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PubMed2004

Structure-based design and characterization of a Novel IL-6 antagonist peptide.

The development of rational methods to design antagonist peptides based on the 3-D structure of protein active region has, to now, been only marginally successful. This has been largely due to the difficulty of constraining the recognition elements of a mimetic structure to the relative conformational and spatial orientations present in the parent molecule. According to the 3-D complex structure of human interleukin-6 (hIL-6) and its receptor (hIL-6R), a novel antagonist peptide (named PT), which possessed potential bioactivity of hIL-6, was designed by the means of distance geometry, molecular modeling and molecular dynamics trajectory analysis. The bioactivity of the designed peptide (i.e. PT) was evaluated using XG-7 cells, a hIL-6-dependent B-cell line. PT possessed potential bioactivity to antagonize the function of hIL-6 and could efficiently induce the growth arrest and apoptosis of XG-7 cells in a dose-dependent manner.

Amino Acid Sequence↗
PubMed2001

Chromatographic analysis of plant sterols in foods and vegetable oils.

This paper reviews recently published chromatographic methods for the analysis of plant sterols in various sample matrices with emphasis on vegetable oils. An overview of structural complexities and biological/nutritional aspects including hypocholesterolemic activities of phytosterols is provided in the Section 1. The principal themes of the review highlight the development and application of chromatographic techniques for the isolation, purification, separation and detection of the title compounds. Pertinent gas chromatographic and high-performance liquid chromatographic methods from the literature are tabulated to illustrate common trends and methodological variability. The review also covers specific analyses of natural/synthetic standard mixtures to shed light on potential applicability in plant sample assays. Examples of combined chromatographic techniques linked in tandem for the analysis of complex samples are included. Elution characteristics of sterol components are discussed in the context of analyte substituent effects, structural factors and stationary/mobile phase considerations.

Food Analysis↗
PubMed2002

Crystal structure and NMR studies of the apo SH2 domains of ZAP-70: two bikes rather than a tandem.

The protein kinase ZAP-70 is involved in T-cell activation, and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs), which are present in three of the subunits of the T-cell receptor. We have studied the tandem SH2 (tSH2) domains of ZAP-70, by both X-ray and NMR. Here, we present the crystal structure of the apoprotein, i.e., the tSH2 domain in the absence of ITAM. Comparison with the previously reported complex structure reveals that binding to the ITAM peptide induces surprisingly large movements between the two SH2 domains and within the actual binding sites. The conformation of the ITAM-free protein is partly governed by a hydrophobic cluster between the linker region and the C-terminal SH2 domain. Our data suggest that the two SH2 domains are able to undergo large interdomain movements. The proposed relative flexibility of the SH2 domains is further supported by the finding that no NMR signals could be detected for the two helices connecting the SH2 domains; these are likely to be broadened beyond detection due to conformational exchange. It is likely that this conformational reorientation induced by ITAM binding is the main signaling event activating the kinase domain in ZAP-70. Another NMR observation was that the N-terminal SH2 domain could bind tetrapeptides derived from the ITAM sequence, apparently without the need to interact with the C-terminal domain. In contrast, the C-terminal domain has little affinity for tetrapeptides. The opposite situation is true for binding to plain phosphotyrosine, where the C-terminal domain has a higher affinity. Distinct features in the crystal structure, showing the interdependence of both domains, explain these binding data.

Amino Acid Motifs↗
PubMed1997

Zinc binding properties of the DNA binding domain of the 1,25-dihydroxyvitamin D3 receptor.

