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Secondary Structure (secondary + structure)
Kinds of Secondary Structure Terms modified by Secondary Structure Selected AbstractsDNA Labeling by Ligand Inducible Secondary StructureCHEMBIOCHEM, Issue 12 2008Tao Peng Dr. Personalized medical solutions: Simple, accurate, and cost effective methods of single nucleotide polymorphism typing would be necessary for personalized medicine. Towards this end, a number of SNP typing methods have been investigated and reported. We report herein, our chemical approach to practical SNP typing based on allele-specific PCR integrated with a new concept of DNA-labeling by ligand-inducible secondary structure. [source] Effect of PbII on the Secondary Structure and Biological Activity of TrypsinCHEMBIOCHEM, Issue 7 2005Lin Yang Prof. Abstract The effects of PbIIon the secondary structure and biological activity of trypsin have been examined by monitoring changes in its conductivity and IR and circular dichroism (CD) spectra. The results show that PbIIreacts with trypsin, and that the binding sites might be OH and NH groups in pepsin. The CD spectra indicate that interaction with PbIIsignificantly affects the secondary structure of trypsin, the ,-sheet-structure content being increased by about 42,%, whilst those of ,-helix and ,-turn structures are decreased by 13,% and 21,%, respectively. The results clearly demonstrate that PbIIaffects the biological activity of trypsin by modifying its secondary structure. Most interesting is that PbIIup-regulates the activity of trypsin at low concentrations while down-regulating it at high concentrations. [source] ChemInform Abstract: Stereospecific Synthesis of Conformationally Constrained ,-Amino Acids: New Foldamer Building Blocks That Support Helical Secondary Structure.CHEMINFORM, Issue 10 2010Li Guo Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] A Water-Soluble, Octacationic Zinc Phthalocyanine as Molecular Probe for Nucleic Acid Secondary StructureCHEMISTRY & BIODIVERSITY, Issue 2 2007An-Ming Zhang Abstract The interaction between CT-DNA and the zinc phthalocyanine ZnPc (1) was studied by UV/VIS and fluorescence titration, as well as by thermal denaturation. ZnPc was found to strongly bind to CT-DNA (Kapp=7.35×105, M,1) in a non-intercalative mode. The photosensitized cleavage of pBR322 DNA was found to efficiently proceed via singlet-oxygen (1O2) production. Further, ZnPc (1) caused site-specific scission of guanine (G) bases around the bulge of the hairpin oligonucleotides OD1,OD3, as clearly shown by gel-electrophoresis experiments. [source] Novel Secondary Structure of Calcitonin in Solid State as Revealed by Circular Dichroism SpectroscopyCHINESE JOURNAL OF CHEMISTRY, Issue 7 2002Hai-Ning Du Abstract The solid-state circular dichroic study reveals that salmon calcitonin presents a typical ,-helical structure while human calcitonin appears to form a ,-sheet in solid state, although both of them adopt random coil structures in aqueous solution. [source] CuI -Catalyzed Azide,Alkyne Intramolecular i -to-(i+4) Side-Chain-to-Side-Chain Cyclization Promotes the Formation of Helix-Like Secondary StructuresEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 3 2010Mario Scrima Abstract A solid-phase assembly of model peptides derived from human parathyroid hormone-related protein (11,19) containing ,-azido- and ,-yl-,-amino acid residues in positions i and i+4 was cyclised in solution by an intramolecular CuI -catalyzed azide,alkyne 1,3-dipolar Huisgen cycloaddition. These series of heterodetic cyclo-nonapeptides varied in the size of the disubstituted 1,2,3-triazolyl-containing bridge, the location and the orientation of the 1,2,3-triazolyl moiety within the bridge. The 1,2,3-triazolyl moiety, presented at either 1,4- or 4,1-orientation, is flanked by side chains containing 1,4 CH2 groups that result in bridges comprised from 4,7 CH2 groups connecting residues 13 and 17. Comprehensive conformational analysis employing CD, NMR and molecular dynamics reveals the conformational propensities of these heterodetic cyclo-nonapeptides. Cyclo-nonapeptides containing either the 7 methylene bridge (VII and VIII) or the 4 methylene bridge (II) are unstructured in structure-promoting solvent. Cyclo-nonapeptide I in which the 1,4-disubstituted 1,2,3-triazolyl is flanked by 3 and 1 CH2 groups in proximity to the respective residues 13 and 17, is stabilized in a non-canonical structure. All the other heterodetic cyclo-nonapeptides (III,VI) in which the 1,2,3-triazolyl is flanked by a total of 5 or 6 CH2 groups nicely accommodate ,-helical structures and reproduce very closely the helical structure stabilized by the analogous cyclo-nonapeptide in which Lys13 and Asp17 are bridged by the isosteric lactam. These studies suggest that the bioorthogonal i -to-(i+4) side-chain-to-side-chain cyclization via the prototypic "click reaction" offers a new and powerful approach for generating stable helix mimetic structures. [source] Synthesis, and Helix or Hairpin-Turn Secondary Structures of ,Mixed' ,/, -Peptides Consisting of Residues with Proteinogenic Side Chains and of 2-Amino-2-methylpropanoic Acid (Aib)HELVETICA CHIMICA ACTA, Issue 9 2006Dieter Seebach Abstract Twelve peptides, 1,12, have been synthesized, which consist of alternating sequences of , - and , -amino acid residues carrying either proteinogenic side chains or geminal dimethyl groups (Aib). Two peptides, 13 and 14, containing 2-methyl-3-aminobutanoic acid residues or a ,random mix' of ,-, ,2 -, and ,3 -amino acid moieties were also prepared. The new compounds were fully characterized by CD (Figs.,1 and 2), and 1H- and 13C-NMR spectroscopy, and high-resolution mass spectrometry (HR-MS). In two cases, 3 and 14, we discovered novel types of turn structures with nine- and ten-membered H-bonded rings forming the actual turns. In two other cases, 8 and 11, we found 14/15 -helices, which had been previously disclosed in mixed ,/, -peptides containing unusual , -amino acids with non-proteinogenic side chains. The helices are formed by peptides containing the amino acid moiety Aib in every other position, and their backbones are primarily not held together by H-bonds, but by the intrinsic conformations of the containing amino acid building blocks. The structures offer new possibilities of mimicking peptide,protein and protein,protein interactions (PPI). [source] DNA and RNA in Anhydrous Media: Duplex, Triplex, and G-Quadruplex Secondary Structures in a Deep Eutectic Solvent,ANGEWANDTE CHEMIE, Issue 36 2010Irena Mamajanov Verlockende Aussichten! Tiefeutektische Solventien (DESs) sind nichtflüchtige Medien, die sich für viele chemische Reaktionen eignen. In einem wasserfreien DES bilden Nucleinsäuren Duplex-, Triplex- und G-Quadruplex-Strukturen (die sich teilweise von denen in wässrigen Medien unterscheiden), sodass katalytische Nucleinsäuren und Enzym-Nucleinsäure-Komplexe vielleicht auch in diesen Solventien genutzt werden können. [source] Secondary structure of lipidated Ras bound to a lipid bilayerFEBS JOURNAL, Issue 23 2008Jörn Güldenhaupt Ras proteins are small guanine nucleotide binding proteins that regulate many cellular processes, including growth control. They undergo distinct post-translational lipid modifications that are required for appropriate targeting to membranes. This, in turn, is critical for Ras biological function. However, most in vitro studies have been conducted on nonlipidated truncated forms of Ras proteins. Here, for the first time, attenuated total reflectance-FTIR studies of lipid-modified membrane-bound N-Ras are performed, and compared with nonlipidated truncated Ras in solution. For these studies, lipidated N-Ras was prepared by linking a farnesylated and hexadecylated N-Ras lipopeptide to a truncated N-Ras protein (residues 1,181). It was then bound to a 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine bilayer tethered on an attenuated total reflectance crystal. The structurally sensitive amide I absorbance band in the IR was detected and analysed to determine the secondary structure of the protein. The NMR three-dimensional structure of truncated Ras was used to calibrate the contributions of the different secondary structural elements to the amide I absorbance band of truncated Ras. Using this novel approach, the correct decomposition was selected from several possible solutions. The same parameter set was then used for the membrane-bound lipidated Ras, and provided a reliable decomposition for the membrane-bound form in comparison with truncated Ras. This comparison indicates that the secondary structure of membrane-bound Ras is similar to that determined for the nonlipidated truncated Ras protein for the highly conserved G-domain. This result validates the multitude of investigations of truncated Ras without anchor in vitro. The novel attenuated total reflectance approach opens the way for detailed studies of the interaction network of the membrane-bound Ras protein. [source] Secondary structure and dynamics of micelle bound ,- and ,-synucleinPROTEIN SCIENCE, Issue 5 2006Yoon-hui Sung Abstract We have used solution state NMR spectroscopy to characterize the secondary structure and backbone dynamics of the proteins ,- and ,-synuclein in their detergent micelle-bound conformations. Comparison of the results with those previously obtained for the Parkinson's disease-linked protein ,-synuclein shows that structural differences between the three homologous synuclein family members are directly related to variations in their primary amino acid sequences. An 11-residue deletion in the lipid-binding domain of ,-synuclein leads to the destabilization of an entire segment of the micelle-bound helical structure containing the deletion site. The acidic