Amino Acid Residues (amino + acid_residue)

Distribution by Scientific Domains
Distribution within Chemistry

Kinds of Amino Acid Residues

  • basic amino acid residue
  • key amino acid residue
  • specific amino acid residue


  • Selected Abstracts


    Amino Acid Residues in GRK1/GRK7 Responsible for Interaction with S-Modulin/Recoverin,

    PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008
    Aya Torisawa
    GRK1 is a visual pigment kinase in rods and is essential for inactivation of light-activated rhodopsin. The GRK1 activity is inhibited by binding of the Ca2+ -bound form of S-modulin/recoverin. We previously identified the S-modulin/recoverin site to interact with GRK1. In the present study, we identified its counterpart in GRK1. We synthesized 29 of GRK1 or GRK7 partial peptides that cover the entire sequence of GRK1/GRK7, and examined whether these peptides inhibit S-modulin/recoverin activity most probably by preoccupying the binding site for GRK1. The inhibition was the greatest with the N-terminal peptide (p1, aa 3,23 in GRK7). On mutation of each of eight amino acid residues highly conserved in the p1 region of more than 10 orthologs, the inhibition was significantly reduced in the mutation of Leu6, Asn12 and Tyr15. We further examined the binding of the peptides, including mutated ones, to S-modulin/recoverin with a resonance mirror biosensor. The binding correlated well with the degree of the inhibition by a peptide. The inhibition, therefore, seemed to be due to a direct binding of the kinase peptide to the binding site of active S-modulin/recoverin. A GRK1 region close to its C-terminus also seemed to be the binding site for S-modulin/recoverin. [source]


    Effects of Three Characteristic Amino Acid Residues of Pharaonis Phoborhodopsin on the Absorption Maximum ,

    PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2000
    Kazumi Shimono
    ABSTRACT Phoborhodopsin (pR or sensory rhodopsin II, sRII) or pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) has a unique absorption maximum (,max) compared with three other archaeal rhodopsins: ,max of pR or ppR at ca 500 nm and others at 560,590 nm. Alignment of amino acid sequences revealed three sites characteristic of the shorter wavelength,absorbing pigments. The amino acids of these three sites are conserved completely among archaeal rhodopsins having longer ,max, and are different from those of pR or ppR. We replaced these amino acids of ppR with amino acids corresponding to those of bacteriorhodopsin, Val-108 to Met, Gly-130 to Ser and Thr-204 to Ala. The ,max of V108M mutant was 502 nm with a slight redshift. G130S and T204A mutants had ,max of 503 and 508 nm, respectively. Thus, each site contributes only a small effect to the color tuning. We then constructed three double mutants and one triple mutant. The opsin-shifts of these mutants suggest that Val-108 and Thr-204 or Gly-130 are synergistic, and that Gly-130 and Thr-204 work additively. Even in the triple mutant, the ,max was 515 nm, an opsin-shift only ca 30% of the shift value from 500 to 560 nm. This means that there is another yet unidentified factor responsible for the color tuning. [source]


    Assembling Heterocycle-Tethered C-Glycosyl and ,-Amino Acid Residues via 1,3-Dipolar Cycloaddition Reactions.

    CHEMINFORM, Issue 50 2004
    Alessandro Dondoni
    Abstract For Abstract see ChemInform Abstract in Full Text. [source]


    Side chain contributions to the interconversion of the topological isomers of guanylin-like peptides

    JOURNAL OF PEPTIDE SCIENCE, Issue 6 2005
    Dr Axel Schulz
    Abstract The peptide hormones guanylin and uroguanylin are ligands of the intestinal guanylyl cyclase-C (GC-C) that is involved in the regulation of epithelial water and electrolyte transport. The small peptides contain 15 and 16 amino acids, respectively, and two disulfide bonds with a 1,3/2,4 connectivity. This structural feature causes the unique existence of two topological isoforms for each peptide in an approximate 3:2 ratio, with only one of the isoforms exhibiting GC-C-activating potential. The two uroguanylin isomers can be separated by HPLC and are of sufficient stability to be studied separately at ambient temperatures while the two guanylin isomers are rapidly interconverting even at low temperatures. Both isomers show clearly distinguishable 1H chemical shifts. To investigate the influence of certain amino acid side chains on this isomerism and interconversion kinetics, derivatives of guanylin and uroguanylin (L -alanine scan and chimeric peptides) were designed and synthesized by Fmoc solid-phase chemistry and compared by HPLC and 2D 1H NMR spectroscopy. Amino acid residues with the most significant effects on the interconversion kinetics were predominantly identified in the COOH-terminal part of both peptides, whereas amino acids in the central part of the peptides only moderately affected the interconversion. Thus, the conformational conversion among the isomers of both peptides is under the control of a COOH-terminal sterical hindrance, providing a detailed model for this dynamic isomerism. Our results demonstrate that kinetic control of the interconversion process can be achieved by the introduction of side chains with a defined sterical profile at suitable sequence positions. This is of potential impact for the future development of GC-C peptide agonists and antagonists. Copyright 2004 European Peptide Society and John Wiley & Sons, Ltd. [source]


    Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin

    PROTEIN SCIENCE, Issue 2 2000
    Oktay K. Gasymov
    Abstract The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a ,-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion. The combination of site-directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X-ray crystallography. [source]


    Degeneration of pontine mossy fibres during cerebellar development in weaver mutant mice

    EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2002
    Miwako Ozaki
    Abstract In weaver mutant mice, substitution of an amino acid residue in the pore region of GIRK2, a subtype of the G-protein-coupled inwardly rectifying K+ channel, changes the properties of the homomeric channel to produce a lethal depolarized state in cerebellar granule cells and dopaminergic neurons in substantia nigra. Degeneration of these types of neurons causes strong ataxia and Parkinsonian phenomena in the mutant mice, respectively. On the other hand, the mutant gene is also expressed in various other brain regions, in which the mutant may have effects on neuronal survival. Among these regions, we focused on the pontine nuclei, the origin of the pontocerebellar mossy fibres, projecting mainly into the central region of the cerebellar cortex. The results of histological analysis showed that by P9 the number of neurons in the nuclei was reduced in the mutant to about one half and by P18 to one third of those in the wild type, whereas until P7 the number were about the same in wild-type and weaver mutant mice. Three-dimensional reconstruction of the nuclei showed a marked reduction in volume and shape of the mutant nuclei, correlating well with the decrease in neuronal number. In addition, DiI (a lipophilic tracer dye) tracing experiments revealed retraction of pontocerebellar mossy fibres from the cerebellar cortex after P5. From these results, we conclude that projecting neurons in the pontine nuclei, as well as cerebellar granule cells and dopaminergic neurons in substantia nigra, strongly degenerate in weaver mutant mice, resulting in elimination of pontocerebellar mossy fibres during cerebellar development. [source]


    Characterization of genomic DNA encoding cecropins from an Aedes albopictus mosquito cell line

    INSECT MOLECULAR BIOLOGY, Issue 1 2002
    D. Sun
    Abstract We used cDNA probes from Aedes albopictus mosquito cecropins AalCecA, B, and C to obtain genomic DNA copies and flanking DNA. Two gene copies (AalCecA1 and A2, AalCecB1 and B2, AalCecC1 and C2) encoding each of the three mature cecropin peptides were recovered. All these genes had a similar organization, into two exons interrupted by a single short intron. AalCecA1 and AalCecA2 encode mature protein products that differ by one amino acid residue, while AalCecB1 and AalCecB2, AalCecC1 and AalCecC2 encode identical mature cecropin peptides, respectively. The AalCecB and C gene pairs each share a common intergenic region of approximately 1 kb, with the two coding regions transcribed in opposite directions. With the exception of small insertions/deletions, the intergenic spacer region was highly conserved between the B1/C1 and B2/C2 clones. In transfected cells, 0.8 kb of upstream sequence was sufficient for inducible expression of AalCecA1. Within this region, a 28 bp sequence at positions ,192 to ,165 upstream of the transcription initiation site was found to contain a potential regulatory element. In electrophoretic mobility shift assays, synthetic double-stranded DNA containing this 28 bp sequence retarded protein in cytoplasmic and nuclear extracts from C7-10 cells. [source]


    Expression and immunocytochemical analysis of Autographa californica nucleopolyhedrovirus (AcMNPV) orf74 gene

    INSECT SCIENCE, Issue 5 2006
    SHI-HENG AN
    Abstract Autographa californica nucleopolyhedrovirus orf74 (Ac74) is located between 62 311 and 63 108bp in the AcMNPV genome, which encodes 265 amino acid residues with a predicted 31 kDa molecular weight. The homologues of Ac74 were searched using BLASTP in protein databases, GenBank/EMBL and SWISS-PROT. The result revealed that deduced Ac74 protein was homologous to the predicted products from 10 lepidoptera NPV ORFs. The multiple sequence alignments of Ac74 and its 10 homologues manifested only one amino acid residue was completely conserved. The transcript analysis revealed that the transcript of Ac74 was detected from 24,72 hours post-infection (hpi). The product of Ac74 was detected at 24 hpi and lasted until 72 hpi by Western blot using anti-Ac74 antiserum, consistent with reverse transcriptase polymerase chain reaction results. These results suggested Ac74 was expressed during the later stages of infection. The product of Ac74 was 31 kDa in size, consistent with predicted molecular weight. The subcellular localization of Ac74 proteins manifested Ac74 protein in the cytoplasm, and was hardly present in the nucleus at 24 hpi. The fluorescence was also observed in polyhedra, except cytoplasm at 72 hpi. Together, Ac74 is a functional protein with 3 1kDa molecular weight and is located in the cytoplasm and the polyhedra. [source]


