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Sample records for acid residue protein

  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.

  2. Dihedral angle preferences of DNA and RNA binding amino acid residues in proteins.

    PubMed

    Ponnuraj, Karthe; Saravanan, Konda Mani

    2017-04-01

    A protein can interact with DNA or RNA molecules to perform various cellular processes. Identifying or analyzing DNA/RNA binding site amino acid residues is important to understand molecular recognition process. It is quite possible to accurately model DNA/RNA binding amino acid residues in experimental protein-DNA/RNA complex by using the electron density map whereas, locating/modeling the binding site amino acid residues in the predicted three dimensional structures of DNA/RNA binding proteins is still a difficult task. Considering the above facts, in the present work, we have carried out a comprehensive analysis of dihedral angle preferences of DNA and RNA binding site amino acid residues by using a classical Ramachandran map. We have computed backbone dihedral angles of non-DNA/RNA binding residues and used as control dataset to make a comparative study. The dihedral angle preference of DNA and RNA binding site residues of twenty amino acid type is presented. Our analysis clearly revealed that the dihedral angles (φ, ψ) of DNA/RNA binding amino acid residues prefer to occupy (-89° to -60°, -59° to -30°) bins. The results presented in this paper will help to model/locate DNA/RNA binding amino acid residues with better accuracy.

  3. Core Amino Acid Residues in the Morphology-Regulating Protein, Mms6, for Intracellular Magnetite Biomineralization

    PubMed Central

    Yamagishi, Ayana; Narumiya, Kaori; Tanaka, Masayoshi; Matsunaga, Tadashi; Arakaki, Atsushi

    2016-01-01

    Living organisms produce finely tuned biomineral architectures with the aid of biomineral-associated proteins. The functional amino acid residues in these proteins have been previously identified using in vitro and in silico experimentation in different biomineralization systems. However, the investigation in living organisms is limited owing to the difficulty in establishing appropriate genetic techniques. Mms6 protein, isolated from the surface of magnetite crystals synthesized in magnetotactic bacteria, was shown to play a key role in the regulation of crystal morphology. In this study, we have demonstrated a defect in the specific region or substituted acidic amino acid residues in the Mms6 protein for observing their effect on magnetite biomineralization in vivo. Analysis of the gene deletion mutants and transformants of Magnetospirillum magneticum AMB-1 expressing partially truncated Mms6 protein revealed that deletions in the N-terminal or C-terminal regions disrupted proper protein localization to the magnetite surface, resulting in a change in the crystal morphology. Moreover, single amino acid substitutions at Asp123, Glu124, or Glu125 in the C-terminal region of Mms6 clearly indicated that these amino acid residues had a direct impact on magnetite crystal morphology. Thus, these consecutive acidic amino acid residues were found to be core residues regulating magnetite crystal morphology. PMID:27759096

  4. 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.)

  5. Entropy reduction in unfolded peptides (and proteins) due to conformational preferences of amino acid residues.

    PubMed

    Schweitzer-Stenner, Reinhard; Toal, Siobhan E

    2014-11-07

    As established by several groups over the last 20 years, amino acid residues in unfolded peptides and proteins do not exhibit the unspecific random distribution as assumed by the classical random coil model. Individual amino acid residues in small peptides were found to exhibit different conformational preferences. Here, we utilize recently obtained conformational distributions of guest amino acid residues in GxG peptides to estimate their conformational entropy, which we find to be significantly lower than the entropy of an assumed random coil like distribution. Only at high temperature do backbone entropies approach random coil like values. We utilized the obtained backbone entropies of the investigated amino acid residues to estimate the loss of conformational entropy caused by a coil → helix transition and identified two subsets of amino acid residues for which the thus calculated entropy losses correlate well with the respective Gibbs energy of helix formation obtained for alanine based host-guest systems. Calculated and experimentally derived entropic losses were found to be in good agreement. For most of the amino acid residues investigated entropic losses derived from our GxG distributions correlate very well with corresponding values recently obtained from MD simulations biased by conformational propensities derived from truncated coil libraries. Both, conformational entropy and the entropy of solvation exhibit a strong, residue specific temperature dependence, which can be expected to substantially affect the stability of unfolded states. Altogether, our results provide strong evidence for the notion that conformational preferences of amino acid residues matter with regard to the thermodynamics of peptide and protein folding.

  6. "Silent" Amino Acid Residues at Key Subunit Interfaces Regulate the Geometry of Protein Nanocages.

    PubMed

    Zhang, Shengli; Zang, Jiachen; Zhang, Xiaorong; Chen, Hai; Mikami, Bunzo; Zhao, Guanghua

    2016-11-22

    Rendering the geometry of protein-based assemblies controllable remains challenging. Protein shell-like nanocages represent particularly interesting targets for designed assembly. Here, we introduce an engineering strategy-key subunit interface redesign (KSIR)-that alters a natural subunit-subunit interface by selective deletion of a small number of "silent" amino acid residues (no participation in interfacial interactions) into one that triggers the generation of a non-native protein cage. We have applied KSIR to construct a non-native 48-mer nanocage from its native 24-mer recombinant human H-chain ferritin (rHuHF). This protein is a heteropolymer composed of equal numbers of two different subunits which are derived from one polypeptide. This strategy has allowed the study of conversion between protein nanocages with different geometries by re-engineering key subunit interfaces and the demonstration of the important role of the above-mentioned specific residues in providing geometric specificity for protein assembly.

  7. Prediction of functionally important residues in globular proteins from unusual central distances of amino acids

    PubMed Central

    2011-01-01

    Background Well-performing automated protein function recognition approaches usually comprise several complementary techniques. Beside constructing better consensus, their predictive power can be improved by either adding or refining independent modules that explore orthogonal features of proteins. In this work, we demonstrated how the exploration of global atomic distributions can be used to indicate functionally important residues. Results Using a set of carefully selected globular proteins, we parametrized continuous probability density functions describing preferred central distances of individual protein atoms. Relative preferred burials were estimated using mixture models of radial density functions dependent on the amino acid composition of a protein under consideration. The unexpectedness of extraordinary locations of atoms was evaluated in the information-theoretic manner and used directly for the identification of key amino acids. In the validation study, we tested capabilities of a tool built upon our approach, called SurpResi, by searching for binding sites interacting with ligands. The tool indicated multiple candidate sites achieving success rates comparable to several geometric methods. We also showed that the unexpectedness is a property of regions involved in protein-protein interactions, and thus can be used for the ranking of protein docking predictions. The computational approach implemented in this work is freely available via a Web interface at http://www.bioinformatics.org/surpresi. Conclusions Probabilistic analysis of atomic central distances in globular proteins is capable of capturing distinct orientational preferences of amino acids as resulting from different sizes, charges and hydrophobic characters of their side chains. When idealized spatial preferences can be inferred from the sole amino acid composition of a protein, residues located in hydrophobically unfavorable environments can be easily detected. Such residues turn out to be

  8. Determination of free acidic and alkaline residues of protein via moving reaction boundary titration in microdevice electrophoresis.

    PubMed

    Wang, Hou-yu; Li, Si; Tang, Yun-yun; Dong, Jing-yu; Fan, Liu-yin; Cao, Cheng-xi

    2013-06-21

    As two important physico-chemical parameters, the acidic and alkaline residues of protein are of evident significance for the evaluation of protein properties and the design of relevant separation and analysis. However, there is still no electrophoretic method used for the direct detection of free acidic and alkaline residues of protein. Herein, we developed the concepts of moving reaction boundary (MRB) and MRB titration, relevant MRB titration theory, and the method of microdevice electrophoresis for the determination of free acidic and alkaline residues of protein. In the MRB titration, the boundary was created with acid or alkali and target protein immobilized via highly cross-linked polyacrylamide gel (PAG). It was theoretically revealed that the number of free acidic or alkaline residues of protein was as a function of MRB displacement in the electrophoretic titration system. As a proof of concept, seven model proteins were chosen for the determination of acidic or alkaline residues of protein via MRB titration. The results showed that the numbers of free acidic and alkaline residues of proteins detected were in good agreement with those obtained from the relevant amino sequences in the NCBI database, demonstrating the feasibility of the developed concept, theory and technique. The general methodology of MRB titration has potential application for inexpensive, facilitative and informative protein structure analysis of free acidic or alkaline residues of protein.

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

    PubMed Central

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

    2016-01-01

    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

  10. Lactobacillus plantarum BL011 cultivation in industrial isolated soybean protein acid residue.

    PubMed

    Coghetto, Chaline Caren; Vasconcelos, Carolina Bettker; Brinques, Graziela Brusch; Ayub, Marco Antônio Záchia

    In this study, physiological aspects of Lactobacillus plantarum BL011 growing in a new, all-animal free medium in bioreactors were evaluated aiming at the production of this important lactic acid bacterium. Cultivations were performed in submerged batch bioreactors using the Plackett-Burman methodology to evaluate the influence of temperature, aeration rate and stirring speed as well as the concentrations of liquid acid protein residue of soybean, soy peptone, corn steep liquor, and raw yeast extract. The results showed that all variables, except for corn steep liquor, significantly influenced biomass production. The best condition was applied to bioreactor cultures, which produced a maximal biomass of 17.87gL(-1), whereas lactic acid, the most important lactic acid bacteria metabolite, peaked at 37.59gL(-1), corresponding to a productivity of 1.46gL(-1)h(-1). This is the first report on the use of liquid acid protein residue of soybean medium for L. plantarum growth. These results support the industrial use of this system as an alternative to produce probiotics without animal-derived ingredients to obtain high biomass concentrations in batch bioreactors.

  11. Relation between the secondary structure of carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and the fluorescence of the protein.

    PubMed

    Albani, Jihad R

    2003-05-01

    We studied in this work the relation that exists between the secondary structure of the glycans of alpha(1)-acid glycoprotein and the fluorescence of the Trp residues of the protein. We calculated for that the efficiency of quenching and the radiative and non-radiative constants. Our results indicate that the glycans display a spatial structure that is modified upon asialylation. The asialylated conformation is closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence parameters of the Trp residues. In fact, the mean quantum yield of Trp residues in sialylated and asialylated alpha(1)-acid glycoprotein are 0.0645 and 0.0385, respectively. Analysis of the fluorescence emission of alpha(1)-acid glycoprotein as the result of two contributions (surface and hydrophobic domains) indicates that quantum yields of both classes of Trp residues are lower when the protein is in the asialylated form. Also, the mean fluorescence lifetime of Trp residues decreases from 2.285 ns in the sialylated protein to 1.948 ns in the asialylated one. The radiative rate constant k(r) of the Trp residues in the sialylated alpha(1)-acid glycoprotein is higher than that in the asialylated protein. Thus, the carbohydrate residues are closer to the Trp residues in the absence of sialic acid. The modification of the spatial conformation of the glycans upon asialylation is confirmed by the decrease of the fluorescence lifetimes of Calcofluor, a fluorophore that binds to the carbohydrate residues. Finally, thermal intensity quenching of Calcofluor bound to alpha(1)-acid glycoprotein shows that the carbohydrate residues have slower residual motions in the absence of sialic acid residues.

  12. Site-Specific Pyrolysis Induced Cleavage at Aspartic Acid Residue in Peptides and Proteins

    PubMed Central

    Zhang, Shaofeng; Basile, Franco

    2011-01-01

    A simple and site-specific non-enzymatic method based on pyrolysis has been developed to cleave peptides and proteins. Pyrolytic cleavage was found to be specific and rapid as it induced a cleavage at the C-terminal side of aspartic acid in the temperature range of 220–250 °C in 10 seconds. Electrospray Ionization (ESI) mass spectrometry (MS) and tandem-MS (MS/MS) were used to characterize and identify pyrolysis cleavage products, confirming that sequence information is conserved after the pyrolysis process in both peptides and protein tested. This suggests that pyrolysis-induced cleavage at aspartyl residues can be used as a rapid protein digestion procedure for the generation of sequence specific protein biomarkers. PMID:17388620

  13. 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

  14. Frequencies of amino acid strings in globular protein sequences indicate suppression of blocks of consecutive hydrophobic residues

    PubMed Central

    Schwartz, Russell; Istrail, Sorin; King, Jonathan

    2001-01-01

    Patterns of hydrophobic and hydrophilic residues play a major role in protein folding and function. Long, predominantly hydrophobic strings of 20–22 amino acids each are associated with transmembrane helices and have been used to identify such sequences. Much less attention has been paid to hydrophobic sequences within globular proteins. In prior work on computer simulations of the competition between on-pathway folding and off-pathway aggregate formation, we found that long sequences of consecutive hydrophobic residues promoted aggregation within the model, even controlling for overall hydrophobic content. We report here on an analysis of the frequencies of different lengths of contiguous blocks of hydrophobic residues in a database of amino acid sequences of proteins of known structure. Sequences of three or more consecutive hydrophobic residues are found to be significantly less common in actual globular proteins than would be predicted if residues were selected independently. The result may reflect selection against long blocks of hydrophobic residues within globular proteins relative to what would be expected if residue hydrophobicities were independent of those of nearby residues in the sequence. PMID:11316883

  15. Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

    PubMed Central

    Wu, Hao; Wang, Wei; Naiyer, Nada; Fichtenbaum, Eric; Qualley, Dominic F.; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic

  16. Site-specific PEGylation of therapeutic proteins via optimization of both accessible reactive amino acid residues and PEG derivatives.

    PubMed

    Zhang, Chun; Yang, Xiao-lan; Yuan, Yong-hua; Pu, Jun; Liao, Fei

    2012-08-01

    Modification of accessible amino acid residues with poly(ethylene glycol) [PEG] is a widely used technique for formulating therapeutic proteins. In practice, site-specific PEGylation of all selected/engineered accessible nonessential reactive residues of therapeutic proteins with common activated PEG derivatives is a promising strategy to concomitantly improve pharmacokinetics, allow retention of activity, alleviate immunogenicity, and avoid modification isomers. Specifically, through molecular engineering of a therapeutic protein, accessible essential residues reactive to an activated PEG derivative are substituted with unreactive residues provided that protein activity is retained, and a limited number of accessible nonessential reactive residues with optimized distributions are selected/introduced. Subsequently, all accessible nonessential reactive residues are completely PEGylated with the activated PEG derivative in great excess. Branched PEG derivatives containing new PEG chains with negligible metabolic toxicity are more desirable for site-specific PEGylation. Accordingly, for the successful formulation of therapeutic proteins, optimization of the number and distributions of accessible nonessential reactive residues via molecular engineering can be integrated with the design of large-sized PEG derivatives to achieve site-specific PEGylation of all selected/engineered accessible reactive residues.

  17. SeqX: a tool to detect, analyze and visualize residue co-locations in protein and nucleic acid structures

    PubMed Central

    Biro, Jan C; Fördös, Gergely

    2005-01-01

    Background The interacting residues of protein and nucleic acid sequences are close to each other – they are co-located. Structure databases (like Protein Data Bank, PDB and Nucleic Acid Data Bank, NDB) contain all information about these co-locations; however it is not an easy task to penetrate this complex information. We developed a JAVA tool, called SeqX for this purpose. Results SeqX tool is useful to detect, analyze and visualize residue co-locations in protein and nucleic acid structures. The user a. selects a structure from PDB; b. chooses an atom that is commonly present in every residues of the nucleic acid and/or protein structure(s) c. defines a distance from these atoms (3–15 Å). The SeqX tool detects every residue that is located within the defined distances from the defined "backbone" atom(s); provides a DotPlot-like visualization (Residues Contact Map), and calculates the frequency of every possible residue pairs (Residue Contact Table) in the observed structure. It is possible to exclude +/- 1 to 10 neighbor residues in the same polymeric chain from detection, which greatly improves the specificity of detections (up to 60% when tested on dsDNA). Results obtained on protein structures showed highly significant correlations with results obtained from literature (p < 0.0001, n = 210, four different subsets). The co-location frequency of physico-chemically compatible amino acids is significantly higher than is calculated and expected in random protein sequences (p < 0.0001, n = 80). Conclusion The tool is simple and easy to use and provides a quick and reliable visualization and analyses of residue co-locations in protein and nucleic acid structures. Availability and requirements SeqX, Java J2SE Runtime Environment 5.0 (available from [see Additional file 1] ) and at least a 1 GHz processor and with a minimum 256 Mb RAM. Source codes are available from the authors. PMID:16011796

  18. Tri-peptide reference structures for the calculation of relative solvent accessible surface area in protein amino acid residues.