To assess the zinc binding stoichiometry and the structural changes induced upon the binding of zinc to the human vitamin D receptor (VDR), we expressed the DNA binding domain (DBD) of the human VDR in bacteria as a soluble glutathione-S-transferase fusion protein at 20 degrees C, and examined the apo-protein and metal-liganded protein by mass spectrometry, and circular dichroism and nuclear magnetic resonance spectroscopy. Following final preparation with a zinc-free buffer, the VDR DBD bound 2 mol of zinc/mol of protein as measured by inductively coupled plasma-mass spectrometry and electrospray ionization-mass spectrometry. When protein preparation was carried out in a zinc containing buffer and zinc content of the protein was assesed by the same methods, VDR DBD bound 4 mol of zinc/mol of protein. Analysis of the protein using circular dichroism spectroscopy demonstrated that the EDTA-treated protein increased in alpha-helical content from 16 to 27% on the addition of zinc. Equilibrium ultracentrifugal analyses of the VDR DBD indicated that the protein was present in solution as a monomer. Gel mobility shift analyses of the VDR DBD with several vitamin D response elements (VDREs) in the absence of accessory proteins such as retinoic acid receptor, showed that VDR DBD was able to form a protein/VDRE DNA structural complex. In the presence of zinc, proton NMR NOESY spectra showed that the protein possessed elements of secondary structure. The addition of VDRE DNA, but not random DNA, caused changes in the proton NMR spectra of VDRE DNA indicating specific interaction between protein and DNA groups. We conclude that the DBD of the VDR binds zinc and DNA and undergoes conformational changes on binding to the metal and DNA.

Amino Acid Sequence↗
PubMed1992

Preparation and structure-activity relationships of simplified analogues of the antifungal agent cilofungin: a total synthesis approach.

The echinocandins are a well-known class of lipopeptides characterized by their potent antifungal activity against Candida species. The mechanism of action of the echinocandins is generally thought to be the inhibition of beta-1,3-glucan synthesis, an important structural component in the cell wall of Candida species. Extensive structure-activity studies on the fatty acid side chain of echinocandin B (1) led to the preparation of the clinical candidate cilofungin (4). However, little is known about the cyclic peptide. We now report the preparation, by solid-phase synthesis, of a series of simplified analogs of cilofungin in which the unusual amino acids found in the echinocandins were replaced with more readily accessible natural amino acids. The solid-phase approach to the total synthesis of these analogs allowed us to conveniently explore structural modifications that could not be accomplished by chemical modification of the natural product. The simplest analog 5 showed no biological activity. Structural complexity was then returned to the system in a systematic fashion so as to reapproach the original cilofungin structure. Antifungal activity and the inhibition of beta-1,3-glucan synthesis were monitored at each step of the process, thereby revealing the basic structure-activity relationships of the amino acids and the minimal structural requirements for biological activity in the echinocandin ring system. The results suggests that the 3-hydroxy-4-methylproline residue enhances activity but the L-homotyrosine residue is crucial for both antifungal activity and the inhibition of beta-1,3-glucan synthesis.

Amino Acid Sequence↗
PubMed1977

Nuclear matrix. Isolation and characterization of a framework structure from rat liver nuclei.

A nuclear framework structure termed the nuclear matrix has been isolated and characterized. This matrix forms the major residual structure of isolated nuclei and consists largely of protein with smaller amounts of RNA, DNA, carbohydrate, and phospholipid. The nuclear matrix can be further resolved by combined treatment with DNase and RNase. The remaining nuclear protein structure, after extraction of 90 percent of the nuclear protein, 99.9 percent of the DNA, and 98 percent of the RNA and phospholipid, is termed the nuclear protein matrix. Electron microscopy of this final nuclear protein matrix reveals an interior framework structure composed of residual nucleolar structures associated with a granular and fibrous internal matrix structure. The internal matrix framework is derived from the interchromatinic structures of the nucleus, and is connected to a surrounding residual nuclear envelope layer containing residual nuclear pore complex structures. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the nuclear matrix proteins demonstrates three major polypeptide fractions, P-1, P-2, and P-3, with average molecular weights of approximately 69,000, 66,000 and 62,000, as well as several minor polypeptides which migrate at approximately 50,000 and at higher molecular weights (>100,000). Polypeptides with molecular weights identical to those of P-1, P-2 and P-3 are also components of isolated nuclear envelopes and nucleoli, whereas isolated chromatin contains no detectable matrix polypeptides. This suggests that the major matrix polypeptides are localized in specific structural regions of the nucleus, i.e., nuclear envelope, nucleoli, and interchromatinic structures. The presence of cytochrome oxidase activity in the isolated nuclear matrix indicates that at least some integral proteins of the nuclear membrane are associated with the matrix.