    No relationship observed between human p53 codon-72 genotype and HPV-associated cervical cancer in a population group with a low arginine-72 allele frequency

    INTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 3 2007
    V. A. Govan
    Summary Infection with high-risk human papillomavirus (HR-HPV) is a necessary but not a sufficient event in the development of cervical cancer, as most infections regress without intervention. Thus, genetic host factors and cellular immune responses could be potential modifiers for the risk of developing cervical cancer. In particular, p53 is considered as the most critical tumour suppressor gene and is involved in regulating cell division. The polymorphism on p53, which encodes either a proline or an arginine amino acid residue at codon 72, has been reported as a possible risk factor for cervical disease. This polymorphism has been shown to differentially affect the efficiency of degradation of p53 protein mediated by HR-HPV E6 oncoprotein. Women with histologically proven cancer of the cervix (n = 111) and hospital-based controls (n = 143) were included in this study. The patients and controls were from the Western Cape Province in South Africa. Genotyping of the p53 polymorphism was conducted using polymerase chain reaction and restriction fragment-length polymorphism method. The distributions of the allelic frequencies were stratified in both patients and controls into two South African ethnic population groups. In this study, we observed no association between the distribution of p53 polymorphism and susceptibility to cervical cancer in the Western Cape Province populations (P = 0.466). However, the frequency of the Pro/Pro residue at codon 72 was increased in the South African population when compared to Caucasians, Indians and Portuguese population groups. Notably, as the distribution of the Pro/Pro at codon 72 of p53 gene was significantly different (P < 0.05) between the control groups of South Africa and other population groups. This result suggests that ethnic disparity may influence the levels of p53 produced. [source]


    Toward direct determination of conformations of protein building units from multidimensional NMR experiments VI.

    JOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 13 2005
    Chemical shift analysis of his to gain 3D structure, protonation state information
    Abstract NMR,chemical shift structure correlations were investigated by using GIAO-RB3LYP/6-311++G(2d,2p) formalism. Geometries and chemical shifts (CSI values) of 103 different conformers of N,-formyl-L-histidinamide were determined including both neutral and charged protonation forms. Correlations between amino acid torsional angle values and chemical shifts were investigated for the first time for an aromatic and polar amino acid residue whose side chain may carry different charges. Linear correlation coefficients of a significant level were determined between chemical shifts and dihedral angles for CSI[1H,]/,, CSI[13C,]/,, and CSI[13C,]/,. Protonation of the imidazole ring induces the upfield shift of CSI[13C,] for positively charged histidines and an opposite effect for the negative residue. We investigated the correspondence of theoretical and experimental 13C,, 13C,, and 1H, chemical shifts and the nine basic conformational building units characteristic for proteins. These three chemical shift values allow the identification of conformational building units at 80% accuracy. These results enable the prediction of additional regular secondary structural elements (e.g., polyProlineII, inverse ,-turns) and loops beyond the assignment of chemical shifts to ,-helices and ,-pleated sheets. Moreover, the location of the His residue can be further specified in a ,-sheet. It is possible to determine whether the appropriate residue is located at the middle or in a first/last ,-strand within a ,-sheet based on calculated CSI values. Thus, the attractive idea of establishing local residue specific backbone folding parameters in peptides and proteins by employing chemical shift information (e.g., 1H, and 13C,) obtained from selected heteronuclear correlation NMR experiments (e.g., 2D-HSQC) is reinforced. 2005 Wiley Periodicals, Inc. J Comput Chem 26: 1307,1317, 2005 [source]


    New energy terms for reduced protein models implemented in an off-lattice force field

    JOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 12 2001
    Tommi Hassinen
    Abstract Parameterization and test calculations of a reduced protein model with new energy terms are presented. The new energy terms retain the steric properties and the most significant degrees of freedom of protein side chains in an efficient way using only one to three virtual atoms per amino acid residue. The energy terms are implemented in a force field containing predefined secondary structure elements as constraints, electrostatic interaction terms, and a solvent-accessible surface area term to include the effect of solvation. In the force field the main-chain peptide units are modeled as electric dipoles, which have constant directions in ,-helices and ,-sheets and variable conformation-dependent directions in loops. Protein secondary structures can be readily modeled using these dipole terms. Parameters of the force field were derived using a large set of experimental protein structures and refined by minimizing RMS errors between the experimental structures and structures generated using molecular dynamics simulations. The final average RMS error was 3.7 for the main-chain virtual atoms (C, atoms) and 4.2 for all virtual atoms for a test set of 10 proteins with 58,294 amino acid residues. The force field was further tested with a substantially larger test set of 608 proteins yielding somewhat lower accuracy. The fold recognition capabilities of the force field were also evaluated using a set of 27,814 misfolded decoy structures. 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1229,1242, 2001 [source]


