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At least 757 records · Page 42Linked to original sources
PubMed1999

Structure and localization of the mouse prolyl oligopeptidase gene.

We have cloned and characterized the genomic structure of the mouse gene for prolyl oligopeptidase that is mapped to chromosome 10B2-B3. The gene is about 92 kilobases in size and contains 15 exons. All exon-intron junction sequences conform to the GT/AG rule. Comparison with the presumed domain structures of the mouse prolyl oligopeptidase indicates that the propeller domain of the enzyme is encoded by exons 3-10, whereas the catalytic domain is encoded by exons 1-3 and 10-15. The catalytic triad residues are encoded by two exons (Ser(554) on exon 13 and His(680) and Asp(642) on exon 15). The 5'-flanking region of the mouse prolyl oligopeptidase gene has structural features found in housekeeping gene promoters, including a GC-rich segment and an absence of TATA and CAAT boxes. A primer extension assay showed the presence of multiple sites for the initiation of transcription. Transient transfection analysis demonstrated that the 5'-flanking region of the gene can direct efficient expression in COS1 cells. Deletion studies revealed that the downstream 125-base pair sequence of the region is required for promoter activity in the cells.

Animals↗
PubMed1989

Fine structure and localization of anionic sites on the surface of microfilaria of Wuchereria bancrofti.

The fine structure of the sheath and the cuticle of microfilaria of Wuchereria bancrofti was analysed and the results obtained were compared with those from other filarial parasites. Cationized ferritin particles bind to the surface of the sheath and the epicuticle. No binding of colloidal iron hydroxyde particles to the microfilaria surface was observed. Reaction product indicative of carbohydrates containing vic-glycol groups was not observed in thin sections of microfilaria submitted to the periodic acid-thiosemicarbazide-silver proteinate technique. The results obtained are discussed in relation to previous studies using lectins.

Animals↗
PubMed2006

The unfolded state of the villin headpiece helical subdomain: computational studies of the role of locally stabilized structure.

The 36 residue villin headpiece helical subdomain (HP36) is one of the fastest cooperatively folding proteins, folding on the microsecond timescale. HP36's simple three helix topology, fast folding and small size have made it an attractive model system for computational and experimental studies of protein folding. Recent experimental studies have explored the denatured state of HP36 using fragment analysis coupled with relatively low-resolution spectroscopic techniques. These studies have shown that there is apparently only a small tendency to form locally stabilized secondary structure. Here, we complement the experimental studies by using replica exchange molecular dynamics with explicit solvent to investigate the structural features of these peptide models of unfolded HP36. To ensure convergence, two sets of simulations for each fragment were performed with different initial structures, and simulations were continued until these generated very similar final ensembles. These simulations reveal low populations of native-like structure and early folding events that cannot be resolved by experiment. For each fragment, calculated J-coupling constants and helical propensities are in good agreement with experimental trends. HP-1, corresponding to residues 41 to 53 and including the first alpha-helix, contains the highest helical population. HP-3, corresponding to residues 62 through 75 and including the third alpha-helix, contains a small population of helical turn residing at the N terminus while HP-2, corresponding to residues 52 through 61 and including the second alpha-helix, formed little to no structure in isolation. Overall, HP-1 was the only fragment to adopt a native-like conformation, but the low population suggests that formation of significant structure only occurs after formation of specific tertiary interactions.

Amino Acids↗
PubMed2000

Genomic structure, chromosomal localization and identification of mutations in the xeroderma pigmentosum variant (XPV) gene.

The xeroderma pigmentosum variant (XP-V) is one of the most common forms of this cancer-prone syndrome. XP groups A through G are characterized by defective nucleotide excision repair, whereas the XP-V phenotype is proficient in this pathway. The XPV gene encodes DNA polymerase eta, which catalyzes an accurate translesion synthesis, indicating that the XPV gene contributes tumor suppression in normal individuals. Here we describe the genomic structure and chromosomal localization of the XPV gene, which includes 11 exons covering the entire coding sequence, lacks a TATA sequence in the upstream region of the transcription-initiation, and is located at the chromosome band 6p21.1-6p12. Analyses of patient-derived XP-V cell lines strongly suggested that three of four cell lines carried homozygous mutations in the XPV gene. The fourth cell line, XP1RO, carried heterozygous point mutations in the XPV gene, one of which was located at the splice acceptor site of exon 2, resulting in the omission of exon 2 from the mature mRNA. These findings provide a basis for diagnosis and therapy of XP-V patients.

