Biomedical subjects

P Wingfield

Publications and source records attributed to P Wingfield.

At least 19 recordsLinked to original sources

PubMed1999

Measurement of 3hJNC' connectivities across hydrogen bonds in a 30 kDa protein.

A method is described which permits detection of 3hJNC' scalar couplings across hydrogen bonds in larger, perdeuterated proteins. The experiment is demonstrated for the uniformly 2H/13C/15N-enriched 30 kDa ribosome inactivating protein MAP30. The 3hJNC' interactions are smaller than 1 Hz, but their detection in an HNCO experiment is made possible through the use of constructive interference between the 15N chemical shift anisotropy and 1H-15N dipole-dipole relaxation mechanisms in a manner similar to that of recently proposed TROSY schemes. Sensitivity of the HNCO experiment depends strongly on the 15N transverse relaxation rate of the downfield 15N multiplet component and on the amide proton T1. In perdeuterated MAP30 at 40 degrees C, the average TROSY T2 was 169 ms at 750 MHz 1H frequency, and a wide range of longitudinal relaxation rates was observed for the amide protons.

Carbon Isotopes↗

PubMed1999

Monomer-trimer equilibrium of the ectodomain of SIV gp41: insight into the mechanism of peptide inhibition of HIV infection.

The monomer-trimer equilibrium of the ectodomain of SIV gp41 (residues 27-149, e-gp41) has been characterized by analytical ultracentrifugation, circular dichroism (CD), and NMR spectroscopy. Based on analytical ultracentrifugation experiments performed at different rotor speeds and protein concentrations, the equilibrium association constant for the SIV e-gp41 trimer is 3.1 x 10(11) M(-2). The presence of intermolecular nuclear Overhauser effects in a mixture of 12C and 13C-labeled e-gp41 prepared under nondenaturing conditions unambiguously demonstrates that there is a dynamic equilibrium between the monomer and trimer. The CD spectra taken as a function of SIV e-gp41 concentration suggest that the helical content of the monomeric state does not change significantly relative to that of the trimeric state. The relevance of the monomer-trimer equilibrium is discussed with respect to gp41 function and the inhibitory properties of gp41 peptides.

Anti-HIV Agents↗

PubMed1997

Conformational selectivity of HIV-1 protease cleavage of X-Pro peptide bonds and its implications.

Kinetic measurements on a fluorescent peptide analog of the p17/p24 cleavage site of the Gag polyprotein demonstrate the conformational selectivity of human immunodeficiency virus, type 1 protease for the trans conformation of the Tyr-Pro bond. A mean cis/trans ratio of 0. 3, and a cis --> trans isomerization rate constant of 0.022 s-1 are determined at T = 22 degrees C. This rate is in excellent agreement with that predicted by 19F NMR studies of structurally analogous peptides containing a fluorine/hydroxyl substitution on the tyrosyl residue. Addition of recombinant human cyclophilin resulted in a significant enhancement of this rate, and it is proposed that this enzyme, which has been shown to be associated with the Gag protein, functions as an auxiliary enzyme for the protease during cleavage in the virion.

Amino Acid Isomerases↗

PubMed1996

Three-dimensional solution structure of the HIV-1 protease complexed with DMP323, a novel cyclic urea-type inhibitor, determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of the HIV-1 protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea-based inhibitor, DMP323, is reported. This is the first solution structure of an HIV protease/inhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 A or 5 degrees, respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 A relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8-A X-ray structure of the protease/DMP323 complex (Chang CH et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 A. As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37-42 and 50-51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4-8 may play a role in protease regulation by facilitating autolysis.

Azepines↗

PubMed1996

Anisotropic rotational diffusion of perdeuterated HIV protease from 15N NMR relaxation measurements at two magnetic fields.