    Nitrogen-to-Protein Conversion Factors for Some Cereal Products in Japan

    JOURNAL OF FOOD SCIENCE, Issue 3 2008
    S. Fujihara
    ABSTRACT:, To evaluate a practical method of determining more accurately conversion factors for calculating the protein contents of foods from the total nitrogen content, 19 cereal products found in Japan were analyzed for total nitrogen, amino acid nitrogen, and amide nitrogen, and then the nitrogen-to-protein conversion factors were calculated. The average conversion factors were 5.75 for rice, 5.81 for wheat, and 5.95 for others. These values, corresponding to the proportion of the amino acid residue to amino acid nitrogen recovered from 20 amino acids, were lower than the currently applied factors to these foods, except for wheat flour and amaranth. The use of this factor for estimating the protein content results in a considerable difference from the estimate based on amino acid residue concentrations, due to the wide variations in amino acid composition and to the presence of a significant level of nonprotein nitrogen. The distribution of the protein nitrogen recovered from the amino acids to total nitrogen averaged 93%. Adjusted conversion factors corresponding to the proportion of the amino acid residue to total nitrogen averaged 5.26 for rice, 5.47 for wheat, and 5.54 for other cereal products. Protein contents estimated using these factors are in good agreement with the contents defined as amino acid residues. [source]


    Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight mass spectrometry: localization of the active site serine,

    JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2004
    Martin Zehl
    Abstract A chemical modification approach combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6-554W. The lipase, purified from a high-level overexpressing strain, was covalently modified by incubation with 3,4-dichloroisocoumarin, a general mechanism-based serine protease inhibitor. MALDI time-of-flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor-modified lipase and to clarify the mechanism of lipase inhibition by 3,4-dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6-dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H]2+ ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor-modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low-energy collision-induced dissociation peptide sequencing of the detected 2-(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly-Asp-Ser-(Leu) motif located close to the N-terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme. Copyright 2004 John Wiley & Sons, Ltd. [source]


    Hepatitis B virus genotypes and HBsAg subtypes in refugees and injection drug users in the United States determined by LiPA and monoclonal EIA

    JOURNAL OF MEDICAL VIROLOGY, Issue 3 2001
    Paul D. Swenson
    Abstract Hepatitis B virus (HBV) genotyping and hepatitis B surface antigen (HBsAg) subtyping were carried out on sera from 196 HBsAg-positive patients, including 151 refugees entering the United States and 45 injection drug users in Seattle. HBsAg subtyping was performed by enzyme immunoassay (EIA) using a panel of monoclonal antibodies and the HBV genotype was determined by polymerase chain reaction (PCR) followed by detection of amplified HBV DNA by a reverse-phase hybridization line probe assay (LiPA) using genotype-specific probes. HBV DNA was detected by PCR in 155 (79%) of the 196 sera and all 155 were genotyped by LiPA. Samples from Southeast Asia were predominantly genotype B/subtype ayw1 and genotype C/adr; samples from the former Soviet Union and eastern Europe were mostly genotype D/ayw2 and genotype D/ayw3; samples from east Africa were mainly genotype A/adw2 and genotype D/ayw2; and samples from injection drug users were mostly genotype D/ayw3 and genotype A/adw2. Some strains of ayw3 gave atypical monoclonal antibody reactivity patterns in the subtyping assay due to a Val/Ala instead of a Thr at amino acid residue 118 and a Thr instead of a Met at residue 125. A strain of ayw2 also gave an atypical monoclonal antibody reactivity pattern due to an Ala instead of a Thr at amino acid residue 123. LiPA genotyping and monoclonal EIA subtyping can provide useful information for epidemiological studies. J. Med. Virol. 64:305,311, 2001. 2001 Wiley-Liss, Inc. [source]


    Kinetic studies on aminopeptidase M-mediated degradation of human hemorphin LVV-H7 and its N -terminally truncated products