Alleles↗
PubMed2005

Band structure and local dynamics of excitons in bacterial light-harvesting complexes revealed by spectrally selective spectroscopy.

Hole-burned absorption and line-narrowed fluorescence spectra are studied at 5 K in wild type and mutant LH1 and LH2 antenna preparations from the photosynthetic purple bacterium Rhodobacter sphaeroides. Evidence was found in all samples, even in intact membranes, of the presence of a broad distribution of bacteriochlorophyll species that are unable to communicate energy between each other and to the exciton states of functional antenna complexes. The distribution maximum of these localized species determined by zero phonon hole action spectroscopy is at 783.5 nm in purified LH1 complexes and at 786.8 nm in B850-only mutant LH2 complexes. A well-resolved peak at 807 nm in LH1 complexes is assigned to the exciton band structure of functional core antenna complexes. Similar structure in LH2 complexes overlaps with the distribution of localized species. Off-diagonal (structural) disorder may be responsible for this exciton band structure. Our data also imply that pair-wise inter-chlorophyll couplings determine the resonance fluorescence lineshape of excitonic polarons.

Light-Harvesting Protein Complexes↗
PubMed2001

Multiple scattering approach to K-edge XANES of Sb-Te systems.

The Sb-Te systems are important for optical memories. The local structures of these Sb-Te systems are crucial to understand the properties. Here we study three different Sb-Te systems, Sb2Te3, c- and a-SbTe (c; crystalline, a; amorphous) by use of XANES analyses. The present calculations for Sb2Te3 system show quite good agreement with the observed spectra. In contrast to Sb2Te3 system, both c- and a-SbTe systems, in which a ratio of Sb to Te is 3, are metastable; both of the structures have not been known yet. We thus investigate these local structures by use of the multiple scattering approach, and propose a possible model for each of c- and a-SbTe.

Journal Article↗
PubMed1995

Probing local secondary structure by fluorescence: time-resolved and circular dichroism studies of highly purified neurotoxins.

The relationship between beta-sheet secondary structure and intrinsic tryptophan fluorescence parameters of erabutoxin b, alpha-cobratoxin, and alpha-bungarotoxin were examined. Nuclear magnetic resonance and x-ray crystallography have shown that these neurotoxins have comparable beta-sheet, beta-turn, and random coil secondary structures. Each toxin contains a single tryptophan (Trp) residue within its beta-sheet. The time-resolved fluorescence properties of native erabutoxin b and alpha-cobratoxin are best described by triple exponential decay kinetics, whereas native alpha-bungarotoxin exhibits more than four lifetimes. The disulphide bonds of each toxin were reduced to facilitate carboxymethylation and amidocarboxymethylation. The two different toxin derivatives of all three neurotoxins displayed triple exponential decay kinetics and were completely denatured as evidenced by circular dichroism (random coil). The concentration (c) values of the three fluorescence decay times (time-resolved fluorescence spectroscopy (TRFS)) were dramatically different from those of the native toxins. Each neurotoxin, treated with different concentrations of guanidinium hydrochloride (GuHCl), was studied both by circular dichroism and TRFS. Disappearance of the beta-sheet secondary structural features with increasing concentrations of GuHCl was accompanied by a shift in the relative contribution (c value) of each fluorescence decay time (TRFS). It was found that certain disulphide residues confer added stability to the beta-sheet secondary structure of these neurotoxins and that the center of the beta-sheet is last to unfold. These titrations show that Trp can be used as a very localized probe of secondary structure.

Animals↗
PubMed2005

Computational investigation of order, structure, and dynamics in modified water models.