    PubMed

    Topham, Christopher M; Smith, Jeremy C

    2015-02-01

    Relative amino acid residue solvent accessibility values allow the quantitative comparison of atomic solvent-accessible surface areas in different residue types and physical environments in proteins and in protein structural alignments. Geometry-optimised tri-peptide structures in extended solvent-exposed reference conformations have been obtained for 43 amino acid residue types at a high level of quantum chemical theory. Significant increases in side-chain solvent accessibility, offset by reductions in main-chain atom solvent exposure, were observed for standard residue types in partially geometry-optimised structures when compared to non-minimised models built from identical sets of proper dihedral angles abstracted from the literature. Optimisation of proper dihedral angles led most notably to marked increases of up to 54% in proline main-chain atom solvent accessibility compared to literature values. Similar effects were observed for fully-optimised tri-peptides in implicit solvent. The relief of internal strain energy was associated with systematic variation in N, C(α) and C(β) atom solvent accessibility across all standard residue types. The results underline the importance of optimisation of 'hard' degrees of freedom (bond lengths and valence bond angles) and improper dihedral angle values from force field or other context-independent reference values, and impact on the use of standardised fixed internal co-ordinate geometry in sampling approaches to the determination of absolute values of protein amino acid residue solvent accessibility. Quantum chemical methods provide a useful and accurate alternative to molecular mechanics methods to perform energy minimisation of peptides containing non-standard (chemically modified) amino acid residues frequently present in experimental protein structure data sets, for which force field parameters may not be available. Reference tri-peptide atomic co-ordinate sets including hydrogen atoms are made freely available.

  19. Influence of bleaching on flavor of 34% whey protein concentrate and residual benzoic acid concentration in dried whey proteins.

    PubMed

    Listiyani, M A D; Campbell, R E; Miracle, R E; Dean, L O; Drake, M A

    2011-09-01

    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 in dried whey products. No legal limit exists in the United States for BP use in whey, but international concerns exist. The objectives of this study were to determine the effect of hydrogen peroxide (HP) or BP bleaching on the flavor of 34% WPC (WPC34) and to evaluate residual BA in commercial and experimental WPC bleached with and without BP. Cheddar whey was manufactured in duplicate. Pasteurized fat-separated whey was subjected to hot bleaching with either HP at 500 mg/kg, BP at 50 or 100 mg/kg, or no bleach. Whey was ultrafiltered and spray dried into WPC34. Color [L*(lightness), a* (red-green), and b* (yellow-blue)] measurements and norbixin extractions were conducted to compare bleaching efficacy. Descriptive sensory and instrumental volatile analyses were used to evaluate bleaching effects on flavor. Benzoic acid was extracted from experimental and commercial WPC34 and 80% WPC (WPC80) and quantified by HPLC. The b* value and norbixin concentration of BP-bleached WPC34 were lower than HP-bleached and control WPC34. Hydrogen peroxide-bleached WPC34 displayed higher cardboard flavor and had higher volatile lipid oxidation products than BP-bleached or control WPC34. Benzoyl peroxide-bleached WPC34 had higher BA concentrations than unbleached and HP-bleached WPC34 and BA concentrations were also higher in BP-bleached WPC80 compared with unbleached and HP-bleached WPC80, with smaller differences than those observed in WPC34. Benzoic acid extraction from permeate showed that WPC80 permeate contained more BA than did WPC34 permeate. Benzoyl peroxide is more effective in color removal of whey and results in fewer flavor side effects compared with HP and residual BA is

  20. Modular organization of residue-level contacts shape the selection pressure on individual amino acid sites of ribosomal proteins.

    PubMed

    Mallik, Saurav; Kundu, Sudip

    2017-02-22

    Understanding the molecular evolution of macromolecular complexes in the light of their structure, assembly and stability is of central importance. Here we address how the modular organization of native molecular contacts shapes the selection pressure on individual residue sites of ribosomal complexes. The bacterial ribosomal complex is represented as a residue contact network where nodes represent amino acid/nucleotide residues and edges represent their van der Waals interactions. We find statistically overrepresented native amino acid-nucleotide contacts (OaantC, one amino acid contacts one or multiple nucleotides, inter-nucleotide contacts are disregarded). Contact number is defined as the number of nucleotides contacted. Involvement of individual amino acids in OaantCs with smaller contact numbers is more random, while only a few amino acids significantly contribute to OaantCs with higher contact numbers. An investigation of structure, stability and assembly of bacterial ribosome depicts the involvement of these OaantCs in diverse biophysical interactions stabilizing the complex, including high-affinity protein-RNA contacts, inter-protein cooperativity, inter-subunit bridge, packing of multiple ribosomal RNA domains etc. Amino acid-nucleotide constituents of OaantCs with higher contact numbers are generally associated with significantly slower substitution rates compared to that of OaantCs with smaller contact numbers. This evolutionary rate heterogeneity emerges from the strong purifying selection pressure that conserves the respective amino acid physicochemical properties relevant to the stabilizing interaction with OaantC nucleotides. An analysis of relative molecular orientations of OaantC residues and their interaction energetics provides the biophysical ground of purifying selection conserving OaantC amino acid physicochemical properties.

  1. Molecular design of glycoprotein mimetics: glycoblotting by engineered proteins with an oxylamino-functionalized amino acid residue.

    PubMed

    Matsubara, Naoki; Oiwa, Kei; Hohsaka, Takahiro; Sadamoto, Reiko; Niikura, Kenichi; Fukuhara, Norio; Takimoto, Akio; Kondo, Hirosato; Nishimura, Shin-Ichiro

    2005-11-18

    The general and efficient method for the site-directed glycosylation of proteins is a key step in order to understand the biological importance of the carbohydrate chains of proteins and to control functional roles of the engineered glycoproteins in terms of the development of improved glycoprotein therapeutics. We have developed a novel method for site-directed glycosylation of proteins based on chemoselective blotting of common reducing sugars by genetically encoded proteins. The oxylamino-functionalized L-homoserine residues, 2-amino-4-O-(N-methylaminooxy) butanoic acid and 2-amino-4-aminooxy butanoic acid, were efficiently incorporated into proteins by using the four-base codon/anticodon pair strategy in Escherichia coli in vitro translation. Direct and chemoselective coupling between unmodified simple sugars and N-methylaminooxy group displayed on the engineered streptavidin allowed for the combinatorial synthesis of novel glycoprotein mimetics.

  2. Early region 1B of adenovirus 2 encodes two coterminal proteins of 495 and 155 amino acid residues.

    PubMed Central

    Anderson, C W; Schmitt, R C; Smart, J E; Lewis, J B

    1984-01-01

    Partial sequence analysis of tryptic peptides has identified the E1B-495R (E1b-57K) (early transcription region 1B of 495 amino acid residues, with an approximate molecular weight of 57,000) protein of adenovirus 2 as encoded by the 495 amino acid open reading frame located in the adenovirus 2 DNA sequence between nucleotides 2016 and 3500. Additional proteins of 16,000 Mr and 18,000 Mr that are related to the E1B-495R protein were identified by cell-free translation of hybridization-selected mRNA. Analysis of [35S]methionine-containing amino terminal tryptic peptides by thin-layer chromatography showed that the E1B-495R, E1B-18K, and E1B-16K proteins all begin at the same initiation codon. The E1B-495R protein from 293 cells also has the same initial tryptic peptide, acetyl-methionyl-glutamyl-arginine. Sequence analysis of E1B-18K tryptic peptides indicated that this protein also has the same carboxy terminus as the E1B-495R protein and that it is derived from an mRNA that is spliced to remove sequences between nucleotides 2250 and 3269, resulting in a protein product of 155 amino acid residues. Analysis of E1B-16K tryptic peptides has not yet revealed the carboxy terminal structure of this protein. Both the E1B-495R and the E1B-155R (E1B-18K) proteins, as well as the E1B-16K protein, were precipitated from cell-free translations and from extracts of infected cells by antiserum against an amino terminal nonapeptide common to these proteins. Images PMID:6323739

  3. Dynamics of linker residues modulate the nucleic acid binding properties of the HIV-1 nucleocapsid protein zinc fingers.

    PubMed

    Zargarian, Loussiné; Tisné, Carine; Barraud, Pierre; Xu, Xiaoqian; Morellet, Nelly; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2014-01-01

    The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity.

  4. Proteomic Investigation of Protein Profile Changes and Amino Acid Residue Level Modification in Cooked Lamb Meat: The Effect of Boiling.

    PubMed

    Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

    2015-10-21

    Hydrothermal treatment (heating in water) is a common method of general food processing and preparation. For red-meat-based foods, boiling is common; however, how the molecular level effects of this treatment correlate to the overall food properties is not yet well-understood. The effects of differing boiling times on lamb meat and the resultant cooking water were here examined through proteomic evaluation. The longer boiling time was found to result in increased protein aggregation involving particularly proteins such as glyceraldehyde-3-phosphate dehydrogenase, as well as truncation in proteins such as in α-actinin-2. Heat-induced protein backbone cleavage was observed adjacent to aspartic acid and asparagine residues. Side-chain modifications of amino acid residues resulting from the heating, including oxidation of phenylalanine and formation of carboxyethyllysine, were characterized in the cooked samples. Actin and myoglobin bands from the cooked meat per se remained visible on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even after significant cooking time. These proteins were also found to be the major source of observed heat-induced modifications. This study provides new insights into molecular-level modifications occurring in lamb meat proteins during boiling and a protein chemistry basis for better understanding the effect of this common treatment on the nutritional and functional properties of red-meat-based foods.

  5. 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

  6. Factors contributing to decreased protein stability when aspartic acid residues are in {beta}-sheet regions.

    SciTech Connect

    Pokkuluri, P. R.; Cai, X.; Raffen, R.; Gu, M.; Stevens, F. J.; Schiffer, M.

    2002-07-01

    Asp residues are significantly under represented in {beta}-sheet regions of proteins, especially in the middle of {beta}-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a {beta}-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V{sub L}) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a {beta}-strand, and that of Q89D, located in the middle of a {beta}-strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V{sub L}-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V{sub L}-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V{sub L}-Len domain. The structures of mutants V{sub L}-Len Q38D and V{sub L}-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these QD mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a {beta}-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a {beta}-strand residue to Asp could prevent the expression of the

  7. Factors contributing to decreased protein stability when aspartic acid residues are in β-sheet regions

    PubMed Central

    Pokkuluri, P.R.; Gu, M.; Cai, X.; Raffen, R.; Stevens, F.J.; Schiffer, M.

    2002-01-01

    Asp residues are significantly under represented in β-sheet regions of proteins, especially in the middle of β-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a β-domain. Two Gln residues of the immunoglobulin light-chain variable domain (VL) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a β-strand, and that of Q89D, located in the middle of a β-strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type VL-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, VL-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type VL-Len domain. The structures of mutants VL-Len Q38D and VL-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 Å resolution. We found no major perturbances in the structures of these Q→D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a β-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a β-strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to

  8. Identification of functionally important amino acid residues in the mitochondria targeting sequence of Hepatitis B virus X protein

    SciTech Connect

    Li, Sai Kam; Ho, Sai Fan; Tsui, Kwok Wing; Fung, Kwok Pui; Waye, M.Y. Mary

    2008-11-10

    Chronic hepatitis B virus (HBV) infection has been strongly associated with hepatocellular carcinoma (HCC) and the X protein (HBx) is thought to mediate the cellular changes associated with carcinogenesis. Recently, isolation of the hepatitis B virus integrants from HCC tissue by others have established the fact that the X gene is often truncated at its C-terminus. Expression of the GFP fusion proteins of HBx and its truncation mutants with a GFP tag in human liver cell-lines in this study revealed that the C-terminus of HBx is indispensable for its specific localization in the mitochondria. A crucial region of seven amino acids at the C-terminus has been mapped out in which the cysteine residue at position 115 serves as the most important residue for the subcellular localization. When cysteine 115 of HBx is mutated to alanine the mitochondria targeting property of HBx is abrogated.

  9. 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...

  10. Influence of the amino acid residue downstream of (Asp)4Lys on enterokinase cleavage of a fusion protein.

    PubMed

    Hosfield, T; Lu, Q

    1999-04-10

    We have studied the cleavage efficiency of the protease enterokinase (EK) using the novel vector pESP4. pESP4 is a yeast expression vector equipped with ligation-independent cloning sites, a GST purification tag, and a FLAG epitope tag. EK is used to cleave the FLAG and GST tags leaving the protein of interest without any extraneously added amino acids. We have found that EK is relatively permissive of the amino acid residue downstream of the recognition sequence (the P'1 position). This makes EK an ideal choice to use as a protease to cleave any protein of interest cloned within the pESP4 yeast expression vector.

  11. Improving volatile fatty acids production by exploiting the residual substrates in post-fermented sludge: Protease catalysis of refractory protein.

    PubMed

    Yin, Bo; Liu, Hongbo; Wang, Yuanyuan; Bai, Jie; Liu, He; Fu, Bo

    2016-03-01

    The real cause to the low yield of volatile fatty acids (VFAs), from inhibition or low biodegradation, is uncertain in sludge anaerobic fermentation. In this study, poor biodegradability of proteins and fast decrease of the indigenous hydrolase activity in the residual post-fermented sludge were found to be the major reasons. With the addition of trypsin or alkaline protease in residual post-fermented sludge after primary alkaline fermentation, degradation efficiency of refractory protein increased by 33.6% and 34.8%, respectively. Accordingly, the VFAs yields were improved by 69.7% and 106.1%, respectively. Furthermore, the activities of added trypsin and alkaline protease could maintain at 13.52 U/mL and 19.11 U/mL in the alkaline fermentation process. This study demonstrated that exploiting the refractory proteins in residual post-fermented sludge by protease addition seems to be a very promising way for improving VFAs yield of conventional alkaline fermentations with waste activated sludge.

  12. Prediction of fatty acid-binding residues on protein surfaces with three-dimensional probability distributions of interacting atoms.

    PubMed

    Mahalingam, Rajasekaran; Peng, Hung-Pin; Yang, An-Suei

    2014-08-01

    Protein-fatty acid interaction is vital for many cellular processes and understanding this interaction is important for functional annotation as well as drug discovery. In this work, we present a method for predicting the fatty acid (FA)-binding residues by using three-dimensional probability density distributions of interacting atoms of FAs on protein surfaces which are derived from the known protein-FA complex structures. A machine learning algorithm was established to learn the characteristic patterns of the probability density maps specific to the FA-binding sites. The predictor was trained with five-fold cross validation on a non-redundant training set and then evaluated with an independent test set as well as on holo-apo pair's dataset. The results showed good accuracy in predicting the FA-binding residues. Further, the predictor developed in this study is implemented as an online server which is freely accessible at the following website, http://ismblab.genomics.sinica.edu.tw/.

  13. Observing Vibrational Energy Flow in a Protein with the Spatial Resolution of a Single Amino Acid Residue.

    PubMed

    Fujii, Naoki; Mizuno, Misao; Ishikawa, Haruto; Mizutani, Yasuhisa

    2014-09-18

    One of the challenges in physical chemistry has been understanding how energy flows in a condensed phase from the microscopic viewpoint. To address this, space-resolved information at the molecular scale is required but has been lacking due to experimental difficulties. We succeeded in the real-time mapping of the vibrational energy flow in a protein with the spatial resolution of a single amino acid residue by combining time-resolved resonance Raman spectroscopy and site-directed single-Trp mutagenesis. Anti-Stokes Raman intensities of the Trp residues at different sites exhibited different temporal evolutions, reflecting propagation of the energy released by the heme group. A classical heat transport model was not able to reproduce the entire experimental data set, showing that we need a molecular-level description to explain the energy flow in a protein. The systematic application of our general methodology to proteins with different structural motifs may provide a greatly increased understanding of the energy flow in proteins.