    JOURNAL OF PEPTIDE SCIENCE, Issue 7 2008
    Harald John
    Abstract The human hemorphin LVV-H7 belongs to the class of -opiod receptor-binding peptides, which also exhibits significant affinity to insulin-regulated aminopeptidase (IRAP) thereby affecting IRAP inhibition. The inhibitory potency towards IRAP is of pharmaceutical interest for the treatment of Alzheimer's disease. Consecutive N -terminal cleavage of the first two amino acid residues of LVV-H7 affects a drastic increase of the binding affinity (V-H7) but ultimately leads to its complete abolition after cleavage of the next amino acid residue (H7). Therefore, we investigated LVV-H7 truncation by aminopeptidase M (AP-M) identified as a LVV-H7 degrading enzyme potentially regulating hemorphin activity towards IRAP in vivo. Using a selective quantitative multi-component capillary zone electrophoretic method (CZE-UV), we analyzed the AP-M-mediated subsequent proteolysis of the hemorphins LVV-H7 (L32 -F41), VV-H7 (V33 -F41), and V-H7 (V34 -F41) in vitro. Incubations were carried out with synthetic hemorphins applied as single substrates or in combination. Maximum velocities (Vmax), catalytic constants (turnover numbers, kcat), and specific enzyme activities (EA) were calculated. L32 cleavage from LVV-H7 happens more than two-times faster (kcat: 140 min,1 9%, EA: 1.0 U/mg 9%) than V33 cleavage from VV-H7 (kcat: 61 min,1 10%, EA: 0.43 U/mg 10%) or V32 deletion from V-H7 (kcat: 62 min,1 8%, EA: 0.46 U/mg 8%). In contrast, we showed that H7 (Y35 -F41) was neither degraded by porcine AP-M nor did it act as an inhibitor for this enzyme. Determined turnover numbers were in the same dimension as those reported for dynorphin degradation. This is the first time that AP-M-mediated truncation of natural underivatized LVV-H7 and its physiological metabolites was analyzed to determine kinetic parameters useful for understanding hemorphin processing and designing hemorphin-derived drug candidates. Copyright 2008 European Peptide Society and John Wiley & Sons, Ltd. [source]


    Post-synthesis incorporation of a lipidic side chain into a peptide on solid support

    JOURNAL OF PEPTIDE SCIENCE, Issue 11 2002
    Cline Douat
    Abstract A new strategy for the synthesis of lipopeptides has been developed. Using Weinreb (N -methoxy, N -methyl) amide as an aldehyde function precursor on the side chains of Asp or Glu residues, this new strategy avoids the synthesis of a lipidic amino acid residue before its incorporation in the peptide sequence. The aldehyde generated on the solid support can react with ylides leading to unsaturated or saturated side chains or with various nucleophiles to yield non-coded amino acid residues incorporated into the sequence. Copyright 2002 European Peptide Society and John Wiley & Sons, Ltd. [source]


    Synthesis of oligopeptides with the sequence SXWS and their chemotactic effects on a ciliated protozoan Tetrahymena pyriformis,

    JOURNAL OF PEPTIDE SCIENCE, Issue 1 2002
    Eszter Illys
    Abstract In this paper, the solid phase synthesis and chemical characterization of members of an SXWS sub-library (SAWS, SDWS and SKWS) as well as the comparison of their chemotactic properties with those of SEWS, which exhibits a prominent effect at 10,12M on a ciliated protozoan, Tetrahymena pyriformis, are described. We found that the chemotaxis of cells induced with the SXWS peptides varied according to the nature of the amino acid residue (Ala, Asp, Lys) in position X. The chemotactic activity of SEWS was not surpassed by any of three new tetrapeptides, although SAWS was also chemoattractant. Interestingly, SDWS, with an acidic side chain at position X, could not elicit any chemotactic response. SKWS, however, showed mild but significant chemorepellent activity over a wide concentration range. Chemotactic selection studies showed that the two chemoattractant peptides (SAWS and SEWS) had an expressed ability to select high-responder offspring cell populations. Peptides with neutral (SDWS) or chemorepellent (SKWS) properties were not able to select such subpopulations from the mixed cultures of Tetrahymena, indicating that the chemotactic response elicited by SXWS peptides is ligand-specific. For ligand-binding experiments N -terminally labelled fluorescent derivatives of SXWS peptides were prepared, applying [4-[7-hydroxycoumaryl]]acetic acid (Hca -OH) or 4-ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone (naOx -OEt) as markers. Hca -OH was introduced using an active ester technique as the last step of SPPS, or after cleavage in solution. The oxazolone naOx -OEt reacted with the amino group of the peptide by liberation of EtOH. The binding characteristics of fixed Tetrahymena cells with the naOx -labelled peptides showed good correlation between binding profiles and chemotactic responsiveness (SEWS > SAWS > SDWS , SKWS). A similar binding pattern was observed in the case of Hca -peptides (SEWS > SAWS > SDWS). Hca -SKWS, however, bound remarkably to the cell surface. The binding activity of the Hca -peptides was less pronounced than that of the naOx -peptides, indicating the importance of the fluorophores applied. Copyright 2002 European Peptide Society and John Wiley & Sons, Ltd. [source]


    Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: Application to the synthesis of phosphorylated p21Max protein(1,101)

