kDa). The composition of the complexes was further characterised by specific antibodies. Besides either hexokinase or creatine kinase molecules the complexes contained porin and adenylate translocator. It was possible to incorporate the complexes into artificial bilayer membranes and to measure conductance in 1 M KCI. The incorporating channels had a high conductance of 6 nS that was asymmetrically voltage dependent. The complexes were also reconstituted in phospholipid vesicles that were loaded with ATP. Complex containing vesicles retained ATP while vesicles reconstituted with pure porin were leaky. The internal ATP could be used by creatine kinase and hexokinase in the complex to phosphorylate external creatine or glucose. This process was inhibited by atractyloside. The hexokinase complex containing vesicles were furthermore loaded with malate or ATP that was gradually released by addition of Ca2+ between 100 and 600 microM. The liberation of malate or ATP by Ca2+ could be inhibited by N-methylVal-4-cyclosporin, suggesting that the porin translocator complex constitutes the permeability transition pore. The results show the physiological existence of kinase porin translocator complexes at the mitochondrial surface. It is assumed that such complexes between inner and outer membrane components are the molecular basis of contact sites observed by electron microscopy. Kinase complex formation may serve three regulatory functions, firstly regulation of the kinase activity, secondly stimulation of oxidative phosphorylation and thirdly regulation of the permeability transition pore.

Adenosine Triphosphate↗
PubMed1996

Structural basis of light harvesting by carotenoids: peridinin-chlorophyll-protein from Amphidinium carterae.

Peridinin-chlorophyll-protein, a water-soluble light-harvesting complex that has a blue-green absorbing carotenoid as its main pigment, is present in most photosynthetic dinoflagellates. Its high-resolution (2.0 angstrom) x-ray structure reveals a noncrystallographic trimer in which each polypeptide contains an unusual jellyroll fold of the alpha-helical amino- and carboxyl-terminal domains. These domains constitute a scaffold with pseudo-twofold symmetry surrounding a hydrophobic cavity filled by two lipid, eight peridinin, and two chlorophyll a molecules. The structural basis for efficient excitonic energy transfer from peridinin to chlorophyll is found in the clustering of peridinins around the chlorophylls at van der Waals distances.

Animals↗
PubMed1996

X-ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from Rhodobacter capsulatus: implications for ion selectivity and single-channel conductance.

The role of charges near the pore mouth has been discussed in theoretical work about ion channels. To introduce new negative charges in a channel protein, amino groups of porin from Rhodobacter capsulatus 37b4 were succinylated with succinic anhydride, and the precise extent and sites of succinylations and structures of the succinylporins determined by mass spectrometry and X-ray crystallography. Molecular weight and peptide mapping analyses using matrix-assisted laser desorption-ionization mass spectrometry identified selective succinylation of three lysine-epsilon-amino groups (Lys-46, Lys-298, Lys-300) and the N-terminal alpha-amino group. The structure of a tetra-succinylated porin (TS-porin) was determined to 2.4 A and was generally found unchanged in comparison to native porin to form a trimeric complex. All succinylated amino groups found in a mono/di-succinylated porin (MS-porin) and a TS-porin are localized at the inner channel surface and are solvent-accessible: Lys-46 is located at the channel constriction site, whereas Lys-298, Lys-300, and the N-terminus are all near the periplasmic entrance of the channel. The Lys-46 residue at the central constriction loop was modeled as succinyl-lysine from the electron density data and shown to bend toward the periplasmic pore mouth. The electrical properties of the MS-and TS-porins were determined by reconstitution into black lipid membranes, and showed a negative charge effect on ion transport and an increased cation selectivity through the porin channel. The properties of a typical general diffusion porin changed to those of a channel that contains point charges near the pore mouth. The single-channel conductance was no longer a linear function of the bulk aqueous salt concentration. The substantially higher cation selectivity of the succinylated porins compared with the native protein is consistent with the increase of negatively charged groups introduced. These results show tertiary structure-selective modification of charged residues as an efficient approach in the structure-function evaluation of ion channels, and X-ray crystallography and mass spectrometry as complementary analytical tools for defining precisely the chemically modified structures.