15N NMR relaxation times in perdeuterated HIV-1 protease, complexed with the sub-nanomolar inhibitor DMP323, have been measured at 600 and 360 MHz 1H frequency. The relative magnitudes of the principal components of the inertia tensor, calculated from the X-ray coordinates of the protein-drug complex, are 1.0:0.85:0.44. The relation between the T1/T2 ratios observed for the individual backbone amides and their N-H orientation within the 3D structure of the protease dimer yields a rotational diffusion tensor oriented nearly collinear to the inertia tensor. The relative magnitudes of its principal components (1.00:1.11:1.42) are also in good agreement with hydrodynamic modeling results. The orientation and magnitude of the diffusion tensors derived from relaxation data obtained at 360 and 600 MHz are nearly identical. The anisotropic nature of the rotational diffusion has little influence on the order parameters derived from the 15N T1 and T2 relaxation times; however, if anisotropy is ignored, this can result in erroneous identification of either exchange broadening or internal motions on a nanosecond time scale. The average ratio of the T1 values measured at 360 and 600 MHz is 0.50 +/- 0.015, which is slightly larger than the value of 0.466 expected for an isotropic rigid rotor with tau c = 10.7 ns. The average ratio of the T2 values measured at 360 and 600 MHz is 1.14 +/- 0.04, which is also slightly larger than the expected ratio of 1.11. This magnetic field dependence of the T1 and T2 relaxation times suggests that the spectral density contribution from fast internal motions is not negligible, and that the chemical shift anisotropy of peptide backbone amides, on average, is larger than the 160 ppm value commonly used in 15N relaxation studies of proteins.

Anisotropy↗

PubMed1994

Biophysical and enzymatic properties of the catalytic domain of HIV-1 integrase.

A deletion derivative of the integrase protein from human immunodeficiency virus type-1 (HIV-1) consisting of the central core domain (amino acids 50-212) has been characterized biophysically and biochemically. This deletion mutant is of particular interest for structural studies as it can carry out the disintegration reaction suggesting the presence of an active site and, under certain conditions, is more soluble than full-length integrase. The circular dichroism and fluorescence of the deletion mutant and the 288-residue full-length integrase were similar, indicating that the core residues maintain similar overall conformations in both proteins. The deletion mutant is approximately 10% more alpha-helical than the full-length protein. Analytical centrifugation demonstrated that both proteins undergo monomer-dimer association although the truncated protein showed slightly less tendency to dimerize; the dissociation constants were 2.5 x 10(-5) M for the full-length protein and 8.0 x 10(-5) M for the truncated protein. The disintegration activity of both proteins was also compared. Although a higher concentration of the truncation mutant was required for optimal activity, the mutant did not have altered pH or Mn2+ requirements relative to the full-length protein. The combined biophysical and enzymatic studies suggest that this truncated form of HIV-1 integrase is likely to be useful for structural studies.

Amino Acid Sequence↗

PubMed1993

Domains of the integrase protein of human immunodeficiency virus type 1 responsible for polynucleotidyl transfer and zinc binding.

The integrase protein of human immunodeficiency virus type 1 carries out a set of polynucleotidyl transfer reactions that result in the covalent attachment of the retroviral cDNA to host DNA. We have analyzed the activities of a set of deletion derivatives of the integrase protein. The analysis reveals that a central domain of only 137 amino acids is sufficient in vitro to catalyze a subset of the reactions carried out by the complete protein. This polypeptide contains an amino acid sequence motif, Asp-Xaa39-58-Asp-Xaa35-Glu (DX39-58DX35E, where X and the subscript indicate the intervening amino acids between the invariant acidic residues), that is found in the integrases of retroviruses and retrotransposons and also the transposase proteins of some bacterial transposable elements. We also find that the integrase protein can bind Zn2+, and the histidine and cysteine residues of another conserved motif (HX3-7HX23-32CX2C) are required for efficient Zn2+ binding. The activities displayed by deletion mutants suggest to us possible functions for the various parts of integrase.

Amino Acid Sequence↗

PubMed1993

Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.