  14. Multivariate analysis of properties of amino acid residues in proteins from a viewpoint of functional site prediction

    NASA Astrophysics Data System (ADS)

    Du, Shiqiao; Sakurai, Minoru

    2010-03-01

    For the prediction of a protein's function from its 3D-structure alone, it is of importance to elucidate by which properties functional site residues in a protein are discriminated from other residues. Here, we calculated five kinds of geometrical or physical properties of each residue in a protein. Those properties were integrated with techniques of multivariate analysis such as principal component analysis (PCA) or kernel PCA. Consequently, functional residues were found to show some distinct distributions in the scatter plot of those integrated data, which led to the proposal of a method for functional site prediction with a good performance.

  15. An active twenty-amino-acid-residue peptide derived from the inhibitor protein of the cyclic AMP-dependent protein kinase.

    PubMed Central

    Cheng, H C; van Patten, S M; Smith, A J; Walsh, D A

    1985-01-01

    Digestion with Staphylococcus aureus V8 proteinase of the inhibitor protein of the cyclic AMP-dependent protein kinase results in the sequential formation of three active inhibitory peptides. The smallest active peptide has the sequence Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- His-Asp . This 20-amino-acid-residue peptide has 20-40% of the activity of the native molecule and a Ki of 0.2 nM. Inhibition, as a minimum, appears to be based upon the inhibitor protein containing the recognition sequences that dictate protein-substrate-specificity. This inhibitory peptide also has sequence homology with the phosphorylation site for a protein kinase other than the cyclic AMP-dependent enzyme. PMID:3000357

  16. Förster energy-transfer studies between Trp residues of alpha1-acid glycoprotein (orosomucoid) and the glycosylation site of the protein.

    PubMed

    Albani, Jihad R

    2003-10-10

    Energy-transfer studies between Trp residues of alpha(1)-acid glycoprotein and the fluorescent probe Calcofluor White were performed. Calcofluor White interacts with carbohydrate residues of the protein, while the three Trp residues are located at the surface (Trp-160) and in hydrophobic domains of the protein (Trp-25 and Trp-122). Binding of Calcofluor to the protein induces a decrease in the fluorescence intensity of the Trp residues accompanied by an increase of that of Calcofluor White. Efficiency (E) of Trp fluorescence quenching was determined to be equal to 45%, and the Förster distance R(o), at which the efficiency of energy transfer is 50%, was calculated to be 18.13 A. This low distance and the value of the efficiency clearly indicate that energy transfer between Trp residues and Calcofluor White is weak.

  17. 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

  18. Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

    PubMed Central

    Worst, Emanuel G.; Exner, Matthias P.; De Simone, Alessandro; Schenkelberger, Marc; Noireaux, Vincent; Budisa, Nediljko; Ott, Albrecht

    2016-01-01

    The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any

  19. Stabilization of an α/β-Hydrolase by Introducing Proline Residues: Salicylic Acid Binding Protein 2 from Tobacco.

    PubMed

    Huang, Jun; Jones, Bryan J; Kazlauskas, Romas J

    2015-07-21

    α/β-Hydrolases are important enzymes for biocatalysis, but their stability often limits their application. We investigated a plant esterase, salicylic acid binding protein 2 (SABP2), as a model α/β-hydrolase. SABP2 shows typical stability to urea (unfolding free energy 6.9 ± 1.5 kcal/mol) and to heat inactivation (T1/2 15min 49.2 ± 0.5 °C). Denaturation in urea occurs in two steps, but heat inactivation occurs in a single step. The first unfolding step in urea eliminates catalytic activity. Surprisingly, we found that the first unfolding likely corresponds to the unfolding of the larger catalytic domain. Replacing selected amino acid residues with proline stabilized SABP2. Proline restricts the flexibility of the unfolded protein, thereby shifting the equilibrium toward the folded conformation. Seven locations for proline substitution were chosen either by amino acid sequence alignment with a more stable homologue or by targeting flexible regions in SABP2. Introducing proline in the catalytic domain stabilized SABP2 to the first unfolding in urea for three of five cases: L46P (+0.2 M urea), S70P (+0.1), and E215P (+0.9). Introducing proline in the cap domain did not stabilize SABP2 (two of two cases), supporting the assignment that the first unfolding corresponds to the catalytic domain. Proline substitutions in both domains stabilized SABP2 to heat inactivation: L46P (ΔT1/2 15min = +6.4 °C), S70P (+5.4), S115P (+1.8), S141P (+4.9), and E215P (+4.2). Combining substitutions did not further increase the stability to urea denaturation, but dramatically increased resistance to heat inactivation: L46P−S70P ΔT1/2 15min = +25.7 °C. This straightforward proline substitution approach may also stabilize other α/β-hydrolases.

  20. Metals in proteins: correlation between the metal-ion type, coordination number and the amino-acid residues involved in the coordination.

    PubMed

    Dokmanić, Ivan; Sikić, Mile; Tomić, Sanja

    2008-03-01

    Metal ions are constituents of many metalloproteins, in which they have either catalytic (metalloenzymes) or structural functions. In this work, the characteristics of various metals were studied (Cu, Zn, Mg, Mn, Fe, Co, Ni, Cd and Ca in proteins with known crystal structure) as well as the specificity of their environments. The analysis was performed on two data sets: the set of protein structures in the Protein Data Bank (PDB) determined with resolution <1.5 A and the set of nonredundant protein structures from the PDB. The former was used to determine the distances between each metal ion and its electron donors and the latter was used to assess the preferred coordination numbers and common combinations of amino-acid residues in the neighbourhood of each metal. Although the metal ions considered predominantly had a valence of two, their preferred coordination number and the type of amino-acid residues that participate in the coordination differed significantly from one metal ion to the next. This study concentrates on finding the specificities of a metal-ion environment, namely the distribution of coordination numbers and the amino-acid residue types that frequently take part in coordination. Furthermore, the correlation between the coordination number and the occurrence of certain amino-acid residues (quartets and triplets) in a metal-ion coordination sphere was analysed. The results obtained are of particular value for the identification and modelling of metal-binding sites in protein structures derived by homology modelling. Knowledge of the geometry and characteristics of the metal-binding sites in metalloproteins of known function can help to more closely determine the biological activity of proteins of unknown function and to aid in design of proteins with specific affinity for certain metals.

  1. Effect of binding of Calcofluor White on the carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) on the structure and dynamics of the protein moiety. A fluorescence study.

    PubMed

    Albani, J R

    2001-08-23

    Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously studied at low and high concentrations of Calcofluor compared to that of the protein. alpha1-Acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. At equimolar concentrations of Calcofluor and alpha1-acid glycoprotein, the fluorophore displays free motions [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94], while at high concentration of Calcofluor, its surrounding microenvironment is rigid, inducing the rigidity of the fluorophore itself [Albani, J. R.; Sillen, A.; Plancke, Y. D.; Coddeville, B.; Engelborghs, Y. Carbohydr. Res. 2000, 327, 333-340]. In the present work, red-edge excitation spectra and steady-state anisotropy studies performed on Trp residues in the presence of Calcofluor, showed that the apparent dynamics of Trp residues are not modified. However, deconvoluting the emission spectra with two different methods into different components, reveals that the structure of the protein matrix has been disrupted in the presence of high Calcofluor concentrations.

  2. Detection of antibodies against therapeutic proteins in the presence of residual therapeutic protein using a solid-phase extraction with acid dissociation (SPEAD) sample treatment prior to ELISA.

    PubMed

    Smith, Holly W; Butterfield, Anthony; Sun, Deqin

    2007-12-01

    The evaluation of the potential immunogenicity of therapeutic proteins in nonclinical safety studies has become complicated by the development of biopharmaceuticals that are dosed at high concentrations and/or have long half lives. These products remain in the circulation of the test system for extended periods of time, resulting in samples containing high concentrations of drug that interfere with standard immunogenicity assays. This protocol describes a novel solid-phase extraction with acid dissociation (SPEAD) sample treatment that removes the interfering therapeutic protein "drug" from the sample prior to performance of a direct immunoassay for detection of anti-drug antibodies (ADA). A biotin-avidin capture technique is used to physically separate ADA and ADA:Drug complexes from the drug and the sample matrix. The acid dissociation step removes the ADA from the biotin-avidin complex, and detection is performed by simple direct enzyme-linked immunoassay (ELISA). The SPEAD treatment allows for detection of ADA in an ELISA format and results in a >10-100-fold increase in residual drug tolerance as compared to an immunoassay without the sample treatment. The method can be used for serum samples from all species, but is presented here as an assay for detection of anti-drug antibodies in cynomolgus monkey serum.

  3. Influence of bleaching on flavor of 34% whey protein concentrate and residual benzoic acid concentration in dried whey products

    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...

  4. Amino acid residues in the GerAB protein important in the function and assembly of the alanine spore germination receptor of Bacillus subtilis 168.

    PubMed

    Cooper, Gareth R; Moir, Anne

    2011-05-01

    The paradigm gerA operon is required for endospore germination in response to c-alanine as the sole germinant, and the three protein products, GerAA, GerAB, and GerAC are predicted to form a receptor complex in the spore inner membrane. GerAB shows homology to the amino acid-polyamine-organocation (APC) family of single-component transporters and is predicted to be an integral membrane protein with 10 membrane-spanning helices. Site-directed mutations were introduced into the gerAB gene at its natural location on the chromosome. Alterations to some charged or potential helix-breaking residues within membrane spans affected receptor function dramatically. In some cases, this is likely to reflect the complete loss of the GerA receptor complex, as judged by the absence of the germinant receptor protein GerAC, which suggests that the altered GerAB protein itself may be unstable or that the altered structure destabilizes the complex. Mutants that have a null phenotype for Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, Av. Real, 1, 24006 León, Spain-alanine germination but retain GerAC protein at near-normal levels are more likely to define amino acid residues of functional, rather than structural, importance. Single-amino-acid substitutions in each of the GerAB and GerAA proteins can prevent incorporation of GerAC protein into the spore; this provides strong evidence that the proteins within a specific receptor interact and that these interactions are required for receptor assembly. The lipoprotein nature of the GerAC receptor subunit is also important; an amino acid change in the prelipoprotein signal sequence in the gerAC1 mutant results in the absence of GerAC protein from the spore.

  5. Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia.

    PubMed

    Hoff, R H; Hengge, A C; Wu, L; Keng, Y F; Zhang, Z Y

    2000-01-11

    General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.

  6. Amino acid residues in the Ler protein critical for derepression of the LEE5 promoter in enteropathogenic E. coli.

    PubMed

    Choi, Su-Mi; Jeong, Jae-Ho; Choy, Hyon E; Shin, Minsang

    2016-08-01

    Enteropathogenic E. coli causes attaching and effacing (A/E) intestinal lesions. The genes involved in the formation of A/E lesions are encoded within a chromosomal island comprising of five major operons, LEE1-5. The global regulator H-NS represses the expression of these operons. Ler, a H-NS homologue, counteracts the H-NS-mediated repression. Using a novel genetic approach, we identified the amino acid residues in Ler that are involved in the interaction with H-NS: I20 and L23 in the C-terminal portion of α-helix 3, and I42 in the following unstructured linker region.

  7. Multiple mutations of the critical amino acid residues for the sweetness of the sweet-tasting protein, brazzein.

    PubMed

    Lee, Joo-Won; Cha, Ji-Eun; Jo, Hyun-Joo; Kong, Kwang-Hoon

    2013-06-01

    We have previously identified critical residues important for sweetness of the sweet protein brazzein by site-directed mutagenesis (Yoon, Kong, Jo, & Kong, 2011). In order to elucidate the interaction mechanisms of brazzein with the sweet taste receptor, we made multiple mutations of three residues (His31 in loop 30-33, Glu36 in β-strand III, and Glu41 in loop 40-43). We found that all double mutations (H31R/E36D, H31R/E41A and E36D/E41A) made the molecules sweeter than des-pE1M-brazzein and three single mutants. Moreover, the triple mutation (H31R/E36D/E41A) made the molecule significantly sweeter than three double mutants. These results strongly support the hypothesis that brazzein binds to the multisite surface of the sweet taste receptor. Our findings also suggest that mutations reducing the overall negative charge and/or increasing the positive charge favour sweet-tasting protein potency.

  8. Stabilization of secondary structure elements by specific combinations of hydrophilic and hydrophobic amino acid residues is more important for proteins encoded by GC-poor genes.

    PubMed

    Khrustalev, Vladislav Victorovich; Barkovsky, Eugene Victorovich

    2012-12-01

    Stabilization of secondary structure elements by specific combinations of hydrophobic and hydrophilic amino acids has been studied by the way of analysis of pentapeptide fragments from twelve partial bacterial proteomes. PDB files describing structures of proteins from species with extremely high and low genomic GC-content, as well as with average G + C were included in the study. Amino acid residues in 78,009 pentapeptides from alpha helices, beta strands and coil regions were classified into hydrophobic and hydrophilic ones. The common propensity scale for 32 possible combinations of hydrophobic and hydrophilic amino acid residues in pentapeptide has been created: specific pentapeptides for helix, sheet and coil were described. The usage of pentapeptides preferably forming alpha helices is decreasing in alpha helices of partial bacterial proteomes with the increase of the average genomic GC-content in first and second codon positions. The usage of pentapeptides preferably forming beta strands is increasing in coil regions and in helices of partial bacterial proteomes with the growth of the average genomic GC-content in first and second codon positions. Due to these circumstances the probability of coil-sheet and helix-sheet transitions should be increased in proteins encoded by GC-rich genes making them prone to form amyloid in certain conditions. Possible causes of the described fact that importance of alpha helix and coil stabilization by specific combinations of hydrophobic and hydrophilic amino acids is growing with the decrease of genomic GC-content have been discussed.

  9. Histidine and Aspartic Acid Residues Important for Immunoglobulin G Endopeptidase Activity of the Group A Streptococcus Opsonophagocytosis-Inhibiting Mac Protein

    PubMed Central

    Lei, Benfang; Liu, Mengyao; Meyers, Elishia G.; Manning, Heather M.; Nagiec, Michael J.; Musser, James M.

    2003-01-01

    The secreted Mac protein made by serotype M1 group A Streptococcus (GAS) (designated Mac5005) inhibits opsonophagocytosis and killing of GAS by human polymorphonuclear neutrophils. This protein also has cysteine endopeptidase activity against human immunoglobulin G (IgG). Site-directed mutagenesis was used to identify histidine and aspartic acid residues important for Mac IgG endopeptidase activity. Replacement of His262 with Ala abolished Mac5005 IgG endopeptidase activity. Asp284Ala and Asp286Ala mutant proteins had compromised enzymatic activity, whereas 21 other Asp-to-Ala mutant proteins cleaved human IgG at the apparent wild-type level. The results suggest that His262 is an active-site residue and that Asp284 and Asp286 are important for the enzymatic activity or structure of Mac protein. These Mac mutants provide new information about structure-activity relationships in this protein and will assist study of the mechanism of inhibition of opsonophagocytosis and killing of GAS by Mac. PMID:12704162

  10. Impaired Acid Catalysis by Mutation of a Protein Loop Hinge Residue in a YopH Mutant Revealed by Crystal Structures

    SciTech Connect

    Brandao, T.; Robinson, H; Johnson, S; Hengge, A

    2009-01-01

    Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presence of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.

  11. Improving the acidic stability of a methyl parathion hydrolase by changing basic residues to acidic residues.

    PubMed

    Huang, Lu; Wang, Ping; Tian, Jian; Jiang, Huachen; Wu, Ningfeng; Yang, Peilong; Yao, Bin; Fan, Yunliu

    2012-06-01

    The acidic stability of a methyl parathion hydrolase (Ochr-MPH) was improved by selectively changing basic amino acids to acidic ones. Mutation sites were selected based on the position-specific amino acid replacement probabilities (more than or equal to 0.2) and the entropy of each site (more than or equal to 0.8). Three mutants (K208E, K277D, and K208E/K277D) were more stable than the wild-type (WT). Their half-lives at pH 5.0 were 64, 68, 65 min, respectively, whereas that of WT was 39 min. The acidic stability of proteins may therefore be improved by changing selected basic amino acid residues to acidic ones.

