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Sample records for acid protein consisting

  1. Comparative characterization of random-sequence proteins consisting of 5, 12, and 20 kinds of amino acids.

    PubMed

    Tanaka, Junko; Doi, Nobuhide; Takashima, Hideaki; Yanagawa, Hiroshi

    2010-04-01

    Screening of functional proteins from a random-sequence library has been used to evolve novel proteins in the field of evolutionary protein engineering. However, random-sequence proteins consisting of the 20 natural amino acids tend to aggregate, and the occurrence rate of functional proteins in a random-sequence library is low. From the viewpoint of the origin of life, it has been proposed that primordial proteins consisted of a limited set of amino acids that could have been abundantly formed early during chemical evolution. We have previously found that members of a random-sequence protein library constructed with five primitive amino acids show high solubility (Doi et al., Protein Eng Des Sel 2005;18:279-284). Although such a library is expected to be appropriate for finding functional proteins, the functionality may be limited, because they have no positively charged amino acid. Here, we constructed three libraries of 120-amino acid, random-sequence proteins using alphabets of 5, 12, and 20 amino acids by preselection using mRNA display (to eliminate sequences containing stop codons and frameshifts) and characterized and compared the structural properties of random-sequence proteins arbitrarily chosen from these libraries. We found that random-sequence proteins constructed with the 12-member alphabet (including five primitive amino acids and positively charged amino acids) have higher solubility than those constructed with the 20-member alphabet, though other biophysical properties are very similar in the two libraries. Thus, a library of moderate complexity constructed from 12 amino acids may be a more appropriate resource for functional screening than one constructed from 20 amino acids. PMID:20162614

  2. Extensive amino acid polymorphism at the pgm locus is consistent with adaptive protein evolution in Drosophila melanogaster.

    PubMed Central

    Verrelli, B C; Eanes, W F

    2000-01-01

    PGM plays a central role in the glycolytic pathway at the branch point leading to glycogen metabolism and is highly polymorphic in allozyme studies of many species. We have characterized the nucleotide diversity across the Pgm gene in Drosophila melanogaster and D. simulans to investigate the role that protein polymorphism plays at this crucial metabolic branch point shared with several other enzymes. Although D. melanogaster and D. simulans share common allozyme mobility alleles, we find these allozymes are the result of many different amino acid changes at the nucleotide level. In addition, specific allozyme classes within species contain several amino acid changes, which may explain the absence of latitudinal clines for PGM allozyme alleles, the lack of association of PGM allozymes with the cosmopolitan In(3L)P inversion, and the failure to detect differences between PGM allozymes in functional studies. We find a significant excess of amino acid polymorphisms within D. melanogaster when compared to the complete absence of fixed replacements with D. simulans. There is also strong linkage disequilibrium across the 2354 bp of the Pgm locus, which may be explained by a specific amino acid haplotype that is high in frequency yet contains an excess of singleton polymorphisms. Like G6pd, Pgm shows strong evidence for a branch point enzyme that exhibits adaptive protein evolution. PMID:11102370

  3. Proteins and Amino Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the most abundant substances in living organisms and cells. All proteins are constructed from the same twenty amino acids that are linked together by covalent bonds. Shorter chains of two or more amino acids can be linked by covalent bonds to form polypeptides. There are twenty amino...

  4. The Defensins Consist of Two Independent, Convergent Protein Superfamilies.

    PubMed

    Shafee, Thomas M A; Lay, Fung T; Hulett, Mark D; Anderson, Marilyn A

    2016-09-01

    The defensin and defensin-like proteins are an extensive group of small, cationic, disulfide-rich proteins found in animals, plants, and fungi and mostly perform roles in host defense. The term defensin was originally used for small mammalian proteins found in neutrophils and was subsequently applied to insect proteins and plant γ-thionins based on their perceived sequence and structural similarity. Defensins are often described as ancient innate immunity molecules and classified as a single superfamily and both sequence alignments and phylogenies have been constructed. Here, we present evidence that the defensins have not all evolved from a single ancestor. Instead, they consist of two analogous superfamilies, and extensive convergent evolution is the source of their similarities. Evidence of common origin necessarily gets weaker for distantly related genes, as is the case for defensins, which are both divergent and small. We show that similarities that have been used as evidence for common origin are all expected by chance in short, constrained, disulfide-rich proteins. Differences in tertiary structure, secondary structure order, and disulfide bond connectivity indicate convergence as the likely source of the similarity. We refer to the two evolutionarily independent groups as the cis-defensins and trans-defensins based on the orientation of the most conserved pair of disulfides. PMID:27297472

  5. Gene stability in mammalian cells and protein consistency.

    PubMed

    Berthold, W

    1994-01-01

    The safety of a patient who is the recipient of protein drugs has to be assured. A "wrong" protein is thought to represent a great risk. The philosophy of testing strategies related to gene stability with product safety will be discussed in the light of experimental data available today. Although all mammalian cell lines used in the production of biologicals including recombinant DNA-derived lines have been produced from individual clones (functional monoclonality) they have been found to be heterogenous with regard to the genomic content (number of chromosomes, characteristics of identifiable chromosomes and position and number of integrated recombinant sequences). The verification of the presence of correct gene in a production cell line constitutes a well accepted and useful test, especially if derived by "population sequencing". A batch not related repeated confirmation of this fact cannot lead to any additional assurance for the correctness of all proteins constituting a given product beyond the level provided by cheminal testing. In contrast to this obvious and unavoidable heterogeneity in cellular genomes, the coding regions of genes have not been shown to change. Evidence is available to demonstrate the consistency of protein products originating from recombinant (and hybridoma) cell lines, e.g. more than 500,000 patients have received and tolerated rtPA well. PMID:7883100

  6. A new drug nanocarrier consisting of polyarginine and hyaluronic acid.

    PubMed

    Oyarzun-Ampuero, Felipe A; Goycoolea, Francisco M; Torres, Dolores; Alonso, Maria J

    2011-09-01

    The purpose of this study was to produce and characterize a variety of nanostructures comprised of the polyaminoacid polyarginine (PArg) and the polysaccharide hyaluronic acid (HA) as a preliminary stage before evaluating their potential application in drug delivery. PArg was combined with high- or low-molecular-weight HA (HMWHA or LMWHA, respectively) to form nanoparticles by simply mixing polymeric aqueous solutions at room temperature. The average size of the resulting nanocarriers was between 116 and 155 nm, and their zeta potential value ranged from +31.3 to -35.9 mV, indicating that the surface composition of the particle could be conveniently modified according to the mass ratio of the polymers. Importantly, the systems prepared with HMWHA remained stable after isolation by centrifugation and in conditions that mimic the physiological medium, whereas particles that incorporated LMWHA were unstable. Transmission electron microscopy showed that the nanostructures made with HMWHA were spherical. Finally, the systems were stable for at least three months at storage conditions (4°C). PMID:21549838

  7. Changes in flexibility upon binding: Application of the self-consistent pair contact probability method to protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Canino, Lawrence S.; Shen, Tongye; McCammon, J. Andrew

    2002-12-01

    We extend the self-consistent pair contact probability method to the evaluation of the partition function for a protein complex at thermodynamic equilibrium. Specifically, we adapt the method for multichain models and introduce a parametrization for amino acid-specific pairwise interactions. This method is similar to the Gaussian network model but allows for the adjusting of the strengths of native state contacts. The method is first validated on a high resolution x-ray crystal structure of bovine Pancreatic Phospholipase A2 by comparing calculated B-factors with reported values. We then examine binding-induced changes in flexibility in protein-protein complexes, comparing computed results with those obtained from x-ray crystal structures and molecular dynamics simulations. In particular, we focus on the mouse acetylcholinesterase:fasciculin II and the human α-thrombin:thrombomodulin complexes.

  8. Human Protein and Amino Acid Requirements.

    PubMed

    Hoffer, L John

    2016-05-01

    Human protein and amino acid nutrition encompasses a wide, complex, frequently misunderstood, and often contentious area of clinical research and practice. This tutorial explains the basic biochemical and physiologic principles that underlie our current understanding of protein and amino acid nutrition. The following topics are discussed: (1) the identity, measurement, and essentiality of nutritional proteins; (2) the definition and determination of minimum requirements; (3) nutrition adaptation; (4) obligatory nitrogen excretion and the minimum protein requirement; (5) minimum versus optimum protein intakes; (6) metabolic responses to surfeit and deficient protein intakes; (7) body composition and protein requirements; (8) labile protein; (9) N balance; (10) the principles of protein and amino acid turnover, including an analysis of the controversial indicator amino acid oxidation technique; (11) general guidelines for evaluating protein turnover articles; (12) amino acid turnover versus clearance; (13) the protein content of hydrated amino acid solutions; (14) protein requirements in special situations, including protein-catabolic critical illness; (15) amino acid supplements and additives, including monosodium glutamate and glutamine; and (16) a perspective on the future of protein and amino acid nutrition research. In addition to providing practical information, this tutorial aims to demonstrate the importance of rigorous physiologic reasoning, stimulate intellectual curiosity, and encourage fresh ideas in this dynamic area of human nutrition. In general, references are provided only for topics that are not well covered in modern textbooks. PMID:26796095

  9. Protein biosynthesis with conformationally restricted amino acids

    SciTech Connect

    Mendel, D. Lawrence Berkeley Lab., CA ); Ellman, J.; Schultz, P.G. )

    1993-05-19

    The incorporation of conformationally constrained amino acids into peptides is a powerful approach for generating structurally defined peptides as conformational probes and bioactive agents. The ability to site-specifically introduce constrained amino acids into large polypeptide chains would provide a similar opportunity to probe the flexibility, conformation, folding and stability of proteins. To this end, we have examined the competence of the Escherichia coli protein biosynthetic machinery to incorporate a number of these unnatural amino acids into the 164 residue protein T4 lysozyme (T4L). Results clearly demonstrate that the protein biosynthetic machinery can accommodate a wide variety of conformationally constrained amino acids. The expansion of structural motifs that can be biosynthetically incorporated into proteins to include a large number of conformationally constrained amino acids significantly increases the power of mutagenesis methods as probes of protein structure and function and provides additional insights into the steric requirements of the translational machinery. 13 refs., 2 figs.

  10. Distinguishing Proteins From Arbitrary Amino Acid Sequences

    PubMed Central

    Yau, Stephen S.-T.; Mao, Wei-Guang; Benson, Max; He, Rong Lucy

    2015-01-01

    What kinds of amino acid sequences could possibly be protein sequences? From all existing databases that we can find, known proteins are only a small fraction of all possible combinations of amino acids. Beginning with Sanger's first detailed determination of a protein sequence in 1952, previous studies have focused on describing the structure of existing protein sequences in order to construct the protein universe. No one, however, has developed a criteria for determining whether an arbitrary amino acid sequence can be a protein. Here we show that when the collection of arbitrary amino acid sequences is viewed in an appropriate geometric context, the protein sequences cluster together. This leads to a new computational test, described here, that has proved to be remarkably accurate at determining whether an arbitrary amino acid sequence can be a protein. Even more, if the results of this test indicate that the sequence can be a protein, and it is indeed a protein sequence, then its identity as a protein sequence is uniquely defined. We anticipate our computational test will be useful for those who are attempting to complete the job of discovering all proteins, or constructing the protein universe. PMID:25609314

  11. Adsorption/desorption in a system consisting of humic acid, heavy metals, and clay minerals

    SciTech Connect

    Liu, A.; Gonzalez, R.D.

    1999-10-01

    Metal adsorption/desorption in a system consisting of humic acid, metal ions, and clay minerals is described. Montmorillonite and purified humic acid were selected as prototype materials for this study. At a constant ionic strength, the amount of humic acid adsorbed on montmorillonite decreases when pH is increased. A slight increase in humic acid adsorption on montmorillonite is observed when there are bivalent metals present in the system. The metal adsorption on montmorillonite does not correlate to the amount of humic acid adsorbed on montmorillonite. Montmorillonite with preadsorbed humic acid does not show a significant change in the capacity of adsorbed metal ions. An increase in the ionic strength at a pH of 6.5 results in an increase in the adsorption of lead on montmorillonite in the presence of humic acid, while at a lower pH, the increase in ionic strength results in a decrease in metal adsorption. The bridging of bivalent metal ions between montmorillonite and humic acid is proposed as the dominant adsorption mechanism.

  12. The Consistency of Isotopologues of Ambient Atmospheric Nitric Acid in Passively Collected Samples

    NASA Astrophysics Data System (ADS)

    Bell, M. D.; Sickman, J. O.; Bytnerowicz, A.; Padgett, P.; Allen, E. B.

    2012-12-01

    Anthropogenic sources of nitrogen oxides have previously been shown to have distinctive isotopic signatures of oxygen and nitrogen. Nylon filters are currently used in passive sampling arrays to measure ambient atmospheric nitric acid concentrations and estimate deposition rates. This experiment measured the ability of nylon filters to consistently collect isotopologues of atmospheric nitric acid in the same ratios as they are present in the atmosphere. Samplers were deployed in continuous stirred tank reactors (CSTR) and at field sites across a nitrogen deposition gradient in Southern California. Filters were exposed over a four week period with individual filters being subjected to 1-4 week exposure times. Extracted nitric acid were measured for δ18O and δ15N ratios and compared for consistency based on length of exposure and amount of HNO3 collected. Filters within the CSTRs collected HNO3 at a consistent rate in both high and low concentration chambers. After two weeks of exposure, the mean δ18O values were within 0.5‰ of the δ18O of the source HNO3 solution. The mean of all weekly exposures were within 0.5‰ of the δ15N of the source solution, but after three weeks, the mean δ15N of adsorbed HNO3 was within 0.2‰. As the length of the exposure increased, the variability of measured delta values decreased for both elements. The field samplers collected HNO3 consistent with previously measured values along a deposition gradient. The mean δ18O at high deposition sites was 52.2‰ compared to 35.7‰ at the low deposition sites. Mean δ15N values were similar at all sites across the deposition gradient. Due to precipitation events occurring during the exposure period, the δ15N and δ18O of nitric acid were highly variable at all field sites. At single sites, changes in δ15N and δ18O were negatively correlated, consistent with two-sourcing mixing dynamics, but the slope of the regressions differed between high and low deposition sites. Anthropogenic

  13. Probing protein stability with unnatural amino acids

    SciTech Connect

    Mendel, D.; Ellman, J.A.; Zhiyuh Chang; Veenstra, D.L.; Kollman, P.A.; Schultz, P.G. )

    1992-06-26

    Unnatural amino acid mutagenesis, in combination with molecular modeling and simulation techniques, was used to probe the effect of side chain structure on protein stability. Specific replacements at position 133 in T4 lysozyme included (1) leucine (wt), norvaline, ethylglycine, and alanine to measure the cost of stepwise removal of methyl groups from the hydrophobic core, (2) norvaline and O-methyl serine to evaluate the effects of side chain solvation, and (3) leucine, S,S-2-amino-4-methylhexanoic acid, and S-2-amino-3-cyclopentylpropanoic acid to measure the influence of packing density and side chain conformational entropy on protein stability. All of these factors (hydrophobicity, packing, conformational entropy, and cavity formation) significantly influence protein stability and must be considered when analyzing any structural change to proteins.

  14. IR-UV photochemistry of protein-nucleic acid systems

    SciTech Connect

    Kozub, J.; Edwards, G.

    1995-12-31

    UV light has often been used to induce the formation of covalent bonds between DNA (or RNA) and tightly-bound protein molecules. However, the internal photoreactions of nucleic acids and proteins limit the yield and complicate the analysis of intermolecular crosslinks. In an ongoing search for improved reaction specificity or new photoreactions in these systems, we have employed UV photons from a Nd:YAG-pumped dye laser and mid-IR photons from the Vanderbilt FEL. Having crosslinked several protein-nucleic acid systems with nanosecond UV laser pulses, we are currently studying the effect of various IR wavelengths on a model system (gene 32 protein and poly[dT]). We have found that irradiation with sufficiently intense FEL macropulses creates an altered form of gene 32 protein which was not observed with UV-only irradiation. The electrophoretic nobility of the product is consistent with the formation of a specific protein-protein crosslink. No evidence of the non-specific protein damage typically induced by UV light is found. The yield of the new photoproduct is apparently enhanced by exposure to FEL macropulses which are synchronized with UV laser pulses. With ideal exposure parameters, the two-color reaction effectively competes with UV-only reactions. Experiments designed to determine the reaction mechanism and to demonstrate FEL-induced reactions in other protein-nucleic acid systems are currently underway.

  15. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    NASA Astrophysics Data System (ADS)

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts.

  16. Modifications of proteins by polyunsaturated fatty acid peroxidation products

    PubMed Central

    Refsgaard, Hanne H. F.; Tsai, Lin; Stadtman, Earl R.

    2000-01-01

    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O2] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (α,β-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts. PMID:10639127

  17. FLU, an amino acid substitution model for influenza proteins

    PubMed Central

    2010-01-01

    Background The amino acid substitution model is the core component of many protein analysis systems such as sequence similarity search, sequence alignment, and phylogenetic inference. Although several general amino acid substitution models have been estimated from large and diverse protein databases, they remain inappropriate for analyzing specific species, e.g., viruses. Emerging epidemics of influenza viruses raise the need for comprehensive studies of these dangerous viruses. We propose an influenza-specific amino acid substitution model to enhance the understanding of the evolution of influenza viruses. Results A maximum likelihood approach was applied to estimate an amino acid substitution model (FLU) from ~113, 000 influenza protein sequences, consisting of ~20 million residues. FLU outperforms 14 widely used models in constructing maximum likelihood phylogenetic trees for the majority of influenza protein alignments. On average, FLU gains ~42 log likelihood points with an alignment of 300 sites. Moreover, topologies of trees constructed using FLU and other models are frequently different. FLU does indeed have an impact on likelihood improvement as well as tree topologies. It was implemented in PhyML and can be downloaded from ftp://ftp.sanger.ac.uk/pub/1000genomes/lsq/FLU or included in PhyML 3.0 server at http://www.atgc-montpellier.fr/phyml/. Conclusions FLU should be useful for any influenza protein analysis system which requires an accurate description of amino acid substitutions. PMID:20384985

  18. Detection of non-protein amino acids in the presence of protein amino acids. II.

    NASA Technical Reports Server (NTRS)

    Shapshak, P.; Okaji, M.

    1972-01-01

    Studies conducted with the JEOL 5AH amino acid analyzer are described. This instrument makes possible the programming of the chromatographic process. Data are presented showing the separations of seventeen non-protein amino acids in the presence of eighteen protein amino acids. It is pointed out that distinct separations could be obtained in the case of a number of chemically similar compounds, such as ornithine and lysine, N-amidino alanine and arginine, and iminodiacetic acid and S-carboxymethyl cysteine and aspartic acid.

  19. Protein and amino Acid supplementation in athletes.

    PubMed

    Armsey, Thomas D; Grime, Todd E

    2002-08-01

    Amino acid supplementation is practiced by numerous individuals with the hope of increasing muscle mass and function by increasing available proteins. Theoretically, this makes a great deal of sense; the scientific facts, however, fail to conclusively prove that ingesting more than the recommended dietary allowance of protein has any effect on otherwise healthy adults. Athletes may be the exception to this rule. This review examines the most current literature pertaining to amino acid supplementation, and reports on the potential benefits and risks of this common practice. PMID:12831703

  20. Using self-consistent fields to bias Monte Carlo methods with applications to designing and sampling protein sequences

    NASA Astrophysics Data System (ADS)

    Zou, Jinming; Saven, Jeffery G.

    2003-02-01

    For complex multidimensional systems, Monte Carlo methods are useful for sampling probable regions of a configuration space and, in the context of annealing, for determining "low energy" or "high scoring" configurations. Such methods have been used in protein design as means to identify amino acid sequences that are energetically compatible with a particular backbone structure. As with many other applications of Monte Carlo methods, such searches can be inefficient if trial configurations (protein sequences) in the Markov chain are chosen randomly. Here a mean-field biased Monte Carlo method (MFBMC) is presented and applied to designing and sampling protein sequences. The MFBMC method uses predetermined sequence identity probabilities wi(α) to bias the sequence selection. The wi(α) are calculated using a self-consistent, mean-field theory that can estimate the number and composition of sequences having predetermined values of energetically related foldability criteria. The MFBMC method is applied to both a simple protein model, the 27-mer lattice model, and an all-atom protein model. Compared to conventional Monte Carlo (MC) and configurational bias Monte Carlo (BMC), the MFBMC method converges faster to low energy sequences and samples such sequences more efficiently. The MFBMC method also tolerates faster cooling rates than the MC and BMC methods. The MFBMC method can be applied not only to protein sequence search, but also to a wide variety of polymeric and condensed phase systems.

  1. Acid Cleavable Surface enhanced Raman Tagging for Protein Detection

    PubMed Central

    Zhang, Dongmao; Vangala, Karthikeshwar; Li, Shaoyong; Yanney, Michael; Xia, Hao; Zou, Sige; Sygula, Andrzej

    2010-01-01

    Dye conjugation is a common strategy improving the surface enhanced Raman detection sensitivity of biomolecules. Reported is a proof-of-concept study of a novel surface enhanced Raman spectroscopic tagging strategy termed as acid-cleavable SERS tag (ACST) method. Using Rhodamine B as the starting material, we prepared the first ACST prototype that consisted of, from the distal end, a SERS tag moiety (STM), an acid-cleavable linker, and a protein reactive moiety. Complete acid cleavage of the ACST tags was achieved at a very mild condition that is 1.5% trifluoroacetic acid (TFA) aqueous solution at room temperature. SERS detection of this ACST tagged protein was demonstrated using bovine serum albumin (BSA) as the model protein. While the SERS spectrum of intact ACST-BSA was entirely dominated by the fluorescent signal of STM, quality SERS spectra can be readily obtained with the acid cleaved ACST-BSA conjugates. Separation of the acid cleaved STM from protein further enhances the SERS sensitivity. Current SERS detection sensitivity, achieved with the acid cleaved ACST-BSA conjugate is ~5 nM in terms of the BSA concentration and ~1.5 nM in ACST content. The linear dynamic range of the cleaved ACST-BSA conjugate spans four orders of magnitudes from ~10 nM to ~100 μM in protein concentrations. Further improvement in the SERS sensitivity can be achieved with resonance Raman acquisition. This cleavable tagging strategy may also be used for elimination of protein interference in fluorescence based biomolecule detection. PMID:21109888

  2. Nucleic acids, proteins, and chirality

    NASA Technical Reports Server (NTRS)

    Usher, D. A.; Profy, A. T.; Walstrum, S. A.; Needels, M. C.; Bulack, S. C.; Lo, K. M.

    1984-01-01

    The present investigation is concerned with experimental results related, in one case, to the chirality of nucleotides, and, in another case, to the possibility of a link between the chirality of nucleic acids, and that of peptides. It has been found that aminoacylation of the 'internal' hydroxyl group of a dinucleoside monophosphate can occur stereoselectively. However, this reaction has not yet been made a part of a working peptide synthesis scheme. The formation and cleavage of oligonucleotides is considered. In the event of the formation of a helical complex between the oligonucleotide and the polymer, 1-prime,5-prime-bonds in the oligomer are found to become more resistant towards cleavage. The conditions required for peptide bond formation are examined, taking into account the known structures of RNA and possible mechanisms for prebiotic peptide bond formation. The possibility is considered that the 2-prime,5-prime-internucleotide linkage could have played an important part in the early days of biological peptide synthesis.

  3. Dihydrolipoic acid reduces cytochrome b561 proteins.

    PubMed

    Bérczi, Alajos; Zimányi, László; Asard, Han

    2013-03-01

    Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins that are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized and the putative tumor suppressor Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins is also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins. PMID:22526465

  4. Consistent Treatment of Hydrophobicity in Protein Lattice Models Accounts for Cold Denaturation

    NASA Astrophysics Data System (ADS)

    van Dijk, Erik; Varilly, Patrick; Knowles, Tuomas P. J.; Frenkel, Daan; Abeln, Sanne

    2016-02-01

    The hydrophobic effect stabilizes the native structure of proteins by minimizing the unfavorable interactions between hydrophobic residues and water through the formation of a hydrophobic core. Here, we include the entropic and enthalpic contributions of the hydrophobic effect explicitly in an implicit solvent model. This allows us to capture two important effects: a length-scale dependence and a temperature dependence for the solvation of a hydrophobic particle. This consistent treatment of the hydrophobic effect explains cold denaturation and heat capacity measurements of solvated proteins.

  5. Consistent Treatment of Hydrophobicity in Protein Lattice Models Accounts for Cold Denaturation.

    PubMed

    van Dijk, Erik; Varilly, Patrick; Knowles, Tuomas P J; Frenkel, Daan; Abeln, Sanne

    2016-02-19

    The hydrophobic effect stabilizes the native structure of proteins by minimizing the unfavorable interactions between hydrophobic residues and water through the formation of a hydrophobic core. Here, we include the entropic and enthalpic contributions of the hydrophobic effect explicitly in an implicit solvent model. This allows us to capture two important effects: a length-scale dependence and a temperature dependence for the solvation of a hydrophobic particle. This consistent treatment of the hydrophobic effect explains cold denaturation and heat capacity measurements of solvated proteins. PMID:26943560

  6. Characterization and amino acid sequence of a fatty acid-binding protein from human heart.

    PubMed

    Offner, G D; Brecher, P; Sawlivich, W B; Costello, C E; Troxler, R F

    1988-05-15

    The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5. PMID:3421901

  7. GPU.proton.DOCK: Genuine Protein Ultrafast proton equilibria consistent DOCKing.

    PubMed

    Kantardjiev, Alexander A

    2011-07-01

    GPU.proton.DOCK (Genuine Protein Ultrafast proton equilibria consistent DOCKing) is a state of the art service for in silico prediction of protein-protein interactions via rigorous and ultrafast docking code. It is unique in providing stringent account of electrostatic interactions self-consistency and proton equilibria mutual effects of docking partners. GPU.proton.DOCK is the first server offering such a crucial supplement to protein docking algorithms--a step toward more reliable and high accuracy docking results. The code (especially the Fast Fourier Transform bottleneck and electrostatic fields computation) is parallelized to run on a GPU supercomputer. The high performance will be of use for large-scale structural bioinformatics and systems biology projects, thus bridging physics of the interactions with analysis of molecular networks. We propose workflows for exploring in silico charge mutagenesis effects. Special emphasis is given to the interface-intuitive and user-friendly. The input is comprised of the atomic coordinate files in PDB format. The advanced user is provided with a special input section for addition of non-polypeptide charges, extra ionogenic groups with intrinsic pK(a) values or fixed ions. The output is comprised of docked complexes in PDB format as well as interactive visualization in a molecular viewer. GPU.proton.DOCK server can be accessed at http://gpudock.orgchm.bas.bg/. PMID:21666258

  8. The Exchangeability of Amino Acids in Proteins

    PubMed Central

    Yampolsky, Lev Y.; Stoltzfus, Arlin

    2005-01-01

    The comparative analysis of protein sequences depends crucially on measures of amino acid similarity or distance. Many such measures exist, yet it is not known how well these measures reflect the operational exchangeability of amino acids in proteins, since most are derived by methods that confound a variety of effects, including effects of mutation. In pursuit of a pure measure of exchangeability, we present (1) a compilation of data on the effects of 9671 amino acid exchanges engineered and assayed in a set of 12 proteins; (2) a statistical procedure to combine results from diverse assays of exchange effects; (3) a matrix of “experimental exchangeability” values EXij derived from applying this procedure to the compiled data; and (4) a set of three tests designed to evaluate the power of an exchangeability measure to (i) predict the effects of amino acid exchanges in the laboratory, (ii) account for the disease-causing potential of missense mutations in the human population, and (iii) model the probability of fixation of missense mutations in evolution. EX not only captures useful information on exchangeability while remaining free of other effects, but also outperforms all measures tested except for the best-performing alignment scoring matrix, which is comparable in performance. PMID:15944362

  9. Protein and Amino Acid Requirements during Pregnancy.

    PubMed

    Elango, Rajavel; Ball, Ronald O

    2016-07-01

    Protein forms an essential component of a healthy diet in humans to support both growth and maintenance. During pregnancy, an exceptional stage of life defined by rapid growth and development, adequate dietary protein is crucial to ensure a healthy outcome. Protein deposition in maternal and fetal tissues increases throughout pregnancy, with most occurring during the third trimester. Dietary protein intake recommendations are based on factorial estimates because the traditional method of determining protein requirements, nitrogen balance, is invasive and undesirable during pregnancy. The current Estimated Average Requirement and RDA recommendations of 0.88 and 1.1 g · kg(-1) · d(-1), respectively, are for all stages of pregnancy. The single recommendation does not take into account the changing needs during different stages of pregnancy. Recently, with the use of the minimally invasive indicator amino acid oxidation method, we defined the requirements to be, on average, 1.2 and 1.52 g · kg(-1) · d(-1) during early (∼16 wk) and late (∼36 wk) stages of pregnancy, respectively. Although the requirements are substantially higher than current recommendations, our values are ∼14-18% of total energy and fit within the Acceptable Macronutrient Distribution Range. Using swine as an animal model we showed that the requirements for several indispensable amino acids increase dramatically during late gestation compared with early gestation. Additional studies should be conducted during pregnancy to confirm the newly determined protein requirements and to determine the indispensable amino acid requirements during pregnancy in humans. PMID:27422521

  10. Kinetic theory of protein filament growth: Self-consistent methods and perturbative techniques

    NASA Astrophysics Data System (ADS)

    Michaels, Thomas C. T.; Knowles, Tuomas P. J.

    2015-12-01

    Filamentous protein structures are of high relevance for the normal functioning of the cell, where they provide the structural component for the cytoskeleton, but are also implicated in the pathogenesis of many disease states. The self-assembly of these supra-molecular structures from monomeric proteins has been studied extensively in the past 50 years and much interest has focused on elucidating the microscopic events that drive linear growth phenomena in a biological setting. Master equations have proven to be particularly fruitful in this context, allowing specific assembly mechanisms to be linked directly to experimental observations of filamentous growth. Recently, these approaches have increasingly been applied to aberrant protein polymerization, elucidating potential implications for controlling or combating the formation of pathological filamentous structures. This article reviews recent theoretical advances in the field of filamentous growth phenomena through the use of the master-equation formalism. We use perturbation and self-consistent methods for obtaining analytical solutions to the rate equations describing fibrillar growth and show how the resulting closed-form expressions can be used to shed light on the general physical laws underlying this complex phenomenon. We also present a connection between the underlying ideas of the self-consistent analysis of filamentous growth and the perturbative renormalization group.

  11. Interference of N-hydroxysuccinimide with bicinchoninic acid protein assay.

    PubMed

    Vashist, Sandeep Kumar; Dixit, Chandra Kumar

    2011-07-29

    We report here substantial interference from N-hydroxysuccinimide (NHS) in the bicinchoninic acid (BCA) protein assay. NHS is one of the most commonly used crosslinking agents in bioanalytical sciences, which can lead to serious potential errors in the BCA protein assay based protein estimation if it is present in the protein analyte solution. It was identified to be a reducing substance, which interferes with the BCA protein assay by reducing Cu(2+) in the BCA working reagent. The absorbance peak and absorbance signal of NHS were very similar to those of bovine serum albumin (BSA), thereby indicating a similar BCA reaction mechanism for NHS and protein. However, the combined absorbance of NHS and BSA was not additive. The time-response measurements of the BCA protein assay showed consistent single-phase kinetics for NHS and gradually decreasing kinetics for BSA. The error in protein estimation due to the presence of NHS was counteracted effectively by plotting additional BCA standard curve for BSA with a fixed concentration of NHS. The difference between the absorbance values of BSA and BSA with a fixed NHS concentration provided the absorbance contributed by NHS, which was then subtracted from the total absorbance of analyte sample to determine the actual absorbance of protein in the analyte sample. PMID:21762678

  12. DBBP: database of binding pairs in protein-nucleic acid interactions

    PubMed Central

    2014-01-01

    Background Interaction of proteins with other molecules plays an important role in many biological activities. As many structures of protein-DNA complexes and protein-RNA complexes have been determined in the past years, several databases have been constructed to provide structure data of the complexes. However, the information on the binding sites between proteins and nucleic acids is not readily available from the structure data since the data consists mostly of the three-dimensional coordinates of the atoms in the complexes. Results We analyzed the huge amount of structure data for the hydrogen bonding interactions between proteins and nucleic acids and developed a database called DBBP (DataBase of Binding Pairs in protein-nucleic acid interactions, http://bclab.inha.ac.kr/dbbp). DBBP contains 44,955 hydrogen bonds (H-bonds) of protein-DNA interactions and 77,947 H-bonds of protein-RNA interactions. Conclusions Analysis of the huge amount of structure data of protein-nucleic acid complexes is labor-intensive, yet provides useful information for studying protein-nucleic acid interactions. DBBP provides the detailed information of hydrogen-bonding interactions between proteins and nucleic acids at various levels from the atomic level to the residue level. The binding information can be used as a valuable resource for developing a computational method aiming at predicting new binding sites in proteins or nucleic acids. PMID:25474259

  13. ff14ipq: A Self-Consistent Force Field for Condensed-Phase Simulations of Proteins.

    PubMed

    Cerutti, David S; Swope, William C; Rice, Julia E; Case, David A

    2014-10-14

    We present the ff14ipq force field, implementing the previously published IPolQ charge set for simulations of complete proteins. Minor modifications to the charge derivation scheme and van der Waals interactions between polar atoms are introduced. Torsion parameters are developed through a generational learning approach, based on gas-phase MP2/cc-pVTZ single-point energies computed of structures optimized by the force field itself rather than the quantum benchmark. In this manner, we sacrifice information about the true quantum minima in order to ensure that the force field maintains optimal agreement with the MP2/cc-pVTZ benchmark for the ensembles it will actually produce in simulations. A means of making the gas-phase torsion parameters compatible with solution-phase IPolQ charges is presented. The ff14ipq model is an alternative to ff99SB and other Amber force fields for protein simulations in programs that accommodate pair-specific Lennard-Jones combining rules. The force field gives strong performance on α-helical and β-sheet oligopeptides as well as globular proteins over microsecond time scale simulations, although it has not yet been tested in conjunction with lipid and nucleic acid models. We show how our choices in parameter development influence the resulting force field and how other choices that may have appeared reasonable would actually have led to poorer results. The tools we developed may also aid in the development of future fixed-charge and even polarizable biomolecular force fields. PMID:25328495

  14. ff14ipq: A Self-Consistent Force Field for Condensed-Phase Simulations of Proteins

    PubMed Central

    2015-01-01

    We present the ff14ipq force field, implementing the previously published IPolQ charge set for simulations of complete proteins. Minor modifications to the charge derivation scheme and van der Waals interactions between polar atoms are introduced. Torsion parameters are developed through a generational learning approach, based on gas-phase MP2/cc-pVTZ single-point energies computed of structures optimized by the force field itself rather than the quantum benchmark. In this manner, we sacrifice information about the true quantum minima in order to ensure that the force field maintains optimal agreement with the MP2/cc-pVTZ benchmark for the ensembles it will actually produce in simulations. A means of making the gas-phase torsion parameters compatible with solution-phase IPolQ charges is presented. The ff14ipq model is an alternative to ff99SB and other Amber force fields for protein simulations in programs that accommodate pair-specific Lennard–Jones combining rules. The force field gives strong performance on α-helical and β-sheet oligopeptides as well as globular proteins over microsecond time scale simulations, although it has not yet been tested in conjunction with lipid and nucleic acid models. We show how our choices in parameter development influence the resulting force field and how other choices that may have appeared reasonable would actually have led to poorer results. The tools we developed may also aid in the development of future fixed-charge and even polarizable biomolecular force fields. PMID:25328495

  15. Fluorinated amino acids: compatibility with native protein structures and effects on protein-protein interactions.

    PubMed

    Salwiczek, Mario; Nyakatura, Elisabeth K; Gerling, Ulla I M; Ye, Shijie; Koksch, Beate

    2012-03-21

    Fluorinated analogues of the canonical α-L-amino acids have gained widespread attention as building blocks that may endow peptides and proteins with advantageous biophysical, chemical and biological properties. This critical review covers the literature dealing with investigations of peptides and proteins containing fluorinated analogues of the canonical amino acids published over the course of the past decade including the late nineties. It focuses on side-chain fluorinated amino acids, the carbon backbone of which is identical to their natural analogues. Each class of amino acids--aliphatic, aromatic, charged and polar as well as proline--is presented in a separate section. General effects of fluorine on essential properties such as hydrophobicity, acidity/basicity and conformation of the specific side chains and the impact of these altered properties on stability, folding kinetics and activity of peptides and proteins are discussed (245 references). PMID:22130572

  16. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  17. Membrane simulations mimicking acidic pH reveal increased thickness and negative curvature in a bilayer consisting of lysophosphatidylcholines and free fatty acids.

    PubMed

    Lähdesmäki, Katariina; Ollila, O H Samuli; Koivuniemi, Artturi; Kovanen, Petri T; Hyvönen, Marja T

    2010-05-01

    Phospholipids are key components of biological membranes and their lipolysis with phospholipase A(2) (PLA(2)) enzymes occurs in different cellular pH environments. Since no studies are available on the effect of pH on PLA(2)-modified phospholipid membranes, we performed 50-ns atomistic molecular dynamics simulations at three different pH conditions (pH 9.0, 7.5, and 5.5) using a fully PLA(2)-hydrolyzed phosphatidylcholine (PC) bilayer which consists solely of lysophosphatidylcholine and free fatty acid molecules. We found that a decrease in pH results in lateral squeezing of the membrane, i.e. in decreased surface area per headgroup. Thus, at the decreased pH, the lipid hydrocarbon chains had larger S(CD) order parameter values, and also enhanced membrane thickness, as seen in the electron density profiles across the membrane. From the lateral pressure profiles, we found that the values of spontaneous curvature of the two opposing monolayers became negative when the pH was decreased. At low pH, protonation of the free fatty acid headgroups reduces their mutual repulsion and accounts for the pH dependence of all the above-mentioned properties. The altered structural characteristics may significantly affect the overall surface properties of biomembranes in cellular vesicles, lipid droplets, and plasma lipoproteins, play an important role in membrane fission and fusion, and modify interactions between membrane lipids and the proteins embedded within them. PMID:20132791

  18. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2011-12-06

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  19. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2011-03-22

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  20. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2008-10-07

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  1. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  2. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2009-04-28

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  3. Studies on the high-sulphur proteins of reduced Merino wool. Amino acid sequence of protein SCMKB-IIIB4

    PubMed Central

    Swart, L. S.; Haylett, T.

    1971-01-01

    The complete amino acid sequence of protein SCMKB-IIIB4 is presented. It is closely related to the sequence of protein SCMKB-IIIB3 (Haylett, Swart & Parris, 1971) differing in only four positions. The peptic and thermolysin peptides of protein SCMKB-IIIB4 were analysed by the dansyl–Edman method (Gray, 1967) and by tritium-labelling of C-terminal residues (Matsuo, Fujimoto & Tatsuno, 1966). This protein is the third member of a group of high-sulphur wool proteins with molecular weight of about 11400. It consists of 98 residues and has acetylalanine and carboxymethylcysteine as N- and C-terminal residues respectively. PMID:4942536

  4. Self-consistent synthesis of the squalene synthase inhibitor zaragozic acid C via controlled oligomerization.

    PubMed

    Nicewicz, David A; Satterfield, Andrew D; Schmitt, Daniel C; Johnson, Jeffrey S

    2008-12-24

    Despite the prevalence of repeating subunits in chiral natural products, stereocontrolled oligomerization is a largely unexplored strategy for construction of carbon skeletal frameworks. This report describes the use of silyl glyoxylates as dipolar glycolic acid synthons in a controlled oligomerization reaction for the efficient construction of the squalene synthase inhibitor zaragozic acid C. This new methodology allows rapid, stereocontrolled formation of the carbon skeleton with a desirable protecting group scheme while minimizing functional group repair and oxidation state manipulations. PMID:19053214

  5. Multifunctional, Biocompatible Supramolecular Hydrogelators Consist Only of Nucleobase, Amino Acid, and Glycoside

    PubMed Central

    Li, Xinming; Kuang, Yi; Shi, Junfeng; Gao, Yuan; Lin, Hsin-Chieh; Xu, Bing

    2011-01-01

    The integration of nucleobase, amino acid, and glycoside into a single molecule results in a novel class of supramolecular hydrogelators, which not only exhibit biocompatibility and biostability, but also facilitate the entry of nucleic acids into cytosol and nuclei of cells. This work illustrates a simple way to generate an unprecedented molecular architecture from the basic biological building blocks for the development of sophisticated soft nanomaterials, including supramolecular hydrogels. PMID:21928792

  6. Microspectrophotometric quantitation of nucleic acid and protein in irradiated epidermis.

    PubMed

    Conti, C J; Giménez, I B; Cabrini, R L

    1976-03-01

    Nucleic acid and proteins of newborn rat tail subjected to local X-irradiation were microspectrophotometrically studied. Feulgen, gallocyanine chrom-alum and naphthol yellow S methods were performed for demonstration of DNA, total nucleic acid and proteins respectively. The amount of proteins and total nucleic acid increases concomitantly with reactional acanthosis. However, the proteins and nucleic acid decrease as from day 3 post-irradiation. A tentative interpretation of the results would point to a giantization of the epidermic cells not only caused by aqueous imbition but also by an actual increase of the cellular protoplasm. PMID:1258094

  7. Relatedness of acyl carrier proteins shown by amino acid compositions.

    PubMed

    Walker, T A; Ernst-Fonberg, M L

    1982-01-01

    1. Relatedness among the following carrier proteins was assessed on the basis of amino acid compositions: eight acyl carrier proteins (ACP's) associated with fatty acid synthesis, ACP's associated with citrate lyase and citramalate lyase, a biotin carboxyl carrier protein and cytochrome 552. Two independent indices of amino acid composition were used. 2. The fatty acid synthesis-associated ACP's of many organisms and the lyase-associated ACP's show a high degree of relatedness among one another. 3. The ACP's show no relatedness to biotin carboxyl carrier protein or cytochrome 552. PMID:7128903

  8. A Novel Red Clover Hydroxycinnamoyl Transferase Has Enzymatic Activities Consistent With a Role in Phaselic Acid Biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red clover (Trifolium pratense L.) leaves accumulate several micromol per g fresh weight of phaselic acid [2-O-(caffeoyl)-L-malate]. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forages like alfalfa (M...

  9. Protein dynamics in the solid state from 2H NMR line shape analysis: a consistent perspective.

    PubMed

    Meirovitch, Eva; Liang, Zhichun; Freed, Jack H

    2015-02-19

    Deuterium line shape analysis of CD3 groups has emerged as a particularly useful tool for studying microsecond-millisecond protein motions in the solid state. The models devised so far consist of several independently conceived simple jump-type motions. They are comprised of physical quantities encoded in their simplest form; improvements are only possible by adding yet another simple motion, thereby changing the model. The various treatments developed are case-specific; hence comparison among the different systems is not possible. Here we develop a new methodology for (2)H NMR line shape analysis free of these limitations. It is based on the microscopic-order-macroscopic-disorder (MOMD) approach. In MOMD motions are described by diffusion tensors, spatial restrictions by potentials/ordering tensors, and geometric features by relative tensor orientations. Jump-type motions are recovered in the limit of large orientational potentials. Model improvement is accomplished by monitoring the magnitude, symmetry, and orientation of the various tensors. The generality of MOMD makes possible comparison among different scenarios. CD3 line shapes from the Chicken Villin Headpiece Subdomain and the Streptomyces Subtilisin Inhibitor are used as experimental examples. All of these spectra are reproduced by using rhombic local potentials constrained for simplicity to be given by the L = 2 spherical harmonics, and by axial diffusion tensors. Potential strength and rhombicity are found to be ca. 2-3 k(B)T. The diffusion tensor is tilted at 120° from the C-CD3 axis. The perpendicular (parallel) correlation times for local motion are 0.1-1.0 ms (3.3-30 μs). Activation energies in the 1.1-8.0 kcal/mol range are estimated. Future prospects include extension to the (2)H relaxation limit, application to the (15)N and (13)C NMR nuclei, and accounting for collective motions and anisotropic media. PMID:25594631

  10. Protein Dynamics in the Solid-State from 2H NMR Lineshape Analysis: a Consistent Perspective

    PubMed Central

    Meirovitch, Eva; Liang, Zhichun; Freed, Jack H.

    2015-01-01

    Deuterium lineshape analysis of CD3 groups has emerged as a particularly useful tool for studying μs - ms protein motions in the solid-state. The models devised so far consist of several independently conceived simple jump-type motions. They are comprised of physical quantities encoded in their simplest form; improvements are only possible by adding yet another simple motion, thereby changing the model. The various treatments developed are case-specific; hence comparison amongst the different systems is not possible. Here we develop a new methodology for 2H NMR lineshape analysis free of these limitations. It is based on the microscopic-order-macroscopic-disorder (MOMD) approach. In MOMD motions are described by diffusion tensors, spatial restrictions by potentials/ordering tensors, and geometric features by relative tensor orientations. Jump-type motions are recovered in the limit of large orientational potentials. Model-improvement is accomplished by monitoring the magnitude, symmetry and orientation of the various tensors. The generality of MOMD makes possible comparison amongst different scenarios. CD3 lineshapes from the Chicken Villin Headpiece Subdomain, and the Streptomyces Subtilisin Inhibitor, are used as experimental examples. All of these spectra are reproduced by using rhombic local potentials constrained for simplicity to be given by the L = 2 spherical harmonics, and axial diffusion tensors. Potential strength and rhombicity are found to be ca. 2 − 3 [kBT]. The diffusion tensor is tilted at 120° from the C−CD3 axis. The perpendicular (parallel) correlation times for local motion are 0.1 − 1.0 ms (3.3 − 30 μs). Activation energies in the 1.1 − 8.0 kcal/mol range are estimated. Future prospects include extension to the 2H relaxation limit, application to the 15N and 13C NMR nuclei, and accounting for collective motions and anisotropic media. PMID:25594631

  11. Strained cycloalkynes as new protein sulfenic acid traps.

    PubMed

    Poole, Thomas H; Reisz, Julie A; Zhao, Weiling; Poole, Leslie B; Furdui, Cristina M; King, S Bruce

    2014-04-30

    Protein sulfenic acids are formed by the reaction of biologically relevant reactive oxygen species with protein thiols. Sulfenic acid formation modulates the function of enzymes and transcription factors either directly or through the subsequent formation of protein disulfide bonds. Identifying the site, timing, and conditions of protein sulfenic acid formation remains crucial to understanding cellular redox regulation. Current methods for trapping and analyzing sulfenic acids involve the use of dimedone and other nucleophilic 1,3-dicarbonyl probes that form covalent adducts with cysteine-derived protein sulfenic acids. As a mechanistic alternative, the present study describes highly strained bicyclo[6.1.0]nonyne (BCN) derivatives as concerted traps of sulfenic acids. These strained cycloalkynes react efficiently with sulfenic acids in proteins and small molecules yielding stable alkenyl sulfoxide products at rates more than 100× greater than 1,3-dicarbonyl reagents enabling kinetic competition with physiological sulfur chemistry. Similar to the 1,3-dicarbonyl reagents, the BCN compounds distinguish the sulfenic acid oxoform from the thiol, disulfide, sulfinic acid, and S-nitrosated forms of cysteine while displaying an acceptable cell toxicity profile. The enhanced rates demonstrated by these strained alkynes identify them as new bioorthogonal probes that should facilitate the discovery of previously unknown sulfenic acid sites and their parent proteins. PMID:24724926

  12. The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes.

    PubMed Central

    Baker, R T; Board, P G

    1991-01-01

    Complementary DNA clones encoding ubiquitin fused to a 52 amino acid tail protein were isolated from human placental and adrenal gland cDNA libraries. The deduced human 52 amino acid tail protein is very similar to the homologous protein from other species, including the conservation of the putative metal-binding, nucleic acid-binding domain observed in these proteins. Northern blot analysis with a tail-specific probe indicated that the previously identified UbA mRNA species most likely represents comigrating transcripts of the 52 amino acid tail (UbA52) and 80 amino acid tail (UbA80) ubiquitin fusion genes. The UbA52 gene was isolated from a human genomic library and consists of five exons distributed over 3400 base pairs. One intron is in the 5' non-coding region, two interrupt the single ubiquitin coding unit, and the fourth intron is within the tail coding region. Several members of the Alu family of repetitive DNA are associated with the gene. The UbA52 promoter has several features in common with mammalian ribosomal protein genes, including its location in a CpG-rich island, initiation of transcription within a polypyrimidine tract, the lack of a consensus TATA motif, and the presence of Sp1 binding sites, observations that are consistent with the recent identification of the ubiquitin-free tail proteins as ribosomal proteins. Thus, in spite of its unusual feature of being translationally fused to ubiquitin, the 52 amino acid tail ribosomal protein is expressed from a structurally typical ribosomal protein gene. Images PMID:1850507

  13. The gyrase inhibitor albicidin consists of p-aminobenzoic acids and cyanoalanine.

    PubMed

    Cociancich, Stéphane; Pesic, Alexander; Petras, Daniel; Uhlmann, Stefanie; Kretz, Julian; Schubert, Vivien; Vieweg, Laura; Duplan, Sandrine; Marguerettaz, Mélanie; Noëll, Julie; Pieretti, Isabelle; Hügelland, Manuela; Kemper, Sebastian; Mainz, Andi; Rott, Philippe; Royer, Monique; Süssmuth, Roderich D

    2015-03-01

    Albicidin is a potent DNA gyrase inhibitor produced by the sugarcane pathogenic bacterium Xanthomonas albilineans. Here we report the elucidation of the hitherto unknown structure of albicidin, revealing a unique polyaromatic oligopeptide mainly composed of p-aminobenzoic acids. In vitro studies provide further insights into the biosynthetic machinery of albicidin. These findings will enable structural investigations on the inhibition mechanism of albicidin and its assessment as a highly effective antibacterial drug. PMID:25599532

  14. LIMB DEFECTS INDUCED BY RETINOIC ACID SIGNALING ANTAGONISM AND SYNTHESIS INHIBITION ARE CONSISTENT WITH ETHANOL-INDUCED LIMB DEFECTS

    EPA Science Inventory

    Limb defects induced by retinoic acid signaling antagonism and synthesis inhibition are consistent with ethanol-induced limb defects

    Johnson CS1, Sulik KK1,2, Hunter, ES III3
    1Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, NC....

  15. Novel Long-Circulating Liposomes Consisting of PEG Modified β-Sitosterol for Gambogic Acid Delivery.

    PubMed

    Yu, Fan; Tang, Xinhui

    2016-03-01

    Long-circulating liposome is an effective formulation in field of cancer treatment. However, high expenditure of formulation and high dose of cholesterol severely restrict its application. In this paper, we developed a method by grafting polyethylene glycol 2000 on β-sitosterol succinic anhydride ester to obtain relatively cheap polyethylene glycol-β-sitosterol conjugates, which were used to prepare long-circulating liposome without cholesterol. Gambogic acid which is an effective antitumor ingredient with very short half-life, was used as a model drug to prepare long-circulating liposome in this research. Meanwhile, the characteristics, pharmacokinetics and distribution of this novel long-circulating liposome were also investigated in comparison with other gambogic acid formulations. Polyethylene glycol-β-sitosterol conjugates were synthesized, different liposomal formulations were also prepared by ethanol injection method, and the obtained nanoparticles were characterized by dynamic light scattering and transmission electron microscope. The long-circulating effect, pharmacokinetics and distribution of gambogic acid in rats were also explored. 1HNMR confirmed that polyethylene glycol-β-sitosterol conjugates were synthesized successfully. Novel long-circulating liposome was successfully prepared by ethanol injection method attaining a entrapment efficiency of 89.4%, exhibiting a homogeneous particle size of 245.2 nm and -24.3 mV zeta potential with smooth continuous surface. This novel long-circulating liposome demonstrated better long-circulating effect than ordinary long-circulating liposome. The novel long-circulating liposome as-prepared not only could reduce cost of grafting polyethylene glycol on macromolecular phospholipid, but also no cholestrol in preparation was applied, expanding the application of liposome as a formulation in the field of lowering blood lipid. Therefore, polyethylene glycol-β-sitosterol conjugates are recommended substitute for

  16. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    PubMed

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

    Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. PMID:26873273

  17. Photoaffinity labeling of retinoic acid-binding proteins.

    PubMed Central

    Bernstein, P S; Choi, S Y; Ho, Y C; Rando, R R

    1995-01-01

    Retinoid-binding proteins are essential mediators of vitamin A function in vertebrate organisms. They solubilize and stabilize retinoids, and they direct the intercellular and intracellular trafficking, transport, and metabolic function of vitamin A compounds in vision and in growth and development. Although many soluble retinoid-binding proteins and receptors have been purified and extensively characterized, relatively few membrane-associated enzymes and other proteins that interact with retinoids have been isolated and studied, due primarily to their inherent instabilities during purification. In an effort to identify and purify previously uncharacterized retinoid-binding proteins, it is shown that radioactively labeled all-trans-retinoic acid can be used as a photoaffinity labeling reagent to specifically tag two known retinoic acid-binding proteins, cellular retinoic acid-binding protein and albumin, in complex mixtures of cytosolic proteins. Additionally, a number of other soluble and membrane-associated proteins that bind all-trans-[11,12-3H]retinoic acid with high specificity are labeled utilizing the same photoaffinity techniques. Most of these labeled proteins have molecular weights that do not correspond to any known retinoid-binding proteins. Thus, photoaffinity labeling with all-trans-retinoic acid and related photoactivatable retinoids is a method that should prove extremely useful in the identification and purification of novel soluble and membrane-associated retinoid-binding proteins from ocular and nonocular tissues. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7846032

  18. Investigation of prototypal MOFs consisting of polyhedral cages with accessible Lewis-acid sites for quinoline synthesis.

    PubMed

    Gao, Wen-Yang; Leng, Kunyue; Cash, Lindsay; Chrzanowski, Matthew; Stackhouse, Chavis A; Sun, Yinyong; Ma, Shengqian

    2015-03-21

    A series of prototypal metal-organic frameworks (MOFs) consisting of polyhedral cages with accessible Lewis-acid sites, have been systematically investigated for Friedländer annulation reaction, a straightforward approach to synthesizing quinoline and its derivatives. Amongst them MMCF-2 demonstrates significantly enhanced catalytic activity compared with the benchmark MOFs, HKUST-1 and MOF-505, as a result of a high-density of accessible Cu(II) Lewis acid sites and large window size in the cuboctahedral cage-based nanoreactor of MMCF-2. PMID:25693429

  19. Phase equilibria in four-component system consisting of water, a nonionic surfactant mixture, and oleic acid

    SciTech Connect

    Matveenko, V.N.; Drovetskii, B.Yu.; Kirasanov, E.A.

    1994-05-01

    The phase diagram of the system consisting of water, Tween 20, Span 80, and oleic acid has been obtained; the coexisting phases have been identified; and the character of the equilibrium of microemulsion, liquid crystal, and molecular solution has been described. In the water-Tween 20-oleic acid system, the ratio of the water volume to the surfactant volume is identical in all of the coexisting phases; this proves the existence of a corresponding field variable in a system with a nonionic surfactant.

  20. Beta-galactosidase and selective neutrality. [amino acid composition of proteins

    NASA Technical Reports Server (NTRS)

    Holmquist, R.

    1979-01-01

    Three hypotheses to explain the amino acid composition of proteins are inconsistent (about 10 to the minus 9th) with the experimental data for beta-galactosidase from Escherichia coli. The exceptional length of this protein, 1021 residues, permits rigorous tests of these hypotheses without complication from statistical artifacts. Either this protein is not at compositional equilibrium, which is unlikely from knowledge about other proteins, or the evolution of this protein and its coding gene have not been selectively neutral. However, the composition of approximately 60% of the molecule is consistent with either a selectively neutral or nonneutral evolutionary process.

  1. Amino acid metabolism and protein synthesis in malarial parasites*

    PubMed Central

    Sherman, I. W.

    1977-01-01

    Malaria-infected red cells and free parasites have limited capabilities for the biosynthesis of amino acids. Therefore, the principal amino acid sources for parasite protein synthesis are the plasma free amino acids and host cell haemoglobin. Infected cells and plasmodia incorporate exogenously supplied amino acids into protein. However, the hypothesis that amino acid utilization (from an external source) is related to availability of that amino acid in haemoglobin is without universal support: it is true for isoleucine and for Plasmodium knowlesi and P. falciparum, but not for methionine, cysteine, and other amino acids, and it does not apply to P. lophurae. More by default than by direct evidence, haemoglobin is believed to be the main amino acid reservoir available to the intraerythrocytic plasmodium. Haemoglobin, ingested via the cytostome, is held in food vacuoles where auto-oxidation takes place. As a consequence, haem is released and accumulates in the vacuole as particulate haemozoin (= malaria pigment). Current evidence favours the view that haemozoin is mainly haematin. Acid and alkaline proteases (identified in crude extracts from mammalian and avian malarias) are presumably secreted directly into the food vacuole. They then digest the denatured globin and the resulting amino acids are incorporated into parasite protein. Cell-free protein synthesizing systems have been developed using P. knowlesi and P. lophurae ribosomes. In the main these systems are typically eukaryotic. Studies of amino acid metabolism are exceedingly limited. Arginine, lysine, methionine, and proline are incorporated into protein, whereas glutamic acid is metabolized via an NADP-specific glutamic dehydrogenase. Glutamate oxidation generates NADPH and auxiliary energy (in the form of α-ketoglutarate). The role of red cell glutathione in the economy of the parasite remains obscure. Important goals for future research should be: quantitative assessment of the relative importance of

  2. Phosphate acceptor amino acid residues in structural proteins of rhabdoviruses.

    PubMed

    Sokol, F; Tan, K B; McFalls, M L; Madore, P

    1974-07-01

    Partial acid hydrolysates of the [(32)P]phosphate- or [(3)H]serine-labeled proteins of purified vesicular stomatitis, rabies, Lagos bat, Mokola, or spring viremia of carp virions and of purified intracellular nucleocapsids of these viruses have been analyzed by paper electrophoresis for the presence of phosphorylated amino acids. Both phosphoserine and phosphothreonine, with the former predominant, were present in virion and nucleocapsid preparations that contained phosphoproteins. An exception was the fish rhabdovirus, which contained only phosphoserine. When vesicular stomatitis or rabies virus proteins were phosphorylated in a cell-free system by the virion-associated protein kinase and analyzed for the presence of phosphorylated amino acid residues, phosphoserine was again found to be more abundant than phosphothreonine. After in vitro protein phosphorylation, another phospho-compound, possibly a third phosphoamino acid, was detected in the partial acid hydrolysates of these viruses. PMID:4365328

  3. Protein and amino acid metabolism and requirements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells of the body. Enzymes, membrane carriers, blood transport molecules, intracellular matrix, and even hair and fingernails are proteins, as are many hormones. Proteins also constitute a major portion of all membranes, and the cons...

  4. Interaction of milk whey protein with common phenolic acids

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Yu, Dandan; Sun, Jing; Guo, Huiyuan; Ding, Qingbo; Liu, Ruihai; Ren, Fazheng

    2014-01-01

    Phenolics-rich foods such as fruit juices and coffee are often consumed with milk. In this study, the interactions of α-lactalbumin and β-lactoglobulin with the phenolic acids (chlorogenic acid, caffeic acid, ferulic acid, and coumalic acid) were examined. Fluorescence, CD, and FTIR spectroscopies were used to analyze the binding modes, binding constants, and the effects of complexation on the conformation of whey protein. The results showed that binding constants of each whey protein-phenolic acid interaction ranged from 4 × 105 to 7 × 106 M-n and the number of binding sites n ranged from 1.28 ± 0.13 to 1.54 ± 0.34. Because of these interactions, the conformation of whey protein was altered, with a significant reduction in the amount of α-helix and an increase in the amounts of β-sheet and turn structures.

