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At least 559 records · Page 31Linked to original sources
PubMed1990

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin.

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val- Gly-Val-Lys, corresponding to sequence 296-316 of thermolysin and thus encompassing the COOH-terminal helical segment 301-312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296-316 was then cleaved with trypsin at Lys307 and Staphylococcus aureus V8 protease at Glu302, producing the additional fragments 296-307, 308-316, 296-302, and 303-316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296-316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301-312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296-316 attains in 90% aqueous trifluoroethanol the same percentage (approximately 58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296-316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule.

Alcohols↗
PubMed1997

Influence of DNA sequence identity on efficiency of targeted gene replacement.

We have developed a system for analyzing recombination between a DNA fragment released in the nucleus from a single-copy plasmid and a genomic target in order to determine the influence of DNA sequence mismatches on the frequency of gene replacement in Saccharomyces cerevisiae. Mismatching was shown to be a potent barrier to efficient gene replacement, but its effect was considerably ameliorated by the presence of DNA sequences that are identical to the genomic target at one end of a chimeric DNA fragment. Disruption of the mismatch repair gene MSH2 greatly reduces but does not eliminate the barrier to recombination between mismatched DNA fragment and genomic target sequences, indicating that the inhibition of gene replacement with mismatched sequences is at least partially under the control of mismatch repair. We also found that mismatched sequences inhibited recombination between a DNA fragment and the genome only when they were close to the edge of the fragment. Together these data indicate that while mismatches can destabilize the relationship between a DNA fragment and a genomic target sequence, they will only do so if they are likely to be in the heteroduplex formed between the recombining molecules.

Base Sequence↗
PubMed1984

Neon-20 depth-dose relations in water.

The dose from heavy ion beams has been calculated using a one-dimensional transport theory and evaluated for 670 MeV/ amu 20Ne beams in water. The result is presented so as to be applicable to arbitrary ions for which the necessary interaction data are known. The present evaluation is based on the Silberberg - Tsao fragmentation parameters augmented with light fragment production from intranuclear cascades, recently calculated nuclear absorption cross sections, and evaluated stopping power data. Comparison with recent experimental data obtained at the Lawrence Berkeley Laboratory reveals the need for more accurate fragmentation data.

Ions↗
PubMed1993

Cloning, nucleotide sequence and expression in Escherichia coli of a gene (ompM) encoding a 25 kDa major outer-membrane protein (MOMP) of legionella pneumophila.

A genomic library derived from a virulent isolate of Legionella pneumophila was constructed in Escherichia coli JM 83 using the cloning vector pUC19. The clones were screened by filter immunoassay using L. pneumophila rabbit polyclonal antisera and in the absence of in situ bacterial lysis one such clone, LP 116, expressed L. pneumophila-specific antigens on the surface of E. coli. Restriction endonuclease digest analysis and agarose gel electrophoresis revealed a fragment measuring approximately 750 bp. Southern hybridization confirmed that the fragment was L. pneumophila DNA. Sequencing data showed that the fragment was 810 bp in length with an open reading frame (ORF) of 678 bp. The outer-membrane profiles of the E. coli parent, the L. pneumophila DNA-contributing strain and clone LP 116 were compared by SDS-PAGE. A protein of 25 kDa was found in outer-membrane preparations of both the clone LP 116 and L. pneumophila but not in E. coli JM 83. This was in agreement with the molecular mass of the deduced peptide of the mature protein. Immunoblots using L. pneumophila-specific polyclonal antiserum confirmed that this 25 kDa outer-membrane protein (OMP) was a L. pneumophila polypeptide. Both direct immunofluorescence assay and immunoblots using the commercially produced monoclonal antibody specific for the common antigen of the major outer-membrane protein (MOMP) confirmed that the 25 kDa protein produced by LP 116 was involved with the MOMP complex. The gene encoding this protein has been designated ompM.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Recognition of defined epitopes by affinity-purified anti-immunoglobulin fab autoantibodies isolated from HIV-infected humans.

