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B Rupp

Publications and source records attributed to B Rupp.

At least 19 recordsLinked to original sources
PubMed2003

The TB structural genomics consortium: a resource for Mycobacterium tuberculosis biology.

The TB Structural Genomics Consortium is an organization devoted to encouraging, coordinating, and facilitating the determination and analysis of structures of proteins from Mycobacterium tuberculosis. The Consortium members hope to work together with other M. tuberculosis researchers to identify M. tuberculosis proteins for which structural information could provide important biological information, to analyze and interpret structures of M. tuberculosis proteins, and to work collaboratively to test ideas about M. tuberculosis protein function that are suggested by structure or related to structural information. This review describes the TB Structural Genomics Consortium and some of the proteins for which the Consortium is in the progress of determining three-dimensional structures.

Amino Acid Sequence↗
PubMed2001

Three-dimensional theory of emittance in Compton scattering and x-ray protein crystallography.

A complete, three-dimensional theory of Compton scattering is described, which fully takes into account the effects of the electron beam emittance and energy spread upon the scattered x-ray spectral brightness. The radiation scattered by an electron subjected to an arbitrary electromagnetic field distribution in vacuum is first derived in the linear regime, and in the absence of radiative corrections; it is found that each vacuum eigenmode gives rise to a single Doppler-shifted classical dipole excitation. This formalism is then applied to Compton scattering in a three-dimensional laser focus, and yields a complete description of the influence of the electron beam phase-space topology on the x-ray spectral brightness; analytical expressions including the effects of emittance and energy spread are also obtained in the one-dimensional limit. Within this framework, the x-ray brightness generated by a 25 MeV electron beam is modeled, fully taking into account the beam emittance and energy spread, as well as the three-dimensional nature of the laser focus; its application to x-ray protein crystallography is outlined. Finally, coherence, harmonics, and radiative corrections are also briefly discussed.

Journal Article↗
PubMed2001

Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: implications for the catalytic mechanism.

The major human abasic endonuclease, Ape1, is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3' replication-blocking moieties, and modulates the DNA binding activity of several transcriptional regulators. We have determined the X-ray structure of the full-length human Ape1 enzyme in two new crystal forms, one at neutral and one at acidic pH. The new structures are generally similar to the previously determined structure of a truncated Ape1 protein, but differ in the conformation of several loop regions and in spans of residues with weak electron density. While only one active-site metal ion is present in the structure determined at low pH, the structure determined from a crystal grown at the pH optimum of Ape1 nuclease activity, pH 7.5, has two metal ions bound 5 A apart in the active site. Enzyme kinetic data indicate that at least two metal-binding sites are functionally important, since Ca(2+) exhibits complex stimulatory and inhibitory effects on the Mg(2+)-dependent catalysis of Ape1, even though Ca(2+) itself does not serve as a cofactor. In conjunction, the structural and kinetic data suggest that Ape1 catalyzes hydrolysis of the DNA backbone through a two metal ion-mediated mechanism.

Binding Sites↗
PubMed2001

Interaction of the N-terminal domain of apolipoprotein E4 with heparin.

Apolipoprotein E (apoE) is an important lipid-transport protein in human plasma and brain. It has three common isoforms (apoE2, apoE3, and apoE4). ApoE is a major genetic risk factor in heart disease and in neurodegenerative disease, including Alzheimer's disease. The interaction of apoE with heparan sulfate proteoglycans plays an important role in lipoprotein remnant uptake and likely in atherogenesis and Alzheimer's disease. Here we report our studies of the interaction of the N-terminal domain of apoE4 (residues 1-191), which contains the major heparin-binding site, with an enzymatically prepared heparin oligosaccharide. Identified by its high affinity for the N-terminal domain of apoE4, this oligosaccharide was determined to be an octasaccharide of the structure DeltaUAp2S(1-->[4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->](3)4)-alpha-D-GlcNpS6S by nuclear magnetic resonance spectroscopy, capillary electrophoresis, and polyacrylamide gel electrophoresis. Kinetic analysis of the interaction between the N-terminal apoE4 fragment and immobilized heparin by surface plasmon resonance yielded a K(d) of 150 nM. A similar binding constant (K(d) = 140 nM) was observed for the interaction between immobilized N-terminal apoE4 and the octasaccharide. Isothermal titration calorimetry revealed a K(d) of 75 nM for the interaction of the N-terminal apoE fragment and the octasaccharide with a binding stoichiometry of approximately 1:1. Using previous studies and molecular modeling, we propose a binding site for this octasaccharide in a basic residue-rich region of helix 4 of the N-terminal fragment. From the X-ray crystal structure of the N-terminal apoE4, we predicted that binding of the octasaccharide at this site would result in a change in intrinsic fluorescence. This prediction was confirmed experimentally by an observed increase in fluorescence intensity with octasaccharide binding corresponding to a K(d) of approximately 1 microM.