  5. A Consistent Experimental and Modeling Approach to Light-Scattering Studies of Protein-Protein Interactions in Solution

    PubMed Central

    Asthagiri, D.; Paliwal, A.; Abras, D.; Lenhoff, A. M.; Paulaitis, M. E.

    2005-01-01

    The osmotic second virial coefficient, B2, obtained by light scattering from protein solutions has two principal components: the Donnan contribution and a contribution due to protein-protein interactions in the limit of infinite dilution. The Donnan contribution accounts for electroneutrality in a multicomponent solution of (poly)electrolytes. The importance of distinguishing this ideal contribution to B2 is emphasized, thereby allowing us to model the interaction part of B2 by molecular computations. The model for protein-protein interactions that we use here extends earlier work (Neal et al., 1998) by accounting for long-range electrostatic interactions and the specific hydration of the protein by strongly associated water molecules. Our model predictions are compared with measurements of B2 for lysozyme at 25°C over pH from 5.0 to 9.0, and 7–60 mM ionic strength. We find that B2 is positive at all solution conditions and decreases with increasing ionic strength, as expected, whereas the interaction part of B2 is negative at all conditions and becomes progressively less negative with increasing ionic strength. Although long-range electrostatic interactions dominate this contribution, particularly at low ionic strength, short-range electrostatic/dispersion interactions with specific hydration are essential for an accurate description of B2 derived from experiment. PMID:15792969

  6. Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

    PubMed

    Sultana, Azmiri; Lee, Jeffrey E

    2015-01-01

    Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample. PMID:25640894

  7. Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type

    PubMed Central

    Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

    2014-01-01

    Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control. PMID:24495932

  8. Enhanced Bio-hydrogen Production from Protein Wastewater by Altering Protein Structure and Amino Acids Acidification Type

    NASA Astrophysics Data System (ADS)

    Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu

    2014-02-01

    Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control.

  9. 3Drefine: Consistent Protein Structure Refinement by Optimizing Hydrogen Bonding Network and Atomic-Level Energy Minimization

    PubMed Central

    Bhattacharya, Debswapna; Cheng, Jianlin

    2013-01-01

    One of the major limitations of computational protein structure prediction is the deviation of predicted models from their experimentally derived true, native structures. The limitations often hinder the possibility of applying computational protein structure prediction methods in biochemical assignment and drug design that are very sensitive to structural details. Refinement of these low-resolution predicted models to high-resolution structures close to the native state, however, has proven to be extremely challenging. Thus, protein structure refinement remains a largely unsolved problem. Critical assessment of techniques for protein structure prediction (CASP) specifically indicated that most predictors participating in the refinement category still did not consistently improve model quality. Here, we propose a two-step refinement protocol, called 3Drefine, to consistently bring the initial model closer to the native structure. The first step is based on optimization of hydrogen bonding (HB) network and the second step applies atomic-level energy minimization on the optimized model using a composite physics and knowledge-based force fields. The approach has been evaluated on the CASP benchmark data and it exhibits consistent improvement over the initial structure in both global and local structural quality measures. 3Drefine method is also computationally inexpensive, consuming only few minutes of CPU time to refine a protein of typical length (300 residues). PMID:22927229

  10. Quality consistency evaluation of Melissa officinalis L. commercial herbs by HPLC fingerprint and quantitation of selected phenolic acids.

    PubMed

    Arceusz, Agnieszka; Wesolowski, Marek

    2013-09-01

    To evaluate the quality consistency of commercial medicinal herbs, a simple and reliable HPLC method with UV-vis detector was developed, both for fingerprint analysis and quantitation of some pharmacologically active constituents (marker compounds). Melissa officinalis L. (lemon balm) was chosen for this study because it is widely used as an aromatic, culinary and medicine remedy. About fifty peaks were found in each chromatogram of a lemon balm extract, including twelve satisfactorily resolved characteristic peaks. A reference chromatographic fingerprint for the studied medicinal herb was calculated using Matlab 9.1 software as a result of analysing all the 19 lemon balm samples obtained from 12 Polish manufacturers. The similarity values and the results of principal component analysis revealed that all the samples were highly correlated with the reference fingerprint and could be accurately classified in relation to their quality consistency. Next, a quantitation of selected phenolic acids in the studied samples was performed. The results have shown that the levels of phenolic acids, i.e. gallic, chlorogenic, syringic, caffeic, ferulic and rosmarinic were as follows (mg/g of dry weight): 0.001-0.067, 0.010-0.333, 0.007-0.553, 0.047-0.705, 0.006-1.589 and 0.158-48.608, respectively. Statistical analysis indicated that rosmarinic acid occurs in M. officinalis at the highest level, whereas gallic acid in the lowest. A detailed inspection of these data has also revealed that reference chromatographic fingerprints combined with quantitation of pharmacologically active constituents of the plant could be used as an efficient strategy for monitoring of the lemon balm quality consistency. PMID:23770780

  11. Balancing selection at the prion protein gene consistent with prehistoric kurulike epidemics.

    PubMed

    Mead, Simon; Stumpf, Michael P H; Whitfield, Jerome; Beck, Jonathan A; Poulter, Mark; Campbell, Tracy; Uphill, James B; Goldstein, David; Alpers, Michael; Fisher, Elizabeth M C; Collinge, John

    2003-04-25

    Kuru is an acquired prion disease largely restricted to the Fore linguistic group of the Papua New Guinea Highlands, which was transmitted during endocannibalistic feasts. Heterozygosity for a common polymorphism in the human prion protein gene (PRNP) confers relative resistance to prion diseases. Elderly survivors of the kuru epidemic, who had multiple exposures at mortuary feasts, are, in marked contrast to younger unexposed Fore, predominantly PRNP 129 heterozygotes. Kuru imposed strong balancing selection on the Fore, essentially eliminating PRNP 129 homozygotes. Worldwide PRNP haplotype diversity and coding allele frequencies suggest that strong balancing selection at this locus occurred during the evolution of modern humans. PMID:12690204

  12. Image analysis reveals that Escherichia coli RecA protein consists of two domains.

    PubMed Central

    Yu, X; Egelman, E H

    1990-01-01

    The Escherichia coli RecA protein catalyzes homologous genetic recombination by forming helical polymers around DNA molecules. These polymers have an ATPase activity, which is essential for the movement of strands between two DNA molecules. One obstacle to structural studies of the RecA filament has been that the ATPase results in a dynamical polymer containing a mixture of states with respect to the bound ATP and its hydrolytic products. We have formed filaments which are trapped in the ADP-Pi state by substituting AIF4- for the Pi, and have used these stable filaments to generate a three-dimensional reconstruction from electron micrographs. The resolution of the reconstruction is sufficient to resolve the 38-k RecA subunit into two nearly equal domains. This reconstruction provides the most detailed view yet of the RecA protein, and serves as a framework within which existing biochemical data on RecA can be understood. Images FIGURE 1 FIGURE 8 FIGURE 12 PMID:2137715

  13. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    SciTech Connect

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  14. Non-protein amino acids and neurodegeneration: the enemy within.

    PubMed

    Rodgers, Kenneth J

    2014-03-01

    Animals, in common with plants and microorganisms, synthesise proteins from a pool of 20 protein amino acids (plus selenocysteine and pyrolysine) (Hendrickson et al., 2004). This represents a small proportion (~2%) of the total number of amino acids known to exist in nature (Bell, 2003). Many 'non-protein' amino acids are synthesised by plants, and in some cases constitute part of their chemical armoury against pathogens, predators or other species competing for the same resources (Fowden et al., 1967). Microorganisms can also use selectively toxic amino acids to gain advantage over competing organisms (Nunn et al., 2010). Since non-protein amino acids (and imino acids) are present in legumes, fruits, seeds and nuts, they are ubiquitous in the diets of human populations around the world. Toxicity to humans is unlikely to have been the selective force for their evolution, but they have the clear potential to adversely affect human health. In this review we explore the links between exposure to non-protein amino acids and neurodegenerative disorders in humans. Environmental factors play a major role in these complex disorders which are predominantly sporadic (Coppede et al., 2006). The discovery of new genes associated with neurodegenerative diseases, many of which code for aggregation-prone proteins, continues at a spectacular pace but little progress is being made in identifying the environmental factors that impact on these disorders. We make the case that insidious entry of non-protein amino acids into the human food chain and their incorporation into protein might be contributing significantly to neurodegenerative damage. PMID:24374297

  15. Heat capacities of amino acids, peptides and proteins.

    PubMed

    Makhatadze, G I

    1998-04-20

    The heat capacity is one of the fundamental parameters describing thermodynamic properties of a system. It has wide applications in a number of areas such as polymer chemistry, protein folding and DNA stability. To aid the scientific community in the analysis of such data, I have compiled a database on the experimentally measured heat capacities of amino acids, polyamino acids, peptides, and proteins in solid state and in aqueous solutions. PMID:9648205

  16. Enamel proteins mitigate mechanical and structural degradations in mature human enamel during acid attack

    NASA Astrophysics Data System (ADS)

    Lubarsky, Gennady V.; Lemoine, Patrick; Meenan, Brian J.; Deb, Sanjukta; Mutreja, Isha; Carolan, Patrick; Petkov, Nikolay

    2014-04-01

    A hydrazine deproteination process was used to investigate the role of enamel proteins in the acid erosion of mature human dental enamel. Bright field high resolution transmission electron micrographs and x-ray diffraction analysis show no crystallographic changes after the hydrazine treatment with similar nanoscale hydroxyapatite crystallite size and orientation for sound and de-proteinated enamel. However, the presence of enamel proteins reduces the erosion depth, the loss of hardness and the loss of structural order in enamel, following exposure to citric acid. Nanoindentation creep is larger for sound enamel than for deproteinated enamel but it reduces in sound enamel after acid attack. These novel results are consistent with calcium ion-mediated visco-elasticty in enamel matrix proteins as described previously for nacre, bone and dental proteins. They are also in good agreement with a previous double layer force spectroscopy study by the authors which found that the proteins electrochemically buffer enamel against acid attack. Finally, this suggests that acid attack, and more specifically dental erosion, is influenced by ionic permeation through the enamel layer and that it is mitigated by the enamel protein matrix.

  17. Phthalic acid chemical probes synthesized for protein-protein interaction analysis.

    PubMed

    Liang, Shih-Shin; Liao, Wei-Ting; Kuo, Chao-Jen; Chou, Chi-Hsien; Wu, Chin-Jen; Wang, Hui-Min

    2013-01-01

    Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid) is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP). According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES) was deposited on silicon dioxides (SiO2) particles and phthalate chemical probes were manufactured from phthalic acid and APTES-SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells) to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA) software showed that these chemical probes were a practical technique for protein-protein interaction analysis. PMID:23797655

  18. HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO. MR Blanton and ES Hunter. Reproductive Toxicology Division, NHEERL, ORD, US EPA, RTP, NC, USA.
    Sponsor: JM Rogers.
    Haloacetic Acids (HAAs) formed during the disinfection process are present in drin...

  19. Conformational Entropy of Intrinsically Disordered Proteins from Amino Acid Triads

    PubMed Central

    Baruah, Anupaul; Rani, Pooja; Biswas, Parbati

    2015-01-01

    This work quantitatively characterizes intrinsic disorder in proteins in terms of sequence composition and backbone conformational entropy. Analysis of the normalized relative composition of the amino acid triads highlights a distinct boundary between globular and disordered proteins. The conformational entropy is calculated from the dihedral angles of the middle amino acid in the amino acid triad for the conformational ensemble of the globular, partially and completely disordered proteins relative to the non-redundant database. Both Monte Carlo (MC) and Molecular Dynamics (MD) simulations are used to characterize the conformational ensemble of the representative proteins of each group. The results show that the globular proteins span approximately half of the allowed conformational states in the Ramachandran space, while the amino acid triads in disordered proteins sample the entire range of the allowed dihedral angle space following Flory’s isolated-pair hypothesis. Therefore, only the sequence information in terms of the relative amino acid triad composition may be sufficient to predict protein disorder and the backbone conformational entropy, even in the absence of well-defined structure. The predicted entropies are found to agree with those calculated using mutual information expansion and the histogram method. PMID:26138206

  20. A Soluble, Folded Protein without Charged Amino Acid Residues.

    PubMed

    Højgaard, Casper; Kofoed, Christian; Espersen, Roall; Johansson, Kristoffer Enøe; Villa, Mara; Willemoës, Martin; Lindorff-Larsen, Kresten; Teilum, Kaare; Winther, Jakob R

    2016-07-19

    Charges are considered an integral part of protein structure and function, enhancing solubility and providing specificity in molecular interactions. We wished to investigate whether charged amino acids are indeed required for protein biogenesis and whether a protein completely free of titratable side chains can maintain solubility, stability, and function. As a model, we used a cellulose-binding domain from Cellulomonas fimi, which, among proteins of more than 100 amino acids, presently is the least charged in the Protein Data Bank, with a total of only four titratable residues. We find that the protein shows a surprising resilience toward extremes of pH, demonstrating stability and function (cellulose binding) in the pH range from 2 to 11. To ask whether the four charged residues present were required for these properties of this protein, we altered them to nontitratable ones. Remarkably, this chargeless protein is produced reasonably well in Escherichia coli, retains its stable three-dimensional structure, and is still capable of strong cellulose binding. To further deprive this protein of charges, we removed the N-terminal charge by acetylation and studied the protein at pH 2, where the C-terminus is effectively protonated. Under these conditions, the protein retains its function and proved to be both soluble and have a reversible folding-unfolding transition. To the best of our knowledge, this is the first time a soluble, functional protein with no titratable side chains has been produced. PMID:27307139

  1. Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs

    SciTech Connect

    Geerds, Christina; Wohlmann, Jens; Haas, Albert; Niemann, Hartmut H.

    2014-06-18

    The structure of VapB, a member of the Vap protein family that is involved in virulence of the bacterial pathogen R. equi, was determined by SAD phasing and reveals an eight-stranded antiparallel β-barrel similar to avidin, suggestive of a binding function. Made up of two Greek-key motifs, the topology of VapB is unusual or even unique. Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology.

  2. A nine-country study of the protein content and amino acid composition of mature human milk

    PubMed Central

    Feng, Ping; Gao, Ming; Burgher, Anita; Zhou, Tian Hui; Pramuk, Kathryn

    2016-01-01

    Background Numerous studies have evaluated protein and amino acid levels in human milk. However, research in this area has been limited by small sample sizes and study populations with little ethnic or racial diversity. Objective Evaluate the protein and amino acid composition of mature (≥30 days) human milk samples collected from a large, multinational study using highly standardized methods for sample collection, storage, and analysis. Design Using a single, centralized laboratory, human milk samples from 220 women (30–188 days postpartum) from nine countries were analyzed for amino acid composition using Waters AccQ-Tag high-performance liquid chromatography and total nitrogen content using the LECO FP-528 nitrogen analyzer. Total protein was calculated as total nitrogen×6.25. True protein, which includes protein, free amino acids, and peptides, was calculated from the total amino acids. Results Mean total protein from individual countries (standard deviation [SD]) ranged from 1,133 (125.5) to 1,366 (341.4) mg/dL; the mean across all countries (SD) was 1,192 (200.9) mg/dL. Total protein, true protein, and amino acid composition were not significantly different across countries except Chile, which had higher total and true protein. Amino acid profiles (percent of total amino acids) did not differ across countries. Total and true protein concentrations and 16 of 18 amino acid concentrations declined with the stage of lactation. Conclusions Total protein, true protein, and individual amino acid concentrations in human milk steadily decline from 30 to 151 days of lactation, and are significantly higher in the second month of lactation compared with the following 4 months. There is a high level of consistency in the protein content and amino acid composition of human milk across geographic locations. The size and diversity of the study population and highly standardized procedures for the collection, storage, and analysis of human milk support the validity and

  3. Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

    PubMed Central

    Blatti, Jillian L.; Beld, Joris; Behnke, Craig A.; Mendez, Michael; Mayfield, Stephen P.; Burkart, Michael D.

    2012-01-01

    Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes. PMID:23028438

  4. Predicting protein disorder by analyzing amino acid sequence

    PubMed Central

    Yang, Jack Y; Yang, Mary Qu

    2008-01-01

    Background Many protein regions and some entire proteins have no definite tertiary structure, presenting instead as dynamic, disorder ensembles under different physiochemical circumstances. These proteins and regions are known as Intrinsically Unstructured Proteins (IUP). IUP have been associated with a wide range of protein functions, along with roles in diseases characterized by protein misfolding and aggregation. Results Identifying IUP is important task in structural and functional genomics. We exact useful features from sequences and develop machine learning algorithms for the above task. We compare our IUP predictor with PONDRs (mainly neural-network-based predictors), disEMBL (also based on neural networks) and Globplot (based on disorder propensity). Conclusion We find that augmenting features derived from physiochemical properties of amino acids (such as hydrophobicity, complexity etc.) and using ensemble method proved beneficial. The IUP predictor is a viable alternative software tool for identifying IUP protein regions and proteins. PMID:18831799

  5. Reducing protein adsorption with polymer-grafted hyaluronic acid coatings.

    PubMed

    Ramadan, Mohamed H; Prata, Joseph E; Karácsony, Orsolya; Dunér, Gunnar; Washburn, Newell R

    2014-07-01

    We report a thermoresponsive chemical modification strategy of hyaluronic acid (HA) for coating onto a broad range of biomaterials without relying on chemical functionalization of the surface. Poly(di(ethylene glycol) methyl ether methacrylate) (PMEO2MA), a polymer with a lower critical solution temperature of 26 °C in water, was grafted onto HA to allow facile formation of biopolymer coatings. While the mechanism for film formation appears to involve a complex combination of homogeneous nucleation followed by heterogeneous film growth, we demonstrate that it resulted in hydrophilic coatings that significantly reduce protein adsorption despite the high fraction of hydrophobic (PMEO2MA). Structural characterization was performed using atomic force microscopy (AFM), which showed the formation of a dense, continuous coating based on 200 nm domains that were stable in protein solutions for at least 15 days. The coatings had a water contact angle of 16°, suggesting the formation of hydrophilic but not fully wetting films. Quartz crystal microbalance with dissipation monitoring (QCM-D) as well as biolayer interferometry (BLI) techniques were used to measure adsorption of bovine serum albumin (BSA), fibrinogen (Fbg), and human immunoglobulin (IgG), with results indicating that HA-PMEO2MA-coated surfaces effectively inhibited adsorption of all three serum proteins. These results are consistent with previous studies demonstrating that this degree of hydrophilicity is sufficient to generate an effectively nonfouling surface and suggest that segregation during the solubility transition resulted in a surface that presented the hydrophilic HA component of the hybrid biopolymer. We conclude that PMEO2MA-grafted HA is a versatile platform for the passivation of hydrophobic biomaterial surfaces without need for substrate functionalization. PMID:24892924

  6. WXG100 Protein Superfamily Consists of Three Subfamilies and Exhibits an α-Helical C-Terminal Conserved Residue Pattern

    PubMed Central

    Poulsen, Christian; Panjikar, Santosh; Holton, Simon J.; Wilmanns, Matthias; Song, Young-Hwa

    2014-01-01

    Members of the WXG100 protein superfamily form homo- or heterodimeric complexes. The most studied proteins among them are the secreted T-cell antigens CFP-10 (10 kDa culture filtrate protein, EsxB) and ESAT-6 (6 kDa early secreted antigen target, EsxA) from Mycobacterium tuberculosis. They are encoded on an operon within a gene cluster, named as ESX-1, that encodes for the Type VII secretion system (T7SS). WXG100 proteins are secreted in a full-length form and it is known that they adopt a four-helix bundle structure. In the current work we discuss the evolutionary relationship between the homo- and heterodimeric WXG100 proteins, the basis of the oligomeric state and the key structural features of the conserved sequence pattern of WXG100 proteins. We performed an iterative bioinformatics analysis of the WXG100 protein superfamily and correlated this with the atomic structures of the representative WXG100 proteins. We find, firstly, that the WXG100 protein superfamily consists of three subfamilies: CFP-10-, ESAT-6- and sagEsxA-like proteins (EsxA proteins similar to that of Streptococcus agalactiae). Secondly, that the heterodimeric complexes probably evolved from a homodimeric precursor. Thirdly, that the genes of hetero-dimeric WXG100 proteins are always encoded in bi-cistronic operons and finally, by combining the sequence alignments with the X-ray data we identify a conserved C-terminal sequence pattern. The side chains of these conserved residues decorate the same side of the C-terminal α-helix and therefore form a distinct surface. Our results lead to a putatively extended T7SS secretion signal which combines two reported T7SS recognition characteristics: Firstly that the T7SS secretion signal is localized at the C-terminus of T7SS substrates and secondly that the conserved residues YxxxD/E are essential for T7SS activity. Furthermore, we propose that the specific α-helical surface formed by the conserved sequence pattern including YxxxD/E motif is a key

  7. [Amino acid composition of rice grain proteins].

    PubMed

    Peruanskiĭ, Iu V; Savich, I M

    1976-01-01

    The composition of the major reserve proteins of rice grain--globulins, prolamines and glutelins--was examined in four rice varieties (Dubovsky 129, Kuban 3, Alakul, Ushtobinsky). Globulins proved to be most heterogeneous whereas glutelins appeared to be least heterogeneous. In regards to the ratio of components globulins showed high variability and glutelins displayed high stability. PMID:1005365

  8. Real-time Measurements of Amino Acid and Protein Hydroperoxides Using Coumarin Boronic Acid*

    PubMed Central

    Michalski, Radoslaw; Zielonka, Jacek; Gapys, Ewa; Marcinek, Andrzej; Joseph, Joy; Kalyanaraman, Balaraman

    2014-01-01

    Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7–23 m−1 s−1) were significantly higher than that measured for H2O2 (1.5 m−1 s−1). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1–1.5 × 103 m−1 s−1. Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems. PMID:24928516

  9. Nucleic acid compositions and the encoding proteins

    DOEpatents

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  10. Efficient protein knockdown of HaloTag-fused proteins using hybrid molecules consisting of IAP antagonist and HaloTag ligand.

    PubMed

    Tomoshige, Shusuke; Hashimoto, Yuichi; Ishikawa, Minoru

    2016-07-15

    We previously reported a protein knockdown system for HaloTag-fused proteins using hybrid small molecules consisting of alkyl chloride, which binds covalently to HaloTag, linked to BE04 (2), a bestatin (3) derivative with an affinity for cellular inhibitor of apoptosis protein 1 (cIAP1, a kind of ubiquitin ligase). This system addressed several limitations of prior protein knockdown technology, and was applied to degrade two HaloTag-fused proteins. However, the degradation activity of these hybrid small molecules was not potent. Therefore, we set out to improve this system. We report here the design, synthesis and biological evaluation of novel hybrid compounds 4a and 4b consisting of alkyl chloride linked to IAP antagonist MV1 (5). Compounds 4a and 4b were confirmed to reduce the levels of HaloTag-fused tumor necrosis factor α (HaloTag-TNFα), HaloTag-fused cell division control protein 42 (HaloTag-Cdc42), and unfused HaloTag protein in living cells more potently than did BE04-linked compound 1b. Analysis of the mode of action revealed that the reduction of HaloTag-TNFα is proteasome-dependent, and is also dependent on the linker structure between MV1 (5) and alkyl chloride. These compounds appear to induce ubiquitination at the HaloTag moiety of HaloTag-fused proteins. Our results indicate that these newly synthesized MV1-type hybrid compounds, 4a and 4b, are efficient tools for protein knockdown for HaloTag-fused proteins. PMID:27236416

  11. Adapting Poisson-Boltzmann to the self-consistent mean field theory: Application to protein side-chain modeling

    NASA Astrophysics Data System (ADS)

    Koehl, Patrice; Orland, Henri; Delarue, Marc

    2011-08-01

    We present an extension of the self-consistent mean field theory for protein side-chain modeling in which solvation effects are included based on the Poisson-Boltzmann (PB) theory. In this approach, the protein is represented with multiple copies of its side chains. Each copy is assigned a weight that is refined iteratively based on the mean field energy generated by the rest of the protein, until self-consistency is reached. At each cycle, the variational free energy of the multi-copy system is computed; this free energy includes the internal energy of the protein that accounts for vdW and electrostatics interactions and a solvation free energy term that is computed using the PB equation. The method converges in only a few cycles and takes only minutes of central processing unit time on a commodity personal computer. The predicted conformation of each residue is then set to be its copy with the highest weight after convergence. We have tested this method on a database of hundred highly refined NMR structures to circumvent the problems of crystal packing inherent to x-ray structures. The use of the PB-derived solvation free energy significantly improves prediction accuracy for surface side chains. For example, the prediction accuracies for χ1 for surface cysteine, serine, and threonine residues improve from 68%, 35%, and 43% to 80%, 53%, and 57%, respectively. A comparison with other side-chain prediction algorithms demonstrates that our approach is consistently better in predicting the conformations of exposed side chains.

  12. Proton transport pathways in an acid-base complex consisting of a phosphonic acid group and a 1,2,3-triazolyl group.

    PubMed

    Yue, Baohua; Yan, Liuming; Han, Shuaiyuan; Xie, Liqing

    2013-07-01

    The proton transport pathways in an acid-base complex consisting of a phosphonic acid group and a 1,2,3-triazolyl group were studied using density functional theory (DFT) calculations in terms of stable configurations and transition states of the molecular or ionic dimers and trimers and verified by proof-of-concept experiments including experimental measurements of overall conductivity and (1)H NMR and FTIR spectroscopy of the methylphosphonic acid (MPA) and 1,2,3-triazole (Tri) complex as well as overall proton conductivity of polymeric blend of poly(vinylphosphonic acid) (PVPA) and poly(4-vinyl-1H-1,2,3-triazole) (PVTri). From the DFT calculations of dimers and trimers composed of ethylphosphonic acid (EPA), Tri, and their deprotonated counterparts, it was concluded that the intermolecular hydrogen bonds of the transition states corresponding to proton transport are much shorter than those of stable configurations, but the O-H and N-H bonds are much longer than those of stable configurations. The tautomerization activation energy decreases from 0.927-1.176 eV in Tri-Tri dimers to 0.336-0.444 eV in the EPA-Tri dimers. From the proof-of-concept experiments, about a 50 fold increase in overall conductivity was observed in the MPA-Tri complex consisting of 10% (molar ratio) MPA compared to pure Tri, and the calculated activation energy is consistent with the experimental activation energy evaluated from temperature dependence of proton conductivity of pure Tri and the MPA-Tri complex. In addition, the fast proton exchange between MPA and Tri, consistent with the DFT calculations, was verified by (1)H NMR and FTIR spectroscopy. Finally, a polymeric blend of PVPA and PVTri was prepared, and its proton conductivity at about 2.1 mS·cm(-1) in anhydrous state at 100 °C was observed to be significantly higher than that of PVPA or of poly(VPA-co-1-vinyl-1,2,4-triazole). The proton conductivity of the polymeric PVPA and PVTri blend in humidity state is in the same range as

  13. Analysis of the Sperm Head Protein Profiles in Fertile Men: Consistency across Time in the Levels of Expression of Heat Shock Proteins and Peroxiredoxins

    PubMed Central

    Kichine, Elsa; Di Falco, Marcos; Hales, Barbara F.; Robaire, Bernard; Chan, Peter

    2013-01-01

    We investigated the identity and quantitative variations of proteins extracted from human sperm heads using a label-free Gel-MS approach. Sperm samples were obtained from three men with high sperm counts at three different time points. This design allowed us to analyse intra-individual and inter-individual variations of the human sperm head proteome. Each time point was analyzed in triplicate to minimize any background artifactual effects of the methodology on the variation analyses. Intra-individual analysis using the spectral counting method revealed that the expression levels of 90% of the common proteins identified in three samples collected at various time-points, separated by several months, had a coefficient of variation of less than 0.5 for each man. Across individuals, the expression level of more than 80% of the proteins had a CV under 0.7. Interestingly, 83 common proteins were found within the core proteome as defined by the intra- and inter-variation analyses set criteria (CV<0.7). Some of these uniformly expressed proteins were chaperones, peroxiredoxins, isomerases, and cytoskeletal proteins. Although there is a significant level of inter-individual variation in the protein profiles of human sperm heads even in a well-defined group of men with high sperm counts, the consistent expression levels of a wide range of proteins points to their essential role during spermatogenesis. PMID:24204839

  14. Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs.

    PubMed

    Geerds, Christina; Wohlmann, Jens; Haas, Albert; Niemann, Hartmut H

    2014-07-01

    Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology. PMID:25005079

  15. Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs

    PubMed Central

    Geerds, Christina; Wohlmann, Jens; Haas, Albert; Niemann, Hartmut H.

    2014-01-01

    Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology. PMID:25005079

  16. The primary structure of fatty-acid-binding protein from nurse shark liver. Structural and evolutionary relationship to the mammalian fatty-acid-binding protein family.

    PubMed

    Medzihradszky, K F; Gibson, B W; Kaur, S; Yu, Z H; Medzihradszky, D; Burlingame, A L; Bass, N M

    1992-02-01

    The primary structure of a fatty-acid-binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high-performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N-terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr = 15124 +/- 2.5) was in good agreement with that calculated from the amino acid sequence (Mr = 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61-53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty-acid/retinoid-binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol-binding proteins I and II, cellular retinoic-acid-binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart-FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid-binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs

  17. Proteins, Peptides and Amino Acids: Role in Infant Nutrition.

    PubMed

    Nutten, Sophie

    2016-01-01

    Proteins are polymers composed of 30 or more amino acids; some of them are essential dietary components, since they are not synthetized by human metabolic processes. They are crucial for healthy growth and development and influence major functions of the body. The infant's first year is a critical time of rapid growth and development, which must be supported by a high rate of protein synthesis. Breast milk, as a single specific food source in the first months of life, is providing the total protein and essential amino acids required. Infant formulas have been designed for infants who cannot be breastfed. They should be similar to breast milk in their composition and their functional outcomes, insuring appropriate growth, optimal development, maturation of the immune system, easy digestion and healthy metabolic programming. By modifying their protein components, specific infant formulas have also been developed for specific needs. For example, partially hydrolyzed (prevention of atopic dermatitis) and extensively hydrolyzed or amino-acid-based infant formulas (reduction in allergy symptoms) have been designed for the management of cow's milk protein allergy. In conclusion, proteins provided via breast milk or infant formula are essential components of the infant's diet; therefore, the specific quality, quantity and conformation of proteins are of utmost importance for healthy growth and development. PMID:27336588

  18. Fatty acid induced remodeling within the human liver fatty acid-binding protein.

    PubMed

    Sharma, Ashwani; Sharma, Amit

    2011-09-01

    We crystallized human liver fatty acid-binding protein (LFABP) in apo, holo, and intermediate states of palmitic acid engagement. Structural snapshots of fatty acid recognition, entry, and docking within LFABP support a heads-in mechanism for ligand entry. Apo-LFABP undergoes structural remodeling, where the first palmitate ingress creates the atomic environment for placement of the second palmitate. These new mechanistic insights will facilitate development of pharmacological agents against LFABP. PMID:21757748

  19. Influence of Polyphosphoric Acid on the Consistency and Composition of Formulated Bitumen: Standard Characterization and NMR Insights.

    PubMed

    Varanda, Catarina; Portugal, Inês; Ribeiro, Jorge; Silva, Artur M S; Silva, Carlos M

    2016-01-01

    Over the recent years, bitumen modification with polymers, acids, or mineral fillers has gained relevance to adjust its performance properties. This work reports the use of polyphosphoric acid (PPA) for the modification of formulated bitumen. With this objective, an in-depth literature review on PPA modification was firstly performed. Subsequently, five individual refinery components were selected for the preparation of bitumen blends, namely, asphaltic residue, vacuum residue, and three lube oils extracts. Seven binary/ternary bitumen blends were prepared and then treated with PPA. Afterwards, the five components and the unmodified and PPA-modified bitumen were characterized by standard methods (penetration, softening point, and penetration index), SARA analysis, elemental analysis, and (31)P and (1)H nuclear magnetic resonance (NMR) spectroscopy. The results evidenced higher asphaltenes and lower saturates/resins contents in PPA-modified bitumen. The NMR data suggest that the paraffinic chains became longer, the content of condensed aromatics increased, more substituted aromatic structures appeared, and α-hydrogen in aromatic structures diminished. These findings disclosed the improved consistency and oxidation stability of PPA-modified bitumen blends. PMID:27579214

  20. Influence of Polyphosphoric Acid on the Consistency and Composition of Formulated Bitumen: Standard Characterization and NMR Insights

    PubMed Central

    Varanda, Catarina; Ribeiro, Jorge

    2016-01-01

    Over the recent years, bitumen modification with polymers, acids, or mineral fillers has gained relevance to adjust its performance properties. This work reports the use of polyphosphoric acid (PPA) for the modification of formulated bitumen. With this objective, an in-depth literature review on PPA modification was firstly performed. Subsequently, five individual refinery components were selected for the preparation of bitumen blends, namely, asphaltic residue, vacuum residue, and three lube oils extracts. Seven binary/ternary bitumen blends were prepared and then treated with PPA. Afterwards, the five components and the unmodified and PPA-modified bitumen were characterized by standard methods (penetration, softening point, and penetration index), SARA analysis, elemental analysis, and 31P and 1H nuclear magnetic resonance (NMR) spectroscopy. The results evidenced higher asphaltenes and lower saturates/resins contents in PPA-modified bitumen. The NMR data suggest that the paraffinic chains became longer, the content of condensed aromatics increased, more substituted aromatic structures appeared, and α-hydrogen in aromatic structures diminished. These findings disclosed the improved consistency and oxidation stability of PPA-modified bitumen blends. PMID:27579214

  1. Nasopharyngeal carcinomas frequently lack the p16/MTS1 tumor suppressor protein but consistently express the retinoblastoma gene product.

    PubMed Central

    Gulley, M. L.; Nicholls, J. M.; Schneider, B. G.; Amin, M. B.; Ro, J. Y.; Geradts, J.

    1998-01-01

    The p16/MTS1 gene is altered by deletion, mutation, or hypermethylation in a wide variety of human cancers. As a result of deficient p16 protein, these cancers lack a critical mechanism for halting G1/S cell cycle progression. In the current study, 59 cases of nasopharyngeal carcinoma were evaluated for expression of the p16 tumor suppressor protein by immunohistochemical analysis of paraffin-embedded tissue. There was no detectable p16 in 38/59 cases (64%), which implies a very high rate of p16 inactivation in this type of cancer. On the other hand, the retinoblastoma gene product, which also regulates the G1 to S phase transition of the cell cycle, was consistently expressed in nasopharyngeal carcinomas by immunohistochemical analysis. These results implicate p16 inactivation but not Rb alteration in the stepwise progression of nasopharyngeal carcinogenesis. Images Figure 1 Figure 2 PMID:9546345

  2. Conformational Changes in the Hepatitis B Virus Core Protein Are Consistent with a Role for Allostery in Virus Assembly

    SciTech Connect

    Packianathan, Charles; Katen, Sarah P.; Dann, III, Charles E.; Zlotnick, Adam

    2010-01-12

    In infected cells, virus components must be organized at the right place and time to ensure assembly of infectious virions. From a different perspective, assembly must be prevented until all components are available. Hypothetically, this can be achieved by allosterically controlling assembly. Consistent with this hypothesis, here we show that the structure of the hepatitis B virus (HBV) core protein dimer, which can spontaneously self-assemble, is incompatible with capsid assembly. Systematic differences between core protein dimer and capsid conformations demonstrate linkage between the intradimer interface and interdimer contact surface. These structures also provide explanations for the capsid-dimer selectivity of some antibodies and the activities of assembly effectors. Solution studies suggest that the assembly-inactive state is more accurately an ensemble of conformations. Simulations show that allostery supports controlled assembly and results in capsids that are resistant to dissociation. We propose that allostery, as demonstrated in HBV, is common to most self-assembling viruses.

  3. A macromolecular delivery vehicle for protein-based vaccines: Acid-degradable protein-loaded microgels

    PubMed Central

    Murthy, Niren; Xu, Mingcheng; Schuck, Stephany; Kunisawa, Jun; Shastri, Nilabh; Fréchet, Jean M. J.

    2003-01-01

    The development of protein-based vaccines remains a major challenge in the fields of immunology and drug delivery. Although numerous protein antigens have been identified that can generate immunity to infectious pathogens, the development of vaccines based on protein antigens has had limited success because of delivery issues. In this article, an acid-sensitive microgel material is synthesized for the development of protein-based vaccines. The chemical design of these microgels is such that they degrade under the mildly acidic conditions found in the phagosomes of antigen-presenting cells (APCs). The rapid cleavage of the microgels leads to phagosomal disruption through a colloid osmotic mechanism, releasing protein antigens into the APC cytoplasm for class I antigen presentation. Ovalbumin was encapsulated in microgel particles, 200–500 nm in diameter, prepared by inverse emulsion polymerization with a synthesized acid-degradable crosslinker. Ovalbumin is released from the acid-degradable microgels in a pH-dependent manner; for example, microgels containing ovalbumin release 80% of their encapsulated proteins after 5 h at pH 5.0, but release only 10% at pH 7.4. APCs that phagocytosed the acid-degradable microgels containing ovalbumin were capable of activating ovalbumin-specific cytoxic T lymphocytes. The acid-degradable microgels developed in this article should therefore find applications as delivery vehicles for vaccines targeted against viruses and tumors, where the activation of cytoxic T lymphocytes is required for the development of immunity. PMID:12704236

  4. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. PMID:21943897

  5. Amino acid repeats and the structure and evolution of proteins.

    PubMed

    Albà, M M; Tompa, P; Veitia, R A

    2007-01-01

    Many proteins have repeats or runs of single amino acids. The pathogenicity of some repeat expansions has fueled proteomic, genomic and structural explorations of homopolymeric runs not only in human but in a wide variety of other organisms. Other types of amino acid repetitive structures exhibit more complex patterns than homopeptides. Irrespective of their precise organization, repetitive sequences are defined as low complexity or simple sequences, as one or a few residues are particularly abundant. Prokaryotes show a relatively low frequency of simple sequences compared to eukaryotes. In the latter the percentage of proteins containing homopolymeric runs varies greatly from one group to another. For instance, within vertebrates, amino acid repeat frequency is much higher in mammals than in amphibians, birds or fishes. For some repeats, this is correlated with the GC-richness of the regions containing the corresponding genes. Homopeptides tend to occur in disordered regions of transcription factors or developmental proteins. They can trigger the formation of protein aggregates, particularly in 'disease' proteins. Simple sequences seem to evolve more rapidly than the rest of the protein/gene and may have a functional impact. Therefore, they are good candidates to promote rapid evolutionary changes. All these diverse facets of homopolymeric runs are explored in this review. PMID:18753788

  6. (-)-Hydroxycitric Acid Nourishes Protein Synthesis via Altering Metabolic Directions of Amino Acids in Male Rats.

    PubMed

    Han, Ningning; Li, Longlong; Peng, Mengling; Ma, Haitian

    2016-08-01

    (-)-Hydroxycitric acid (HCA), a major active ingredient of Garcinia Cambogia extracts, had shown to suppress body weight gain and fat accumulation in animals and humans. While, the underlying mechanism of (-)-HCA has not fully understood. Thus, this study was aimed to investigate the effects of long-term supplement with (-)-HCA on body weight gain and variances of amino acid content in rats. Results showed that (-)-HCA treatment reduced body weight gain and increased feed conversion ratio in rats. The content of hepatic glycogen, muscle glycogen, and serum T4 , T3 , insulin, and Leptin were increased in (-)-HCA treatment groups. Protein content in liver and muscle were significantly increased in (-)-HCA treatment groups. Amino acid profile analysis indicated that most of amino acid contents in serum and liver, especially aromatic amino acid and branched amino acid, were higher in (-)-HCA treatment groups. However, most of the amino acid contents in muscle, especially aromatic amino acid and branched amino acid, were reduced in (-)-HCA treatment groups. These results indicated that (-)-HCA treatment could reduce body weight gain through promoting energy expenditure via regulation of thyroid hormone levels. In addition, (-)-HCA treatment could promote protein synthesis by altering the metabolic directions of amino acids. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27145492

  7. Suppression of muscle protein turnover and amino acid degradation by dietary protein deficiency

    NASA Technical Reports Server (NTRS)

    Tawa, N. E. Jr; Goldberg, A. L.

    1992-01-01

    To define the adaptations that conserve amino acids and muscle protein when dietary protein intake is inadequate, rats (60-70 g final wt) were fed a normal or protein-deficient (PD) diet (18 or 1% lactalbumin), and their muscles were studied in vitro. After 7 days on the PD diet, both protein degradation and synthesis fell 30-40% in skeletal muscles and atria. This fall in proteolysis did not result from reduced amino acid supply to the muscle and preceded any clear decrease in plasma amino acids. Oxidation of branched-chain amino acids, glutamine and alanine synthesis, and uptake of alpha-aminoisobutyrate also fell by 30-50% in muscles and adipose tissue of PD rats. After 1 day on the PD diet, muscle protein synthesis and amino acid uptake decreased by 25-40%, and after 3 days proteolysis and leucine oxidation fell 30-45%. Upon refeeding with the normal diet, protein synthesis also rose more rapidly (+30% by 1 day) than proteolysis, which increased significantly after 3 days (+60%). These different time courses suggest distinct endocrine signals for these responses. The high rate of protein synthesis and low rate of proteolysis during the first 3 days of refeeding a normal diet to PD rats contributes to the rapid weight gain ("catch-up growth") of such animals.

  8. Structural Assessment of the Effects of Amino Acid Substitutions on Protein Stability and Protein-Protein Interaction

    PubMed Central

    Teng, Shaolei; Wang, Liangjiang; Srivastava, Anand K.; Schwartz, Charles E.; Alexov, Emil

    2012-01-01

    A structure-based approach is described for predicting the effects of amino acid substitutions on protein function. Structures were predicted using a homology modelling method. Folding and binding energy differences between wild-type and mutant structures were computed to quantitatively assess the effects of amino acid substitutions on protein stability and protein–protein interaction, respectively. We demonstrated that pathogenic mutations at the interaction interface could affect binding energy and destabilise protein complex, whereas mutations at the non-interface might reduce folding energy and destabilise monomer structure. The results suggest that the structure-based analysis can provide useful information for understanding the molecular mechanisms of diseases. PMID:21297231

  9. INCAP studies of energy, amino acids, and protein.

    PubMed

    Viteri, Fernando E

    2010-03-01

    This Special Issue summarizes the results of several studies aimed at providing information on a series of questions related to the adequate protein and energy intakes that allow adequate growth and function in children and work performance and productivity in adults. The effect of different sources of protein on nitrogen balance and the requirements of essential amino acids in young children were also explored in fully recovered, previously malnourished children housed in the Metabolic Ward of the Biomedical Division of INCAP. The following are the main results of these investigations: Animal experiments and studies in children recovering from protein-energy malnutrition (PEM) strongly suggest that even when requirements of all nutrients are satisfied, inactivity reduces the rate of linear growth and physical activity improves it as well as lean body mass repletion. The effects of different energy intakes on nitrogen balance demonstrated how energy intake modifies the need to ingest different amounts of protein to satisfy protein requirements. Insensible nitrogen losses in preschool children and their relation to protein intake was demonstrated. The quality of even "good protein sources" modifies the amount needed to satisfy nitrogen requirements, and corn and bean-based diets can satisfy protein needs for health and even growth of young children. Essential amino acid requirements of 2-year-old children was assessed by diverse measurements of nitrogen metabolism and amino acid levels in blood, and were found lower than those recommended by FAO-WHO. In rural adult populations the relationship between energy and protein intake, productivity and body composition, and the impact of environmental hygiene on nitrogen balance was demonstrated and measured. PMID:20461903

  10. Studies on fatty acid-binding proteins. The diurnal variation shown by rat liver fatty acid-binding protein.

    PubMed Central

    Wilkinson, T C; Wilton, D C

    1987-01-01

    The concentration of fatty acid-binding protein in rat liver was examined by SDS/polyacrylamide-gel electrophoresis, by Western blotting and by quantifying the fluorescence enhancement achieved on the binding of the fluorescent probe 11-(dansylamino)undecanoic acid. A 2-3-fold increase in the concentration of this protein produced by treatment of rats with the peroxisome proliferator tiadenol was readily detected; however, only a small variation in the concentration of the protein due to a diurnal rhythm was observed. This result contradicts the 7-10-fold variation previously reported for this protein [Hargis, Olson, Clarke & Dempsey (1986) J. Biol. Chem. 261, 1988-1991]. Images Fig. 1. Fig. 3. PMID:3593284

  11. Model of β-Sheet of Muscle Fatty Acid Binding Protein of Locusta migratoria Displays Characteristic Topology

    PubMed Central

    Kizilbash, Nadeem A; Hai, Abdul; Alruwaili, Jamal

    2013-01-01

    The β-sheet of muscle fatty acid binding protein of Locusta migratoria (Lm-FABP) was modeled by employing 2-D NMR data and the Rigid Body Assembly method. The model shows the β-sheet to comprise ten β-strands arranged anti-parallel to each other. There is a β-bulge between Ser 13 and Gln 14 which is a difference from the published structure of β-sheet of bovine heart Fatty Acid Binding Protein. Also, a hydrophobic patch consisting of Ile 45, Phe 51, Phe 64 and Phe 66 is present on the surface which is characteristic of most Fatty Acid Binding Proteins. A “gap” is present between βD and βE that provides evidence for the presence of a portal or opening between the polypeptide chains which allows ligand fatty acids to enter the protein cavity and bind to the protein. PMID:24497726

  12. Protein and amino acid metabolism in the human newborn

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Birth and adaptation to extrauterine life involve major shifts in the protein and energy metabolism of the human newborn. These include a shift from a state of continuous supply of nutrients including amino acids from the mother to cyclic periodic oral intake, a change in the redox state of organs, ...

  13. Fundamental differences between the nucleic acid chaperone activities of HIV-1 nucleocapsid protein and Gag or Gag-derived proteins: Biological implications

    PubMed Central

    Wu, Tiyun; Datta, Siddhartha A.K.; Mitra, Mithun; Gorelick, Robert J.; Rein, Alan; Levin, Judith G.

    2010-01-01

    The HIV-1 Gag polyprotein precursor has multiple domains including nucleocapsid (NC). Although mature NC and NC embedded in Gag are nucleic acid chaperones (proteins that remodel nucleic acid structure), few studies include detailed analysis of the chaperone activity of partially processed Gag proteins and comparison with NC and Gag. Here we address this issue by using a reconstituted minus-strand transfer system. NC and NC-containing Gag proteins exhibited annealing and duplex destabilizing activities required for strand transfer. Surprisingly, unlike NC, with increasing concentrations, Gag proteins drastically inhibited the DNA elongation step. This result is consistent with “nucleic acid-driven multimerization” of Gag and the reported slow dissociation of Gag from bound nucleic acid, which prevent reverse transcriptase from traversing the template (“roadblock” mechanism). Our findings illustrate one reason why NC (and not Gag) has evolved as a critical cofactor in reverse transcription, a paradigm that might also extend to other retrovirus systems. PMID:20655566

  14. Protein location prediction using atomic composition and global features of the amino acid sequence

    SciTech Connect

    Cherian, Betsy Sheena; Nair, Achuthsankar S.

    2010-01-22

    Subcellular location of protein is constructive information in determining its function, screening for drug candidates, vaccine design, annotation of gene products and in selecting relevant proteins for further studies. Computational prediction of subcellular localization deals with predicting the location of a protein from its amino acid sequence. For a computational localization prediction method to be more accurate, it should exploit all possible relevant biological features that contribute to the subcellular localization. In this work, we extracted the biological features from the full length protein sequence to incorporate more biological information. A new biological feature, distribution of atomic composition is effectively used with, multiple physiochemical properties, amino acid composition, three part amino acid composition, and sequence similarity for predicting the subcellular location of the protein. Support Vector Machines are designed for four modules and prediction is made by a weighted voting system. Our system makes prediction with an accuracy of 100, 82.47, 88.81 for self-consistency test, jackknife test and independent data test respectively. Our results provide evidence that the prediction based on the biological features derived from the full length amino acid sequence gives better accuracy than those derived from N-terminal alone. Considering the features as a distribution within the entire sequence will bring out underlying property distribution to a greater detail to enhance the prediction accuracy.

  15. Nucleic acid-protein interactions: Wedding for love or circumstances?

    PubMed

    Lavelle, Christophe; Buckle, Malcolm

    2009-08-01

    The sixth Figeac meeting on nucleic acid-protein interactions was held in Figeac, France, from September 26th to October 1st, 2008. It was organized by the working group "nucleic acid-protein interactions and gene expression" from the French Society for Biochemistry and Molecular Biology. This report briefly summarizes the presentations by 40 speakers during the four plenary sessions, which were organised as follows: (1) nucleic acids: targets and tools, (2) RNA superstar, (3) nuclear structure and dynamics, and (4) new concepts - new approaches. A total of 22 plenary lectures, 18 oral communications and 40 posters were presented over the 5 days, providing a highly stimulating environment for scientific exchange between the approximately 80 participants (biochemists, physicists, bio-informaticians and molecular and cellular biologists). PMID:19422875

  16. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    PubMed

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins. PMID:26774272

  17. Amino acid and protein changes in tilapia and Spanish mackerel after irradiation and storage

    NASA Astrophysics Data System (ADS)

    Al-Kahtani, Hassan A.; Abu-Tarboush, Hamza M.; Atia, Mohamed; Bajaber, Adnan S.; Ahmed, Mohamed A.; El-Mojaddidi, Mohamed A.

    1998-01-01

    Some amino acids in tilapia decreased while some others increased when subjected to doses up to 10.0 kGy. However, 10 kGy contributed to a significant reduction in all amino acids of Spanish mackerel. Variations in amino acid contents continued during post-irradiation storage with no consistant trend of increase or decrease. SDS-PAGE of protein from both fish showed 27 bands of subunits with MW < 14.0-94.0 KD. Isoelectric focusing patterns of sarcoplasmic protein of unirradiated and irradiated fish showed no charge in the number of bands, while some changes were observed in the intensities of the anodic and cathodic bands depending on isoelectric points (pIs).

  18. Acid extraction and purification of recombinant spider silk proteins.

    PubMed

    Mello, Charlene M; Soares, Jason W; Arcidiacono, Steven; Butler, Michelle M

    2004-01-01

    A procedure has been developed for the isolation of recombinant spider silk proteins based upon their unique stability and solubilization characteristics. Three recombinant silk proteins, (SpI)7, NcDS, and [(SpI)4/(SpII)1]4, were purified by extraction with organic acids followed by affinity or ion exchange chromatography resulting in 90-95% pure silk solutions. The protein yield of NcDS (15 mg/L culture) and (SpI)7 (35 mg/L) increased 4- and 5-fold, respectively, from previously reported values presumably due to a more complete solubilization of the expressed recombinant protein. [(SpI)4/(SpII)1]4, a hybrid protein based on the repeat sequences of spidroin I and spidroin II, had a yield of 12.4 mg/L. This method is an effective, reproducible technique that has broad applicability for a variety of silk proteins as well as other acid stable biopolymers. PMID:15360297

  19. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. PMID:27080133

  20. Buffer interference with protein dynamics: a case study on human liver fatty acid binding protein.

    PubMed

    Long, Dong; Yang, Daiwen

    2009-02-18

    Selection of suitable buffer types is often a crucial step for generating appropriate protein samples for NMR and x-ray crystallographic studies. Although the possible interaction between MES buffer (2-(N-morpholino)ethanesulfonic acid) and proteins has been discussed previously, the interaction is usually thought to have no significant effects on the structures of proteins. In this study, we demonstrate the direct, albeit weak, interaction between MES and human liver fatty acid binding protein (hLFABP). Rather than affecting the structure of hLFABP, we found that the dynamics of hLFABP, which were previously proposed to be relevant to its functions, were significantly affected by the binding of hLFABP with MES. Buffer interference with protein dynamics was also demonstrated with Bis-Tris buffer, which is quite different from MES and fatty acids in terms of their molecular structures and properties. This result, to our knowledge, is the first published report on buffer interference with protein dynamics on a microsecond to millisecond timescale and could represent a generic problem in the studies of functionally relevant protein dynamics. Although being a fortuity, our finding of buffer-induced changes in protein dynamics offers a clue to how hLFABP accommodates its ligands. PMID:19217864

  1. Advances in protein-amino acid nutrition of poultry.

    PubMed

    Baker, David H

    2009-05-01

    The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn-soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn-soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary L: -cysteine (2.5% or higher) is lethal for young chicks, but a similar level of DL: -methionine, L: -cystine or N-acetyl-L: -cysteine causes no mortality. A supplemental dietary level of 3.0% L: -cysteine (7x requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks. PMID:19009229

  2. Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids.

    PubMed Central

    Dynan, W S; Yoo, S

    1998-01-01

    The Ku protein-DNA-dependent protein kinase system is one of the major pathways by which cells of higher eukaryotes respond to double-strand DNA breaks. The components of the system are evolutionarily conserved and homologs are known from a number of organisms. The Ku protein component binds directly to DNA ends and may help align them for ligation. Binding of Ku protein to DNA also nucleates formation of an active enzyme complex containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The interaction between Ku protein, DNA-PKcs and nucleic acids has been extensively investigated. This review summarizes the results of these biochemical investigations and relates them to recent molecular genetic studies that reveal highly characteristic repair and recombination defects in mutant cells lacking Ku protein or DNA-PKcs. PMID:9512523

  3. Protein inter-domain linker prediction using Random Forest and amino acid physiochemical properties

    PubMed Central

    2014-01-01

    Background Protein chains are generally long and consist of multiple domains. Domains are distinct structural units of a protein that can evolve and function independently. The accurate prediction of protein domain linkers and boundaries is often regarded as the initial step of protein tertiary structure and function predictions. Such information not only enhances protein-targeted drug development but also reduces the experimental cost of protein analysis by allowing researchers to work on a set of smaller and independent units. In this study, we propose a novel and accurate domain-linker prediction approach based on protein primary structure information only. We utilize a nature-inspired machine-learning model called Random Forest along with a novel domain-linker profile that contains physiochemical and domain-linker information of amino acid sequences. Results The proposed approach was tested on two well-known benchmark protein datasets and achieved 68% sensitivity and 99% precision, which is better than any existing protein domain-linker predictor. Without applying any data balancing technique such as class weighting and data re-sampling, the proposed approach is able to accurately classify inter-domain linkers from highly imbalanced datasets. Conclusion Our experimental results prove that the proposed approach is useful for domain-linker identification in highly imbalanced single- and multi-domain proteins. PMID:25521329

  4. Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins.

    PubMed

    Wang, Jilite; Shimada, Masaya; Kato, Yukina; Kusada, Mio; Nagaoka, Satoshi

    2015-01-01

    Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins. PMID:25374002

  5. Leukocyte Protease Binding to Nucleic Acids Promotes Nuclear Localization and Cleavage of Nucleic Acid Binding Proteins

    PubMed Central

    Thomas, Marshall P.; Whangbo, Jennifer; McCrossan, Geoffrey; Deutsch, Aaron; Martinod, Kimberly; Walch, Michael; Lieberman, Judy

    2014-01-01

    Killer lymphocyte granzyme (Gzm) serine proteases induce apoptosis of pathogen-infected cells and tumor cells. Many known Gzm substrates are nucleic acid binding proteins, and the Gzms accumulate in the target cell nucleus by an unknown mechanism. Here we show that human Gzms bind to DNA and RNA with nanomolar affinity. Gzms cleave their substrates most efficiently when both are bound to nucleic acids. RNase treatment of cell lysates reduces Gzm cleavage of RNA binding protein (RBP) targets, while adding RNA to recombinant RBP substrates increases in vitro cleavage. Binding to nucleic acids also influences Gzm trafficking within target cells. Pre-incubation with competitor DNA and DNase treatment both reduce Gzm nuclear localization. The Gzms are closely related to neutrophil proteases, including neutrophil elastase (NE) and cathepsin G (CATG). During neutrophil activation, NE translocates to the nucleus to initiate DNA extrusion into neutrophil extracellular traps (NETs), which bind NE and CATG. These myeloid cell proteases, but not digestive serine proteases, also bind DNA strongly and localize to nuclei and NETs in a DNA-dependent manner. Thus, high affinity nucleic acid binding is a conserved and functionally important property specific to leukocyte serine proteases. Furthermore, nucleic acid binding provides an elegant and simple mechanism to confer specificity of these proteases for cleavage of nucleic acid binding protein substrates that play essential roles in cellular gene expression and cell proliferation. PMID:24771851

  6. Modeling nucleic acid structure in the presence of single-stranded binding proteins

    NASA Astrophysics Data System (ADS)

    Forties, Robert; Bundschuh, Ralf

    2009-03-01

    There are many important proteins which bind single-stranded nucleic acids, such as the nucleocapsid protein in HIV, the RecA DNA repair protein in bacteria, and all proteins involved in mRNA splicing and translation. We extend the Vienna Package for quantitatively modeling the secondary structure of nucleic acids to include proteins which bind to unpaired portions of the nucleic acid. All parameters needed to model nucleic acid secondary structures in the absence of proteins have been previously measured. This leaves the footprint and sequence dependent binding affinity of the protein as adjustable parameters of our model. Using this model we are able to predict the probability of the protein binding at any position in the nucleic acid sequence, the impact of the protein on nucleic acid base pairing, the end-to-end distance distribution for the nucleic acid, and FRET distributions for fluorophores attached to the nucleic acid.

  7. Polyunsaturated Branched-Chain Fatty Acid Geranylgeranoic Acid Induces Unfolded Protein Response in Human Hepatoma Cells

    PubMed Central

    Iwao, Chieko; Shidoji, Yoshihiro

    2015-01-01

    The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells. PMID:26186544

  8. Protein sequence analysis by incorporating modified chaos game and physicochemical properties into Chou's general pseudo amino acid composition.