Model liquids have been constructed to study the role of local structure in the anomalous properties of liquid water. The intermolecular potentials were modified by increasing the weight of the Lennard-Jones term relative to the electrostatic term in the SPC/E model for water. The resulting family of liquids varies from SPC/E water to a Lennard-Jones-like liquid. Properties were measured as a function of density and temperature. The local structure was described by two order parameters, one measuring the tetrahedral order and the other measuring the translational order. The translational order parameter was found to be large for both tetrahedral and Lennard-Jones liquids, but to go through a minimum as the potentials were modified, demonstrating that the two types of structure are incompatible. Just as in water several properties (e.g., the translational diffusion coefficient, entropy) exhibit anomalous density dependence as a result of the breakdown of local tetrahedrality, we observed nonmonotonic behavior of the translational diffusion constant and reorientational relaxation rate as the fluids were transformed from tetrahedral to Lennard-Jones-like. This is also an indication of the incompatibility between Lennard-Jones and water-like structure.

Journal Article↗
PubMed2001

Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties.

Abnormalities in cellular differentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell differentiation. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful effects occur when mda-7 is expressed in normal epithelial or fibroblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Hu-mda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-beta plus MEZ. Mda-7 expression is also induced during megakaryocyte differentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-beta+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-beta+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in significant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a differentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers.

Antigens, Neoplasm↗
PubMed2001

Gene structure, chromosomal localization and immunolocalization of chicken centromere proteins CENP-C and ZW10.

We determined the genomic structures and complete sequences of the coding regions of the chicken CENP-C and ZW10 genes. These two genes encode proteins that are thought to be involved in maintaining the fidelity of chromosome segregation. The chicken CENP-C gene is 30 kb in length and contains 19 exons. The chicken ZW10 gene spans 10 kb and contains 15 exons. The 5'-untranslated regions of these genes contain several binding sites for transcription factors such as Sp-1, E2F, p300, and members of the GATA family. By fluorescence in situ hybridization (FISH) analysis, the CENP-C was mapped to chromosome 4 and the ZW10 gene was mapped to a microchromosome. Antibodies against the chicken ZW10 protein revealed a cell cycle-dependent staining pattern in DT40 cells. ZW10 protein was distributed throughout the cytoplasm of DT40 cells during interphase. In most metaphase cells, ZW10 proteins appeared equally divided between the centromere and the spindle apparatus. During anaphase, chicken ZW10 proteins were no longer localized near chromosomes or the mitotic apparatus but were present diffusely in the cytoplasm.

5' Untranslated Regions↗
PubMed1975

The fine structure and localization of alkaline phosphatase activity of the small intestinal epithelium in the postnatal developing rat.

The morphological structure and the development of alkaline phosphatase activity of the small intestine of neo-natal rats were investigated by means of light and electron microscopy. Until around the 14th day of neo-natal life alkaline phosphatase activity of the duodenal and jejunal epithelial cells was observed on the microvilli, the apical vacuoles and tubules, and the tubular invagination. After the 15th day, the tubulo-vacuolar system below the microvilli disappears from the duodenal and jejunal cells. Also the enzyme activity becomes localized solely on the microvillous membrane. This cytochemical pattern in the jejunum shows no change with the advance of age. Around the 23rd day, alkaline phosphatase activities of the duodenal epithelium were observed in the same sites as those in the full grown rat, i.e. on the microvilli, the lateral cell membrane, and the Golgi apparatus. During the suckling period ultrastructural localization of alkaline phosphatase in the ileal cells can be seen in the microvillous membrane, the lateral cell membrane, the tubulo-vacuolar system, and the supranuclear vacuole. At the weaning period, however, the apical tubulo-vacuolar system and the supranuclear vacuole disappear, and all the absorptive cells in the ileal epithelium are free of reaction products as has been shown to be the case in the adult rat.

Age Factors↗
PubMed2004

Domain swapping localizes the structural determinants of regioselectivity in membrane-bound fatty acid desaturases of Caenorhabditis elegans.