Amino Acids↗
PubMed2003

U1A RNA-binding domain at 1.8 A resolution.

The human U1A RNA-binding domain (RBD1) adopts one of the most common protein folds, the RNA-recognition motif, and is a paradigm for understanding RNA-protein interactions. A 2.8 A resolution structure of the unbound RBD1 has previously been determined [Nagai et al. (1990). Nature (London), 348, 515-520] and revealed a well defined alpha/beta core with disordered termini. Using a longer construct, a 1.8 A resolution structure of the unbound domain was determined that reveals an ordered C-terminal helix. The presence of this helix is consistent with a solution structure of the free domain [Avis et al. (1996). J. Mol. Biol. 257, 398-411]; however, in the solution structure the helix occludes the RNA-binding surface. In the present structure, the helix occupies a position similar to that seen in a 1.9 A resolution RNA-RBD1 complex structure [Oubridge et al. (1994). Nature (London), 372, 432-438]. The crystals in this study were grown from 2.2 M sodium malonate. It is possible that the high salt concentration helps to orient the C-terminal helix in the RNA-bound conformation by strengthening hydrophobic interactions between the buried face of the helix and the alpha/beta core of the protein. Alternatively, the malonate (several molecules of which are bound in the vicinity of the RNA-binding surface) may mimic RNA.

Amino Acid Motifs↗
PubMed1993

Application of the minimal principle to peptide structures.

A new direct-methods procedure has been devised which consists of phase refinement via the minimal function, R(phi), alternated with Fourier summation and real space filtering. All phases are initially assigned values by computing structure factors for a randomly positioned set of atoms. These phases are then refined by using a parameter shift method to minimize R(phi). The refined phases are Fourier transformed, and a specified number of the largest peaks in the electron-density function are found and used as a new trial structure. The probability of a trial structure converging to a solution appears to depend on structural complexity and a number of refinement parameters. This procedure shows potential for providing fully automatic routine solutions for structures in the 200-400 atom range.

Journal Article↗
PubMed2000

Estimation of fuzzy structure parameters for continuous junctions

The fuzzy structure theory introduced 15 years ago is designed to predict the frequency response functions of structures and structural acoustic systems with structural complexity, in the low- and medium-frequency ranges. This paper constitutes a first validation of the fuzzy structure theory for continuous junctions between the master structure and the fuzzy substructures. In addition, a method to estimate the fuzzy structure parameters introduced in the fuzzy structure theory is presented and it is validated for the case of continuous junctions. This validation obtained by numerical simulations opens the field of experimental identifications.

Journal Article↗
PubMed2003

Regulation of alpha-globin mRNA stability.

mRNA stability is a critical determinant of normal red blood cell development and function. The long half-life of globin mRNA is central to the continued synthesis of globin proteins throughout all stages of erythropoiesis, even as the cells undergo programmed transcriptional arrest during terminal differentiation. Studies of a naturally occurring alpha-thalassemic mutation that triggers marked destabilization of alpha-globin mRNA first led investigators to search for a stability determinant in the 3'-untranslated region (3'UTR). Analysis of this region identified three cytosine-rich (C-rich) segments that contributed to alpha-globin mRNA stability when studied in transfected erythroid cells. Subsequently, in vitro studies demonstrated assembly of a sequence-specific ribonucleic-protein (RNP) complex at this site. Mutations in the 3'UTR that blocked formation of this "alpha-complex" in vitro resulted in a parallel destabilization of alpha-globin mRNA in transfected cells. Members of the alpha-globin poly(C)-binding protein (alphaCP) subfamily of heteronuclear (hn) RNP K homology (KH) domain RNA-binding proteins have been identified as essential protein components of the alpha-complex. In vitro studies suggested that additional proteins may also contribute to alpha-complex structure and/or function. Surveys of additional highly stable mRNAs point to a general function for the alpha-complex in mRNA stabilization. In vitro and in vivo analyses indicated that the alpha-complex stabilizes alpha-globin mRNA by two mechanisms: control of 3'-terminal deadenylation and steric protection of an endoribonuclease-sensitive site. Confirmation of these pathways, determination of their relative importance, and generalization of these findings to additional systems await future studies.