  12. High Affinity Small Protein Inhibitors of Human Chymotrypsin C (CTRC) Selected by Phage Display Reveal Unusual Preference for P4′ Acidic Residues*

    PubMed Central

    Szabó, András; Héja, Dávid; Szakács, Dávid; Zboray, Katalin; Kékesi, Katalin A.; Radisky, Evette S.; Sahin-Tóth, Miklós; Pál, Gábor

    2011-01-01

    Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2′ (Leu or Asp), and P4′ (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2′ (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4′ (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (KD 20 pm) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a KD of 110 pm and a selectivity ranging from 225- to 112,664-fold against other human chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys51, Arg56, and Arg80) on the surface of human CTRC that interact with the P4′ acidic residue of the inhibitor. The acidic preference of CTRC at P4′ is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation. PMID:21515688

  13. Red fluorescent protein DsRed: parametrization of its chromophore as an amino acid residue for computer modeling in the OPLS-AA force field.

    PubMed

    Dmitrienko, D V; Vrzheshch, E P; Drutsa, V L; Vrzheshch, P V

    2006-10-01

    Topology of the neutral form of the DsRed fluorescent protein chromophore as a residue of [(4-cis)-2-[(1-cis)-4-amino-4-oxobutanimidoyl]-4-(4-hydroxybenzylidene)-5-oxo-4,5-dihydro-1H-imidazol-1-yl]acetic acid was calculated with OPLS-AA force field. Use of this topology and molecular dynamics simulation allows calculating the parameters of proteins that contain such residue in their polypeptide chains. The chromophore parameters were obtained by ab initio (RHF/6-31G**) quantum chemical calculations applying density functional theory (B3LYP). Using this chromophore, we have calculated the molecular dynamics trajectory of tetrameric fluorescent protein DsRed in solution at 300 K (4 nsec). Correctness of the chromophore parametrization was revealed by comparison of quantitative characteristics of the chromophore structure obtained from the molecular dynamic simulations of DsRed protein with the quantitative characteristics of the chromophore based on the crystallographic X-ray data of fluorescent protein DsRed (PDB ID: 1ZGO, 1G7K, and 1GGX), and also with the quantitative characteristics of the chromophore obtained by quantum chemical calculations. Inclusion of the neutral form of DsRed protein chromophore topology into the OPLS-AA force field yielded the extended force field OPLS-AA/DsRed. This force field can be used for molecular dynamics calculations of proteins containing the DsRed chromophore. The parameter set presented in this study can be applied for similar extension in any other force fields.

  14. Developing an Acidic Residue Reactive and Sulfoxide-Containing MS-Cleavable Homobifunctional Cross-Linker for Probing Protein–Protein Interactions

    PubMed Central

    2016-01-01

    Cross-linking mass spectrometry (XL-MS) has become a powerful strategy for defining protein–protein interactions and elucidating architectures of large protein complexes. However, one of the inherent challenges in MS analysis of cross-linked peptides is their unambiguous identification. To facilitate this process, we have previously developed a series of amine-reactive sulfoxide-containing MS-cleavable cross-linkers. These MS-cleavable reagents have allowed us to establish a common robust XL-MS workflow that enables fast and accurate identification of cross-linked peptides using multistage tandem mass spectrometry (MSn). Although amine-reactive reagents targeting lysine residues have been successful, it remains difficult to characterize protein interaction interfaces with little or no lysine residues. To expand the coverage of protein interaction regions, we present here the development of a new acidic residue-targeting sulfoxide-containing MS-cleavable homobifunctional cross-linker, dihydrazide sulfoxide (DHSO). We demonstrate that DHSO cross-linked peptides display the same predictable and characteristic fragmentation pattern during collision induced dissociation as amine-reactive sulfoxide-containing MS-cleavable cross-linked peptides, thus permitting their simplified analysis and unambiguous identification by MSn. Additionally, we show that DHSO can provide complementary data to amine-reactive reagents. Collectively, this work not only enlarges the range of the application of XL-MS approaches but also further demonstrates the robustness and applicability of sulfoxide-based MS-cleavability in conjunction with various cross-linking chemistries. PMID:27417384

  15. Neutralization of acidic residues in helix II stabilizes the folded conformation of acyl carrier protein and variably alters its function with different enzymes.

    PubMed

    Gong, Huansheng; Murphy, Anne; McMaster, Christopher R; Byers, David M

    2007-02-16

    Acyl carrier protein (ACP), a small protein essential for bacterial growth and pathogenesis, interacts with diverse enzymes during the biosynthesis of fatty acids, phospholipids, and other specialized products such as lipid A. NMR and hydrodynamic studies have previously shown that divalent cations stabilize native helical ACP conformation by binding to conserved acidic residues at two sites (A and B) at either end of the "recognition" helix II. To examine the roles of these amino acids in ACP structure and function, site-directed mutagenesis was used to replace individual site A (Asp-30, Asp-35, Asp-38) and site B (Glu-47, Glu-53, Asp-56) residues in recombinant Vibrio harveyi ACP with the corresponding amides, along with combined mutations at each site (SA, SB) or both sites (SA/SB). Like native V. harveyi ACP, all individual mutants were unfolded at neutral pH but adopted a helical conformation in the presence of millimolar Mg(2+) or upon fatty acylation. Mg(2+) binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg(2+), suggesting that charge neutralization is largely responsible for ACP stabilization by divalent cations. Asp-35 in site A was critical for holo-ACP synthase activity, while acyl-ACP synthetase and UDP-N-acetylglucosamine acyltransferase (LpxA) activities were more affected by mutations in site B. Both sites were required for fatty acid synthase activity. Overall, our results indicate that divalent cation binding site mutations have predicted effects on ACP conformation but unpredicted and variable consequences on ACP function with different enzymes.

  16. Proteomic investigation of protein profile changes and amino acid residue-level modification in cooked lamb longissimus thoracis et lumborum: The effect of roasting.

    PubMed

    Yu, Tzer-Yang; Morton, James D; Clerens, Stefan; Dyer, Jolon M

    2016-09-01

    Protein modifications of meat cooked by typical dry-heat methods (e.g., roasting) are currently not well understood. The present study utilised a shotgun proteomic approach to examine the molecular-level effect of roasting on thin lamb longissimus thoracis et lumborum patties, in terms of changes to both the protein profile and amino acid residue side-chain modifications. Cooking caused aggregation of actin, myosin heavy chains and sarcoplasmic proteins. Longer roasting time resulted in significantly reduced protein extractability as well as protein truncation involving particularly a number of myofibrillar and sarcoplasmic proteins, e.g., 6-phosphofructokinase, beta-enolase, l-lactate dehydrogenase A chain, alpha-actinin-3, actin and possibly myosin heavy chains. Modifications that have potential influence on nutritional properties, including carboxyethyllysine and a potentially glucose-derived N-terminal Amadori compound, were observed in actin and myoglobin after roasting. This study provided new insights into molecular changes resulting from the dry-heat treatment of meat, such as commonly used in food preparation.

  17. Direct coevolutionary couplings reflect biophysical residue interactions in proteins

    NASA Astrophysics Data System (ADS)

    Coucke, Alice; Uguzzoni, Guido; Oteri, Francesco; Cocco, Simona; Monasson, Remi; Weigt, Martin

    2016-11-01

    Coevolution of residues in contact imposes strong statistical constraints on the sequence variability between homologous proteins. Direct-Coupling Analysis (DCA), a global statistical inference method, successfully models this variability across homologous protein families to infer structural information about proteins. For each residue pair, DCA infers 21 × 21 matrices describing the coevolutionary coupling for each pair of amino acids (or gaps). To achieve the residue-residue contact prediction, these matrices are mapped onto simple scalar parameters; the full information they contain gets lost. Here, we perform a detailed spectral analysis of the coupling matrices resulting from 70 protein families, to show that they contain quantitative information about the physico-chemical properties of amino-acid interactions. Results for protein families are corroborated by the analysis of synthetic data from lattice-protein models, which emphasizes the critical effect of sampling quality and regularization on the biochemical features of the statistical coupling matrices.

  18. Direct coevolutionary couplings reflect biophysical residue interactions in proteins.

    PubMed

    Coucke, Alice; Uguzzoni, Guido; Oteri, Francesco; Cocco, Simona; Monasson, Remi; Weigt, Martin

    2016-11-07

    Coevolution of residues in contact imposes strong statistical constraints on the sequence variability between homologous proteins. Direct-Coupling Analysis (DCA), a global statistical inference method, successfully models this variability across homologous protein families to infer structural information about proteins. For each residue pair, DCA infers 21 × 21 matrices describing the coevolutionary coupling for each pair of amino acids (or gaps). To achieve the residue-residue contact prediction, these matrices are mapped onto simple scalar parameters; the full information they contain gets lost. Here, we perform a detailed spectral analysis of the coupling matrices resulting from 70 protein families, to show that they contain quantitative information about the physico-chemical properties of amino-acid interactions. Results for protein families are corroborated by the analysis of synthetic data from lattice-protein models, which emphasizes the critical effect of sampling quality and regularization on the biochemical features of the statistical coupling matrices.

  19. Protein Thermostability Is Owing to Their Preferences to Non-Polar Smaller Volume Amino Acids, Variations in Residual Physico-Chemical Properties and More Salt-Bridges

    PubMed Central

    Panja, Anindya Sundar; Bandopadhyay, Bidyut; Maiti, Smarajit

    2015-01-01

    Introduction Protein thermostability is an important field for its evolutionary perspective of mesophilic versus thermophilic relationship and for its industrial/ therapeutic applications. Methods Presently, a total 400 (200 thermophilic and 200 mesophilic homologue) proteins were studied utilizing several software/databases to evaluate their amino acid preferences. Randomly selected 50 homologous proteins with available PDB-structure of each group were explored for the understanding of the protein charges, isoelectric-points, hydrophilicity, hydrophobicity, tyrosine phosphorylation and salt-bridge occurrences. These 100 proteins were further probed to generate Ramachandran plot/data for the gross secondary structure prediction in and comparison between the thermophilic and mesophilic proteins. Results Present results strongly suggest that nonpolar smaller volume amino acids Ala (χ2 = 238.54, p<0.001) and Gly (χ2 = 73.35, p<0.001) are highly and Val moderately (χ2 = 144.43, p<0.001) occurring in the 85% of thermophilic proteins. Phospho-regulated Tyr and redox-sensitive Cys are also moderately distributed (χ2~20.0, p<0.01) in a larger number of thermophilic proteins. A consistent lower distribution of thermophilicity and discretely higher distribution of hydrophobicity is noticed in a large number of thermophilic versus their mesophilic protein homolog. The mean differences of isoelectric points and charges are found to be significantly less (7.11 vs. 6.39, p<0.05 and 1 vs. -0.6, p<0.01, respectively) in thermophilic proteins compared to their mesophilic counterpart. The possible sites for Tyr phosphorylation are noticed to be 25% higher (p<0.05) in thermophilic proteins. The 60% thermophiles are found with higher number of salt bridges in this study. The average percentage of salt-bridge of thermophiles is found to be higher by 20% than their mesophilic homologue. The GLU-HIS and GLU-LYS salt-bridge dyads are calculated to be significantly higher (p<0.05 and p

  20. Structure-based engineering of histidine residues in the catalytic domain of α-amylase from Bacillus subtilis for improved protein stability and catalytic efficiency under acidic conditions.

    PubMed

    Yang, Haiquan; Liu, Long; Shin, Hyun-dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-03-10

    This work aims to improve the protein stability and catalytic efficiency of α-amylase from Bacillus subtilis under acidic conditions by site-directed mutagenesis. Based on the analysis of a three dimensional structure model, four basic histidine (His) residues His(222), His(275), His(293), and His(310) in the catalytic domain were selected as the mutation sites and were further replaced with acidic aspartic acid (Asp), respectively, yielding four mutants H222D, H275D, H293D, H310D. The mutant H222D was inactive. Double and triple mutations were further conducted and four mutants H275/293D, H275/310D, H293/310D, and H275/293/310D were obtained. The acidic stability of enzyme was significantly enhanced after mutation, and 45-92% of initial activity of mutants was retained after incubation at pH 4.5 and 25°C for 24h, while that for wild-type was only 39.5%. At pH 4.5, the specific activity of wild-type and mutants H275D, H293D, H310D, H275/293D, H275/310D, H293/310D, and H275/293/310D were 108.2, 131.8, 138.9, 196.6, 156.3, 204.6, and 216.2U/mg, respectively. The catalytic efficiency for each active mutant was much higher than that of wild-type at low pH. The kcat/Km values of the mutants H275D, H293D, H310D, H275/293D, H275/310D, H293/310D, and H275/293/310D at pH 4.5 were 3.3-, 4.3-, 6.5-, 4.5-, 11.0-, 14.5-, and 16.7-fold higher, respectively, than that of the wild-type. As revealed by the structure models of the wild-type and mutant enzymes, the hydrogen bonds and salt bridges were increased after mutation, and an obvious shift of the basic limb toward acidity was observed for mutants. These changes around the catalytic domain contributed to the significantly improved protein stability and catalytic efficiency at low pH. This work provides an effective strategy to improve the catalytic activity and stability of α-amylase under acidic conditions, and the results obtained here may be useful for the improvement of acid-resistant ability of other enzymes.

  1. Overexpression of hydroxynitrile lyase in cassava roots elevates protein and free amino acids while reducing residual cyanogen levels.

    PubMed

    Narayanan, Narayanan N; Ihemere, Uzoma; Ellery, Claire; Sayre, Richard T

    2011-01-01

    Cassava is the major source of calories for more than 250 million Sub-Saharan Africans, however, it has the lowest protein-to-energy ratio of any major staple food crop in the world. A cassava-based diet provides less than 30% of the minimum daily requirement for protein. Moreover, both leaves and roots contain potentially toxic levels of cyanogenic glucosides. The major cyanogen in cassava is linamarin which is stored in the vacuole. Upon tissue disruption linamarin is deglycosylated by the apolplastic enzyme, linamarase, producing acetone cyanohydrin. Acetone cyanohydrin can spontaneously decompose at pHs >5.0 or temperatures >35°C, or is enzymatically broken down by hydroxynitrile lyase (HNL) to produce acetone and free cyanide which is then volatilized. Unlike leaves, cassava roots have little HNL activity. The lack of HNL activity in roots is associated with the accumulation of potentially toxic levels of acetone cyanohydrin in poorly processed roots. We hypothesized that the over-expression of HNL in cassava roots under the control of a root-specific, patatin promoter would not only accelerate cyanogenesis during food processing, resulting in a safer food product, but lead to increased root protein levels since HNL is sequestered in the cell wall. Transgenic lines expressing a patatin-driven HNL gene construct exhibited a 2-20 fold increase in relative HNL mRNA levels in roots when compared with wild type resulting in a threefold increase in total root protein in 7 month old plants. After food processing, HNL overexpressing lines had substantially reduced acetone cyanohydrin and cyanide levels in roots relative to wild-type roots. Furthermore, steady state linamarin levels in intact tissues were reduced by 80% in transgenic cassava roots. These results suggest that enhanced linamarin metabolism contributed to the elevated root protein levels.

  2. The functional importance of co-evolving residues in proteins.

    PubMed

    Sandler, Inga; Zigdon, Nitzan; Levy, Efrat; Aharoni, Amir

    2014-02-01

    Computational approaches for detecting co-evolution in proteins allow for the identification of protein-protein interaction networks in different organisms and the assignment of function to under-explored proteins. The detection of co-variation of amino acids within or between proteins, moreover, allows for the discovery of residue-residue contacts and highlights functional residues that can affect the binding affinity, catalytic activity, or substrate specificity of a protein. To explore the functional impact of co-evolutionary changes in proteins, a combined experimental and computational approach must be recruited. Here, we review recent studies that apply computational and experimental tools to obtain novel insight into the structure, function, and evolution of proteins. Specifically, we describe the application of co-evolutionary analysis for predicting high-resolution three-dimensional structures of proteins. In addition, we describe computational approaches followed by experimental analysis for identifying specificity-determining residues in proteins. Finally, we discuss studies addressing the importance of such residues in terms of the functional divergence of proteins, allowing proteins to evolve new functions while avoiding crosstalk with existing cellular pathways or forming reproductive barriers and hence promoting speciation.

  3. Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2).

    PubMed

    Kobayashi, Michie; Yamamoto-Katou, Ayako; Katou, Shinpei; Hirai, Katsuyuki; Meshi, Tetsuo; Ohashi, Yuko; Mitsuhara, Ichiro

    2011-07-01

    The Tm-2 gene of tomato and its allelic gene, Tm-2(2), confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2(2), Tm-2(2) confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2(2). Although resistance induced by Tm-2 and Tm-2(2) is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2(2) induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2(2) but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2(2) is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2(2) are involved in HR cell death.