During acute human immunodeficiency virus type 1 (HIV-1) infection or after transfection of the tat gene, Tat protein is released into the cell culture supernatant. In this extracellular form, Tat stimulates both HIV-1 gene expression and the growth of cells derived from Kaposi's sarcoma (KS) lesions of HIV-1-infected individuals (AIDS-KS cells). Tat protein and its biological activities appear in the cell supernatants at the peak of Tat expression, when the rate of cell death is low (infection) or cell death is undetectable (transfection) and increased levels of cytoplasmic Tat are present. Tat-containing supernatants stimulate maximal AIDS-KS cell growth but only low to moderate levels of HIV-1 gene expression. This is due to the different concentrations of exogenous Tat required for the two effects. The cell growth-promoting effects of Tat peak at between 0.1 and 1 ng of purified recombinant protein per ml in the cell growth medium and do not increase with concentration. In contrast, both the detection of nuclear-localized Tat taken up by cells and the induction of HIV-1 gene expression or replication require higher Tat concentrations (> or = 100 ng/ml), and all increase linearly with increasing amounts of the exogenous protein. These data suggest that Tat can be released by a mechanism(s) other than cell death and that the cell growth-promoting activity and the virus-transactivating effect of extracellular Tat are mediated by different pathways.

Animals↗

PubMed1991

Biochemical and biological comparison of HIV-1 NEF and ras gene products.

Human immunodeficiency virus type 1 (HIV-1) NEF protein has been reported to share certain biochemical and structural properties with known oncoproteins like src or rats. To determine whether this is a general property of NEF from various HIV isolates, three different NEF proteins were expressed in Escherichia coli using a thermoinducible expression system previously exploited to overproduce functionally active p21 ras proteins. ras and NEF proteins expressed in this manner were evaluated in parallel to compare their biochemical and biological properties. In contrast to ras, our NEF protein preparations had no detectable GTP binding but showed autophosphorylation activity when incubated in the presence of either GTP or ATP. This putative autokinase activity was higher in NEF proteins containing threonine at position 15 than in those carrying alanine at that position. Two different NEF genes also failed to induce oncogenic transformation of permanently transfected NIH 3T3 cells under conditions that led to oncogenic transformation using activated ras genes. Also, unlike ras, the NEF gene products failed to induce meiotic maturation when injected into fully grown Xenopus oocytes.

Adenosine Triphosphate↗

PubMed1991

Specific complex of human immunodeficiency virus type 1 rev and nucleolar B23 proteins: dissociation by the Rev response element.

The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.

Binding Sites↗

PubMed1990

Evidence that high mannose glycopeptides are able to functionally interact with recombinant tumor necrosis factor and recombinant interleukin 1.

Both recombinant tumor necrosis factor (rTNF) and recombinant interleukin 1 (rIL-1) are able to mediate vascular collapse and death in a previously described murine model, using galactosamine to enhance the toxicity of these cytokines. Unexpectedly, both acid-treated tumor necrosis factor (TNF) and a site-specifically mutagenized form of interleukin 1 (IL-1) (His-30----Arg-30), which fails to bind to the IL-1 receptor, retain full in vivo toxicity in this model of TNF- and IL-1-mediated shock. Previous studies have shown that rTNF and rIL-1 exhibit two functionally distinct binding regions. Both cytokines bind to their respective cell surface receptors and they also express lectin like binding specificity (Muchmore and Decker, J. Biol. Chem., 261: 13404-13407, 1986; Muchmore and Decker, J. Immunol., 138: 2541-2546, 1987) for defined oligosaccharides. The specificity of these two types of interactions is quite different. Cell surface receptors for IL-1 and TNF demonstrate essentially no cross-reactivity, whereas, in the case of carbohydrate binding, competition studies reveal an almost identical carbohydrate specificity for the structure Man5(6)GlcNAc2-Asn. Man5(6)GlcNAc2-Asn binding is either unaffected or actually enhanced by either acid treatment of rTNF or mutation at His-30 for rIL-1. Both deoxymannojirimycin and swainsonine, inhibitors of glycoprotein processing, raise intracellular levels of Man5-9GlcNAc2 and enhance the in vitro biological activity of both rTNF and rIL-1. Conversely, castanosperimine, a glucosidase I inhibitor which blocks the synthesis of mature high mannose structures, inhibits the biological activity of IL-1. These observations support the hypothesis that some effects of IL-1 and TNF may involve interaction with high mannose-substituted glycoproteins.

Animals↗

PubMed1990

Structure and partial amino acid sequence of calf thymus DNA topoisomerase II: comparison with other type II enzymes.