Amino Acid Sequence↗
PubMed1996

Differences between porin isolated from Rhodobacter capsulatus B10 and 37b4.

Porin was isolated from Rhodobacter (Rb.) capsulatus wild type B10, as well as from Rb. capsulatus 37b4 as a control. The porin from Rb. capsulatus B10 shows significant differences to that of Rb. capsulatus 37b4 in N-terminal sequence, amino acid composition and molecular mass. The apparent molecular mass of the purified porin from Rb. capsulatus B10 is about 28 kDa by SDS-PAGE, whereas the native porin trimer migrates at 75 kDa. For Rb. capsulatus 37b4 the molecular mass of the momomer is about 30 kDa and for the trimer about 76 kDa. These differences may be related to the morphological differences between the two wild type strains. Rb. capsulatus B10 has an extracellular capsule whereas Rb. capsulatus 37b4 is capsuleless. The native proteins from both wild types showed similar single channel conductance measurements in black lipid membranes. The B10 porin has been crystallised using the detergent beta-d-octyl-gluco-pyranoside. The crystals diffracted to 3 A and belong to the orthorhombic space-group P212121. The crystal packing could be elucidated by molecular replacement.

Amino Acids↗
PubMed1996

[Management of psychiatric patients in the community--developments up to now, current problems and future perspectives exemplified by the Tübingen district].

The development of the care of mentally ill persons in the last 25 years is shown by the example of the district of Tübingen with about 200,000 inhabitants, a district that did not take part in the model program of the Government. Considering the current care situation, the problems of cooperation and coordination as well as the ensuring of quality, which are actually most important, are discussed; approaches for solutions are outlined. We try a view on the coming 25 years.

Activities of Daily Living↗
PubMed1995

Molecular and crystal structure of an amphiphile: 4'-propoxybiphenyl-4-methyl-N,N-dimethylamineoxide dihydrate.

A novel amphiphile, 4'-propoxybiphenyl-4-methyl-N,N-dimethylamineoxide, has been synthesized, crystallized (P2(1)/a, a = 9.084 A, b = 8.911 A, c = 22.460 A, beta = 96.224 degrees) and its crystal structure was determined. The amphiphile forms a bilayer in which the amineoxide oxygen of each molecule binds two water molecules. In the hydrophobic part of the bilayer the biphenyls form edge-to-face contacts, in the polar layer there is a hydrogen bonding network. The potential use of the compound as a detergent for membrane proteins has been demonstrated and the relevance of the amineoxide hydrate for other detergents discussed.

Biphenyl Compounds↗
PubMed1995

Purification, characterization, crystallization, and preliminary X-ray results from Paracoccus denitrificans porin.

The porin from Paracoccus denitrificans ATCC 13543 was purified and crystallized. Two crystal forms were obtained from porin solutions with beta-d-octylglucopyranoside as detergent. Crystals of form I belong to the monoclinic spacegroup C2 with unit cell dimensions a = 112.2 A, b = 193.8 A, c = 100.5 A and beta = 129.2 degrees. There is 1 trimer per asymmetric unit. Crystals of form II are triclinic with a = 89.7 A, b = 98.8 A, c = 112.5 A, alpha = 112.5 degrees, beta = 101.8 degrees, gamma = 106.7 degrees (2 trimers per asymmetric unit). Both crystal forms diffract to 3 A.

Crystallization↗
PubMed1994

Influence of the lipid matrix on incorporation and function of LPS-free porin from Paracoccus denitrificans.