C-terminal tail region of ,-synuclein, which displays extensive sequence divergence, is more highly disordered than the corresponding regions in the other two family members. The observed structural differences are likely to mediate functional variations between the three proteins, with differences between ,- and ,-synuclein expected to revolve around their lipid interactions, while differences in ,-synuclein function are expected to result from different protein,protein interactions mediated by its unique C-terminal tail. [source] Systematic replacement of lysine with glutamine and alanine in Escherichia coli malate synthase G: effect on crystallizationACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2005David M. Anstrom Two proposals recommend substitution of surface lysine residues as a means to improve the quality of protein crystals. In proposal I, substitution of lysine by alanine has been suggested to improve crystallization by reducing the entropic cost of ordering flexible side chains at crystal contacts. In proposal II, substitution of lysine by residues more commonly found in crystal contacts, such as glutamine, has been proposed to improve crystallization. 15 lysine residues on the surface of Escherichia coli malate synthase G, distributed over a variety of secondary structures, were individually mutated to both alanine and glutamine. For 28 variants, detailed studies of the effect on enzymatic activity and crystallization were conducted. This has permitted direct comparison of the relative effects of the two types of mutations. While none of the variants produced crystals suitable for X-ray structural determination, small crystals were obtained in a wide variety of conditions, in support of the general approach. Glutamine substitutions were found to be more effective than alanine in producing crystals, in support of proposal II. Secondary structure at the site of mutation does not appear to play a major role in determining the rate of success. [source] DFold: PCR design that minimizes secondary structure and optimizes downstream genotyping applications,HUMAN MUTATION, Issue 1 2004David Fredman Abstract Secondary structures in polymerase chain reaction (PCR) target sequences have a negative impact on amplification success rates and on downstream uses of PCR products. For example, signal strength and allele discrimination in single nucleotide polymorphism (SNP) genotyping methods can be compromised by allele-biased amplification and/or by PCR product folding that limits access of interrogating probes. To increase the fidelity and robustness of PCR, and to aid follow-on applications, we have developed DFold (http://dfold.cgb.ki.se),a generalized software solution that creates PCR oligonucleotide primer designs devoid of stable secondary structures. We demonstrate the effectiveness of the tool by applying it to a range of dynamic allele-specific hybridization (DASH) assay designs, many of which we evaluate in the laboratory. We further consider how the system throughput may be made sufficiently high for use upon millions of target sequences in order to support whole-genome analyses. Hum Mutat 24:1,8, 2004. © 2004 Wiley-Liss, Inc. [source] Comparative analysis of mt LSU rRNA secondary structures of Odonates: structural variability and phylogenetic signalINSECT MOLECULAR BIOLOGY, Issue 6 2003B. Misof Abstract Secondary structures of the most conserved part of the mt 16S rRNA gene, domains IV and V, have been recently analysed in a comparative study. However, full secondary structures of the mt LSU rRNA molecule are published for only a few insect species. The present study presents full secondary structures of domains I, II, IV and V of Odonates and one representative of mayflies, Ephemera sp. The reconstructions are based on a comparative approach and minimal consensus structures derived from sequence alignments. The inferred structures exhibit remarkable similarities to the published Drosophila melanogaster model, which increases confidence in these structures. Structural variance within Odonates is homoplastic, and neighbour-joining trees based on tree edit distances do not correspond to any of the phylogenetically expected patterns. However, despite homoplastic quantitative structural variation, many similarities between Odonates and Ephemera sp. suggest promising character sets for higher order insect systematics that merit further investigations. [source] The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5,-untranslated part of mRNAMICROBIAL BIOTECHNOLOGY, Issue 3 2009Laila Berg Summary Secondary structures and the short Shine,Dalgarno sequence in the 5,-untranslated region of bacterial mRNAs (UTR) are known to affect gene expression at the level of translation. Here we report the use of random combinatorial DNA sequence libraries to study UTR function, applying the strong, ,32/,38 -dependent, and positively