The partial amino acid sequence of p140 calf thymus DNA topoisomerase II was determined by analysis of cyanogen bromide peptides. Five peptides were aligned and shared extensive homology with sequences derived from cDNA clones for the human topoisomerase II isoenzyme forms. Less homology was seen with the Drosophila, yeast and bacterial type II enzymes. Calf and human enzymes shared epitopes allowing isolation of a cDNA clone to human topoisomerase II isoenzyme alpha. Our results indicate that calf thymus p140 topoisomerase II is an active N-terminal proteolytic fragment of the native p180 enzyme and demonstrate that mammalian type II enzymes exhibit close sequence similarity.

Amino Acid Sequence↗

PubMed1990

Tumor necrosis factor inhibitor: purification, NH2-terminal amino acid sequence and evidence for anti-inflammatory and immunomodulatory activities.

The urine of some febrile patients has been shown to contain a tumor necrosis factor-alpha-inhibiting activity (TNF-alpha INH) when tested in a cytotoxicity assay using the TNF-susceptible cell line L-929. The inhibitor was purified to homogeneity using a simple three-step procedure which included a TNF-alpha affinity column, cation exchange and reverse-phase chromatography. The NH2-terminal amino acid sequence of the inhibitor showed no sequence similarity with proteins in the data bases used. Using gel filtration, it was shown that TNF-alpha and the inhibitor form a stable complex which eluted with a molecular weight of about 75,000. This value corresponds to the sum of the inhibitor (approximately 30,000) and TNF-alpha (approximately 45,000-50,000) molecular weight. The TNF-alpha INH blocked prostaglandin E2 production by dermal fibroblasts in a dose-dependent manner, providing evidence for antiinflammatory activity. TNF-alpha INH also blocked class I antigen expression in a dose-dependent manner as measured using the human Colo 205 tumor cell line. Furthermore, TNF-alpha INH affected TNF-alpha synergism with IFN-gamma-induced HLA-DR antigen expression but had no effect on IFN-gamma activity. The data presented demonstrate that TNF-alpha bioactivity can be regulated at the protein level.

Adjuvants, Immunologic↗

PubMed1990

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene.

The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely--35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.

Alleles↗

PubMed1990

Human immunodeficiency virus rev protein recognizes a target sequence in rev-responsive element RNA within the context of RNA secondary structure.

Human immunodeficiency virus type 1 Rev protein modulates the distribution of viral mRNAs from the nucleus to the cytoplasm by interaction with a highly structured viral RNA sequence, the Rev-responsive element (RRE). To identify the minimal functional elements of RRE, we evaluated mutant RREs for Rev binding in vitro and Rev response in vivo in the context of a Gag expression plasmid. The critical functional elements fold into a structure composed of a stem-loop A, formed by the ends of the RRE, joined to a branched stem-loop B/B1/B2, between bases 49 and 113. The 5' 132 nucleotides of RRE, RREDDE, which possessed a similar structure, bound Rev efficiently but were nonfunctional in vivo, implying separate binding and functional domains within the RRE. Excision of stem-loop A reduced Rev binding significantly and abolished the in vivo Rev response. The B2 branch could be removed without severe impairment of binding, but deletions in the B1 branch significantly reduced binding and function. However, deletion of 12 nucleotides, including the 5' strand of stem B, abolished both binding and function, while excision of the 3' strand of stem B only reduced them. Maintenance of the native RRE secondary structure alone was not sufficient for Rev recognition. Many mutations that altered the primary structure of the critical region while preserving the original RNA conformation were Rev responsive. However, mutations that changed a 5'..CACUAUGGG..3' sequence in the B stem, without affecting the overall structure abolished both in vitro Rev binding and the in vivo Rev response.

Base Sequence↗

PubMed1989

A proton nuclear magnetic resonance assignment and secondary structure determination of recombinant human thioredoxin.