We have studied the role of lipopolysaccharide (LPS) for the insertion of LPS-free porin from Paracoccus denitrificans into planar lipid bilayers and its function therein. For this, we reconstituted the porin into different asymmetric planar lipid bilayers with or without LPS and into symmetric phospholipid bilayers. LPS-free porin added to the various bilayer systems was found to induce a step-wise increase in membrane conductance with different incorporation rates, depending on the presence of LPS in the bilayer leaflet opposite to porin addition. The incorporation rate into asymmetric LPS/phospholipid membranes from the phospholipid side was more than 10-fold higher than that observed for pure phospholipid membranes. The porin formed general diffusion pores without any salt specificity. The mean single-channel conductance did not depend on the presence of LPS and was about 4.2 nS for a subphase containing 1 M KCl in all systems tested. At certain applied transmembrane voltages, which depended on membrane composition and were approximately greater than 100 mV for the LPS/phospholipid system, single-channel closing in three steps was observed. Differences in the voltage dependence of porin-channel closing could be correlated with the surface charge of the bilayer. From the voltage-dependent gating behaviour proof for an oriented incorporation of the porin molecules, depending on the side of porin addition, and evidence for their orientation could be derived. Measurements at temperatures above and below the beta<==>alpha phase transition temperature of LPS gave evidence for the influence of membrane rigidity on the gating behaviour.

Carbohydrate Sequence↗
PubMed1994

Detergent organisation in solutions and in crystals of membrane proteins.

The use of neutron scattering in studying the organisation of detergents in pure micelles, in protein/detergent mixed micelles and in crystals of membrane proteins, is reviewed. Small angle scattering has been used to study the size, shape and composition of pure and mixed protein/detergent micelles as well as the effects of adding small amphiphiles. The technique of contrast variation applied to single crystals is described and its application to the determination of the organization of detergent in single crystals of membrane proteins is discussed. A better understanding of protein/detergent interactions should help in producing crystals of membrane proteins more easily as well as clues to the nature of protein/lipid interactions in vivo.

Journal Article↗
PubMed1993

Crystallization and preliminary X-ray diffraction analysis of ScrY, a specific bacterial outer membrane porin.

The sucrose-specific outer membrane porin ScrY of Salmonella typhimurium was isolated from Escherichia coli K-12 strain KS 26 containing the plasmid pPSO112. The protein was purified to homogeneity by differential extraction of the cell envelope in the presence of the detergents sodium dodecyl sulfate and lauryl (dimethyl)-amine oxide (LDAO). The porin had apparent molecular weights of 58 kDa and 120 kDa for the monomer and for the trimer, respectively, on SDS/PAGE. The purified trimers were crystallized using poly(ethylene glycol) 2000 and the detergents octylglucoside (OG) and hexyl-(dimethyl)-amine oxide (C6DAO). X-ray diffraction of the crystals showed reflections to 2.3 A. The space group of the crystals was R3 and the lattice constants of the hexagonal axes were a = b = 112.85 A and c = 149.9 A. The crystal volume per unit of protein molecular weight was 3.47 A3/Da.

Bacterial Outer Membrane Proteins↗
PubMed1992

Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanobacterium Synechococcus elongatus.

Immobilized metal affinity chromatography (IMAC) of solubilized, photosystem II (PS II) enriched particles from the thermophilic cyanobacterium Synechococcus elongatus was studied. A chelating Sepharose Fast Flow column was charged with various metal ions (Mn2+, Fe2+, Fe3+, Ni2+, Co2+, Ca2+, Sr2+, Zn2+ and Cu2+) and their affinity to photosystem I (PS I) and PS II was examined. Among all the metal ions tested, only copper was able to bind the two protein complexes. For elution of the column, a pH gradient, a pH step gradient and gradients of imidazole, amino acids, organic acids and various other eluents were tested; only the pH step gradient, which selectively eluted PS II at a pH between 6 and 5, was useful for the separation of PS I and PS II. All other gradients proved to be inappropriate for the separation of these two photosystems. Mechanisms of protein elution by these compounds are discussed. Alternatively, a separation of PS I and PS II at pH 7.5 could be achieved when an IMAC column was used on which the free coordination positions of the bound copper ions were occupied by imidazole. When solubilized photosystems were loaded on to this column, PS I replaced imidazole and remained bound on the column, whereas PS II was highly enriched in the effluent.

Centrifugation↗