regulated Pm promoter as a model. All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site. The libraries were screened using the ampicillin-resistance gene (bla) as reporter, allowing easy identification of UTR mutants that display high levels of expression (up to 20-fold increase relative to the wild-type at the protein level). Studies of the two UTR mutants identified by a modified screening procedure showed that their expression is stimulated to a similar extent at both the transcript and protein product levels. For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18-fold higher than the wild type), while there was no evidence of a difference in transcript stability. The two UTR sequences also stimulated expression from a constitutive ,70 -dependent promoter (P1/Panti-tet), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription. [source] Structured Nucleic Acid Probes for Electrochemical DevicesELECTROANALYSIS, Issue 19 2009Rebeca Miranda-Castro Abstract The use of nucleic acid with a specific sequence and a highly ordered secondary structure such as hairpins, quadruplexes and pseudoknots as biological recognition elements and switches in biosensors is rapidly increasing because of their improved features (e.g. selectivity) when compared with the traditional linear probes. Owing to the novelty, a critical outlook of their characteristics and a compilation of the latest advances are lacking. This article describes the potential of those nucleic acids probes whose molecular recognition ability relies on a conformational change (e.g. folding/unfolding mechanism) in electrochemical sensing. It provides an overview of the toolbox of assays using these probes for genosensors and aptasensors, highlighting its performance characteristics and the prospects and challenges for biosensor design. [source] Using DNA sequencing electrophoresis compression artifacts as reporters of stable mRNA structures affecting gene expressionELECTROPHORESIS, Issue 21 2007Divya Kapoor Abstract The formation of secondary structure in oligonucleotide DNA is known to lead to "compression" artifacts in electropherograms produced through DNA sequencing. Separately, the formation of secondary structure in mRNA is known to suppress translation; in particular, when such structures form in a region covered by the ribosome either during, or shortly after, initiation of translation. Here, we demonstrate how a DNA sequencing compression artifact provides important clues to the location(s) of translation-suppressing secondary structural elements in mRNA. Our study involves an engineered version of a gene sourced from Rhodothermus marinus encoding an enzyme called Cel12A. We introduced this gene into Escherichia coli with the intention of overexpressing it, but found that it expressed extremely poorly. Intriguingly, the gene displayed a remarkable compression artifact during DNA sequencing electrophoresis. Selected "designer" silent mutations destroyed the artifact. They also simultaneously greatly enhanced the expression of the cel12A gene, presumably by destroying stable mRNA structures that otherwise suppress translation. We propose that this method of finding problem mRNA sequences is superior to software-based analyses, especially if combined with low-temperature CE. [source] Molecular Interaction between a Gadolinium,Polyoxometalate and Human Serum AlbuminEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 34 2009Li Zheng Abstract Polyoxometalates (POMs) show promising antibacterial, antiviral (particularly anti-HIV), antitumor, and anticancer activities, but the mechanism of these potential therapeutic effects remains to be elucidated at the molecular level. The interaction between the Gd-containing tungstosilicate [Gd(,2 -SiW11O39)2]13, and human serum albumin (HSA) was studied by several techniques. Fluorescence spectroscopy showed an energy transfer between the single tryptophan residue of HSA and the POM. Circular dichroism led to the conclusion that the POM significantly altered the secondary structure of HSA. Isothermal titration calorimetry revealed an enthalpy-driven binding reaction between HSA and the POM, resulting in the formation of a 1:1 complex.