    JOURNAL OF PEPTIDE SCIENCE, Issue 9 2001
    Toru Kawakami
    Abstract An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C -terminal building block, could be prepared from a recombinant protein; its N -terminal amino acid residue was transaminated to an ,-oxoacyl group, the side-chain amino groups were then protected with t -butoxycarbonyl (Boc) groups, and, finally, the ,-oxoacyl group was removed. On the other hand, an O -phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N -dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2,11]-p21Max(1,101), was successfully synthesized. Copyright 2001 European Peptide Society and John Wiley & Sons, Ltd. [source]


    Structural Coupling of 11- cis -7-Methyl-retinal and Amino Acids at the Ligand Binding Pocket of Rhodopsin,

    PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2009
    Mnica Aguil
    It was previously shown that opsin can be regenerated with the newly synthesized 11- cis -7-methyl-retinal forming an artificial visual pigment. We now extend this study to include mutants at positions close to the retinal to further dissect the interactions of native and artificial chromophores with opsin. Several mutants at M207, W265 and Y268 have been obtained and regenerated with 11- cis -retinal and the 7-methyl analog. M207 is the site of the point mutation M207R associated with the retinal degenerative disease retinitis pigmentosa. All the studied mutants regenerated with 11- cis -retinal except for M207C which proved to be completely misfolded. The naturally occurring M207R mutant formed a pigment with an unprotonated Schiff base linkage, altered photobleaching and low MetarhodopsinII stability. Mutants regenerated with the 7-methyl analog showed altered photobleaching reflecting a structural perturbation in the vicinity of M207. The newly obtained mutants at M207 also showed reduced levels of transducin activation with M207R showing essentially no transducin activation. Our results highlight the tight coupling of the vicinity of C7 of retinal and M207 and support the involvement of this amino acid residue in the conformational changes associated with rhodopsin photoactivation. [source]


    Gelatinization temperature of rice explained by polymorphisms in starch synthase

    PLANT BIOTECHNOLOGY JOURNAL, Issue 1 2006
    Daniel L. E. Waters
    Summary The cooking quality of rice is associated with the starch gelatinization temperature (GT). Rice genotypes with low GT have probably been selected for their cooking quality by humans during domestication. We now report polymorphisms in starch synthase IIa (SSIIa) that explain the variation in rice starch GT. Sequence analysis of the eight exons of SSIIa identified significant polymorphism in only exon 8. These single nucleotide polymorphisms (SNPs) were determined in 70 diverse genotypes of rice. Two SNPs could classify all 70 genotypes into either high GT or low GT types which differed in GT by 8 C. ,A' rather than ,G' at base 2412 determined whether a methionine or valine was present at the corresponding amino acid residue in SSIIa, whilst two adjacent SNPs at bases 2543 and 2544 coded for either leucine (GC) or phenylalanine (TT). Rice varieties with high GT starch had a combination of valine and leucine at these residues. In contrast, rice varieties with low GT starch had a combination of either methionine and leucine or valine and phenylalanine at these same residues. At least two distinct polymorphisms have apparently been selected for their desirable cooking qualities in the domestication of rice. [source]


    Structure of human brain fructose 1,6-(bis)phosphate aldolase: Linking isozyme structure with function

    PROTEIN SCIENCE, Issue 12 2004
    Tracy L. Arakaki
    Abstract Fructose-1,6-(bis)phosphate aldolase is a ubiquitous enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-(bis)phosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceral-dehyde-3-phosphate or glyceraldehyde, respectively. Vertebrate aldolases exist as three isozymes with different tissue distributions and kinetics: aldolase A (muscle and red blood cell), aldolase B (liver, kidney, and small intestine), and aldolase C (brain and neuronal tissue). The structures of human aldolases A and B are known and herein we report the first structure of the human aldolase C, solved by X-ray crystallography at 3.0 resolution. Structural differences between the isozymes were expected to account for isozyme-specific activity. However, the structures of isozymes A, B, and C are the same in their overall fold and active site structure. The subtle changes observed in active site residues Arg42, Lys146, and Arg303 are insufficient to completely account for the tissue-specific isozymic differences. Consequently, the structural analysis has been extended to the isozyme-specific residues (ISRs), those residues conserved among paralogs. A complete analysis of the ISRs in the context of this structure demonstrates that in several cases an amino acid residue that is conserved among aldolase C orthologs prevents an interaction that occurs in paralogs. In addition, the structure confirms the clustering of ISRs into discrete patches on the surface and reveals the existence in aldolase C of a patch of electronegative residues localized near the C terminus. Together, these structural changes highlight the differences required for the tissue and kinetic specificity among aldolase isozymes. [source]