    PubMed

    Xu, Chunrui; Sun, Dandan; Liu, Shenghui; Zhang, Yusen

    2016-10-01

    In this contribution we introduced a novel graphical method to compare protein sequences. By mapping a protein sequence into 3D space based on codons and physicochemical properties of 20 amino acids, we are able to get a unique P-vector from the 3D curve. This approach is consistent with wobble theory of amino acids. We compute the distance between sequences by their P-vectors to measure similarities/dissimilarities among protein sequences. Finally, we use our method to analyze four datasets and get better results compared with previous approaches. PMID:27375218

  9. Hyperdimensional analysis of amino acid pair distributions in proteins.

    PubMed

    Henriksen, Svend B; Mortensen, Rasmus J; Geertz-Hansen, Henrik M; Neves-Petersen, Maria Teresa; Arnason, Omar; Söring, Jón; Petersen, Steffen B

    2011-01-01

    Our manuscript presents a novel approach to protein structure analyses. We have organized an 8-dimensional data cube with protein 3D-structural information from 8706 high-resolution non-redundant protein-chains with the aim of identifying packing rules at the amino acid pair level. The cube contains information about amino acid type, solvent accessibility, spatial and sequence distance, secondary structure and sequence length. We are able to pose structural queries to the data cube using program ProPack. The response is a 1, 2 or 3D graph. Whereas the response is of a statistical nature, the user can obtain an instant list of all PDB-structures where such pair is found. The user may select a particular structure, which is displayed highlighting the pair in question. The user may pose millions of different queries and for each one he will receive the answer in a few seconds. In order to demonstrate the capabilities of the data cube as well as the programs, we have selected well known structural features, disulphide bridges and salt bridges, where we illustrate how the queries are posed, and how answers are given. Motifs involving cysteines such as disulphide bridges, zinc-fingers and iron-sulfur clusters are clearly identified and differentiated. ProPack also reveals that whereas pairs of Lys residues virtually never appear in close spatial proximity, pairs of Arg are abundant and appear at close spatial distance, contrasting the belief that electrostatic repulsion would prevent this juxtaposition and that Arg-Lys is perceived as a conservative mutation. The presented programs can find and visualize novel packing preferences in proteins structures allowing the user to unravel correlations between pairs of amino acids. The new tools allow the user to view statistical information and visualize instantly the structures that underpin the statistical information, which is far from trivial with most other SW tools for protein structure analysis. PMID:22174733

  10. Probing interactions between plant virus movement proteins and nucleic acids.

    PubMed

    Tzfira, Tzvi; Citovsky, Vitaly

    2008-01-01

    Most plant viruses move between plant cells with the help of their movement proteins (MPs). MPs are multifunctional proteins, and one of their functions is almost invariably binding to nucleic acids. Presumably, the MP-nucleic acid interaction is directly involved in formation of nucleoprotein complexes that function as intermediates in the cell-to-cell transport of many plant viruses. Thus, when studying a viral MP, it is important to determine whether or not it binds nucleic acids, and to characterize the hallmark parameters of such binding, i.e., preference for single- or double-stranded nucleic acids and binding cooperativity and sequence specificity. Here, we present two major experimental approaches, native gel mobility shift assay and ultra violet (UV) light cross-linking, for detection and characterization of MP binding to DNA and RNA molecules. We also describe protocols for purification of recombinant viral MPs over-expressed in bacteria and production of different DNA and RNA probes for these binding assays. PMID:18370264

  11. Fatty acids exacerbate tubulointerstitial injury in protein-overload proteinuria.

    PubMed

    Thomas, Mark E; Harris, Kevin P G; Walls, John; Furness, Peter N; Brunskill, Nigel J

    2002-10-01

    The role of the albumin-carried fatty acids in the induction of tubulointerstitial injury was studied in protein-overload proteinuria. Rats were injected with fatty acid-carrying BSA [FA(+)BSA], fatty acid-depleted BSA [FA(-)BSA], or saline. Macrophage infiltration was measured by immunohistochemical staining, apoptotic cells were detected by in situ end labeling, and proliferating cells were identified by in situ hybridization for histone mRNA. Macrophage infiltration was significantly greater in the FA(+)BSA group than in the FA(-)BSA and saline groups. The infiltrate was largely restricted to the outer cortex. Apoptosis was greater in the FA(+)BSA group than in the FA(-)BSA and saline groups. Compared with the saline group, apoptosis was significantly increased in the FA(+)BSA group but not in the FA(-)BSA group. Cortical cells proliferated significantly more in the FA(+)BSA and FA(-)BSA groups than in the saline group. FA(+)BSA is therefore a more potent inducer of macrophage infiltration and cell death than FA(-)BSA. The fatty acids carried on albumin may be the chief instigators of tubulointerstitial injury in protein-overload proteinuria. PMID:12217854

  12. The biological activities of protein/oleic acid complexes reside in the fatty acid.

    PubMed

    Fontana, Angelo; Spolaore, Barbara; Polverino de Laureto, Patrizia

    2013-06-01

    A complex formed by human α-lactalbumin (α-LA) and oleic acid (OA), named HAMLET, has been shown to have an apoptotic activity leading to the selective death of tumor cells. In numerous publications it has been reported that in the complex α-LA is monomeric and adopts a partly folded or "molten globule" state, leading to the idea that partly folded proteins can have "beneficial effects". The protein/OA molar ratio initially has been reported to be 1:1, while recent data have indicated that the OA-complex is given by an oligomeric protein capable of binding numerous OA molecules per protein monomer. Proteolytic fragments of α-LA, as well as other proteins unrelated to α-LA, can form OA-complexes with biological activities similar to those of HAMLET, thus indicating that a generic protein can form a cytotoxic complex under suitable experimental conditions. Moreover, even the selective tumoricidal activity of HAMLET-like complexes has been questioned. There is recent evidence that the biological activity of long chain unsaturated fatty acids, including OA, can be ascribed to their effect of perturbing the structure of biological membranes and consequently the function of membrane-bound proteins. In general, it has been observed that the cytotoxic effects exerted by HAMLET-like complexes are similar to those reported for OA alone. Overall, these findings can be interpreted by considering that the protein moiety does not have a toxic effect on its own, but merely acts as a solubilising agent for the inherently toxic fatty acid. PMID:23499846

  13. The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins.

    PubMed Central

    Foster, J W

    1993-01-01

    Although Salmonella typhimurium prefers neutral-pH environments, it can adapt to survive conditions of severe low-pH stress (pH 3.3). The process, termed the acid tolerance response (ATR), includes two distinct stages. The first stage, called pre-acid shock, is induced at pH 5.8 and involves the production of an inducible pH homeostasis system functional at external pH values below 4.0. The second stage occurs following an acid shock shift to pH 4.5 or below and is called the post-acid shock stage. During this stage of the ATR, 43 acid shock proteins (ASPs) are synthesized. The present data reveal that several ASPs important for pH 3.3 acid tolerance are only transiently produced. Their disappearance after 30 to 40 min of pH 4.4 acid shock coincides with an inability to survive subsequent pH 3.3 acid challenge. Clearly, an essential feature of inducible acid tolerance is an ability to synthesize these key ASPs. The pre-acid shock stage, with its inducible pH homeostasis system, offers the cell an enhanced ability to synthesize ASPs following rapid shifts to conditions below pH 4.0, an external pH that normally prevents ASP synthesis. The data also address possible signals for ASP synthesis. The inducing signal for 22 ASPs appears to be internal acidification, while external pH serves to induce 13 others. Of the 14 transient ASPs, 10 are induced in response to changes in internal pH. Mutations in the fur (ferric uptake regulator) locus that produce an Atr- acid-sensitive phenotype also eliminate induction of six transiently induced ASPs. Images PMID:8458840

  14. Amino acid alphabet reduction preserves fold information contained in contact interactions in proteins.

    PubMed

    Solis, Armando D

    2015-12-01

    To reduce complexity, understand generalized rules of protein folding, and facilitate de novo protein design, the 20-letter amino acid alphabet is commonly reduced to a smaller alphabet by clustering amino acids based on some measure of similarity. In this work, we seek the optimal alphabet that preserves as much of the structural information found in long-range (contact) interactions among amino acids in natively-folded proteins. We employ the Information Maximization Device, based on information theory, to partition the amino acids into well-defined clusters. Numbering from 2 to 19 groups, these optimal clusters of amino acids, while generated automatically, embody well-known properties of amino acids such as hydrophobicity/polarity, charge, size, and aromaticity, and are demonstrated to maintain the discriminative power of long-range interactions with minimal loss of mutual information. Our measurements suggest that reduced alphabets (of less than 10) are able to capture virtually all of the information residing in native contacts and may be sufficient for fold recognition, as demonstrated by extensive threading tests. In an expansive survey of the literature, we observe that alphabets derived from various approaches-including those derived from physicochemical intuition, local structure considerations, and sequence alignments of remote homologs-fare consistently well in preserving contact interaction information, highlighting a convergence in the various factors thought to be relevant to the folding code. Moreover, we find that alphabets commonly used in experimental protein design are nearly optimal and are largely coherent with observations that have arisen in this work. PMID:26407535

  15. Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells.

    PubMed

    Qin, Ying; Naito, Yuji; Handa, Osamu; Hayashi, Natsuko; Kuki, Aiko; Mizushima, Katsura; Omatsu, Tatsushi; Tanimura, Yuko; Morita, Mayuko; Adachi, Satoko; Fukui, Akifumi; Hirata, Ikuhiro; Kishimoto, Etsuko; Nishikawa, Taichiro; Uchiyama, Kazuhiko; Ishikawa, Takeshi; Takagi, Tomohisa; Yagi, Nobuaki; Kokura, Satoshi; Yoshikawa, Toshikazu

    2011-11-01

    Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic

  16. Effect of Ethionine on the Ribonucleic Acid, Deoxyribonucleic Acid, and Protein Content of Escherichia coli

    PubMed Central

    Smith, Robert C.; Salmon, W. D.

    1965-01-01

    Smith, Robert C. (Auburn University, Auburn, Ala.), and W. D. Salmon. Effect of ethionine on the ribonucleic acid, deoxyribonucleic acid, and protein content of Escherichia coli. J. Bacteriol. 89:687–692. 1965.—The addition of ethionine to cultures of Escherichia coli K-12 W6, a methionine-requiring auxotroph, led to inhibition of the rate of increase in optical density when the ratio of ethionine to methionine was 200:1. When the ratio was 600:1, the increase in optical density became linear. When ethionine was substituted for methionine in the medium, the optical density of the culture increased, and there was a parallel increase in protein content. There was no cell division in these cultures. The rate of synthesis of ribonucleic acid (RNA) in a culture containing ethionine was similar to that of a culture deprived of methionine, but the synthesis of deoxyribonucleic acid in a culture with ethionine was about twice that of a culture deprived of methionine. No detectable radioactivity from ethionine-ethyl-1-C14 was incorporated into RNA. Ethionine-ethyl-1-C14 was readily incorporated into the protein fraction. PMID:14273646

  17. The requirements of protein & amino acid during acute & chronic infections.

    PubMed

    Kurpad, Anura V

    2006-08-01

    Nutrition and infection interact with each other in a synergistic vicious cycle, leading to an adverse nutritional status and increased susceptibility to infection. Infectious episodes result in hypermetabolism and a negative nitrogen balance which is modulated by hormones, cytokines and other pro-inflammatory mediators, and is compounded by a reduced food intake. The extent of the negative nitrogen balance varies with the type of infection and its duration; however, it is reasonable to suggest that the loss of body protein could be minimized by the provision of dietary nitrogen, although anorexia will limit this. Further, distinctions need to be made about the provision of nutrients or protein during the catabolic and anabolic or recovery phase of the infection, since the capacity of the body to retain protein is enhanced in the anabolic recovery phase. Meeting the increased requirement for protein (and other nutrients) in infection does not imply a complete therapeutic strategy. Infections need to be treated appropriately, with nutrition as an adjunct to the treatment. Prior undernutrition could also impair the body's response to infection, although the weight of the evidence would suggest that this happens more particularly in oedematous undernutrition. In general, the amount of extra protein that would appear to be needed is of the order of 20-25 per cent of the recommended intake, for most infections. In acute infections, this is particularly relevant during the convalescence period. Community trials have suggested that lysine supplementation to the level required for normal daily nutriture, in predominantly wheat eating or potentially lysine deficient communities, improves immune function among other functional nutritional parameters; however, there is as yet insufficient evidence to suggest a specific requirement for amino acids in infections over and above the normal daily requirement as based on recent evidence. Some clinical studies that have showed

  18. Retinoic acid binding protein in normal and neopolastic rat prostate.

    PubMed

    Gesell, M S; Brandes, M J; Arnold, E A; Isaacs, J T; Ueda, H; Millan, J C; Brandes, D

    1982-01-01

    Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation. PMID:6283503

  19. Foamy Virus Protein-Nucleic Acid Interactions during Particle Morphogenesis.

    PubMed

    Hamann, Martin V; Lindemann, Dirk

    2016-01-01

    Compared with orthoretroviruses, our understanding of the molecular and cellular replication mechanism of foamy viruses (FVs), a subfamily of retroviruses, is less advanced. The FV replication cycle differs in several key aspects from orthoretroviruses, which leaves established retroviral models debatable for FVs. Here, we review the general aspect of the FV protein-nucleic acid interactions during virus morphogenesis. We provide a summary of the current knowledge of the FV genome structure and essential sequence motifs required for RNA encapsidation as well as Gag and Pol binding in combination with details about the Gag and Pol biosynthesis. This leads us to address open questions in FV RNA engagement, binding and packaging. Based on recent findings, we propose to shift the point of view from individual glycine-arginine-rich motifs having functions in RNA interactions towards envisioning the FV Gag C-terminus as a general RNA binding protein module. We encourage further investigating a potential new retroviral RNA packaging mechanism, which seems more complex in terms of the components that need to be gathered to form an infectious particle. Additional molecular insights into retroviral protein-nucleic acid interactions help us to develop safer, more specific and more efficient vectors in an era of booming genome engineering and gene therapy approaches. PMID:27589786

  20. Human Skeletal Muscle Protein Metabolism Responses to Amino Acid Nutrition.

    PubMed

    Mitchell, W Kyle; Wilkinson, Daniel J; Phillips, Bethan E; Lund, Jonathan N; Smith, Kenneth; Atherton, Philip J

    2016-07-01

    Healthy individuals maintain remarkably constant skeletal muscle mass across much of adult life, suggesting the existence of robust homeostatic mechanisms. Muscle exists in dynamic equilibrium whereby the influx of amino acids (AAs) and the resulting increases in muscle protein synthesis (MPS) associated with the intake of dietary proteins cancel out the efflux of AAs from muscle protein breakdown that occurs between meals. Dysregulated proteostasis is evident with aging, especially beyond the sixth decade of life. Women and men aged 75 y lose muscle mass at a rate of ∼0.7% and 1%/y, respectively (sarcopenia), and lose strength 2- to 5-fold faster (dynapenia) as muscle "quality" decreases. Factors contributing to the disruption of an otherwise robust proteostatic system represent targets for potential therapies that promote healthy aging. Understanding age-related impairments in anabolic responses to AAs and identifying strategies to mitigate these factors constitute major areas of interest. Numerous studies have aimed to identify 1) the influence of distinct protein sources on absorption kinetics and muscle anabolism, 2) the latency and time course of MPS responses to protein/AAs, 3) the impacts of protein/AA intake on muscle microvascular recruitment, and 4) the role of certain AAs (e.g., leucine) as signaling molecules, which are able to trigger anabolic pathways in tissues. This review aims to discuss these 4 issues listed, to provide historical and modern perspectives of AAs as modulators of human skeletal muscle protein metabolism, to describe how advances in stable isotope/mass spectrometric approaches and instrumentation have underpinned these advances, and to highlight relevant differences between young adults and older individuals. Whenever possible, observations are based on human studies, with additional consideration of relevant nonhuman studies. PMID:27422520

  1. Amino acid profiles and digestible indispensable amino acid scores of proteins from the prioritized key foods in Bangladesh.

    PubMed

    Shaheen, Nazma; Islam, Saiful; Munmun, Sarah; Mohiduzzaman, Md; Longvah, Thingnganing

    2016-12-15

    Concentrations of standard amino acids were determined in the composite samples (representing 30 agro-ecological zones of Bangladesh) of six prioritized key dietary protein sources: Oryza sativa (rice), Triticum aestivum (wheat flour), Lens culinaris (lentils), Pangusius pangusius (pangas), Labeo rohita (rohu) and Oreochromis mossambicus (tilapia). Digestible indispensable amino acid scores (DIAAS) was calculated using published data on amino acids' digestibility to evaluate the protein quality of these foods. Indispensable amino acid (IAA) contents (mg IAA/g protein), found to be highest in pangas (430) and lowest in wheat (336), of all these analyzed foods exceeded the FAO recommended daily allowance (277mg IAA/g protein) and contributed on average 40% to total amino acid contents. Untruncated DIAAS values ranged from 51% (lysine) in wheat to 106% (histidine) in pangas and distinguished pangas, rohu, and tilapia containing 'excellent quality' protein (DIAAS>100%) with potential to complement lower quality protein of cereals, fruits, and vegetables. PMID:27451158

  2. Studies on fatty acid-binding proteins. The detection and quantification of the protein from rat liver by using a fluorescent fatty acid analogue.

    PubMed Central

    Wilkinson, T C; Wilton, D C

    1986-01-01

    Fatty acid-binding protein from rat liver is shown to bind the fluorescent fatty acid probe dansyl undecanoic acid. Binding is accompanied by a shift in the fluorescence emission maximum from 550 nm to 500 nm and a 60-fold fluorescence enhancement at 500 nm. These spectral properties have allowed the use of this probe to detect and quantify microgram amounts of liver fatty acid-binding protein during purification procedures. In conjunction with h.p.l.c. the method allows the rapid estimation of liver fatty acid-binding protein in biological samples. The validity of the method is demonstrated by measuring the concentration of fatty acid-binding protein in livers from control and hypolipidaemic-drug-treated rats. The dramatic diurnal rhythm previously reported for this protein [Dempsey (1984) Curr. Top. Cell. Regul. 24, 63-86] was not observed with this method. Images Fig. 1. PMID:3800946

  3. Effects of protein deficiency and food restriction on lung ascorbic acid and glutathione in rats exposed to ozone

    SciTech Connect

    Dubick, M.A.; Heng, H.; Rucker, R.B.

    1985-08-01

    Weanling (52 +/- 4 g) or adult (259 +/- 16 g) male Sprague-Dawley rats were fed ad libitum casein-based diets containing 4 or 16% protein. A third group (food restricted) was fed daily the 16% protein diet, but at the food intake level of the 4% protein group. After 3 wk (weanling) or 5 wk (adults), half of the rats in each group were continuously exposed to 0.64 ppm ozone for 7 d. Ascorbic acid and reduced glutathione levels were then measured. In the heart and liver from weanling rats, ascorbic acid concentrations were lower in the protein-deficient group than in either control group. In the liver from weanling rats glutathione concentrations were also reduced in response to protein deficiency. Exposure to ozone produced no additional response. For adult rats the response for liver glutathione was similar to that of the weanlings. The liver ascorbate concentration, however, was consistently lower in adult rats compared to weanlings exposed to ozone. In lungs from adult rats, the ascorbic acid concentration was lower in the protein-deficient group than in either control group. On a whole-organ basis, both ascorbic acid and glutathione were usually higher in lungs from rats exposed to ozone than from those exposed to air. Interestingly, protein deficiency did not appear to compromise the lung's ability to maintain, in relative terms, the ascorbic acid or glutathione concentration in response to ozone.

  4. Cloning and characterization of a complementary deoxyribonucleic acid encoding haploid-specific alanine-rich acidic protein located on chromosome-X.

    PubMed

    Uchida, K; Tsuchida, J; Tanaka, H; Koga, M; Nishina, Y; Nozaki, M; Yoshinaga, K; Toshimori, K; Matsumiya, K; Okuyama, A; Nishimune, Y

    2000-10-01

    We have isolated a cDNA clone encoding a germ cell-specific protein from an expression cDNA library prepared from the mouse testis using testis-specific polyclonal antibodies. Northern blot analysis showed a transcript of 1.1 kilobases exclusively expressed in haploid germ cells of the testis. Sequence analysis of the cDNA revealed one long open reading frame consisting of 238 deduced amino acids, rich in basic amino acids in the N-terminal one-third that also contained the nuclear localization signal, and rich in acidic amino acids, including two type of acidic alanine-rich repeats, in the rest of the deduced protein. The protein having a molecular weight of approximately 55 kDa and an isoelectric point of pH 4.3-4.7 was also exclusively detected in the testis by Western blot analysis. As the cDNA was located on chromosome-X, Halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) was proposed for the name of the protein encoded by the cDNA. Immunohistochemical observation revealed that the Halap-X protein was predominantly present in the nucleoplasm of round spermatids but gradually decreased as spermatids matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids. Thus, the Halap-X protein was transferred from the nuclei to the cytoplasm during the spermatid maturation when the chromatin condensation and transformation of the nuclei occurred. The Halap-X may facilitate specific association of nuclear DNA with some basic chromosomal proteins and play important roles in the process of chromatin condensation. PMID:10993819

  5. Amino acid sequence of the Amur tiger prion protein.

    PubMed

    Wu, Changde; Pang, Wanyong; Zhao, Deming

    2006-10-01

    Prion diseases are fatal neurodegenerative disorders in human and animal associated with conformational conversion of a cellular prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)). Various data indicate that the polymorphisms within the open reading frame (ORF) of PrP are associated with the susceptibility and control the species barrier in prion diseases. In the present study, partial Prnp from 25 Amur tigers (tPrnp) were cloned and screened for polymorphisms. Four single nucleotide polymorphisms (T423C, A501G, C511A, A610G) were found; the C511A and A610G nucleotide substitutions resulted in the amino acid changes Lysine171Glutamine and Alanine204Threoine, respectively. The tPrnp amino acid sequence is similar to house cat (Felis catus ) and sheep, but differs significantly from other two cat Prnp sequences that were previously deposited in GenBank. PMID:16780982

  6. Immunocytochemical localization of glial fibrillary acidic protein (GFAP) in the area postrema of the cat - Light and electron microscopic study

    NASA Technical Reports Server (NTRS)

    Damelio, F. E.; Gibbs, M. A.; Mehler, W. R.; Eng, L. F.

    1985-01-01

    Glial fibrillary acidic protein (GFAP) was demonstrated in the cytoplasm and processes of ependymal cells and astroglial components of the area postrema of the cat. These observations differ from the findings in the ependyma of the ventricular cavities which are consistently negative for the protein. Since some studies have suggested sensory functions of the glial cells in this emetic chemoreceptor trigger zone, a careful consideration of morphological and biochemical attributes of these cells seems appropriate.

  7. Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein.

    PubMed

    Longo, Liam M; Lee, Jihun; Blaber, Michael

    2013-02-01

    A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 "prebiotic" α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a "foldable set"--that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two "primitive" versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment. PMID:23341608

  8. Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein

    PubMed Central

    Longo, Liam M.; Lee, Jihun; Blaber, Michael

    2013-01-01

    A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 “prebiotic” α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a “foldable set”—that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two “primitive” versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment. PMID:23341608

  9. Photolabeling of brain membrane proteins by lysergic acid diethylamide.

    PubMed

    Mahon, A C; Hartig, P R

    1982-04-01

    3H-Lysergic acid diethylamide (3H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a subpopulation of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. 3H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays. PMID:7087658

  10. Photolabeling of brain membrane proteins by lysergic acid diethylamide

    SciTech Connect

    Mahon, A.C.; Hartig, P.R.

    1982-04-05

    /sup 3/H-Lysergic acid diethylamide (/sup 3/H-LSD) is irreversibly incorporated into bovine caudate membranes during ultraviolet light illumination. The incorporated radioligand apparently forms a covalent bond with a sub-population of the membrane proteins. Although the photolabeling pattern differs significantly from the Coomassie blue staining pattern on SDS gels, the photolabeling is apparently not specific for LSD binding sites associated with neurotransmitter receptors. /sup 3/H-LSD photolabeling can occur during prolonged exposure of membrane samples to room lighting and thus may introduce artifacts into receptor binding assays.

  11. Acid-base chemistry of frustrated water at protein interfaces.

    PubMed

    Fernández, Ariel

    2016-01-01

    Water molecules at a protein interface are often frustrated in hydrogen-bonding opportunities due to subnanoscale confinement. As shown, this condition makes them behave as a general base that may titrate side-chain ammonium and guanidinium cations. Frustration-based chemistry is captured by a quantum mechanical treatment of proton transference and shown to remove same-charge uncompensated anticontacts at the interface found in the crystallographic record and in other spectroscopic information on the aqueous interface. Such observations are untenable within classical arguments, as hydronium is a stronger acid than ammonium or guanidinium. Frustration enables a directed Grotthuss mechanism for proton transference stabilizing same-charge anticontacts. PMID:26762189

  12. Nucleic acid and protein synthesis during lateral root initiation in Marsilea quadrifolia (Marsileaceae)

    NASA Technical Reports Server (NTRS)

    Lin, B. L.; Raghavan, V.

    1991-01-01

    The pattern of DNA, RNA, and protein synthesis during lateral root initiation in Marsilea quadrifolia L. was monitored by autoradiography of incorporated of 3H-thymidine, 3H-uridine, and 3H-leucine, respectively. DNA synthesis was associated with the enlargement of the lateral root initial prior to its division. Consistent with histological studies, derivatives of the lateral root initial as well as the cells of the adjacent inner cortex and pericycle of the parent root also continued to synthesize DNA. RNA and protein synthetic activities were found to be higher in the lateral root initials than in the endodermal initials of the same longitudinal layer. The data suggest a role for nucleic acid and protein synthesis during cytodifferentiation of a potential endodermal cell into a lateral root initial.

  13. Clustering of protein families into functional subtypes using Relative Complexity Measure with reduced amino acid alphabets

    PubMed Central

    2010-01-01

    Background Phylogenetic analysis can be used to divide a protein family into subfamilies in the absence of experimental information. Most phylogenetic analysis methods utilize multiple alignment of sequences and are based on an evolutionary model. However, multiple alignment is not an automated procedure and requires human intervention to maintain alignment integrity and to produce phylogenies consistent with the functional splits in underlying sequences. To address this problem, we propose to use the alignment-free Relative Complexity Measure (RCM) combined with reduced amino acid alphabets to cluster protein families into functional subtypes purely on sequence criteria. Comparison with an alignment-based approach was also carried out to test the quality of the clustering. Results We demonstrate the robustness of RCM with reduced alphabets in clustering of protein sequences into families in a simulated dataset and seven well-characterized protein datasets. On protein datasets, crotonases, mandelate racemases, nucleotidyl cyclases and glycoside hydrolase family 2 were clustered into subfamilies with 100% accuracy whereas acyl transferase domains, haloacid dehalogenases, and vicinal oxygen chelates could be assigned to subfamilies with 97.2%, 96.9% and 92.2% accuracies, respectively. Conclusions The overall combination of methods in this paper is useful for clustering protein families into subtypes based on solely protein sequence information. The method is also flexible and computationally fast because it does not require multiple alignment of sequences. PMID:20718947

  14. Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

    NASA Astrophysics Data System (ADS)

    Hong, Seok Hoon; Kwon, Yong-Chan; Jewett, Michael

    2014-06-01

    Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

  15. DNA binding proteins that alter nucleic acid flexibility

    NASA Astrophysics Data System (ADS)

    McCauley, Micah; Hardwidge, Philip R.; Maher, L. J., III; Williams, Mark C.

    2007-09-01

    Dual - beam optical tweezers experiments subject single molecules of DNA to high forces (~ 300 pN) with 0.1 pN accuracy, probing the energy and specificity of nucleic acid - ligand structures. Stretching phage λ-DNA reveals an increase in the applied force up to a critical force known as the overstretching transition. In this region, base pairing and stacking are disrupted as double stranded DNA (dsDNA) is melted. Proteins that bind to the double strand will tend to stabilize dsDNA, and melting will occur at higher forces. Proteins that bind to single stranded DNA (ssDNA) destabilize melting, provided that the rate of association is comparable to the pulling rate of the experiment. Many proteins, however, exhibit some affinity for both dsDNA and ssDNA. We describe experiments upon DNA + HMGB2 (box A), a nuclear protein that is believed to facilitate transcription. By characterizing changes in the structure of dsDNA with a polymer model of elasticity, we have determined the equilibrium association constant for HMGB2 to be K ds = 0.15 +/- 0.7 10 9 M -1 for dsDNA binding. Analysis of the melting transition reveals an equilibrium association constant for HMGB2 to ssDNA to be K ss = 0.039 +/- 0.019 10 9 M -1 for ssDNA binding.

  16. Salicylic acid interferes with clathrin-mediated endocytic protein trafficking.

    PubMed

    Du, Yunlong; Tejos, Ricardo; Beck, Martina; Himschoot, Ellie; Li, Hongjiang; Robatzek, Silke; Vanneste, Steffen; Friml, Jirí

    2013-05-01

    Removal of cargos from the cell surface via endocytosis is an efficient mechanism to regulate activities of plasma membrane (PM)-resident proteins, such as receptors or transporters. Salicylic acid (SA) is an important plant hormone that is traditionally associated with pathogen defense. Here, we describe an unanticipated effect of SA on subcellular endocytic cycling of proteins. Both exogenous treatments and endogenously enhanced SA levels repressed endocytosis of different PM proteins. The SA effect on endocytosis did not involve transcription or known components of the SA signaling pathway for transcriptional regulation. SA likely targets an endocytic mechanism that involves the coat protein clathrin, because SA interfered with the clathrin incidence at the PM and clathrin-deficient mutants were less sensitive to the impact of SA on the auxin distribution and root bending during the gravitropic response. By contrast, SA did not affect the ligand-induced endocytosis of the flagellin sensing2 (FLS2) receptor during pathogen responses. Our data suggest that the established SA impact on transcription in plant immunity and the nontranscriptional effect of SA on clathrin-mediated endocytosis are independent mechanisms by which SA regulates distinct aspects of plant physiology. PMID:23613581

  17. Dependence of intestinal amino acid uptake on dietary protein or amino acid levels

    SciTech Connect

    Karasov, W.H.; Solberg, D.H.; Diamond, J.M.

    1987-05-01

    To understand how intestinal amino acid (AA) transport is regulated by dietary substrate levels, the authors measured uptake of seven radioactively-labelled AAs and glucose across the jejunal brush-border membrane of mice kept on one of three isocaloric rations differing in nitrogen content. In the high-protein ration, uptake increased by 77-81% for the nonessential, less toxic AAs, proline, and aspartate but only by 32-61% for the more toxic essential AAs tested. In the nitrogen-deficient ration, uptake decreased for the nonessential aspartate and proline but stayed constant or increased for essential AAs and for the nonessential alanine. These patterns imply independent regulation of the intestine's various AA transporters. With decreasing dietary AA (or protein), the imino acid and acidic AA private transporters are repressed, while activities of the basic AA transporter and the neutral AA public transporter decrease to an asymptote or else go through a minimum. These regulatory patterns can be understood as a compromise among conflicting constraints imposed by protein's multiple roles as a source of calories, nitrogen, and essential AAs and by the toxicity of essential AAs at high concentrations.

  18. Ribonucleic Acid Regulation in Permeabilized Cells of Escherichia coli Capable of Ribonucleic Acid and Protein Synthesis1

    PubMed Central

    Atherly, Alan G.

    1974-01-01

    A cell permeabilization procedure is described that reduces viability less than 10% and does not significantly reduce the rates of ribonucleic acid and protein synthesis when appropriately supplemented. Permeabilization abolishes the normal stringent coupling of protein and ribonucleic acid synthesis. PMID:4364330

  19. The natural non-protein amino acid N-β-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis.

    PubMed

    Glover, W Broc; Mash, Deborah C; Murch, Susan J

    2014-11-01

    N-β-methylamino-L-alanine (BMAA) is an amino acid produced by cyanobacteria and accumulated through trophic levels in the environment and natural food webs. Human exposure to BMAA has been linked to progressive neurodegenerative diseases, potentially due to incorporation of BMAA into protein. The insertion of BMAA and other non-protein amino acids into proteins may trigger protein misfunction, misfolding and/or aggregation. However, the specific mechanism by which BMAA is associated with proteins remained unidentified. Such studies are challenging because of the complexity of biological systems and samples. A cell-free in vitro protein synthesis system offers an excellent approach for investigation of changing amino acid composition in protein. In this study, we report that BMAA incorporates into protein as an error in synthesis when a template DNA sequence is used. Bicinchoninic acid assay of total protein synthesis determined that BMAA effectively substituted for alanine and serine in protein product. LC-MS/MS confirmed that BMAA was selectively inserted into proteins in place of other amino acids, but isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) did not share this characteristic. Incorporation of BMAA into proteins was significantly higher when genomic DNA from post-mortem brain was the template. About half of BMAA in the synthetic proteins was released with denaturation with sodium dodecylsulfonate and dithiothreitol, but the remaining BMAA could only be released by acid hydrolysis. Together these data demonstrate that BMAA is incorporated into the amino acid backbone of proteins during synthesis and also associated with proteins through non-covalent bonding. PMID:25096519

  20. Physiologic hyperinsulinemia stimulates protein synthesis and enhances transport of selected amino acids in human skeletal muscle.

    PubMed Central

    Biolo, G; Declan Fleming, R Y; Wolfe, R R

    1995-01-01

    We have investigated the mechanisms of the anabolic effect of insulin on muscle protein metabolism in healthy volunteers, using stable isotopic tracers of amino acids. Calculations of muscle protein synthesis, breakdown, and amino acid transport were based on data obtained with the leg arteriovenous catheterization and muscle biopsy. Insulin was infused (0.15 mU/min per 100 ml leg) into the femoral artery to increase femoral venous insulin concentration (from 10 +/- 2 to 77 +/- 9 microU/ml) with minimal systemic perturbations. Tissue concentrations of free essential amino acids decreased (P < 0.05) after insulin. The fractional synthesis rate of muscle protein (precursor-product approach) increased (P < 0.01) after insulin from 0.0401 +/- 0.0072 to 0.0677 +/- 0.0101%/h. Consistent with this observation, rates of utilization for protein synthesis of intracellular phenylalanine and lysine (arteriovenous balance approach) also increased from 40 +/- 8 to 59 +/- 8 (P < 0.05) and from 219 +/- 21 to 298 +/- 37 (P < 0.08) nmol/min per 100 ml leg, respectively. Release from protein breakdown of phenylalanine, leucine, and lysine was not significantly modified by insulin. Local hyperinsulinemia increased (P < 0.05) the rates of inward transport of leucine, lysine, and alanine, from 164 +/- 22 to 200 +/- 25, from 126 +/- 11 to 221 +/- 30, and from 403 +/- 64 to 595 +/- 106 nmol/min per 100 ml leg, respectively. Transport of phenylalanine did not change significantly. We conclude that insulin promoted muscle anabolism, primarily by stimulating protein synthesis independently of any effect on transmembrane transport. Images PMID:7860765

  1. From thiol to sulfonic acid: modeling the oxidation pathway of protein thiols by hydrogen peroxide.

    PubMed

    van Bergen, Laura A H; Roos, Goedele; De Proft, Frank

    2014-08-01

    Hydrogen peroxide is a natural oxidant that can oxidize protein thiols (RSH) via sulfenic acid (RSOH) and sulfinic acid (RSO2H) to sulfonic acid (RSO3H). In this paper, we study the complete anionic and neutral oxidation pathway from thiol to sulfonic acid. Reaction barriers and reaction free energies for all three oxidation steps are computed, both for the isolated substrates and for the substrates in the presence of different model ligands (CH4, H2O, NH3) mimicking the enzymatic environment. We found for all three barriers that the anionic thiolate is more reactive than the neutral thiol. However, the assistance of the environment in the neutral pathway in a solvent-assisted proton-exchange (SAPE) mechanism can lower the reaction barrier noticeably. Polar ligands can decrease the reaction barriers, whereas apolar ligands do not influence the barrier heights. The same holds for the reaction energies: they decrease (become more negative) in the presence of polar ligands whereas apolar ligands do not have an influence. The consistently negative consecutive reaction energies for the oxidation in the anionic pathway when going from thiolate over sulfenic and sulfinic acid to sulfonic acid are in agreement with biological reversibility. PMID:25036614

  2. Self-consistent field theory investigation of the behavior of hyaluronic acid chains in aqueous salt solutions

    NASA Astrophysics Data System (ADS)

    Nogovitsin, E. A.; Budkov, Yu. A.

    2012-04-01

    In this work we continue to develop a field-theoretic methodology, which combines the technique of Gaussian equivalent representation for the calculation of functional integrals with the continuous Gaussian thread model of flexible polymers for solving statistical-mechanical problems of polyelectrolyte solutions. We present new analytic expressions for the osmotic pressure, the potential of mean force, and the monomer-monomer pair distribution function, and employ them to investigate the structural and thermodynamic quantities of the polyelectrolyte system. We demonstrate the applicability of the method for systems of polyelectrolyte chains in which the monomers interact via a Yukawa-type pair potential. As a specific example, the present work focuses on aqueous solutions of hyaluronic acid with added salts NaCl and CaCl2. Hyaluronic acid is a high molecular weight linear polysaccharide, which has a multitude of roles in biological tissues. We conclude that the effect of sodium chloride and calcium chloride on the osmotic properties of hyaluronic acid solutions can be accounted for by their contributions to the ionic strength. Nevertheless, the effects of coiling and self-association can be stimulated in solution by added salt.

  3. Using self-consistent Gibbs free energy surfaces to calculate size distributions of neutral and charged clusters for the sulfuric acid-water binary system

    NASA Astrophysics Data System (ADS)

    Smith, J. A.; Froyd, K. D.; Toon, O. B.

    2012-12-01

    We construct tables of reaction enthalpies and entropies for the association reactions involving sulfuric acid vapor, water vapor, and the bisulfate ion. These tables are created from experimental measurements and quantum chemical calculations for molecular clusters and a classical thermodynamic model for larger clusters. These initial tables are not thermodynamically consistent. For example, the Gibbs free energy of associating a cluster consisting of one acid molecule and two water molecules depends on the order in which the cluster was assembled: add two waters and then the acid or add an acid and a water and then the second water. We adjust the values within the tables using the method of Lagrange multipliers to minimize the adjustments and produce self-consistent Gibbs free energy surfaces for the neutral clusters and the charged clusters. With the self-consistent Gibbs free energy surfaces, we calculate size distributions of neutral and charged clusters for a variety of atmospheric conditions. Depending on the conditions, nucleation can be dominated by growth along the neutral channel or growth along the ion channel followed by ion-ion recombination.

  4. Are proteins ideal mixtures of amino acids? Analysis of energy parameter sets.

    PubMed Central

    Godzik, A.; Koliński, A.; Skolnick, J.

    1995-01-01

    Various existing derivations of the effective potentials of mean force for the two-body interactions between amino acid side chains in proteins are reviewed and compared to each other. The differences between different parameter sets can be traced to the reference state used to define the zero of energy. Depending on the reference state, the transfer free energy or other pseudo-one-body contributions can be present to various extents in two-body parameter sets. It is, however, possible to compare various derivations directly by concentrating on the "excess" energy-a term that describes the difference between a real protein and an ideal solution of amino acids. Furthermore, the number of protein structures available for analysis allows one to check the consistency of the derivation and the errors by comparing parameters derived from various subsets of the whole database. It is shown that pair interaction preferences are very consistent throughout the database. Independently derived parameter sets have correlation coefficients on the order of 0.8, with the mean difference between equivalent entries of 0.1 kT. Also, the low-quality (low resolution, little or no refinement) structures show similar regularities. There are, however, large differences between interaction parameters derived on the basis of crystallographic structures and structures obtained by the NMR refinement. The origin of the latter difference is not yet understood. PMID:8535247

  5. Specific high-affinity binding of fatty acids to epidermal cytosolic proteins

    SciTech Connect

    Raza, H.; Chung, W.L.; Mukhtar, H. )

    1991-08-01

    Cytosol from rat, mouse, and human skin or rat epidermis was incubated with (3H)arachidonic acid, (14C)retinoic acid, (14C)oleic acid, (3H)leukotriene A4, (3H)prostaglandin E2 (PGE2) or (3H) 15-hydroxyeicosatetraenoic acid (15-HETE), and protein-bound ligands were separated using Lipidex-1000 at 4C to assess the binding specificity. The binding of oleic acid and arachidonic acid with rat epidermal cytosol was rapid, saturable, and reversible. Binding of oleic acid was competed out with the simultaneous addition of other ligands and found to be in the following order: arachidonic acid greater than oleic acid greater than linoleic acid greater than lauric acid greater than leukotriene A4 greater than 15-HETE = PGE1 greater than PGE2 = PGF2. Scatchard analysis of the binding with arachidonic acid, oleic acid, and retinoic acid revealed high-affinity binding sites with the dissociation constant in the nM range. SDS-PAGE analysis of the oleic acid-bound epidermal cytosolic protein(s) revealed maximum binding at the 14.5 kDa region. The presence of the fatty acid-binding protein in epidermal cytosol and its binding to fatty acids and retinoic acid may be of significance both in the trafficking and the metabolism of fatty acids and retinoids across the skin.

  6. Shedding light on proteins, nucleic acids, cells, humans and fish.

    PubMed

    Setlow, Richard B

    2002-03-01

    I was trained as a physicist in graduate school. Hence, when I decided to go into the field of biophysics, it was natural that I concentrated on the effects of light on relatively simple biological systems, such as proteins. The wavelengths absorbed by the amino acid subunits of proteins are in the ultraviolet (UV). The wavelengths that affect the biological activities, the action spectra, also are in the UV, but are not necessarily parallel to the absorption spectra. Understanding these differences led me to investigate the action spectra for affecting nucleic acids, and the effects of UV on viruses and cells. The latter studies led me to the discovery of the important molecular nature of the damages affecting DNA (cyclobutane pyrimidine dimers) and to the discovery of nucleotide excision repair. Individuals with the genetic disease xeroderma pigmentosum (XP) are extraordinarily sensitive to sunlight-induced skin cancer. The finding, by James Cleaver, that their skin cells were defective in DNA repair strongly suggested that DNA damage was a key step in carcinogenesis. Such information was important for estimating the wavelengths in sunlight responsible for human skin cancer and for predicting the effects of ozone depletion on the incidence of non-melanoma skin cancer. It took experiments with backcross hybrid fish to call attention to the probable role of the longer UV wavelengths not absorbed by DNA in the induction of melanoma. These reflections trace the biophysicist's path from molecules to melanoma. PMID:11906839

  7. Shedding light on proteins, nucleic acids, cells, humans and fish

    NASA Technical Reports Server (NTRS)

    Setlow, Richard B.

    2002-01-01

    I was trained as a physicist in graduate school. Hence, when I decided to go into the field of biophysics, it was natural that I concentrated on the effects of light on relatively simple biological systems, such as proteins. The wavelengths absorbed by the amino acid subunits of proteins are in the ultraviolet (UV). The wavelengths that affect the biological activities, the action spectra, also are in the UV, but are not necessarily parallel to the absorption spectra. Understanding these differences led me to investigate the action spectra for affecting nucleic acids, and the effects of UV on viruses and cells. The latter studies led me to the discovery of the important molecular nature of the damages affecting DNA (cyclobutane pyrimidine dimers) and to the discovery of nucleotide excision repair. Individuals with the genetic disease xeroderma pigmentosum (XP) are extraordinarily sensitive to sunlight-induced skin cancer. The finding, by James Cleaver, that their skin cells were defective in DNA repair strongly suggested that DNA damage was a key step in carcinogenesis. Such information was important for estimating the wavelengths in sunlight responsible for human skin cancer and for predicting the effects of ozone depletion on the incidence of non-melanoma skin cancer. It took experiments with backcross hybrid fish to call attention to the probable role of the longer UV wavelengths not absorbed by DNA in the induction of melanoma. These reflections trace the biophysicist's path from molecules to melanoma.

  8. Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community ▿ †

    PubMed Central

    Jiao, Yongqin; D'haeseleer, Patrik; Dill, Brian D.; Shah, Manesh; VerBerkmoes, Nathan C.; Hettich, Robert L.; Banfield, Jillian F.; Thelen, Michael P.

    2011-01-01

    In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by ∼2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as β-N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development. PMID:21685158

  9. Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community

    SciTech Connect

    Jiao, Yongqin; D'Haeseleer, Patrik M; Dill, Brian; Shah, Manesh B; Verberkmoes, Nathan C; Hettich, Robert {Bob} L; Banfield, Jillian F.; Thelen, Michael P.

    2011-01-01

    In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by 2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as -N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.

  10. Proteomic Upregulation of Fatty Acid Synthase and Fatty Acid Binding Protein 5 and Identification of Cancer- and Race-Specific Pathway Associations in Human Prostate Cancer Tissues

    PubMed Central

    Myers, Jennifer S.; von Lersner, Ariana K.; Sang, Qing-Xiang Amy

    2016-01-01

    Protein profiling studies of prostate cancer have been widely used to characterize molecular differences between diseased and non-diseased tissues. When combined with pathway analysis, profiling approaches are able to identify molecular mechanisms of prostate cancer, group patients by cancer subtype, and predict prognosis. This strategy can also be implemented to study prostate cancer in very specific populations, such as African Americans who have higher rates of prostate cancer incidence and mortality than other racial groups in the United States. In this study, age-, stage-, and Gleason score-matched prostate tumor specimen from African American and Caucasian American men, along with non-malignant adjacent prostate tissue from these same patients, were compared. Protein expression changes and altered pathway associations were identified in prostate cancer generally and in African American prostate cancer specifically. In comparing tumor to non-malignant samples, 45 proteins were significantly cancer-associated and 3 proteins were significantly downregulated in tumor samples. Notably, fatty acid synthase (FASN) and epidermal fatty acid-binding protein (FABP5) were upregulated in human prostate cancer tissues, consistent with their known functions in prostate cancer progression. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3) was also upregulated in tumor samples. The Metastasis Associated Protein 3 (MTA3) pathway was significantly enriched in tumor samples compared to non-malignant samples. While the current experiment was unable to detect statistically significant differences in protein expression between African American and Caucasian American samples, differences in overrepresentation and pathway enrichment were found. Structural components (Cytoskeletal Proteins and Extracellular Matrix Protein protein classes, and Biological Adhesion Gene Ontology (GO) annotation) were overrepresented in African American but not Caucasian American tumors. Additionally, 5

  11. Ileal apical sodium-dependent bile acid transporter protein levels are down-regulated through ubiquitin-dependent protein degradation induced by bile acids.

    PubMed

    Miyata, Masaaki; Yamakawa, Hiroki; Hayashi, Kenjiro; Kuribayashi, Hideaki; Yamazoe, Yasushi; Yoshinari, Kouichi

    2013-08-15

    The ileal apical sodium-dependent bile acid transporter (ASBT or SLC10A2) has a crucial role in intestinal bile acid absorption. We previously reported that enterobacteria-mediated bile acid conversion was involved in the alteration of ileal ASBT expression levels. In the present study, to investigate the hypothesis that ileal ASBT protein levels are post-translationally regulated by enterobacteria-associated bile acids, alteration of ileal ASBT protein levels was analysed in mice 12 h and 24 h after anti-bacterial drug ampicillin (ABPC) treatment (100 mg/kg, single shot) that altered bile acid composition in the intestinal lumen. In ABPC-treated mice, enterobacteria-biotransformed bile acid, taurodeoxycholic acid (TDCA) and cholic acid (CA) levels were decreased, whereas taurocholic acid (TCA) and tauro-β-muricholic acid levels were increased in the intestinal lumen. Ileal ASBT protein levels in brush-border membrane vesicles (BBMVs), but not ileal Asbt mRNA levels, were significantly increased in the ABPC-treated mice, and the extent of ubiquitination of the ileal ASBT protein was reduced in the ABPC-treated mice. Treatment of ABPC-pretreated mice with CA or TDCA, but not TCA, significantly decreased ileal ASBT protein levels and increased the extent of ubiquitination of ileal ASBT protein. Treatment of mice with the lysosome inhibitor, chloroquine, or the proteasome inhibitor, MG132, increased ileal ASBT protein levels in BBMVs. CA-mediated reduction of ASBT protein levels in the ABPC-pretreated mice was attenuated by co-treatment with chloroquine or MG132. These results suggest that ileal ASBT protein is degraded by a ubiquitin-dependent pathway in response to enterobacteria-associated bile acids. PMID:23872411

  12. Mutagenesis Mapping of the Protein-Protein Interaction Underlying FusB-Type Fusidic Acid Resistance

    PubMed Central

    Cox, Georgina; Edwards, Thomas A.

    2013-01-01

    FusB-type proteins represent the predominant mechanism of resistance to fusidic acid in staphylococci and act by binding to and modulating the function of the drug target (elongation factor G [EF-G]). To gain further insight into this antibiotic resistance mechanism, we sought to identify residues important for the interaction of FusB with EF-G and thereby delineate the binding interface within the FusB–EF-G complex. Replacement with alanine of any one of four conserved residues within the C-terminal domain of FusB (F156, K184, Y187, and F208) abrogated the ability of the protein to confer resistance to fusidic acid; the purified mutant proteins also lost the ability to bind S. aureus EF-G in vitro. E. coli EF-G, which is not ordinarily able to bind FusB-type proteins, was rendered competent for binding to FusB following deletion of a 3-residue tract (529SNP531) from domain IV of the protein. This study has identified key regions of both FusB and EF-G that are important for the interaction between the proteins, findings which corroborate our previous in silico prediction for the architecture of the complex formed between the resistance protein and the drug target (G. Cox, G. S. Thompson, H. T. Jenkins, F. Peske, A. Savelsbergh, M. V. Rodnina, W. Wintermeyer, S. W. Homans, T. A. Edwards, and A. J. O'Neill, Proc. Natl. Acad. Sci. U. S. A. 109:2102-2107, 2012). PMID:23836182

  13. Mutagenesis mapping of the protein-protein interaction underlying FusB-type fusidic acid resistance.

    PubMed

    Cox, Georgina; Edwards, Thomas A; O'Neill, Alex J

    2013-10-01

    FusB-type proteins represent the predominant mechanism of resistance to fusidic acid in staphylococci and act by binding to and modulating the function of the drug target (elongation factor G [EF-G]). To gain further insight into this antibiotic resistance mechanism, we sought to identify residues important for the interaction of FusB with EF-G and thereby delineate the binding interface within the FusB-EF-G complex. Replacement with alanine of any one of four conserved residues within the C-terminal domain of FusB (F156, K184, Y187, and F208) abrogated the ability of the protein to confer resistance to fusidic acid; the purified mutant proteins also lost the ability to bind S. aureus EF-G in vitro. E. coli EF-G, which is not ordinarily able to bind FusB-type proteins, was rendered competent for binding to FusB following deletion of a 3-residue tract (529SNP531) from domain IV of the protein. This study has identified key regions of both FusB and EF-G that are important for the interaction between the proteins, findings which corroborate our previous in silico prediction for the architecture of the complex formed between the resistance protein and the drug target (G. Cox, G. S. Thompson, H. T. Jenkins, F. Peske, A. Savelsbergh, M. V. Rodnina, W. Wintermeyer, S. W. Homans, T. A. Edwards, and A. J. O'Neill, Proc. Natl. Acad. Sci. U. S. A. 109:2102-2107, 2012). PMID:23836182

  14. Bile salt recognition by human liver fatty acid binding protein.

    PubMed

    Favretto, Filippo; Santambrogio, Carlo; D'Onofrio, Mariapina; Molinari, Henriette; Grandori, Rita; Assfalg, Michael

    2015-04-01

    Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules, and play a role in global metabolism regulation. Liver FABP (L-FABP) is prominent among FABPs for its wide ligand repertoire, which includes long-chain fatty acids as well as bile acids (BAs). In this work, we performed a detailed molecular- and atomic-level analysis of the interactions established by human L-FABP with nine BAs to understand the binding specificity for this important class of cholesterol-derived metabolites. Protein-ligand complex formation was monitored using heteronuclear NMR, steady-state fluorescence spectroscopy, and mass spectrometry. BAs were found to interact with L-FABP with dissociation constants in the narrow range of 0.6-7 μm; however, the diverse substitution patterns of the sterol nucleus and the presence of side-chain conjugation resulted in complexes endowed with various degrees of conformational heterogeneity. Trihydroxylated BAs formed monomeric complexes in which single ligand molecules occupied similar internal binding sites, based on chemical-shift perturbation data. Analysis of NMR line shapes upon progressive addition of taurocholate indicated that the binding mechanism departed from a simple binary association equilibrium, and instead involved intermediates along the binding path. The co-linear chemical shift behavior observed for L-FABP complexes with cholate derivatives added insight into conformational dynamics in the presence of ligands. The observed spectroscopic features of L-FABP/BA complexes, discussed in relation to ligand chemistry, suggest possible molecular determinants of recognition, with implications regarding intracellular BA transport. Our findings suggest that human L-FABP is a poorly selective, universal BA binder. PMID:25639618

  15. An efficient protocol for incorporation of an unnatural amino acid in perdeuterated recombinant proteins using glucose-based media.

    PubMed

    Venditti, Vincenzo; Fawzi, Nicolas L; Clore, G Marius

    2012-03-01

    The in vivo incorporation of unnatural amino acids into proteins is a well-established technique requiring an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is incorporated at a position encoded by a TAG amber codon. Although this technology provides unique opportunities to engineer protein structures, poor protein yields are usually obtained in deuterated media, hampering its application in the protein NMR field. Here, we describe a novel protocol for incorporating unnatural amino acids into fully deuterated proteins using glucose-based media (which are relevant to the production, for example, of amino acid-specific methyl-labeled proteins used in the study of large molecular weight systems). The method consists of pre-induction of the pEVOL plasmid encoding the tRNA/aminoacyl-tRNA synthetase pair in a rich, H(2)O-based medium prior to exchanging the culture into a D(2)O-based medium. Our protocol results in high level of isotopic incorporation (~95%) and retains the high expression level of the target protein observed in Luria-Bertani medium. PMID:22350951

  16. Prediction of protein-protein interactions with clustered amino acids and weighted sparse representation.

    PubMed

    Huang, Qiaoying; You, Zhuhong; Zhang, Xiaofeng; Zhou, Yong

    2015-01-01

    With the completion of the Human Genome Project, bioscience has entered into the era of the genome and proteome. Therefore, protein-protein interactions (PPIs) research is becoming more and more important. Life activities and the protein-protein interactions are inseparable, such as DNA synthesis, gene transcription activation, protein translation, etc. Though many methods based on biological experiments and machine learning have been proposed, they all spent a long time to learn and obtained an imprecise accuracy. How to efficiently and accurately predict PPIs is still a big challenge. To take up such a challenge, we developed a new predictor by incorporating the reduced amino acid alphabet (RAAA) information into the general form of pseudo-amino acid composition (PseAAC) and with the weighted sparse representation-based classification (WSRC). The remarkable advantages of introducing the reduced amino acid alphabet is being able to avoid the notorious dimensionality disaster or overfitting problem in statistical prediction. Additionally, experiments have proven that our method achieved good performance in both a low- and high-dimensional feature space. Among all of the experiments performed on the PPIs data of Saccharomyces cerevisiae, the best one achieved 90.91% accuracy, 94.17% sensitivity, 87.22% precision and a 83.43% Matthews correlation coefficient (MCC) value. In order to evaluate the prediction ability of our method, extensive experiments are performed to compare with the state-of-the-art technique, support vector machine (SVM). The achieved results show that the proposed approach is very promising for predicting PPIs, and it can be a helpful supplement for PPIs prediction. PMID:25984606

  17. Synthesis and phosphorylation of the glial fibrillary acidic protein during brain development: A tissue slice study

    SciTech Connect

    Noetzel, M.J. )

    1990-01-01

    Brain slices were incubated with either (3H) amino acids or (32P) orthophosphate in order to characterize the synthesis and phosphorylation of the glial fibrillary acidic protein (GFAP) in the rat nervous system. The incorporation of (3H) amino acids into GFAP was found to increase significantly during early postnatal development, reaching a peak of activity on day 5 of life and then declining over the next 2 weeks. Concomitant with this peak of synthetic activity the content of GFAP in rat brain was also observed to increase dramatically. GFAP continued to accumulate in brain through postnatal day 30 despite a decrease in the synthesis of the protein. These results indicate that the increase in GFAP during the first month of life cannot be ascribed solely to the rate of GFAP synthesis. The findings are consistent with the hypothesis that during later stages of astrocytic development the accumulation of GFAP may be primarily dependent upon a low rate of protein degradation. The pattern of GFAP phosphorylation in the developing rat brain differed from that observed for the incorporation of (3H) amino acids. The peak incorporation of 32P into GFAP occurred on postnatal day 10 at a time when synthesis of the protein had declined by 43%. These findings suggest that during development phosphorylation of GFAP is mediated by factors different from those directing its synthesis. In addition, phosphorylation of GFAP did not alter its solubility in cytoskeletal preparations indicating that GFAP phosphorylation is probably not a major regulatory mechanism in disassembly of the astroglial filaments.

  18. Thermophysical properties of starch and whey protein composite prepared in presence of organic acid and esters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously, we prepared starch and protein composite by reactive mixing in presence of various organic acids and found that use of these acid esters resulted in composites with good mechanical properties. In this study, concentration (% w/w) of acid citrates in the starch-protein composites were var...

  19. Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid.

    PubMed

    Pan, Yijun; Scanlon, Martin J; Owada, Yuji; Yamamoto, Yui; Porter, Christopher J H; Nicolazzo, Joseph A

    2015-12-01

    The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice

  20. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2011-08-30

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  1. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2009-02-24

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  2. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  3. Site-specific incorporation of redox active amino acids into proteins

    DOEpatents

    Alfonta; Lital , Schultz; Peter G. , Zhang; Zhiwen

    2010-10-12

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

  4. Design and expression of fusion protein consists of HBsAg and Polyepitope of HCV as an HCV potential vaccine

    PubMed Central

    Gholizadeh, Monireh; Khanahmad, Hossein; Memarnejadian, Arash; Aghasadeghi, Mohammad Reza; Roohvand, Farzin; Sadat, Seyed Mehdi; Cohan, Reza Ahangari; Nazemi, Ali; Motevalli, Fatemeh; Asgary, Vahid; Arezumand, Roghaye

    2015-01-01

    Background: Hepatitis C virus (HCV) infection is a serious public health threat worldwide. Cellular immune responses, especially cytotoxic T-lymphocytes (CTLs), play a critical role in immune response toward the HCV clearance. Since polytope vaccines have the ability to stimulate the cellular immunity, a recombinant fusion protein was developed in this study. Materials and Methods: The designed fusion protein is composed of hepatitis B surface antigen (HBsAg), as an immunocarrier, fused to an HCV polytope sequence. The polytope containing five immunogenic epitopes of HCV was designed to induce specific CTL responses. The construct was cloned into the pET-28a, and its expression was investigated in BL21 (DE3), BL21 pLysS, BL21 pLysE, and BL21 AI Escherichia coli strains using 12% gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the identity of expressed fusion protein was confirmed by Western blotting using anti-His monoclonal antibody and affinity chromatography was applied to purify the expressed protein. Results: The accuracy of the construct was confirmed by restriction map analysis and sequencing. The transformation of the construct into the BL21 (DE3), pLysS, and pLysE E. coli strains did not lead to any expression. The fusion protein was found to be toxic for E. coli DE3. By applying two steps inhibition, the fusion protein was successfully expressed in BL21 (AI) E. coli strain. Conclusion: The HBsAg-polytope fusion protein expressed in this study can be further evaluated for its immunogenicity in animal models. PMID:26682209

  5. A fusion protein consisting of the exopeptidases PepN and PepX-production, characterization, and application.

    PubMed

    Stressler, Timo; Pfahler, Nina; Merz, Michael; Hubschneider, Larissa; Lutz-Wahl, Sabine; Claaßen, Wolfgang; Fischer, Lutz

    2016-09-01

    Nowadays, general and specific aminopeptidases are of great interest, especially for protein hydrolysis in the food industry. As shown previously, it is confirmed that the general aminopeptidase N (PepN; EC 3.4.11.2) and the proline-specific peptidase PepX (EC 3.4.14.11) from Lactobacillus helveticus ATCC 12046 show a synergistic effect during protein hydrolysis which results in high degrees of hydrolysis and reduced bitterness. To combine both activities, the enzymes were linked and a fusion protein called PepN-L1-PepX (FUS-PepN-PepX) was created. After production and purification, the fusion protein was characterized. Some of its biochemical characteristics were altered in favor for an application compared to the single enzymes. As an example, the optimum temperature for the PepN activity increased from 30 °C for the single enzyme to 35 °C for FUS-PepN. In addition, the temperature stability of PepX was higher for FUS-PepX than for the single enzyme (50 % compared to 40 % residual activity at 50 °C after 14 days, respectively). In addition, the disulfide bridge-reducing reagent β-mercaptoethanol did not longer inactivate the FUS-PepN activity. Furthermore, the K M values decreased for both enzyme activities in the fusion protein. Finally, it was found that the synergistic hydrolysis performance in a casein hydrolysis was not reduced for the fusion protein. The increase of the relative degree of hydrolysis of a prehydrolyzed casein solution was the same as it was for the single enzymes. As a benefit, the resulting hydrolysate showed a strong antioxidative capacity (ABTS-IC50 value: 5.81 μg mL(-1)). PMID:27037692

  6. Perlwapin, an abalone nacre protein with three four-disulfide core (whey acidic protein) domains, inhibits the growth of calcium carbonate crystals.