Animals↗
PubMed2000

Differences in stability among the human apolipoprotein E isoforms determined by the amino-terminal domain.

Denaturation by guanidine-HCl, urea, or heating was performed on the common isoforms of human apolipoprotein (apo) E (apoE2, apoE3, and apoE4) and their 22-kDa and 10-kDa fragments in order to investigate the effects of the cysteine/arginine interchanges at residues 112 and 158. Previous physical characterization of apoE3 established that apoE contains two domains, the 10-kDa carboxyl-terminal and 22-kDa amino-terminal domains, which unfold independently and exhibit large differences in stability. However, the physical properties of apoE2, apoE3, and apoE4 have not been compared before. Analysis by circular dichroism showed that the different isoforms have identical alpha-helical contents and guanidine-HCl denaturation confirmed that the two domains unfold independently in all three isoforms. However, guanidine-HCl, urea, and thermal denaturation showed differences in stability among the 22-kDa amino-terminal fragments of the apoE isoforms (apoE4 < apoE3 < apoE2). Furthermore, guanidine-HCl denaturation monitored by circular dichroism and fluorescence suggested the presence of a folding intermediate in apoE, most prominently in apoE4. Thus, these studies reveal that the major isoforms of apoE, which are associated with different pathological consequences, exhibit significant differences in stability.

Apolipoproteins E↗
PubMed2000

Binding of an antibody mimetic of the human low density lipoprotein receptor to apolipoprotein E is governed through electrostatic forces. Studies using site-directed mutagenesis and molecular modeling.

Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.

Antibodies, Monoclonal↗
PubMed2000

Reversible expression of tryptases in continuous L138.8A mast cells.

It has been established that mast cells can alter their expression of granule chymases and tryptases in vivo. In vitro, a reversible cytokine regulation has so far only been demonstrated for chymases. We now show a reversible and cytokine-regulated expression of the tryptases MMCP-6 and MMCP-7 and of the chymases MMCP-1, MMCP-2 and MMCP-4 in the continuous murine mast cell line L138.8A. The L138.8A mast cells lacked expression of mRNA for mast cell-specific proteases when cultured in IL-3, and only 49% and 41% of the cells were c-kit+ and FcepsilonRI+, respectively, by flow cytometry. Kit-ligand/stem cell factor induced synthesis of the chymase MMCP-4 and the tryptases MMCP-6 and MMCP-7 and increased the fraction of c-kit+ and FcepsilonRI+ L138.8A cells to >70%. Kit-ligand-induced tryptase expression was suppressed in the presence of IL-3 or IL-9, and reversed after withdrawal of kit-ligand. IL-9 or IL-3/IL-10 promoted the formation of Alcian blue+ granules and increased the fraction of c-kit+ and FcepsilonRI+ L138.8A cells to >90%. IL-9 further induced the expression of the chymases MMCP-1, MMCP-2 and MMCP-4. Thus, the immature mast cell line L138.8A has the capacity to modulate both tryptase and chymase expression and represents the first model system to analyze the molecular regulation of tryptase expression in vitro.

Animals↗
PubMed2000

Cloning, expression, and one-step purification of the minimal essential domain of the light chain of botulinum neurotoxin type A.

A truncated but functional form of the botulinum neurotoxin A light chain (Tyr 9-Leu 415) has been cloned into the three bacterial expression vectors, pET 28, pET 30, and PGEX-2T, and produced as fusion proteins. This 406-amino-acid light chain was expressed with 1 six-histidine tag (LC-pET28), 2 six histidine tags and a S-tag (LC-pET30), or a six-histidine tag and a glutathione S-transferase tag (LC-pGEX-2T). The three fusion proteins have been overexpressed in Escherichia coli, purified in a soluble form, and tested for protease activity. All three recombinant proteins were found to have similar enzymatic activity, comparable to the light chain purified from the whole toxin. The LC-pET30 protein was the most soluble and stable of the three fusion proteins, and it could be purified using a one-step affinity chromatography protocol. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. This protein has been crystallized and initial X-ray data show that the crystals diffract to 1.8 A.

Botulinum Toxins, Type A↗
PubMed2000

Conformational flexibility in the apolipoprotein E amino-terminal domain structure determined from three new crystal forms: implications for lipid binding.