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] Comparison of the aggregation properties, secondary structure and apoptotic effects of wild-type, Flemish and Dutch N-terminally truncated amyloid , peptidesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2001N. Demeester Abstract The Dutch (E22Q) and Flemish (A21G) mutations in the ,APP region of the amyloid precursor protein (APP) are associated with familial forms of Alzheimer dementia. However, patients with these mutations express substantially different clinical phenotypes. Therefore, secondary structure and cytotoxic effects of the three A,(12,42) variants [wild-type (WT), Dutch and Flemish] were tested. At a concentration of 5 µm the aggregation of these peptides followed the order: A,(1,42) WT > A,(12,42) WT > A,(12,42) Flemish >,A,(12,42) Dutch. The stability of the secondary structure of these peptides upon decreasing the trifluoroethanol (TFE) concentration in the buffer was followed by circular dichroism measurements. WT peptides progressively lost their ,-helical structure; this change occurred faster for both the Flemish and Dutch peptides, and at higher percentages of TFE in the buffer, and was accompanied by an increase in ,-sheet and random coil content. Apoptosis was induced in neuronal cells by the A,(12,42) WT and Flemish peptides at concentrations as low as 1,5 µm, as evidenced by propidium iodide (PI) staining, DNA laddering and caspase-3 activity measurements. Even when longer incubation times and higher peptide concentrations were applied the N-truncated Dutch peptide did not induce apoptosis. Apoptosis induced by the full length A,(1,42) peptide was weaker than that induced by its N-truncated variant. These data suggest that N-truncation enhanced the cytotoxic effects of A, WT and Flemish peptides, which may play a role in the accelerated progression of dementia. [source] Structure of the HIV-1 Rev response element alone and in complex with regulator of virion (Rev) studied by atomic force microscopyFEBS JOURNAL, Issue 15 2009Jesper Pallesen The interaction of multiple HIV-1 regulator of virion (Rev) proteins with the viral RNA target, the Rev response element (RRE), is critical for nuclear export of incompletely spliced and unspliced viral RNA, and for the onset of the late phase in the viral replication cycle. The heterogeneity of the Rev,RRE complex has made it difficult to study using conventional structural methods. In the present study, atomic force microscopy is applied to directly visualize the tertiary structure of the RRE RNA alone and in complex with Rev proteins. The appearance of the RRE is compatible with the earlier proposed RRE secondary structure in dimensions and overall shape, including a stalk and a head interpreted as stem I, and stem-loops II,V in the secondary structure model, respectively. Atomic force microscopy imaging of the Rev,RRE complex revealed an increased height of the structure both in the stalk and head regions, which is in accordance with a binding model in which Rev binding to a high affinity site in stem IIB triggers oligomerization of Rev proteins through cooperative binding along stem I in RRE. The present study demonstrates that atomic force microscopy comprises a useful technique to study complex biological structures of nucleic acids at high resolution. [source] Secondary structure of lipidated Ras bound to a lipid bilayerFEBS JOURNAL, Issue 23 2008Jörn Güldenhaupt Ras proteins are small guanine nucleotide binding proteins that regulate many cellular processes, including growth control. They undergo distinct post-translational lipid modifications that are required for appropriate targeting to membranes. This, in turn, is critical for Ras biological function. However, most in vitro studies have been conducted on nonlipidated truncated forms of Ras proteins. Here, for the first time, attenuated total reflectance-FTIR studies of lipid-modified membrane-bound N-Ras are performed, and compared with nonlipidated truncated Ras in solution. For these studies, lipidated N-Ras was prepared by linking a farnesylated and hexadecylated N-Ras lipopeptide to a truncated N-Ras protein (residues 1,181). It was then bound to a 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine bilayer tethered on an attenuated total reflectance crystal. The structurally sensitive amide I absorbance band in the IR was detected and analysed to determine the secondary structure of the protein. The NMR three-dimensional structure of truncated Ras was used to calibrate the contributions of the different secondary structural elements to the amide I absorbance band of truncated Ras. Using this novel approach, the correct decomposition was selected from several possible solutions. The same parameter set was then used for the membrane-bound lipidated Ras, and provided a reliable decomposition for the membrane-bound form in comparison with truncated Ras. This comparison indicates that the secondary structure of membrane-bound Ras is similar to that determined for the nonlipidated truncated Ras protein for the highly conserved G-domain. This result validates the multitude of investigations of truncated Ras without anchor in vitro. The novel attenuated total reflectance approach opens the way for detailed studies of the interaction network of the membrane-bound Ras protein. [source] Full-length prion protein aggregates to amyloid fibrils and spherical particles by distinct pathwaysFEBS JOURNAL, Issue 9 2008Driss El Moustaine As limited structural information is available on prion protein (PrP) misfolding and aggregation, a causative