    PubMed

    Treccani, Laura; Mann, Karlheinz; Heinemann, Fabian; Fritz, Monika

    2006-10-01

    We have isolated a new protein from the nacreous layer of the shell of the sea snail Haliotis laevigata (abalone). Amino acid sequence analysis showed the protein to consist of 134 amino acids and to contain three sequence repeats of approximately 40 amino acids which were very similar to the well-known whey acidic protein domains of other proteins. The new protein was therefore named perlwapin. In addition to the major sequence, we identified several minor variants. Atomic force microscopy was used to explore the interaction of perlwapin with calcite crystals. Monomolecular layers of calcite crystals dissolve very slowly in deionized water and recrystallize in supersaturated calcium carbonate solution. When perlwapin was dissolved in the supersaturated calcium carbonate solution, growth of the crystal was inhibited immediately. Perlwapin molecules bound tightly to distinct step edges, preventing the crystal layers from growing. Using lower concentrations of perlwapin in a saturated calcium carbonate solution, we could distinguish native, active perlwapin molecules from denaturated ones. These observations showed that perlwapin can act as a growth inhibitor for calcium carbonate crystals in saturated calcium carbonate solution. The function of perlwapin in nacre growth may be to inhibit the growth of certain crystallographic planes in the mineral phase of the polymer/mineral composite nacre. PMID:16861275

  7. Perlwapin, an Abalone Nacre Protein with Three Four-Disulfide Core (Whey Acidic Protein) Domains, Inhibits the Growth of Calcium Carbonate Crystals

    PubMed Central

    Treccani, Laura; Mann, Karlheinz; Heinemann, Fabian; Fritz, Monika

    2006-01-01

    We have isolated a new protein from the nacreous layer of the shell of the sea snail Haliotis laevigata (abalone). Amino acid sequence analysis showed the protein to consist of 134 amino acids and to contain three sequence repeats of ∼40 amino acids which were very similar to the well-known whey acidic protein domains of other proteins. The new protein was therefore named perlwapin. In addition to the major sequence, we identified several minor variants. Atomic force microscopy was used to explore the interaction of perlwapin with calcite crystals. Monomolecular layers of calcite crystals dissolve very slowly in deionized water and recrystallize in supersaturated calcium carbonate solution. When perlwapin was dissolved in the supersaturated calcium carbonate solution, growth of the crystal was inhibited immediately. Perlwapin molecules bound tightly to distinct step edges, preventing the crystal layers from growing. Using lower concentrations of perlwapin in a saturated calcium carbonate solution, we could distinguish native, active perlwapin molecules from denaturated ones. These observations showed that perlwapin can act as a growth inhibitor for calcium carbonate crystals in saturated calcium carbonate solution. The function of perlwapin in nacre growth may be to inhibit the growth of certain crystallographic planes in the mineral phase of the polymer/mineral composite nacre. PMID:16861275

  8. Quantitative proteomics by amino acid labeling identifies novel NHR-49 regulated proteins in C. elegans

    PubMed Central

    Fredens, Julius; Færgeman, Nils J.

    2012-01-01

    Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology to quantitatively examine metabolic and signaling pathways in yeast, fruit flies, plants, cell cultures and mice. However, only metabolic labeling using 15N has been applied to examine such events in the nematode Caenorhabditis elegans. We have recently shown that C. elegans can be completely labeled with heavy-labeled lysine by feeding worms on prelabeled lysine auxotroph Escherichia coli for just one generation. We applied this methodology to examine the organismal response to functional loss or RNAi mediated knock down of the transcription factor NHR-49, and found numerous proteins involved in lipid metabolism to be downregulated, which is consistent with its previously proposed function as a transcriptional regulator of fatty acid metabolism. The combined use of quantitative proteomics and selective gene knockdown by RNAi provides a powerful tool with broad implications for C. elegans biology. PMID:24058826

  9. Formation and stability of interpenetrating polymer network hydrogels consisting of fibrin and hyaluronic acid for tissue engineering.

    PubMed

    Lee, Fan; Kurisawa, Motoichi

    2013-02-01

    Fibrin gel is widely used as a tissue engineering scaffold. However, it has poor mechanical properties, which often result in rapid contraction and degradation of the scaffold. An interpenetrating polymer network (IPN) hydrogel composed of fibrin and hyaluronic acid-tyramine (HA-Tyr) was developed to improve the mechanical properties. The fibrin network was formed by cleaving fibrinogen with thrombin, producing fibrin monomers that rapidly polymerize. The HA network was formed through the coupling of tyramine moieties using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The degree of crosslinking of the HA-Tyr network can be tuned by varying the H₂O₂ concentration, producing IPN hydrogels with different storage moduli (G'). While fibrin gels were completely degraded in the presence of plasmin and contracted when embedded with cells, the shape of the IPN hydrogels was maintained due to structural support by the HA-Tyr networks. Cell proliferation and capillary formation occurred in IPN hydrogels and were found to decrease with increasing G' of the hydrogels. The results suggest that fibrin-HA-Tyr IPN hydrogels are a potential alternative to fibrin gels as scaffolds for tissue engineering applications that require shape stability. PMID:22943886

  10. Human odontoblasts express transient receptor protein and acid-sensing ion channel mechanosensor proteins.

    PubMed

    Solé-Magdalena, Antonio; Revuelta, Enrique G; Menénez-Díaz, Ivan; Calavia, Marta G; Cobo, Teresa; García-Suárez, Olivia; Pérez-Piñera, Pablo; De Carlos, Felix; Cobo, Juan; Vega, Jose A

    2011-05-01

    Diverse proteins of the denegerin/epithelial sodium channel (DEG/ENa(+) C) superfamily, in particular those belonging to the acid-sensing ion channel (ASIC) family, as well as some members of the transient receptor protein (TRP) channel, function as mechanosensors or may be required for mechanosensation in a diverse range of species and cell types. Therefore, we investigated the putative mechanosensitive function of human odontoblasts using immunohistochemistry to detect ENa(+) C subunits (α, β, and γ) and ASIC (1, 2, 3, and 4) proteins, as well as TRPV4, in these cells. Positive and specific immunoreactivity in the odontoblast soma and/or processes was detected for all proteins studied except α-ENa(+) C. The intensity of immunostaining was high for β-ENa(+) C and ASIC2, whereas it was low for ASIC1, ASIC3, γ-ENa(+) C, and TRPV4, being absent for α-ENa(+) C and ASIC4. These results suggest that human odontoblasts in situ express proteins related to mechanosensitive channels that probably participate in the mechanisms involved in teeth sensory transmission. PMID:20836083

  11. A self-consistent, microenvironment modulated screened coulomb potential approximation to calculate pH-dependent electrostatic effects in proteins.

    PubMed

    Mehler, E L; Guarnieri, F

    1999-07-01

    An improved approach is presented for calculating pH-dependent electrostatic effects in proteins using sigmoidally screened Coulomb potentials (SCP). It is hypothesized that a key determinant of seemingly aberrant behavior in pKa shifts is due to the properties of the unique microenvironment around each residue. To help demonstrate this proposal, an approach is developed to characterize the microenvironments using the local hydrophobicity/hydrophilicity around each residue of the protein. The quantitative characterization of the microenvironments shows that the protein is a complex mosaic of differing dielectric regions that provides a physical basis for modifying the dielectric screening functions: in more hydrophobic microenvironments the screening decreases whereas the converse applies to more hydrophilic regions. The approach was applied to seven proteins providing more than 100 measured pKa values and yielded a root mean square deviation of 0.5 between calculated and experimental values. The incorporation of the local hydrophobicity characteristics into the algorithm allowed the resolution of some of the more intractable problems in the calculation of pKa. Thus, the divergent shifts of the pKa of Glu-35 and Asp-66 in hen egg white lysozyme, which are both about 90% buried, was correctly predicted. Mechanistically, the divergence occurs because Glu-35 is in a hydrophobic microenvironment, while Asp-66 is in a hydrophilic microenvironment. Furthermore, because the calculation of the microenvironmental effects takes very little CPU time, the computational speed of the SCP formulation is conserved. Finally, results from different crystal structures of a given protein were compared, and it is shown that the reliability of the calculated pKa values is sufficient to allow identification of conformations that may be more relevant for the solution structure. PMID:10388736

  12. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes.

    PubMed

    Leitner, Alexander; Joachimiak, Lukasz A; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-07-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  13. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

    PubMed Central

    Leitner, Alexander; Joachimiak, Lukasz A.; Unverdorben, Pia; Walzthoeni, Thomas; Frydman, Judith; Förster, Friedrich; Aebersold, Ruedi

    2014-01-01

    The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches. PMID:24938783

  14. Coffee bean arabinogalactans: acidic polymers covalently linked to protein.

    PubMed

    Redgwell, Robert J; Curti, Delphine; Fischer, Monica; Nicolas, Pierre; Fay, Laurent B

    2002-02-11

    The arabinogalactan content of green coffee beans (Coffea arabica var. Yellow Caturra) was released by a combination of chemical extraction and enzymatic hydrolysis of the mannan-cellulose component of the wall. Several arabinogalactan fractions were isolated, purified by gel-permeation and ion-exchange chromatography and characterised by compositional and linkage analysis. The AG fractions contained between 6 and 8% glucuronic acid, and gave a positive test for the beta-glucosyl-Yariv reagent, a stain specific for arabinogalactan-proteins. The protein component accounted for between 0.5 and 2.0% of the AGPs and contained between 7 and 12% hydroxyproline. The AG moieties displayed considerable heterogeneity with regard to their degree of arabinosylation and the extent and composition of their side-chains. They possessed a MW average of 650 kDa which ranged between 150 and 2000 kDa. An investigation of the structural features of the major AG fraction, released following enzymatic hydrolysis of the mannan-cellulose polymers, allowed a partial structure of coffee arabinogalactan to be proposed. PMID:11844494

  15. Small acid soluble proteins for rapid spore identification.

    SciTech Connect

    Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.; Jokerst, Amanda S.

    2006-12-01

    This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.

  16. Crystal growth of proteins, nucleic acids, and viruses in gels.

    PubMed

    Lorber, Bernard; Sauter, Claude; Théobald-Dietrich, Anne; Moreno, Abel; Schellenberger, Pascale; Robert, Marie-Claire; Capelle, Bernard; Sanglier, Sarah; Potier, Noëlle; Giegé, Richard

    2009-11-01

    Medium-sized single crystals with perfect habits and no defect producing intense and well-resolved diffraction patterns are the dream of every protein crystallographer. Crystals of biological macromolecules possessing these characteristics can be prepared within a medium in which mass transport is restricted to diffusion. Chemical gels (like polysiloxane) and physical gels (such as agarose) provide such an environment and are therefore suitable for the crystallisation of biological macromolecules. Instructions for the preparation of each type of gel are given to urge crystal growers to apply diffusive media for enhancing crystallographic quality of their crystals. Examples of quality enhancement achieved with silica and agarose gels are given. Results obtained with other substances forming gel-like media (such as lipidic phases and cellulose derivatives) are presented. Finally, the use of gels in combination with capillary tubes for counter-diffusion experiments is discussed. Methods and techniques implemented with proteins can also be applied to nucleic acids and nucleoprotein assemblies such as viruses. PMID:20005247

  17. An amphipathic polypeptide derived from poly-γ-glutamic acid for the stabilization of membrane proteins

    PubMed Central

    Han, Seong-Gu; Na, Jung-Hyun; Lee, Won-Kyu; Park, Dongkook; Oh, Jihye; Yoon, Sung-Ho; Lee, Cheng-Kang; Sung, Moon-Hee; Shin, Yeon-Kyun; Yu, Yeon Gyu

    2014-01-01

    Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly-γ-glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self-assembles as mono-disperse oligomers consisted of 4–5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)-solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ETA) in their active conformations. Furthermore, ETA in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins. PMID:25283538

  18. Prolonged fasting identifies heat shock protein 10 as a Sirtuin 3 substrate: elucidating a new mechanism linking mitochondrial protein acetylation to fatty acid oxidation enzyme folding and function.

    PubMed

    Lu, Zhongping; Chen, Yong; Aponte, Angel M; Battaglia, Valentina; Gucek, Marjan; Sack, Michael N

    2015-01-23

    Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function. PMID:25505263

  19. Generation of a functional monomolecular protein lattice consisting of an s-layer fusion protein comprising the variable domain of a camel heavy chain antibody.

    PubMed

    Pleschberger, Magdalena; Neubauer, Angela; Egelseer, Eva M; Weigert, Stefan; Lindner, Brigitte; Sleytr, Uwe B; Muyldermans, Serge; Sára, Margit

    2003-01-01

    Crystalline bacterial cell surface layer (S-layer) proteins are composed of a single protein or glycoprotein species. Isolated S-layer subunits frequently recrystallize into monomolecular protein lattices on various types of solid supports. For generating a functional protein lattice, a chimeric protein was constructed, which comprised the secondary cell wall polymer-binding region and the self-assembly domain of the S-layer protein SbpA from Bacillus sphaericus CCM 2177, and a single variable region of a heavy chain camel antibody (cAb-Lys3) recognizing lysozyme as antigen. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-Lys3. The functionality of the fused cAb-Lys3 in the S-layer fusion protein was proved by surface plasmon resonance measurements. Dot blot assays revealed that the accessibility of the fused functional sequence for the antigen was independent of the use of soluble or assembled S-layer fusion protein. Recrystallization of the S-layer fusion protein into the square lattice structure was observed on peptidoglycan-containing sacculi of B. sphaericus CCM 2177, on polystyrene or on gold chips precoated with thiolated secondary cell wall polymer, which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Thereby, the fused cAb-Lys3 remained located on the outer S-layer surface and accessible for lysozyme binding. Together with solid supports precoated with secondary cell wall polymers, S-layer fusion proteins comprising rSbpA(31)(-)(1068) and cAbs directed against various antigens shall be exploited for building up monomolecular functional protein lattices as required for applications in nanobiotechnology. PMID:12643755

  20. Latency, duration and dose response relationships of amino acid effects on human muscle protein synthesis.

    PubMed

    Rennie, Michael J; Bohé, Julien; Wolfe, Robert R

    2002-10-01

    The components of the stimulatory effect of food on net deposition of protein are beginning to be identified and separated. One of the most important of these appears to be the effect of amino acids per se in stimulating muscle anabolism. Amino acids appear to have a linear stimulatory effect within the range of normal diurnal plasma concentrations from postabsorptive to postprandial. Within this range, muscle protein synthesis (measured by incorporation of stable isotope tracers of amino acids into biopsied muscle protein) appears to be stimulated approximately twofold; however, little further increase occurs when very high concentrations of amino acids (>2.5 times the normal postabsorptive plasma concentration) are made available. Amino acids provided in surfeit of the ability of the system to synthesize protein are disposed of by oxidation, ureagenesis and gluconeogenesis. The stimulatory effect of amino acids appears to be time dependent; a square wave increase in the availability of amino acids causes muscle protein synthesis to be stimulated and to fall back to basal values, despite continued amino acid availability. The relationship between muscle protein synthesis and insulin availability suggests that most of the stimulatory effects occur at low insulin concentrations, with large increases having no effect. These findings may have implications for our understanding of the body's requirements for protein. The maximal capacity for storage of amino acids as muscle protein probably sets an upper value on the extent to which amino acids can be stored after a single meal. PMID:12368422

  1. Pitfalls in protein quantitation using acid-catalyzed O18 labeling: hydrolysis-driven deamidation

    PubMed Central

    Wang, Shunhai; Bobst, Cedric E.; Kaltashov, Igor A.

    2011-01-01

    Proteolysis combined with O18 labeling emerged recently as a powerful tool for quantitation of proteins for which suitable internal standards cannot be produced using molecular biology methods. Several recent reports suggested that acid-catalyzed O18 labeling may be superior to the commonly accepted enzymatic protocol, as it may allow more significant spacing between the isotopic clusters of labeled and unlabeled peptides, thereby eliminating signal interference and enhancing the quality of quantitation. However, careful examination of this procedure reveals that the results of protein quantitation assisted by acid-catalyzed O18 labeling are highly peptide-dependent. The inconsistency was found to be caused by deamidation of Asn, Gln and carbamidomethylated Cys residues during prolonged exposure of the proteolytic fragments to the acidic environment of the labeling reaction, which translates into a loss in signal for theses peptides. Taking deamidation into account leads to a significant improvement in the consistency of quantitation across a range of different proteolytic fragments. PMID:21819098

  2. ASSESSMENT OF NEUROTOXICITY: USE OF GLIAL FIBRILLARY ACIDIC PROTEIN AS A BIOMARKER

    EPA Science Inventory

    Diverse neurotoxic insults results in proliferation and hypertrophy of astrocytes. he hallmark of this response is enhanced expression of the major intermediate filament protein of astrocytes, glial fibrillary acidic protein (GFAP). hese observations suggest that GFAP may be a us...

  3. SEED PROTEIN QUANTITIES OF FIELD-GROWN SOYBEANS EXPOSED TO SIMULATED ACIDIC RAIN

    EPA Science Inventory

    Analysis of seeds harvested from field-grown soybeans demonstrated that simulated acidic rainfalls from two experimental protocols can significantly decrease total protein contents of soybeans. Statistically significant differences in protein content per seed mass were obtained i...

  4. Solvent accessible surface area-based hot-spot detection methods for protein-protein and protein-nucleic acid interfaces.

    PubMed

    Munteanu, Cristian R; Pimenta, António C; Fernandez-Lozano, Carlos; Melo, André; Cordeiro, Maria N D S; Moreira, Irina S

    2015-05-26

    Due to the importance of hot-spots (HS) detection and the efficiency of computational methodologies, several HS detecting approaches have been developed. The current paper presents new models to predict HS for protein-protein and protein-nucleic acid interactions with better statistics compared with the ones currently reported in literature. These models are based on solvent accessible surface area (SASA) and genetic conservation features subjected to simple Bayes networks (protein-protein systems) and a more complex multi-objective genetic algorithm-support vector machine algorithms (protein-nucleic acid systems). The best models for these interactions have been implemented in two free Web tools. PMID:25845030

  5. Experimental Evolution of a Green Fluorescent Protein Composed of 19 Unique Amino Acids without Tryptophan

    NASA Astrophysics Data System (ADS)

    Kawahara-Kobayashi, Akio; Hitotsuyanagi, Mitsuhiro; Amikura, Kazuaki; Kiga, Daisuke

    2014-04-01

    At some stage of evolution, genes of organisms may have encoded proteins that were synthesized using fewer than 20 unique amino acids. Similar to evolution of the natural 19-amino-acid proteins GroEL/ES, proteins composed of 19 unique amino acids would have been able to evolve by accumulating beneficial mutations within the 19-amino-acid repertoire encoded in an ancestral genetic code. Because Trp is thought to be the last amino acid included in the canonical 20-amino-acid repertoire, this late stage of protein evolution could be mimicked by experimental evolution of 19-amino-acid proteins without tryptophan (Trp). To further understand the evolution of proteins, we tried to mimic the evolution of a 19-amino-acid protein involving the accumulation of beneficial mutations using directed evolution by random mutagenesis on the whole targeted gene sequence. We created active 19-amino-acid green fluorescent proteins (GFPs) without Trp from a poorly fluorescent 19-amino-acid mutant, S1-W57F, by using directed evolution with two rounds of mutagenesis and selection. The N105I and S205T mutations showed beneficial effects on the S1-W57F mutant. When these two mutations were combined on S1-W57F, we observed an additive effect on the fluorescence intensity. In contrast, these mutations showed no clear improvement individually or in combination on GFPS1, which is the parental GFP mutant composed of 20 amino acids. Our results provide an additional example for the experimental evolution of 19-amino-acid proteins without Trp, and would help understand the mechanisms underlying the evolution of 19-amino-acid proteins. (236 words)

  6. Hydroxyindole Carboxylic Acid-Based Inhibitors for Receptor-Type Protein Tyrosine Protein Phosphatase Beta

    PubMed Central

    Zeng, Li-Fan; Zhang, Ruo-Yu; Bai, Yunpeng; Wu, Li; Gunawan, Andrea M.

    2014-01-01

    Abstract Aims: Protein tyrosine phosphatases (PTPs) play an important role in regulating a wide range of cellular processes. Understanding the role of PTPs within these processes has been hampered by a lack of potent and selective PTP inhibitors. Generating potent and selective probes for PTPs remains a significant challenge because of the highly conserved and positively charged PTP active site that also harbors a redox-sensitive Cys residue. Results: We describe a facile method that uses an appropriate hydroxyindole carboxylic acid to anchor the inhibitor to the PTP active site and relies on the secondary binding elements introduced through an amide-focused library to enhance binding affinity for the target PTP and to impart selectivity against off-target phosphatases. Here, we disclose a novel series of hydroxyindole carboxylic acid-based inhibitors for receptor-type tyrosine protein phosphatase beta (RPTPβ), a potential target that is implicated in blood vessel development. The representative RPTPβ inhibitor 8b-1 (L87B44) has an IC50 of 0.38 μM and at least 14-fold selectivity for RPTPβ over a large panel of PTPs. Moreover, 8b-1 also exhibits excellent cellular activity and augments growth factor signaling in HEK293, MDA-MB-468, and human umbilical vein endothelial cells. Innovation: The bicyclic salicylic acid pharmacophore-based focused library approach may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Conclusion: A novel method is described for the development of bioavailable PTP inhibitors that utilizes bicyclic salicylic acid to anchor the inhibitors to the active site and peripheral site interactions to enhance binding affinity and selectivity. Antioxid. Redox Signal. 20, 2130–2140. PMID:24180557

  7. Effects of starvation for potassium and other inorganic ions on protein degradation and ribonucleic acid synthesis in Escherichia coli.

    PubMed

    St John, A C; Goldberg, A L

    1980-09-01

    Starvation of Escherichia coli for potassium, phosphate, or magnesium ions leads to a reversible increase in the rate of protein degradation and an inhibition of ribonucleic acid (RNA) synthesis. In cells deprived of potassium, the breakdown of the more stable cell proteins increased two- to threefold, whereas the hydrolysis of short-lived proteins, both normal ones and analog-containing polypeptides, did not change. The mechanisms initiating the enhancement of proteolysis during starvation for these ions were examined. Upon starvation for amino acids or amino acyl-transfer RNA (tRNA), protein breakdown increases in relA+ (but not relA) cells as a result of the rapid synthesis of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). However, a lack of amino acyl-tRNA does not appear to be responsible for the increased protein breakdown in cells starved for inorganic ions, since protein breakdown increased in the absence of these ions in both relA+ and relA cultures, and since a large excess of amino acids did not affect this response. In bacteria in which energy production is restricted, ppGpp levels also rise, and protein breakdown increases. The ion-deprived cultures did show a 40 to 75% reduction in adenosine-5'-triphosphate levels,l similar to that seen upon glucose starvation. However, this decrease in ATP content does not appear to cause the increase in protein breakdown or lead to an accumulation of ppGpp. No consistent change in intracellular ppGpp levels was found in relA+ or relA cells starved for these ions. In addition, in relX mutants, removal of these ions led to accelerated protein degradation even though relX cells are unable to increase ppGpp levels or proteolysis when deprived of a carbon source. In the potassium-, phosphate-, and magnesium-deprived cultures, the addition of choramphenicol or tetracycline caused a reduction in protein breakdown toward basal levels. Such findings, however, do not indicate that protein synthesis is essential for the

  8. Fatty acid transport protein 1 can compensate for fatty acid transport protein 4 in the developing mouse epidermis.

    PubMed

    Lin, Meei-Hua; Miner, Jeffrey H

    2015-02-01

    Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very-long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective barrier; they die neonatally because of dehydration and restricted movements. Mutations in SLC27A4, the gene encoding FATP4, cause ichthyosis prematurity syndrome (IPS), characterized by premature birth, respiratory distress, and edematous skin with severe ichthyotic scaling. Symptoms of surviving patients become mild, although atopic manifestations are common. We previously showed that suprabasal keratinocyte expression of a Fatp4 transgene in Fatp4 mutant skin rescues the lethality and ameliorates the skin phenotype. Here we tested the hypothesis that FATP1, the closest FATP4 homolog, can compensate for the lack of FATP4 in our mouse model of IPS, as it might do postnatally in IPS patients. Transgenic expression of FATP1 in suprabasal keratinocytes rescued the phenotype of Fatp4 mutants, and FATP1 sorted to the same intracellular organelles as endogenous FATP4. Thus, FATP1 and FATP4 likely have overlapping substrate specificities, enzymatic activities, and biological functions. These results suggest that increasing expression of FATP1 in suprabasal keratinocytes could normalize the skin of IPS patients and perhaps prevent the atopic manifestations. PMID:25184958

  9. D-amino acids for the enhancement of a binary biocide cocktail consisting of THPS and EDDS against an SRB biofilm.

    PubMed

    Xu, D; Wen, J; Fu, W; Gu, T; Raad, I

    2012-04-01

    Biofilms of sulfate reducing bacteria (SRB) are often responsible for Microbiologically Influenced Corrosion (MIC) that is a major problem in the oil and gas industry as well as water utilities and other industries. This work was inspired by recent reports that some D: -amino acids may be useful in the control of microbial biofilms. A D: -amino acid mixture with equimolar D: -tyrosine, D: -methionine, D: -tryptophan and D: -leucine was tested in this work for their enhancement of a biocide cocktail containing tetrakis (hydroxymethyl) phosphonium sulfate (THPS) and ethylenediamine-N,N'-disuccinic acid (EDDS). Desulfovibrio vulgaris (ATCC 7757) was cultured in ATCC 1249 medium. Its biofilm was grown on C1018 carbon steel coupons. Experimental results indicated that the triple biocide cocktail consisting of 30 ppm THPS, 500 ppm EDDS and 6.6 ppm D: -amino acid mixture (with equimolar D: -tyrosine, D: -methionine, D: -tryptophan and D: -leucine) was far more effective than THPS and EDDS alone and their binary combination. The triple biocide cocktail effectively prevented SRB biofilm establishment and removed the established SRB biofilm. The D: -amino acid mixture alone did not show significant effects in the two tasks even at 660 ppm. PMID:22805946

  10. Impact of antinutritional factors in food proteins on the digestibility of protein and the bioavailability of amino acids and on protein quality.

    PubMed

    Sarwar Gilani, G; Wu Xiao, Chao; Cockell, Kevin A

    2012-08-01

    Dietary antinutritional factors have been reported to adversely affect the digestibility of protein, bioavailability of amino acids and protein quality of foods. Published data on these negative effects of major dietary antinutritional factors are summarized in this manuscript. Digestibility and the quality of mixed diets in developing countries are considerably lower than of those in developed regions. For example, the digestibility of protein in traditional diets from developing countries such as India, Guatemala and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94 %). Poor digestibility of protein in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, is due to the presence of less digestible protein fractions, high levels of insoluble fibre, and/or high concentrations of antinutritional factors present endogenously or formed during processing. Examples of naturally occurring antinutritional factors include glucosinolates in mustard and canola protein products, trypsin inhibitors and haemagglutinins in legumes, tannins in legumes and cereals, gossypol in cottonseed protein products, and uricogenic nucleobases in yeast protein products. Heat/alkaline treatments of protein products may yield Maillard reaction compounds, oxidized forms of sulphur amino acids, D-amino acids and lysinoalanine (LAL, an unnatural nephrotoxic amino acid derivative). Among common food and feed protein products, soyabeans are the most concentrated source of trypsin inhibitors. The presence of high levels of dietary trypsin inhibitors from soyabeans, kidney beans or other grain legumes have been reported to cause substantial reductions in protein and amino acid digestibility (up to 50 %) and protein quality (up to 100 %) in rats and/or pigs. Similarly, the presence of high levels of tannins in sorghum and other cereals, fababean and other grain legumes can cause

  11. Prebiotic Synthesis of Hydrophobic and Protein Amino Acids

    PubMed Central

    Ring, David; Wolman, Yecheskel; Friedmann, Nadav; Miller, Stanley L.

    1972-01-01

    The formation of amino acids by the action of electric discharges on a mixture of methane, nitrogen, and water with traces of ammonia was studied in detail. The presence of glycine, alanine, α-amino-n-butyric acid, α-aminoisobutyric acid, valine, norvaline, isovaline, leucine, isoleucine, alloisoleucine, norleucine, proline, aspartic acid, glutamic acid, serine, threonine, allothreonine, α-hydroxy-γ-aminobutyric acid, and α,γ-diaminobutyric acid was confirmed by ion-exchange chromatography and gas chromatography-mass spectrometry. All of the primary α-amino acids found in the Murchison Meteorite have been synthesized by this electric discharge experiment. PMID:4501592

  12. Gene Activation in Eukaryotes: Are Nuclear Acidic Proteins the Cause or the Effect?

    PubMed Central

    Pederson, Thoru

    1974-01-01

    Nuclear acidic proteins have been implicated in the positive control of gene transcription in eukaryotes. This hypothesis was examined in greater detail by analysis of these proteins during experimental gene activation by a technique for fractionating nuclei into chromatin and the ribonucleoprotein particles that contain heterogeneous nuclear RNA. When synthesis of rat-liver heterogeneous nuclear RNA was stimulated by administration of hydrocortisone, there was a parallel increase in the labeling of acidic proteins in ribonucleoprotein particles. However, there was no detectable effect on the labeling of either acidic chromatin proteins or histones. Thus, the nuclear acidic proteins that respond to the hormone are concerned with a post-transcriptional event, namely the assembly and processing of ribonucleoprotein particles that contain heterogeneous RNA, rather than with direct gene activation. Increases in synthesis of “chromatin” acidic proteins during gene activation observed by others may reflect the presence of these ribonucleoprotein particles in crude chromatin preparations. Images PMID:4522777

  13. Synthesis of alanyl nucleobase amino acids and their incorporation into proteins.

    PubMed

    Talukder, Poulami; Dedkova, Larisa M; Ellington, Andrew D; Yakovchuk, Petro; Lim, Jaebum; Anslyn, Eric V; Hecht, Sidney M

    2016-09-15

    Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail. PMID:27452282

  14. Identification of Dynamic Changes in Proteins Associated with the Cellular Cytoskeleton after Exposure to Okadaic Acid

    PubMed Central

    Opsahl, Jill A.; Ljostveit, Sonja; Solstad, Therese; Risa, Kristin; Roepstorff, Peter; Fladmark, Kari E.

    2013-01-01

    Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100–500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment. PMID:23708184

  15. Betulinic Acid Suppresses STAT3 Activation Pathway Through Induction of Protein Tyrosine Phosphatase SHP-1 in Human Multiple Myeloma Cells

    PubMed Central

    Pandey, Manoj K.; Sung, Bokyung; Aggarwal, Bharat B.

    2009-01-01

    STAT3 activation has been associated with survival, proliferation and invasion of various human cancers. Whether betulinic acid, a pentacyclic triterpene, can modulates the STAT3 pathway, was investigated in human multiple myeloma (MM) cells. We found that betulinic acid inhibited constitutive activation of STAT3, Src kinase, JAK1 and JAK2. Pervanadate reversed the betulinic acid -induced down regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, betulinic acid induced the expression of the PTP SHP-1 and silencing of the SHP-1 gene abolished the ability of betulinic acid to inhibit STAT3 activation and rescues betulinic acid-induced cell death. Betulinic acid also downregulated the expression of STAT3-regulated gene products such as bcl-xL, bcl-2, cyclin D1, and survivin. This correlated with an increase in apoptosis as indicated by an increase in the sub-G1 cell population and an increase in caspase-3–induced PARP cleavage. Consistent with these results, over expression of constitutive active STAT3 significantly reduced the betulinic acid-induced apoptosis. Betulinic acid also enhanced the apoptosis induced by thalidomide (from 10% to 55%) and bortezomib (from 5% to 70%) in MM cells. Overall, our results suggest that betulinic acid down regulates STAT3 activation through upregulation of SHP-1 and this may have potential in sensitization of STAT3 over expressing tumors to chemotherapeutic agents. PMID:19937797

  16. Comparative Analysis of Barophily-Related Amino Acid Content in Protein Domains of Pyrococcus abyssi and Pyrococcus furiosus

    PubMed Central

    Yafremava, Liudmila S.; Di Giulio, Massimo; Caetano-Anollés, Gustavo

    2013-01-01

    Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code. PMID:24187517

  17. Amino Acid Flux from Metabolic Network Benefits Protein Translation: the Role of Resource Availability

    PubMed Central

    Hu, Xiao-Pan; Yang, Yi; Ma, Bin-Guang

    2015-01-01

    Protein translation is a central step in gene expression and affected by many factors such as codon usage bias, mRNA folding energy and tRNA abundance. Despite intensive previous studies, how metabolic amino acid supply correlates with protein translation efficiency remains unknown. In this work, we estimated the amino acid flux from metabolic network for each protein in Escherichia coli and Saccharomyces cerevisiae by using Flux Balance Analysis. Integrated with the mRNA expression level, protein abundance and ribosome profiling data, we provided a detailed description of the role of amino acid supply in protein translation. Our results showed that amino acid supply positively correlates with translation efficiency and ribosome density. Moreover, with the rank-based regression model, we found that metabolic amino acid supply facilitates ribosome utilization. Based on the fact that the ribosome density change of well-amino-acid-supplied genes is smaller than poorly-amino-acid-supply genes under amino acid starvation, we reached the conclusion that amino acid supply may buffer ribosome density change against amino acid starvation and benefit maintaining a relatively stable translation environment. Our work provided new insights into the connection between metabolic amino acid supply and protein translation process by revealing a new regulation strategy that is dependent on resource availability. PMID:26056817

  18. The pH profile for acid-induced elongation of coleoptile and epicotyl sections is consistent with the acid-growth theory

    NASA Technical Reports Server (NTRS)

    Cleland, R. E.; Buckley, G.; Nowbar, S.; Lew, N. M.; Stinemetz, C.; Evans, M. L.; Rayle, D. L.

    1991-01-01

    The acid-growth theory predicts that a solution with a pH identical to that of the apoplast of auxin-treated tissues (4.5.-5.0) should induce elongation at a rate comparable to that of auxin. Different pH profiles for elongation have been obtained, however, depending on the type of pretreatment between harvest of the sections and the start of the pH-incubations. To determine the acid sensitivity under in vivo conditions, oat (Avena sativa L.) coleoptile, maize (Zea mays L.) coleoptile and pea (Pisum sativum L.) epicotyl sections were abraded so that exogenous buffers could penetrate the free space, and placed in buffered solutions of pH 3.5-6.5 without any preincubation. The extension, without auxin, was measured over the first 3 h. Experiments conducted in three laboratories produced similar results. For all three species, sections placed in buffer without pretreatment elongated at least threefold faster at pH 5.0 than at 6.0 or 6.5, and the rate elongation at pH 5.0 was comparable to that induced by auxin. Pretreatment of abraded sections with pH-6.5 buffer or distilled water adjusted to pH 6.5 or above gave similar results. We conclude that the pH present in the apoplast of auxin-treated coleoptile and stems is sufficiently low to account for the initial growth response to auxin.

  19. BILE ACIDS REGULATE THE ONTOGENIC EXPRESSION OF ILEAL BILE ACID BINDING PROTEIN IN THE RAT VIA THE FARNESOID X RECEPTOR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the rat, an increase in ileal bile acid binding protein (IBABP) expression occurs during the third postnatal week. In vitro studies suggest that bile acids (BAs) increase IBABP transcription by activating the BA receptor, farnesoid X receptor (FXR). Thus, we investigated the role of BAs on the on...

  20. [Effect of proteolysis inhibitors on the incorporation of labelled amino acids into proteins].

    PubMed

    Konikova, A S; Korotkina, R N

    1975-01-01

    Role of peptide bond breaks in the incorporation of amino acids into proteins in a "protein--amino acid" system is investigated. For this purpose the incorporation of labelled amino acids into trypsin under the inhibition of its autolysis by a specific inhibitor from soybean and epsilon-amino-caproic acid is studied. The trypsin inhibitor from soybean is found to suppress considerably the incorporation of 14C-glycine, 14C-lysine and 14C-methionine into crystal trypsin and not to affect the incorporation of labelled amino acids into chomotrypsin, papain and carboxypeptidase. Epsilon-Aminocaproic acid inhibited 14C-glycine incorporation into crystal trypsin by 40% and did not change its incorporation level into serum albumin. The dependency of amino acid incorporation level into trypsin on the activity of autolysis in the "protein--amino acid" system is demonstrated. PMID:1212456

  1. Retinoic acid-binding protein, rhombomeres and the neural crest.

    PubMed

    Maden, M; Hunt, P; Eriksson, U; Kuroiwa, A; Krumlauf, R; Summerbell, D

    1991-01-01

    We have investigated by immunocytochemistry the spatial and temporal distribution of cellular retinoic acid-binding protein (CRABP) in the developing nervous system of the chick embryo in order to answer two specific questions: do neural crest cells contain CRABP and where and when do CRABP-positive neuroblasts first arise in the neural tube? With regard to the neural crest, we have compared CRABP staining with HNK-1 staining (a marker of migrating neural crest) and found that they do indeed co-localise, but cephalic and trunk crest behave slightly differently. In the cephalic region in tissues such as the frontonasal mass and branchial arches, HNK-1 immunoreactivity is intense at early stages, but it disappears as CRABP immunoreactivity appears. Thus the two staining patterns do not overlap, but are complementary. In the trunk, HNK-1 and CRABP stain the same cell populations at the same time, such as those migrating through the anterior halves of the somites. In the neural tube, CRABP-positive neuroblasts first appear in the rhombencephalon just after the neural folds close and then a particular pattern of immunoreactivity appears within the rhombomeres of the hindbrain. Labelled cells are present in the future spinal cord, the posterior rhombencephalon up to rhombomere 6 and in rhombomere 4 thus producing a single stripe pattern. This pattern is dynamic and gradually changes as anterior rhombomeres begin to label. The similarity of this initial pattern to the arrangement of certain homeobox genes in the mouse stimulated us to examine the expression of the chicken Hox-2.9 gene. We show that at stage 15 the pattern of expression of this gene is closely related to that of CRABP. The relationship between retinoic acid, CRABP and homeobox genes is discussed. PMID:1707786

  2. The amino acid sequence of protein CM-3 from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Joubert, F J

    1985-01-01

    Protein CM-3 from Dendroaspis polylepis polylepis venom was purified by gel filtration and ion exchange chromatography. It comprises 65 amino acids including eight half-cystines. The complete amino acid sequence of protein CM-3 has been elucidated. The sequence (residues 1-50) resembles that of the N-terminal sequence of the subunits of a synergistic type protein and residues 51-65 that of the C-terminal sequence of an angusticeps type protein. Mixtures of protein CM-3 and angusticeps type proteins showed no apparent synergistic effect, in that their toxicity in combination was no greater than the sum of their individual toxicities. PMID:4029488

  3. Characterization and Localization of Iron-Oxidizing Proteins in Acid Mine Drainage Biofilms

    NASA Astrophysics Data System (ADS)

    Chan, C. S.; Thelen, M. P.; Hwang, M.; Banfield, J. F.

    2005-12-01

    As molecular geomicrobiologists, we are interested in the microbially-produced molecules that effect geochemical transformations, particularly proteins involved in lithotrophic energy generation. We have identified two such proteins produced by Leptospirillum group II microbes, which dominate biofilms floating on acidic waters in the Richmond Mine at Iron Mountain, CA. Leptospirillum generates energy by iron oxidation, producing the ferric iron catalyst responsible for pyrite oxidation, subsequent acid generation and toxic metal release. We have shown that a small (~16 kDa) soluble protein, cytochrome-579, extracted from environmental biofilm samples is capable of iron oxidation in vitro, consistent with prior studies on similar cytochromes from L. ferriphilum and ferrooxidans (Blake et al., 1993; Hart et al., 1991). The abundance of cyt579 and its ability to oxidize iron makes it a key link between microbial metabolism and acid mine drainage. Given the importance of cyt579 in biofilm sustenance as well as acid generation, we want to understand more about its distribution and also the architecture of the biofilm environment in which it functions. Using transmission electron microscopy (TEM) on ultrathin sections, we observe biofilms as thin as 15 microns with densely-packed cells in a matrix of polymers. To localize cyt579 in the biofilm, we purified the protein and developed antibodies for immunolabeling. The antibodies were shown to be highly specific for cyt579 using Western blots of whole biofilm lysate. Fluorescence- and gold-labeled secondary antibodies were used to visualize immunolabeled biofilms by confocal laser scanning microscopy and TEM, respectively. Preliminary results suggest that the cytochrome is on the bacterial cell surface or in the periplasm but not throughout the biofilm, as we had postulated due to the abundance of cytochrome in extracellular fractions of biofilm samples. These localization studies will be helpful in determining the

  4. Effects of antinutritional factors on protein digestibility and amino acid availability in foods.

    PubMed

    Gilani, G Sarwar; Cockell, Kevin A; Sepehr, Estatira

    2005-01-01

    Digestibility of protein in traditional diets from developing countries such as India, Guatemala, and Brazil is considerably lower compared to that of protein in typical North American diets (54-78 versus 88-94%). The presence of less digestible protein fractions, high levels of insoluble fiber, and high concentrations of antinutritional factors in the diets of developing countries, which are based on less refined cereals and grain legumes as major sources of protein, are responsible for poor digestibility of protein. The effects of the presence of some of the important antinutritional factors on protein and amino digestibilities of food and feed products are reviewed in this chapter. Food and feed products may contain a number of antinutritional factors that may adversely affect protein digestibility and amino acid availability. Antinutritional factors may occur naturally, such as glucosinolates in mustard and rapeseed protein products, trypsin inhibitors and hemagglutinins in legumes, tannins in legumes and cereals, phytates in cereals and oilseeds, and gossypol in cottonseed protein products. Antinutritional factors may also be formed during heat/alkaline processing of protein products, yielding Maillard compounds, oxidized forms of sulfur amino acids, D-amino acids, and lysinoalanine (LAL, an unnatural amino acid derivative). The presence of high levels of dietary trypsin inhibitors from soybeans, kidney beans, or other grain legumes can cause substantial reductions in protein and amino acid digestibilities (up to 50%) in rats and pigs. Similarly, the presence of high levels of tannins in cereals, such as sorghum, and grain legumes, such as fababean (Vicia faba L.), can result in significantly reduced protein and amino acid digestibilities (up to 23%) in rats, poultry, and pigs. Studies involving phytase supplementation of production rations for swine or poultry have provided indirect evidence that normally encountered levels of phytates in cereals and legumes

  5. Effects of dietary cottonseed oil and tannin supplements on protein and fatty acid composition of bovine milk.

    PubMed

    Aprianita, Aprianita; Donkor, Osaana N; Moate, Peter J; Williams, S Richard O; Auldist, Martin J; Greenwood, Jae S; Hannah, Murray C; Wales, William J; Vasiljevic, Todor

    2014-05-01

    This experiment was conducted to determine the effects of diets supplemented with cottonseed oil, Acacia mearnsii-condensed tannin extract, and a combination of both on composition of bovine milk. Treatment diets included addition of cottonseed oil (800 g/d; CSO), condensed tannin from Acacia mearnsii (400 g/d; TAN) or a combination of cottonseed oil (800 g/d) and condensed tannin (400 g/d; CPT) with a diet consisting of 6·0 kg dry matter (DM) of concentrates and alfalfa hay ad libitum, which also served as the control diet (CON). Relative to the CON diet, feeding CSO and CPT diets had a minor impact on feed intake and yield of lactose in milk. These diets increased yields of milk and protein in milk. In contrast to the TAN diet, the CSO and CPT diets significantly decreased milk fat concentration and altered milk fatty acid composition by decreasing the proportion of saturated fatty acids but increasing proportions of monounsaturated and polyunsaturated fatty acids. The CPT diet had a similar effect to the CSO diet in modifying fatty acid profile. Overall, reduction in milk fat concentration and changes in milk fatty acid profile were probably due to supplementation of linoleic acid-rich cottonseed oil. The TAN diet had no effect on feed intake, milk yield and milk protein concentration. However, a reduction in the yields of protein and lactose occurred when cows were fed this diet. Supplemented tannin had no significant effect on fat concentration and changes in fatty acid profile in milk. All supplemented diets did not affect protein concentration or composition, nitrogen concentration, or casein to total protein ratio of the resulting milk. PMID:24594257

  6. Sorption behavior of polyaramides in relation to isolation of nucleic acids and proteins.

    PubMed

    Liaw, Der-Jang; Yagudaeva, Elena; Prostyakova, Anna; Lazov, Michael; Zybin, Dmitry; Ischenko, Anatoly; Zubov, Vitaly; Chang, Cheng-Hung; Huang, Ying-Chi; Kapustin, Dmitry

    2016-09-01

    The effect of chemical composition and morphology of the surface layers of new polyaramide-containing sorbents on the mechanism of selective sorption of nucleic acids and proteins was investigated as compared to the previously studied sorbents modified with fluoropolymers and polyaniline (high-throughput materials providing one-step isolation of DNA from biological mixtures). A series of silica-based sorbents modified with polyaramides having consistently varying structure and containing the set of "key" structural elements (aromatic units and nitrogen atoms in the backbone, fluorinated groups), and various donor and acceptor moieties was prepared. The chemical composition of the polymer coatings was evaluated by X-ray photoelectron spectroscopy. The surface morphology was studied by scanning probe microscopy. The sorption properties were investigated by passing the mixtures containing DNA, RNA and proteins of different nature through the cartridges containing the obtained sorbents. All the investigated materials weakly retain double-stranded DNA but effectively retain RNA and proteins. The sorption capacity of the sorbents depends on the protein nature. The observed sorption behavior was shown to be determined by the chemical structure and not by the morphology of the polymer coating. It was proposed that similarity of the sorption properties of the series of chemically different polymers could be determined by similar total input of different sorption mechanisms. PMID:27341305

  7. Amino acid repletion does not decrease muscle protein catabolism during hemodialysis.

    PubMed

    Raj, Dominic S C; Adeniyi, Oladipo; Dominic, Elizabeth A; Boivin, Michel A; McClelland, Sandra; Tzamaloukas, Antonios H; Morgan, Nancy; Gonzales, Lawrence; Wolfe, Robert; Ferrando, Arny

    2007-06-01

    Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P < 0.001). Net balance (nmol.min(-1).100 ml (-1)) was more negative during HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P < 0.001). Despite an abundant supply of amino acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P < 0.01). Thus amino acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown. PMID:17264222

  8. Translocator Protein (TSPO) Affects Mitochondrial Fatty Acid Oxidation in Steroidogenic Cells.

    PubMed

    Tu, Lan N; Zhao, Amy H; Hussein, Mahmoud; Stocco, Douglas M; Selvaraj, Vimal

    2016-03-01

    Translocator protein (TSPO), also known as the peripheral benzodiazepine receptor, is a highly conserved outer mitochondrial membrane protein present in specific subpopulations of cells within different tissues. In recent studies, the presumptive model depicting mammalian TSPO as a critical cholesterol transporter for steroidogenesis has been refuted by studies examining effects of Tspo gene deletion in vivo and in vitro, biochemical testing of TSPO cholesterol transport function, and specificity of TSPO-mediated pharmacological responses. Nevertheless, high TSPO expression in steroid-producing cells seemed to indicate an alternate function for this protein in steroidogenic mitochondria. To seek an explanation, we used CRISPR/Cas9-mediated TSPO knockout steroidogenic MA-10 Leydig cell (MA-10:TspoΔ/Δ) clones to examine changes to core mitochondrial functions resulting from TSPO deficiency. We observed that 1) MA-10:TspoΔ/Δ cells had a shift in substrate utilization for energy production from glucose to fatty acids with significantly higher mitochondrial fatty acid oxidation (FAO), and increased reactive oxygen species production; and 2) oxygen consumption rate, mitochondrial membrane potential, and proton leak were not different between MA-10:TspoΔ/Δ and MA-10:Tspo+/+ control cells. Consistent with this finding, TSPO-deficient adrenal glands from global TSPO knockout (Tspo(-/-)) mice also showed up-regulation of genes involved in FAO compared with the TSPO floxed (Tspo(fl/fl)) controls. These results demonstrate the first experimental evidence that TSPO can affect mitochondrial energy homeostasis through modulation of FAO, a function that appears to be consistent with high levels of TSPO expression observed in cell types active in lipid storage/metabolism. PMID:26741196

  9. Experimentally Testing the Hypothesis of a Limited Amino Acid Repertoire in Primitive Proteins

    NASA Astrophysics Data System (ADS)

    Akanuma, S.; Nakajima, Y.; Yokobori, S.; Yamagishi, A.

    2013-11-01

    It has been argued that a fewer amino acids were used in primitive proteins and later the repertoire increased up to 20. To test this hypothesis experimentally, we restricted the amino acid usage of a reconstructed, ancestral protein to reduced sets.

  10. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    DOEpatents

    Xie, Jianming; Wang, Lei; Wu, Ning; Schultz, Peter G.

    2008-07-15

    Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

  11. Intra-molecular cross-linking of acidic residues for protein structure studies.

    PubMed

    Novak, Petr; Kruppa, Gary H

    2008-01-01

    Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would help to develop structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine (lysine, the amino terminus) selective reagents. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution and solvent accessibility of the lysines in the protein sequence. To overcome these limitations, we have investigated the use of cross-linking reagents that can react with other reactive side chains in proteins. We used 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E) and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO side chains can react to form "zero-length" cross-links with nearby primary amine containing residues, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO side chains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker arm of variable length. Using these reagents, we have found three new "zero-length" cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18 and K63-E64). Using the dihydrazide cross-linkers, we have identified two new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 A. These results show that additional structural information can be obtained by exploiting new cross-linker chemistry

  12. Intra-molecular cross-linking of acidic residues for protein structure studies.

    SciTech Connect

    Kruppa, Gary Hermann; Young, Malin M.; Novak, Petr; Schoeniger, Joseph S.

    2005-03-01

    Intra-molecular cross-linking has been suggested as a method of obtaining distance constraints that would be useful in developing structural models of proteins. Recent work published on intra-molecular cross-linking for protein structural studies has employed commercially available primary amine selective reagents that can cross-link lysine residues to other lysine residues or the amino terminus. Previous work using these cross-linkers has shown that for several proteins of known structure, the number of cross-links that can be obtained experimentally may be small compared to what would be expected from the known structure, due to the relative reactivity, distribution, and solvent accessibility of the lysines in the protein sequence. To overcome these limitations we have investigated the use of cross-linking reagents that can react with other reactive sidechains in proteins. We used 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to activate the carboxylic acid containing residues, aspartic acid (D), glutamic acid (E), and the carboxy terminus (O), for cross-linking reactions. Once activated, the DEO sidechains can react to form 'zero-length' cross-links with nearby primary amine containing resides, lysines (K) and the amino terminus (X), via the formation of a new amide bond. We also show that the EDC-activated DEO sidechains can be cross-linked to each other using dihydrazides, two hydrazide moieties connected by an alkyl cross-linker ann of variable length. Using these reagents, we have found three new 'zero-length' cross-links in ubiquitin consistent with its known structure (M1-E16, M1-E18, and K63-E64). Using the dihydrazide cross-linkers, we have identified 2 new cross-links (D21-D32 and E24-D32) unambiguously. Using a library of dihydrazide cross-linkers with varying arm length, we have shown that there is a minimum arm length required for the DEO-DEO cross-links of 5.8 angstroms. These results show that additional structural information

  13. Interaction of perfluoroalkyl acids with human liver fatty acid-binding protein.

    PubMed

    Sheng, Nan; Li, Juan; Liu, Hui; Zhang, Aiqian; Dai, Jiayin

    2016-01-01

    Perfluoroalkyl acids (PFAAs) are highly persistent and bioaccumulative, resulting in their broad distribution in humans and the environment. The liver is an important target for PFAAs, but the mechanisms behind PFAAs interaction with hepatocyte proteins remain poorly understood. We characterized the binding of PFAAs to human liver fatty acid-binding protein (hL-FABP) and identified critical structural features in their interaction. The binding interaction of PFAAs with hL-FABP was determined by fluorescence displacement and isothermal titration calorimetry (ITC) assay. Molecular simulation was conducted to define interactions at the binding sites. ITC measurement revealed that PFOA/PFNA displayed a moderate affinity for hL-FABP at a 1:1 molar ratio, a weak binding affinity for PFHxS and no binding for PFHxA. Moreover, the interaction was mainly mediated by electrostatic attraction and hydrogen bonding. Substitution of Asn111 with Asp caused loss of binding affinity to PFAA, indicating its crucial role for the initial PFAA binding to the outer binding site. Substitution of Arg122 with Gly caused only one molecule of PFAA to bind to hL-FABP. Molecular simulation showed that substitution of Arg122 increased the volume of the outer binding pocket, making it impossible to form intensive hydrophobic stacking and hydrogen bonds with PFOA, and highlighting its crucial role in the binding process. The binding affinity of PFAAs increased significantly with their carbon number. Arg122 and Asn111 played a pivotal role in these interactions. Our findings may help understand the distribution pattern, bioaccumulation, elimination, and toxicity of PFAAs in humans. PMID:25370009

  14. Amino Acid and Protein Metabolism in Bermuda Grass During Water Stress 12

    PubMed Central

    Barnett, N. M.; Naylor, A. W.

    1966-01-01

    The ability of Arizona Common and Coastal Bermuda grass [Cynodon dactylon (L.) Pers.] to synthesize amino acids and proteins during water stress was investigated. Amino acids were continually synthesized during the water stress treatments, but protein synthesis was inhibited and protein levels decreased. Water stress induced a 10- to 100-fold accumulation of free proline in shoots and a 2- to 6-fold accumulation of free asparagine, both of which are characteristic responses of water-stressed plants. Valine levels increased, and glutamic acid and alanine levels decreased. 14C labeling experiments showed that free proline turns over more slowly than any other free amino acid during water stress. This proline is readily synthesized and accumulated from glutamic acid. It is suggested that during water stress free proline functions as a storage compound. No significant differences were found in the amino acid and protein metabolism of the 2 varieties of Bermuda grass. PMID:16656387

  15. Snake venom. The amino acid sequence of protein A from Dendroaspis polylepis polylepis (black mamba) venom.

    PubMed

    Joubert, F J; Strydom, D J

    1980-12-01

    Protein A from Dendroaspis polylepis polylepis venom comprises 81 amino acids, including ten half-cystine residues. The complete primary structures of protein A and its variant A' were elucidated. The sequences of proteins A and A', which differ in a single position, show no homology with various neurotoxins and non-neurotoxic proteins and represent a new type of elapid venom protein. PMID:7461607

  16. Identification of protein-protein binding sites by incorporating the physicochemical properties and stationary wavelet transforms into pseudo amino acid composition.

    PubMed

    Jia, Jianhua; Liu, Zi; Xiao, Xuan; Liu, Bingxiang; Chou, Kuo-Chen

    2016-09-01

    With the explosive growth of protein sequences entering into protein data banks in the post-genomic era, it is highly demanded to develop automated methods for rapidly and effectively identifying the protein-protein binding sites (PPBSs) based on the sequence information alone. To address this problem, we proposed a predictor called iPPBS-PseAAC, in which each amino acid residue site of the proteins concerned was treated as a 15-tuple peptide segment generated by sliding a window along the protein chains with its center aligned with the target residue. The working peptide segment is further formulated by a general form of pseudo amino acid composition via the following procedures: (1) it is converted into a numerical series via the physicochemical properties of amino acids; (2) the numerical series is subsequently converted into a 20-D feature vector by means of the stationary wavelet transform technique. Formed by many individual "Random Forest" classifiers, the operation engine to run prediction is a two-layer ensemble classifier, with the 1st-layer voting out the best training data-set from many bootstrap systems and the 2nd-layer voting out the most relevant one from seven physicochemical properties. Cross-validation tests indicate that the new predictor is very promising, meaning that many important key features, which are deeply hidden in complicated protein sequences, can be extracted via the wavelets transform approach, quite consistent with the facts that many important biological functions of proteins can be elucidated with their low-frequency internal motions. The web server of iPPBS-PseAAC is accessible at http://www.jci-bioinfo.cn/iPPBS-PseAAC , by which users can easily acquire their desired results without the need to follow the complicated mathematical equations involved. PMID:26375780

  17. A Novel Fatty Acid-Binding Protein-Like Carotenoid-Binding Protein from the Gonad of the New Zealand Sea Urchin Evechinus chloroticus

    PubMed Central

    Pilbrow, Jodi; Sabherwal, Manya; Garama, Daniel; Carne, Alan

    2014-01-01

    A previously uncharacterized protein with a carotenoid-binding function has been isolated and characterized from the gonad of the New Zealand sea urchin Evechinus chloroticus. The main carotenoid bound to the protein was determined by reversed phase-high performance liquid chromatography to be 9′-cis-echinenone and hence this 15 kDa protein has been called an echinenone-binding protein (EBP). Purification of the EBP in quantity from the natural source proved to be challenging. However, analysis of EBP by mass spectrometry combined with information from the Strongylocentrotus purpuratus genome sequence and the recently published E. chloroticus transcriptome database, enabled recombinant expression of wild type EBP and also of a cysteine61 to serine mutant that had improved solubility characteristics. Circular dichroism data and ab initio structure prediction suggests that the EBP adopts a 10-stranded β-barrel fold consistent with that of fatty acid-binding proteins. Therefore, EBP may represent the first report of a fatty acid-binding protein in complex with a carotenoid. PMID:25192378

  18. A novel fatty acid-binding protein-like carotenoid-binding protein from the gonad of the New Zealand sea urchin Evechinus chloroticus.

    PubMed

    Pilbrow, Jodi; Sabherwal, Manya; Garama, Daniel; Carne, Alan

    2014-01-01

    A previously uncharacterized protein with a carotenoid-binding function has been isolated and characterized from the gonad of the New Zealand sea urchin Evechinus chloroticus. The main carotenoid bound to the protein was determined by reversed phase-high performance liquid chromatography to be 9'-cis-echinenone and hence this 15 kDa protein has been called an echinenone-binding protein (EBP). Purification of the EBP in quantity from the natural source proved to be challenging. However, analysis of EBP by mass spectrometry combined with information from the Strongylocentrotus purpuratus genome sequence and the recently published E. chloroticus transcriptome database, enabled recombinant expression of wild type EBP and also of a cysteine61 to serine mutant that had improved solubility characteristics. Circular dichroism data and ab initio structure prediction suggests that the EBP adopts a 10-stranded β-barrel fold consistent with that of fatty acid-binding proteins. Therefore, EBP may represent the first report of a fatty acid-binding protein in complex with a carotenoid. PMID:25192378

  19. Influence of Bleaching on Flavor of 34% Whey Protein Concentrate and Residual Benzoic Acid Concentration in Dried Whey Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies have shown that bleaching negatively affects the flavor of 70% whey protein concentrate (WPC70), but bleaching effects on lower-protein products have not been established. Benzoyl peroxide (BP), a whey bleaching agent, degrades to benzoic acid (BA) and may elevate BA concentrations...