Most fatty acid desaturases are members of a large superfamily of integral membrane, O(2)-dependent, iron-containing enzymes that catalyze a variety of oxidative modifications to lipids. Sharing a similar primary structure and membrane topology, these enzymes are broadly categorized according to their positional specificity or regioselectivity, which designates the preferred position for substrate modification. To investigate the structural basis of regioselectivity in membrane-bound desaturases, the Caenorhabditis elegans omega-3 (FAT-1) and "Delta12" (FAT-2) desaturases were used as a model system. With the use of unnatural substrates, the regioselectivity of C. elegans FAT-2 was clearly defined as nu+3, i.e. it "measures" three carbons from an existing double bond. The structural basis for nu+3 and omega-3 regioselectivities was examined through construction and expression of chimeric DNA sequences based on FAT-1 and FAT-2. Each sequence was divided into seven domains, and chimeras were constructed in which specific domains were replaced with sequence from the other desaturase. When tested by expression in yeast using exogenously supplied substrates, chimeric sequences were found in which domain swapping resulted in a change of regioselectivity from nu+3 to omega-3 and vice versa. In this way, the structural determinants of regioselectivity in FAT-1 and FAT-2 have been localized to two interdependent regions: a relatively hydrophobic region between the first two histidine boxes and the carboxyl-terminal region.

Amino Acid Sequence↗
PubMed2004

Urea-dependent unfolding of murine adenosine deaminase: sequential destabilization as measured by 19F NMR.

Murine adenosine deaminase (mADA) is a 40 kDa (beta/alpha)(8)-barrel protein consisting of eight central beta-strands and eight peripheral alpha-helices containing four tryptophan residues. In this study, we investigated the urea-dependent behavior of the protein labeled with 6-fluorotryptophan (6-(19)F-Trp). The (19)F NMR spectrum of 6-(19)F-Trp-labeled mADA reveals four distinct resonances in the native state and three partly overlapped resonances in the unfolded state. The resonances were assigned unambiguously by site-directed mutagenesis. Equilibrium unfolding of 6-(19)F-Trp-labeled mADA was monitored using (19)F NMR based on these assignments. The changes in intensity of folded and unfolded resonances as a function of urea concentration show transition midpoints consistent with data observed by far-UV CD and fluorescence spectroscopy, indicating that conformational changes in mADA during urea unfolding can be followed by (19)F NMR. Chemical shifts of the (19)F resonances exhibited different changes between 1.0 and 6.0 M urea, indicating that local structures around 6-(19)F-Trp residues change differently. The urea-induced changes in local structure around four 6-(19)F-Trp residues of mADA were analyzed on the basis of the tertiary structure and chemical shifts of folded resonances. The results reveal that different local regions in mADA have different urea-dependent behavior, and that local regions of mADA change sequentially from native to intermediate topologies on the unfolding pathway.

Adenosine Deaminase↗
PubMed2000

Direct 3D imaging of Al70.4Pd21Mn8.6 quasicrystal local atomic structure by X-ray holography

Inverse x-ray fluorescence holography was used to explore the local atomic order of a nearly perfect quasicrystal with composition Al70. 4Pd21Mn8.6. We have demonstrated the possibility of direct 3D imaging of the atomic decoration in a quasicrystal. We have obtained the average 3D environment of selected coordination shells around the Mn atoms. These results open the way to obtaining further and more complete information about the various coordination shells in complex materials by measuring multiple energy x-ray holograms at different sites.

Journal Article↗
PubMed2006

Multiscale detection of localized anomalous structure in aggregate disease incidence data.

We present a modelling framework for detection of potentially anomalous structure in aggregate spatial disease incidence data in a manner sensitive to localization at multiple scales and/or positions. The key technical contribution is the re-casting of the components of a multiscale disease mapping methodology, recently introduced by the authors in an earlier paper, into a form appropriate for hypothesis testing. In particular, we describe how hypotheses of spatially clustered variations in disease incidence may be linked in one-to-one correspondence with collections of hypotheses on the values of certain multiscale parameters associated with a user-defined hierarchy of nested partitions of an overall spatial region. A Bayesian hypothesis testing methodology is developed in the context of a standard Poisson measurement model, over the collection of possible multiscale hypotheses. We discuss the specification of hyper parameters and prior distributions on the space of models. The methodology is illustrated on both simulated and real data.

Cluster Analysis↗
PubMed1998

Calculations on folding of segment B1 of streptococcal protein G.