Infection of humans with HIV-1 has previously been independently shown to result in the generation of autoantibodies (AAbs) reactive with immunoglobulin Fab fragments (Heidelberg), and with autoantibodies to T-cell receptors (TCRs) (Tucson). Here, we carry out epitope mapping studies of affinity-purified AAbs to Fab fragments prepared from individual HIV-positive patients for their capacity to bind recombinant constructs and peptide-defined epitopes modeling TCR and Ig light chains. Some affinity-purified autoantibodies reacted strongly with TCRs expressed by intact T-cells, and recombinant Valpha/Vbeta constructs as well as with certain synthetic peptide epitopes. The binding reactions of affinity-purified AAbs of individual patients were distinct, and the AAb preparations consisted of populations of polyclonal lgs as reflected in specificity and isotype. AAb pools from individual patients all bound particular regions of TCR and Ig chains defined by comprehensive peptide synthesis including the CDR1 and Fr3 segments of the variable domains and the joining segment/switch peptide. In addition, other reactivities to restricted regions of alpha, beta and lambda light chains were documented. These results substantiate the cross-reactivity of TCR and Ig-Fab determinants, and are consistent with the hypothesis that autoantibodies arising as a consequence of HIV infection can have an immunomodulatory role.

Amino Acid Sequence↗
PubMed1993

A three-dimensional plot for the display of transverse pore gradient gel electrophoresis data: application to a kinetoplast DNA fragment of planar circular conformation.

A 219 bp fragment of kinetoplast DNA from Crithidia fasciculata has previously been shown to exhibit a gel concentration dependent, anomalously low migration rate in 3-10% polyacrylamide transverse pore gradient gels relative to that of straight standards. The anomaly was interpreted in accordance with previous results of Marini et al. [1] as being due to the planar circular conformation of the fragment. The transverse pore gradient gel pattern is displayed here as a three-dimensional plot of migration distance vs. gel concentration and DNA length for the kinetoplast fragment and straight DNA standards. The plot yields a smooth surface for the standards clearly separated from the curve of the kinetoplast DNA fragment. Thus, transverse gradient gel electrophoresis in conjunction with the three-dimensional plot provides a unique method for the recognition of abnormal conformational features of DNA on the basis of electrophoretic mobility in a pore-size gradient.

Animals↗
PubMed1992

Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed.

Amino Acid Sequence↗
PubMed1993

Crystallization and preliminary X-ray diffraction data of two heparin-binding fragments of human fibronectin.

Two different heparin-binding fragments of human fibronectin have been crystallized in forms which are suitable for crystal structure analyses. The 30 kDa hep-2A fragment, consisting of type III domains 12-14, was crystallized from solutions containing ammonium sulfate or polyethylene glycol 6000. The crystals grown in ammonium sulfate solutions were orthorhombic with space group I222 or I2(1)2(1)2(1) with a = 68.1 A, b = 88.6 A, and c = 144.9 A. The crystals grown in polyethylene glycol solutions are hexagonal with space group P6(1)22 or P6(5)22 with a = b = 66.7 A and c = 245.7 A. The 40 kDa hep-2B fragment, consisting of type III domains 12-15, was also crystallized from solutions containing ammonium sulfate with the addition of glycerol. Glycerol proved an effective agent for reducing the number of crystals in the crystallization experiments, and thus, increasing the size of the crystals in these experiments. This crystal form is nearly isomorphous to the orthorhombic form of the hep-2A fragment with space group I222 or I2(1)2(1)2(1) and a = 67.5 A, b = 87.0 A, and c = 144.3 A. All crystal forms diffract to at least 3.5 A resolution and contain a single molecule in the asymmetric unit.

Crystallization↗
PubMed1997

A monoclonal antibody to avidin dissociates quaternary structure and curtails biotin binding to avidin and streptavidin.