An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.

Animals↗
PubMed1999

Structure of a monoclonal 2E8 Fab antibody fragment specific for the low-density lipoprotein-receptor binding region of apolipoprotein E refined at 1.9 A.

The crystal structure of the Fab fragment of 2E8, the monoclonal IgG1,kappa antibody specific for the low-density lipoprotein (LDL) receptor-binding region of apolipoprotein E (apoE), has been solved by molecular replacement and refined at 1.9 A resolution (PDB entry 12E8). Two 2E8 Fab molecules in the asymmetric unit are related by noncrystallographic symmetry and are hydrogen bonded through a beta-sheet-like intermolecular contact between the heavy-chain complementarity-determining regions 3 (CDRH3) of each molecule. The structure has been refined to an R value of 0.22 (Rfree = 0.27). The initially ill-defined heavy-chain constant domain (CH1) of 2E8 has been retraced with the aid of automatic refinement, confirming the beta-sheet tracing independently of any starting models. As a resolution better than 2 A is not common for Fab fragments, this model represents a well defined Fab structure and should prove useful in MR solution of other Fab fragments. Furthermore, in the absence of an LDL-receptor structure, the homology of the 2E8 CDRH2 to the ligand-binding domain of the LDL receptor has been exploited to model the apoE-LDL-receptor interaction.

Animals↗
PubMed1999

Purification, characterization, and crystallization of the distal BRCT domain of the human XRCC1 DNA repair protein.

The XRCC1 DNA repair protein contains two regions of approximately 100 amino acids each that share homology with the BRCT (BRCA1 carboxyl terminus) domain superfamily. These two regions of XRCC1 have been shown to interact independently with DNA ligase III and poly(ADP-ribose)polymerase as part of a mechanism involved in the repair of DNA single-strand breaks. To understand how these BRCT regions specify protein-protein interactions and contribute to DNA repair function, we have overexpressed and purified the distal BRCT domain of XRCC1 with the goal of structure determination. The cDNA encoding this BRCT region (X1BRCTb) was inserted into the pET29 bacterial expression vector; the polypeptide was expressed in mostly soluble form and then purified by anion-exchange and gel filtration chromatography. Crystallization screening with the purified material resulted in the formation of large bipyramidal crystals. Crystals formed within several hours at room temperature from salt solutions of ammonium sulfate. Crystals diffract to approximately 2.85 A and were found to be in space group P4(1)2(1)2 (or its enantiomorph P4(3)2(1)2) with unit cell dimensions a = 100.43 A, c = 105.62 A. Crystals of similar character have also been obtained after incorporation of selenomethionine during expression of the protein. Efforts are now under way to determine the molecular structure of the X1BRCTb domain. These studies are likely to give insight into the interaction between XRCC1 and DNA ligase III and into general structural features of BRCT domains that exist in many other proteins.

Base Sequence↗
PubMed1999

Carboxyl-terminal domain of human apolipoprotein E: expression, purification, and crystallization.

Thioredoxin fusion expression vectors for two carboxyl-terminal fragments of human apolipoprotein (apo) E (residues 223-272 and 223-299) were generated from an apoE cDNA with the objective of obtaining structural information on this functionally important region of apoE by X-ray crystallography. A thrombin cleavage recognition site was positioned at the fusion junction to release the apoE fragments from the fusion protein. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates by nickel-affinity column chromatography, and cleaved with thrombin. After gel filtration and ion exchange chromatography, yields of each fragment were approximately 14 mg/L. Both fragments bind to the phospholipid dimyristoylphosphatidylcholine in a manner similar to that of the 216-299 fragment of apoE isolated from plasma, which represents the major lipid-binding region of the protein. Orthorhombic crystals of the apoE 223-272 fragment that diffracted to 1.8 A were obtained in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH 7.0-7.5), containing 30% glycerol. The space group is C222 with cell dimensions of a = 35.17 A, b = 38.95 A, and c = 133.27 A.

Amino Acid Motifs↗
PubMed1999

Suppression of T1-receptor expression by antisense RNA abrogates differentiation of osteogenic osteosarcoma cells.