  4. The C-terminal 50 Amino Acid Residues of Dengue NS3 Protein Are Important for NS3-NS5 Interaction and Viral Replication*

    PubMed Central

    Tay, Moon Y. F.; Saw, Wuan Geok; Zhao, Yongqian; Chan, Kitti W. K.; Singh, Daljit; Chong, Yuwen; Forwood, Jade K.; Ooi, Eng Eong; Grüber, Gerhard; Lescar, Julien; Luo, Dahai; Vasudevan, Subhash G.

    2015-01-01

    Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5′-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566–585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172–618) helicase and covalently linked NS3(172–618)-NS5(320–341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320–341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis. PMID:25488659

  5. Serine 157, a retinoic acid receptor alpha residue phosphorylated by protein kinase C in vitro, is involved in RXR.RARalpha heterodimerization and transcriptional activity.

    PubMed

    Delmotte, M H; Tahayato, A; Formstecher, P; Lefebvre, P

    1999-12-31

    Retinoic acid (RA) regulation of cellular proliferation and differentiation is mediated, at least in part, through two related nuclear receptors, RAR and RXR. RA-induced modulation of gene expression leads generally to cellular differentiation, whereas stimulation of the protein kinase C (PKC) signaling pathway is associated with cellular proliferation. Pursuant to our discovery that prolonged activation of PKCs induced a strong decrease in RA responsiveness of a retinoid-inducible reporter gene, we have further investigated the connections between these two signaling pathways. We demonstrate that PKC isoforms alpha and gamma are able to phosphorylate human RARalpha (hRARalpha) in vitro on a single serine residue located in the extended DNA binding domain (T box). The introduction of a negative charge at this position (serine 157) strongly decreased hRARalpha transcriptional activity, whereas a similar mutation at other PKC consensus phosphorylation sites had no effect. The effect on transcriptional activation was correlated with a decrease in the capacity of hRARalpha to heterodimerize with hRXRalpha. Thus hRARalpha is a direct target for PKCalpha and gamma, which may control retinoid receptor transcriptional activities during cellular proliferation and differentiation.

  6. Analysis of SAT Type Foot-And-Mouth Disease Virus Capsid Proteins and the Identification of Putative Amino Acid Residues Affecting Virus Stability

    PubMed Central

    Maree, Francois F.; Blignaut, Belinda; de Beer, Tjaart A. P.; Rieder, Elizabeth

    2013-01-01

    Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces. PMID:23717387

  7. Analysis of SAT type foot-and-mouth disease virus capsid proteins and the identification of putative amino acid residues affecting virus stability.

    PubMed

    Maree, Francois F; Blignaut, Belinda; de Beer, Tjaart A P; Rieder, Elizabeth

    2013-01-01

    Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces.

  8. In-Frame Amber Stop Codon Replacement Mutagenesis for the Directed Evolution of Proteins Containing Non-Canonical Amino Acids: Identification of Residues Open to Bio-Orthogonal Modification

    PubMed Central

    Arpino, James A. J.; Baldwin, Amy J.; McGarrity, Adam R.; Tippmann, Eric M.; Jones, D. Dafydd

    2015-01-01

    Expanded genetic code approaches are a powerful means to add new and useful chemistry to proteins at defined residues positions. One such use is the introduction of non-biological reactive chemical handles for site-specific biocompatible orthogonal conjugation of proteins. Due to our currently limited information on the impact of non-canonical amino acids (nAAs) on the protein structure-function relationship, rational protein engineering is a “hit and miss” approach to selecting suitable sites. Furthermore, dogma suggests surface exposed native residues should be the primary focus for introducing new conjugation chemistry. Here we describe a directed evolution approach to introduce and select for in-frame codon replacement to facilitate engineering proteins with nAAs. To demonstrate the approach, the commonly reprogrammed amber stop codon (TAG) was randomly introduced in-frame in two different proteins: the bionanotechnologically important cyt b562 and therapeutic protein KGF. The target protein is linked at the gene level to sfGFP via a TEV protease site. In absence of a nAA, an in-frame TAG will terminate translation resulting in a non-fluorescent cell phenotype. In the presence of a nAA, TAG will encode for nAA incorporation so instilling a green fluorescence phenotype on E. coli. The presence of endogenously expressed TEV proteases separates in vivo target protein from its fusion to sfGFP if expressed as a soluble fusion product. Using this approach, we incorporated an azide reactive handle and identified residue positions amenable to conjugation with a fluorescence dye via strain-promoted azide-alkyne cycloaddition (SPAAC). Interestingly, best positions for efficient conjugation via SPAAC were residues whose native side chain were buried through analysis of their determined 3D structures and thus may not have been chosen through rational protein engineering. Molecular modeling suggests these buried native residues could become partially exposed on

  9. On the relation between residue flexibility and residue interactions in proteins.

    PubMed

    Yin, Hui; Li, Yi-Zhou; Li, Meng-Long

    2011-05-01

    B-factor from X-ray crystal structure can well measure protein structural flexibility, which plays an important role in different biological processes, such as catalysis, binding and molecular recognition. Understanding the essence of flexibility can be helpful for the further study of the protein function. In this study, we attempted to correlate the flexibility of a residue to its interactions with other residues by representing the protein structure as a residue contact network. Here, several well established network topological parameters were employed to feature such interactions. A prediction model was constructed for B-factor of a residue by using support vector regression (SVR). Pearson correlation coefficient (CC) was used as the performance measure. CC values were 0.63 and 0.62 for single amino acid and for the whole sequence, respectively. Our results revealed well correlations between B-factors and network topological parameters. This suggests that the protein structural flexibility could be well characterized by the inter-amino acid interactions in a protein.

  10. Chemical modification of lysozyme, glucose 6-phosphate dehydrogenase, and bovine eye lens proteins induced by peroxyl radicals: role of oxidizable amino acid residues.

    PubMed

    Arenas, Andrea; López-Alarcón, Camilo; Kogan, Marcelo; Lissi, Eduardo; Davies, Michael J; Silva, Eduardo

    2013-01-18

    Chemical and structural alterations to lysozyme (LYSO), glucose 6-phosphate dehydrogenase (G6PD), and bovine eye lens proteins (BLP) promoted by peroxyl radicals generated by the thermal decomposition of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) under aerobic conditions were investigated. SDS-PAGE analysis of the AAPH-treated proteins revealed the occurrence of protein aggregation, cross-linking, and fragmentation; BLP, which are naturally organized in globular assemblies, were the most affected proteins. Transmission electron microscopy (TEM) analysis of BLP shows the formation of complex protein aggregates after treatment with AAPH. These structural modifications were accompanied by the formation of protein carbonyl groups and protein hydroperoxides. The yield of carbonyls was lower than that for protein hydroperoxide generation and was unrelated to protein fragmentation. The oxidized proteins were also characterized by significant oxidation of Met, Trp, and Tyr (but not other) residues, and low levels of dityrosine. As the dityrosine yield is too low to account for the observed cross-linking, we propose that aggregation is associated with tryptophan oxidation and Trp-derived cross-links. It is also proposed that Trp oxidation products play a fundamental role in nonrandom fragmentation and carbonyl group formation particularly for LYSO and G6PD. These data point to a complex mechanism of peroxyl-radical mediated modification of proteins with monomeric (LYSO), dimeric (G6PD), and multimeric (BLP) structural organization, which not only results in oxidation of protein side chains but also gives rise to radical-mediated protein cross-links and fragmentation, with Trp species being critical intermediates.

  11. Sequence-Specific Solvent Accessibilities of Protein Residues in Unfolded Protein Ensembles

    PubMed Central

    Bernadó, Pau; Blackledge, Martin; Sancho, Javier

    2006-01-01

    Protein stability cannot be understood without the correct description of the unfolded state. We present here an efficient method for accurate calculation of atomic solvent exposures for denatured protein ensembles. The method used to generate the ensembles has been shown to reproduce diverse biophysical experimental data corresponding to natively and chemically unfolded proteins. Using a data set of 19 nonhomologous proteins containing from 98 to 579 residues, we report average accessibilities for all residue types. These averaged accessibilities are considerably lower than those previously reported for tripeptides and close to the lower limit reported by Creamer and co-workers. Of importance, we observe remarkable sequence dependence for the exposure to solvent of all residue types, which indicates that average residue solvent exposures can be inappropriate to interpret mutational studies. In addition, we observe smaller influences of both protein size and protein amino acid composition in the averaged residue solvent exposures for individual proteins. Calculating residue-specific solvent accessibilities within the context of real sequences is thus necessary and feasible. The approach presented here may allow a more precise parameterization of protein energetics as a function of polar- and apolar-area burial and opens new ways to investigate the energetics of the unfolded state of proteins. PMID:17012314

  12. Amino acid residues that define both the isoprenoid and CAAX preferences of the Saccharomyces cerevisiae protein farnesyltransferase. Creating the perfect farnesyltransferase.

    PubMed

    Caplin, B E; Ohya, Y; Marshall, M S

    1998-04-17

    Studies of the yeast protein farnesyltransferase (FTase) have shown that the enzyme preferentially farnesylates proteins ending in CAAX (C = cysteine, A = aliphatic residue, X = cysteine, serine, methionine, alanine) and to a lesser degree CAAL. Furthermore, like the type I protein geranylgeranyltransferase (GGTase-I), FTase can also geranylgeranylate methionine- and leucine-ending substrates both in vitro and in vivo. Substrate overlap of FTase and GGTase I has not been determined to be biologically significant. In this study, specific residues that influence the substrate preferences of FTase have been identified using site-directed mutagenesis. Three of the mutations altered the substrate preferences of the wild type enzyme significantly. The ram1p-74D FTase farnesylated only Ras-CIIS and not Ras-CII(M,L), and it geranylgeranylated all three substrates as well or better than wild type. The ram1p-206DDLF FTase farnesylated Ras-CII(S,M,L) at wild type levels but could no longer geranylgeranylate the Ras-CII(M,L) substrates. The ram1p-351FSKN FTase farnesylated Ras-CIIS and Ras-CIIM but not Ras-CIIL. The ram1p-351FSKN FTase was not capable of geranylgeranylating the Ras-CII(M,L) substrates, giving this mutant the attributes of the dogmatic FTase that only farnesylates non-leucine-ending CAAX substrates and does not geranylgeranylate any substrate. These results suggest that the isoprenoid and protein substrate specificities of FTase are interrelated. The availability of a mutant FTase that lacked substrate overlap with the protein GGTase-I made possible an analysis of the role of substrate overlap in normal cellular processes of yeast, such as mating and growth at elevated temperatures. Our findings suggest that neither farnesylation of leucine-ending CAAX substrates nor geranylgeranylation by the FTase is necessary for these cellular processes.

  13. Correlated Evolution of Nearby Residues in Drosophilid Proteins

    PubMed Central

    Callahan, Benjamin; Neher, Richard A.; Bachtrog, Doris; Andolfatto, Peter; Shraiman, Boris I.

    2011-01-01

    Here we investigate the correlations between coding sequence substitutions as a function of their separation along the protein sequence. We consider both substitutions between the reference genomes of several Drosophilids as well as polymorphisms in a population sample of Zimbabwean Drosophila melanogaster. We find that amino acid substitutions are “clustered” along the protein sequence, that is, the frequency of additional substitutions is strongly enhanced within ≈10 residues of a first such substitution. No such clustering is observed for synonymous substitutions, supporting a “correlation length” associated with selection on proteins as the causative mechanism. Clustering is stronger between substitutions that arose in the same lineage than it is between substitutions that arose in different lineages. We consider several possible origins of clustering, concluding that epistasis (interactions between amino acids within a protein that affect function) and positional heterogeneity in the strength of purifying selection are primarily responsible. The role of epistasis is directly supported by the tendency of nearby substitutions that arose on the same lineage to preserve the total charge of the residues within the correlation length and by the preferential cosegregation of neighboring derived alleles in our population sample. We interpret the observed length scale of clustering as a statistical reflection of the functional locality (or modularity) of proteins: amino acids that are near each other on the protein backbone are more likely to contribute to, and collaborate toward, a common subfunction. PMID:21383965

  14. Measuring residue associations in protein structures. Possible implications for protein folding.

    PubMed

    Karlin, S; Zuker, M; Brocchieri, L

    1994-06-03

    We propose a number of distance measures between residues in protein structures based on average, minimum and maximum distances of all atom (backbone and side-chain) coordinates or with respect to side-chain atom coordinates only. The d1-distance (D1-distance) refers to the average distance between side-chain (backbone and side-chain) atoms of a residue pair in a given structure. The dm-distance (Dm-distance) refers to the minimum distance between side-chain atoms (non-trivial minimum distance between all atoms of a residue pair). For each distance measure, averaging and normalizing over representative protein structures, association values and closeness orderings for all amino acid types are determined. The expected associations of side-chain interactions between oppositely charged residues, among hydrophobic residues and of cysteine with cysteine are confirmed. Several surprising associations are observed relative to (1) the aromatic residues tyrosine and tryptophan, but not phenylalanine; (2) multiple histidine residues; (3) asymmetries of arginine versus lysine, aspartate versus glutamate, alanine versus glycine, and asparagine versus glutamine; (4) absence of correlations of alpha-carbon distances with side-chain distances. The all atoms D1-distance attractions are dominated by steric relationships, with glycine and alanine significantly close to all amino acids, whereas large residues are under-associated with all residue types. In contrast, for the closeness ordering corresponding to the minimum side-chain dm-distance, glycine and alanine are among the least associated. However, in the d1-distance alanine is significantly close to all hydrophobic residues with the exception of tryptophan. The dm-distance preferences display a pervasive attraction for tyrosine by almost all residue types, the prominence of tyrosine and tryptophan in cation-aromatic interactions, and the versatility of histidine in functionality. The principal findings suggest a new

  15. Implication of Terminal Residues at Protein-Protein and Protein-DNA Interfaces.

    PubMed

    Martin, Olivier M F; Etheve, Loïc; Launay, Guillaume; Martin, Juliette

    2016-01-01

    Terminal residues of protein chains are charged and more flexible than other residues since they are constrained only on one side. Do they play a particular role in protein-protein and protein-DNA interfaces? To answer this question, we considered large sets of non-redundant protein-protein and protein-DNA complexes and analyzed the status of terminal residues and their involvement in interfaces. In protein-protein complexes, we found that more than half of terminal residues (62%) are either modified by attachment of a tag peptide (10%) or have missing coordinates in the analyzed structures (52%). Terminal residues are almost exclusively located at the surface of proteins (94%). Contrary to charged residues, they are not over or under-represented in protein-protein interfaces, but strongly prefer the peripheral region of interfaces when present at the interface (83% of terminal residues). The almost exclusive location of terminal residues at the surface of the proteins or in the rim regions of interfaces explains that experimental methods relying on tail hybridization can be successfully applied without disrupting the complexes under study. Concerning conformational rearrangement in protein-protein complexes, despite their expected flexibility, terminal residues adopt similar locations between the free and bound forms of the docking benchmark. In protein-DNA complexes, N-terminal residues are twice more frequent than C-terminal residues at interfaces. Both N-terminal and C-terminal residues are under-represented in interfaces, in contrast to positively charged residues, which are strongly favored. When located in protein-DNA interfaces, terminal residues prefer the periphery. N-terminal and C-terminal residues thus have particular properties with regard to interfaces, which cannot be reduced to their charged nature.