  20. Protein–Protein Interactions in Dilute to Concentrated Solutions: α-Chymotrypsinogen in Acidic Conditions

    PubMed Central

    2015-01-01

    Protein–protein interactions were investigated for α-chymotrypsinogen by static and dynamic light scattering (SLS and DLS, respectively), as well as small-angle neutron scattering (SANS), as a function of protein and salt concentration at acidic conditions. Net protein–protein interactions were probed via the Kirkwood–Buff integral G22 and the static structure factor S(q) from SLS and SANS data. G22 was obtained by regressing the Rayleigh ratio versus protein concentration with a local Taylor series approach, which does not require one to assume the underlying form or nature of intermolecular interactions. In addition, G22 and S(q) were further analyzed by traditional methods involving fits to effective interaction potentials. Although the fitted model parameters were not always physically realistic, the numerical values for G22 and S(q → 0) were in good agreement from SLS and SANS as a function of protein concentration. In the dilute regime, fitted G22 values agreed with those obtained via the osmotic second virial coefficient B22 and showed that electrostatic interactions are the dominant contribution for colloidal interactions in α-chymotrypsinogen solutions. However, as protein concentration increases, the strength of protein–protein interactions decreases, with a more pronounced decrease at low salt concentrations. The results are consistent with an effective “crowding” or excluded volume contribution to G22 due to the long-ranged electrostatic repulsions that are prominent even at the moderate range of protein concentrations used here (<40 g/L). These apparent crowding effects were confirmed and quantified by assessing the hydrodynamic factor H(q → 0), which is obtained by combining measurements of the collective diffusion coefficient from DLS data with measurements of S(q → 0). H(q → 0) was significantly less than that for a corresponding hard-sphere system and showed that hydrodynamic nonidealities can lead to qualitatively incorrect

  1. Allied Health Chemistry Laboratory: Amino Acids, Insulin, Proteins, and Skin

    ERIC Educational Resources Information Center

    Dever, David F.

    1975-01-01

    Presents a laboratory experiment specifically designed for allied health students. The students construct molecular models of amino acids, extract amino acids from their skin with hot water, and chromatographically analyze the skin extract and hydrolyzed insulin. (MLH)

  2. Altered expression level of Escherichia coli proteins in response to treatment with the antifouling agent zosteric acid sodium salt.

    PubMed

    Villa, Federica; Remelli, William; Forlani, Fabio; Vitali, Alberto; Cappitelli, Francesca

    2012-07-01

    Zosteric acid sodium salt is a powerful antifouling agent. However, the mode of its antifouling action has not yet been fully elucidated. Whole cell proteome of Escherichia coli was analysed to study the different protein patterns expressed by the surface-exposed planktonic cells without and with sublethal concentrations of the zosteric acid sodium salt. Proteomic analysis revealed that at least 27 proteins showed a significant (19 upregulated and 8 downregulated, P < 0.001) altered expression level in response to the antifoulant. The proteomic signatures of zosteric acid sodium salt-treated cells are characterized by stress-associated (e.g. AhpC, OsmC, SodB, GroES, IscU, DnaK), motility-related (FliC), quorum-sensing-associated (LuxS) and metabolism/biosynthesis-related (e.g. PptA, AroA, FabD, FabB, GapA) proteins. Consistent with the overexpression of LuxS enzyme, the antifouling agent increased autoinducer-2 (AI-2) concentration by twofold. Moreover, treated cells experienced a statistically significant but modest increase of reactive oxygen species (+ 23%), tryptophanase (1.2-fold) and indole (1.2-fold) synthesis. Overall, our data suggest that zosteric acid sodium salt acts as environmental cue leading to global stress on E. coli cells, which favours the expression of various protective proteins, the AI-2 production and the synthesis of flagella, to escape from adverse conditions. PMID:22176949

  3. Optimizing Scoring Function of Protein-Nucleic Acid Interactions with Both Affinity and Specificity

    PubMed Central

    Yan, Zhiqiang; Wang, Jin

    2013-01-01

    Protein-nucleic acid (protein-DNA and protein-RNA) recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions) for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions. PMID:24098651

  4. Properties of low-moisture viscoplastic materials consisting of oil droplets dispersed in a protein-carbohydrate-glycerol matrix: effect of oil concentration.

    PubMed

    Gu, Yeun Suk; Corradini, Maria G; McClements, D Julian; DesRochers, Julia

    2007-10-31

    The influence of oil concentration and baking on the properties of low-moisture composites consisting of oil droplets dispersed in a protein-carbohydrate-glycerol matrix was investigated. These composites were produced by blending canola oil, whey protein concentrate (WPC), corn syrup, and glycerol together using a high-speed mixer. The resulting system consisted of oil droplets dispersed in a polar matrix. Some composites were analyzed directly after preparation, while others were analyzed after being heated at 176 degrees C for 10 min to simulate baking. The "lightness" of the composites was greater before baking (higher L value), but the color was more intense after baking (higher a and b values). The lightness and color intensity of the composites decreased as the oil concentration increased, with the effects being more pronounced in the baked samples. The zeta potential of the oil droplets (measured after dilution at pH 6) was highly negative (approximately -40 mV), indicating that whey protein was adsorbed to the droplet surfaces. The mean particle diameter (measured after dilution at pH 6) increased appreciably after baking, which was attributed to droplet flocculation. The rheological properties of the unbaked and baked materials were characterized by squeezing flow viscometry, which showed that the measurements associated with consistency and yield stress increased with increasing oil concentration and with baking. PMID:17902626

  5. The "Jekyll and Hyde" Actions of Nucleic Acids on the Prion-like Aggregation of Proteins.

    PubMed

    Silva, Jerson L; Cordeiro, Yraima

    2016-07-22

    Protein misfolding results in devastating degenerative diseases and cancer. Among the culprits involved in these illnesses are prions and prion-like proteins, which can propagate by converting normal proteins to the wrong conformation. For spongiform encephalopathies, a real prion can be transmitted among individuals. In other disorders, the bona fide prion characteristics are still under investigation. Besides inducing misfolding of native proteins, prions bind nucleic acids and other polyanions. Here, we discuss how nucleic acid binding might influence protein misfolding for both disease-related and benign, functional prions and why the line between bad and good amyloids might be more subtle than previously thought. PMID:27288413

  6. Biophysical and computational methods to analyze amino acid interaction networks in proteins.

    PubMed

    O'Rourke, Kathleen F; Gorman, Scott D; Boehr, David D

    2016-01-01

    Globular proteins are held together by interacting networks of amino acid residues. A number of different structural and computational methods have been developed to interrogate these amino acid networks. In this review, we describe some of these methods, including analyses of X-ray crystallographic data and structures, computer simulations, NMR data, and covariation among protein sequences, and indicate the critical insights that such methods provide into protein function. This information can be leveraged towards the design of new allosteric drugs, and the engineering of new protein function and protein regulation strategies. PMID:27441044

  7. Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

    DOEpatents

    Wang, Jiangyun; Schultz, Peter G.

    2012-07-10

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

  8. Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

    DOEpatents

    Wang, Jiangyun; Schultz, Peter G.

    2010-09-07

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel synthetase molecules, methods for identifying and making the novel synthetases, methods for producing proteins containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lipidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

  9. Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity

    PubMed Central

    James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

    2015-01-01

    Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury. PMID:26208104

  10. Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity.

    PubMed

    James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

    2015-01-01

    Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury. PMID:26208104

  11. AMINO ACIDS AUGMENT MUSCLE PROTEIN SYNTHESIS IN NEONATAL PIGS DURING ENDOTOXEMIA BY MODULATING TRANSLATION INITIATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In adults, sepsis reduces protein synthesis in skeletal muscle by restraining translation. The effect of sepsis on amino acid-stimulated muscle protein synthesis has not been determined in neonates, a population who is highly anabolic and whose muscle protein synthesis rates are uniquely sensitive ...

  12. Improved method for simultaneous isolation of proteins and nucleic acids.

    PubMed

    Chey, Soroth; Claus, Claudia; Liebert, Uwe Gerd

    2011-04-01

    Guanidinium thiocyanate-phenol-chloroform extraction (GTPC extraction) is widely used in molecular biology for isolating DNA, RNA, and proteins. Protein isolation by commercially available GTPC solutions is time consuming and the resulting pellets are only incompletely soluble. In this study ethanol-bromochloropropane-water was used for precipitation of proteins from the phenol-ethanol phase after GTPC extraction of RNA and DNA. The precipitated proteins can be readily dissolved in 4% SDS for subsequent analysis. This technique allows a fast (30min) and efficient (with a protein recovery of up to 95%) extraction of proteins for the study of transcriptional and posttranscriptional events from the same sample. PMID:21094121

  13. Novel humic acid-bonded magnetite nanoparticles for protein immobilization.

    PubMed

    Bayrakci, Mevlut; Gezici, Orhan; Bas, Salih Zeki; Ozmen, Mustafa; Maltas, Esra

    2014-09-01

    The present paper is the first report that introduces (i) a useful methodology for chemical immobilization of humic acid (HA) to aminopropyltriethoxysilane-functionalized magnetite iron oxide nanoparticles (APS-MNPs) and (ii) human serum albumin (HSA) binding to the obtained material (HA-APS-MNPs). The newly prepared magnetite nanoparticle was characterized by using Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and elemental analysis. Results indicated that surface modification of the bare magnetite nanoparticles (MNPs) with aminopropyltriethoxysilane (APS) and HA was successfully performed. The protein binding studies that were evaluated in batch mode exhibited that HA-APS-MNPs could be efficiently used as a substrate for the binding of HSA from aqueous solutions. Usually, recovery values higher than 90% were found to be feasible by HA-APS-MNPs, while that value was around 2% and 70% in the cases of MNPs and APS-MNPs, respectively. Hence, the capacity of MNPs was found to be significantly improved by immobilization of HA. Furthermore, thermal degradation of HA-APS-MNPs and HSA bonded HA-APS-MNPs was evaluated in terms of the Horowitz-Metzger equation in order to determine kinetic parameters for thermal decomposition. Activation energies calculated for HA-APS-MNPs (20.74 kJmol(-1)) and HSA bonded HA-APS-MNPs (33.42 kJmol(-1)) implied chemical immobilization of HA to APS-MNPs, and tight interactions between HA and HA-APS-MNPs. PMID:25063152

  14. Expression of liver fatty acid binding protein in hepatocellular carcinoma.

    PubMed

    Cho, Soo-Jin; Ferrell, Linda D; Gill, Ryan M

    2016-04-01

    Loss of expression of liver fatty acid binding protein (LFABP) by immunohistochemistry has been shown to be characteristic of a subset of hepatocellular adenomas (HCAs) in which HNF1A is inactivated. Transformation to hepatocellular carcinoma is thought to be a very rare phenomenon in the HNF1A-inactivated variant of HCA. However, we recently observed 2 cases at our institution, 1 definite hepatocellular carcinoma and 1 possible hepatocellular carcinoma, with loss of LFABP staining, raising the possibility that LFABP down-regulation may be associated with hepatocellular carcinogenesis. Our aim was to evaluate hepatocellular carcinomas arising in various backgrounds and with varying degrees of differentiation for loss of LFABP staining. Twenty total cases of hepatocellular carcinoma were examined. Thirteen cases arose in a background of cirrhosis due to hepatitis C (n = 8) or steatohepatitis (n = 5); 7 cases arose in a noncirrhotic background, with 2 cases arising within HNF1A-inactivated variant HCA and 2 cases arising within inflammatory variant HCA. Complete loss of expression of LFABP was seen in 6 of 20 cases, including 2 cases of hepatocellular carcinoma arising within HNF1A-inactivated variant HCA. Thus, loss of staining for LFABP appears to be common in hepatocellular carcinoma and may be seen in well-differentiated hepatocellular carcinoma. Therefore, LFABP loss should not be interpreted as evidence for hepatocellular adenoma over carcinoma, when other features support a diagnosis of hepatocellular carcinoma. The findings raise consideration for a role of HNF1A inactivation in hepatocellular carcinogenesis, particularly in less differentiated tumors. PMID:26997447

  15. Fast computational methods for predicting protein structure from primary amino acid sequence

    DOEpatents

    Agarwal, Pratul Kumar

    2011-07-19

    The present invention provides a method utilizing primary amino acid sequence of a protein, energy minimization, molecular dynamics and protein vibrational modes to predict three-dimensional structure of a protein. The present invention also determines possible intermediates in the protein folding pathway. The present invention has important applications to the design of novel drugs as well as protein engineering. The present invention predicts the three-dimensional structure of a protein independent of size of the protein, overcoming a significant limitation in the prior art.

  16. Drosophila Fatty Acid Transport Protein Regulates Rhodopsin-1 Metabolism and Is Required for Photoreceptor Neuron Survival

    PubMed Central

    Dourlen, Pierre; Bertin, Benjamin; Chatelain, Gilles; Robin, Marion; Napoletano, Francesco; Roux, Michel J.; Mollereau, Bertrand

    2012-01-01

    Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance. PMID:22844251

  17. Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

    PubMed Central

    Hu, Wenbing; Walters, Benjamin T.; Kan, Zhong-Yuan; Mayne, Leland; Rosen, Laura E.; Marqusee, Susan; Englander, S. Walter

    2013-01-01

    The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a “foldon.” Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed “sequential stabilization” based on native-like interfoldon interactions orders the pathway. PMID:23603271

  18. Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry.

    PubMed

    Hu, Wenbing; Walters, Benjamin T; Kan, Zhong-Yuan; Mayne, Leland; Rosen, Laura E; Marqusee, Susan; Englander, S Walter

    2013-05-01

    The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on native-like interfoldon interactions orders the pathway. PMID:23603271

  19. Target-driven self-assembly of stacking deoxyribonucleic acids for highly sensitive assay of proteins.

    PubMed

    Cao, Ya; Chen, Weiwei; Han, Peng; Wang, Zhuxin; Li, Genxi

    2015-08-26

    In this paper, we report a new signal amplification strategy for highly sensitive and enzyme-free method to assay proteins based on the target-driven self-assembly of stacking deoxyribonucleic acids (DNA) on an electrode surface. In the sensing procedure, binding of target protein with the aptamer probe is used as a starting point for a scheduled cycle of DNA hairpin assembly, which consists of hybridization, displacement and target regeneration. Following numbers of the assembly repeats, a great deal of DNA duplexes can accordingly be formed on the electrode surface, and then switch on a succeeding propagation of self-assembled DNA concatemers that provide further signal enhancement. In this way, each target binding event can bring out two cascaded DNA self-assembly processes, namely, stacking DNA self-assembly, and therefore can be converted into remarkably intensified electrochemical signals by associating with silver nanoparticle-based readout. Consequently, highly sensitive detection of target proteins can be achieved. Using interferon-gamma as a model, the assay method displays a linear range from 1 to 500 pM with a detection limit of 0.57 pM, which is comparable or even superior to other reported amplified assays. Moreover, the proposed method eliminates the involvement of any enzymes, thereby enhancing the feasibility in clinical diagnosis. PMID:26347164

  20. The immune evasion function of J and Beilong virus V proteins is distinct from that of other paramyxoviruses, consistent with their inclusion in the proposed genus Jeilongvirus.

    PubMed

    Audsley, Michelle D; Marsh, Glenn A; Lieu, Kim G; Tachedjian, Mary; Joubert, D Albert; Wang, Lin-Fa; Jans, David A; Moseley, Gregory W

    2016-03-01

    IFN-antagonist function is a major determinant of pathogenicity and cross-species infection by viruses, but remains poorly defined for many potentially zoonotic viruses resident in animal species. The paramyxovirus family contains several zoonotic viruses, including highly pathogenic viruses such as Nipah virus and Hendra virus, and an increasing number of largely uncharacterized animal viruses. Here, we report the characterization of IFN antagonism by the rodent viruses J virus (JPV) and Beilong virus (BeiPV) of the proposed genus Jeilongvirus of the paramyxoviruses. Infection of cells by JPV and BeiPV was found to inhibit IFN-activated nuclear translocation of signal transducer and activator of transcription 1 (STAT1). However, in contrast to most other paramyxoviruses, the JPV and BeiPV V proteins did not interact with or inhibit signalling by STAT1 or STAT2, suggesting that JPV/BeiPV use an atypical V protein-independent strategy to target STATs, consistent with their inclusion in a separate genus. Nevertheless, the V proteins of both viruses interacted with melanoma differentiation-associated protein 5 (MDA5) and robustly inhibited MDA5-dependent activation of the IFN-β promoter. This supports a growing body of evidence that MDA5 is a universal target of paramyxovirus V proteins, such that the V-MDA5 interaction represents a potential target for broad-spectrum antiviral approaches. PMID:26703878

  1. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-06-23

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins. PMID:25940624

  2. Prediction of nucleic acid binding probability in proteins: a neighboring residue network based score

    PubMed Central

    Miao, Zhichao; Westhof, Eric

    2015-01-01

    We describe a general binding score for predicting the nucleic acid binding probability in proteins. The score is directly derived from physicochemical and evolutionary features and integrates a residue neighboring network approach. Our process achieves stable and high accuracies on both DNA- and RNA-binding proteins and illustrates how the main driving forces for nucleic acid binding are common. Because of the effective integration of the synergetic effects of the network of neighboring residues and the fact that the prediction yields a hierarchical scoring on the protein surface, energy funnels for nucleic acid binding appear on protein surfaces, pointing to the dynamic process occurring in the binding of nucleic acids to proteins. PMID:25940624

  3. Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

    PubMed Central

    Hellman, Lance M.; Fried, Michael G.

    2009-01-01

    The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195

  4. Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

    PubMed Central

    McCormack, M; Brecher, P

    1987-01-01

    Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes. PMID:3446187

  5. Improved Experimental Techniques for Analyzing Nucleic Acid Transport Through Protein Nanopores in Planar Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Costa, Justin A.

    The translocation of nucleic acid polymers across cell membranes is a fundamental requirement for complex life and has greatly contributed to genomic molecular evolution. The diversity of pathways that have evolved to transport DNA and RNA across membranes include protein receptors, active and passive transporters, endocytic and pinocytic processes, and various types of nucleic acid conducting channels known as nanopores. We have developed a series of experimental techniques, collectively known as "Wicking", that greatly improves the biophysical analysis of nucleic acid transport through protein nanopores in planar lipid bilayers. We have verified the Wicking method using numerous types of classical ion channels including the well-studied chloride selective channel, CLIC1. We used the Wicking technique to reconstitute α-hemolysin and found that DNA translocation events of types A and B could be routinely observed using this method. Furthermore, measurable differences were observed in the duration of blockade events as DNA length and composition was varied, consistent with previous reports. Finally, we tested the ability of the Wicking technology to reconstitute the dsRNA transporter Sid-1. Exposure to dsRNAs of increasing length and complexity showed measurable differences in the current transitions suggesting that the charge carrier was dsRNA. However, the translocation events occurred so infrequently that a meaningful electrophysiological analysis was not possible. Alterations in the lipid composition of the bilayer had a minor effect on the frequency of translocation events but not to such a degree as to permit rigorous statistical analysis. We conclude that in many instances the Wicking method is a significant improvement to the lipid bilayer technique, but is not an optimal method for analyzing transport through Sid-1. Further refinements to the Wicking method might have future applications in high throughput DNA sequencing, DNA computation, and

  6. Tsetse Salivary Gland Proteins 1 and 2 Are High Affinity Nucleic Acid Binding Proteins with Residual Nuclease Activity

    PubMed Central

    Caljon, Guy; Ridder, Karin De; Stijlemans, Benoît; Coosemans, Marc; Magez, Stefan; De Baetselier, Patrick; Van Den Abbeele, Jan

    2012-01-01

    Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans) saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2) display DNA/RNA non-specific, high affinity nucleic acid binding with KD values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents. PMID:23110062

  7. Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats.

    PubMed

    Pelassa, Ilaria; Fiumara, Ferdinando

    2015-01-01

    Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as "junk" sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in shaping

  8. Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids.

    PubMed

    Lim, Sung In; Kwon, Inchan

    2016-10-01

    The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering. PMID:26036278

  9. Proximity-enabled protein crosslinking through genetically encoding haloalkane unnatural amino acids.

    PubMed

    Xiang, Zheng; Lacey, Vanessa K; Ren, Haiyan; Xu, Jing; Burban, David J; Jennings, Patricia A; Wang, Lei

    2014-02-17

    The selective generation of covalent bonds between and within proteins would provide new avenues for studying protein function and engineering proteins with new properties. New covalent bonds were genetically introduced into proteins by enabling an unnatural amino acid (Uaa) to selectively react with a proximal natural residue. This proximity-enabled bioreactivity was expanded to a series of haloalkane Uaas. Orthogonal tRNA/synthetase pairs were evolved to incorporate these Uaas, which only form a covalent thioether bond with cysteine when positioned in close proximity. By using the Uaa and cysteine, spontaneous covalent bond formation was demonstrated between an affibody and its substrate Z protein, thereby leading to irreversible binding, and within the affibody to increase its thermostability. This strategy of proximity-enabled protein crosslinking (PEPC) may be generally expanded to target different natural amino acids, thus providing diversity and flexibility in covalent bond formation for protein research and protein engineering. PMID:24449339

  10. Cellular retinol-binding protein and retinoic acid-binding protein in rat testes: effect of retinol depletion.

    PubMed

    Ong, D E; Tsai, C H; Chytil, F

    1976-02-01

    Testes of rats contain two cellular binding proteins of interest in vitamin A metabolism. One protein binds retinoic acid with high specificity; the other binds retinol with high specificity. When the cellular retinol-binding protein was partially purified from rat testes, it exhibited fluorescence excitation and emission spectra similar to that of all-trans-retinol in hexane. Exposure of this preparation to UV light destroyed this fluorescence but spectra identical to the original were obtained after addition of retinol. Hexane extracts of the binding protein had fluorescence spectra identical to all-trans-retinol, suggesting that this compound is bound to the protein in vivo. Extracts of testes from retinol depleted rats were submitted to gel filtration but failed to show a retinol-like fluorescence at the elution position of retinol binding protein. This fluorescence was observed in the preparations from pair fed control animals. However, after addition of all-trans-retinol to the extracts from the depleted rats, fluorescence at that elution position was observed. This indicates that in testes of retinol depleted rats the cellular retinol binding protein is present but without bound retinol, in contrast to the non-depleted rats where 30-43% of the binding protein had bound retinol. The amounts of cellular retinol binding protein and retinoic acid binding protein in testes, as determined by sucrose gradient centrifugation, were found to be similar for retinol depleted and pair fed control rats. PMID:942996

  11. Possible involvement of lipoic acid in binding protein-dependent transport systems in Escherichia coli.

    PubMed

    Richarme, G

    1985-04-01

    We describe the properties of the binding protein dependent-transport of ribose, galactose, and maltose and of the lactose permease, and the phosphoenolpyruvate-glucose phosphotransferase transport systems in a strain of Escherichia coli which is deficient in the synthesis of lipoic acid, a cofactor involved in alpha-keto acid dehydrogenation. Such a strain can grow in the absence of lipoic acid in minimal medium supplemented with acetate and succinate. Although the lactose permease and the phosphoenolypyruvate-glucose phosphotransferase are not affected by lipoic acid deprivation, the binding protein-dependent transports are reduced by 70% in conditions of lipoic acid deprivation when compared with their activity in conditions of lipoic acid supply. The remaining transport is not affected by arsenate but is inhibited by the uncoupler carbonylcyanide-m-chlorophenylhydrazone; however the lipoic acid-dependent transport is completely inhibited by arsenate and only weakly inhibited by carbonylcyanide-m-chlorophenylhydrazone. The known inhibitor of alpha-keto acid dehydrogenases, 5-methoxyindole-2-carboxylic acid, completely inhibits all binding protein-dependent transports whether in conditions of lipoic supply or deprivation; the results suggest a possible relation between binding protein-dependent transport and alpha-keto acid dehydrogenases and shed light on the inhibition of these transports by arsenicals and uncouplers. PMID:3920206

  12. Amino Acid Signature Enables Proteins to Recognize Modified tRNA

    PubMed Central

    2015-01-01

    Human tRNALys3UUU is the primer for HIV replication. The HIV-1 nucleocapsid protein, NCp7, facilitates htRNALys3UUU recruitment from the host cell by binding to and remodeling the tRNA structure. Human tRNALys3UUU is post-transcriptionally modified, but until recently, the importance of those modifications in tRNA recognition by NCp7 was unknown. Modifications such as the 5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34 and 2-methylthio-N6-threonylcarbamoyladenosine, adjacent to the anticodon at position-37, are important to the recognition of htRNALys3UUU by NCp7. Several short peptides selected from phage display libraries were found to also preferentially recognize these modifications. Evolutionary algorithms (Monte Carlo and self-consistent mean field) and assisted model building with energy refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify the in silico results and elucidate a 15-amino acid signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic) that recognized the tRNA’s fully modified anticodon stem and loop domain, hASLLys3UUU. Peptides of this sequence specifically recognized and bound modified htRNALys3UUU with an affinity 10-fold higher than that of the starting sequence. Thus, this approach provides an effective means of predicting sequences of RNA binding peptides that have better binding properties. Such peptides can be used in cell and molecular biology as well as biochemistry to explore RNA binding proteins and to inhibit those protein functions. PMID:24483944

  13. Towards the elucidation of molecular determinants of cooperativity in the liver bile acid binding protein.

    PubMed

    Pedò, Massimo; D'Onofrio, Mariapina; Ferranti, Pasquale; Molinari, Henriette; Assfalg, Michael

    2009-11-15

    Bile acid binding proteins (BABPs) are cytosolic lipid chaperones contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Liver BABPs act in parallel with ileal transporters to ensure vectorial transport of bile salts in hepatocytes and enterocytes, respectively. We describe the investigation of ligand binding to liver BABP, an essential step in the understanding of intracellular bile salt transport. Binding site occupancies were monitored in NMR titration experiments using (15)N-labelled ligand, while the relative populations of differently bound BABP forms were assessed by mass spectrometry. This site-specific information allowed the determination of intrinsic thermodynamic parameters and the identification of an extremely high cooperativity between two binding sites. Protein-observed NMR experiments revealed a global structural rearrangement which suggests an allosteric mechanism at the basis of the observed cooperativity. The view of a molecular tool capable of buffering against significant concentrations of free bile salts in a large range of solution conditions emerges from the observed pH-dependence of binding. We set to determine the molecular determinants of cooperativity by analysing the binding properties of a protein containing a mutated internal histidine. Both mass spectrometry and NMR experiments are consistent with an overall decreased binding affinity of the mutant, while the measured diffusion coefficients of ligand species reveal that the affinity loss concerns essentially one of the two binding sites. We therefore identified a mutation able to disrupt energetic communication functional to efficient binding and conclude that the buried histidine establishes contacts that stabilize the ternary complex. PMID:19603488

  14. Comparison of Quantum Mechanics and Molecular Mechanics Dimerization Energy Landscapes for Pairs of Ring-Containing Amino Acids in Proteins

    SciTech Connect

    Morozov, Alexandre V.; Misura M. S., Kira; Tsemekhman, Kiril; Baker, David

    2004-06-17

    A promising approach to developing improved potential functions for modeling macromolecular interactions consists of combining protein structural analysis, quantum mechanical calculations on small molecule models, and molecular mechanics potential decomposition. Here we apply this approach to the interactions of pairs of ring-containing amino acids in proteins. We find reasonable qualitative agreement between molecular mechanics and quantum chemistry calculations, both over one-dimensional projections of the binding free energy landscape for amino acid homodimers and over a set of homodimers and heterodimers from experimentally observed protein crystal structures. The molecular mechanics landscapes are a sum of charge-charge and Lennard-Jones contributions; short-range quantum mechanical effects such as charge transfer appear not to be significant in ring side chain interactions. We also find a reasonable degree of correlation between the molecular mechanics energy landscapes and the distributions of dimer geometries observed in protein structures, suggesting that the intrinsic dimer interaction energies do contribute to packing of side chains in proteins rather than being overwhelmed by the numerous interactions with other protein atoms and solvent. These results demonstrate that interactions involving aromatic residues and proline can be fairly well modeled using current molecular mechanics force fields, but there is still room for improvement, particularly for interactions involving proline and tyrosine.

  15. The role of putrescine in the regulation of proteins and fatty acids of thylakoid membranes under salt stress.

    PubMed

    Shu, Sheng; Yuan, Yinghui; Chen, Jie; Sun, Jin; Zhang, Wenhua; Tang, Yuanyuan; Zhong, Min; Guo, Shirong

    2015-01-01

    Polyamines can alleviate the inhibitory effects of salinity on plant growth by regulating photosynthetic efficiency. However, little information is available to explain the specific mechanisms underlying the contribution of polyamines to salt tolerance of the photosynthetic apparatus. Here, we investigated the role of putrescine (Put) on the photosynthetic apparatus of cucumber seedlings under salt stress. We found that NaCl stress resulted in severe ion toxicity and oxidative stress in cucumber chloroplasts. In addition, salinity caused a significant increase in the saturated fatty acid contents of thylakoid membranes. Put altered unsaturated fatty acid content, thereby alleviating the disintegration of thylakoid grana lamellae and reducing the number of plastoglobuli in thylakoid membranes. BN-PAGE revealed Put up-regulated the expression of ATP synthase, CP47, D1, Qb, and psbA proteins and down-regulated CP24, D2, and LHCII type III in NaCl-stressed thylakoid membranes. qRT-PCR analysis of gene expression was used to compare transcript and protein accumulation among 10 candidate proteins. For five of these proteins, induced transcript accumulation was consistent with the pattern of induced protein accumulation. Our results suggest that Put regulates protein expression at transcriptional and translational levels by increasing endogenous polyamines levels in thylakoid membranes, which may stabilise photosynthetic apparatus under salt stress. PMID:26435404

  16. Amino acid residue doublet propensity in the protein-RNA interface and its application to RNA interface prediction.

    PubMed

    Kim, Oanh T P; Yura, Kei; Go, Nobuhiro

    2006-01-01

    Protein-RNA interactions play essential roles in a number of regulatory mechanisms for gene expression such as RNA splicing, transport, translation and post-transcriptional control. As the number of available protein-RNA complex 3D structures has increased, it is now possible to statistically examine protein-RNA interactions based on 3D structures. We performed computational analyses of 86 representative protein-RNA complexes retrieved from the Protein Data Bank. Interface residue propensity, a measure of the relative importance of different amino acid residues in the RNA interface, was calculated for each amino acid residue type (residue singlet interface propensity). In addition to the residue singlet propensity, we introduce a new residue-based propensity, which gives a measure of residue pairing preferences in the RNA interface of a protein (residue doublet interface propensity). The residue doublet interface propensity contains much more information than the sum of two singlet propensities alone. The prediction of the RNA interface using the two types of propensities plus a position-specific multiple sequence profile can achieve a specificity of about 80%. The prediction method was then applied to the 3D structure of two mRNA export factors, TAP (Mex67) and UAP56 (Sub2). The prediction enables us to point out candidate RNA interfaces, part of which are consistent with previous experimental studies and may contribute to elucidation of atomic mechanisms of mRNA export. PMID:17130160

  17. The role of putrescine in the regulation of proteins and fatty acids of thylakoid membranes under salt stress

    PubMed Central

    Shu, Sheng; Yuan, Yinghui; Chen, Jie; Sun, Jin; Zhang, Wenhua; Tang, Yuanyuan; Zhong, Min; Guo, Shirong

    2015-01-01

    Polyamines can alleviate the inhibitory effects of salinity on plant growth by regulating photosynthetic efficiency. However, little information is available to explain the specific mechanisms underlying the contribution of polyamines to salt tolerance of the photosynthetic apparatus. Here, we investigated the role of putrescine (Put) on the photosynthetic apparatus of cucumber seedlings under salt stress. We found that NaCl stress resulted in severe ion toxicity and oxidative stress in cucumber chloroplasts. In addition, salinity caused a significant increase in the saturated fatty acid contents of thylakoid membranes. Put altered unsaturated fatty acid content, thereby alleviating the disintegration of thylakoid grana lamellae and reducing the number of plastoglobuli in thylakoid membranes. BN-PAGE revealed Put up-regulated the expression of ATP synthase, CP47, D1, Qb, and psbA proteins and down-regulated CP24, D2, and LHCII type III in NaCl-stressed thylakoid membranes. qRT-PCR analysis of gene expression was used to compare transcript and protein accumulation among 10 candidate proteins. For five of these proteins, induced transcript accumulation was consistent with the pattern of induced protein accumulation. Our results suggest that Put regulates protein expression at transcriptional and translational levels by increasing endogenous polyamines levels in thylakoid membranes, which may stabilise photosynthetic apparatus under salt stress. PMID:26435404

  18. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  19. Safety and Health Benefits of Novel Dietary Supplements Consisting Multiple Phytochemicals, Vitamins, Minerals and Essential Fatty Acids in High Fat Diet Fed Rats.

    PubMed

    Ramprasath, Vanu Ramkumar; Jones, Peter J H

    2016-01-01

    The objective was to determine safety and efficacy of health supplements "Beyond Tangy Tangerine," a multivitamin/mineral complex and combination of multivitamin/mineral complex, "Osteofx," a bone healthy supplement and "Ultimate Essential Fatty Acids" in Sprague Dawley rats consuming high-fat diets. Initially a pilot study was conducted which confirmed palatability and acceptability of supplements. In a second study, rats (n = 15/group) were randomized to Control; Multivitamin/mineral complex (2 g/kg BW) or Combination (2 g Multivitamin/mineral complex, 1.5 g Bone healthy supplement and 0.34 g Essential fatty acids/kg BW). No differences were observed in BW change, feed intake, organ weights or bone mineral composition with supplementations compared to control. Multivitamin/mineral complex supplementation decreased abdominal white adipose tissue weights (WAT) (p = .005), total (p = .033) and fat mass (p = .040), plasma IL-6 (p = .016) and ALKP (p = .038) and elevated plasma calcium (p < .001), phosphorus (p = .038), total protein (p = .002), albumin (p = .014) and globulin (p = .018), compared to control. Similarly, combination supplementation reduced WAT (p < .001), total (p = .023) and fat mass (p = .045), plasma triglycerides (p = .018), IL-6 (p = .002) and ALKP (p < .001) with increases in plasma calcium (p = .031), phosphorus (p < .001) compared to control. Results indicate that consuming either supplement can be considered safe and improves overall health by reducing inflammation, abdominal fat mass and plasma triglycerides, as well as promote bone health. PMID:26317447

  20. Quantifying Protein-Fatty Acid Interactions Using Electrospray Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Liu, Lan; Kitova, Elena N.; Klassen, John S.

    2011-02-01

    The application of the direct electrospray ionization mass spectrometry (ESI-MS) assay to quantify interactions between bovine β-lactoglobulin (Lg) and a series of fatty acids (FA), CH3(CH2)xCOOH, where x = 6 (caprylic acid, CpA), 8 (capric acid, CA), 10 (lauric acid, LA), 12 (myristic acid, MA), 14 (palmitic acid, PA) and 16 (stearic acid, SA), is described. Control ESI-MS binding measurements performed on the Lg-PA interaction revealed that both the protonated and deprotonated gas phase ions of the (Lg + PA) complex are prone to dissociate in the ion source, which leads to artificially small association constants ( K a ). The addition of imidazole, a stabilizing solution additive, at high concentration (10 mM) increased the relative abundance of (Lg + PA) complex measured by ESI-MS in both positive and negative ion modes. The K a value measured in negative ion mode and using sampling conditions that minimize in-source dissociation is in good agreement with a value determined using a competitive fluorescence assay. The K a values measured by ESI-MS for the Lg interactions with MA and SA are also consistent with values expected based on the fluorescence measurements. However, the K a values measured using optimal sampling conditions in positive ion mode are significantly lower than those measured in negative ion mode for all of the FAs investigated. It is concluded that the protonated gaseous ions of the (Lg + FA) complexes are kinetically less stable than the deprotonated ions. In-source dissociation was significant for the complexes of Lg with the shorter FAs (CpA, CA, and LA) in both modes and, in the case of CpA, no binding could be detected by ESI-MS. The affinities of Lg for CpA, CA, and LA determined using the reference ligand ESI-MS assay, a method for quantifying labile protein-ligand complexes that are prone to in-source dissociation, were found to be in good agreement with reported values.

  1. Method for nucleic acid hybridization using single-stranded DNA binding protein

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

    Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

  2. AFAL: a web service for profiling amino acids surrounding ligands in proteins.

    PubMed

    Arenas-Salinas, Mauricio; Ortega-Salazar, Samuel; Gonzales-Nilo, Fernando; Pohl, Ehmke; Holmes, David S; Quatrini, Raquel

    2014-11-01

    With advancements in crystallographic technology and the increasing wealth of information populating structural databases, there is an increasing need for prediction tools based on spatial information that will support the characterization of proteins and protein-ligand interactions. Herein, a new web service is presented termed amino acid frequency around ligand (AFAL) for determining amino acids type and frequencies surrounding ligands within proteins deposited in the Protein Data Bank and for assessing the atoms and atom-ligand distances involved in each interaction (availability: http://structuralbio.utalca.cl/AFAL/index.html ). AFAL allows the user to define a wide variety of filtering criteria (protein family, source organism, resolution, sequence redundancy and distance) in order to uncover trends and evolutionary differences in amino acid preferences that define interactions with particular ligands. Results obtained from AFAL provide valuable statistical information about amino acids that may be responsible for establishing particular ligand-protein interactions. The analysis will enable investigators to compare ligand-binding sites of different proteins and to uncover general as well as specific interaction patterns from existing data. Such patterns can be used subsequently to predict ligand binding in proteins that currently have no structural information and to refine the interpretation of existing protein models. The application of AFAL is illustrated by the analysis of proteins interacting with adenosine-5'-triphosphate. PMID:25085083

  3. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    NASA Astrophysics Data System (ADS)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  4. Natural vs. random protein sequences: Discovering combinatorics properties on amino acid words.

    PubMed

    Santoni, Daniele; Felici, Giovanni; Vergni, Davide

    2016-02-21

    Casual mutations and natural selection have driven the evolution of protein amino acid sequences that we observe at present in nature. The question about which is the dominant force of proteins evolution is still lacking of an unambiguous answer. Casual mutations tend to randomize protein sequences while, in order to have the correct functionality, one expects that selection mechanisms impose rigid constraints on amino acid sequences. Moreover, one also has to consider that the space of all possible amino acid sequences is so astonishingly large that it could be reasonable to have a well tuned amino acid sequence indistinguishable from a random one. In order to study the possibility to discriminate between random and natural amino acid sequences, we introduce different measures of association between pairs of amino acids in a sequence, and apply them to a dataset of 1047 natural protein sequences and 10,470 random sequences, carefully generated in order to preserve the relative length and amino acid distribution of the natural proteins. We analyze the multidimensional measures with machine learning techniques and show that, to a reasonable extent, natural protein sequences can be differentiated from random ones. PMID:26656109

  5. One-Pot Procedure for Recovery of Gallic Acid from Wastewater and Encapsulation within Protein Particles.

    PubMed

    Nourbakhsh, Himan; Madadlou, Ashkan; Emam-Djomeh, Zahra; Wang, Yi-Cheng; Gunasekaran, Sundaram; Mousavi, Mohammad E

    2016-02-24

    A whey protein isolate solution was heat-denatured and treated with the enzyme transglutaminase, which cross-linked ≈26% of the amino groups and increased the magnitude of the ζ-potential value. The protein solution was microemulsified, and then the resulting water-in-oil microemulsion was dispersed within a gallic acid-rich model wastewater. Gallic acid extraction by the outlined microemulsion liquid membrane (MLM) from the exterior aqueous phase (wastewater) and accumulation within the internal aqueous nanodroplets induced protein cold-set gelation and resulted in the formation of gallic acid-enveloping nanoparticles. Measurements with a strain-controlled rheometer indicated a progressive increase in the MLM viscosity during gallic acid recovery corresponding to particle formation. The mean hydrodynamic size of the nanoparticles made from the heat-denatured and preheated enzymatically cross-linked proteins was 137 and 122 nm, respectively. The enzymatic cross-linking of whey proteins led to a higher gallic acid recovery yield and increased the glass transition enthalpy and temperature. A similar impact on glass transition indices was observed by the gallic acid-induced nanoparticulation of proteins. Scanning electron microscopy showed the existence of numerous jammed/fused nanoparticles. It was suggested on the basis of the results of Fourier transform infrared spectroscopy that the in situ nanoparticulation of proteins shifted the C-N stretching and C-H bending peaks to higher wavenumbers. X-ray diffraction results proposed a decreased β-sheet content for proteins because of the acid-induced particulation. The nanoparticles made from the enzymatically cross-linked protein were more stable against the in vitro gastrointestinal digestion and retained almost 19% of the entrapped gallic acid after 300 min sequential gastric and intestinal digestions. PMID:26862880

  6. Genetically programmed expression of proteins containing the unnatural amino acid phenylselenocysteine

    DOEpatents

    Wang, Jiangyun; Schultz, Peter G.

    2013-03-12

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetase that can incorporate the unnatural amino acid phenylselenocysteine into proteins produced in eubacterial host cells such as E. coli. The invention provides, for example but not limited to, novel orthogonal aminoacyl-tRNA synthetases, polynucleotides encoding the novel sythetases molecules, methods for identifying and making the novel synthetases, methods for producing containing the unnatural amino acid phenylselenocysteine and translation systems. The invention further provides methods for producing modified proteins (e.g., lapidated proteins) through targeted modification of the phenylselenocysteine residue in a protein.

  7. Properties of low moisture composite materials consisting of oil droplets dispersed in a protein-carbohydrate-glycerol matrix: effect of continuous phase composition.

    PubMed

    Gu, Yeun Suk; Corradini, Maria G; McClements, D Julian; Desrochers, Julia

    2006-01-25

    The influence of continuous phase composition on the properties of low moisture (<3% water) composite materials consisting of oil droplets dispersed in a protein-carbohydrate-glycerol matrix was investigated. These composites were produced by blending canola oil (62.3%), whey protein concentrate (1.7%, WPC), and corn syrup and glycerol together (36.0% combined) using a high speed mixer equipped with a whisk. The polyol composition was varied by changing the ratio of corn syrup to glycerol in the system while keeping the total concentration of these two polyol components constant. Some composites were analyzed directly after preparation ("unbaked"), while others were analyzed after heating at 176 degrees C for 10 min to simulate baking of a food product ("baked"). The "lightness" of the composites was greater before baking (higher L value), but the color intensity of the composites was greater after baking (higher b value), which was attributed to Maillard browning reactions. The brownness of the baked composites increased with increasing corn syrup concentration, which was attributed to Maillard browning reactions. Squeezing flow viscometry indicated that the consistency and yield stress of the composites increased with baking, which was attributed to whey protein unfolding and aggregation. These rheological parameters also increased with increasing corn syrup concentration, which was attributed to its influence on the continuous phase rheology and on the interactions between the whey proteins. This study shows that the continuous phase composition and thermal history of low moisture composite materials have a large impact on their final physicochemical properties. PMID:16417299

  8. Fish protein hydrolysates: proximate composition, amino acid composition, antioxidant activities and applications: a review.

    PubMed

    Chalamaiah, M; Dinesh Kumar, B; Hemalatha, R; Jyothirmayi, T

    2012-12-15

    The fish processing industry produces more than 60% by-products as waste, which includes skin, head, viscera, trimmings, liver, frames, bones, and roes. These by-product wastes contain good amount of protein rich material that are normally processed into low market-value products, such as animal feed, fish meal and fertilizer. In view of utilizing these fish industry wastes, and for increasing the value to several underutilised fish species, protein hydrolysates from fish proteins are being prepared by several researchers all over the world. Fish protein hydrolysates are breakdown products of enzymatic conversion of fish proteins into smaller peptides, which normally contain 2-20 amino acids. In recent years, fish protein hydrolysates have attracted much attention of food biotechnologists due to the availability of large quantities of raw material for the process, and presence of high protein content with good amino acid balance and bioactive peptides (antioxidant, antihypertensive, immunomodulatory and antimicrobial peptides). PMID:22980905

  9. Single-molecule pull-down for investigating protein-nucleic acid interactions.

    PubMed

    Fareh, Mohamed; Loeff, Luuk; Szczepaniak, Malwina; Haagsma, Anna C; Yeom, Kyu-Hyeon; Joo, Chirlmin

    2016-08-01

    The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes. PMID:27017911

  10. Conservation of Shannon's redundancy for proteins. [information theory applied to amino acid sequences

    NASA Technical Reports Server (NTRS)

    Gatlin, L. L.

    1974-01-01

    Concepts of information theory are applied to examine various proteins in terms of their redundancy in natural originators such as animals and plants. The Monte Carlo method is used to derive information parameters for random protein sequences. Real protein sequence parameters are compared with the standard parameters of protein sequences having a specific length. The tendency of a chain to contain some amino acids more frequently than others and the tendency of a chain to contain certain amino acid pairs more frequently than other pairs are used as randomness measures of individual protein sequences. Non-periodic proteins are generally found to have random Shannon redundancies except in cases of constraints due to short chain length and genetic codes. Redundant characteristics of highly periodic proteins are discussed. A degree of periodicity parameter is derived.

  11. GLUCOCORTICOIDS REGULATE THE CONCENTRATION OF GLIAL FIBRILLARY ACIDIC PROTEIN THROUGHOUT THE BRAIN

    EPA Science Inventory

    The role of glucocorticoids in the in vivo regulation of glial fibrillary acidic protein was examined. orticosterone administration to adult rats resulted in decreased levels of GFAP throughout the brain whereas adrenalectomy caused levels of GFAP to increase. orticosterone admin...

  12. GONADAL STEROIDS REGULATED THE EXPRESSION OF GLIAL FIBRILLARY ACIDIC PROTEIN IN THE ADULT MALE RAT HIPPOCAMPUS

    EPA Science Inventory

    This study demonstrates that gonadal steroids (estradiol, testosterone, dihydrotestosterone) can inhibit the expression of glial fibrillary acidic protein and it MRNA in the adult male rat brain. esticular hormones may influence the activity of astrocytes in the intact and lesion...

  13. Identify Secretory Protein of Malaria Parasite with Modified Quadratic Discriminant Algorithm and Amino Acid Composition.

    PubMed

    Feng, Yong-E

    2016-06-01

    Malaria parasite secretes various proteins in infected red blood cell for its growth and survival. Thus identification of these secretory proteins is important for developing vaccine or drug against malaria. In this study, the modified method of quadratic discriminant analysis is presented for predicting the secretory proteins. Firstly, 20 amino acids are divided into five types according to the physical and chemical characteristics of amino acids. Then, we used five types of amino acids compositions as inputs of the modified quadratic discriminant algorithm. Finally, the best prediction performance is obtained by using 20 amino acid compositions, the sensitivity of 96 %, the specificity of 92 % with 0.88 of Mathew's correlation coefficient in fivefold cross-validation test. The results are also compared with those of existing prediction methods. The compared results shown our method are prominent in the prediction of secretory proteins. PMID:26286010

  14. Study of Stationary Phase Metabolism Via Isotopomer Analysis of Amino Acids from an Isolated Protein

    SciTech Connect

    Shaikh, AfshanS.; Tang, YinjieJ.; Mukhopadhyay, Aindrila; Martin, Hector Garcia; Gin, Jennifer; Benke, Peter; Keasling, Jay D.

    2009-09-14

    Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully 13C-labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase.

  15. Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

    PubMed Central

    Mendoza, Vanessa Leah; Vachet, Richard W.

    2009-01-01

    For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

  16. Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria.

    PubMed

    Landete, José M; Langa, Susana; Revilla, Concepción; Margolles, Abelardo; Medina, Margarita; Arqués, Juan L

    2015-08-01

    Lactic acid bacteria (LAB) are commonly used in the production of fermented and probiotic foods. Development of molecular tools to discriminate the strains of interest from the endogenous microbiota in complex environments like food or gut is of high interest. Green fluorescent protein (GFP)-like chromophores strictly requires molecular oxygen for maturation of fluorescence, which restrict the study of microorganisms in low-oxygen environments. In this work, we have developed a noninvasive cyan-green fluorescent based reporter system for real-time tracking of LAB that is functional under anoxic conditions. The evoglow-Pp1 was cloned downstream from the promoters D-alanyl-D-alanine carboxypeptidase and elongation factor Tu of Lactobacillus reuteri CECT925 using pNZ8048 and downstream of the lactococcal P1 promoter using pT1NX. The classical gfp was also cloned in pT1NX. These recombinant expression vectors were electroporated into Lactococccus, Lactobacillus, and Enterococcus strains with biotechnological and/or probiotic interests to assess and compare their functionality under different conditions of oxygen and pH. The expression was analyzed by imaging and fluorometric methods as well as by flow cytometry. We demonstrate that reporter systems pNZ:TuR-aFP and pT1-aFP are two versatile molecular markers for monitoring LAB in food and fecal environments without the potential problems caused by oxygen and pH limitations, which could be exploited for in vivo studies. Production of the fluorescent protein did not disturb any important physiological properties of the parental strains, such as growth rate, reuterin, or bacteriocin production. PMID:26129953

  17. Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli.

    PubMed

    Joshi, Bharat H; Puri, Raj K

    2005-02-01

    We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material

  18. Isolation and characterization of a fatty acid- and retinoid-binding protein from the cereal cyst nematode Heterodera avenae.

    PubMed

    Le, Xiuhu; Wang, Xuan; Guan, Tinglong; Ju, Yuliang; Li, Hongmei

    2016-08-01

    A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy. PMID:27240755

  19. Hypochlorite-induced oxidation of amino acids, peptides and proteins.

    PubMed

    Hawkins, C L; Pattison, D I; Davies, M J

    2003-12-01

    Activated phagocytes generate the potent oxidant hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is known to react with a number of biological targets including proteins, DNA, lipids and cholesterol. Proteins are likely to be major targets for reaction with HOCl within a cell due to their abundance and high reactivity with HOCl. This review summarizes information on the rate of reaction of HOCl with proteins, the nature of the intermediates formed, the mechanisms involved in protein oxidation and the products of these reactions. The predicted targets for reaction with HOCl from kinetic modeling studies and the consequences of HOCl-induced protein oxidation are also discussed. PMID:14661089

  20. A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.

    PubMed Central

    Neupert, B; Menotti, E; Kühn, L C

    1995-01-01

    We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151. Images PMID:7544459

  1. Tables of critical values for examining compositional non-randomness in proteins and nucleic acids

    NASA Technical Reports Server (NTRS)

    Laird, M.; Holmquist, R.

    1975-01-01

    A binomially distributed statistic is defined to show whether or not the proportion of a particular amino acid in a protein deviates from random expectation. An analogous statistic is derived for nucleotides in nucleic acids. These new statistics are simply related to the classical chi-squared test. They explicitly account for the compositional fluctuations imposed by the finite length of proteins, and they are more accurate than previous tables.

  2. Highly efficient extraction of cellular nucleic acid associated proteins in vitro with magnetic oxidized carbon nanotubes.

    PubMed

    Zhang, Yi; Hu, Zhengyan; Qin, Hongqiang; Wei, Xiaoluan; Cheng, Kai; Liu, Fangjie; Wu, Ren'an; Zou, Hanfa

    2012-12-01

    Nucleic acid associated proteins (NAaP) play the essential roles in gene regulation and protein expression. The global analysis of cellular NAaP would give a broad insight to understand the interaction between nucleic acids and the associated proteins, such as the important proteinous regulation factors on nucleic acids. Proteomic analysis presents a novel strategy to investigate a group of proteins. However, the large scale analysis of NAaP is yet impossible due to the lack of approaches to harvest target protein groups with a high efficiency. Herein, a simple and efficient method was developed to collect cellular NAaP using magnetic oxidized carbon nanotubes based on the strong interaction between carbon nanotubes and nucleic acids along with corresponding associated proteins. We found that the magnetic oxidized carbon nanotubes demonstrated a nearly 100% extraction efficiency for intracellular nucleic acids from cells in vitro. Importantly, the proteins associated on nucleic acids could be highly efficiently harvested using magnetic oxidized carbon nanotubes due to the binding of NAaP on nucleic acids. 1594 groups of nuclear NAaP and 2595 groups of cellular NAaP were extracted and identified from about 1,000,000 cells, and 803 groups of NAaP were analyzed with only about 10,000 cells, showing a promising performance for the proteomic analysis of NAaP from minute cellular samples. This highly efficient extraction strategy for NAaP is a simple approach to identify cellular nucleic acid associated proteome, and we believed this strategy could be further applied in systems biology to understand the gene expression and regulation. PMID:23121485

  3. Glutathione peroxidase's reaction intermediate selenenic acid is stabilized by the protein microenvironment.

    PubMed

    Li, Fei; Liu, Jun; Rozovsky, Sharon

    2014-11-01

    Selenenic acids are highly reactive intermediates of selenoproteins' enzymatic reactions. Knowledge of how the protein environment protects and stabilizes them is fundamental not only to descriptions of selenoproteins' reactivity but also potentially for proteomics and therapeutics. However, selenenic acids are considered particularly short-lived and are not yet identified in wild-type selenoproteins. Here, we report trapping the selenenic acid in glutathione peroxidase, an antioxidant enzyme that efficiently eliminates hydroperoxides. It has long been thought that selenium-containing glutathione peroxidases form a selenenic acid intermediate. However, this putative species has eluded detection. Here, we report its identification. The selenenic acid in bovine glutathione peroxidase 1 was chemically trapped using dimedone, an alkylating agent specific to sulfenic and selenenic acids. The alkylation of the catalytic selenocysteine was verified by electrospray ionization mass spectrometry. In the presence of glutathione, the selenocysteine was not alkylated because the selenenic acid condenses faster with glutathione than the alkylation reaction. In the absence of thiols, the selenenic acid was surprisingly long-lived with 95% of the protein still able to react with dimedone 10 min after hydrogen peroxide was removed, indicating that the protein environment stabilizes the selenenic acid by shielding it from reactive groups in the protein. After 30 min, the selenocysteine was no longer modified but became accessible once the protein was exposed to reducing agents. This suggests that the selenenic acid reacted with a protein's amide or amine to form a selenylamide bond. Such a modification may play a role in protecting glutathione peroxidase׳' reactivity. PMID:25124921

  4. Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

    SciTech Connect

    Garrison, W. M.

    1981-12-01

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides, and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the ..cap alpha..,..cap alpha..'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The ..cap alpha..,..cap alpha..'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the ..cap alpha..,..cap alpha..'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized.

  5. Metabolism of Nonessential N15-Labeled Amino Acids and the Measurement of Human Whole-Body Protein Synthesis Rates

    NASA Technical Reports Server (NTRS)

    Stein, T. P.; Settle, R. G.; Albina, J. A.; Dempsey, D. T.; Melnick, G.

    1991-01-01

    Eight N-15 labeled nonessential amino acids plus (15)NH4Cl were administered over a 10 h period to four healthy adult males using a primed-constant dosage regimen. The amount of N-15 excreted in the urine and the urinary ammonia, hippuric acid, and plasma alanine N-15 enrichments were measured. There was a high degree of consistency across subjects in the ordering of the nine compounds based on the fraction of N-15 excreted (Kendall coefficient of concordance W = 0.83, P is less than 0.01). Protein synthesis rates were calculated from the urinary ammonia plateau enrichment and the cumulative excretion of N-15. Glycine was one of the few amino acids that gave similar values by both methods.

  6. Enterocyte Fatty Acid Binding Proteins (FABPs): Different Functions of Liver- and Intestinal- FABPs in the Intestine

    PubMed Central

    Gajda, Angela M.; Storch, Judith

    2014-01-01

    SUMMARY Fatty acid binding proteins (FABP) are highly abundant cytosolic proteins that are expressed in most mammalian tissues. In the intestinal enterocyte, both Liver- (LFABP; FABP1) and Intestinal-fatty acid binding proteins (IFABP; FABP2) are expressed. These proteins display high affinity binding for long chain fatty acids (FA) and other hydrophobic ligands, thus they are believed to be involved with uptake and trafficking of lipids in the intestine. In vitro studies have identified differences in ligand binding stoichiometry and specificity, and in mechanisms of FA transfer to membranes, and it has been hypothesized that LFABP and IFABP have difference functions in the enterocyte. Studies directly comparing LFABP- and IFABP-null mice have revealed markedly different phenotypes, indicating that these proteins indeed have different functions in intestinal lipid metabolism and whole body energy homeostasis. In this review, we discuss the evolving knowledge of the functions of LFABP and IFABP in the intestinal enterocyte. PMID:25458898

  7. Super-resolution Microscopy of Clickable Amino Acids Reveals the Effects of Fluorescent Protein Tagging on Protein Assemblies.