Soluble and membrane-associated variants of the orphan T1-receptor, a homolog of interleukin-1 receptor type I, are expressed in proliferating preosteoblasts in differentiating bone. Recent evidence reveals that T1-receptor synthesis is retained in osteogenic osteosarcoma cells. Here we report that the suppression of T1-receptor expression by mouse osteosarcoma cells using a T1 -antisense expression vector results in the abrogation of the osteogenic potential of the tumor cells. T1-antisense-expressing tumor cells formed anaplastic tumors in vivo and failed to express the osteoblast-specific genes collagen type 1, alkaline phosphatase, and osteocalcin when cultured in a 3-dimensional collagen type I matrix in vitro. Suppression of T1-receptor synthesis did not affect the expression of the essential bone cell-specific transcription factor AML3/CBFA1 in the osteosarcoma cells. These data provide the first evidence that T1-receptor plays a key role in osteogenic differentiation.

Animals↗
PubMed1998

Cadmium-induced crystallization of proteins: II. Crystallization of the Salmonella typhimurium histidine-binding protein in complex with L-histidine, L-arginine, or L-lysine.

To further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine-binding protein were crystallized with varying concentrations of cadmium salts. For each of the three histidine-binding protein complexes, cadmium cations were found to promote or improve crystallization. The optimal cadmium concentration is ligand specific and falls within a narrow concentration range. In each case, crystals grown in the presence of cadmium diffract to better than 2.0 angstroms resolution and belong to the orthorhombic space group P2(1)2(1)2(1). From our results and from the analysis of cadmium sites in well-refined protein structures, we propose that cadmium addition provides a generally useful technique to modify crystal morphology and to improve diffraction quality.

Arginine↗
PubMed1998

The diencephalon and pretectum of the white sturgeon (Acipenser transmontanus): a cytoarchitectonic study.

The cytoarchitecture of the diencephalon and pretectum of the white sturgeon, Acipenser transmontanus, was studied utilizing cresyl violet stained serial paraffin sections. The identified cell groups were assigned to the preoptic area, hypothalamus, thalamus and posterior tubercle, epithalamus, synencephalon and pretectum. The outlines of the diencephalic and pretectal nuclei were projected graphically onto a midsagittal section of the brain, thus providing a reconstruction of the relative positions of the major cell groups. This facilitated comparisons with the diencephalic and pretectal nuclei of other ray-finned fishes. Our cytoarchitectural analysis indicates that the diencephalon and pretectum of the white sturgeon is intermediate to that described for cladistians and neopterygians. The preoptic area in Acipenser is relatively conservative compared to other ray-finned fishes but lacks distinct subdivisions in the magnocellular periventricular preoptic nucleus and includes a unique migrated rostral accessory nucleus. The hypothalamic walls are rather thin, and due to the presence of extensive lateral and posterior recesses and the lack of migrated nuclei, they superficially resemble the condition seen in sharks. The dorsal and ventral thalamic nuclei do not exhibit much variation compared to other ray-finned fishes, except for the presence of a small lateral posterior thalamic nucleus, the absence of a distinct ventrolateral thalamic nucleus, and slight differences in the internal organization of the ventromedial thalamic nucleus. The posterior tubercle in Acipenser clearly comprises more migrated cell groups than that of Polypterus, however, these cell groups are considerably less well defined than in neopterygians. As in other nonteleost actinopterygians, the habenular nuclei are highly asymmetrical with the right side larger than the left side. The cytoarchitectonic complexity of the pretectum in Acipenser is intermediate to that observed in Polypterus and neopterygians in that a magnocellular component within the superficial pretectal nucleus is clearly present but cannot be delineated as a distinct magnocellular superficial pretectal nucleus. Also, the posterior pretectal nucleus which is absent in Polypterus but which has been identified as a small nucleus both in Lepisosteus and Amia is represented in Acipenser by a small group of scattered cells.

Animals↗
PubMed1998

Discordant effects of activator protein-1 transcription factor on gene regulation, invasion, and metastasis in spontaneous, radiation-induced, and fos-induced osteosarcomas.

Bone cells are a prime target for the biological function of the fos/jun (activating protein-1 (AP-1)) transcription factor complex. Deregulated expression of c-fos or v-fos in bone cells induces tumorigenicity and the formation of non-metastatic osteosarcomas. In contrast, fos oncogenes transform fibroblasts to an invasive phenotype accompanied by the expression of various invasion- and metastasis-associated genes. Here we compared the expression of AP-1-dependent genes and AP-1 activity in cell lines from fos-induced, radiation-induced, and spontaneous osteosarcomas. We showed that the presence of high AP-1 activity was not sufficient for the induction of invasion- and metastasis-associated AP-1-dependent genes in transformed bone cells. Further, we identified the collagenase I and stromelysin 1 gene promoters as suitable tools for the analysis of other factors regulating metastatic progression of osteosarcoma.

Animals↗