link between the specific (supra)molecular structure of PrP and transmissible spongiform encephalopathies remains to be elucidated. In this study, high pressure was utilized, as an approach to perturb protein structure, to characterize different morphological and structural PrP aggregates. It was shown that full-length recombinant PrP undergoes ,-sheet aggregation on high-pressure-induced destabilization. By tuning the physicochemical conditions, the assembly process evolves through two distinct pathways leading to the irreversible formation of spherical particles or amyloid fibrils, respectively. When the PrP aggregation propensity is enhanced, high pressure induces the formation of a partially unfolded aggregated protein, AggHP, which relaxes at ambient pressure to form amorphous aggregates. The latter largely retain the native secondary structure. On prolonged incubation at high pressure, followed by depressurization, AggHP transforms to a monodisperse population of spherical particles of about 20 nm in diameter, characterized by an essentially ,-sheet secondary structure. When the PrP aggregation propensity is decreased, an oligomeric reaction intermediate, IHP, is formed under high pressure. After pressure release, IHP relaxes to the original native structure. However, on prolonged incubation at high pressure and subsequent depressurization, it transforms to amyloid fibrils. Structural evaluation, using optical spectroscopic methods, demonstrates that the conformation adopted by the subfibrillar oligomeric intermediate, IHP, constitutes a necessary prerequisite for the formation of amyloids. The use of high-pressure perturbation thus provides an insight into the molecular mechanism of the first stages of PrP misfolding into amyloids. [source] Secondary structure assignment of mouse SOCS3 by NMR defines the domain boundaries and identifies an unstructured insertion in the SH2 domainFEBS JOURNAL, Issue 23 2005Jeffrey J. Babon SOCS3 is a negative regulator of cytokine signalling that inhibits Janus kinase-signal transduction and activator of transcription (JAK-STAT) mediated signal tranduction by binding to phosphorylated tyrosine residues on intracellular subunits of various cytokine receptors, as well as possibly the JAK proteins. SOCS3 consists of a short N-terminal sequence followed by a kinase inhibitory region, an extended SH2 domain and a C-terminal suppressor of cytokine signalling (SOCS) box. SOCS3 and the related protein, cytokine-inducible SH2-containing protein, are unique among the SOCS family of proteins in containing a region of mostly low complexity sequence, between the SH2 domain and the C-terminal SOCS box. Using NMR, we assigned and determined the secondary structure of a murine SOCS3 construct. The SH2 domain, unusually, consists of 140 residues, including an unstructured insertion of 35 residues. This insertion fits the criteria for a PEST sequence and is not required for phosphotyrosine binding, as shown by isothermal titration calorimetry. Instead, we propose that the PEST sequence has a functional role unrelated to phosphotyrosine binding, possibly mediating efficient proteolytic degradation of the protein. The latter half of the kinase inhibitory region and the entire extended SH2 subdomain form a single ,-helix. The mapping of the true SH2 domain, and the location of its C terminus more than 50 residues further downstream than predicted by sequence homology, explains a number of previously unexpected results that have shown the importance of residues close to the SOCS box for phosphotyrosine binding. [source] Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificityFEBS JOURNAL, Issue 12 2005Vitor Oliveira The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). The constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. In the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function. [source] Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharideFEBS JOURNAL, Issue 10 2004Jörg Andrä Endotoxin-neutralizing protein (ENP) of the horseshoe crab is one of the most potent neutralizers of endotoxins [bacterial lipopolysaccharide (LPS)]. Here, we report on the interaction of LPS with recombinant ENP using a variety of physical and biological techniques. In biological assays (Limulus amebocyte lysate and tumour necrosis factor-, induction in human mononuclear cells), ENP causes a strong reduction of the immunostimulatory ability of LPS in a dose-dependent manner. Concomitantly, the accessible negative surface charges of LPS and lipid A (zeta potential) are neutralized and even converted into positive values. The gel to liquid crystalline phase transitions of LPS and lipid A shift to higher temperatures indicative of a rigidification of the acyl chains, however, the only slight enhancement of the transition enthalpy indicates that the hydrophobic moiety is not strongly disturbed. The aggregate