    PubMed

    Vreja, Ingrid C; Nikić, Ivana; Göttfert, Fabian; Bates, Mark; Kröhnert, Katharina; Outeiro, Tiago F; Hell, Stefan W; Lemke, Edward A; Rizzoli, Silvio O

    2015-11-24

    The advent of super-resolution microscopy (nanoscopy) has set high standards for fluorescence tagging. Fluorescent proteins (FPs) are convenient tags in conventional imaging, but their use in nanoscopy has been questioned due to their relatively large size and propensity to form multimers. Here, we compared the nanoscale organization of proteins with or without FP tags by introducing the unnatural amino acid propargyl-L-lysine (PRK) in 26 proteins known to form multimolecular arrangements and into their FP-tagged variants. We revealed the proteins by coupling synthetic fluorophores to PRK via click chemistry and visualized them using ground-state depletion microscopy followed by individual molecule return, as well as stimulated emission depletion microscopy. The arrangements formed by the FP-tagged and nontagged proteins were similar. Mild, but statistically significant differences were observed for only six proteins (23% of all proteins tested). This suggests that FP-based nanoscopy is generally reliable. Unnatural amino acids should be a reliable alternative for the few proteins that are sensitive to FP tagging. PMID:26498474

  8. Sialic Acid within the Glycosylphosphatidylinositol Anchor Targets the Cellular Prion Protein to Synapses.

    PubMed

    Bate, Clive; Nolan, William; McHale-Owen, Harriet; Williams, Alun

    2016-08-12

    Although the cellular prion protein (PrP(C)) is concentrated at synapses, the factors that target PrP(C) to synapses are not understood. Here we demonstrate that exogenous PrP(C) was rapidly targeted to synapses in recipient neurons derived from Prnp knock-out((0/0)) mice. The targeting of PrP(C) to synapses was dependent upon both neuronal cholesterol concentrations and the lipid and glycan composition of its glycosylphosphatidylinositol (GPI) anchor. Thus, the removal of either an acyl chain or sialic acid from the GPI anchor reduced the targeting of PrP(C) to synapses. Isolated GPIs (derived from PrP(C)) were also targeted to synapses, as was IgG conjugated to these GPIs. The removal of sialic acid from GPIs prevented the targeting of either the isolated GPIs or the IgG-GPI conjugate to synapses. Competition studies showed that pretreatment with sialylated GPIs prevented the targeting of PrP(C) to synapses. These results are consistent with the hypothesis that the sialylated GPI anchor attached to PrP(C) acts as a synapse homing signal. PMID:27325697

  9. The distinct C-terminal acidic domains of HMGB proteins are functionally relevant in Schistosoma mansoni.

    PubMed

    de Abreu da Silva, Isabel Caetano; Carneiro, Vitor Coutinho; Vicentino, Amanda Roberta Revoredo; Aguilera, Estefania Anahi; Mohana-Borges, Ronaldo; Thiengo, Silvana; Fernandez, Monica Ammon; Fantappié, Marcelo Rosado

    2016-04-01

    The Schistosoma mansoni High Mobility Group Box (HMGB) proteins SmHMGB1, SmHMGB2 and SmHMGB3 share highly conserved HMG box DNA binding domains but have significantly different C-terminal acidic tails. Here, we used three full-length and tailless forms of the S. mansoni HMGB proteins to examine the functional roles of their acidic tails. DNA binding assays revealed that the different lengths of the acidic tails among the three SmHMGB proteins significantly and distinctively influenced their DNA transactions. Spectroscopic analyses indicated that the longest acidic tail of SmHMGB3 contributes to the structural stabilisation of this protein. Using immunohistochemical analysis, we showed distinct patterns of SmHMGB1, SmHMGB2 and SmHMGB3 expression in different tissues of adult worms. RNA interference approaches indicated a role for SmHMGB2 and SmHMGB3 in the reproductive system of female worms, whereas for SmHMGB1 no clear phenotype was observed. Schistosome HMGB proteins can be phosphorylated, acetylated and methylated. Importantly, the acetylation and methylation of schistosome HMGBs were greatly enhanced upon removal of the acidic tail. These data support the notion that the C-terminal acidic tails dictate the differences in the structure, expression and function of schistosome HMGB proteins. PMID:26820302

  10. Distinctive amino acid composition profiles in salivary proteins of the tick Ixodes scapularis

    PubMed Central

    Hughes, Austin L.; Friedman, Robert

    2012-01-01

    Bioinformatic analysis of the amino acid composition of proteins of the tick Ixodes scapularis showed that, in comparison to other secreted proteins, salivary proteins in general have higher frequencies of polar residues and lower frequencies of the non-polar residues leucine and valine. Computer prediction of linear B-cell epitopes showed that polar residues were associated with the presence of high-quality epitopes and that tick salivary proteins included significantly more proteins with predicted high-quality epitopes than did other secreted proteins. The results provided no evidence that salivary proteins as a whole have evolved characteristics minimizing their antigenicity to the vertebrate host. Certain salivary proteins may indeed have evolved low antigenicity, but the I. scapularis sialome include at least some apparently antigenic proteins that might be tested experimentally to determine whether they would be suitable candidates for anti-tick vaccines. PMID:22108016

  11. NPPD: A Protein-Protein Docking Scoring Function Based on Dyadic Differences in Networks of Hydrophobic and Hydrophilic Amino Acid Residues

    PubMed Central

    Shih, Edward S. C.; Hwang, Ming-Jing

    2015-01-01

    Protein-protein docking (PPD) predictions usually rely on the use of a scoring function to rank docking models generated by exhaustive sampling. To rank good models higher than bad ones, a large number of scoring functions have been developed and evaluated, but the methods used for the computation of PPD predictions remain largely unsatisfactory. Here, we report a network-based PPD scoring function, the NPPD, in which the network consists of two types of network nodes, one for hydrophobic and the other for hydrophilic amino acid residues, and the nodes are connected when the residues they represent are within a certain contact distance. We showed that network parameters that compute dyadic interactions and those that compute heterophilic interactions of the amino acid networks thus constructed allowed NPPD to perform well in a benchmark evaluation of 115 PPD scoring functions, most of which, unlike NPPD, are based on some sort of protein-protein interaction energy. We also showed that NPPD was highly complementary to these energy-based scoring functions, suggesting that the combined use of conventional scoring functions and NPPD might significantly improve the accuracy of current PPD predictions. PMID:25811640

  12. [Recombinant proteins containing amino acid sequences of two ectatomin chains].

    PubMed

    Esipov, R S; Gurevich, A I; Kaiushin, A L; Korosteleva, M D; Miroshnikov, A I; Shevchenko, L V; Pluzhnikov, K A; Grishin, E V

    1997-12-01

    Artificial genes for chains A and B of ectatomin, an Ectatomma tuberculatum ant toxin, were obtained by chemical and enzymic synthesis and cloned into new plasmid vectors. Expression plasmids with the genes of hybrid proteins were constructed containing human interleukin-3 or its terminal 63-mer fragment as well as chains A and B of ectatomin, which are linked via a region containing the cleavage site of specific protease, enterokinase (hybrid proteins IL3ETOXA, IL3ETOXB, ILETOXA, and ILETOXB). Escherichia coli producer strains providing a high yield of IL3ETOXA and IL3ETOXB proteins as inclusion bodies were obtained. PMID:9499370

  13. Long-chain n-3 fatty acids - New anabolic compounds improving protein metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous animal studies demonstrated that chronic feeding of long-chain n-3 polyunsaturated fatty acids (LCn-3PUFA) that modifies muscle membrane fatty acid composition promotes protein anabolism by blunting the age-associated deterioration in insulin sensitivity. The current study assessed, as a pr...

  14. Prolonged stimulation of muscle protein synthesis by leucine in neonates is dependent on amino acid availability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rise in amino acids and insulin after a meal independently stimulate protein synthesis in skeletal muscle of neonates by activating the intracellular signalling pathways that regulate mRNA translation. Leucine, in particular, is important in mediating the response to amino acids. Previously, w...

  15. Amino acid composition and crude protein values of some Cyanobacteria from Çanakkale (Turkey).

    PubMed

    Akgül, Rıza; Kızılkaya, Bayram; Akgül, Füsun; Erduğan, Hüseyin

    2015-09-01

    Cyanobacteria (blue-green algae) form an important component of integrated nutrient managements in agriculture and are exploited in commercial biotechnological ventures. In this study, Rivularia bullata (Poir) Berkeley ex Bornet & Flahault, Nostocs pongiaeforme C. Agardh ex Bornet & Flahault were researched for their amino acid composition and crude protein values. R. bullata was collected from coastal zones of the Gulf of Saros and N. spongiaeforme from the Ayazma Stream. The levels of amino acids were measured in algae samples using EZ: fast kits (EZ: fast GC/FID Protein Hydrolysate Amino Acid Kit) by gas chromatography. The crude proteins of samples were determined by the Kjeldahl method and were calculated using a nitrogen conversion factor of 6.25. Thirty-two amino acids were investigated, for N. spongiaeforme eight free essential amino acids (EAA), eight free non-essential amino acids (NEAA) and eleven other amino acids (OAA); for R. bullata eight EAA, eight NEAA and eight OAA were detected. Aspartic acid is the major constituent for both species. The total protein percents were determined for N. spongiaeforme as % 19.83 and for R. bullata as % 6.15. When considering the increasing world population and reducing natural products; Cyanobacteria will benew feed sources for all living. PMID:26408895

  16. COMPARATIVE PATHOGENESIS OF HALOACETIC ACID AND PROTEIN KINASE INHIBITOR EMBRYOTOXICITY IN MOUSE WHOLE EMBRYO CULTURE

    EPA Science Inventory

    Comparative pathogenesis of haloacetic acid and protein kinase inhibitor embryotoxicity in mouse whole embryo culture.

    Ward KW, Rogers EH, Hunter ES 3rd.

    Curriculum in Toxicology, University of North Carolina at Chapel Hill, 27599-7270, USA.

    Haloacetic acids ...

  17. Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

    PubMed Central

    Hesse, Almut

    2016-01-01

    Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

  18. Nucleic acid chaperons: a theory of an RNA-assisted protein folding

    PubMed Central

    Biro, Jan C

    2005-01-01

    Background Proteins are assumed to contain all the information necessary for unambiguous folding (Anfinsen's principle). However, ab initio structure prediction is often not successful because the amino acid sequence itself is not sufficient to guide between endless folding possibilities. It seems to be a logical to try to find the "missing" information in nucleic acids, in the redundant codon base. Results mRNA energy dot plots and protein residue contact maps were found to be rather similar. The structure of mRNA is also conserved if the protein structure is conserved, even if the sequence similarity is low. These observations led me to suppose that some similarity might exist between nucleic acid and protein folding. I found that amino acid pairs, which are co-located in the protein structure, are preferentially coded by complementary codons. This codon complementarity is not perfect; it is suboptimal where the 1st and 3rd codon residues are complementary to each other in reverse orientation, while the 2nd codon letters may be, but are not necessarily, complementary. Conclusion Partial complementary coding of co-locating amino acids in protein structures suggests that mRNA assists in protein folding and functions not only as a template but even as a chaperon during translation. This function explains the role of wobble bases and answers the mystery of why we have a redundant codon base. PMID:16137324

  19. Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids.

    PubMed

    Hesse, Almut; Weller, Michael G

    2016-01-01

    Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports. PMID:27559481

  20. Subunit-specific inhibition of acid sensing ion channels by stomatin-like protein 1

    PubMed Central

    Kozlenkov, Alexey; Lapatsina, Liudmila; Lewin, Gary R; Smith, Ewan St John

    2014-01-01

    There are five mammalian stomatin-domain genes, all of which encode peripheral membrane proteins that can modulate ion channel function. Here we examined the ability of stomatin-like protein 1 (STOML1) to modulate the proton-sensitive members of the acid-sensing ion channel (ASIC) family. STOML1 profoundly inhibits ASIC1a, but has no effect on the splice variant ASIC1b. The inactivation time constant of ASIC3 is also accelerated by STOML1. We examined STOML1 null mutant mice with a β-galactosidase-neomycin cassette gene-trap reporter driven from the STOML1 gene locus, which indicated that STOML1 is expressed in at least 50% of dorsal root ganglion (DRG) neurones. Patch clamp recordings from mouse DRG neurones identified a trend for larger proton-gated currents in neurones lacking STOML1, which was due to a contribution of effects upon both transient and sustained currents, at different pH, a finding consistent with an endogenous inhibitory function for STOML1. PMID:24247984

  1. Reasons for the occurrence of the twenty coded protein amino acids

    NASA Technical Reports Server (NTRS)

    Weber, A. L.; Miller, S. L.

    1981-01-01

    Factors involved in the selection of the 20 protein L-alpha-amino acids during chemical evolution and the early stages of Darwinian evolution are discussed. The selection is considered on the basis of the availability in the primitive ocean, function in proteins, the stability of the amino acid and its peptides, stability to racemization, and stability on the transfer RNA. It is concluded that aspartic acid, glutamic acid, arginine, lysine, serine and possibly threonine are the best choices for acidic, basic and hydroxy amino acids. The hydrophobic amino acids are reasonable choices, except for the puzzling absences of alpha-amino-n-butyric acid, norvaline and norleucine. The choices of the sulfur and aromatic amino acids seem reasonable, but are not compelling. Asparagine and glutamine are apparently not primitive. If life were to arise on another planet, it would be expected that the catalysts would be poly-alpha-amino acids and that about 75% of the amino acids would be the same as on the earth.

  2. Proteomic analysis of proteins selectively associated with hydroxyapatite, brushite, and uric acid crystals precipitated from human urine.

    PubMed

    Thurgood, Lauren A; Ryall, Rosemary L

    2010-10-01

    The aim of this study was to compare the intracrystalline protein profiles of hydroxyapatite (HA), brushite (BR), and uric acid (UA) crystals precipitated from the same urine samples. HA, BR, and UA crystals were precipitated on two different occasions from the same pooled healthy urine. Crystals were washed to remove surface-bound proteins, and their composition was confirmed using Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) coupled with energy dispersive X-ray analysis (EDAX). SDS-PAGE was used for visual comparison of the protein content of the demineralised crystal extracts, which were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). HA comprised nanosized particles interspersed with organic material, which was absent from the BR and UA crystals. The number and type of individual proteins differed between the 3 minerals: 45 proteins were detected in the HA crystal extracts and 77 in the BR crystals, including a number of keratins, which were regarded as methodological contaminants. After excluding the keratins, 21 proteins were common to both HA and BR crystals. Seven nonkeratin proteins were identified in the UA extracts. Several proteins consistently detected in the HA and BR crystal extracts have been previously implicated in kidney stone disease, including osteopontin, prothrombin, protein S100A9 (calgranulin B), inter-α-inhibitor, α1-microglobulin bikunin (AMBP), heparan sulfate proteoglycan, and Tamm-Horsfall glycoprotein, all of which are strong calcium binders. We concluded that the association of proteins with HA, BR, and UA crystals formed in healthy urine is selective and that only a few of the numerous proteins present in healthy urine are likely to play any significant role in preventing stone pathogenesis. PMID:20795672

  3. INCREASE IN GLIAL FIBRILLARY ACIDIC PROTEIN FOLLOWS BRAIN HYPERTHERMIA IN RATS

    EPA Science Inventory

    Previously, the authors have demonstrated that an increase in the astrocyte-associated protein, glial fibrillary acidic protein (GFAP), accompanies brain injury induced by a variety of chemical insults. In the present study the authors examined the effects of microwave-induced hy...

  4. Effect of microfluidized and stearic acid modified soy protein in natural rubber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microfluidized and stearic acid modified soy protein aggregates were used to reinforced natural rubber. The size of soy protein particles was reduced with a microfluidizing and ball milling process. Filler size reduction with longer ball milling time tends to increase tensile strength of the rubber ...

  5. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions

    PubMed Central

    Vickers, Timothy A.; Crooke, Stanley T.

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  6. Isolation and partial characterization of a fatty acid binding protein in rat liver plasma membranes.

    PubMed Central

    Stremmel, W; Strohmeyer, G; Borchard, F; Kochwa, S; Berk, P D

    1985-01-01

    When [14C]oleate-bovine serum albumin complexes were incubated in vitro with rat liver plasma membranes (LPM), specific, saturable binding of oleate to the membranes was observed. Maximal heat-sensitive (i.e., specific) binding was 3.2 nmol/mg of membrane protein. Oleate-agarose affinity chromatography of Triton X-100-solubilized LPM was used to isolate a single 40-kDa protein with high affinity for oleate. On gel filtration, the protein comigrated with various fatty acids but not with [14C]bilirubin, [35S]sulfobromophthalein, [14C]taurocholate, [14C]phosphatidylcholine, or [14C]cholesteryloleate. A rabbit antibody to this membrane fatty acid-binding protein gave a single precipitin line with the antigen but no reactivity with concentrated cytosolic proteins, LPM bilirubin/sulfobromophthalein-binding protein, or rat albumin or other rat plasma proteins. The antibody selectively inhibited heat-sensitive binding of [14C]oleate to LPM. Immunofluorescence studies localized the antigen in liver-cell plasma membranes as well as in other major sites of fatty acid transport. These data are compatible with the hypothesis that this protein may act as a receptor in a hepatocellular uptake mechanism for fatty acids. Images PMID:3881757

  7. Long-term leucine induced stimulation of muscle protein synthesis is amino acid dependent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infusing leucine for 1 h increases skeletal muscle protein synthesis in the neonate, but this is not sustained for 2 h unless the corresponding fall in amino acids is prevented. This study aimed to determine whether a continuous leucine infusion can stimulate protein synthesis for a prolonged period...

  8. Acid diet (high meat protein) effects on calcium metabolism and bone health

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Purpose of review: Update recent advancements regarding the effect of high animal protein on calcium utilization and bone health. Recent findings: Increased potential renal acid load resulting from a high protein (meat) intake has been closely associated with increased urinary calcium excretion. How...

  9. Semisynthetic DNA-protein conjugates for fabrication of nucleic acid based nanostructures

    NASA Astrophysics Data System (ADS)

    Rabe, Kersten S.; Feldkamp, Udo; Niemeyer, Christof M.

    2008-10-01

    We here report on the developments of semisynthetic DNA-protein conjugates and their assembly into multi-component nanostructures. We describe the improvement of the DNA sequences embedded in such nanostructures by computational and analytical methods. Moreover, we report on the exploration of novel DNA conjugates of streptavidin or redox proteins with improved properties for the assembly of nucleic acid based nanostructures.

  10. CONCENTRATION OF GLIAL FIBRILLARY ACIDIC PROTEIN INCREASES WITH AGE IN THE MOUSE AND RAT BRAIN

    EPA Science Inventory

    The role of aging in the expression of the astrocyte protein, glial fibrillary acidic protein (GFAP), was examined. n both mice and rats the concentration of GFAP increased throughout the brain as a function of aging. he largest increase (2-fold) was observed in striatum for both...

  11. Insulin and amino acids stimulate whole body protein synthesis in neonates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin and amino acids (AA) stimulate muscle protein synthesis in neonatal pigs. To determine the effects of insulin and AA on whole body protein turnover, hyperinsulinemic (0 and 100 ng/(kg[0.66]/min))-euglycemic-AA clamps were performed during euaminoacidemia or hyperaminoacidemia in fasted 7-d-...

  12. Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the curre...

  13. Umami taste amino acids produced by hydrolyzing extracted protein from tomato seed meal

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymatic hydrolysis was performed for extracting protein to prepare umami taste amino acids from defatted tomato seed meal (DTSM) which is a by-product of tomato processing. Papain was used as an enzyme for the hydrolysis of DTSM. The particle size distribution of DTSM, protein concentration and fr...

  14. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

    PubMed

    Vickers, Timothy A; Crooke, Stanley T

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  15. Isoelectric Point, Electric Charge, and Nomenclature of the Acid-Base Residues of Proteins

    ERIC Educational Resources Information Center

    Maldonado, Andres A.; Ribeiro, Joao M.; Sillero, Antonio

    2010-01-01

    The main object of this work is to present the pedagogical usefulness of the theoretical methods, developed in this laboratory, for the determination of the isoelectric point (pI) and the net electric charge of proteins together with some comments on the naming of the acid-base residues of proteins. (Contains 8 figures and 4 tables.)

  16. Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

    PubMed Central

    Freire, João Miguel; Veiga, Ana Salomé; Conceição, Thaís M.; Kowalczyk, Wioleta; Mohana-Borges, Ronaldo; Andreu, David; Santos, Nuno C.; Da Poian, Andrea T.; Castanho, Miguel A. R. B.

    2013-01-01

    Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins. PMID:24339931

  17. Nucleic acid encoding DS-CAM proteins and products related thereto

    SciTech Connect

    Korenberg, Julie R.

    2005-11-01

    In accordance with the present invention, there are provided Down Syndrome-Cell Adhesion Molecule (DS-CAM) proteins. Nucleic acid sequences encoding such proteins and assays employing same are also disclosed. The invention DS-CAM proteins can be employed in a variety of ways, for example, for the production of anti-DS-CAM antibodies thereto, in therapeutic compositions and methods employing such proteins and/or antibodies. DS-CAM proteins are also useful in bioassays to identify agonists and antagonists thereto.

  18. Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment

    PubMed Central

    Gruber, David F.; Gaffney, Jean P.; Mehr, Shaadi; DeSalle, Rob; Sparks, John S.; Platisa, Jelena; Pieribone, Vincent A.

    2015-01-01

    We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein’s fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment. PMID:26561348

  19. Glutathione Peroxidase’s Reaction Intermediate Selenenic Acid is Stabilized by the Protein Microenvironment

    PubMed Central

    Li, Fei; Liu, Jun; Rozovsky, Sharon

    2014-01-01

    Selenenic acids are highly reactive intermediates of selenoproteins’ enzymatic reactions. Knowledge of how the protein environment protects and stabilizes them is fundamental not only to descriptions of selenoproteins’ reactivity but also potentially for proteomics and therapeutics. However, selenenic acids are considered particularly short-lived and were not yet identified in wild-type selenoproteins. Here, we report trapping the selenenic acid in glutathione peroxidase, an anti-oxidant enzyme that efficiently eliminates hydroperoxides. It has long been thought that selenium-containing glutathione peroxidases form a selenenic acid intermediate. However, this putative species has eluded detection. Here, we report its identification. The selenenic acid in bovine glutathione peroxidase 1 was chemically trapped using dimedone, an alkylating agent specific to sulfenic and selenenic acids. The alkylation of the catalytic selenocysteine was verified by electrospray ionization mass spectrometry. In the presence of glutathione, the selenocysteine was not alkylated because the selenenic acid condenses faster with glutathione than the alkylation reaction. In the absence of thiols, the selenenic acid was surprisingly long-lived with 95% of the protein still able to react with dimedone 10 min after hydrogen peroxide was removed, indicating that the protein environment stabilizes the selenenic acid by shielding it from reactive groups in the protein. After 30 min, the selenocysteine was no longer modified but became accessible once the protein was exposed to reducing agents. This suggests that the selenenic acid reacted with a protein’s amide or amine to form a selenylamide bond. Such a modification may play a role in protecting glutathione peroxidase’s reactivity. PMID:25124921

  20. Isotopomer distributions in amino acids from a highly expressed protein as a proxy for those from total protein

    SciTech Connect

    Shaikh, Afshan; Shaikh, Afshan S.; Tang, Yinjie; Mukhopadhyay, Aindrila; Keasling, Jay D.

    2008-06-27

    {sup 13}C-based metabolic flux analysis provides valuable information about bacterial physiology. Though many biological processes rely on the synergistic functions of microbial communities, study of individual organisms in a mixed culture using existing flux analysis methods is difficult. Isotopomer-based flux analysis typically relies on hydrolyzed amino acids from a homogeneous biomass. Thus metabolic flux analysis of a given organism in a mixed culture requires its separation from the mixed culture. Swift and efficient cell separation is difficult and a major hurdle for isotopomer-based flux analysis of mixed cultures. Here we demonstrate the use of a single highly-expressed protein to analyze the isotopomer distribution of amino acids from one organism. Using the model organism E. coli expressing a plasmid-borne, his-tagged Green Fluorescent Protein (GFP), we show that induction of GFP does not affect E. coli growth kinetics or the isotopomer distribution in nine key metabolites. Further, the isotopomer labeling patterns of amino acids derived from purified GFP and total cell protein are indistinguishable, indicating that amino acids from a purified protein can be used to infer metabolic fluxes of targeted organisms in a mixed culture. This study provides the foundation to extend isotopomer-based flux analysis to study metabolism of individual strains in microbial communities.

  1. Protein adsorption on piezoelectric poly(L-lactic) acid thin films by scanning probe microscopy

    NASA Astrophysics Data System (ADS)

    Barroca, Nathalie; Vilarinho, Paula M.; Daniel-da-Silva, Ana Luisa; Wu, Aiying; Fernandes, Maria Helena; Gruverman, Alexei

    2011-03-01

    Up until now, no direct evidence of protein adsorption processes associated with polar activity of a piezoelectric has been reported. This work presents the experimental evidence of the protein adsorption process' dependence on the surface polarization of a piezoelectric by showing at the local scale that the process of protein adsorption is highly favored in the poled areas of a piezoelectric polymer such as poly(L-lactic) acid.

  2. Novel biomimetic tripolymer scaffolds consisting of chitosan, collagen type 1, and hyaluronic acid for bone marrow-derived human mesenchymal stem cells-based bone tissue engineering.

    PubMed

    Mathews, Smitha; Bhonde, Ramesh; Gupta, Pawan Kumar; Totey, Satish

    2014-11-01

    Human bone marrow-derived mesenchymal stem cells (hMSCs) are an ideal osteogenic cell source for bone tissue engineering (BTE). A scaffold, in the context of BTE, is the extracellular matrix (ECM) that provides the unique microenvironment and play significant role in regulating cell behavior, differentiation, and development in an in vitro culture system. In this study, we have developed novel biomimetic tripolymer scaffolds for BTE using an ECM protein, collagen type 1; an ECM glycosaminoglycan, hyaluronic acid; and a natural osteoconductive polymer, chitosan. The scaffolds were characterized by scanning electron microscopy (SEM) and swelling ratio. The scaffolds were seeded with hMSCs and tested for cytocompatibility and osteogenic potential. The scaffolds supported cell adhesion, enhanced cell proliferation, promoted cell migration, showed good cell viability, and osteogenic potential. The cells were able to migrate out from the scaffolds in favorable conditions. SEM, alkaline phosphatase assay, and immunofluorescent staining confirmed the differentiation of hMSCs to osteogenic lineage in the scaffolds. In conclusion, we have successfully developed biomimetic scaffolds that supported the proliferation and differentiation of hMSCs. These scaffolds hold great promise as a cell-delivery vehicle for regenerative therapies and as a support system for enhancing bone regeneration. PMID:24723571

  3. Vaccination of Gerbils with Bm-103 and Bm-RAL-2 Concurrently or as a Fusion Protein Confers Consistent and Improved Protection against Brugia malayi Infection

    PubMed Central

    Arumugam, Sridhar; Wei, Junfei; Liu, Zhuyun; Abraham, David; Bell, Aaron; Bottazzi, Maria Elena; Hotez, Peter J.; Zhan, Bin; Lustigman, Sara; Klei, Thomas R.

    2016-01-01

    Background The Brugia malayi Bm-103 and Bm-RAL-2 proteins are orthologous to Onchocerca volvulus Ov-103 and Ov-RAL-2, and which were selected as the best candidates for the development of an O. volvulus vaccine. The B. malayi gerbil model was used to confirm the efficacy of these Ov vaccine candidates on adult worms and to determine whether their combination is more efficacious. Methodology and Principle Findings Vaccine efficacy of recombinant Bm-103 and Bm-RAL-2 administered individually, concurrently or as a fusion protein were tested in gerbils using alum as adjuvant. Vaccination with Bm-103 resulted in worm reductions of 39%, 34% and 22% on 42, 120 and 150 days post infection (dpi), respectively, and vaccination with Bm-RAL-2 resulted in worm reductions of 42%, 22% and 46% on 42, 120 and 150 dpi, respectively. Vaccination with a fusion protein comprised of Bm-103 and Bm-RAL-2 resulted in improved efficacy with significant reduction of worm burden of 51% and 49% at 90 dpi, as did the concurrent vaccination with Bm-103 and Bm-RAL-2, with worm reduction of 61% and 56% at 90 dpi. Vaccination with Bm-103 and Bm-RAL-2 as a fusion protein or concurrently not only induced a significant worm reduction of 61% and 42%, respectively, at 150 dpi, but also significantly reduced the fecundity of female worms as determined by embryograms. Elevated levels of antigen-specific IgG were observed in all vaccinated gerbils. Serum from gerbils vaccinated with Bm-103 and Bm-RAL-2 individually, concurrently or as a fusion protein killed third stage larvae in vitro when combined with peritoneal exudate cells. Conclusion Although vaccination with Bm-103 and Bm-RAL-2 individually conferred protection against B. malayi infection in gerbils, a more consistent and enhanced protection was induced by vaccination with Bm-103 and Bm-RAL-2 fusion protein and when they were used concurrently. Further characterization and optimization of these filarial vaccines are warranted. PMID:27045170

  4. Surface lysine residues modulate the collisional transfer of fatty acid from adipocyte fatty acid binding protein to membranes.

    PubMed

    Herr, F M; Matarese, V; Bernlohr, D A; Storch, J

    1995-09-19

    The transfer of unesterified fatty acids (FA) from adipocyte fatty acid binding protein (A-FABP) to phospholipid membranes is proposed to occur via a collisional mechanism involving transient ionic and hydrophobic interactions [Wootan & Storch (1994) J. Biol. Chem. 269, 10517-10523]. In particular, it was suggested that membrane acidic phospholipids might specifically interact with basic residues on the surface of A-FABP. Here we addressed whether lysine residues on the surface of the protein are involved in this collisional transfer mechanism. Recombinant A-FABP was acetylated to neutralize all positively charged surface lysine residues. Protein fluorescence, CD spectra, and chemical denaturant data indicate that acetylation did not substantially alter the conformational integrity of the protein, and nearly identical affinities were obtained for binding of the fluorescently labeled FA [12-(9-anthroyloxy)oleate] to native and acetylated protein. Transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated A-FABP to small unilamellar vesicles (SUV) was 35-fold slower than from native protein. In addition, whereas the 2AP transfer rate from native A-FABP was directly dependent on SUV concentration, 2AP transfer from acetylated protein was independent on the concentration of acceptor membranes. Factors which alter aqueous-phase solubility of FA, such as ionic strength and acyl chain length and saturation, affected the AOFA transfer rate from acetylated but not native A-FABP. Finally, an increase in the negative charge density of the acceptor SUV resulted in a marked increase in the rate of transfer from native A-FABP but did not increase the rate from acetylated A-FABP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7547918

  5. Rapid Online Non-Enzymatic Protein Digestion Combining Microwave Heating Acid Hydrolysis and Electrochemical Oxidation

    PubMed Central

    Basile, Franco; Hauser, Nicolas

    2010-01-01

    We report an online non-enzymatic method for site-specific digestion of proteins to yield peptides that are well suited for collision induced dissociation (CID) tandem mass spectrometry (MS/MS). The method combines online microwave heating acid hydrolysis at aspartic acid and online electrochemical oxidation at tryptophan and tyrosine. The combined microwave/electrochemical (microwave/echem) digestion is reproducible and produces peptides with an average sequence length of 10 amino acids. This peptide length is similar to the average peptide length of 9 amino acids obtained by digestion of proteins with the enzyme trypsin. As a result, the peptides produced by this novel non-enzymatic digestion method, when analyzed by ESI-MS, produce protonated molecules with mostly +1 and +2 charge states. The combination of these two non-enzymatic methods overcomes shortcomings with each individual method in that: i) peptides generated by the microwave-hydrolysis method have an average amino acid length of 16 amino acids, and ii) the inability of the electrochemical-cleavage method to reproducibly digest proteins with molecular masses above 4 kDa. Preliminary results are presented on the application and utility of this rapid online digestion (total of 6 min digestion time) on a series of standard peptides and proteins as well as an E. coli protein extract. PMID:21138252

  6. Production of hydrophobic amino acids from biobased resources: wheat gluten and rubber seed proteins.

    PubMed

    Widyarani; Sari, Yessie W; Ratnaningsih, Enny; Sanders, Johan P M; Bruins, Marieke E

    2016-09-01

    Protein hydrolysis enables production of peptides and free amino acids that are suitable for usage in food and feed or can be used as precursors for bulk chemicals. Several essential amino acids for food and feed have hydrophobic side chains; this property may also be exploited for subsequent separation. Here, we present methods for selective production of hydrophobic amino acids from proteins. Selectivity can be achieved by selection of starting material, selection of hydrolysis conditions, and separation of achieved hydrolysate. Several protease combinations were applied for hydrolysis of rubber seed protein concentrate, wheat gluten, and bovine serum albumin (BSA). High degree of hydrolysis (>50 %) could be achieved. Hydrophobic selectivity was influenced by the combination of proteases and by the extent of hydrolysis. Combination of Pronase and Peptidase R showed the highest selectivity towards hydrophobic amino acids, roughly doubling the content of hydrophobic amino acids in the products compared to the original substrates. Hydrophobic selectivity of 0.6 mol-hydrophobic/mol-total free amino acids was observed after 6 h hydrolysis of wheat gluten and 24 h hydrolysis of rubber seed proteins and BSA. The results of experiments with rubber seed proteins and wheat gluten suggest that this process can be applied to agro-industrial residues. PMID:27118013

  7. Biological chemistry and functionality of protein sulfenic acids and related thiol modifications.

    PubMed

    Devarie-Baez, Nelmi O; Silva Lopez, Elsa I; Furdui, Cristina M

    2016-01-01

    Selective modification of proteins at cysteine residues by reactive oxygen, nitrogen or sulfur species formed under physiological and pathological states is emerging as a critical regulator of protein activity impacting cellular function. This review focuses primarily on protein sulfenylation (-SOH), a metastable reversible modification connecting reduced cysteine thiols to many products of cysteine oxidation. An overview is first provided on the chemistry principles underlining synthesis, stability and reactivity of sulfenic acids in model compounds and proteins, followed by a brief description of analytical methods currently employed to characterize these oxidative species. The following chapters present a selection of redox-regulated proteins for which the -SOH formation was experimentally confirmed and linked to protein function. These chapters are organized based on the participation of these proteins in the regulation of signaling, metabolism and epigenetics. The last chapter discusses the therapeutic implications of altered redox microenvironment and protein oxidation in disease. PMID:26340608

  8. Genetically Encoding an Electrophilic Amino Acid for Protein Stapling and Covalent Binding to Native Receptors

    PubMed Central

    2015-01-01

    Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology. In addition to efficient cross-linking of proteins inter- and intramolecularly, this Uaa permits direct stapling of a protein α-helix in a recombinant manner and covalent binding of native membrane receptors in live cells. The target diversity, recombinant stapling, and covalent targeting of endogenous proteins enabled by this versatile Uaa should prove valuable in developing novel research tools, biological diagnostics, and therapeutics by exploiting covalent protein linkages for specificity, irreversibility, and stability. PMID:25010185

  9. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running.

    PubMed

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d₃-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  10. Leucine-Enriched Essential Amino Acids Augment Mixed Protein Synthesis, But Not Collagen Protein Synthesis, in Rat Skeletal Muscle after Downhill Running

    PubMed Central

    Kato, Hiroyuki; Suzuki, Hiromi; Inoue, Yoshiko; Suzuki, Katsuya; Kobayashi, Hisamine

    2016-01-01

    Mixed and collagen protein synthesis is elevated for as many as 3 days following exercise. Immediately after exercise, enhanced amino acid availability increases synthesis of mixed muscle protein, but not muscle collagen protein. However, the potential for synergic effects of amino acid ingestion with exercise on both mixed and collagen protein synthesis remains unclear. We investigated muscle collagen protein synthesis in rats following post-exercise ingestion of leucine-enriched essential amino acids. We determined fractional protein synthesis rates (FSR) at different time points following exercise. Mixed protein and collagen protein FSRs in skeletal muscle were determined by measuring protein-bound enrichments of hydroxyproline and proline, and by measuring the intracellular enrichment of proline, using injections of flooding d3-proline doses. A leucine-enriched mixture of essential amino acids (or distilled water as a control) was administrated 30 min or 1 day post-exercise. The collagen protein synthesis in the vastus lateralis was elevated for 2 days after exercise. Although amino acid administration did not increase muscle collagen protein synthesis, it did lead to augmented mixed muscle protein synthesis 1 day following exercise. Thus, contrary to the regulation of mixed muscle protein synthesis, muscle collagen protein synthesis is not affected by amino acid availability after damage-inducing exercise. PMID:27367725

  11. Mosaic protein and nucleic acid vaccines against hepatitis C virus

    DOEpatents

    Yusim, Karina; Korber, Bette T. M.; Kuiken, Carla L.; Fischer, William M.

    2013-06-11

    The invention relates to immunogenic compositions useful as HCV vaccines. Provided are HCV mosaic polypeptide and nucleic acid compositions which provide higher levels of T-cell epitope coverage while minimizing the occurrence of unnatural and rare epitopes compared to natural HCV polypeptides and consensus HCV sequences.

  12. Macronutrient requirement for growth: Protein/amino acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Current recommendations by the Institute of Medicine on amino acid requirements in healthy children older than 6 months and for children and adolescents have been established using the factorial approach, which takes into account: i) maintenance for obligatory losses, which is estimated by regressio...

  13. Glycation inhibits trichloroacetic acid (TCA)-induced whey protein precipitation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Four different WPI saccharide conjugates were successfully prepared to test whether glycation could inhibit WPI precipitation induced by trichloroacetic acid (TCA). Conjugates molecular weights after glycation were analyzed with SDS-PAGE. No significant secondary structure change due to glycation wa...

  14. Protein and Essential Amino Acids to Protect Musculoskeletal Health during Spaceflight: Evidence of a Paradox?

    PubMed

    Hackney, Kyle J; English, Kirk L

    2014-01-01

    Long-duration spaceflight results in muscle atrophy and a loss of bone mineral density. In skeletal muscle tissue, acute exercise and protein (e.g., essential amino acids) stimulate anabolic pathways (e.g., muscle protein synthesis) both independently and synergistically to maintain neutral or positive net muscle protein balance. Protein intake in space is recommended to be 12%-15% of total energy intake (≤1.4 g∙kg-1∙day-1) and spaceflight is associated with reduced energy intake (~20%), which enhances muscle catabolism. Increasing protein intake to 1.5-2.0 g∙kg-1∙day-1 may be beneficial for skeletal muscle tissue and could be accomplished with essential amino acid supplementation. However, increased consumption of sulfur-containing amino acids is associated with increased bone resorption, which creates a dilemma for musculoskeletal countermeasures, whereby optimizing skeletal muscle parameters via essential amino acid supplementation may worsen bone outcomes. To protect both muscle and bone health, future unloading studies should evaluate increased protein intake via non-sulfur containing essential amino acids or leucine in combination with exercise countermeasures and the concomitant influence of reduced energy intake. PMID:25370374

  15. Protein and Essential Amino Acids to Protect Musculoskeletal Health during Spaceflight: Evidence of a Paradox?

    PubMed Central

    Hackney, Kyle J.; English, Kirk L.

    2014-01-01

    Long-duration spaceflight results in muscle atrophy and a loss of bone mineral density. In skeletal muscle tissue, acute exercise and protein (e.g., essential amino acids) stimulate anabolic pathways (e.g., muscle protein synthesis) both independently and synergistically to maintain neutral or positive net muscle protein balance. Protein intake in space is recommended to be 12%–15% of total energy intake (≤1.4 g∙kg−1∙day−1) and spaceflight is associated with reduced energy intake (~20%), which enhances muscle catabolism. Increasing protein intake to 1.5–2.0 g∙kg−1∙day−1 may be beneficial for skeletal muscle tissue and could be accomplished with essential amino acid supplementation. However, increased consumption of sulfur-containing amino acids is associated with increased bone resorption, which creates a dilemma for musculoskeletal countermeasures, whereby optimizing skeletal muscle parameters via essential amino acid supplementation may worsen bone outcomes. To protect both muscle and bone health, future unloading studies should evaluate increased protein intake via non-sulfur containing essential amino acids or leucine in combination with exercise countermeasures and the concomitant influence of reduced energy intake. PMID:25370374

  16. Lipid binding protein response to a bile acid library: a combined NMR and statistical approach.

    PubMed

    Tomaselli, Simona; Pagano, Katiuscia; Boulton, Stephen; Zanzoni, Serena; Melacini, Giuseppe; Molinari, Henriette; Ragona, Laura

    2015-11-01

    Primary bile acids, differing in hydroxylation pattern, are synthesized from cholesterol in the liver and, once formed, can undergo extensive enzyme-catalysed glycine/taurine conjugation, giving rise to a complex mixture, the bile acid pool. Composition and concentration of the bile acid pool may be altered in diseases, posing a general question on the response of the carrier (bile acid binding protein) to the binding of ligands with different hydrophobic and steric profiles. A collection of NMR experiments (H/D exchange, HET-SOFAST, ePHOGSY NOESY/ROESY and (15) N relaxation measurements) was thus performed on apo and five different holo proteins, to monitor the binding pocket accessibility and dynamics. The ensemble of obtained data could be rationalized by a statistical approach, based on chemical shift covariance analysis, in terms of residue-specific correlations and collective protein response to ligand binding. The results indicate that the same residues are influenced by diverse chemical stresses: ligand binding always induces silencing of motions at the protein portal with a concomitant conformational rearrangement of a network of residues, located at the protein anti-portal region. This network of amino acids, which do not belong to the binding site, forms a contiguous surface, sensing the presence of the bound lipids, with a signalling role in switching protein-membrane interactions on and off. PMID:26260520

  17. Determination of seleno-amino acids bound to proteins in extra virgin olive oils.

    PubMed

    Torres, Sabier; Gil, Raul; Silva, María Fernanda; Pacheco, Pablo

    2016-04-15

    An analytical method has been developed to determine seleno-amino acids in proteins extracted from extra virgin olive oils (EVOOs). Different aqueous/organic solvents were tested to isolate proteins, an acetone:n-hexane combination being the best protein precipitant. In a first dimension chromatography, extracted proteins were analysed by size exclusion chromatography (SEC) coupled to inductively coupled plasma mass spectrometry (ICP-MS) to identify S and Se associations as proteins marker. Two fractions of 66 kDa (A) and 443 kDa (B) were identified. These fractions were submitted to microwave-assisted acid hydrolysis (MAAH) to release seleno-amino acids. In a second dimension chromatography seleno-amino acids were determined by reversed-phase chromatography (RPC) coupled to ICP-MS. Seleno-methylselenocysteine was determined with values ranging from 1.03-2.03±0.2 μg kg(-1) and selenocysteine at a concentration of 1.47±0.1 μg kg(-1). Variations of protein and seleno-amino acid concentrations were observed between EVOO varieties, contributing to EVOO cultivar differentiation. PMID:26616967

  18. Influence of various nitrogen applications on protein and amino acid profiles of amaranth and quinoa.

    PubMed

    Thanapornpoonpong, Sa-nguansak; Vearasilp, Suchada; Pawelzik, Elke; Gorinstein, Shela

    2008-12-10

    The effect of nitrogen application levels (0.16 and 0.24 g N kg(-1) soil) on seed proteins and their amino acid compositions of amaranth (Amaranthus spp.) and quinoa (Chenopodium quinoa Willd) was studied. Total proteins of amaranth and quinoa had high contents of lysine (6.3-8.2 g 100 g(-1) protein) but low contents of methionine (1.2-1.8 g 100 g(-1) protein). Seed proteins were fractionated on the basis of different solubility in water, saline, and buffer as albumin-1 (Albu-1), albumin-2 (Albu-2), globulin (Glob), and glutelin (Glu) and were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Albu-1 was high in lysine (5.4-8.6 g 100 g(-1) protein), while Albu-2, which is a part of storage proteins, had a high leucine content (7.2-8.9 g 100 g(-1) protein) as an effect of different nitrogen application levels. Glu fractions were well-balanced in their essential amino acids with the exception of methionine. In conclusion, nitrogen application can be used for the nutritional improvement in human diet by increasing and maintaining protein and essential amino acid contents. PMID:19006392

  19. Okadaic acid-sensitive protein phosphatases constrain phrenic long-term facilitation after sustained hypoxia.

    PubMed

    Wilkerson, Julia E R; Satriotomo, Irawan; Baker-Herman, Tracy L; Watters, Jyoti J; Mitchell, Gordon S

    2008-03-12

    Phrenic long-term facilitation (pLTF) is a serotonin-dependent form of pattern-sensitive respiratory plasticity induced by intermittent hypoxia (IH), but not sustained hypoxia (SH). The mechanism(s) underlying pLTF pattern sensitivity are unknown. SH and IH may differentially regulate serine/threonine protein phosphatase activity, thereby inhibiting relevant protein phosphatases uniquely during IH and conferring pattern sensitivity to pLTF. We hypothesized that spinal protein phosphatase inhibition would relieve this braking action of protein phosphatases, thereby revealing pLTF after SH. Anesthetized rats received intrathecal (C4) okadaic acid (25 nm) before SH (25 min, 11% O(2)). Unlike (vehicle) control rats, SH induced a significant pLTF in okadaic acid-treated rats that was indistinguishable from rats exposed to IH (three 5 min episodes, 11% O(2)). IH and SH with okadaic acid may elicit pLTF by similar, serotonin-dependent mechanisms, because intravenous methysergide blocks pLTF in rats receiving IH or okadaic acid plus SH. Okadaic acid did not alter IH-induced pLTF. In summary, pattern sensitivity in pLTF may reflect differential regulation of okadaic acid-sensitive serine/threonine phosphatases; presumably, these phosphatases are less active during/after IH versus SH. The specific okadaic acid-sensitive phosphatase(s) constraining pLTF and their spatiotemporal dynamics during and/or after IH and SH remain to be determined. PMID:18337426

  20. Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes.

    PubMed

    De Gerónimo, Eduardo; Hagan, Robert M; Wilton, David C; Córsico, Betina

    2010-09-01

    Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates. PMID:20541621

  1. Phytanic acid, a novel activator of uncoupling protein-1 gene transcription and brown adipocyte differentiation.

    PubMed Central

    Schlüter, Agatha; Barberá, Maria José; Iglesias, Roser; Giralt, Marta; Villarroya, Francesc

    2002-01-01

    Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5' enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis. PMID:11829740

  2. Structure of the photoactive yellow protein reconstituted with caffeic acid at 1.16 A resolution.

    PubMed

    van Aalten, Daan M F; Crielaard, Wim; Hellingwerf, Klaas J; Joshua-Tor, Leemor

    2002-04-01

    A structural study is described of the photoactive yellow protein (PYP) reconstituted with the chromophore derivative 3,4-dihydroxycinnamic acid. The crystal structure of PYP reconstituted with this chromophore at 1.16 A resolution is reported in space group P6(5). This is the first high-resolution structure of a photoreceptor containing a modified chromophore. The introduction of an extra hydroxyl group in the native chromophore (i.e. p-coumaric acid) appears to perturb the structure of the hybrid yellow protein only slightly. The chromophore is bound by the protein in two different conformations, separated by a rotation of 180 degrees of the catechol ring. In combination with available spectroscopic data, it is concluded that the caffeic acid chromophore binds to the protein in a strained conformation, which leads to a faster ejection from the chromophore-binding pocket upon pB formation. PMID:11914481

  3. Incorporation of Noncanonical Amino Acids into Rosetta and Use in Computational Protein-Peptide Interface Design

    PubMed Central

    Renfrew, P. Douglas; Choi, Eun Jung; Bonneau, Richard; Kuhlman, Brian

    2012-01-01

    Noncanonical amino acids (NCAAs) can be used in a variety of protein design contexts. For example, they can be used in place of the canonical amino acids (CAAs) to improve the biophysical properties of peptides that target protein interfaces. We describe the incorporation of 114 NCAAs into the protein-modeling suite Rosetta. We describe our methods for building backbone dependent rotamer libraries and the parameterization and construction of a scoring function that can be used to score NCAA containing peptides and proteins. We validate these additions to Rosetta and our NCAA-rotamer libraries by showing that we can improve the binding of a calpastatin derived peptides to calpain-1 by substituting NCAAs for native amino acids using Rosetta. Rosetta (executables and source), auxiliary scripts and code, and documentation can be found at (http://www.rosettacommons.org/). PMID:22431978

  4. In planta changes in protein phosphorylation induced by the plant hormone abscisic acid

    PubMed Central

    Kline, Kelli G.; Barrett-Wilt, Gregory A.; Sussman, Michael R.

    2010-01-01

    Abscisic acid (ABA) is a hormone that controls seed dormancy and germination as well as the overall plant response to important environmental stresses such as drought. Recent studies have demonstrated that the ABA-bound receptor binds to and inhibits a class of protein phosphatases. To identify more broadly the phosphoproteins affected by this hormone in vivo, we used 14N/15N metabolic labeling to perform a quantitative untargeted mass spectrometric analysis of the Arabidopsis thaliana phosphoproteome following ABA treatment. We found that 50 different phosphopeptides had their phosphorylation state significantly altered by ABA over a treatment period lasting 5–30 min. Among these changes were increases in phosphorylation of subfamily 2 SNF1-related kinases and ABA-responsive basic leucine zipper transcription factors implicated in ABA signaling by previous in vitro studies. Furthermore, four members of the aquaporin family showed decreased phosphorylation at a carboxy-terminal serine which is predicted to cause closure of the water-transporting aquaporin gate, consistent with ABA's role in ameliorating the effect of drought. Finally, more than 20 proteins not previously known to be involved with ABA were found to have significantly altered phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that an expanded model of ABA signaling, beyond simple phosphatase inhibition, may be necessary. This quantitative proteomics dataset provides a more comprehensive, albeit incomplete, view both of the protein targets whose biochemical activities are likely to be controlled by ABA and of the nature of the emerging phosphorylation and dephosphorylation cascades triggered by this hormone. PMID:20733066

  5. Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium[S

    PubMed Central

    Rudolph, Michael C.; Wellberg, Elizabeth A.; Lewis, Andrew S.; Terrell, Kristina L.; Merz, Andrea L.; Maluf, N. Karl; Serkova, Natalie J.; Anderson, Steven M.

    2014-01-01

    Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefined during lactation. Spot14-null mice produce milk deficient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When [13C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed. PMID:24771867

  6. Distinct nucleic acid interaction properties of HIV-1 nucleocapsid protein precursor NCp15 explain reduced viral infectivity

    PubMed Central

    Wang, Wei; Naiyer, Nada; Mitra, Mithun; Li, Jialin; Williams, Mark C.; Rouzina, Ioulia; Gorelick, Robert J.; Wu, Zhengrong; Musier-Forsyth, Karin

    2014-01-01

    During human immunodeficiency virus type 1 (HIV-1) maturation, three different forms of nucleocapsid (NC) protein—NCp15 (p9 + p6), NCp9 (p7 + SP2) and NCp7—appear successively. A mutant virus expressing NCp15 shows greatly reduced infectivity. Mature NCp7 is a chaperone protein that facilitates remodeling of nucleic acids (NAs) during reverse transcription. To understand the strict requirement for NCp15 processing, we compared the chaperone function of the three forms of NC. NCp15 anneals tRNA to the primer-binding site at a similar rate as NCp7, whereas NCp9 is the most efficient annealing protein. Assays to measure NA destabilization show a similar trend. Dynamic light scattering studies reveal that NCp15 forms much smaller aggregates relative to those formed by NCp7 and NCp9. Nuclear magnetic resonance studies suggest that the acidic p6 domain of HIV-1 NCp15 folds back and interacts with the basic zinc fingers. Neutralizing the acidic residues in p6 improves the annealing and aggregation activity of NCp15 to the level of NCp9 and increases the protein–NA aggregate size. Slower NCp15 dissociation kinetics is observed by single-molecule DNA stretching, consistent with the formation of electrostatic inter-protein contacts, which likely contribute to the distinct aggregate morphology, irregular HIV-1 core formation and non-infectious virus. PMID:24813443

  7. Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis.

    PubMed

    Chen, Hsi-Chuan; Li, Quanzi; Shuford, Christopher M; Liu, Jie; Muddiman, David C; Sederoff, Ronald R; Chiang, Vincent L

    2011-12-27

    The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (V(max)/k(m)) for any of the complexes is 70-6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex-mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis. PMID:22160716

  8. Single-Cell Protein Production by the Acid-Tolerant Fungus Scytalidium acidophilum from Acid Hydrolysates of Waste Paper †

    PubMed Central

    Ivarson, K. C.; Morita, H.

    1982-01-01

    The bioconversion of waste paper to single-cell protein at pH <1 by Scytalidium acidophilum is described. Waste paper pretreated with 72% H2SO4 at 4°C was diluted with water to a pH of <0.1 and hydrolyzed. This yielded an adequate sugar-containing substrate for the growth of the fungus. A total of 97% of the sugars (glucose, galactose, mannose, xylose, arabinose) in the hydrolysates were converted to cell biomass. Microbial contamination was not observed. Based on the sugars consumed, S. acidophilum produced higher yields in shake cultures than many other Fungi Imperfecti. In aerated cultures, productivity increased, and yields of 43 to 46% containing 44 to 47% crude protein were obtained. This compares favorably with Candida utilis, a yeast used commercially to produce single-cell protein. The chemical constituents and the essential amino acids of the fungal cells were similar to those of other fungi. The nucleic acid content was characteristic of microbes containing low levels of nucleic acid. The advantages of using S. acidophilum for single-cell protein production are discussed. PMID:16345970

  9. Single-cell protein production by the acid-tolerant fungus Scytalidium acidophilum from acid hydrolysates of waste paper

    SciTech Connect

    Ivarson, K.C.; Morita, H.

    1982-03-01

    The bioconversion of waste paper to single-cell protein at pH less than 1 by Scytalidium acidophilum is described. Waste paper pretreated with 72% H/sub 2/SO/sub 4/ at 4 degrees C was diluted with water to a pH of less than 0.1 and hydrolyzed. This yielded an adequate sugar-containing substrate for the growth of the fungus. A total of 97% of the sugars (glucose, galactose, mannose, xylose, arabinose) in the hydrolysates were converted to cell biomass. Microbial contamination was not observed. Based on the sugars consumed, S. acidophilum produced higher yields in shake cultures than many other Fungi Imperfecti. In aerated cultures, productivity increased, and yields of 43 to 46% containing 44 to 47% crude protein were obtained. This compares favorably with Candida utilis, a yeast used commercially to produce single-cell protein. The chemical constituents and the essential amino acids of the fungal cells were similar to those of other fungi. The nucleic acid content was characteristic of microbes containing low levels of nucleic acid. The advantages of using S. acidophilum for single-cell protein production are discussed. (Refs. 30).

  10. Maternal Low Quality Protein Diet Alters Plasma Amino Acid Concentrations of Weaning Rats

    PubMed Central

    Kabasakal Cetin, Arzu; Dasgin, Halil; Gülec, Atila; Onbasilar, İlyas; Akyol, Asli

    2015-01-01

    Several studies have indicated the influence of a maternal low protein diet on the fetus. However, the effect of a maternal low quality protein diet on fetal growth and development is largely unknown. Wistar rats (11 weeks old) were mated and maintained on either a chow diet with 20% casein (n = 6) as the control group (C), or a low quality protein diet with 20% wheat gluten (n = 7) as the experimental group (WG) through gestation and lactation. Maternal body weights were similar in both groups throughout the study. Birth weights were not influenced by maternal diet and offspring body weights during lactation were similar between the groups. Offspring’s plasma amino acid profiles showed that plasma methionine, glutamine and lysine were significantly lower and aspartic acid, ornithine and glycine-proline were significantly higher in the WG. Plant based protein comprises an important part of protein intake in developing countries. It is well-known that these diets can be inadequate in terms of essential amino acids. The current study shows differential effects of a maternal low quality protein diet on the offspring’s plasma amino acids. Future studies will examine further aspects of the influence of maternal low quality protein diets on fetal growth and development. PMID:26633475

  11. Cinnamic Acid and Its Derivatives Inhibit Fructose-Mediated Protein Glycation

    PubMed Central

    Adisakwattana, Sirichai; Sompong, Weerachat; Meeprom, Aramsri; Ngamukote, Sathaporn; Yibchok-anun, Sirintorn

    2012-01-01

    Cinnamic acid and its derivatives have shown a variety of pharmacologic properties. However, little is known about the antiglycation properties of cinnamic acid and its derivatives. The present study sought to characterize the protein glycation inhibitory activity of cinnamic acid and its derivatives in a bovine serum albumin (BSA)/fructose system. The results demonstrated that cinnamic acid and its derivatives significantly inhibited the formation of advanced glycation end products (AGEs) by approximately 11.96–63.36% at a concentration of 1 mM. The strongest inhibitory activity against the formation of AGEs was shown by cinnamic acid. Furthermore, cinnamic acid and its derivatives reduced the level of fructosamine, the formation of Nɛ-(carboxymethyl) lysine (CML), and the level of amyloid cross β-structure. Cinnamic acid and its derivatives also prevented oxidative protein damages, including effects on protein carbonyl formation and thiol oxidation of BSA. Our findings may lead to the possibility of using cinnamic acid and its derivatives for preventing AGE-mediated diabetic complications. PMID:22408423

  12. Accurate ab initio prediction of NMR chemical shifts of nucleic acids and nucleic acids/protein complexes

    PubMed Central

    Victora, Andrea; Möller, Heiko M.; Exner, Thomas E.

    2014-01-01

    NMR chemical shift predictions based on empirical methods are nowadays indispensable tools during resonance assignment and 3D structure calculation of proteins. However, owing to the very limited statistical data basis, such methods are still in their infancy in the field of nucleic acids, especially when non-canonical structures and nucleic acid complexes are considered. Here, we present an ab initio approach for predicting proton chemical shifts of arbitrary nucleic acid structures based on state-of-the-art fragment-based quantum chemical calculations. We tested our prediction method on a diverse set of nucleic acid structures including double-stranded DNA, hairpins, DNA/protein complexes and chemically-modified DNA. Overall, our quantum chemical calculations yield highly/very accurate predictions with mean absolute deviations of 0.3–0.6 ppm and correlation coefficients (r2) usually above 0.9. This will allow for identifying misassignments and validating 3D structures. Furthermore, our calculations reveal that chemical shifts of protons involved in hydrogen bonding are predicted significantly less accurately. This is in part caused by insufficient inclusion of solvation effects. However, it also points toward shortcomings of current force fields used for structure determination of nucleic acids. Our quantum chemical calculations could therefore provide input for force field optimization. PMID:25404135

  13. Accurate ab initio prediction of NMR chemical shifts of nucleic acids and nucleic acids/protein complexes.

    PubMed

    Victora, Andrea; Möller, Heiko M; Exner, Thomas E

    2014-12-16

    NMR chemical shift predictions based on empirical methods are nowadays indispensable tools during resonance assignment and 3D structure calculation of proteins. However, owing to the very limited statistical data basis, such methods are still in their infancy in the field of nucleic acids, especially when non-canonical structures and nucleic acid complexes are considered. Here, we present an ab initio approach for predicting proton chemical shifts of arbitrary nucleic acid structures based on state-of-the-art fragment-based quantum chemical calculations. We tested our prediction method on a diverse set of nucleic acid structures including double-stranded DNA, hairpins, DNA/protein complexes and chemically-modified DNA. Overall, our quantum chemical calculations yield highly/very accurate predictions with mean absolute deviations of 0.3-0.6 ppm and correlation coefficients (r(2)) usually above 0.9. This will allow for identifying misassignments and validating 3D structures. Furthermore, our calculations reveal that chemical shifts of protons involved in hydrogen bonding are predicted significantly less accurately. This is in part caused by insufficient inclusion of solvation effects. However, it also points toward shortcomings of current force fields used for structure determination of nucleic acids. Our quantum chemical calculations could therefore provide input for force field optimization. PMID:25404135

  14. Amino acid alignment of cholinesterases, esterases, lipases, and related proteins

    SciTech Connect

    Gentry, M.K.; Doctor, B.P.

    1995-12-31

    The alignments previously published (Gentry Doctor, 1991; Cygler et al., 1993), nine and 32 sequences respectively, have been further expanded by the addition of 22 newly-found sequences. References and protein sequences were found by searching on the term acetylcholinesterase using the software package Entrez, an integrated citation and sequence retrieval system (National Center for Biotechnology Information, NLM, Bethesda, MD).

  15. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  16. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  17. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  18. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  19. 40 CFR 79.67 - Glial fibrillary acidic protein assay.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... addition to information meeting the requirements stated under 40 CFR 79.60, the following specific...), 5 mM Hepes, pH 7.4, 0.7 percent Triton X-100) to a final concentration of 0.25 mg total protein per... the desired dilution in blocking solution containing 0.1 percent Triton X-100). Serum anti-bovine...

  20. Thermoformed protein based composites in presence of organic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    World industrialization has generated substantial quantities of petroleum-based plastics over many years, which are non biodegradable. There is a growing demand for the use of renewable agricultural sources to develop eco-friendly biobased composites. Agriculture-sourced proteins and starches are b...

  1. Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone.

    PubMed

    Armas, Pablo; Nasif, Sofía; Calcaterra, Nora B

    2008-02-15

    Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. PMID:17661353

  2. Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis.