We present an investigation of the folding thermodynamics and mechanism of segment B1 of streptococcal protein G. Molecular dynamics simulations of the fully solvated protein are used to probe thermodynamically significant states at different stages of folding. We performed several unfolding simulations to generate a database of initial conditions. The database is analyzed and clustered. The cluster centers extracted from this database were then used as starting points for umbrella sampling of the folding free energy landscape under folding conditions. The resulting sampling was combined with the weighted histogram analysis method. One and two-dimensional free energy surfaces were constructed along several order parameters and used to analyze the folding process. Our findings indicate that an initial collapse precedes the formation of significant native structure. Elements of local structure originate in the regions of the protein shown to have higher H/2H exchange protection factors in early stages of folding. A non-native contact, observed experimentally at the N terminus of the alpha-helix in a peptide excised from the protein, is seen to pre-organize the chain in early stages of folding. Collapse and early structure formation yields a compact globule with a significant number of water molecules present. Desolvation of the protein core is coincident with the final stages of folding from the compact state.

Bacterial Proteins↗
PubMed1992

Nuclear magnetic resonance solution structure of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis).

The three-dimensional structure in solution of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis) has been determined by nuclear magnetic resonance spectroscopy. A high quality structure for this 60-residue protein was obtained from 656 NOE distance constraints and 143 dihedral angle constraints, using the distance geometry program DIANA for the structure calculation and AMBER for restrained energy minimization. For a group of 20 conformers used to represent the solution structure, the average root-mean-square deviation value calculated for the polypeptide backbone heavy atoms relative to the mean structure was 0.45 A. The protein consists of a core region from which three finger-like loops extend outwards. It includes a short, two-stranded antiparallel beta-sheet of residues 1-5 and 13-17, a three-stranded antiparallel beta-sheet involving residues 23-31, 34-42 and 51-55, and four disulfide bridges in the core region. There is also extensive non-regular hydrogen bonding between the carboxy-terminal tail of the polypeptide chain and the rest of the core region. Comparison with the crystal structure of erabutoxin-b indicates that the structure of alpha-neurotoxin is quite similar to other neurotoxin structures, but that local structural differences are seen in regions thought to be important for binding of neurotoxins to the acetylcholine receptor. For two regions of the alpha-neurotoxin structure there is evidence for an equilibrium between multiple conformations, which might be related to conformational rearrangements upon binding to the receptor. Overall, the alpha-neurotoxin presents itself as a protein with a stable core and flexible surface areas that interact with the acetylcholine receptor in such a way that high affinity binding is achieved by conformational rearrangements of the deformable regions of the neurotoxin structure.

Amides↗
PubMed1994

Equilibrium intermediates in the denaturation of human insulin and two monomeric insulin analogs.

The equilibrium denaturation of human insulin in a monomer-inducing solvent and of two monomeric insulin analogs, lysB28proB29 insulin and aspB10desB28-30 insulin, was reexamined [Brems, D. N., Brown, P. L., Heckenlaible, L. A., & Frank, B. H. (1990) Biochemistry 29,9289-9293] by circular dichroism (CD) at additional wavelengths in the near-UV region. Previous denaturation studies were limited by the solubility of guanidine hydrochloride being only slightly greater than the level of denaturant required to fully unfold human insulin; therefore, only a few data points were available for construction of the post-transitional baseline. In the present study, we report the use of an unfolded mimic created by enzymatic digestion of insulin to confirm the slope of the post-transitional baseline. Evidence for equilibrium unfolding intermediates for each of these insulins was indicated by noncoincidence of the denaturation transitions as monitored by tyrosine and helical-dependent CD bands (270 and 224 nm, respectively). Additional evidence for intermediates through multiphasic denaturation transitions was obtained at a wavelength likely related to disulfide conformation, 251 nm. The results suggest that for each of the insulins, at least two intermediates are significantly populated. An unfolding model is proposed in which the conformation of the least stable intermediate is slightly unfolded only in the C-terminal segment of the B chain. A second more stable intermediate retains minimal secondary structure while containing localized structure proximal to one or more of the disulfide groups. The presence of equilibrium intermediates has important implications for the folding pathway of insulin, in pharmaceutical applications such as formulation stability, and for conformational transitions that accompany receptor binding.

Animals↗