structure of lipid A is converted from a cubic into a multilamellar phase upon ENP binding, whereas the secondary structure of ENP does not change due to the interaction with LPS. ENP contains a hydrophobic binding site to which the dye 1-anilino-8-sulfonic acid binds at a Kd of 19 µm, which is displaced by LPS. Because lipopolysaccharide-binding protein (LBP) is not able to bind to LPS when ENP and LPS are preincubated, tight binding of ENP to LPS can be deduced with a Kd in the low nonomolar range. Importantly, ENP is able to incorporate by itself into target phospholipid liposomes, and is also able to mediate the intercalation of LPS into the liposomes thus acting as a transport protein in a manner similar to LBP. Thus, LPS,ENP complexes might enter target membranes of immunocompetent cells, but are not able to activate due to the ability of ENP to change LPS aggregates from an active into an inactive form. [source] Biophysical characterization of the interaction of high-density lipoprotein (HDL) with endotoxinsFEBS JOURNAL, Issue 23 2002Klaus Brandenburg The interaction of bacterial endotoxins [lipopolysaccharide (LPS) and the ,endotoxic principle' lipid A], with high-density lipoprotein (HDL) from serum was investigated with a variety of physical techniques and biological assays. HDL exhibited an increase in the gel to liquid crystalline phase transition temperature Tc and a rigidification of the acyl chains of the endotoxins as measured by Fourier-transform infrared spectroscopy and differential scanning calorimetry. The functional groups of the endotoxins interacting with HDL are the phosphates and the diglucosamine backbone. The finding of phosphates as target groups is in accordance to measurements of the electrophoretic mobility showing that the zeta potential decreases from ,50 to ,60 mV to ,20 mV at binding saturation. The importance of the sugar backbone as further target structure is in accordance with the remaining negative potential and competition experiments with polymyxin B (PMB) and phase transition data of the system PMB/dephosphorylated LPS. Furthermore, endotoxin binding to HDL influences the secondary structure of the latter manifesting in a change from a mixed ,-helical/,-sheet structure to a predominantly ,-helical structure. The aggregate structure of the lipid A moiety of the endotoxins as determined by small-angle X-ray scattering shows a change of a unilamellar/inverted cubic into a multilamellar structure in the presence of HDL. Fluorescence resonance energy transfer data indicate an intercalation of pure HDL, and of [LPS],[HDL] complexes into phospholipid liposomes. Furthermore, HDL may enhance the lipopolysaccharide-binding protein-induced intercalation of LPS into phospholipid liposomes. Parallel to these observations, the LPS-induced cytokine production of human mononuclear cells and the reactivity in the Limulus test are strongly reduced by the addition of HDL. These data allow to develop a model of the [endotoxin]/[HDL] interaction. [source] The refolding of type II shikimate kinase from Erwinia chrysanthemi after denaturation in ureaFEBS JOURNAL, Issue 8 2002Eleonora Cerasoli Shikimate kinase was chosen as a convenient representative example of the subclass of ,/, proteins with which to examine the mechanism of protein folding. In this paper we report on the refolding of the enzyme after denaturation in urea. As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism (CD) and fluorescence, respectively, the enzyme was fully unfolded in 4 m urea. From an analysis of the unfolding curve in terms of the two-state model, the stability of the folded state could be estimated as 17 kJ·mol,1. Approximately 95% of the enzyme activity could be recovered on dilution of the urea from 4 to 0.36 m. The results of spectroscopic studies indicated that refolding occurred in at least four kinetic phases, the slowest of which (k = 0.009 s,1) corresponded with the regain of shikimate binding and of enzyme activity. The two most rapid phases were associated with a substantial increase in the binding of 8-anilino-1-naphthalenesulfonic acid with only modest changes in the far UV CD, indicating that a collapsed intermediate with only partial native secondary structure was formed rapidly. The relevance of the results to the folding of other ,/, domain proteins is discussed. [source] NMR investigations of subunit c of the ATP synthase from Propionigenium modestum in chloroform/methanol/water (4 : 4 : 1)FEBS JOURNAL, Issue 7 2002Ulrich Matthey The subunit c from the ATP synthase of Propionigenium modestum was studied by NMR in chloroform/methanol/water (4 : 4 : 1). In this solvent, subunit c consists of two helical segments, comprised of residues L5 to I26 and G29 to N82, respectively. On comparing the secondary structure of subunit c from P. modestum in the organic solvent mixture with that in dodecylsulfate micelles