  16. Role of lysine and acidic amino acid residues on the insecticidal activity of Jackbean urease.

    PubMed

    Real-Guerra, Rafael; Carlini, Célia Regina; Stanisçuaski, Fernanda

    2013-09-01

    Canavalia ensiformis has three isoforms of urease: Jackbean urease (JBU), Jackbean urease II and canatoxin. These isoforms present several biological activities, independent from the enzymatic property, such as entomotoxicity and antifungal properties. The entomotoxic activity is a property of the whole protein, as well as of a 10 kDa peptide released by insect digestive enzymes. Here we have used chemical modification to observe the influence of lysines and acidic residues on JBU enzymatic and insecticidal activities. Chemical modification of lysine residues was performed with dimethylamine-borane complex and formaldehyde, and acidic residues were modified by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and ethylenediamine. Derivatized ureases, called JBU-Lys (lysine-modified) and JBU-Ac (acidic residues-modified), were assayed for their biochemical and insecticidal properties. Neither modification altered significantly the kinetic parameters analyzed, indicating that no residue critical for the enzyme activity was affected and that the modifications did not incur in any significant structural alteration. On the other hand, both modifications reduced the toxic activity of the native protein fed to Dysdercus peruvianus. The changes observed in the entomotoxic property of the derivatized proteins reflect alterations in different steps of JBU's toxicity towards insects. JBU-Ac is not susceptible to hydrolysis by insect digestive enzymes, hence impairing the release of toxic peptide(s), while JBU-Lys is processed as the native protein. On the other hand, the antidiuretic effect of JBU on Rhodnius prolixus is altered in JBU-Lys, but not in JBU-Ac. Altogether, these data emphasize the role of lysine and acidic residues on the insecticidal properties of ureases.

  17. Molecular insights into protein synthesis with proline residues.

    PubMed

    Melnikov, Sergey; Mailliot, Justine; Rigger, Lukas; Neuner, Sandro; Shin, Byung-Sik; Yusupova, Gulnara; Dever, Thomas E; Micura, Ronald; Yusupov, Marat

    2016-12-01

    Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono- and diprolyl-tRNAs. These structures provide a high-resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.

  18. Non-local residue-residue contacts in proteins are more conserved than local ones.

    PubMed

    Noivirt-Brik, Orly; Hazan, Gershon; Unger, Ron; Ofran, Yanay

    2013-02-01

    Non-covalent residue-residue contacts drive the folding of proteins and stabilize them. They may be local-i.e. involve residues that are close in sequence, or non-local. It has been suggested that, in most proteins, local contacts drive protein folding by providing crucial constraints of the conformational space, thus allowing proteins to fold. We compared residues that are involved in local contacts to residues that are involved in non-local contacts and found that, in most proteins, residues in non-local contacts are significantly more conserved evolutionarily than residues in local contacts. Moreover, non-local contacts are more structurally conserved: a contact between positions that are distant in sequence is more likely to exist in many structural homologues compared with a contact between positions that are close in sequence. These results provide new insights into the mechanisms of protein folding and may allow for better prediction of critical intra-chain contacts.

  19. Mutational analysis of the major coat protein of M13 identifies residues that control protein display.

    PubMed Central

    Weiss, G. A.; Wells, J. A.; Sidhu, S. S.

    2000-01-01

    We have reported variants of the M13 bacteriophage major coat protein (P8) that enable high copy display of monomeric and oligomeric proteins, such as human growth hormone and steptavidin, on the surface of phage particles (Sidhu SS, Weiss GA, Wells JA. 2000. High copy display of large proteins on phage for functional selections. J Mol Biol 296:487-495). Here, we explore how an optimized P8 variant (opti-P8) could evolve the ability to efficiently display a protein fused to its N-terminus. Reversion of individual opti-P8 residues back to the wild-type P8 residue identifies a limited set of hydrophobic residues responsible for the high copy protein display. These hydrophobic amino acids bracket a conserved hydrophobic face on the P8 alpha helix thought to be in contact with the phage coat. Mutations additively combine to promote high copy protein display, which was further enhanced by optimization of the linker between the phage coat and the fusion protein. These data are consistent with a model in which protein display-enhancing mutations allow for better packing of the fusion protein into the phage coat. The high tolerance for phage coat protein mutations observed here suggests that filamentous phage coat proteins could readily evolve new capabilities. PMID:10794407

  20. Prediction of binding modes between protein L-isoaspartyl (D-aspartyl) O-methyltransferase and peptide substrates including isomerized aspartic acid residues using in silico analytic methods for the substrate screening.

    PubMed

    Oda, Akifumi; Noji, Ikuhiko; Fukuyoshi, Shuichi; Takahashi, Ohgi

    2015-12-10

    Because the aspartic acid (Asp) residues in proteins are occasionally isomerized in the human body, not only l-α-Asp but also l-β-Asp, D-α-Asp and D-β-Asp are found in human proteins. In these isomerized aspartic acids, the proportion of D-β-Asp is the largest and the proportions of l-β-Asp and D-α-Asp found in human proteins are comparatively small. To explain the proportions of aspartic acid isomers, the possibility of an enzyme able to repair l-β-Asp and D-α-Asp is frequently considered. The protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) is considered one of the possible repair enzymes for l-β-Asp and D-α-Asp. Human PIMT is an enzyme that recognizes both l-β-Asp and D-α-Asp, and catalyzes the methylation of their side chains. In this study, the binding modes between PIMT and peptide substrates containing l-β-Asp or D-α-Asp residues were investigated using computational protein-ligand docking and molecular dynamics simulations. The results indicate that carboxyl groups of both l-β-Asp and D-α-Asp were recognized in similar modes by PIMT and that the C-terminal regions of substrate peptides were located in similar positions on PIMT for both the l-β-Asp and D-α-Asp peptides. In contrast, for peptides containing l-α-Asp or D-β-Asp residues, which are not substrates of PIMT, the computationally constructed binding modes between PIMT and peptides greatly differed from those between PIMT and substrates. In the nonsubstrate peptides, not inter- but intra-molecular hydrogen bonds were observed, and the conformations of peptides were more rigid than those of substrates. Thus, the in silico analytical methods were able to distinguish substrates from nonsubstrates and the computational methods are expected to complement experimental analytical methods.

  1. 40 CFR 180.289 - Methanearsonic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... residues. 180.289 Section 180.289 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.289 Methanearsonic acid; tolerances for residues. (a) General. Tolerances are established...

  2. 40 CFR 180.311 - Cacodylic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... residues. 180.311 Section 180.311 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.311 Cacodylic acid; tolerances for residues. (a) General. Tolerances are established for...

  3. 40 CFR 180.289 - Methanearsonic acid; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Methanearsonic acid; tolerances for...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.289 Methanearsonic acid; tolerances for residues. (a) General. Tolerances are established...

  4. 40 CFR 180.289 - Methanearsonic acid; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Methanearsonic acid; tolerances for...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.289 Methanearsonic acid; tolerances for residues. (a) General. Tolerances are established...

  5. 40 CFR 180.289 - Methanearsonic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Methanearsonic acid; tolerances for...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.289 Methanearsonic acid; tolerances for residues. (a) General. Tolerances are established...

  6. 40 CFR 180.289 - Methanearsonic acid; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Methanearsonic acid; tolerances for...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.289 Methanearsonic acid; tolerances for residues. (a) General. Tolerances are established...

  7. The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residue in ficin.

    PubMed

    Husain, S S; Lowe, G

    1970-04-01

    Ficin that had been prepared from the latex of Ficus glabrata by salt fractionation and chromatography on carboxymethylcellulose was completely and irreversibly inhibited with 1,3-dibromo[2-(14)C]acetone and then treated with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide in 6m-guanidinium chloride. After reduction and carboxymethylation of the labelled protein, it was digested with trypsin and alpha-chymotrypsin. Two radioactive peptides and two coloured peptides were isolated chromatographically and their sequences determined. The radioactive peptides revealed the amino acid sequences around the active-site cysteine and histidine residues and showed a high degree of homology with the omino acid sequence around the active-site cysteine and histidine residues in papain. The coloured peptides allowed the amino acid sequence around the buried cysteine residue in ficin to be determined.

  8. Mutation of G234 amino acid residue in Candida albicans drug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport

    PubMed Central

    Zhang, Shi-Qun; Miao, Qi; Li, Li-Ping; Zhang, Lu-lu; Yan, Lan; Jia, Yu; Cao, Yong-Bing; Jiang, Yuan-Ying

    2015-01-01

    Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca2+ did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport. PMID:26220356

  9. Mutation of G234 amino acid residue in candida albicans drug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport.

    PubMed

    Zhang, Shi-Qun; Miao, Qi; Li, Li-Ping; Zhang, Lu-Lu; Yan, Lan; Jia, Yu; Cao, Yong-Bing; Jiang, Yuan-Ying

    2015-01-01

    Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca(2+) did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport.

  10. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery.

    PubMed

    Kalescky, Robert; Zhou, Hongyu; Liu, Jin; Tao, Peng

    2016-04-01

    Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery.

  11. Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery

    PubMed Central

    Liu, Jin

    2016-01-01

    Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Châtelier’s principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery. PMID:27115535

  12. Protein packing: dependence on protein size, secondary structure and amino acid composition.

    PubMed

    Fleming, P J; Richards, F M

    2000-06-02

    We have used the occluded surface algorithm to estimate the packing of both buried and exposed amino acid residues in protein structures. This method works equally well for buried residues and solvent-exposed residues in contrast to the commonly used Voronoi method that works directly only on buried residues. The atomic packing of individual globular proteins may vary significantly from the average packing of a large data set of globular proteins. Here, we demonstrate that these variations in protein packing are due to a complex combination of protein size, secondary structure composition and amino acid composition. Differences in protein packing are conserved in protein families of similar structure despite significant sequence differences. This conclusion indicates that quality assessments of packing in protein structures should include a consideration of various parameters including the packing of known homologous proteins. Also, modeling of protein structures based on homologous templates should take into account the packing of the template protein structure.

  13. Graphical models of residue coupling in protein families.

    PubMed

    Thomas, John; Ramakrishnan, Naren; Bailey-Kellogg, Chris

    2008-01-01

    Many statistical measures and algorithmic techniques have been proposed for studying residue coupling in protein families. Generally speaking, two residue positions are considered coupled if, in the sequence record, some of their amino acid type combinations are significantly more common than others. While the proposed approaches have proven useful in finding and describing coupling, a significant missing component is a formal probabilistic model that explicates and compactly represents the coupling, integrates information about sequence,structure, and function, and supports inferential procedures for analysis, diagnosis, and prediction.We present an approach to learning and using probabilistic graphical models of residue coupling. These models capture significant conservation and coupling constraints observable ina multiply-aligned set of sequences. Our approach can place a structural prior on considered couplings, so that all identified relationships have direct mechanistic explanations. It can also incorporate information about functional classes, and thereby learn a differential graphical model that distinguishes constraints common to all classes from those unique to individual classes. Such differential models separately account for class-specific conservation and family-wide coupling, two different sources of sequence covariation. They are then able to perform interpretable functional classification of new sequences, explaining classification decisions in terms of the underlying conservation and coupling constraints. We apply our approach in studies of both G protein-coupled receptors and PDZ domains, identifying and analyzing family-wide and class-specific constraints, and performing functional classification. The results demonstrate that graphical models of residue coupling provide a powerful tool for uncovering, representing, and utilizing significant sequence structure-function relationships in protein families.

  14. Effect of the secondary structure of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) on the local dynamics of Trp residues.

    PubMed

    Albani, Jihad René

    2004-01-01

    We studied in this work the relation between the secondary structure of the carbohydrate residues of alpha1-acid glycoprotein and the local motions of Trp residues of the protein. We measured for this purpose the fluorescence emission intensity and anisotropy of the Trp residues between -46 and +30 degrees of the sialylated and asialylated protein. Our results indicate that, in both forms, the global profile of the emission intensity with temperature shows that Trp residues display static and collisional interaction with the neighboring amino acids. However, the profile of the asialylated form is more structured than that observed for the sialylated protein. The Y-plot analysis of the emission-anisotropy results indicated that the frictional resistance to rotation of the surface Trp residue is less important in the sialylated protein than in the asialylated form. This result is in good agreement with the fact that, in the asialylated conformation, the carbohydrate residues are closer to the protein surface than in the sialylated form, thereby increasing the contact of the surface Trp residue with the neighboring amino acids. Also, the interaction between the carbohydrate residues and the surface Trp residue contributes to the modification of the frictional resistance to rotation of the fluorophore.

  15. Identification of amino-acid residues in the V protein of peste des petits ruminants essential for interference and suppression of STAT-mediated interferon signaling

    SciTech Connect

    Ma, Xusheng; Yang, Xing; Nian, Xiaofeng; Zhang, Zhidong; Dou, Yongxi; Zhang, Xuehu; Luo, Xuenong; Su, Junhong; Zhu, Qiyun; Cai, Xuepeng

    2015-09-15

    Peste des petits ruminants virus (PPRV) causes a fatal disease in small ruminants. V protein of PPRV plays a pivotal role in interfering with host innate immunity by blocking IFNs signaling through interacting with STAT1 and STAT2. In the present study, the results demonstrated that PPRV V protein blocks IFN actions in a dose dependent manner and restrains the translocation of STAT1/2 proteins. We speculate that the translocation inhibition might be caused by the interfering of the downstream of STAT protein. Mutagenesis defines that Cys cluster and Trp motif of PPRV V protein are essential for STAT-mediated IFN signaling. These findings give a new sight for the further studies to understand the delicate mechanism of PPRV to escape the IFN signaling. - Highlights: • PPRV V protein inhibits type I IFN production and blocks its activation. • PPRV V protein negatively regulates activation of ISRE and GAS promoter. • PPRV V protein inhibits nuclear translocation of STAT protein by non-degradation. • PNT and VCT domain of PPRV V protein inhibit IFN transduction. • PPRV V protein binds with STAT protein via some conserved motifs.

  16. On the distribution of amino acid residues in transmembrane alpha-helix bundles.

    PubMed Central

    Samatey, F A; Xu, C; Popot, J L

    1995-01-01

    The periodic distribution of residues in the sequence of 469 putative transmembrane alpha-helices from eukaryotic plasma membrane polytopic proteins has been analyzed with correlation matrices. The method does not involve any a priori assumption about the secondary structure of the segments or about the physicochemical properties of individual amino acid residues. Maximal correlation is observed at 3.6 residues per period, characteristic of alpha-helices. A scale extracted from the data describes the propensity of the various residues to lie on the same or on opposite helix faces. The most polar face of transmembrane helices, presumably that buried in the protein core, shows a strong enrichment in aromatic residues, while residues likely to face the fatty acyl chains of lipids are largely aliphatic. PMID:7753846

  17. Prediction of functional residues in water channels and related proteins.

    PubMed Central

    Froger, A.; Tallur, B.; Thomas, D.; Delamarche, C.

    1998-01-01

    In this paper, we present an updated classification of the ubiquitous MIP (Major Intrinsic Protein) family proteins, including 153 fully or partially sequenced members available in public databases. Presently, about 30 of these proteins have been functionally characterized, exhibiting essentially two distinct types of channel properties: (1) specific water transport by the aquaporins, and (2) small neutral solutes transport, such as glycerol by the glycerol facilitators. Sequence alignments were used to predict amino acids and motifs discriminant in channel specificity. The protein sequences were also analyzed using statistical tools (comparisons of means and correspondence analysis). Five key positions were clearly identified where the residues are specific for each functional subgroup and exhibit high dissimilar physico-chemical properties. Moreover, we have found that the putative channels for small neutral solutes clearly differ from the aquaporins by the amino acid content and the length of predicted loop regions, suggesting a substrate filter function for these loops. From these results, we propose a signature pattern for water transport. PMID:9655351

  18. Missing strings of residues in protein crystal structures.

    PubMed

    Djinovic-Carugo, Kristina; Carugo, Oliviero

    2015-01-01

    A large fraction of the protein crystal structures deposited in the Protein Data Bank are incomplete, since the position of one or more residues is not reported, despite these residues are part of the material that was analyzed. This may bias the use of the protein crystal structures by molecular biologists. Here we observe that in the large majority of the protein crystal structures strings of residues are missing. Polar residues incline to occur in missing strings together with glycine, while apolar and aromatic residues tend to avoid them. Particularly flexible residues, as shown by their extremely high B-factors, by their exposure to the solvent and by their secondary structures, flank the missing strings. These data should be a helpful guideline for crystallographers that encounter regions of flat and uninterpretable electron density as well as end-users of crystal structures.