    PubMed

    Ye, Shixin; Köhrer, Caroline; Huber, Thomas; Kazmi, Manija; Sachdev, Pallavi; Yan, Elsa C Y; Bhagat, Aditi; RajBhandary, Uttam L; Sakmar, Thomas P

    2008-01-18

    G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors. PMID:17993461

  3. Interactions of myelin basic protein with mixed dodecylphosphocholine/palmitoyllysophosphatidic acid micelles

    SciTech Connect

    Mendz, G.L. ); Brown, L.R. ); Martenson, R.E. )

    1990-03-06

    The interactions of myelin basic protein and peptides derived from it with detergent micelles of lysophosphatidylglycerol, lysophosphatidylserine, palmitoyllysophosphatidic acid, and sodium lauryl sulfate, and with mixed micelles of the neutral detergent dodecylphosphocholine and the negatively charged detergent palmitoyllysophosphatidic acid, were investigated by {sup 1}H NMR spectroscopy and circular dichroic spectropolarimetry. The results with single detergents suggested that there are discrete interaction sites in the protein molecule for neutral and anionic detergent micelles and that at least some of these sites are different for each type of detergent. The data on the binding of the protein and peptides to mixed detergent micelles suggested that intramolecular interactions in the intact protein and in one of the longer peptides limited the formation of helices and also that a balance between hydrophobic and ionic forces is achieved in the interactions of the peptides with the detergents. At high detergent/protein molar ratios, hydrophobic interactions appeared to be favored.

  4. A strategy to select suitable physicochemical attributes of amino acids for protein fold recognition

    PubMed Central

    2013-01-01

    Background Assigning a protein into one of its folds is a transitional step for discovering three dimensional protein structure, which is a challenging task in bimolecular (biological) science. The present research focuses on: 1) the development of classifiers, and 2) the development of feature extraction techniques based on syntactic and/or physicochemical properties. Results Apart from the above two main categories of research, we have shown that the selection of physicochemical attributes of the amino acids is an important step in protein fold recognition and has not been explored adequately. We have presented a multi-dimensional successive feature selection (MD-SFS) approach to systematically select attributes. The proposed method is applied on protein sequence data and an improvement of around 24% in fold recognition has been noted when selecting attributes appropriately. Conclusion The MD-SFS has been applied successfully in selecting physicochemical attributes of the amino acids. The selected attributes show improved protein fold recognition performance. PMID:23879571

  5. A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein binding

    SciTech Connect

    Kaye, F.J.; Gerster, J.L. Uniformed Services Univ. of Health Sciences, Bethesda, MD ); Kratzke, R.A. ); Horowitz, J.M. )

    1990-09-01

    The authors have previously identified a small-cell lung cancer cell line (NCI-H209) that expresses an aberrant, underphosphorylated form of the retinoblastoma protein RB1. Molecular analysis of RB1 mRNA from this cell line revealed a single point mutation within exon 21 that resulted in a nonconservative amino acid substitution (cysteine to phenylalanine) at codon 706. Stable expression of this mutant RB1 cDNA in a human cell line lacking endogenous RB1 demonstrated that this amino acid change was sufficient to inhibit phosphorylation. In addition, this cysteine-to-phenylalanine substitution also resulted in loss of RB1 binding to the simian virus 40 large tumor and adenovirus E1A transforming proteins. These results confirm the importance of exon 21 coding sequences and suggest that the cysteine residue at codon 706 may play a role in achieving a specific protein conformation essential for protein-protein interactions.

  6. Antitumor effects of an engineered and energized fusion protein consisting of an anti-CD20 scFv fragment and lidamycin.

    PubMed

    Fang, Hong; Miao, Qingfang; Zhang, Shenghua; Cheng, Xin; Xiong, Dongsheng; Zhen, Yongsu

    2011-03-01

    Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases. CD20 antigen, which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymphoma, is an attractive target for the therapy of B-lymphoid malignancies. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic that now has entered phase II clinical trials. In this study, we prepared an engineered fusion protein, scFv-LDP, consisting of an anti-CD20 scFv fragment and the apoprotein LDP of LDM using DNA recombination. After purification and refolding, scFv-LDP was found to bind specifically to CD20-positive lymphoma cells using ELISA and indirect immunofluorescent cytochemical staining assays. The energized fusion protein scFv-LDP-AE was obtained using molecular reconstitution of the active chromophore AE of LDM and scFv-LDP. MTT assay revealed potent cytotoxicity of scFv-LDP-AE to CD20-positive Raji and Daudi cells, with IC(50) values of 1.21×10(-11) and 6.24×10(-11) mol L(-1), respectively. An in vivo subcutaneous xenograft model of CD20-positive B cell lymphoma in BALB/c (nu/nu) mice was also utilized. Drugs were given intravenously on day 14 and 21 after tumor transplantation. In terms of maximal tolerated doses, scFv-LDP-AE at 0.3 mg kg(-1) suppressed tumor growth by 79.3%, and LDM at 0.05 mg kg(-1) by 68.6% (P<0.05). Results suggested scFv-LDP-AE could be a potential candidate for tumor-targeting therapy. PMID:21416325

  7. N-Homocysteinylation Induces Different Structural and Functional Consequences on Acidic and Basic Proteins

    PubMed Central

    Sharma, Gurumayum Suraj; Kumar, Tarun; Singh, Laishram Rajendrakumar

    2014-01-01

    One of the proposed mechanisms of homocysteine toxicity in human is the modification of proteins by the metabolite of Hcy, homocysteine thilolactone (HTL). Incubation of proteins with HTL has earlier been shown to form covalent adducts with ε-amino group of lysine residues of protein (called N-homocysteinylation). It has been believed that protein N-homocysteinylation is the pathological hallmark of cardiovascular and neurodegenerative disorders as homocysteinylation induces structural and functional alterations in proteins. In the present study, reactivity of HTL towards proteins with different physico-chemical properties and hence their structural and functional alterations were studied using different spectroscopic approaches. We found that N-homocysteinylation has opposite consequences on acidic and basic proteins suggesting that pI of the protein determines the extent of homocysteinylation, and the structural and functional consequences due to homocysteinylation. Mechanistically, pI of protein determines the extent of N-homocysteinylation and the associated structural and functional alterations. The study suggests the role of HTL primarily targeting acidic proteins in eliciting its toxicity that could yield mechanistic insights for the associated neurodegeneration. PMID:25551634

  8. Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-(1')anilinonaphthalene binding to intestinal fatty acid binding protein.

    PubMed Central

    Kirk, W R; Kurian, E; Prendergast, F G

    1996-01-01

    1-Sulfonato-8-(1')anilinonaphthalene (1,8-ANS) was employed as a fluorescent probe of the fatty acid binding site of recombinant rat intestinal fatty acid binding protein (1-FABP). The enhancement of fluorescence upon binding allowed direct determination of binding affinity by fluorescence titration experiments, and measurement of the effects on that affinity of temperature, pH, and ionic strength. Solvent isotope effects were also determined. These data were compared to results from isothermal titration calorimetry. We obtained values for the enthalpy and entropy of this interaction at a variety of temperatures, and hence determined the change in heat capacity of the system consequent upon binding. The ANS-1-FABP is enthalpically driven; above approximately 14 degrees C it is entropically opposed, but below this temperature the entropy makes a positive contribution to the binding. The changes we observe in both enthalpy and entropy of binding with temperature can be derived from the change in heat capacity upon binding by integration, which demonstrates the internal consistency of our results. Bound ANS is displaced by fatty acids and can itself displace fatty acids bound to I-FABP. The binding site for ANS appears to be inside the solvent-containing cavity observed in the x-ray crystal structure, the same cavity occupied by fatty acid. From the fluorescence spectrum and from an inversion of the Debye-Hueckel formula for the activity coefficients as a function of added salt, we inferred that this cavity is fairly polar in character, which is in keeping with inferences drawn from the x-ray structure. The binding affinity of ANS is considered to be a consequence of both electrostatic and conditional hydrophobic effects. We speculate that the observed change in heat capacity is produced mainly by the displacement of strongly hydrogen-bonded waters from the protein cavity. PMID:8770188

  9. Effects of postruminal protein on fatty acid digestibility in dairy cows.

    PubMed

    Goodling, L E; Grummer, R R

    1998-06-01

    Eight ruminally cannulated Holstein cows (four multiparous and four primiparous) were used in a replicated 4 x 4 Latin square with an extra period to determine the effects of postruminal protein on fatty acid digestibility. Samples were collected during the last 4 d of each 14-d period. Total mixed rations were composed of 41% alfalfa haylage, 42% corn silage, 12% concentrate based on corn, and 5% tallow. Cows were fed at 90% of ad libitum intake. Treatments were abomasal infusion of guar gum or guar gum plus urea, corn gluten meal, or blood meal in 12 L of water. The basal ration contained 12% crude protein (CP), and infusion of N sources increased CP to approximately 14%. Dry matter intake was similar for cows on all treatments. Milk, 3.5% fat-corrected milk, milk protein, and milk fat yields increased for cows receiving postruminal CP. Postruminal CP did not affect milk fat and protein percentages. Dry matter, organic matter, and CP digestibilities were greater in cows receiving postruminal CP. Total fatty acid and total C18 fatty acid digestibilities were not affected by treatment. Total C16 fatty acid and C18:0 fatty acid digestibilities were greater for cows receiving nonprotein N than for those receiving true protein. PMID:9684169

  10. Enamel erosion in dietary acids: inhibition by food proteins in vitro.

    PubMed

    Hemingway, C A; White, A J; Shellis, R P; Addy, M; Parker, D M; Barbour, M E

    2010-01-01

    The aim of this study was to investigate the effects of two common food proteins on human enamel erosion in vitro. Erosion was measured by non-contact profilometry in citric, malic and lactic acids at pH 2.8, 3.2 and 3.8 and five commercially available soft drinks, in the presence of a salivary pellicle. Whole milk casein or hen egg ovalbumin was added to the acid solutions and drinks at 0.2% w/v, and the effect on erosion was determined by comparison with the corresponding solution without protein. Casein significantly reduced erosion in all but two solutions. The effects of the individual subfractions of casein in citric acid at pH 3.2 were similar to that of whole casein. Ovalbumin reduced erosion in some solutions, but the magnitude of the reduction was less than that with casein. A greater proportional reduction in erosion was seen in citric acid than in malic or lactic acids. We postulate that the mechanism involves adsorption of proteins to the pellicle or the enamel surface, forming a protein film with enhanced erosion-inhibiting properties. The citrate ion may play an active stabilising role, since erosion reduction was less in the other acids. In conclusion, casein and, to a lesser extent, ovalbumin show promise as potential anti-erosive additives to drinks. PMID:20980757

  11. Uniformly sup 13 C-labeled algal protein used to determine amino acid essentiality in vivo

    SciTech Connect

    Berthold, H.K.; Hachey, D.L.; Reeds, P.J.; Klein, P.D. ); Thomas, O.P. ); Hoeksema, S. )

    1991-09-15

    The edible alga Spirulina platensis was uniformly labeled with {sup 13}C by growth in an atmosphere of pure {sup 13}CO{sub 2}. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly {sup 13}C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo.

  12. Search for conserved amino acid residues of the [Formula: see text]-crystallin proteins of vertebrates.

    PubMed

    Shiliaev, Nikita G; Selivanova, Olga M; Galzitskaya, Oxana V

    2016-04-01

    [Formula: see text]-crystallin is the major eye lens protein and a member of the small heat-shock protein (sHsp) family. [Formula: see text]-crystallins have been shown to support lens clarity by preventing the aggregation of lens proteins. We performed the bioinformatics analysis of [Formula: see text]-crystallin sequences from vertebrates to find conserved amino acid residues as the three-dimensional (3D) structure of [Formula: see text]-crystallin is not identified yet. We are the first who demonstrated that the N-terminal region is conservative along with the central domain for vertebrate organisms. We have found that there is correlation between the conserved and structured regions. Moreover, amyloidogenic regions also correspond to the structured regions. We analyzed the amino acid composition of [Formula: see text]-crystallin A and B chains. Analyzing the occurrence of each individual amino acid residue, we have found that such amino acid residues as leucine, serine, lysine, proline, phenylalanine, histidine, isoleucine, glutamic acid, and valine change their content simultaneously in A and B chains in different classes of vertebrates. Aromatic amino acids occur more often in [Formula: see text]-crystallins from vertebrates than on the average in proteins among 17 animal proteomes. We obtained that the identity between A and B chains in the mammalian group is 0.35, which is lower than the published 0.60. PMID:26972563

  13. Uniformly 13C-labeled algal protein used to determine amino acid essentiality in vivo.

    PubMed Central

    Berthold, H K; Hachey, D L; Reeds, P J; Thomas, O P; Hoeksema, S; Klein, P D

    1991-01-01

    The edible alga Spirulina platensis was uniformly labeled with 13C by growth in an atmosphere of pure 13CO2. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly 13C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo. Images PMID:11607211

  14. Controlled protein release from polyethyleneimine-coated poly(L-lactic acid)/pluronic blend matrices.

    PubMed

    Park, T G; Cohen, S; Langer, R

    1992-01-01

    Protein release from degradable polymer matrices, composed of poly(L-lactic acid) and its blends with Pluronic surfactant, was investigated with and without the aqueous coating of an adsorptive water-soluble polymer, polyethyleneimine (PEI). PEI is a highly branched cationic polymer containing primary, secondary, and tertiary amino groups in its backbone. The treatment of PEI for PLA/Pluronic blend films exhibited a remarkable decrease in the "burst" release of protein at an initial stage and a significant extension in the protein release period. Protein release profiles could be controlled by varying PEI treatment time and its concentration. Our results suggest that PEI diffuses into the polymer matrices and crosslinks protein molecules by ionic interactions. Such a PEI-protein network near the surface region of matrix may act as a diffusional barrier for further release of protein molecules. PMID:1589407

  15. Dendronylation: Residue-specific chemoselective attachment of oligoglycerol dendrimers on proteins with noncanonical amino acids.

    PubMed

    Ma, Ying; Thota, Bala N S; Haag, Rainer; Budisa, Nediljko

    2015-11-15

    Polyglycerol dendrimers as an important class of polymeric materials especially attractive for covalent attachment to therapeutic proteins as a useful alternative to traditional PEGylation procedures. Herein, we combine in vivo noncanonical amino acid (ncAA) incorporation and chemoselective conjugation in vitro to produce novel hybrid protein-dendrimer conjugates with the defined architectures. We incorporated Azidohomoalanine (Aha) as methionine substitute in vivo into various protein scaffolds to allow non-invasive dendrimer conjugations (dendronylation). Our approach makes recombinant proteins accessible for the design of multivalent dendrimer conjugates since it enables the preparation of many sequences with various positions for regioselective dendronylation. PMID:26483199

  16. Stabilisation of proteins via mixtures of amino acids during spray drying.

    PubMed

    Ajmera, Ankur; Scherließ, Regina

    2014-03-10

    Biologicals are often formulated as solids in an effort to preserve stability which generally requires stabilising excipients for proper drying. The purpose of this study was to screen amino acids and their combinations for their stabilising effect on proteins during spray drying. Catalase, as model protein, was spray dried in 1+1 or 1+2 ratios with amino acids. Some amino acids namely arginine, glycine and histidine showed good retention of catalase functionality after spray drying and subsequent storage stress. A 1+1 combination of arginine and glycine in a 1+2 ratio with catalase resulted in a tremendously good stabilising effect. Storage at high temperature/humidity also showed beneficial effects of this combination. To evaluate whether this was a general principle, these findings were transferred to an antigenic protein of comparable size and supramolecular structure (haemagglutinin) as well as to a smaller enzyme (lysozyme). Upon spray drying with the combination of amino acids it could be shown that both proteins remain more stable especially after storage compared to the unprotected protein. The combination of arginine and glycine is tailored to the needs of protein stabilisation during spray drying and may hence be utilised in dry powder formulation of biomolecules with superior stability characteristics. PMID:24412336

  17. The effect of whey acidic protein fractions on bone loss in the ovariectomised rat.

    PubMed

    Kruger, Marlena C; Plimmer, Gabrielle G; Schollum, Linda M; Haggarty, Neill; Ram, Satyendra; Palmano, Kate

    2005-08-01

    Bovine milk has been shown to contain bioactive components with bone-protective properties. Earlier studies on bovine milk whey protein showed that it suppressed bone resorption in the female ovariectomised rat. A new osteotropic component was subsequently identified in the whey basic protein fraction, but bone bioactivity may also be associated with other whey fractions. In the present study, we investigated whether acidic protein fractions isolated from bovine milk whey could prevent bone loss in mature ovariectomised female rats. Six-month-old female rats were ovariectomised (OVX) or left intact (sham). The OVX rats were randomised into four groups. One group remained the control (OVX), whereas three groups were fed various whey acidic protein fractions from milk whey as 3 g/kg diet for 4 months. Outcomes were bone mineral density, bone biomechanics and markers of bone turnover. Bone mineral density of the femurs indicated that one of the whey AF over time caused a recovery of bone lost from OVX. Plasma C-telopeptide of type I collagen decreased significantly in all groups except OVX control over time, indicating an anti-resorptive effect of whey acidic protein. Biomechanical data showed that the AF may affect bone architecture as elasticity was increased by one of the whey AF. The femurs of AF-supplemented rats all showed an increase in organic matter. This is the first report of an acidic whey protein fraction isolated from milk whey that may support the recovery of bone loss in vivo. PMID:16115359

  18. Four Trypanosoma brucei fatty acyl-CoA synthetases: fatty acid specificity of the recombinant proteins.

    PubMed Central

    Jiang, D W; Englund, P T

    2001-01-01

    As part of our investigation of fatty acid metabolism in Trypanosoma brucei, we have expressed four acyl-CoA synthetase (TbACS) genes in Esherichia coli. The recombinant proteins, with His-tags on their C-termini, were purified to near homogeneity using nickel-chelate affinity chromatography. Although these enzymes are highly homologous, they have distinct specificities for fatty acid chain length. TbACS1 prefers saturated fatty acids in the range C(11:0) to C(14:0) and TbACS2 prefers shorter fatty acids, mainly C(10:0). TbACS3 and 4, which have 95% sequence identity, have similar specificities, favouring fatty acids between C(14:0) and C(17:0). In addition, TbACS1, 3 and 4 function well with a variety of unsaturated fatty acids. PMID:11535136

  19. Comparative nutrition and metabolism: Explication of open questions with emphasis on protein and amino acids

    PubMed Central

    Baker, David H.

    2005-01-01

    The 20th century saw numerous important discoveries in the nutritional sciences. Nonetheless, many unresolved questions still remain. Fifteen questions dealing with amino acid nutrition and metabolism are posed in this review. The first six deal with the functionality of sulfur amino acids (methionine and cysteine) and related compounds. Other unresolved problems that are discussed include priorities of use for amino acids having multiple functions; interactions among lysine, niacin and tryptophan; amino acid contributions to requirements from gut biosynthesis; the potential for gluconeogenesis to divert amino acids away from protein synthesis; the unique nutritional and metabolic idiosyncrasies of feline species, with emphasis on arginine; controversies surrounding human amino acid requirements; and the potential for maternal diet to influence sex ratio of offspring. PMID:16326801

  20. Effect of increased protein intake on renal acid load and renal hemodynamic responses.

    PubMed

    Teunissen-Beekman, Karianna F M; Dopheide, Janneke; Geleijnse, Johanna M; Bakker, Stephan J L; Brink, Elizabeth J; de Leeuw, Peter W; van Baak, Marleen A

    2016-03-01

    Increased protein intake versus maltodextrin intake for 4 weeks lowers blood pressure. Concerns exist that high-protein diets reduce renal function. Effects of acute and 4-week protein intake versus maltodextrin intake on renal acid load, glomerular filtration rate and related parameters were compared in this study. Seventy-nine overweight individuals with untreated elevated blood pressure and normal kidney function were randomized to consume a mix of protein isolates (60 g/day) or maltodextrin (60 g/day) for 4 weeks in energy balance. Twenty-four-hour urinary potential renal acid load (uPRAL) was compared between groups. A subgroup (maltodextrin N = 27, protein mix N = 25) participated in extra test days investigating fasting levels and postprandial effects of meals supplemented with a moderate protein- or maltodextrin-load on glomerular filtration rate, effective renal plasma flow, plasma renin, aldosterone, pH, and bicarbonate. uPRAL was significantly higher in the protein group after 4 weeks (P ≤ 0.001). Postprandial filtration fraction decreased further after the protein-supplemented breakfast than after the maltodextrin-supplemented breakfast after 4 weeks of supplementation (P ≤ 0.001). Fasting and postprandial levels of glomerular filtration rate, effective renal plasma flow, renin, aldosterone, angiotensin-converting enzyme, pH and bicarbonate did not differ between groups. In conclusion, 4 weeks on an increased protein diet (25% of energy intake) increased renal acid load, but did not affect renal function. Postprandial changes, except for filtration fraction, also did not differ between groups. These data suggest that a moderate increase in protein intake by consumption of a protein mix for 4 weeks causes no (undesirable) effects on kidney function in overweight and obese individuals with normal kidney function. PMID:26997623

  1. Neural network consistent empirical physical formula construction for density functional theory based nonlinear vibrational absorbance and intensity of 6-choloronicotinic acid molecule

    NASA Astrophysics Data System (ADS)

    Yildiz, Nihat; Karabacak, Mehmet; Kurt, Mustafa; Akkoyun, Serkan

    2012-05-01

    Being directly related to the electric charge distributions in a molecule, the vibrational spectra intensities are both experimentally and theoretically important physical quantities. However, these intensities are inherently highly nonlinear and of complex pattern. Therefore, in particular for unknown detailed spatial molecular structures, it is difficult to make ab initio intensity calculations to compare with new experimental data. In this respect, we very recently initiated entirely novel layered feedforward neural network (LFNN) approach to construct empirical physical formulas (EPFs) for density functional theory (DFT) vibrational spectra of some molecules. In this paper, as a new and far improved contribution to our novel molecular vibrational spectra LFNN-EPF approach, we constructed LFFN-EPFs for absorbances and intensities of 6-choloronicotinic acid (6-CNA) molecule. The 6-CNA data, borrowed from our previous study, was entirely different and much larger than the vibrational intensity data of our formerly used LFNN-EPF molecules. In line with our another previous work which theoretically proved the LFNN relevance to EPFs, although the 6-CNA DFT absorbance and intensity were inherently highly nonlinear and sharply fluctuating in character, still the optimally constructed train set LFFN-EPFs very successfully fitted the absorbances and intensities. Moreover, test set (i.e. yet-to-be measured experimental data) LFNN-EPFs consistently and successfully predicted the absorbance and intensity data. This simply means that the physical law embedded in the 6-CNA vibrational data was successfully extracted by the LFNN-EPFs. In conclusion, these vibrational LFNN-EPFs are of explicit form. Therefore, by various suitable operations of mathematical analysis, they can be used to estimate the electronic charge distributions of the unknown molecule of the significant complexity. Additionally, these estimations can be combined with those of theoretical DFT atomic polar

  2. Protein Analysis of Sapienic Acid-Treated Porphyromonas gingivalis Suggests Differential Regulation of Multiple Metabolic Pathways

    PubMed Central

    Dawson, Deborah V.; Blanchette, Derek R.; Drake, David R.; Wertz, Philip W.; Brogden, Kim A.

    2015-01-01

    ABSTRACT Lipids endogenous to skin and mucosal surfaces exhibit potent antimicrobial activity against Porphyromonas gingivalis, an important colonizer of the oral cavity implicated in periodontitis. Our previous work demonstrated the antimicrobial activity of the fatty acid sapienic acid (C16:1Δ6) against P. gingivalis and found that sapienic acid treatment alters both protein and lipid composition from those in controls. In this study, we further examined whole-cell protein differences between sapienic acid-treated bacteria and untreated controls, and we utilized open-source functional association and annotation programs to explore potential mechanisms for the antimicrobial activity of sapienic acid. Our analyses indicated that sapienic acid treatment induces a unique stress response in P. gingivalis resulting in differential expression of proteins involved in a variety of metabolic pathways. This network of differentially regulated proteins was enriched in protein-protein interactions (P = 2.98 × 10−8), including six KEGG pathways (P value ranges, 2.30 × 10−5 to 0.05) and four Gene Ontology (GO) molecular functions (P value ranges, 0.02 to 0.04), with multiple suggestive enriched relationships in KEGG pathways and GO molecular functions. Upregulated metabolic pathways suggest increases in energy production, lipid metabolism, iron acquisition and processing, and respiration. Combined with a suggested preferential metabolism of serine, which is necessary for fatty acid biosynthesis, these data support our previous findings that the site of sapienic acid antimicrobial activity is likely at the bacterial membrane. IMPORTANCE P. gingivalis is an important opportunistic pathogen implicated in periodontitis. Affecting nearly 50% of the population, periodontitis is treatable, but the resulting damage is irreversible and eventually progresses to tooth loss. There is a great need for natural products that can be used to treat and/or prevent the overgrowth of

  3. Analysis of soybean root proteins affected by gibberellic acid treatment under flooding stress.

    PubMed

    Oh, Myeong Won; Nanjo, Yohei; Komatsu, Setsuko

    2014-01-01

    Flooding is a serious abiotic stress for soybean because it restricts growth and reduces grain yields. To investigate the effect of gibberellic acid (GA) on soybean under flooding stress, root proteins were analyzed using a gel-free proteomic technique. Proteins were extracted from the roots of 4-days-old soybean seedlings exposed to flooding stress in the presence and absence of exogenous GA3 for 2 days. A total of 307, 324, and 250 proteins were identified from untreated, and flooding-treated soybean seedlings without or with GA3, respectively. Secondary metabolism- and cell-related proteins, and proteins involved in protein degradation/synthesis were decreased by flooding stress; however, the levels of these proteins were restored by GA3 supplementation under flooding. Fermentation- and cell wall-related proteins were not affected by GA3 supplementation. Furthermore, putative GA-responsive proteins, which were identified by the presence of a GA-responsive element in the promoter region, were less abundant by flooding stress; however, these proteins were more abundant by GA3 supplementation under flooding. Taken together, these results suggest that GA3 affects the abundance of proteins involved in secondary metabolism, cell cycle, and protein degradation/synthesis in soybeans under flooding stress. PMID:24702262

  4. Swfoldrate: predicting protein folding rates from amino acid sequence with sliding window method.

    PubMed

    Cheng, Xiang; Xiao, Xuan; Wu, Zhi-cheng; Wang, Pu; Lin, Wei-zhong

    2013-01-01

    Protein folding is the process by which a protein processes from its denatured state to its specific biologically active conformation. Understanding the relationship between sequences and the folding rates of proteins remains an important challenge. Most previous methods of predicting protein folding rate require the tertiary structure of a protein as an input. In this study, the long-range and short-range contact in protein were used to derive extended version of the pseudo amino acid composition based on sliding window method. This method is capable of predicting the protein folding rates just from the amino acid sequence without the aid of any structural class information. We systematically studied the contributions of individual features to folding rate prediction. The optimal feature selection procedures are adopted by means of combining the forward feature selection and sequential backward selection method. Using the jackknife cross validation test, the method was demonstrated on the large dataset. The predictor was achieved on the basis of multitudinous physicochemical features and statistical features from protein using nonlinear support vector machine (SVM) regression model, the method obtained an excellent agreement between predicted and experimentally observed folding rates of proteins. The correlation coefficient is 0.9313 and the standard error is 2.2692. The prediction server is freely available at http://www.jci-bioinfo.cn/swfrate/input.jsp. PMID:22933332

  5. Amyloid protein precursor stimulates excitatory amino acid transport. Implications for roles in neuroprotection and pathogenesis.

    PubMed

    Masliah, E; Raber, J; Alford, M; Mallory, M; Mattson, M P; Yang, D; Wong, D; Mucke, L

    1998-05-15

    Excitatory neurotransmitters such as glutamate are required for the normal functioning of the central nervous system but can trigger excitotoxic neuronal injury if allowed to accumulate to abnormally high levels. Their extracellular levels are controlled primarily by transmitter uptake into astrocytes. Here, we demonstrate that the amyloid protein precursor may participate in the regulation of this important process. The amyloid protein precursor has been well conserved through evolution, and a number of studies indicate that it may function as an endogenous excitoprotectant. However, the mechanisms underlying this neuroprotective capacity remain largely unknown. At moderate levels of expression, human amyloid protein precursors increased glutamate/aspartate uptake in brains of transgenic mice, with the 751-amino acid isoform showing greater potency than the 695-amino acid isoform. Cerebral glutamate/aspartate transporter protein levels were higher in transgenic mice than in non-transgenic controls, whereas transporter mRNA levels were unchanged. Amyloid protein precursor-dependent stimulation of aspartate uptake by cultured primary astrocytes was associated with increases in protein kinase A and C activity and could be blocked by inhibitors of these kinases. The stimulation of astroglial excitatory amino acid transport by amyloid protein precursors could protect the brain against excitotoxicity and may play an important role in neurotransmission. PMID:9575214

  6. Rumen Degradability and Small Intestinal Digestibility of the Amino Acids in Four Protein Supplements

    PubMed Central

    Wang, Y.; Jin, L.; Wen, Q. N.; Kopparapu, N. K.; Liu, J.; Liu, X. L.; Zhang, Y. G.

    2016-01-01

    The supplementation of livestock feed with animal protein is a present cause for public concern, and plant protein shortages have become increasingly prominent in China. This conflict may be resolved by fully utilizing currently available sources of plant protein. We estimated the rumen degradability and the small intestinal digestibility of the amino acids (AA) in rapeseed meal (RSM), soybean meal (SBM), sunflower seed meal (SFM) and sesame meal (SSM) using the mobile nylon bag method to determine the absorbable AA content of these protein supplements as a guide towards dietary formulations for the dairy industry. Overall, this study aimed to utilize protein supplements effectively to guide dietary formulations to increase milk yield and save plant protein resources. To this end, we studied four cows with a permanent rumen fistula and duodenal T-shape fistula in a 4×4 Latin square experimental design. The results showed that the total small intestine absorbable amino acids and small intestine absorbable essential amino acids were higher in the SBM (26.34% and 13.11% dry matter [DM], respectively) than in the SFM (13.97% and 6.89% DM, respectively). The small intestine absorbable Lys contents of the SFM, SSM, RSM and SBM were 0.86%, 0.88%, 1.43%, and 2.12% (DM basis), respectively, and the absorbable Met contents of these meals were 0.28%, 1.03%, 0.52%, and 0.47% (DM basis), respectively. Among the examined food sources, the milk protein score of the SBM (0.181) was highest followed by those of the RSM (0.136), SSM (0.108) and SFM (0.106). The absorbable amino acid contents of the protein supplements accurately reflected protein availability, which is an important indicator of the balance of feed formulation. Therefore, a database detailing the absorbable AA should be established. PMID:26732449

  7. Rumen Degradability and Small Intestinal Digestibility of the Amino Acids in Four Protein Supplements.

    PubMed

    Wang, Y; Jin, L; Wen, Q N; Kopparapu, N K; Liu, J; Liu, X L; Zhang, Y G

    2016-02-01

    The supplementation of livestock feed with animal protein is a present cause for public concern, and plant protein shortages have become increasingly prominent in China. This conflict may be resolved by fully utilizing currently available sources of plant protein. We estimated the rumen degradability and the small intestinal digestibility of the amino acids (AA) in rapeseed meal (RSM), soybean meal (SBM), sunflower seed meal (SFM) and sesame meal (SSM) using the mobile nylon bag method to determine the absorbable AA content of these protein supplements as a guide towards dietary formulations for the dairy industry. Overall, this study aimed to utilize protein supplements effectively to guide dietary formulations to increase milk yield and save plant protein resources. To this end, we studied four cows with a permanent rumen fistula and duodenal T-shape fistula in a 4×4 Latin square experimental design. The results showed that the total small intestine absorbable amino acids and small intestine absorbable essential amino acids were higher in the SBM (26.34% and 13.11% dry matter [DM], respectively) than in the SFM (13.97% and 6.89% DM, respectively). The small intestine absorbable Lys contents of the SFM, SSM, RSM and SBM were 0.86%, 0.88%, 1.43%, and 2.12% (DM basis), respectively, and the absorbable Met contents of these meals were 0.28%, 1.03%, 0.52%, and 0.47% (DM basis), respectively. Among the examined food sources, the milk protein score of the SBM (0.181) was highest followed by those of the RSM (0.136), SSM (0.108) and SFM (0.106). The absorbable amino acid contents of the protein supplements accurately reflected protein availability, which is an important indicator of the balance of feed formulation. Therefore, a database detailing the absorbable AA should be established. PMID:26732449

  8. Multiplexed microfluidic blotting of proteins and nucleic acids by parallel, serpentine microchannels.

    PubMed

    He, Sha; Zhang, Yi; Wang, Pei; Xu, Xingzhi; Zhu, Kui; Pan, Wenying; Liu, Wenwen; Cai, Kaiyong; Sun, Jiashu; Zhang, Wei; Jiang, Xingyu

    2015-01-01

    This work develops a high-throughput, high-efficiency and straightforward microfluidic blotting method for analyzing proteins and nucleic acids. Sample solutions containing antibodies (for protein detection) or hybridization probes (for nucleic acid detection) are introduced into the parallel, serpentine microchannels to specifically recognize the immobilized targets on the substrate, achieving the identification of multiple targets in multiple samples simultaneously. The loading control, molecular weight markers, and antigen/antibody titration are designed and integrated into the microfluidic chip, thus allowing for the quantification of proteins and nucleic acids. Importantly, we could easily distinguish the adjacent blotting bands inside parallel microchannels, which may be difficult to achieve in conventional blotting. The small dimensions of microfluidic channels also help to reduce the amount of probing molecules and to accelerate the biochemical reaction. Our microfluidic blotting could bypass the steps of blocking and washing, further reducing the operation time and complexity. PMID:25342223

  9. PROTEIN NUCLEIC ACID INTERACTIONS GRANT # DE-FG02-96ER62166 FINAL REPORT

    SciTech Connect

    Berman, Helen M.; Thornton, Janet

    2005-02-17

    The overall goal of this collaborative project is to develop methods for analyzing protein-nucleic acid interactions. Nucleic acid-binding proteins have a central role in all aspects of genetic activity within an organism, such as transcription, replication, and repair. Thus, it is extremely important to examine the nature of complexes that are formed between proteins and nucleic acids, as they form the basis of our understanding of how these processes take place. Over the past decade, the world has witnessed a great expansion in the determination of high-quality structures of nucleic acid-binding proteins. As a result, the number of such structures has seen a constant increase in the Protein Data Bank (PDB) (1) and the Nucleic Acid Database (NDB) (2). These structures, especially those of proteins in complex with DNA, have provided valuable insight into the stereochemical principles of binding, including how particular base sequences are recognized and how the nucleic acid structure is quite often modified on binding. In this project, we designed several approaches to characterize and classify the properties of both protein-DNA and protein-RNA complexes. In work done in the previous grant period, we developed methods to use experimental data to evaluate nucleic acid crystal structures in order to ensure that the structures utilized in future studies would be of high quality. The methodology was collated in the standalone software package SFCHECK (3) [A], and an applied survey of structures in the NDB produced very positive results. With this quality control mechanism in place, we then analyzed DNA-binding sites on proteins by studying the distortions observed in DNA structures bound to protein. From our observations, we found that DNA-binding proteins present a very different binding surface to those that bind other proteins and defined three modes of protein binding [B]. Following this survey, we classified DNA-binding proteins into eight different structural

  10. Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins. Purification and characterization of toxin A and toxin B.

    PubMed

    Guo, L; Fatig, R O; Orr, G L; Schafer, B W; Strickland, J A; Sukhapinda, K; Woodsworth, A T; Petell, J K

    1999-04-01

    Both the bacterium Photorhabdus luminescens alone and its symbiotic Photorhabdus-nematode complex are known to be highly pathogenic to insects. The nature of the insecticidal activity of Photorhabdus bacteria was investigated for its potential application as an insect control agent. It was found that in the fermentation broth of P. luminescens strain W-14, at least two proteins, toxin A and toxin B, independently contributed to the oral insecticidal activity against Southern corn rootworm. Purified toxin A and toxin B exhibited single bands on native polyacrylamide gel electrophoresis and two peptides of 208 and 63 kDa on SDS-polyacrylamide gel electrophoresis. The native molecular weight of both the toxin A and toxin B was determined to be approximately 860 kDa, suggesting that they are tetrameric. NH2-terminal amino acid sequencing and Western analysis using monospecific antibodies to each toxin demonstrated that the two toxins were distinct but homologous. The oral potency (LD50) of toxin A and toxin B against Southern corn rootworm larvae was determined to be similar to that observed with highly potent Bt toxins against lepidopteran pests. In addition, it was found that the two peptides present in toxin B could be processed in vitro from a 281-kDa protoxin by endogenous P. luminescens proteases. Proteolytic processing was shown to enhance insecticidal activity. PMID:10092674

  11. Analysis of protein-protein interactions in MCF-7 and MDA-MB-231 cell lines using phthalic acid chemical probes.

    PubMed

    Liang, Shih-Shin; Wang, Tsu-Nai; Tsai, Eing-Mei

    2014-01-01

    Phthalates are a class of plasticizers that have been characterized as endocrine disrupters, and are associated with genital diseases, cardiotoxicity, hepatotoxicity, and nephrotoxicity in the GeneOntology gene/protein database. In this study, we synthesized phthalic acid chemical probes and demonstrated differing protein-protein interactions between MCF-7 cells and MDA-MB-231 breast cancer cell lines. Phthalic acid chemical probes were synthesized using silicon dioxide particle carriers, which were modified using the silanized linker 3-aminopropyl triethoxyslane (APTES). Incubation with cell lysates from breast cancer cell lines revealed interactions between phthalic acid and cellular proteins in MCF-7 and MDA-MB-231 cells. Subsequent proteomics analyses indicated 22 phthalic acid-binding proteins in both cell types, including heat shock cognate 71-kDa protein, ATP synthase subunit beta, and heat shock protein HSP 90-beta. In addition, 21 MCF-7-specific and 32 MDA-MB-231 specific phthalic acid-binding proteins were identified, including related proteasome proteins, heat shock 70-kDa protein, and NADPH dehydrogenase and ribosomal correlated proteins, ras-related proteins, and members of the heat shock protein family, respectively. PMID:25402641

  12. Protein content and amino acids profile of pseudocereals.

    PubMed

    Mota, Carla; Santos, Mariana; Mauro, Raul; Samman, Norma; Matos, Ana Sofia; Torres, Duarte; Castanheira, Isabel

    2016-02-15

    Quinoa (Chenopodium quinoa), amaranth (Amaranthus caudatus) and buckwheat (Fagopyrum esculentum) represent the main protein source in several diets, although these pseudocereals are not currently present in the FCDB nutrient profile information. The aim of this work is to characterise the AA profile of these pseudocereals and compare them with rice. Total protein content revealed to vary from 16.3g/100g (quinoa Salta) to 13.1g/100g (buckwheat) and lower values were found in rice samples (6.7g/100g). For pseudocereals the most abundant essential AA was leucine. Quinoa-Salta evidences the highest leucine content (1013mg/100g) and the minor methionine content (199mg/100g). Buckwheat was the cereal with the highest phenylalanine content (862mg/100g). Rice (Oryza sativa) presents the lowest content for all AA. Results showed pseudocereals as the best source of AA. EuroFIR guidelines where strictly followed and proved to be a crucial tool to guarantee data interchangeability and comparability. PMID:26433287

  13. d-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis

    PubMed Central

    Leiman, Sara A.; May, Janine M.; Lebar, Matthew D.; Kahne, Daniel; Kolter, Roberto

    2013-01-01

    The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis. PMID:24097941

  14. D-amino acids indirectly inhibit biofilm formation in Bacillus subtilis by interfering with protein synthesis.

    PubMed

    Leiman, Sara A; May, Janine M; Lebar, Matthew D; Kahne, Daniel; Kolter, Roberto; Losick, Richard

    2013-12-01

    The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of D-leucine, D-methionine, D-tryptophan, and D-tyrosine and was reported to inhibit biofilm formation via the incorporation of these D-amino acids into the cell wall. Here, we show that L-amino acids were able to specifically reverse the inhibitory effects of their cognate D-amino acids. We also show that D-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding D-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of D-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of D-amino acids without losing the ability to incorporate at least one noncanonical D-amino acid, D-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of D-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis. PMID:24097941

  15. Fatty acid binding protein 7 and n-3 poly unsaturated fatty acid supply in early rat brain development.

    PubMed

    Maximin, Elise; Langelier, Bénédicte; Aïoun, Josiane; Al-Gubory, Kaïs H; Bordat, Christian; Lavialle, Monique; Heberden, Christine

    2016-03-01

    Fatty acid binding protein 7 (FABP7), abundant in the embryonic brain, binds with the highest affinity to docosahexaenoic acid (DHA) and is expressed in the early stages of embryogenesis. Here, we have examined the consequences of the exposure to different DHA levels and of the in utero depletion of FABP7 on early rat brain development. Neurodevelopment was evaluated through the contents of two proteins, connexin 43 (Cx43) and cyclin-dependent kinase 5 (CDK5), both involved in neuroblast proliferation, differentiation, and migration. The dams were fed with diets presenting different DHA contents, from deficiency to supplementation. DHA brain embryos contents already differed at embryonic day 11.5 and the differences kept increasing with time. Cx43 and CDK5 contents were positively associated with the brain DHA levels. When FABP7 was depleted in vivo by injections of siRNA in the telencephalon, the enhancement of the contents of both proteins was lost in supplemented animals, but FABP7 depletion did not modify phospholipid compositions regardless of the diets. Thus, FABP7 is a necessary mediator of the effect of DHA on these proteins synthesis, but its role in DHA uptake is not critical, although FABP7 is localized in phospholipid-rich areas. Our study shows that high contents of DHA associated with FABP7 are necessary to promote early brain development, which prompted us to recommend DHA supplementation early in pregnancy. PMID:26037116

  16. Ruminal protein metabolism and intestinal amino acid utilization as affected by dietary protein and carbohydrate sources in sheep.

    PubMed

    Hussein, H S; Jordan, R M; Stern, M D

    1991-05-01

    Eight wether lambs fitted with ruminal, duodenal, and ileal cannulas were used in a replicated 4 x 4 Latin square design to study the effects of carbohydrate and protein sources on ruminal protein metabolism and carbohydrate fermentation and intestinal amino acid (AA) absorption. Treatments were arranged as a 2 x 2 factorial. Carbohydrate sources were corn and barley; protein sources were soybean meal (SBM) and fish meal (FM). Diets contained 15.5% CP, of which 40% was supplied by SBM or FM. Corn or barley provided 39% of dietary DM that contained equal amounts of grass hay and wheat straw. Fish meal diets produced a lower (P less than .05) ruminal NH3 concentration and resulted in less CP degradation and bacterial protein flow to the duodenum than did SBM diets. Replacing SBM with FM increased (P less than .05) ruminal digestion of all fiber fractions. In addition, cellulose and hemicellulose digestibilities in the rumen tended to increase (P greater than .05) when barley replaced corn in the FM diets. Carbohydrate x protein interactions (P less than .05) were observed for OM digestion in the rumen and AA absorption in the small intestine (percentage of AA entering); these interactions were highest for the barley-FM diet. These results suggest that feeding FM with barley, which is high in both degradable carbohydrate and protein, might benefit ruminants more than feeding FM with corn, which is high in degradable carbohydrate but relatively low in degradable protein. PMID:1648551

  17. Crystal structure of phenylacetic acid degradation protein PaaG from Thermus thermophilus HB8.

    PubMed

    Kichise, Tomoyasu; Hisano, Tamao; Takeda, Kazuki; Miki, Kunio

    2009-09-01

    Microbial degradation of phenylacetic acid proceeds via the hybrid pathway that includes formation of a coenzyme A thioester, ring hydroxylation, non-oxygenolytic ring opening, and beta-oxidation-like reactions. A phenylacetic acid degradation protein PaaG is a member of the crotonase superfamily, and is a candidate non-oxygenolytic ring-opening enzyme. The crystal structure of PaaG from Thermus thermophilus HB8 was determined at a resolution of 1.85 A. PaaG consists of three identical subunits related by local three-fold symmetry. The monomer is comprised of a spiral and a helical domain with a fold characteristic of the crotonase superfamily. A putative active site residue, Asp136, is situated in an active site cavity and surrounded by several hydrophobic and hydrophilic residues. The active site cavity is sufficiently large to accommodate a ring substrate. Two conformations are observed for helix H2 located adjacent to the active site. Helix H2 is kinked at Asn81 in two subunits, whereas it is kinked at Leu77 in the other subunit, and the side chain of Tyr80 is closer to Asp136. This indicates that catalytic reaction of PaaG may proceed with large conformational changes at the active site. Asp136 is the only conserved polar residue in the active site. It is located at the same position as those of 4-chlorobenzoyl-CoA dehalogenase and peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase, indicating that PaaG may undergo isomerization or a ring-opening reaction via a Delta(3),Delta(2)-enoyl-CoA isomerase-like mechanism. PMID:19452559

  18. Radiolytic Modification of Sulfur Containing Acidic Amino Residues in Model Peptides: Fundamental Studies for Protein Footprinting

    SciTech Connect

    Xu,G.; Chance, M.

    2005-01-01

    Protein footprinting based on hydroxyl radical-mediated modification and quantitative mass spectroscopic analysis is a proven technique for examining protein structure, protein-ligand interactions, and structural allostery upon protein complex formation. The reactive and solvent-accessible amino acid side chains function as structural probes; however, correct structural analysis depends on the identification and quantification of all the relevant oxidative modifications within the protein sequence. Sulfur-containing amino acids are oxidized readily and the mechanisms of oxidation are particularly complex, although they have been extensively investigated by EPR and other spectroscopic methods. Here we have undertaken a detailed mass spectrometry study (using electrospray ionization mass spectrometry and tandem mass spectrometry) of model peptides containing cysteine (Cys-SH), cystine (disulfide bonded Cys), and methionine after oxidation using {gamma}-rays or synchrotron X-rays and have compared these results to those expected from oxidation mechanisms proposed in the literature. Radiolysis of cysteine leads to cysteine sulfonic acid (+48 Da mass shift) and cystine as the major products; other minor products including cysteine sulfinic acid (+32 Da mass shift) and serine (-16 Da mass shift) are observed. Radiolysis of cystine results in the oxidative opening of the disulfide bond and generation of cysteine sulfonic acid and sulfinic acid; however, the rate of oxidation is significantly less than that for cysteine. Radiolysis of methionine gives rise primarily to methionine sulfoxide (+16 Da mass shift); this can be further oxidized to methionine sulfone (+32 Da mass shift) or another product with a -32 Da mass shift likely due to aldehyde formation at the {gamma}-carbon. Due to the high reactivity of sulfur-containing amino acids, the extent of oxidation is easily influenced by secondary oxidation events or the presence of redox reagents used in standard proteolytic

  19. Thermodynamic principles for the engineering of pH-driven conformational switches and acid insensitive proteins

    PubMed Central

    Bell-Upp, Peregrine; Robinson, Aaron C.; Whitten, Steven; Wheeler, Erika L.; Lin, Janine; Stites, Wesley E.; García-Moreno E, Bertrand

    2012-01-01

    The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions. Acid unfolding is a structural transition driven by preferential H+ binding to the acid unfolded state (U) over the native (N) state of a protein. It is the result of carboxylic groups that titrate with more normal pKa values in the U state than in the N state. Acid unfolding profiles of proteins reflect a balance between electrostatic and non-electrostatic contributions to stability. Several strategies were used in attempts to turn SNase into an acid insensitive protein: (1) enhancing global stability of the protein with mutagenesis or with osmolytes, (2) use of high salt concentrations to screen Coulomb interactions, (3) stabilizing the N state through specific anion effects, (4) removing Asp or Glu residues that titrate with depressed pKa values in the N state, and (5) removing basic residues that might have strong repulsive interactions in the N state at low pH. The only effective way to engineer acid resistance in SNase is not through modulation of pKa values of Asp/Glu but by enhancing the global stability of the protein. Modulation of pH-driven conformational transitions by selective manipulation of the electrostatic component of the switch is an extremely difficult undertaking. PMID:21802194

  20. Ascorbic acid and protein glycation in vitro.

    PubMed

    Sadowska-Bartosz, Izabela; Bartosz, Grzegorz

    2015-10-01

    The aim of the study was to compare the effects of ascorbic acid (AA) in vitro in the absence and in the presence of cell-dependent recycling. In a cell-free system, AA enhanced glycoxidation of bovine serum albumin (BSA) by glucose and induced BSA glycation in the absence of sugars. On the other hand, AA did not affect erythrocyte hemolysis, glycation of hemoglobin and erythrocyte membranes, and inactivation of catalase, protected against inactivation of acetylcholinesterase of erythrocytes incubated with high glucose concentrations and enhanced the loss of glutathione. These results can be explained by assumption that AA acts as a proglycating agent in the absence of recycling while is an antiglycating agent when metabolic recycling occurs. PMID:26163454

  1. Role of Ingested Amino Acids and Protein in the Promotion of Resistance Exercise-Induced Muscle Protein Anabolism.

    PubMed

    Reidy, Paul T; Rasmussen, Blake B

    2016-02-01

    The goal of this critical review is to comprehensively assess the evidence for the molecular, physiologic, and phenotypic skeletal muscle responses to resistance exercise (RE) combined with the nutritional intervention of protein and/or amino acid (AA) ingestion in young adults. We gathered the literature regarding the translational response in human skeletal muscle to acute exposure to RE and protein/AA supplements and the literature describing the phenotypic skeletal muscle adaptation to RE and nutritional interventions. Supplementation of protein/AAs with RE exhibited clear protein dose-dependent effects on translational regulation (protein synthesis) through mammalian target of rapamycin complex 1 (mTORC1) signaling, which was most apparent through increases in p70 ribosomal protein S6 kinase 1 (S6K1) phosphorylation, compared with postexercise recovery in the fasted or carbohydrate-fed state. These acute findings were critically tested via long-term exposure to RE training (RET) and protein/AA supplementation, and it was determined that a diminishing protein/AA supplement effect occurs over a prolonged exposure stimulus after exercise training. Furthermore, we found that protein/AA supplements, combined with RET, produced a positive, albeit minor, effect on the promotion of lean mass growth (when assessed in >20 participants/treatment); a negligible effect on muscle mass; and a negligible to no additional effect on strength. A potential concern we discovered was that the majority of the exercise training studies were underpowered in their ability to discern effects of protein/AA supplementation. Regardless, even when using optimal methodology and large sample sizes, it is clear that the effect size for protein/AA supplementation is low and likely limited to a subset of individuals because the individual variability is high. With regard to nutritional intakes, total protein intake per day, rather than protein timing or quality, appears to be more of a factor on

  2. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  3. Assessing amino acid racemization variability in coral intra-crystalline protein for geochronological applications

    NASA Astrophysics Data System (ADS)

    Hendy, Erica J.; Tomiak, Peter J.; Collins, Matthew J.; Hellstrom, John; Tudhope, Alexander W.; Lough, Janice M.; Penkman, Kirsty E. H.

    2012-06-01

    Over 500 Free Amino Acid (FAA) and corresponding Total Hydrolysed Amino Acid (THAA) analyses were completed from eight independently-dated, multi-century coral cores of massive Porites sp. colonies. This dataset allows us to re-evaluate the application of amino acid racemization (AAR) for dating late Holocene coral material, 20 years after Goodfriend et al. (GCA56 (1992), 3847) first showed AAR had promise for developing chronologies in coral cores. This re-assessment incorporates recent method improvements, including measurement by RP-HPLC, new quality control approaches (e.g. sampling and sub-sampling protocols, statistically-based data screening criteria), and cleaning steps to isolate the intra-crystalline skeletal protein. We show that the removal of the extra-crystalline contaminants and matrix protein is the most critical step for reproducible results and recommend a protocol of bleaching samples in NaOCl for 48 h to maximise removal of open system proteins while minimising the induced racemization. We demonstrate that AAR follows closed system behaviour in the intra-crystalline fraction of the coral skeletal proteins. Our study is the first to assess the natural variability in intra-crystalline AAR between colonies, and we use coral cores taken from the Great Barrier Reef, Australia, and Jarvis Island in the equatorial Pacific to explore variability associated with different environmental conditions and thermal histories. Chronologies were developed from THAA Asx D/L, Ala D/L, Glx D/L and FAA Asx D/L for each core and least squares Monte Carlo modelling applied in order to quantify uncertainty of AAR age determinations and assess the level of dating resolution possible over the last 5 centuries. AAR within colonies follow consistent stratigraphic aging. However, there are systematic differences in rates between the colonies, which would preclude direct comparison from one colony to another for accurate age estimation. When AAR age models are developed from

  4. Assessing amino acid racemization variability in coral intra-crystalline protein for geochronological applications.

    PubMed

    Hendy, Erica J; Tomiak, Peter J; Collins, Matthew J; Hellstrom, John; Tudhope, Alexander W; Lough, Janice M; Penkman, Kirsty E H

    2012-06-01

    Over 500 Free Amino Acid (FAA) and corresponding Total Hydrolysed Amino Acid (THAA) analyses were completed from eight independently-dated, multi-century coral cores of massive Porites sp. colonies. This dataset allows us to re-evaluate the application of amino acid racemization (AAR) for dating late Holocene coral material, 20 years after Goodfriend et al. (GCA 56 (1992), 3847) first showed AAR had promise for developing chronologies in coral cores. This re-assessment incorporates recent method improvements, including measurement by RP-HPLC, new quality control approaches (e.g. sampling and sub-sampling protocols, statistically-based data screening criteria), and cleaning steps to isolate the intra-crystalline skeletal protein. We show that the removal of the extra-crystalline contaminants and matrix protein is the most critical step for reproducible results and recommend a protocol of bleaching samples in NaOCl for 48 h to maximise removal of open system proteins while minimising the induced racemization. We demonstrate that AAR follows closed system behaviour in the intra-crystalline fraction of the coral skeletal proteins. Our study is the first to assess the natural variability in intra-crystalline AAR between colonies, and we use coral cores taken from the Great Barrier Reef, Australia, and Jarvis Island in the equatorial Pacific to explore variability associated with different environmental conditions and thermal histories. Chronologies were developed from THAA Asx D/L, Ala D/L, Glx D/L and FAA Asx D/L for each core and least squares Monte Carlo modelling applied in order to quantify uncertainty of AAR age determinations and assess the level of dating resolution possible over the last 5 centuries. AAR within colonies follow consistent stratigraphic aging. However, there are systematic differences in rates between the colonies, which would preclude direct comparison from one colony to another for accurate age estimation. When AAR age models are developed

  5. Inhibition of Peptidoglycan, Ribonucleic Acid, and Protein Synthesis in Tolerant Strains of Streptococcus mutans

    PubMed Central

    Mychajlonka, Myron; McDowell, Thomas D.; Shockman, Gerald D.

    1980-01-01

    Exposure of exponentially growing cultures of Streptococcus mutans strains FA-1 and GS-5 to various concentrations of benzylpenicillin (Pen G) resulted in inhibition of turbidity increases at low concentrations (0.02 to 0.04 μg/ml). However, in contrast to some other streptococcal species, growth inhibition was not accompanied by cellular lysis or by a rapid loss of viability. In both strains, synthesis of insoluble cell wall peptidoglycan was very sensitive to Pen G inhibition and responded in a dose-dependent manner to concentrations of about 0.2 and 0.5 μg/ml for strains GS-5 and FA-1, respectively. Higher Pen G concentrations failed to inhibit further either growth or insoluble peptidoglycan assembly. Somewhat surprisingly, Pen G also inhibited both ribonucleic acid (RNA) and protein syntheses, each in a dose-dependent manner. Compared with inhibition of peptidoglycan synthesis, inhibition of RNA and protein syntheses by Pen G was less rapid and less extensive. Maximum amounts of radiolabeled Pen G were specifically bound to intact cells upon exposure to about 0.2 and 0.5 μg/ml of Pen G for strains GS-5 and FA-1, respectively, concentrations consistent with those that resulted in maximum or near-maximum inhibitions of the synthesis of cellular peptidoglycan, RNA, and protein. Five polypeptide bands that had a very high affinity for [14C]Pen G were detected in a crude cell envelope preparation of strain FA-1. After exposure of cultures of strain FA-1 to the effects of saturating concentrations of the drug for up to 3 h, addition of penicillinase was followed by recovery of growth after a lag. The length of the lag before regrowth depended on both Pen G concentration and time of exposure. On the basis of these and other observations, it is proposed that the secondary inhibitions of cellular RNA or protein synthesis, or both, are involved in the tolerance of these organisms to lysis and killing by Pen G and other inhibitors of insoluble peptidoglycan assembly

  6. Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination

    PubMed Central

    Field, Anjalie; Field, Jeffrey

    2010-01-01

    In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food caused the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Bradford and Ninhydrin assays, two common dye-based assays for protein, as well as by ammonia release, the most common assay used in the food industry. Neither compound was detected in the Ninhydrin and Bradford assays at concentrations of >100 μg/ml. The ammonia assay detected melamine but was inconclusive with respect to cyanuric acid. To develop an accurate test for food that would not detect either chemical as a protein, assays were run on cat food and reconstituted milk powder. The Bradford assay readily measured the protein content of each food, and importantly, the addition of melamine or cyanuric acid to reconstituted milk did not affect the readings. The protein concentrations obtained for reconstituted milk powder were as expected, but those for the cat food were 10 to 30-fold lower, due to its low solubility. We conclude that dye-binding assays can be employed to detect protein in food without interference from melamine and cyanuric acid, thus reducing the incentive to use them as additives. PMID:20228949

  7. Flavonoids inhibit iNOS production via mitogen activated proteins in lipoteichoic acid stimulated cardiomyoblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Ventura-Arroyo, Jairo Agustín; Arreguín-Cano, Juan Antonio; Ostoa-Pérez, María Fernanda

    2014-08-01

    Infective endocarditis is caused by oral commensal bacteria which are important etiologic agents in this disease and can induce release of nitric oxide (NO), promoting an inflammatory response in the endocardium. In this study, we investigated the properties of kaempherol, epigallocatechin, apigenin, and naringin in embryonic mouse heart cells (H9c2) treated with lipoteichoic acid (LTA) obtained from Streptococcus sanguinis. NO production was measured with the Griess method. Expression of inducible nitric oxide synthase (iNOS) was detected by reverse transcriptase polymerase chain reaction (RT-PCR). In addition, western blot assays and immunofluorescence staining were used to assess translocation of nuclear factor kappa beta (NF-κB), degradation of IκB, and activity of the mitogen activated protein (MAP) kinases extracellular signal-regulated kinase (ERK 1/2), p38, and c-Jun N-terminal kinase (JNK). And the effects of these flavonoids on cell viability were also assessed. Our results showed that flavonoids blocked activation of ERK, JNK, and p38 in cardiomyocytes treated with LTA. Moreover, the flavonoids showed no cytotoxic effects and blocked NF-κB translocation and IκB degradation and inhibited LTA-induced NF-κB promoter activity, iNOS expression and NO production. In conclusion these effects are consistent with some of the observed anti-inflammatory properties of other flavonoids. PMID:24768712

  8. Local Unfolding of Fatty Acid Binding Protein to Allow Ligand Entry for Binding.

    PubMed

    Xiao, Tianshu; Fan, Jing-Song; Zhou, Hu; Lin, Qingsong; Yang, Daiwen

    2016-06-01

    Fatty acid binding proteins are responsible for the transportation of fatty acids in biology. Despite intensive studies, the molecular mechanism of fatty acid entry to and exit from the protein cavity is still unclear. Here a cap-closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the helical cap is locked to the β-barrel by a disulfide linkage. Structure determination shows that this variant adopts a closed conformation, but still uptakes fatty acids. Stopped-flow experiments indicate that a rate-limiting step exists before the ligand association and this step corresponds to the conversion of the closed form to the open one. NMR relaxation dispersion and H-D exchange data demonstrate the presence of two excited states: one is native-like, but the other adopts a locally unfolded structure. Local unfolding of helix 2 generates an opening for ligands to enter the protein cavity, and thus controls the ligand association rate. PMID:27105780

  9. "Click" Chemistry-Tethered Hyaluronic Acid-Based Contact Lens Coatings Improve Lens Wettability and Lower Protein Adsorption.