several deviations became apparent: in the organic solvent, the interruption of the ,,helical structure within the conserved GXGXGXGX motif was shortened from five to two residues, the prominent interruption of the ,,helical structure in the cystoplasmic loop region was not apparent, and neither was there a break in the ,,helix after the sodium ion-binding Glu65 residue. The folding of subunit c of P. modestum in the organic solvent also deviated from that of Escherichia coli in the same environment, the most important difference being that subunit c of P. modestum did not adopt a stable hairpin structure like subunit c of E. coli. [source] Stepwise proteolytic removal of the , subdomain in ,-lactalbuminFEBS JOURNAL, Issue 15 2001The protein remains folded, can form the molten globule in acid solution Bovine ,-lactalbumin (,-LA) is an ,/, protein which adopts partly folded states when dissolved at low pH (A-state), by removal of the protein-bound calcium at neutral pH and low salt concentration (apo-state), as well as in aqueous trifluoroethanol. Previous spectroscopic studies have indicated that the A-state of ,-LA at pH 2.0, considered a prototype molten globule, has a native-like fold in which the helical core is mostly retained, while the , subdomain is less structured. Here, we investigate the conformational features of three derivatives of ,-LA characterized by a single peptide bond fission or a deletion of 12 or 19/22 amino-acid residues of the , subdomain of the native protein (approximately from residue 34 to 57). These ,-LA derivatives were obtained by limited proteolysis of the protein in its partly folded state(s). A nicked ,-LA species consisting of fragments 1-,3,40 and 41,123 (nicked-LA) was prepared by thermolytic digestion of the 123-residue chain of ,-LA in 50% (v/v) aqueous trifluoroethanol. Two truncated or gapped protein species given by fragments 1,40 and 53,123 (des,1-LA) or fragments 1,34 and 54-,57,123 (des,2-LA) were obtained by digestion of ,-LA with pepsin in acid or with proteinase K at neutral pH in its apo-state, respectively. The two protein fragments of nicked or gapped ,-LA are covalently linked by the four disulfide bridges of the native protein. CD measurements revealed that, in aqueous solution at neutral pH and in the presence of calcium, the three protein species maintain the helical secondary structure of intact ,-LA, while the tertiary structure is strongly affected by the proteolytic cleavages of the chain. Temperature effects of CD signals in the far- and near-UV region reveal a much more labile tertiary structure in the ,-LA derivatives, while the secondary structure is mostly retained even upon heating. In acid solution at pH 2.0, the three ,-LA variants adopt a conformational state essentially identical to the molten globule displayed by intact ,-LA, as demonstrated by CD measurements. Moreover, they bind strongly the fluorescent dye 8-anilinonaphthalene-1-sulfonate, which is considered a diagnostic feature of the molten globule of proteins. Therefore, the , subdomain can be removed from the ,-LA molecule without impairing the capability of the rest of the chain to adopt a molten globule state. The results of this protein dissection study provide direct experimental evidence that in the ,-LA molten globule only the , domain is structured. [source] Thermally induced conformational changes in horseradish peroxidaseFEBS JOURNAL, Issue 1 2001David G. Pina Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated. [source] The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA-dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 °CFEBS JOURNAL, Issue 4 2000Maria Spies The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recombinational DNA repair. We cloned the radA gene from a hyperthermophilic archaeon, Pyrobaculum islandicum, overproduced the radA gene product in Escherichia coli and purified it to homogeneity. The purified P. islandicum RadA protein maintained its secondary structure and activities in vitro at high temperatures, up to 87 °C. It also showed high stability of 18.3 kcal·mol,1 (76.5 kJ·mol,1) at 25 °C and neutral pH. P. islandicum RadA exhibited activities typical of the family of RecA-like proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA-dependent manner and to catalyze DNA strand exchange. At 75 °C, all DNAs tested stimulated ATPase activity of the RadA. The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 °C. The cooperativity of ATP hydrolysis and ssDNA-binding ability of the protein above 75 °C were higher than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature. These results suggest that the ssDNA-dependent ATPase activity of P. islandicum RadA displays a temperature-dependent capacity to exist in two different catalytic modes, with 75 °C being the critical threshold temperature. [source] | ||||||||||||||||||||||||||||||||||||||||||||||