An anti-avidin mAb, viz., H12G4, is shown to release bound biotin in a dose-dependent manner from holoavidin and holostreptavidin and inhibit the binding of ligand to the two apoproteins. The release of biotin by this mAb is accompanied by quenching of ligand-induced enhanced fluorescence of the FITC-avidin conjugate. In terms of mechanism of release of bound biotin, we demonstrate that on binding to the Fab fragment of the mAb, the native tetrameric holoavidin undergoes dissociation progressively with time to monomers with no bound biotin associated with the latter. Based on the immunoreactivity associated with defined overlapping fragments of avidin obtained by chemical cleavage, the epitope recognized by mAb H12G4 has been localized to residues 58-96 of the primary sequence. By pepscan method of epitope mapping, this mAb is shown to identify a minimal core sequence of 87RNGK90 in avidin and a corresponding sequence of 84RNAH87 in streptavidin.

Amino Acid Sequence↗
PubMed2001

Molecular phylogeny of the subfamilies in Geometridae (Geometroidea: Lepidoptera).

Molecular sequence data from three gene fragments were used to examine critically a provisional phylogenetic classification based on morphological characters of the Geometridae, one of the most species-rich families of moths. The sister group relationship between Geometridae and Drepanidae gained further support from the molecular analysis, which was based on the ND1 mitochondrial gene and the first and second expansion segments of the 28S ribosomal RNA gene. Although the alignment of the second expansion segment contained regions with many gaps, it provided the most resolution of the gene fragments. Parsimony analysis of the combined data resulted in a cladogram in which species belonging to Drepanidae, Larentiinae, and Sterrhinae formed monophyletic groups. The Ennominae did not form a monophyletic group but rather were contained within a broader monophyletic group including Archiearinae, Geometrinae, and Alsophilinae (represented by only one species per group in the present study). The molecular results were used to explore further the relationship between Sterrhinae and Larentiinae, the question as to whether Ennominae actually represent a monophyletic group, and the relationships between Ennominae and some of the other subfamilies.

Amino Acid Sequence↗
PubMed2006

Identification of target-specific bioisosteric fragments from ligand-protein crystallographic data.

Bioisosteres are functional groups or atoms that are structurally different but that can form similar intermolecular interactions. Potential bioisosteres were identified here from analysing the X-ray crystallographic structures for sets of different ligands complexed with a fixed protein. The protein was used to align the ligands with each other, and then pairs of ligands compared to identify substructural features with high volume overlap that occurred in approximately the same region of geometric space. The resulting pairs of substructural features can suggest potential bioisosteric replacements for use in lead-optimisation studies. Experiments with 12 sets of ligand-protein complexes from the Protein Data Bank demonstrate the effectiveness of the procedure.

Crystallography, X-Ray↗
PubMed1994

Simplified conjugation chemistry for coupling peptides to F(ab') fragments: autologous red cell agglutination assay for HIV-1 antibodies.

The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on agglutination of autologous red blood cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579-613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous red cell agglutination test is described. F(ab')2 fragments of the anti-glycophorin antibody were prepared by pepsin digestion and reduced to F(ab') fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous reduction of the F(ab') fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579-613) [Cys-Acm 598, Lys-BrAc 604] containing epsilon-bromoacetyl-lysine at residue 604 to the resultant F(ab') fragment. Conjugation to the F(ab') fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab'). The resultant F(ab')-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous red cell agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

Amino Acid Sequence↗
PubMed1986

Preliminary X-ray data for an N-terminal fragment of rabbit serum transferrin.

An iron-containing fragment (Mr approximately 39,000) of rabbit serum transferrin has been crystallized from a solution of 25% (w/v) polyethylene glycol 6000, 50 mM-disodium piperazine-N,N'bis(2-ethanesulphonate) adjusted to pH 6.0 at 4 degrees C. The space group is P3(1)21 (or the enantiomorph) with a = b = 66.8(1) A, c = 137.5(3) A and Z = 6. The crystals appear as hexagonal plates, with the unique axis perpendicular to the plate. The crystals, kept at 4 degrees C, are stable in the X-ray beam for at least 130 hours and diffract to better than 1.8 A resolution.

Animals↗
PubMed1990

A bifunctional fusion protein containing Fc-binding fragment B of staphylococcal protein A amino terminal to antidigoxin single-chain Fv.