  19. Missing strings of residues in protein crystal structures

    PubMed Central

    Djinovic-Carugo, Kristina; Carugo, Oliviero

    2015-01-01

    A large fraction of the protein crystal structures deposited in the Protein Data Bank are incomplete, since the position of one or more residues is not reported, despite these residues are part of the material that was analyzed. This may bias the use of the protein crystal structures by molecular biologists. Here we observe that in the large majority of the protein crystal structures strings of residues are missing. Polar residues incline to occur in missing strings together with glycine, while apolar and aromatic residues tend to avoid them. Particularly flexible residues, as shown by their extremely high B-factors, by their exposure to the solvent and by their secondary structures, flank the missing strings. These data should be a helpful guideline for crystallographers that encounter regions of flat and uninterpretable electron density as well as end-users of crystal structures.

  20. Recovery of mercury from acid waste residues

    DOEpatents

    Greenhalgh, Wilbur O.

    1989-12-05

    Mercury can be recovered from nitric acid-containing fluids by reacting the fluid with aluminum metal to produce mercury metal, and then quenching the reactivity of the nitric acid prior to nitration of the mercury metal.

  1. Recovery of mercury from acid waste residues

    DOEpatents

    Greenhalgh, W.O.

    1987-02-27

    Mercury can be recovered from nitric acid-containing fluids by reacting the fluid with aluminum metal to produce mercury metal, and thence quenching the reactivity of the nitric acid prior to nitration of the mercury metal. 1 fig.

  2. Recovery of mercury from acid waste residues

    DOEpatents

    Greenhalgh, Wilbur O.

    1989-01-01

    Mercury can be recovered from nitric acid-containing fluids by reacting the fluid with aluminum metal to produce mercury metal, and then quenching the reactivity of the nitric acid prior to nitration of the mercury metal.

  3. 40 CFR 180.180 - Orthoarsenic acid; tolerance for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Orthoarsenic acid; tolerance for residues. 180.180 Section 180.180 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific...

  4. Fatty Acid Structure and Degradation Analysis in Fingerprint Residues.

    PubMed

    Pleik, Stefanie; Spengler, Bernhard; Schäfer, Thomas; Urbach, Dieter; Luhn, Steven; Kirsch, Dieter

    2016-09-01

    GC-MS investigations were carried out to elucidate the aging behavior of unsaturated fatty acids in fingerprint residues and to identify their degradation products in aged samples. For this purpose, a new sample preparation technique for fingerprint residues was developed that allows producing N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) derivatives of the analyzed unsaturated fatty acids and their degradation products. MSTFA derivatization catalyzed by iodotrimethylsilane enables the reliable identification of aldehydes and oxoacids as characteristic MSTFA derivatives in GCMS. The obtained results elucidate the degradation pathway of unsaturated fatty acids. Our study of aged fingerprint residues reveals that decanal is the main degradation product of the observed unsaturated fatty acids. Furthermore, oxoacids with different chain lengths are detected as specific degradation products of the unsaturated fatty acids. The detection of the degradation products and their chain length is a simple and effective method to determine the double bond position in unsaturated compounds. We can show that the hexadecenoic and octadecenoic acids found in fingerprint residues are not the pervasive fatty acids Δ9-hexadecenoic (palmitoleic acid) and Δ9-octadecenoic (oleic acid) acid but Δ6-hexadecenoic acid (sapienic acid) and Δ8-octadecenoic acid. The present study focuses on the structure identification of human sebum-specific unsaturated fatty acids in fingerprint residues based on the identification of their degradation products. These results are discussed for further investigations and method developments for age determination of fingerprints, which is still a tremendous challenge because of several factors affecting the aging behavior of individual compounds in fingerprints. Graphical Abstract ᅟ.

  5. Fatty Acid Structure and Degradation Analysis in Fingerprint Residues

    NASA Astrophysics Data System (ADS)

    Pleik, Stefanie; Spengler, Bernhard; Schäfer, Thomas; Urbach, Dieter; Luhn, Steven; Kirsch, Dieter

    2016-09-01

    GC-MS investigations were carried out to elucidate the aging behavior of unsaturated fatty acids in fingerprint residues and to identify their degradation products in aged samples. For this purpose, a new sample preparation technique for fingerprint residues was developed that allows producing N-methyl- N-trimethylsilyl-trifluoroacetamide (MSTFA) derivatives of the analyzed unsaturated fatty acids and their degradation products. MSTFA derivatization catalyzed by iodotrimethylsilane enables the reliable identification of aldehydes and oxoacids as characteristic MSTFA derivatives in GCMS. The obtained results elucidate the degradation pathway of unsaturated fatty acids. Our study of aged fingerprint residues reveals that decanal is the main degradation product of the observed unsaturated fatty acids. Furthermore, oxoacids with different chain lengths are detected as specific degradation products of the unsaturated fatty acids. The detection of the degradation products and their chain length is a simple and effective method to determine the double bond position in unsaturated compounds. We can show that the hexadecenoic and octadecenoic acids found in fingerprint residues are not the pervasive fatty acids Δ9-hexadecenoic (palmitoleic acid) and Δ9-octadecenoic (oleic acid) acid but Δ6-hexadecenoic acid (sapienic acid) and Δ8-octadecenoic acid. The present study focuses on the structure identification of human sebum-specific unsaturated fatty acids in fingerprint residues based on the identification of their degradation products. These results are discussed for further investigations and method developments for age determination of fingerprints, which is still a tremendous challenge because of several factors affecting the aging behavior of individual compounds in fingerprints.

  6. 40 CFR 180.550 - Arsanilic acid [(4-aminophenyl) arsonic acid]; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... for residues of the plant growth regulator arsanilic acid , in or on the following food commodities in...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific...

  7. 40 CFR 180.550 - Arsanilic acid [(4-aminophenyl) arsonic acid]; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... for residues of the plant growth regulator arsanilic acid , in or on the following food commodities in...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific...

  8. 40 CFR 180.550 - Arsanilic acid [(4-aminophenyl) arsonic acid]; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... for residues of the plant growth regulator arsanilic acid , in or on the following food commodities in...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific...

  9. Residue contact-count potentials are as effective as residue-residue contact-type potentials for ranking protein decoys

    PubMed Central

    Bolser, Dan M; Filippis, Ioannis; Stehr, Henning; Duarte, Jose; Lappe, Michael

    2008-01-01

    Background For over 30 years potentials of mean force have been used to evaluate the relative energy of protein structures. The most commonly used potentials define the energy of residue-residue interactions and are derived from the empirical analysis of the known protein structures. However, single-body residue 'environment' potentials, although widely used in protein structure analysis, have not been rigorously compared to these classical two-body residue-residue interaction potentials. Here we do not try to combine the two different types of residue interaction potential, but rather to assess their independent contribution to scoring protein structures. Results A data set of nearly three thousand monomers was used to compare pairwise residue-residue 'contact-type' propensities to single-body residue 'contact-count' propensities. Using a large and standard set of protein decoys we performed an in-depth comparison of these two types of residue interaction propensities. The scores derived from the contact-type and contact-count propensities were assessed using two different performance metrics and were compared using 90 different definitions of residue-residue contact. Our findings show that both types of score perform equally well on the task of discriminating between near-native protein decoys. However, in a statistical sense, the contact-count based scores were found to carry more information than the contact-type based scores. Conclusion Our analysis has shown that the performance of either type of score is very similar on a range of different decoys. This similarity suggests a common underlying biophysical principle for both types of residue interaction propensity. However, several features of the contact-count based propensity suggests that it should be used in preference to the contact-type based propensity. Specifically, it has been shown that contact-counts can be predicted from sequence information alone. In addition, the use of a single-body term allows

  10. The prediction of the degree of exposure to solvent of amino acid residues via genetic programming

    SciTech Connect

    Handley, S.

    1994-12-31

    In this paper I evolve programs that predict the degree of exposure to solvent (the buriedness) of amino acid residues given only the primary structure. I use genetic programming to evolve programs that take as input the primary structure and that output the buriedness of each residue. I trained these programs on a set of 82 proteins from the Brookhaven Protein Data Bank (PDB) and cross-validated them on a separate testing set of 40 proteins, also from the PDB. The best program evolved had a correlation of 0.434 between the predicted and observed buriednesses on the testing set.

  11. Protein and amino acid nutrition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dairy cow protein and amino acid nutrition have a significant role in sustainable dairying. Protein, amino acids, and nitrogen are inextricably linked through effects in the rumen, metabolism of the cow, and environmental nutrient management. Feeding systems have been making progress toward emphasiz...

  12. Microscopic residues of bone from dissolving human remains in acids.

    PubMed

    Vermeij, Erwin; Zoon, Peter; van Wijk, Mayonne; Gerretsen, Reza

    2015-05-01

    Dissolving bodies is a current method of disposing of human remains and has been practiced throughout the years. During the last decade in the Netherlands, two cases have emerged in which human remains were treated with acid. In the first case, the remains of a cremated body were treated with hydrofluoric acid. In the second case, two complete bodies were dissolved in a mixture of hydrochloric and sulfuric acid. In both cases, a great variety of evidence was collected at the scene of crime, part of which was embedded in resin, polished, and investigated using SEM/EDX. Apart from macroscopic findings like residual bone and artificial teeth, in both cases, distinct microscopic residues of bone were found as follows: (partly) digested bone, thin-walled structures, and recrystallized calcium phosphate. Although some may believe it is possible to dissolve a body in acid completely, at least some of these microscopic residues will always be found.

  13. Protein splicing of inteins with atypical glutamine and aspartate C-terminal residues.

    PubMed

    Amitai, Gil; Dassa, Bareket; Pietrokovski, Shmuel

    2004-01-30

    Inteins are protein-splicing domains present in many proteins. They self-catalyze their excision from the host protein, ligating their former flanks by a peptide bond. The C-terminal residue of inteins is typically an asparagine (Asn). Cyclization of this residue to succinimide causes the final detachment of inteins from their hosts. We studied protein-splicing activity of two inteins with atypical C-terminal residues. One having a C-terminal glutamine (Gln), isolated from Chilo iridescent virus (CIV), and another unique intein, first reported here, with a C-terminal aspartate, isolated from Carboxydothermus hydrogenoformans (Chy). Protein-splicing activity was examined in the wild-type inteins and in several mutants with N- and C-terminal amino acid substitutions. We demonstrate that both wild-type inteins can protein splice, probably by new variations of the typical protein-splicing mechanism. Substituting the atypical C-terminal residue to the typical Asn retained protein-splicing only in the CIV intein. All diverse C-terminal substitutions in the Chy intein (Asp(345) to Asn, Gln, Glu, and Ala) abolished protein-splicing and generated N- and C-terminal cleavage. The observed C-terminal cleavage in the Chy intein ending with Ala cannot be explained by cyclization of this residue. We present and discuss several new models for reactions in the protein-splicing pathway.

  14. Progesterone binding to the tryptophan residues of human alpha1-acid glycoprotein.

    PubMed

    Albani, J R

    2006-11-06

    Binding studies between progesterone and alpha1-acid glycoprotein allowed us to demonstrate that the binding site of progesterone contains one hydrophobic tryptophan residue and that the structure of the protein is not altered upon binding. The data obtained at saturated concentrations of progesterone clearly reveal the type of interaction at physiological levels.

  15. Acid transformation of bauxite residue: Conversion of its alkaline characteristics.

    PubMed

    Kong, Xiangfeng; Li, Meng; Xue, Shengguo; Hartley, William; Chen, Chengrong; Wu, Chuan; Li, Xiaofei; Li, Yiwei

    2017-02-15

    Bauxite residue (BR) is a highly alkaline solid hazardous waste produced from bauxite processing for alumina production. Alkaline transformation appears to reduce the environmental risk of bauxite residue disposal areas (BRDAs) whilst potentially providing opportunities for the sustainable reuse and on-going management of BR. Mineral acids, a novel citric acid and a hybrid combination of acid-gypsum treatments were investigated for their potential to reduce residue pH and total alkalinity and transform the alkaline mineral phase. XRD results revealed that with the exception of andradite, the primary alkaline solid phases of cancrinite, grossular and calcite were transformed into discriminative products based on the transformation used. Supernatants separated from BR and transformed bauxite residue (TBR) displayed distinct changes in soluble Na, Ca and Al, and a reduction in pH and total alkalinity. SEM images suggest that mineral acid transformations promote macro-aggregate formation, and the positive promotion of citric acid, confirming the removal or reduction in soluble and exchangeable Na. NEXAFS analysis of Na K-edge revealed that the chemical speciation of Na in TBRs was consistent with BR. Three acid treatments and gypsum combination had no effect on Na speciation, which affects the distribution of Na revealed by sodium STXM imaging.

  16. Electron microscopy of carbonaceous matter in Allende acid residues

    NASA Technical Reports Server (NTRS)

    Lumpkin, G. R.

    1982-01-01

    On the basis of characteristic diffuse ring diffraction patterns, much of the carbonaceous matter in a large suite of Allende acid residues has been identified as a variety of turbostratic carbon. Crystallites of this phase contain randomly stacked sp(2) hybridized carbon layers and diffraction patterns resemble those from carbon black and glassy carbon. Carbynes are probably absent, and are certainly restricted to less than 0.5% of these acid residues. The work of Ott et al. (1981) provides a basis for the possibility that turbostratic carbon is a carrier of noble gases, but an additional component - amorphous carbon - may be necessary to explain the high release temperatures of noble gases as well as the glassy character of many of the carbonaceous particles. Carbynes are considered to be questionable as important carriers of noble gases in the Allende acid residues.

  17. Oxidation and inactivation of SERCA by selective reaction of cysteine residues with amino acid peroxides.

    PubMed

    Dremina, Elena S; Sharov, Victor S; Davies, Michael J; Schöneich, Christian

    2007-10-01

    The oxidative modification of proteins plays an important role in a wide range of pathological processes and aging. Proteins are modified by numerous biologic oxidants including hydrogen peroxide, peroxynitrite, singlet oxygen, and oxygen- and nitrogen-centered radicals. More recently, an additional class of physiologically important oxidants has been identified, peptide and protein peroxides. The latter react quite rapidly and selectively with protein cysteine residues. The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is reversibly regulated through NO-dependent S-glutathiolation of specific cysteine residues. The irreversible oxidation of these cysteine residues could, therefore, impair NO-dependent muscle relaxation. Here, we show that specific protein-derived (amino acid) peroxides react selectively with a subset of the 22 reduced cysteine residues of SERCA1, including a peptide-containing Cys674 and Cys675, where Cys674 (in SERCA2) represents one of the targets for NO-dependent S-glutathiolation. Out of 11 tested amino acid, peptide, and protein peroxides, those derived from free tryptophan and free tyrosine showed the highest reactivity towards SERCA, while no oxidation under similar experimental conditions was detected through hydrogen peroxide. Among the peroxides from tryptophan, those of free tryptophan showed a significantly higher reactivity as compared to those from N- and C-terminally blocked tryptophan. Quantitative HPLC-MS/MS analysis demonstrated that the highest reactivity of the tryptophan-derived peroxides was observed for Cys774 and Cys938, cysteine residues, which are embedded within the transmembrane domains of SERCA1. This unusual reactivity of transmembrane domains cannot be solely rationalized by the hydrophobicity of the oxidant, as the peroxide from dl-tryptophan shows considerable higher reactivity as compared to the one derived from N-acetyl-tryptophan methyl ester. Our data demonstrate a potential role of peptide- and protein

  18. Amino acid sequences of proteins from Leptospira serovar pomona.

    PubMed

    Alves, S F; Lefebvre, R B; Probert, W

    2000-01-01

    This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.

  19. The functions of tryptophan residues in membrane proteins

    SciTech Connect

    Schiffer, M.; Chang, C.H.; Stevens, F.J.

    1994-08-01

    Membrane proteins in general have a significantly higher Trp content than do soluble proteins. This is especially true for the M and L subunits of the photosynthetic reaction center from purple bacteria. The Trp residues are located mostly in the segments that connect the transmembrane helices. Further, they are concentrated at the periplasmic side of the complex. Within the protein subunits, many form hydrogen bonds with carbonyl oxygens of the main chain, thereby stabilizing the protein. On the surface of the molecule, they are correctly positioned to form hydrogen bonds with the lipid head groups while their hydrophobic rings are immersed in the lipid part of the bilayer. We suggest that Trp residues are involved in the translocation of protein through the membrane and that following translocation, Trp residues serve as anchors on the periplasmic side of the membrane.

  20. Prediction of residue-residue contact matrix for protein-protein interaction with Fisher score features and deep learning.