Two-dimensional 1H NMR spectroscopy has been applied to a structural analysis of the reduced form of a recombinant human thioredoxin, a ubiquitous dithiol oxidoreductase recently isolated from an immunocompetent lymphoblastoid cell line. The sequential assignment of the spectrum, including all proline residues, has been accomplished by using experiments to demonstrate through-bond and through-space connectivities. The secondary structure has been determined by a qualitative interpretation of nuclear Overhauser effects, NH exchange data, and 3JHN alpha coupling constants. The secondary structure was found to be similar to that of the X-ray structure of Escherichia coli thioredoxin, consisting of a mixed five-stranded beta-sheet surrounded by four alpha-helices. The assignment and structural characterization of human thioredoxin was facilitated by the increased resolution and sensitivity afforded by a magnetic field strength of 600 MHz and required the use of two temperatures and two pH conditions to resolve ambiguities caused by a duplication of resonances. This duplication, extending from Phe-41 to Val-59, and including Lys-3-Ile-5, Val-24, Val-25, Asn-39, and Ile-101-Glu-103, appears to be due to heterogeneity arising from the presence or absence of the N-terminal methionine.

Amino Acid Sequence↗

PubMed1989

Rabbit IL-1. Cloning, expression, biologic properties, and transcription during endotoxemia.

The cloning, sequencing, expression, and biologic activities of rabbit IL-1 alpha and beta are described. A cDNA library was constructed in lambda gt10 by using polyadenylated RNA extracted from rabbit adherent splenic macrophages 4 h after stimulation with endotoxin. By using the cDNA for human IL-1 beta and IL-1 alpha as hybridization probes, cDNA for both forms of rabbit IL-1 were isolated. The cDNA for rabbit IL-1 beta encodes a precursor polypeptide of 268 amino acids with an overall homology to human IL-1 beta of 74% (81% in the mature region coding for a 17.5 kDa carboxyl-terminal protein). The similarity between the two rabbit IL-1 forms is 31% for the entire molecule and 34% for the mature protein. The mature polypeptides of both forms were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity and tested in a variety of biologic assays. Both forms produced typical endogenous pyrogen fevers in rabbits and augmented murine thymocyte and Th cell proliferation. Rabbit IL-1 alpha and beta were more pyrogenic in rabbits than human rIL-1 beta, whereas human rIL-1 alpha and beta were slightly more potent lymphocyte-activating factors. The recombinant rabbit proteins induced PGE and IL-1 production from human PBMC in vitro. A RIA for human IL-1 alpha did not recognize rabbit IL-1 alpha or beta, but rabbit IL-1 beta cross-reacted (as much as 30%) in a RIA for human IL-1 beta. Rabbits were injected with endotoxin and mRNA for both forms of IL-1 were observed primarily in the spleen and liver. The mRNA reached maximal levels after 60 min, then declined rapidly over the next 3 h, but were still present after 24 h. Liver tissue removed 4 h after endotoxin infusion produced lymphocyte-activating factors which were neutralized by more than 90% with a combination of goat anti-rabbit IL-1 alpha and anti-IL-1 beta.

Amino Acid Sequence↗

PubMed1989

Purification and characterization of a methionine-specific aminopeptidase from Salmonella typhimurium.

An aminopeptidase specific for methionine (peptidase M) has been purified from wild-type and mutant Salmonella typhimurium strains. Recombinant peptidase M was also purified from Escherichia coli. These preparations were characterized with respect to their physicochemical properties using analytical ultracentrifugation, SDS/PAGE, isoelectric focusing, titration curve analysis, amino acid analysis, N-and C-terminal sequencing and various spectroscopic methods. Peptidase M activity is stimulated by Co2+, in agreement with previous studies using crude extracts of Salmonella. The purified preparations did not contain significant amounts of any metal. Enzymically important metal is loosely associated and lost during enzyme purification. Peptidase M was shown to contain seven free sulphydryl residues none of which are involved in either intra-or inter-molecular disulphide bonds. Most appear solvent-accessible as evidenced by their reactivity under native conditions. Limited modification of the sulphydryl residues with either iodoacetamide or 5,5'-dithiobis(2-nitrobenzoic acid) led to inactivation. Several cysteines were shown to be labelled to various degrees by peptide mapping of inactivated S-[14C]carboxymethylated protein. Whether cysteine modification affects enzymic activity directly (blocking an active site) or indirectly (by causing conformational change) remains to be established.

Amino Acid Sequence↗