A bifunctional molecule was genetically engineered which contained an amino-terminal effector domain that bound immunoglobulin Fc (fragment B of staphylococcal protein A) and a carboxyl-terminal domain that bound digoxin [a single-chain Fv (sFv)]. Effector and sFv binding properties were virtually identical with those of the parent molecules, despite the proximity of the FB to the sFv combining site. This finding is unprecedented since in all molecules of the natural immunoglobulin superfamily, the antigen binding domain is amino terminal to the effector domain. The FB-sFv sequence was encoded in a single synthetic gene and expressed as a 33,106 molecular weight protein in Escherichia coli. After purification, renaturation, and affinity isolation, yield of active fusion protein were 110 mg/L of fermented cells (18.5-g cell paste). Bifunctionality was confirmed by the ability of FB-sFv to cross-link IgG to digoxin-bovine serum albumin, as measured by plate assays and by Ouchterlony analysis. Analysis of the expressed fusion protein suggests that the sFv holds promise for the development of multifunctional, targetable single-chain proteins.

Amino Acid Sequence↗
PubMed2001

Thermal destabilization of rhodopsin and opsin by proteolytic cleavage in bovine rod outer segment disk membranes.

The G-protein coupled receptor, rhodopsin, consists of seven transmembrane helices which are buried in the lipid bilayer and are connected by loop domains extending out of the hydrophobic core. The thermal stability of rhodopsin and its bleached form, opsin, was investigated using differential scanning calorimetry (DSC). The thermal transitions were asymmetric, and the temperatures of the thermal transitions were scan rate dependent. This dependence exhibited characteristics of a two-state irreversible denaturation in which intermediate states rapidly proceed to the final irreversible state. These studies suggest that the denaturation of both rhodopsin and opsin is kinetically controlled. The denaturation of the intact protein was compared to three proteolytically cleaved forms of the protein. Trypsin removed nine residues of the carboxyl terminus, papain removed 28 residues of the carboxyl terminus and a portion of the third cytoplasmic loop, and chymotrypsin cleaved cytoplasmic loops 2 and 3. In each of these cases the fragments remained associated as a complex in the membrane. DSC studies were carried out on each of the fragmented proteins. In all of the samples the scan rate dependence of the Tm indicated that the transition was kinetically controlled. Trypsin-proteolyzed protein differed little from the intact protein. However, the activation energy for denaturation was decreased when cytoplasmic loop 3 was cleaved by papain or chymotrypsin. This was observed for both bleached and unbleached samples. In the presence of the chromophore, 11-cis-retinal, the noncovalent interactions among the proteolytic fragments produced by papain and chymotrypsin cleavage were sufficiently strong such that each of the complexes denatured as a unit. Upon bleaching, the papain fragments exhibited a single thermal transition. However, after bleaching, the chymotrypsin fragments exhibited two calorimetric transitions. These data suggest that the loops of rhodopsin exert a stabilizing effect on the protein.

Amino Acid Sequence↗
PubMed2002

Generation of a recombinant Fab antibody reactive with the Alzheimer's disease-related Abeta peptide.

A recombinant Fab antibody, designated 1E8-4b, which reacts with the Alzheimer's disease (AD)-related Abeta peptides, Abeta[1-40], Abeta[1-42] and Abeta[1-43] has been developed. The 1E8-4b Fab was constructed by cloning the V(H)C(H1) and V(L)C(L) domains from the parent hybridoma 1E8 antibody, reported previously to recognize these Abeta peptides. Briefly, a C-terminal Flag tag sequence was incorporated into this construct, which was ligated into the vector pHFA2 and expressed in Escherichia coli. Following purification on an M2 anti-Flag affinity column, the 1E8-4b recombinant Fab antibody was shown to bind plaques within sections of brain tissue from CERAD-defined AD patients by immunohistochemistry. ELISA, epitope mapping and immunoblotting confirmed the recognition of the Abeta1-40/42/43] peptides by the 1E8-4b Fab. The 1E8-4b Fab did not recognize APP695 or APP770 which contain the Abeta sequence. The Abeta specificity of the recombinant 1E8-4b Fab antibody was identical to the parent 1E8 monoclonal antibody.

Alzheimer Disease↗