3' Untranslated Regions↗
PubMed1981

[The cybernetic circuit of the regulation of antibody formation].

The current theory concerning the formation of antibodies is no more sustainable against the recent view on the formation of complex structures, that involves, together with a random variability, also an ordered variability derived from information interactions. This paper aims at showing that the formation of antibodies requires the existence of a complex sensor which can be excited by the presence of antigenic structures of any shape. The stimulation of this sensor originates both a mass type feedback and a form type feedback; the latter consisting in the activation of a conformational genetic variability that turns into a form variability of the immunoglobulinic active site. This site can thus take a form capable to react with the antigen. The reaction with the antigen acts back negatively upon the sensor and causes the permanent acquisition in repertory of the active form for the subsequent disappearance of the form variability in the immunoglobulinic active site.

Animals↗
PubMed1983

Structural homology between different archaebacterial DNA-dependent RNA polymerases analyzed by immunological comparison of their components.

The archaebacterial DNA-dependent RNA polymerases have a complex structure containing eight or more components. Immunochemical analysis shows an extensive homology between the components of the enzymes of nine different species. Two enzyme subtypes can be distinguished: that of the thermoacidophilic and/or sulfur-metabolizing archaebacteria with the composition BACDEFGHIJ and that of the methanogenic plus halophilic archaebacteria with the composition ABB'C(D).... Components B and B' of the latter subtype probably evolved by the division of the large component B of the BACD... type enzyme. The existence of the two subtypes corroborates the division of the archaebacteria into two phylogenetic main branches.

Journal Article↗
PubMed1997

Design and synthesis of benzoic acid derivatives as influenza neuraminidase inhibitors using structure-based drug design.

A series of 94 benzoic acid derivatives was synthesized and tested for its ability to inhibit influenza neuraminidase. The enzyme-inhibitor complex structure was determined by X-ray crystallographic analysis for compounds which inhibited the enzyme. The most potent compound tested in vitro, 5 (4-acetylamino)-3-guanidinobenzoic acid), had an IC50 = 2.5 x 10(-6) M against N9 neuraminidase. Compound 5 was oriented in the active site of the neuraminidase in a manner that was not predicted from the reported active site binding of GANA (4) with neuraminidase. In a mouse model of influenza, 5 did not protect the mice from weight loss due to the influenza virus when dosed intranasally.

Animals↗
PubMed2003

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA.

Translation of the hepatitis C genome is mediated by internal ribosome entry on the structurally complex 5' untranslated region of the large viral RNA. Initiation of protein synthesis by this mechanism is independent of the cap-binding factor eIF4E, but activity of the initiator Met-tRNA(f)-binding factor eIF2 is still required. HCV protein synthesis is thus potentially sensitive to the inhibition of eIF2 activity that can result from the phosphorylation of the latter by the interferon-inducible, double-stranded RNA-activated protein kinase PKR. Two virally encoded proteins, NS5A and E2, have been shown to reduce this inhibitory effect of PKR by impairing the activation of the kinase. Here we present evidence for a third viral strategy for PKR inhibition. A region of the viral RNA comprising part of the internal ribosome entry site (IRES) is able to bind to PKR in competition with double-stranded RNA and can prevent autophosphorylation and activation of the kinase in vitro. The HCV IRES itself has no PKR-activating ability. Consistent with these findings, cotransfection experiments employing a bicistronic reporter construct and wild-type PKR indicate that expression of the protein kinase is less inhibitory towards HCV IRES-driven protein synthesis than towards cap-dependent protein synthesis. These data suggest a dual function for the viral IRES, with both a structural role in promoting initiation complex formation and a regulatory role in preventing inhibition of initiation by PKR.