    Characterization of heterotrimeric G protein complexes in rice plasma membrane

    THE PLANT JOURNAL, Issue 2 2004
    Chiyuki Kato
    Summary Two genes in the rice genome were identified as those encoding the , subunits, ,1 and ,2, of heterotrimeric G proteins. Using antibodies against the recombinant proteins for the ,, ,, ,1, and ,2 subunits of the G protein complexes, all of the subunits were proven to be localized in the plasma membrane in rice. Gel filtration of solubilized plasma membrane proteins showed that all of the , subunits were present in large protein complexes (about 400 kDa) containing the other subunits, ,, ,1, and ,2, and probably also some other proteins, whereas large amounts of the , and , (,1 and ,2) subunits were freed from the large complexes and took a 60-kDa form. A yeast two-hybrid assay and co-immunoprecipitation experiments showed that the , subunit interacted tightly with the ,1 and ,2 subunits, and so the , and , subunits appeared to form dimers in rice cells. Some dimers were associated with the , subunit, because few ,, ,1, and ,2 subunits were present in the 400-kDa complexes in a rice mutant, d1, which was lacking in the , subunit. When a constitutively active form of the , subunit was prepared by the exchange of one amino acid residue and introduced into d1, the mutagenized subunit was localized in the plasma membrane of the transformants and took a free, and not the 400-kDa, form. [source]


    Haplotype analysis of the human ,2-HS glycoprotein (fetuin) gene

    ANNALS OF HUMAN GENETICS, Issue 1 2001
    M. OSAWA
    Alpha2-HS glycoprotein (AHSG), which is equivalent to fetuin in other species, is a protein found in human plasma. AHSG is polymorphic with two common alleles and many variants. To examine the intragenic haplotypes and their diversity at this locus, a contiguous genomic DNA sequence (103 kb) was analyzed in 20 samples (40 chromosomes), and haplotypes were determined for 309 subjects. Judging from the aligned nucleotide sequences and the conserved amino acid residues comparing human and chimpanzee AHSG, it was concluded that the type 1 allele is probably older and has evolved into four major suballeles. The type 2 allele was generated from one branch of the type 1 allele. AHSG*3 and *5 variants were each found to have a single nucleotide change in exon 7, resulting in the change of an amino acid residue from Arg299 to Cys and from Asp258 to Asn, respectively. It was noted that the AHSG*3 mutation gives rise to an additional cysteine residue, which possibly affects the conformation of the protein. The AHSG gene was found to have a low mutation rate and no apparent recombination events. Furthermore, the detected substitutions were nonhomogeneously distributed at this locus. In particular, four nonsynonymous substitutions were concentrated in the carboxyl-terminal domain. [source]


    Analysis of the conformational profile of trishomocubane amino acid dipeptide

    BIOPOLYMERS, Issue 5 2006
    Krishna Bisetty
    Abstract 4-Amino-(D3)-trishomocubane-4-carboxylic acid is a constrained ,-amino acid residue that exhibits promising conformational characteristics, i.e., helical and ,-turns. As part of the development of conformational guidelines for the design of peptides and protein surrogates, the conformational energy calculations on trishomocubane using molecular mechanics and ab initio methods are presented. The C, carbon of trishomocubane forms part of the cyclic structure, and consequently a peptidic environment was simulated with an acetyl group on its N-terminus and a methylamide group on its C-terminus. Ramachandran maps computed at the molecular mechanics level using the standard AMBER (parm94) force field libraries compared reasonably well with the corresponding maps computed at the Hartree Fock level, using the 6-31G* basis set. Trishomocubane peptide (Ac-Tris-NHMe) is characterized by four low energy conformers corresponding to the C7ax, C7eq, 310, and ,L helical structures. 2005 Wiley Periodicals, Inc. Biopolymers 81: 339,349, 2006 This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source]


    Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris

    CHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2001
    J.B. Kim
    Abstract: Six peptides with antimicrobial activity were isolated from an extract of freeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin-1Oa FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-1Ob FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-1Oc FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-1Od FLPLLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic ,-helical conformation but it is significant that temporin-1Od, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 m compared with 2 m for temporin-1Oa). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity. [source]


    A Novel Heavy-Atom Label for Side-Specific Peptide Iodination: Synthesis, Membrane Incorporation and X-ray Reflectivity