    PubMed

    Deng, Xudong; Korogiannaki, Myrto; Rastegari, Banafsheh; Zhang, Jianfeng; Chen, Mengsu; Fu, Qiang; Sheardown, Heather; Filipe, Carlos D M; Hoare, Todd

    2016-08-31

    Improving the wettability of and reducing the protein adsorption to contact lenses may be beneficial for improving wearer comfort. Herein, we describe a simple "click" chemistry approach to surface functionalize poly(2-hydroxyethyl methacrylate) (pHEMA)-based contact lenses with hyaluronic acid (HA), a carbohydrate naturally contributing to the wettability of the native tear film. A two-step preparation technique consisting of laccase/TEMPO-mediated oxidation followed by covalent grafting of hydrazide-functionalized HA via simple immersion resulted in a model lens surface that is significantly more wettable, more water retentive, and less protein binding than unmodified pHEMA while maintaining the favorable transparency, refractive, and mechanical properties of a native lens. The dipping/coating method we developed to covalently tether the HA wetting agent is simple, readily scalable, and a highly efficient route for contact lens modification. PMID:27509015

  10. The endothelial cell binding site for advanced glycation end products consists of a complex: an integral membrane protein and a lactoferrin-like polypeptide.

    PubMed

    Schmidt, A M; Mora, R; Cao, R; Yan, S D; Brett, J; Ramakrishnan, R; Tsang, T C; Simionescu, M; Stern, D

    1994-04-01

    Advanced glycation end products (AGEs), formed as the result of the extended interaction of proteins with ketoses, modulate central properties of endothelial cells and mononuclear phagocytes by interacting with a cell surface binding site comprised of a novel integral membrane protein (receptor for AGE = RAGE) and a lactoferrin-like polypeptide (LF-L), the latter having sequence identity to lactoferrin (LF). To further understand this cellular binding site, the interaction of RAGE with LF-L and LF was characterized. By ligand blotting and a solid state competitive binding assay, 125I-LF-L and 125I-LF bound to RAGE immobilized on nitrocellulose membranes or polypropylene tubes in a time-dependent and reversible manner, demonstrating a high affinity component with Kd approximately 100 pM. The interaction of 125I-LF-L and 125I-LF with RAGE was independent of iron in LF and was competed by addition of an excess of unlabeled carboxyl-terminal portion of LF. Cross-linking studies with purified 125I-LF-L and RAGE, in the presence of disuccinimidyl suberate, showed a new, slowly migrating band, corresponding to a complex of RAGE and LF-L, and cross-linking on mouse aortic endothelial cells showed two new slowly migrating bands on immunoblotting visualized with both anti-RAGE IgG and anti-LF-L IgG. These data lead us to propose that the endothelial cell surface binding site for AGEs consists of LF-L bound noncovalently to RAGE anchored in the cell membrane. PMID:8144581

  11. Nitric Oxide Regulates Mitochondrial Fatty Acid Metabolism through Reversible Protein S-Nitrosylation **

    PubMed Central

    Doulias, Paschalis-Thomas; Tenopoulou, Margarita; Greene, Jennifer L.; Raju, Karthik; Ischiropoulos, Harry

    2014-01-01

    Cysteine S-nitrosylation is a posttranslational modification by which nitric oxide regulates protein function and signaling. Studies of individual proteins have elucidated specific functional roles for S-nitrosylation, but knowledge of the extent of endogenous S-nitrosylation, the sites that are nitrosylated, and the regulatory consequences of S-nitrosylation remains limited. We used mass spectrometry-based methodologies to identify 1011 S-nitrosocysteine residues in 647 proteins in various mouse tissues. We uncovered selective S-nitrosylation of enzymes participating in glycolysis, gluconeogenesis, tricarboxylic acid cycle, and oxidative phosphorylation, indicating that this posttranslational modification may regulate metabolism and mitochondrial bioenergetics. S-nitrosylation of the liver enzyme VLCAD (very long acyl-CoA dehydrogenase) at Cys238, which was absent in mice lacking endothelial nitric oxide synthase, improved its catalytic efficiency. These data implicate protein S-nitrosylation in the regulation of β-oxidation of fatty acids in mitochondria. PMID:23281369

  12. Covalent protein crosslinks: general detection, quantitation, and characterization via modification with diphenylborinic acid.

    PubMed

    Graham, L; Gallop, P M

    1994-03-01

    Progressive crosslinking of proteins appears to be a general phenomenon in aging cells and tissues. Crosslinked proteins can form insoluble aggregates which become increasingly resistant to proteolysis as more crosslinks form. However, most evidence for progressive crosslinking with age is indirect, and little is known about the chemical mechanisms involved. We have therefore developed a method for detection and isolation of any type of stable covalent crosslink from protein hydrolysates which requires no prior knowledge of the molecular structure of whatever crosslink(s) may be present. It utilizes the specificity of the diphenylborinic acid reagent for alpha-amino acid groups and the chromatographic properties and uv absorbance of the crosslink derivatives. The method is demonstrated using eight different crosslinks from collagen and fibrin, and a general procedure is given for detection of any type of crosslink in a protein hydrolysate. PMID:8203759

  13. Alkali and Acid Solubilization Effects on Rheological Properties of Horse Mackerel Muscle Proteins

    NASA Astrophysics Data System (ADS)

    Campo-Deaño, L.; Tovar, C. A.

    2008-07-01

    Influence of the acid (Type A) and alkali (Type B) solubilization of muscle proteins in the viscoelastic properties of surimi and surimi gels made from horse mackerel (Trachurus trachurus) muscle were evaluated. Stress and frequency sweep tests showed that surimi from method B presents higher viscoelastic moduli, lowest values of phase angle and minimum viscoelastic moduli dependence with frequency than surimi A. These results show a high inicial protein aggregation in surimi B, that could explain the greater firmness and hardness of this sample, showing a more compact network structure. From static and dynamic tests, gel developed from alkali solubilization resulted in higher gel strength and more rigid network than that from acidic pH, despite the incial protein aggregation of surimi B its protein keeps better gelation capacity. The less structural quality of GA gel is likely due to the more lipid content on the surimi as compared to alkali treatment.

  14. The Prevalence of STIV c92-Like Proteins in Acidic Thermal Environments.

    PubMed

    Snyder, Jamie C; Bolduc, Benjamin; Bateson, Mary M; Young, Mark J

    2011-01-01

    A new type of viral-induced lysis system has recently been discovered for two unrelated archaeal viruses, STIV and SIRV2. Prior to the lysis of the infected host cell, unique pyramid-like lysis structures are formed on the cell surface by the protrusion of the underlying cell membrane through the overlying external S-layer. It is through these pyramid structures that assembled virions are released during lysis. The STIV viral protein c92 is responsible for the formation of these lysis structures. We searched for c92-like proteins in viral sequences present in multiple viral and cellular metagenomic libraries from Yellowstone National Park acidic hot spring environments. Phylogenetic analysis of these proteins demonstrates that, although c92-like proteins are detected in these environments, some are quite divergent and may represent new viral families. We hypothesize that this new viral lysis system is common within diverse archaeal viral populations found within acidic hot springs. PMID:22312348

  15. Amino acids

    MedlinePlus

    Amino acids are organic compounds that combine to form proteins . Amino acids and proteins are the building blocks of life. When proteins are digested or broken down, amino acids are left. The human body uses amino acids ...

  16. Structure of the ordered hydration of amino acids in proteins: analysis of crystal structures

    SciTech Connect

    Biedermannová, Lada Schneider, Bohdan

    2015-10-27

    The hydration of protein crystal structures was studied at the level of individual amino acids. The dependence of the number of water molecules and their preferred spatial localization on various parameters, such as solvent accessibility, secondary structure and side-chain conformation, was determined. Crystallography provides unique information about the arrangement of water molecules near protein surfaces. Using a nonredundant set of 2818 protein crystal structures with a resolution of better than 1.8 Å, the extent and structure of the hydration shell of all 20 standard amino-acid residues were analyzed as function of the residue conformation, secondary structure and solvent accessibility. The results show how hydration depends on the amino-acid conformation and the environment in which it occurs. After conformational clustering of individual residues, the density distribution of water molecules was compiled and the preferred hydration sites were determined as maxima in the pseudo-electron-density representation of water distributions. Many hydration sites interact with both main-chain and side-chain amino-acid atoms, and several occurrences of hydration sites with less canonical contacts, such as carbon–donor hydrogen bonds, OH–π interactions and off-plane interactions with aromatic heteroatoms, are also reported. Information about the location and relative importance of the empirically determined preferred hydration sites in proteins has applications in improving the current methods of hydration-site prediction in molecular replacement, ab initio protein structure prediction and the set-up of molecular-dynamics simulations.

  17. Flexibility-rigidity index for protein-nucleic acid flexibility and fluctuation analysis.

    PubMed

    Opron, Kristopher; Xia, Kelin; Burton, Zach; Wei, Guo-Wei

    2016-05-30

    Protein-nucleic acid complexes are important for many cellular processes including the most essential functions such as transcription and translation. For many protein-nucleic acid complexes, flexibility of both macromolecules has been shown to be critical for specificity and/or function. The flexibility-rigidity index (FRI) has been proposed as an accurate and efficient approach for protein flexibility analysis. In this article, we introduce FRI for the flexibility analysis of protein-nucleic acid complexes. We demonstrate that a multiscale strategy, which incorporates multiple kernels to capture various length scales in biomolecular collective motions, is able to significantly improve the state of art in the flexibility analysis of protein-nucleic acid complexes. We take the advantage of the high accuracy and O(N) computational complexity of our multiscale FRI method to investigate the flexibility of ribosomal subunits, which are difficult to analyze by alternative approaches. An anisotropic FRI approach, which involves localized Hessian matrices, is utilized to study the translocation dynamics in an RNA polymerase. © 2016 Wiley Periodicals, Inc. PMID:26927815

  18. Effect of glycine supplementation in low protein diets with amino acids from soy protein isolate or free amino acids on broiler growth and nitrogen utilisation.

    PubMed

    Siegert, W; Wild, K J; Schollenberger, M; Helmbrecht, A; Rodehutscord, M

    2016-06-01

    Here, it was investigated whether substitution of amino acids (AA) from soy protein isolate with free AA in low crude protein diets influences the growth performance and N utilisation in broilers, and whether interactions with dietary glycine equivalent (Glyequi) concentration exist. Birds were distributed in two 2 × 2 factorial arrangements of 48 floor pens containing 10 birds each, plus 48 metabolism cages containing two birds each. Experimental feed was provided for ad libitum consumption from d 7 to 22. Diets contained either a soy protein isolate at 79 g/kg or a mix of free AA, which supplied the same amount of 18 proteinogenic AA. A mix of free glycine and l-serine was used to obtain low and high (12.0 and 20.5 g/kg dry matter) levels of dietary Glyequi. Substitution of soy protein isolate with free AA reduced the average daily gain and feed efficiency, mainly due to reduced feed intake. Efficiency of N accretion was not influenced by the AA source or Glyequi concentration on d 21, possibly due to the lower AA digestibility of soy protein isolate and higher urinary excretion of nitrogenous substances in the treatments with the AA mix. The average daily weight gain of the treatments with high Glyequi concentration was higher for both AA sources. This increase was due to higher average daily feed intake by broilers in the treatments with soy protein isolate and due to the increased feed efficiency in the treatments with the AA mix. Broilers exhibited different growth responses to dietary Glyequi between the AA sources; however, these responses could not be attributed to the different utilisation of Glyequi for uric acid synthesis. PMID:26955743

  19. The non-protein amino acid BMAA is misincorporated into human proteins in place of L-serine causing protein misfolding and aggregation.

    PubMed

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNA(Ala) with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of L-serine into human proteins. We also report that this misincorporation can be inhibited by L-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

  20. The Non-Protein Amino Acid BMAA Is Misincorporated into Human Proteins in Place of l-Serine Causing Protein Misfolding and Aggregation

    PubMed Central

    Dunlop, Rachael Anne; Cox, Paul Alan; Banack, Sandra Anne; Rodgers, Kenneth John

    2013-01-01

    Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNAAla with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of l-serine into human proteins. We also report that this misincorporation can be inhibited by l-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS. PMID:24086518

  1. Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein nutrition.

    PubMed

    Wu, Guoyao

    2014-01-01

    Amino acids are building blocks for proteins in all animals. Based on growth or nitrogen balance, amino acids were traditionally classified as nutritionally essential or nonessential for mammals, birds and fish. It was assumed that all the "nutritionally nonessential amino acids (NEAA)" were synthesized sufficiently in the body to meet the needs for maximal growth and optimal health. However, careful analysis of the scientific literature reveals that over the past century there has not been compelling experimental evidence to support this assumption. NEAA (e.g., glutamine, glutamate, proline, glycine and arginine) play important roles in regulating gene expression, cell signaling, antioxidative responses, fertility, neurotransmission, and immunity. Additionally, glutamate, glutamine and aspartate are major metabolic fuels for the small intestine to maintain its digestive function and to protect the integrity of the intestinal mucosa. Thus, diets for animals must contain all NEAA to optimize their survival, growth, development, reproduction, and health. Furthermore, NEAA should be taken into consideration in revising the "ideal protein" concept that is currently used to formulate swine and poultry diets. Adequate provision of all amino acids (including NEAA) in diets enhances the efficiency of animal production. In this regard, amino acids should not be classified as nutritionally essential or nonessential in animal or human nutrition. The new Texas A&M University's optimal ratios of dietary amino acids for swine and chickens are expected to beneficially reduce dietary protein content and improve the efficiency of their nutrient utilization, growth, and production performance. PMID:24999386

  2. Purification and complete amino acid sequence of a new type of sweet protein taste-modifying activity, curculin.

    PubMed

    Yamashita, H; Theerasilp, S; Aiuchi, T; Nakaya, K; Nakamura, Y; Kurihara, Y

    1990-09-15

    A new taste-modifying protein named curculin was extracted with 0.5 M NaCl from the fruits of Curculigo latifolia and purified by ammonium sulfate fractionation, CM-Sepharose ion-exchange chromatography, and gel filtration. Purified curculin thus obtained gave a single band having a Mr of 12,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 8 M urea. The molecular weight determined by low-angle laser light scattering was 27,800. These results suggest that native curculin is a dimer of a 12,000-Da polypeptide. The complete amino acid sequence of curculin was determined by automatic Edman degradation. Curculin consists of 114 residues. Curculin itself elicits a sweet taste. After curculin, water elicits a sweet taste, and sour substances induce a stronger sense of sweetness. No protein with both sweet-tasting and taste-modifying activities has ever been found. There are five sets of tripeptides common to miraculin (a taste-modifying protein), six sets of tripeptides common to thaumatin (a sweet protein), and two sets of tripeptides common to monellin (a sweet protein). Anti-miraculin serum was not immunologically reactive with curculin. The mechanism of the taste-modifying action of curculin is discussed. PMID:2394746

  3. Site-specific analysis of protein hydration based on unnatural amino acid fluorescence.

    PubMed

    Amaro, Mariana; Brezovský, Jan; Kováčová, Silvia; Sýkora, Jan; Bednář, David; Němec, Václav; Lišková, Veronika; Kurumbang, Nagendra Prasad; Beerens, Koen; Chaloupková, Radka; Paruch, Kamil; Hof, Martin; Damborský, Jiří

    2015-04-22

    Hydration of proteins profoundly affects their functions. We describe a simple and general method for site-specific analysis of protein hydration based on the in vivo incorporation of fluorescent unnatural amino acids and their analysis by steady-state fluorescence spectroscopy. Using this method, we investigate the hydration of functionally important regions of dehalogenases. The experimental results are compared to findings from molecular dynamics simulations. PMID:25815779

  4. Adhesion and structure properties of protein nanomaterials containing hydrophobic and charged amino acids.

    PubMed

    Shen, Xinchun; Mo, Xiaoqun; Moore, Robyn; Frazier, Shawnalea J; Iwamoto, Takeo; Tomich, John M; Sun, Xiuzhi Susan

    2006-03-01

    Protein polymers are being used or considered for biobased adhesives and coating materials. Most adhesives derived from macro protein molecules work through receptors or cross-links to bring about adhesion. The adhesion mechanism of protein polymers would lead to better understanding of adhesives and the discovery of new practical properties of protein polymers at both nano- and macro-scales. The objective of this research work was to study adhesion properties of protein polymers at nanoscale (a peptide adhesive with nanometer-scale units that range in size of several nanometers, defined as protein nanomaterial). Seven protein nanomaterial samples with different degrees of adhesive strength were designed and synthesized using solid phase chemistries. All protein nanomaterials contain a common hydrophobic core flanked by charged amino acid sequences. The adhesion properties of the protein nanomaterials were investigated at different pH values and curing temperatures. The protein nanomaterials self aggregate and interact with the wood surface. The protein nanomaterial KKK-FLIVIGSII-KKK identified in this study had high adhesive strength toward wood. It had the highest shear strength at pH 12, with an amino acid sequence that was very hydrophobic and uncharged. This protein nanomaterial underwent structural analyses using circular dichroism, laser-Fourier transform infrared, and laser desorption mass spectrometry. At pH 12 this peptide adopted a pH-induced beta-like conformation. Adhesive strength reflects contributions of both hydrogen bonding and van der Waals interactions. Ionic and covalent bonds do not appear to be significant factors for adhesion in this study. PMID:16573147

  5. A modified PCR protocol for consistent amplification of fatty acid desaturase (FAD) alleles in marker-assisted backcross breeding for high oleic trait in peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High oleic acid, such as is found in olive oil, is desirable for the healthy cholesterol-lowering benefits. The oxidative stability of the oil with high oleic acid also gives longer “shelve life” for peanut products. These benefits drive the breeding effort toward developing high oleic peanuts worl...

  6. Identification of β-phenylalanine as a non-protein amino acid in cultivated rice, Oryza sativa

    PubMed Central

    Yokoo, Takayuki; Takata, Ryo; Yan, Jian; Matsumoto, Fuka; Teraishi, Masayoshi; Okumoto, Yutaka; Jander, Georg; Mori, Naoki

    2015-01-01

    Non-protein amino acids, often analogs of the standard 20 protein amino acids, have been discovered in many plant species. Recent research with cultivated rice (Oryza sativa) identified (3R)-β-tyrosine, as well as a tyrosine amino mutase that synthesizes (3R)-β-tyrosine from the protein amino acid (2S)-α-tyrosine. Gas chromatography-mass spectrometry (GC-MS) assays and comparison to an authentic standard showed that β-phenylalanine is also a relatively abundant non-protein amino acid in rice leaves and that its biosynthesis occurs independently from that of β-tyrosine. PMID:27066169

  7. Studies on the protein and sulfur amino acid requirements of young bobwhite quail

    USGS Publications Warehouse

    Serafin, J.A.

    1977-01-01

    Four experiments were conducted with purified diets to examine the influence of protein level and to estimate the sulfur amino acid (S.A.A.) requirement of young Bobwhite quail (Colinus virginianus). These studies demonstrated (I) that 26% protein was sufficient for rapid growth when the diet was supplemented with methionine; (2) that diets containing higher levels of protein (29.3% and 31.3%) failed to support satisfactory growth unless they contained supplemental methionine; and (3) that young Bobwhite quail require no more than 1.0% sulfur-containing amino acids for optimal growth and efficiency of feed utilization. A fifth experiment was conducted to examine the protein and S.A.A. requirements of young Bobwhite quail using practical rations and to compare results with those obtained with purified diets. Diets containing 24%, 26% and 28% protein were supplied with and without supplemental methionine in a five week study. Results showed significant growth responses to protein and supplemental methionine. Responses showed that Bobwhite quail require no more than 26% protein for maximum growth and efficiency of feed utilization when the S.A.A. level of the diet was approximately 1.0%. The results were in close agreement with those obtained with purified diets. These findings define more precisely than had been known the quantitative requirements of young Bobwhite quail for protein and for the S.A.A. necessary for optimal growth.

  8. Meal composition and plasma amino acid ratios: Effect of various proteins or carbohydrates, and of various protein concentrations

    NASA Technical Reports Server (NTRS)

    Yokogoshi, Hidehiko; Wurtman, Richard J.

    1986-01-01

    The effects of meals containing various proteins and carbohydrates, and of those containing various proportions of protein (0 percent to 20 percent of a meal, by weight) or of carbohydrate (0 percent to 75 percent), on plasma levels of certain large neutral amino acids (LNAA) in rats previously fasted for 19 hours were examined. Also the plasma tryptophan ratios (the ratio of the plasma trytophan concentration to the summed concentrations of the other large neutral amino acids) and other plasma amino acid ratios were calculated. (The plasma tryptophan ratio has been shown to determine brain tryptophan levels and, thereby, to affect the synthesis and release of the neurotransmitter serotonin). A meal containing 70 percent to 75 percent of an insulin-secreting carbohydrate (dextrose or dextrin) increased plasma insulin levels and the tryptophan ratio; those containing 0 percent or 25 percent carbohydrate failed to do so. Addition of as little as 5 percent casein to a 70 percent carbohydrate meal fully blocked the increase in the plasma tryptophan ratio without affecting the secretion of insulin - probably by contributing much larger quantities of the other LNAA than of tryptophan to the blood. Dietary proteins differed in their ability to suppress the carbohydrate-induced rise in the plasma tryptophan ratio. Addition of 10 percent casein, peanut meal, or gelatin fully blocked this increase, but lactalbumin failed to do so, and egg white did so only partially. (Consumption of the 10 percent gelatin meal also produced a major reduction in the plasma tyrosine ratio, and may thereby have affected brain tyrosine levels and catecholamine synthesis.) These observations suggest that serotonin-releasing neurons in brains of fasted rats are capable of distinguishing (by their metabolic effects) between meals poor in protein but rich in carbohydrates that elicit insulin secretion, and all other meals. The changes in brain serotonin caused by carbohydrate-rich, protein

  9. Resolution-enhanced native acidic gel electrophoresis: a method for resolving, sizing, and quantifying prion protein oligomers.

    PubMed

    Ladner, Carol L; Wishart, David S

    2012-07-01

    The formation of β-sheet-rich prion protein (PrP(β)) oligomers from native or cellular PrP(c) is thought to be a key step in the development of prion diseases. To assist in this characterization process we have developed a rapid and remarkably high resolution gel electrophoresis technique called RENAGE (resolution-enhanced native acidic gel electrophoresis) for separating, sizing, and quantifying oligomeric PrP(β) complexes. PrP(β) oligomers formed via either urea/salt or acid conversion can be resolved by RENAGE into a clear set of oligomeric bands differing by just one subunit. Calibration of the size of the PrP(β) oligomer bands was made possible with a cross-linked mouse PrP(90-232) ladder (1- to 11-mer) generated using ruthenium bipyridyl-based photoinduced cross-linking of unmodified proteins (PICUP). This PrP PICUP ladder allowed the size and abundance of PrP(β) oligomers formed from urea/salt and acid conversion to be determined. This distribution consists of 7-, 8-, 9-, 10-, and 11-mers, with the most abundant species being the 8-mer. The high-resolution separation afforded by RENAGE has allowed us to investigate distinctive size and population changes in PrP(β) oligomers formed under various conversion conditions, with various construct lengths, from various species or in the presence of anti-prion compounds. PMID:22490465

  10. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    PubMed Central

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-01-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/. PMID:26482832

  11. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    NASA Astrophysics Data System (ADS)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  12. The Hypothesis that the Genetic Code Originated in Coupled Synthesis of Proteins and the Evolutionary Predecessors of Nucleic Acids in Primitive Cells

    PubMed Central

    Francis, Brian R.

    2015-01-01

    polypeptides is proposed. The cell consists of an iron-sulfide particle enclosed by tholin, a heterogeneous organic material that is produced by Miller-Urey type experiments that simulate conditions on the early Earth. As the synthesis of nucleic acids evolved from β-linked polyesters, the singlet coding system for replication evolved into a four nucleotide/four amino acid process (AMP = aspartic acid, GMP = glycine, UMP = valine, CMP = alanine) and then into the triplet ribosomal process that permitted multiple copies of protein to be synthesized independent of replication. This hypothesis reconciles the “genetics first” and “metabolism first” approaches to the origin of life and explains why there are four bases in the genetic alphabet. PMID:25679748

  13. The Hypothesis that the Genetic Code Originated in Coupled Synthesis of Proteins and the Evolutionary Predecessors of Nucleic Acids in Primitive Cells.

    PubMed

    Francis, Brian R

    2015-01-01

    polypeptides is proposed. The cell consists of an iron-sulfide particle enclosed by tholin, a heterogeneous organic material that is produced by Miller-Urey type experiments that simulate conditions on the early Earth. As the synthesis of nucleic acids evolved from β-linked polyesters, the singlet coding system for replication evolved into a four nucleotide/four amino acid process (AMP = aspartic acid, GMP = glycine, UMP = valine, CMP = alanine) and then into the triplet ribosomal process that permitted multiple copies of protein to be synthesized independent of replication. This hypothesis reconciles the "genetics first" and "metabolism first" approaches to the origin of life and explains why there are four bases in the genetic alphabet. PMID:25679748

  14. The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins.

    PubMed Central

    Oberer, Monika; Zangger, Klaus; Prytulla, Stefan; Keller, Walter

    2002-01-01

    NMR and CD spectroscopy have been used to characterize, both structurally and dynamically, the 82-amino-acid ParD protein of the post-segregational killing module of the broad-host-range plasmid RP4/RK2. ParD occurs as a dimer in solution and exercises two different control functions; an autoregulatory function by binding to its own promoter P(parDE) and a plasmid-stabilizing function by inhibiting ParE toxicity in cells that express ParD and ParE. Analysis of the secondary structure based on the chemical-shift indices, sequential nuclear Overhauser enhancements (NOEs) and (3)J(Halpha-NH) scalar coupling constants showed that the N-terminal domain of ParD consists of a short beta-ribbon followed by three alpha-helices, demonstrating that ParD contains a ribbon-helix-helix fold, a DNA-binding motif found in a family of small prokaryotic repressors. (15)N longitudinal (T(1)) and transverse (T(2)) relaxation measurements and hetero nuclear NOEs showed that ParD is divided into two separate domains, a well-ordered N-terminal domain and a very flexible C-terminal domain. An increase in secondary structure was observed upon addition of trifluoroethanol, suggested to result from the formation of structured stretches in the C-terminal part of the protein. This is the first experimental evidence that the DNA-binding domain of ParD belongs to the ribbon-helix-helix fold family, and this structural motif is proposed to be present in functionally similar antidote proteins. PMID:11743881

  15. Ab initio detection of fuzzy amino acid tandem repeats in protein sequences

    PubMed Central

    2012-01-01

    Background Tandem repetitions within protein amino acid sequences often correspond to regular secondary structures and form multi-repeat 3D assemblies of varied size and function. Developing internal repetitions is one of the evolutionary mechanisms that proteins employ to adapt their structure and function under evolutionary pressure. While there is keen interest in understanding such phenomena, detection of repeating structures based only on sequence analysis is considered an arduous task, since structure and function is often preserved even under considerable sequence divergence (fuzzy tandem repeats). Results In this paper we present PTRStalker, a new algorithm for ab-initio detection of fuzzy tandem repeats in protein amino acid sequences. In the reported results we show that by feeding PTRStalker with amino acid sequences from the UniProtKB/Swiss-Prot database we detect novel tandemly repeated structures not captured by other state-of-the-art tools. Experiments with membrane proteins indicate that PTRStalker can detect global symmetries in the primary structure which are then reflected in the tertiary structure. Conclusions PTRStalker is able to detect fuzzy tandem repeating structures in protein sequences, with performance beyond the current state-of-the art. Such a tool may be a valuable support to investigating protein structural properties when tertiary X-ray data is not available. PMID:22536906

  16. Expression in the human brain of retinoic acid induced 1, a protein associated with neurobehavioural disorders.

    PubMed

    Fragoso, Yara Dadalti; Stoney, Patrick N; Shearer, Kirsty D; Sementilli, Angelo; Nanescu, Sonia E; Sementilli, Pietro; McCaffery, Peter

    2015-03-01

    Retinoic acid induced 1 (RAI1) is a protein of uncertain mechanism of action which nevertheless has been the focus of attention because it is a major contributing factor in several human developmental disorders including Smith-Magenis and Potocki-Lupski syndromes. Further, RAI1 may be linked to adult neural disorders with developmental origins such as schizophrenia and autism. The protein has been extensively examined in the rodent but very little is known about its distribution in the human central nervous system. This study demonstrated the presence of RAI1 transcript in multiple regions of the human brain. The cellular expression of RAI1 protein in the human brain was found to be similar to that described in the mouse, with high levels in neurons, but not glia, of the dentate gyrus and cornus ammonis of the hippocampus. In the cerebellum, a second region of high expression, RAI1 was present in Purkinje cells, but not granule cells. RAI1 was also found in neurons of the occipital cortex. The expression of this retinoic acid-induced protein matched well in the hippocampus with expression of the retinoic acid receptors. The subcellular distribution of human neuronal RAI1 indicated its presence in both cytoplasm and nucleus. Overall, human RAI1 protein was found to be a highly expressed neuronal protein whose distribution matches well with its role in cognitive and motor skills. PMID:24519454

  17. Distinct single amino acid replacements in the control of virulence regulator protein differentially impact streptococcal pathogenesis.

    PubMed

    Horstmann, Nicola; Sahasrabhojane, Pranoti; Suber, Bryce; Kumaraswami, Muthiah; Olsen, Randall J; Flores, Anthony; Musser, James M; Brennan, Richard G; Shelburne, Samuel A

    2011-10-01

    Sequencing of invasive strains of group A streptococci (GAS) has revealed a diverse array of single nucleotide polymorphisms in the gene encoding the control of virulence regulator (CovR) protein. However, there is limited information regarding the molecular mechanisms by which CovR single amino acid replacements impact GAS pathogenesis. The crystal structure of the CovR C-terminal DNA-binding domain was determined to 1.50 Å resolution and revealed a three-stranded β-sheet followed by a winged helix-turn-helix DNA binding motif. Modeling of the CovR protein-DNA complex indicated that CovR single amino acid replacements observed in clinical GAS isolates could directly alter protein-DNA interaction and impact protein structure. Isoallelic GAS strains that varied by a single amino acid replacement in the CovR DNA binding domain had significantly different transcriptomes compared to wild-type and to each other. Similarly, distinct recombinant CovR variants had differential binding affinity for DNA from the promoter regions of several virulence factor-encoding genes. Finally, mice that were challenged with GAS CovR isoallelic strains had significantly different survival times, which correlated with the transcriptome and protein-DNA binding studies. Taken together, these data provide structural and functional insights into the critical and distinct effects of variation in the CovR protein on GAS pathogenesis. PMID:22028655

  18. Mass Spectrometric and Spectrofluorometric Studies of the Interaction of Aristolochic Acids with Proteins

    PubMed Central

    Li, Weiwei; Hu, Qin; Chan, Wan

    2015-01-01

    Aristolochic acid (AA) is a potent carcinogen and nephrotoxin and is associated with the development of “Chinese herb nephropathy” and Balkan endemic nephropathy. Despite decades of research, the specific mechanism of the observed nephrotoxicity has remained elusive and the potential effects on proteins due to the observed toxicity of AA are not well-understood. To better understand the pharmacotoxicological features of AA, we investigated the non-covalent interactions of AA with proteins. The protein-binding properties of AA with bovine serum albumin (BSA) and lysozyme were characterized using spectrofluorometric and mass spectrometric (MS) techniques. Moreover, the protein-AA complexes were clearly identified by high-resolution MS analyses. To the best of our knowledge, this is the first direct evidence of non-covalently bound protein-AA complexes. An analysis of the spectrofluorometric data by a modified Stern−Volmer plot model also revealed that both aristolochic acid I (AAI) and aristolochic acid II (AAII) were bound to BSA and lysozyme in 1:1 stoichiometries. A significantly stronger protein binding property was observed in AAII than in AAI as evidenced by the spectrofluorometric and MS analyses, which may explain the observed higher mutagenicity of AAII. PMID:26471474

  19. Revealing the amino acid composition of proteins within an expanded genetic code

    PubMed Central

    Aerni, Hans R.; Shifman, Mark A.; Rogulina, Svetlana; O'Donoghue, Patrick; Rinehart, Jesse

    2015-01-01

    The genetic code can be manipulated to reassign codons for the incorporation of non-standard amino acids (NSAA). Deletion of release factor 1 in Escherichia coli enhances translation of UAG (Stop) codons, yet may also extended protein synthesis at natural UAG terminated messenger RNAs. The fidelity of protein synthesis at reassigned UAG codons and the purity of the NSAA containing proteins produced require careful examination. Proteomics would be an ideal tool for these tasks, but conventional proteomic analyses cannot readily identify the extended proteins and accurately discover multiple amino acid (AA) insertions at a single UAG. To address these challenges, we created a new proteomic workflow that enabled the detection of UAG readthrough in native proteins in E. coli strains in which UAG was reassigned to encode phosphoserine. The method also enabled quantitation of NSAA and natural AA incorporation at UAG in a recombinant reporter protein. As a proof-of-principle, we measured the fidelity and purity of the phosphoserine orthogonal translation system (OTS) and used this information to improve its performance. Our results show a surprising diversity of natural AAs at reassigned stop codons. Our method can be used to improve OTSs and to quantify amino acid purity at reassigned codons in organisms with expanded genetic codes. PMID:25378305

  20. Predicting the Types of J-Proteins Using Clustered Amino Acids

    PubMed Central

    Feng, Pengmian; Zuo, Yongchun

    2014-01-01

    J-proteins are molecular chaperones and present in a wide variety of organisms from prokaryote to eukaryote. Based on their domain organizations, J-proteins can be classified into 4 types, that is, Type I, Type II, Type III, and Type IV. Different types of J-proteins play distinct roles in influencing cancer properties and cell death. Thus, reliably annotating the types of J-proteins is essential to better understand their molecular functions. In the present work, a support vector machine based method was developed to identify the types of J-proteins using the tripeptide composition of reduced amino acid alphabet. In the jackknife cross-validation, the maximum overall accuracy of 94% was achieved on a stringent benchmark dataset. We also analyzed the amino acid compositions by using analysis of variance and found the distinct distributions of amino acids in each family of the J-proteins. To enhance the value of the practical applications of the proposed model, an online web server was developed and can be freely accessed. PMID:24804260

  1. Structural frameworks for considering microbial protein and nucleic-acid dependent motor ATPases

    PubMed Central

    Thomsen, Nathan D.

    2008-01-01

    Many fundamental cellular processes depend on enzymes that utilize chemical energy to catalyze unfavourable reactions. Certain classes of ATPases provide a particularly vivid example of the process of energy conversion, employing cycles of nucleotide turnover to move and/or rearrange biological polymers such as proteins and nucleic acids. Four well characterized classes of ATP-dependent protein/nucleic acid translocases and remodelling factors are found in all three domains of life (bacteria, archaea, and eukarya): ASCE P-loop NTPases, GHL proteins, actin-fold enzymes, and chaperonins. These unrelated protein superfamilies have each evolved the ability to couple ATP binding and hydrolysis to the generation of motion and force along or within their substrates. The past several years have witnessed the emergence of a wealth of structural data that help explain how such molecular engines couple nucleotide turnover to conformational change. In this review, we highlight several recent advances to illustrate some of the mechanisms by which each family of ATP-dependent motors facilitates the rearrangement and movement of proteins, protein complexes and nucleic acids. PMID:18647240

  2. Revealing the amino acid composition of proteins within an expanded genetic code.

    PubMed

    Aerni, Hans R; Shifman, Mark A; Rogulina, Svetlana; O'Donoghue, Patrick; Rinehart, Jesse

    2015-01-01

    The genetic code can be manipulated to reassign codons for the incorporation of non-standard amino acids (NSAA). Deletion of release factor 1 in Escherichia coli enhances translation of UAG (Stop) codons, yet may also extended protein synthesis at natural UAG terminated messenger RNAs. The fidelity of protein synthesis at reassigned UAG codons and the purity of the NSAA containing proteins produced require careful examination. Proteomics would be an ideal tool for these tasks, but conventional proteomic analyses cannot readily identify the extended proteins and accurately discover multiple amino acid (AA) insertions at a single UAG. To address these challenges, we created a new proteomic workflow that enabled the detection of UAG readthrough in native proteins in E. coli strains in which UAG was reassigned to encode phosphoserine. The method also enabled quantitation of NSAA and natural AA incorporation at UAG in a recombinant reporter protein. As a proof-of-principle, we measured the fidelity and purity of the phosphoserine orthogonal translation system (OTS) and used this information to improve its performance. Our results show a surprising diversity of natural AAs at reassigned stop codons. Our method can be used to improve OTSs and to quantify amino acid purity at reassigned codons in organisms with expanded genetic codes. PMID:25378305

  3. Mass Spectrometric and Spectrofluorometric Studies of the Interaction of Aristolochic Acids with Proteins

    NASA Astrophysics Data System (ADS)

    Li, Weiwei; Hu, Qin; Chan, Wan

    2015-10-01

    Aristolochic acid (AA) is a potent carcinogen and nephrotoxin and is associated with the development of “Chinese herb nephropathy” and Balkan endemic nephropathy. Despite decades of research, the specific mechanism of the observed nephrotoxicity has remained elusive and the potential effects on proteins due to the observed toxicity of AA are not well-understood. To better understand the pharmacotoxicological features of AA, we investigated the non-covalent interactions of AA with proteins. The protein-binding properties of AA with bovine serum albumin (BSA) and lysozyme were characterized using spectrofluorometric and mass spectrometric (MS) techniques. Moreover, the protein-AA complexes were clearly identified by high-resolution MS analyses. To the best of our knowledge, this is the first direct evidence of non-covalently bound protein-AA complexes. An analysis of the spectrofluorometric data by a modified Stern-Volmer plot model also revealed that both aristolochic acid I (AAI) and aristolochic acid II (AAII) were bound to BSA and lysozyme in 1:1 stoichiometries. A significantly stronger protein binding property was observed in AAII than in AAI as evidenced by the spectrofluorometric and MS analyses, which may explain the observed higher mutagenicity of AAII.

  4. Distinct Single Amino Acid Replacements in the Control of Virulence Regulator Protein Differentially Impact Streptococcal Pathogenesis

    PubMed Central

    Horstmann, Nicola; Sahasrabhojane, Pranoti; Suber, Bryce; Kumaraswami, Muthiah; Olsen, Randall J.; Flores, Anthony; Musser, James M.; Brennan, Richard G.; Shelburne, Samuel A.

    2011-01-01

    Sequencing of invasive strains of group A streptococci (GAS) has revealed a diverse array of single nucleotide polymorphisms in the gene encoding the control of virulence regulator (CovR) protein. However, there is limited information regarding the molecular mechanisms by which CovR single amino acid replacements impact GAS pathogenesis. The crystal structure of the CovR C-terminal DNA-binding domain was determined to 1.50 Å resolution and revealed a three-stranded β-sheet followed by a winged helix-turn-helix DNA binding motif. Modeling of the CovR protein-DNA complex indicated that CovR single amino acid replacements observed in clinical GAS isolates could directly alter protein-DNA interaction and impact protein structure. Isoallelic GAS strains that varied by a single amino acid replacement in the CovR DNA binding domain had significantly different transcriptomes compared to wild-type and to each other. Similarly, distinct recombinant CovR variants had differential binding affinity for DNA from the promoter regions of several virulence factor-encoding genes. Finally, mice that were challenged with GAS CovR isoallelic strains had significantly different survival times, which correlated with the transcriptome and protein-DNA binding studies. Taken together, these data provide structural and functional insights into the critical and distinct effects of variation in the CovR protein on GAS pathogenesis. PMID:22028655

  5. Bile acid binding capacity of fish protein hydrolysates from discard species of the West Mediterranean Sea.

    PubMed

    Pérez-Gálvez, Raúl; García-Moreno, Pedro J; Morales-Medina, Rocío; Guadix, Antonio; Guadix, Emilia M

    2015-04-01

    Fish protein hydrolysates (FPH), produced from the six main discard species from the West Mediterranean Sea (sardine, horse mackerel, axillary seabream, bogue, small-spotted catshark and blue whiting) were tested for their bile acid binding capacity. This capacity is directly linked to the ability to inhibit bile reabsorption in the ileum and therefore to lower cholesterol levels in the bloodstream. From each species, FPH were obtained by three different enzymatic treatments employing two serine endoproteases (subtilisin and trypsin) sequentially or in combination. The results show statistically significant differences among the fish species, attaining interesting average values of bile acid binding capacity for blue whiting (27.32% relative to cholestyramine on an equal protein basis) and horse mackerel (27.42% relative to cholestyramine on an equal protein basis). The enzymatic treatments did not significantly affect the ability of a given species to bind bile acids. These results are similar to other protein sources, such as soy protein or casein, of proven hypocholesterolemic effect. It can be concluded that fish protein hydrolysates from these discard species are suitable as ingredients in the formulation of cholesterol-lowering supplements. PMID:25756593

  6. Endogenous fatty acids in olfactory hairs influence pheromone binding protein structure and function in Lymantria dispar.

    PubMed

    Nardella, Jason; Terrado, Mailyn; Honson, Nicolette S; Plettner, Erika

    2015-08-01

    The gypsy moth utilizes a pheromone, (7R,8S)-2-methyl-7,8-epoxyoctadecane, for mate location. The pheromone is detected by sensory hairs (sensilla) on the antennae of adult males. Sensilla contain the dendrites of olfactory neurons bathed in lymph, which contains pheromone binding proteins (PBPs). We have extracted and identified free fatty acids from lymph of sensory hairs, and we demonstrate that these function as endogenous ligands for gypsy moth PBP1 and PBP2. Homology modeling of both PBPs, and docking of fatty acids reveal multiple binding sites: one internal, the others external. Pheromone binding assays suggest that these fatty acids increase PBP-pheromone binding affinity. We show that fatty acid binding causes an increase in α-helix content in the N-terminal domain, but not in the C-terminal peptide of both proteins. The C-terminal peptide was shown to form a α-helix in a hydrophobic, homogeneous environment, but not in the presence of fatty acid micelles. Through partition assays we show that the fatty acids prevent adsorption of the pheromone on hydrophobic surfaces and facilitate pheromone partition into an aqueous phase. We propose that lymph is an emulsion of fatty acids and PBP that influence each other and thereby control the partition equilibria of hydrophobic odorants. PMID:26032337

  7. Improved Proteomic Analysis Following Trichloroacetic Acid Extraction of Bacillus anthracis Spore Proteins

    SciTech Connect

    Kaiser, Brooke LD; Wunschel, David S.; Sydor, Michael A.; Warner, Marvin G.; Wahl, Karen L.; Hutchison, Janine R.

    2015-08-07

    Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Proteomic analysis is dependent upon efficient extraction of proteins from bacterial samples without introducing bias toward extraction of particular protein classes. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrich for certain classes of proteins. The method presented here is technically simple and does not require specialized equipment such as a mechanical disrupter. Our data reveal that for particularly challenging samples, such as B. anthracis Sterne spores, trichloroacetic acid extraction improved the number of proteins identified within a sample compared to bead beating (714 vs 660, respectively). Further, TCA extraction enriched for 103 known spore specific proteins whereas bead beating resulted in 49 unique proteins. Analysis of C. botulinum samples grown to 5 days, composed of vegetative biomass and spores, showed a similar trend with improved protein yields and identification using our method compared to bead beating. Interestingly, easily lysed samples, such as B. anthracis vegetative cells, were equally as effectively processed via TCA and bead beating, but TCA extraction remains the easiest and most cost effective option. As with all assays, supplemental methods such as implementation of an alternative preparation method may provide additional insight to the protein biology of the bacteria being studied.

  8. Spaceflight effects on adult rat muscle protein, nucleic acids, and amino acids

    NASA Technical Reports Server (NTRS)

    Steffen, J. M.; Musacchia, X. J.

    1986-01-01

    Exposure to conditions of weightlessness has been associated with decrements in muscle mass and strength. The present studies were undertaken to determine muscle responses at the cellular level. Male Sprague-Dawley rats (360-410 g) were exposed to 7 days of weightlessness during the Spacelab-3 shuttle flight (May 1985). Animals were killed 12 h postflight, and soleus (S), gastrocnemius (G), and extensor digitorum longus (EDL) muscles were excised. Muscle protein, RNA, and DNA were extracted and quantified. Differential muscle atrophy was accompanied by a significant (P less than 0.05) reduction in total protein only in S muscles. There were no significant changes in protein concentration (mg/g) in the muscles examined. In S muscles from flight animals, sarcoplasmic protein accounted for a significantly greater proportion of total protein that in ground controls (37.5 vs. 32.5%). Tissue concentrations (nmol/g) of asparagine-aspartate, glutamine-glutamate, glycine, histidine, and lysine were significantly reduced (from 17 to 63%) in S muscles from flight animals, but only glutamine-glutamate levels were decreased in the G and EDL. Muscle DNA content (microgram) was unchanged in the tissues examined, but S muscle DNA concentration (micrograms/mg) increased 27%. RNA content (micrograms) was significantly (P less than 0.025) reduced in S (-28%) and G(-22%) muscles following spaceflight. These results identify specific alterations in rat skeletal muscle during short term (7-day) exposure to weightlessness and compare favorably with observations previously obtained from ground-based suspension simulations.

  9. Atomic force microscopy and anodic porous allumina of nucleic acid programmable protein arrays.

    PubMed

    Nicolini, Claudio; Correia, Tercio Bezerra; Stura, Enrico; Larosa, Claudio; Spera, Rosanna; Pechkova, Eugenia

    2013-08-01

    The methodological aspects are here presented for the NAPPA (Nucleic Acid Programmable Protein Arrays) characterization by atomic force microscopy and anodic porous alumina. Anodic Porous Alumina represents also an advanced on chip laboratory for gene expression contained in an engineered plasmid vector. The results obtained with CdK2, CDKN1A, p53 and Jun test genes expressed on NAPPA and the future developments are discussed in terms of our pertinent and recent Patents and of their possibility to overcome some limitations of present fluorescence detection in probing protein-protein interaction in both basic sciences and clinical studies. PMID:23848275

  10. Glycation by Ascorbic Acid Oxidation Products Leads to the Aggregation of Lens Proteins

    PubMed Central

    Linetsky, Mikhail; Shipova, Ekaterina; Cheng, Rongzhu; Ortwerth, Beryl J.

    2008-01-01

    Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 × 106 up to 3.0 × 108 Da. This compared to proteins ranging from 1.8 × 106 up to 3.6 × 108 Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-14C] ascorbate was incorporated into the aggregate fraction and because CNBH3, which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of α-crystallin, significant aggregation of βH-crystallin, but rapid precipitation of purified βL- and γ-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, α-crystallin prevented the precipitation of the purified βL- and γ-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with βL-, or γ-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from

  11. Protein induced fluorescence enhancement (PIFE) for probing protein–nucleic acid interactions

    PubMed Central

    Hwang, Helen

    2014-01-01

    Single molecule studies of protein–nucleic acid interactions shed light on molecular mechanisms and kinetics involved in protein binding, translocation, and unwinding of DNA and RNA substrates. In this review, we provide an overview of a single molecule fluorescence method, termed “protein induced fluorescence enhancement” (PIFE). Unlike FRET where two dyes are required, PIFE employs a single dye attached to DNA or RNA to which an unlabeled protein is applied. We discuss both ensemble and single molecule studies in which PIFE was utilized. PMID:24056732

  12. FAX1, a Novel Membrane Protein Mediating Plastid Fatty Acid Export

    PubMed Central

    Li, Nannan; Gügel, Irene Luise; Giavalisco, Patrick; Zeisler, Viktoria; Schreiber, Lukas; Soll, Jürgen; Philippar, Katrin

    2015-01-01

    Fatty acid synthesis in plants occurs in plastids, and thus, export for subsequent acyl editing and lipid assembly in the cytosol and endoplasmatic reticulum is required. Yet, the transport mechanism for plastid fatty acids still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel protein, which inserts into the chloroplast inner envelope by α-helical membrane-spanning domains. Detailed phenotypic and ultrastructural analyses of FAX1 mutants in Arabidopsis thaliana showed that FAX1 function is crucial for biomass production, male fertility and synthesis of fatty acid-derived compounds such as lipids, ketone waxes, or pollen cell wall material. Determination of lipid, fatty acid, and wax contents by mass spectrometry revealed that endoplasmatic reticulum (ER)-derived lipids decreased when FAX1 was missing, but levels of several plastid-produced species increased. FAX1 over-expressing lines showed the opposite behavior, including a pronounced increase of triacyglycerol oils in flowers and leaves. Furthermore, the cuticular layer of stems from fax1 knockout lines was specifically reduced in C29 ketone wax compounds. Differential gene expression in FAX1 mutants as determined by DNA microarray analysis confirmed phenotypes and metabolic imbalances. Since in yeast FAX1 could complement for fatty acid transport, we concluded that FAX1 mediates fatty acid export from plastids. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems involved in vertebrate fatty acid and lipid metabolism. PMID:25646734

  13. Amino acids and proteins at ZnO-water interfaces in molecular dynamics simulations.

    PubMed

    Nawrocki, Grzegorz; Cieplak, Marek

    2013-08-28

    We determine potentials of the mean force for interactions of amino acids with four common surfaces of ZnO in aqueous solutions. The method involves all-atom molecular dynamics simulations combined with the umbrella sampling technique. The profiled nature of the density of water with the strongly adsorbed first layer affects the approach of amino acids to the surface and generates either repulsion or weak binding. The largest binding energy is found for tyrosine interacting with the surface in which the Zn ions are at the top. It is equal to 7 kJ mol(-1) which is comparable to that of the hydrogen bonds in a protein. This makes the adsorption of amino acids onto the ZnO surface much weaker than onto the well studied surface of gold. Under vacuum, binding energies are more than 40 times stronger (for one of the surfaces). The precise manner in which water molecules interact with a given surface influences the binding energies in a way that depends on the surface. Among the four considered surfaces the one with Zn at the top is recognized as binding almost all amino acids with an average binding energy of 2.60 kJ mol(-1). Another (O at the top) is non-binding for most amino acids. For binding situations the average energy is 0.66 kJ mol(-1). The remaining two surfaces bind nearly as many amino acids as they do not and the average binding energies are 1.46 and 1.22 kJ mol(-1). For all of the surfaces the binding energies vary between amino acids significantly: the dispersion in the range of 68-154% of the mean. A small protein is shown to adsorb onto ZnO only intermittently and with only a small deformation. Various adsorption events lead to different patterns in mobilities of amino acids within the protein. PMID:23836065

  14. FAX1, a novel membrane protein mediating plastid fatty acid export.

    PubMed

    Li, Nannan; Gügel, Irene Luise; Giavalisco, Patrick; Zeisler, Viktoria; Schreiber, Lukas; Soll, Jürgen; Philippar, Katrin

    2015-02-01

    Fatty acid synthesis in plants occurs in plastids, and thus, export for subsequent acyl editing and lipid assembly in the cytosol and endoplasmatic reticulum is required. Yet, the transport mechanism for plastid fatty acids still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel protein, which inserts into the chloroplast inner envelope by α-helical membrane-spanning domains. Detailed phenotypic and ultrastructural analyses of FAX1 mutants in Arabidopsis thaliana showed that FAX1 function is crucial for biomass production, male fertility and synthesis of fatty acid-derived compounds such as lipids, ketone waxes, or pollen cell wall material. Determination of lipid, fatty acid, and wax contents by mass spectrometry revealed that endoplasmatic reticulum (ER)-derived lipids decreased when FAX1 was missing, but levels of several plastid-produced species increased. FAX1 over-expressing lines showed the opposite behavior, including a pronounced increase of triacyglycerol oils in flowers and leaves. Furthermore, the cuticular layer of stems from fax1 knockout lines was specifically reduced in C29 ketone wax compounds. Differential gene expression in FAX1 mutants as determined by DNA microarray analysis confirmed phenotypes and metabolic imbalances. Since in yeast FAX1 could complement for fatty acid transport, we concluded that FAX1 mediates fatty acid export from plastids. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems involved in vertebrate fatty acid and lipid metabolism. PMID:25646734

  15. ENTPRISE: An Algorithm for Predicting Human Disease-Associated Amino Acid Substitutions from Sequence Entropy and Predicted Protein Structures

    PubMed Central

    Zhou, Hongyi; Gao, Mu; Skolnick, Jeffrey

    2016-01-01

    The advance of next-generation sequencing technologies has made exome sequencing rapid and relatively inexpensive. A major application of exome sequencing is the identification of genetic variations likely to cause Mendelian diseases. This requires processing large amounts of sequence information and therefore computational approaches that can accurately and efficiently identify the subset of disease-associated variations are needed. The accuracy and high false positive rates of existing computational tools leave much room for improvement. Here, we develop a boosted tree regression machine-learning approach to predict human disease-associated amino acid variations by utilizing a comprehensive combination of protein sequence and structure features. On comparing our method, ENTPRISE, to the state-of-the-art methods SIFT, PolyPhen-2, MUTATIONASSESSOR, MUTATIONTASTER, FATHMM, ENTPRISE exhibits significant improvement. In particular, on a testing dataset consisting of only proteins with balanced disease-associated and neutral variations defined as having the ratio of neutral/disease-associated variations between 0.3 and 3, the Mathews Correlation Coefficient by ENTPRISE is 0.493 as compared to 0.432 by PPH2-HumVar, 0.406 by SIFT, 0.403 by MUTATIONASSESSOR, 0.402 by PPH2-HumDiv, 0.305 by MUTATIONTASTER, and 0.181 by FATHMM. ENTPRISE is then applied to nucleic acid binding proteins in the human proteome. Disease-associated predictions are shown to be highly correlated with the number of protein-protein interactions. Both these predictions and the ENTPRISE server are freely available for academic users as a web service at http://cssb.biology.gatech.edu/entprise/. PMID:26982818

  16. A comparative study of serum protein-bound sialic acid in benign and malignant prostatic growth: possible role of oxidative stress in sialic acid homeostasis.

    PubMed

    Goswami, K; Nandeesha, H; Koner, B C; Nandakumar, D N

    2007-01-01

    Benign and malignant prostatic growths are associated with an increase in sialoconjugates (e.g. prostate-specific antigen (PSA)) in blood. Oxidative stress plays a crucial role in pathogenesis of various malignancies. The objective of this study was to evaluate oxidative stress parameters and protein-bound sialic acid level in sera of prostatic tumor cases and to asses for any association between them. Sera samples were collected and estimated for carbonylation of proteins, lipid peroxidation products, PSA and protein-bound sialic acid from 10 patients in each group with prostatic carcinoma (Ca prostate) and benign prostatic hyperplasia (BPH) along with 10 healthy male subjects of similar age group as control. In carcinoma prostate cases, lipid peroxides, protein carbonyls, protein-bound sialic acid and PSA were significantly increased compared to BPH and controls. There was significant association between oxidative stress parameters (lipid peroxide and protein carbonyl) and sialoconjugates (PSA and protein-bound sialic acid). In BPH cases, serum lipid peroxides and protein-bound sialic acid were significantly higher in comparison to controls and protein carbonyls were correlated with protein-bound sialic acid. ROC curve for sialic acid showed that it can be used as a marker to differentiate carcinoma prostate from benign growth of prostate at a cutoff level of 11.38 mug/mg protein with a sensitivity of 100% and specificity of 80%. We conclude that oxidative stress might be associated with the degree of sialylation of protein and graded changes in these parameters possibly unveil the pathogenic demarcation from benign to malignant condition of prostate. PMID:17404581

  17. Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

    PubMed

    Rowland, Andrew; Hallifax, David; Nussio, Matthew R; Shapter, Joseph G; Mackenzie, Peter I; Brian Houston, J; Knights, Kathleen M; Miners, John O

    2015-01-01

    1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu < 0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP. PMID:25801059

  18. Molecular cloning and primary structure of human glial fibrillary acidic protein

    SciTech Connect

    Reeves, S.A.; Helman, L.J.; Allison, A.; Israel, M.A. )

    1989-07-01

    Glial fibrillary acidic protein (GFAP) is an intermediate-filament (IF) protein that is highly specific for cells of astroglial lineage, although its tissue-specific role is speculative. Determination of the primary structure of this protein should be of importance for understanding the functional role it plays in astroglia. Therefore, the authors isolated a cDNA clone encoding this protein and determined its nucleotide sequence. The predicted amino acid sequence indicates that GFAP shares structural similarities-particularly in the central rod domain and to a lesser degree in the carboxyl-terminal domain-with other IF proteins found in nonepithelial cell types. Considerable sequence divergence in the amino-terminal region of GFAP suggests that the tissue-specific functions of this IF protein might be mediated through this region of the molecule. In contrast, conservation of structural characteristics and a moderate degree of sequence conservation in the carboxyl-terminal region suggest functional similarities. Blot hybridization analysis using the GFAP cDNA as a probe failed to detect GFAP mRNA in both normal and neoplastic human tissues in which IF proteins other than GFAP are known to be expressed.

  19. The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1

    PubMed Central

    Keller, Michael; Taskin, Asli A.; Horvath, Susanne E.; Guan, Xue Li; Prinz, Claudia; Opalińska, Magdalena; Zorzin, Carina; van der Laan, Martin; Wenk, Markus R.; Schubert, Rolf; Wiedemann, Nils; Holzer, Martin

    2015-01-01

    Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein. PMID:26347140

  20. Affinity regression predicts the recognition code of nucleic acid binding proteins

    PubMed Central

    Pelossof, Raphael; Singh, Irtisha; Yang, Julie L.; Weirauch, Matthew T.; Hughes, Timothy R.; Leslie, Christina S.

    2016-01-01

    Predicting the affinity profiles of nucleic acid-binding proteins directly from the protein sequence is a major unsolved problem. We present a statistical approach for learning the recognition code of a family of transcription factors (TFs) or RNA-binding proteins (RBPs) from high-throughput binding assays. Our method, called affinity regression, trains on protein binding microarray (PBM) or RNA compete experiments to learn an interaction model between proteins and nucleic acids, using only protein domain and probe sequences as inputs. By training on mouse homeodomain PBM profiles, our model correctly identifies residues that confer DNA-binding specificity and accurately predicts binding motifs for an independent set of divergent homeodomains. Similarly, learning from RNA compete profiles for diverse RBPs, our model can predict the binding affinities of held-out proteins and identify key RNA-binding residues. More broadly, we envision applying our method to model and predict biological interactions in any setting where there is a high-throughput ‘affinity’ readout. PMID:26571099

  1. Stimulation of muscle protein synthesis by leucine is dependent on plasma amino acid availability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have reported that a physiological increase in plasma leucine increased translation initiation factor activity during 60- and 120-min leucine infusion. Muscle protein synthesis was stimulated at 60 min but not at 120 min, perhaps due to the decrease (-50%) in plasma essential amino acids (AA). ...

  2. Extraction of protein and amino acids from deoiled rice bran by subcritical water hydrolysis.

    PubMed

    Sereewatthanawut, Issara; Prapintip, Surawit; Watchiraruji, Katemanee; Goto, Motonobu; Sasaki, Mitsuru; Shotipruk, Artiwan

    2008-02-01

    This study investigated the production of value-added protein and amino acids from deoiled rice bran by hydrolysis in subcritical water (SW) in the temperature range between 100 and 220 degrees C for 0-30 min. The results suggested that SW could effectively be used to hydrolyze deoiled rice bran to produce useful protein