Animals↗
PubMed1998

A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs.

Initiation of translation of hepatitis C virus and classical swine fever virus mRNAs results from internal ribosomal entry. We reconstituted internal ribosomal entry in vitro from purified translation components and monitored assembly of 48S ribosomal preinitiation complexes by toe-printing. Ribosomal subunits (40S) formed stable binary complexes on both mRNAs. The complex structure of these RNAs determined the correct positioning of the initiation codon in the ribosomal "P" site in binary complexes. Ribosomal binding and positioning on these mRNAs did not require the initiation factors eIF3, eIF4A, eIF4B, and eIF4F and translation of these mRNAs was not inhibited by a trans-dominant eIF4A mutant. Addition of Met-tRNAiMet, eIF2, and GTP to these binary ribosomal complexes resulted in formation of 48S preinitiation complexes. The striking similarities between this eukaryotic initiation mechanism and the mechanism of translation initiation in prokaryotes are discussed.

Animals↗
PubMed1988

Lucifer yellow-CH identification of a primary neuron connecting the first segmental nerve to the three giant axons in the nerve cord of the earthworm Amynthas hawayanus.

This is the first step in the identification of a primary or first-order neuron connecting terminal organs, such as receptors or muscle, to interneurons, such as giant axons. The preparation employed here was a single segment of the ventral nerve cord between the 30th and the 40th segment, with a single first segmental nerve connected. A small drop of 7% Lucifer Yellow-CH was placed at the cut end of the first nerve in the absence of detergent or electric current. A structured complex could be seen in the cord anterior to the first nerve after one or two min backfilling. Several long processes extend from this structure to the contralateral side of the nerve cord and terminate in button-like structures near the medial giant axon and the contralateral lateral giant axon. The afferent or efferent function of this neural complex remains to be determined.

Animals↗
PubMed1988

The molecular structure of the complex of Hoechst 33258 and the DNA dodecamer d(CGCGAATTCGCG).

The crystal structure of the complex between the dodecamer d(CGCGAATTCGCG) and a synthetic dye molecule Hoechst 33258 was solved by X-ray diffraction analysis and refined to an R-factor of 15.7% at 2.25 A resolution. The crescent-shaped Hoechst compound is found to bind to the central four AATT base pairs in the narrow minor groove of the B-DNA double helix. The piperazine ring of the drug has its flat face almost parallel to the aromatic bisbenzimidazole ring and lies sideways in the minor groove. No evidence of disordered structure of the drug is seen in the complex. The binding of Hoechst to DNA is stabilized by a combination of hydrogen bonding, van der Waals interaction and electrostatic interactions. The binding preference for AT base pairs by the drug is the result of the close contact between the Hoechst molecule and the C2 hydrogen atoms of adenine. The nature of these contacts precludes the binding of the drug to G-C base pairs due to the presence of N2 amino groups of guanines. The present crystal structural information agrees well with the data obtained from chemical footprinting experiments.

Benzimidazoles↗
PubMed1998

Structure and dynamics of ribosomal RNA.

Over the past two years, progress in X-ray crystallography, NMR spectroscopy and electron microscopy has begun to reveal the complex structure of the RNA within the ribosome. The structures of ribosomal proteins L11 and S15, among others, show how RNA-protein interactions organize the conformation of the junctions between ribosomal RNA helices. Genetic and biochemical methods have also identified a three base-pair switch within the 16S rRNA that is linked to mRNA decoding.

Base Sequence↗