    CHEMPHYSCHEM, Issue 9-10 2009
    Philipp E. Schneggenburger
    Abstract A novel iodine peptide label for X-ray analysis of membrane-active peptide structures is applied to solid-phase peptide synthesis. The resulting pore-structured labeled peptide as well as a non-labeled reference were reconstituted in lipid bilayer stacks (see scheme). The results indicate the exhibition of a membrane-spanning ,5.6 -double helical peptide structure and illustrate the quality of the new label. Structural parameters, such as conformation, orientation and penetration depth of membrane-bound peptides and proteins that may function as channels, pores or biocatalysts, are of persistent interest and have to be probed in the native fluid state of a membrane. X-ray scattering in combination with heavy-atom labeling is a powerful and highly appropriate method to reveal the position of a certain amino acid residue within a lipid bilayer with respect to the membrane normal axis up to a resolution of several ngstrm. Herein, we report the synthesis of a new iodine-labeled amino acid building block. This building block is intended for peptide incorporation to provide high intensities for electron density difference analysis of X-ray reflectivity data and improve the labeling potential for the lipid bilayer head-group and water region. The novel building block as well as the commercially available non-iodinated analogue, required for X-ray scattering, was implemented in a transmembrane peptide motif via manual solid-phase peptide synthesis (SPPS) following the fluorenylmethyloxycarbonyl (Fmoc)-strategy. The derived peptides were reconstituted in lipid vesicles as well as in highly aligned multilamellar lipid stacks and investigated via circular dichroism (CD) and X-ray reflectivity. Thereby, it has been revealed that the bulky iodine probe neither causes conformational change of the peptide structure nor lamellar disordering of the membrane complexes. [source]


    Chiral recognition of dipeptide methyl esters by an anionic ,-cyclodextrin

    CHIRALITY, Issue 8 2001
    Koji Kano
    Abstract Chiral recognition of dipeptide methyl esters by anionic heptakis[6-carboxymethylthio-6-deoxy]-,-cyclodextrin (per-CO2, -,-CD) was studied in D2O at pD 7.0 by means of 1H NMR spectroscopy. The methyl esters of alanylalanine (Ala-Ala-OMe), alanylleucine (Ala-Leu-OMe), alanyltryptophan (Ala-Trp-OMe), glycyltryptophan (Gly-Trp-OMe), valyltryptophan (Val-Trp-OMe), leucyltryptophan (Leu-Trp-OMe), and tryptophylalanine (Trp-Ala-OMe) were used as the dipeptides. The binding constant (K) determined from NMR titration increases in the order Ala-Ala-OMe < Ala-Leu-OMe < Ala-Trp-OMe, suggesting that van der Waals interactions between the host and the guest participate in complexation. Coulomb interactions between the protonated dipeptide methyl esters and the anionic host seem to be another attractive force. Per-CO2, -,-CD interacts with the (R,R)-enantiomers of the dipeptide methyl esters more strongly than the (S,S)-enantiomers. Such enantioselectivity corresponds to that for ,-amino acid methyl esters such as Leu-OMe and Trp-OMe, whose (R)-enantiomers are the preferable guests. The enantioselectivity is mainly dominated by amino acid residue at the C -terminal and chirality at the N -terminal residue plays an assistant role. An asymmetrically twisted shape of the host cavity may be essential for chiral recognition. Chirality 13:474,482, 2001. 2001 Wiley-Liss, Inc. [source]


    Cloning and characterization of cDNA for syndecan core protein in sea urchin embryos

    DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2000
    Kazuo Tomita
    The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription,polymerase chain reaction (RT-PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan-1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae. [source]


    Polylysine-Catalyzed Hydrogen Evolution at Mercury Electrodes

    ELECTROANALYSIS, Issue 17-18 2010
    Marko, ivanovi
    Abstract It has been shown that peptides and proteins produce at nanomolar concentrations a structure-sensitive chronopotentiometric peak H at mercury electrodes, which is due to the catalytic hydrogen evolution reaction (HER). Herein, we use for the first time poly(amino acids) to obtain information about the role of individual amino acid residues in the HER. At pH,6 polylysine (polyLys) and polyarginine,tryptophan yield a peak H, in agreement with their ionization state, while polyglutamic acid gives no catalytic response. PolyLys catalyzes hydrogen evolution in its adsorbed state. Even at potentials negative to the potential of zero charge, hydrophobic interactions could be involved in polyLys adsorption. [source]


    Cloning and Characterization of the cDNA Encoding the Masquerade-like Serine Proteinase Homologue Gene of the Silkworm, Bombyx mori

    ENTOMOLOGICAL RESEARCH, Issue 3 2002
    Doo-Sang PARK
    ABSTRACT From Bombyx mori larvae, RT-PCR and cDNA library screening isolated masquerade-like serine proteinase homologue cDNA gene, proposed to be related to insect immunity and its characteristics were examined. The isolated gene is composed of 1.3 kb of nucleotide and 420 amino acid residues were encoded. According to the results of database search, the isolated gene showed high sequence homology with Holotrichia and Tenebrio's 45 kDa protein, Drosophila CG5390 gene. Moreover, it is composed of regulatory domain and catalytic domain, which is characteristic of serine proteinase that can be found in the insect immune reaction and embryonic development processes. Enzyme activation site by proteolytic cleavage and the sequence of three amino acids participate in the catalytic triad of enzyme and 14 cystein residues used in disulfide bridges are well conserved with the compared genes. The mRNA expression was increased following E. coli injection and constitutive expression was also observed before injection by Northern blot analysis. [source]