    PubMed

    Du, Tianchuan; Liao, Li; Wu, Cathy H; Sun, Bilin

    2016-11-01

    Protein-protein interactions play essential roles in many biological processes. Acquiring knowledge of the residue-residue contact information of two interacting proteins is not only helpful in annotating functions for proteins, but also critical for structure-based drug design. The prediction of the protein residue-residue contact matrix of the interfacial regions is challenging. In this work, we introduced deep learning techniques (specifically, stacked autoencoders) to build deep neural network models to tackled the residue-residue contact prediction problem. In tandem with interaction profile Hidden Markov Models, which was used first to extract Fisher score features from protein sequences, stacked autoencoders were deployed to extract and learn hidden abstract features. The deep learning model showed significant improvement over the traditional machine learning model, Support Vector Machines (SVM), with the overall accuracy increased by 15% from 65.40% to 80.82%. We showed that the stacked autoencoders could extract novel features, which can be utilized by deep neural networks and other classifiers to enhance learning, out of the Fisher score features. It is further shown that deep neural networks have significant advantages over SVM in making use of the newly extracted features.

  1. Proline and lysine residues provide modulatory switches in amyloid formation: Insights from prion protein.

    PubMed

    Kraus, Allison

    2016-01-01

    Amyloidogenic proteins have an increased propensity to reorganize into the highly structured, β sheet rich structures that characterize amyloid. The probability of attaining these highly structured assemblies is influenced by multiple factors, including amino acid composition and environmental conditions. Evolutionary selection for amino acid sequences that prevent amyloid formation could further modulate amyloid-forming propensity. Indeed, we have recently identified specific proline and lysine residues, contained within a highly conserved central region of prion protein (PrP), that impede PrP amyloid formation in vitro. These prolines are mutated in certain forms of the human familial genetic disease, Gerstmann-Straüssler-Schneiker (GSS) syndrome. Here, I discuss the influence of these proline and lysine residues on PrP amyloid formation and how such anti-amyloidogenic primary amino acid sequences might be modulated to influence protein amyloidogenicity.

  2. Electrocatalysis in proteins, nucleic acids and carbohydrates.

    PubMed

    Paleček, Emil; Bartošík, Martin; Ostatná, Veronika; Trefulka, Mojmír

    2012-02-01

    The ability of proteins to catalyze hydrogen evolution has been known for more than 80 years, but the poorly developed d.c. polarographic "pre-sodium wave" was of little analytical use. Recently, we have shown that by using constant current chronopotentiometric stripping analysis, proteins produce a well-developed peak H at hanging mercury drop and solid amalgam electrodes. Peak H sensitively reflects changes in protein structures due to protein denaturation, single amino acid exchange, etc. at the picomole level. Unmodified DNA and RNA do not yield such a peak, but they produce electrocatalytic voltammetric signals after modification with osmium tetroxide complexes with nitrogen ligands [Os(VIII)L], binding covalently to pyrimidine bases in nucleic acids. Recently, it has been shown that six-valent [Os(VI)L] complexes bind to 1,2-diols in polysaccharides and oligosaccharides, producing voltammetric responses similar to those of DNA-Os(VIII)L adducts. Electrocatalytic peaks produced by Os-modified nucleic acids, proteins (reaction with tryptophan residues) and carbohydrates are due to the catalytic hydrogen evolution, allowing determination of oligomers at the picomolar level.

  3. 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.

  4. Toward fluorescence detection of protein residues on surgical instruments

    NASA Astrophysics Data System (ADS)

    Richardson, Patricia R.; Jones, Anita C.; Baxter, Robert L.; Baxter, Helen C.; Whittaker, A. Gavin; Campbell, Gaynor A.

    2004-06-01

    Prion proteins are the infectious agents that cause Creutzfeldt-Jakob Disease (CJD) in humans. These proteins are particularly resistant to normal sterilization procedures, and the theoretical risk of prion transmission via surgical instruments is of current public and professional concern. We are currently investigating fluorescence methods for the detection of proteins on surfaces, with a view to developing an optical-fiber-based system for routine, online monitoring of residual protein contamination on surgical instruments, in hospital sterilization departments. This paper presents preliminary results on the detection of femtomole amounts of fluorescently labelled protein on surgical steel and discusses some of the problems involved in the detection of fluorescence from metal samples.

  5. Lactic Acid and Biosurfactants Production from Residual Cellulose Films.

    PubMed

    Portilla Rivera, Oscar Manuel; Arzate Martínez, Guillermo; Jarquín Enríquez, Lorenzo; Vázquez Landaverde, Pedro Alberto; Domínguez González, José Manuel

    2015-11-01

    The increasing amounts of residual cellulose films generated as wastes all over the world represent a big scale problem for the meat industry regarding to environmental and economic issues. The use of residual cellulose films as a feedstock of glucose-containing solutions by acid hydrolysis and further fermentation into lactic acid and biosurfactants was evaluated as a method to diminish and revalorize these wastes. Under a treatment consisting in sulfuric acid 6% (v/v); reaction time 2 h; solid liquid ratio 9 g of film/100 mL of acid solution, and temperature 130 °C, 35 g/L of glucose and 49% of solubilized film was obtained. From five lactic acid strains, Lactobacillus plantarum was the most suitable for metabolizing the glucose generated. The process was scaled up under optimized conditions in a 2-L bioreactor, producing 3.4 g/L of biomass, 18 g/L of lactic acid, and 15 units of surface tension reduction of a buffer phosphate solution. Around 50% of the cellulose was degraded by the treatment applied, and the liqueurs generated were useful for an efficient production of lactic acid and biosurfactants using L. plantarum. Lactobacillus bacteria can efficiently utilize glucose from cellulose films hydrolysis without the need of clarification of the liqueurs.

  6. Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.

    PubMed Central

    Ibáñez, C F; Hallböök, F; Ebendal, T; Persson, H

    1990-01-01

    Selected amino acid residues in chicken nerve growth factor (NGF) were replaced by site-directed mutagenesis. Mutated NGF sequences were transiently expressed in COS cells and the yield of NGF protein in conditioned medium was quantified by Western blotting. Binding of each mutant to NGF receptors on PC12 cells was evaluated in a competition assay. The biological activity was determined by measuring stimulation of neurite outgrowth from chick sympathetic ganglia. The residues homologous to the proposed receptor binding site of insulin (Ser18, Met19, Val21, Asp23) were substituted by Ala. Replacement of Ser18, Met19 and Asp23 did not affect NGF activity. Modification of Val21 notably reduced both receptor binding and biological activity, suggesting that this residue is important to retain a fully active NGF. The highly conserved Tyr51 and Arg99 were converted into Phe and Lys respectively, without changing the biological properties of the molecule. However, binding and biological activity were greatly impaired after the simultaneous replacement of both Arg99 and Arg102 by Gly. The three conserved Trp residues at positions 20, 75 and 98 were substituted by Phe. The Trp mutated proteins retained 15-60% of receptor binding and 40-80% of biological activity, indicating that the Trp residues are not essential for NGF activity. However, replacement of Trp20 significantly reduced the amount of NGF in the medium, suggesting that this residue may be important for protein stability. Images Fig. 4. PMID:2328722

  7. Graphlet kernels for prediction of functional residues in protein structures.

    PubMed

    Vacic, Vladimir; Iakoucheva, Lilia M; Lonardi, Stefano; Radivojac, Predrag

    2010-01-01

    We introduce a novel graph-based kernel method for annotating functional residues in protein structures. A structure is first modeled as a protein contact graph, where nodes correspond to residues and edges connect spatially neighboring residues. Each vertex in the graph is then represented as a vector of counts of labeled non-isomorphic subgraphs (graphlets), centered on the vertex of interest. A similarity measure between two vertices is expressed as the inner product of their respective count vectors and is used in a supervised learning framework to classify protein residues. We evaluated our method on two function prediction problems: identification of catalytic residues in proteins, which is a well-studied problem suitable for benchmarking, and a much less explored problem of predicting phosphorylation sites in protein structures. The performance of the graphlet kernel approach was then compared against two alternative methods, a sequence-based predictor and our implementation of the FEATURE framework. On both tasks, the graphlet kernel performed favorably; however, the margin of difference was considerably higher on the problem of phosphorylation site prediction. While there is data that phosphorylation sites are preferentially positioned in intrinsically disordered regions, we provide evidence that for the sites that are located in structured regions, neither the surface accessibility alone nor the averaged measures calculated from the residue microenvironments utilized by FEATURE were sufficient to achieve high accuracy. The key benefit of the graphlet representation is its ability to capture neighborhood similarities in protein structures via enumerating the patterns of local connectivity in the corresponding labeled graphs.

  8. Modeling the oxidation of methionine residues by peroxides in proteins.

    PubMed

    Chennamsetty, Naresh; Quan, Yong; Nashine, Vishal; Sadineni, Vikram; Lyngberg, Olav; Krystek, Stanley

    2015-04-01

    We report the use of molecular modeling to predict the oxidation propensity of methionine residues in proteins. Oxidation of methionine to the sulfoxide form is one of the major degradation pathways for therapeutic proteins. Oxidation can occur during production, formulation, or storage of pharmaceuticals and it often reduces or eliminates biological activity. We use a molecular model based on atomistic simulations called 2-shell water coordination number to predict the oxidation rates for several model proteins and therapeutic candidates. In addition, we implement models that are based on static and simulation average of the solvent-accessible area (SAA) for either the side chain or the sulfur atom in the methionine residue. We then compare the results from the different models against the experimentally measured relative rates of methionine oxidation. We find that both the 2-shell model and the simulation-averaged SAA models are accurate in predicting the oxidation propensity of methionine residues for the proteins tested. We also find the appropriate parameter ranges where the models are most accurate. These models have significant predictive power and can be used to enable further protein engineering or to guide formulation approaches in stabilizing the unstable methionine residues.

  9. Polymorphisms at Amino Acid Residues 141 and 154 Influence Conformational Variation in Ovine PrP

    PubMed Central

    Yang, Sujeong; Thackray, Alana M.; Hopkins, Lee; Monie, Tom P.; Burke, David F.; Bujdoso, Raymond

    2014-01-01

    Polymorphisms in ovine PrP at amino acid residues 141 and 154 are associated with susceptibility to ovine prion disease: Leu141Arg154 with classical scrapie and Phe141Arg154 and Leu141His154 with atypical scrapie. Classical scrapie is naturally transmissible between sheep, whereas this may not be the case with atypical scrapie. Critical amino acid residues will determine the range or stability of structural changes within the ovine prion protein or its functional interaction with potential cofactors, during conversion of PrPC to PrPSc in these different forms of scrapie disease. Here we computationally identified that regions of ovine PrP, including those near amino acid residues 141 and 154, displayed more conservation than expected based on local structural environment. Molecular dynamics simulations showed these conserved regions of ovine PrP displayed genotypic differences in conformational repertoire and amino acid side-chain interactions. Significantly, Leu141Arg154 PrP adopted an extended beta sheet arrangement in the N-terminal palindromic region more frequently than the Phe141Arg154 and Leu141His154 variants. We supported these computational observations experimentally using circular dichroism spectroscopy and immunobiochemical studies on ovine recombinant PrP. Collectively, our observations show amino acid residues 141 and 154 influence secondary structure and conformational change in ovine PrP that may correlate with different forms of scrapie. PMID:25126555

  10. Oxidative modification of native protein residues using cerium(IV) ammonium nitrate.

    PubMed

    Seim, Kristen L; Obermeyer, Allie C; Francis, Matthew B

    2011-10-26

    A new protein modification strategy has been developed that is based on an oxidative coupling reaction that targets electron-rich amino acids. This strategy relies on cerium(IV) ammonium nitrate (CAN) as an oxidation reagent and results in the coupling of tyrosine and tryptophan residues to phenylene diamine and anisidine derivatives. The methodology was first identified and characterized on peptides and small molecules, and was subsequently adapted for protein modification by determining appropriate buffer conditions. Using the optimized procedure, native and introduced solvent-accessible residues on proteins were selectively modified with polyethylene glycol (PEG) and small peptides. This unprecedented bioconjugation strategy targets these under-utilized amino acids with excellent chemoselectivity and affords good-to-high yields using low concentrations of the oxidant and coupling partners, short reaction times, and mild conditions.

  11. Mutagenesis of conserved amino acids of Helicobacter pylori fur reveals residues important for function.

    PubMed

    Carpenter, Beth M; Gancz, Hanan; Benoit, Stéphane L; Evans, Sarah; Olsen, Cara H; Michel, Sarah L J; Maier, Robert J; Merrell, D Scott

    2010-10-01

    The ferric uptake regulator (Fur) of the medically important pathogen Helicobacter pylori is unique in that it has been shown to function as a repressor both in the presence of an Fe2+ cofactor and in its apo (non-Fe2+-bound) form. However, virtually nothing is known concerning the amino acid residues that are important for Fur functioning. Therefore, mutations in six conserved amino acid residues of H. pylori Fur were constructed and analyzed for their impact on both iron-bound and apo repression. In addition, accumulation of the mutant proteins, protein secondary structure, DNA binding ability, iron binding capacity, and the ability to form higher-order structures were also examined for each mutant protein. While none of the mutated residues completely abrogated the function of Fur, we were able to identify residues that were critical for both iron-bound and apo-Fur repression. One mutation, V64A, did not alter regulation of any target genes. However, each of the five remaining mutations showed an effect on either iron-bound or apo regulation. Of these, H96A, E110A, and E117A mutations altered iron-bound Fur regulation and were all shown to influence iron binding to different extents. Additionally, the H96A mutation was shown to alter Fur oligomerization, and the E110A mutation was shown to impact oligomerization and DNA binding. Conversely, the H134A mutant exhibited changes in apo-Fur regulation that were the result of alterations in DNA binding. Although the E90A mutant exhibited alterations in apo-Fur regulation, this mutation did not affect any of the assessed protein functions. This study is the first for H. pylori to analyze the roles of specific amino acid residues of Fur in function and continues to highlight the complexity of Fur regulation in this organism.

  12. Solvent accessibility, residue charge and residue volume, the three ingredients of a robust amino acid substitution matrix.

    PubMed

    Goodarzi, Hani; Katanforoush, Ali; Torabi, Noorossadat; Najafabadi, Hamed Shateri

    2007-04-21

    Cost measure matrices or different amino acid indices have been widely used for studies in many fields of biology. One major criticism of these studies might be based on the unavailability of an unbiased and yet effective amino acid substitution matrix. Throughout this study we have devised a cost measure matrix based on the solvent accessibility, residue charge, and residue volume indices. Performed analyses on this novel substitution matrix (i.e. solvent accessibility charge volume (SCV) matrix) support the uncontaminated nature of this matrix regarding the genetic code. Although highly similar to a number of previously available cost measure matrices, the SCV matrix results in a more significant optimality in the error-buffering capacity of the genetic code when compared to many other amino acid substitution matrices. Besides, a method to compare an SCV-based scoring matrix with a number of widely used matrices has been devised, the results of which highlights the robustness of this matrix in protein family discrimination.

  13. FastRNABindR: Fast and Accurate Prediction of Protein-RNA Interface Residues.

    PubMed

    El-Manzalawy, Yasser; Abbas, Mostafa; Malluhi, Qutaibah; Honavar, Vasant

    2016-01-01

    A wide range of biological processes, including regulation of gene expression, protein synthesis, and replication and assembly of many viruses are mediated by RNA-protein interactions. However, experimental determination of the structures of protein-RNA complexes is expensive and technically challenging. Hence, a number of computational tools have been developed for predicting protein-RNA interfaces. Some of the state-of-the-art protein-RNA interface predictors rely on position-specific scoring matrix (PSSM)-based encoding of the protein sequences. The computational efforts needed for generating PSSMs severely limits the practical utility of protein-RNA interface prediction servers. In this work, we experiment with two approaches, random sampling and sequence similarity reduction, for extracting a representative reference database of protein sequences from more than 50 million protein sequences in UniRef100. Our results suggest that random sampled databases produce better PSSM profiles (in terms of the number of hits used to generate the profile and the distance of the generated profile to the corresponding profile generated using the entire UniRef100 data as well as the accuracy of the machine learning classifier trained using these profiles). Based on our results, we developed FastRNABindR, an improved version of RNABindR for predicting protein-RNA interface residues using PSSM profiles generated using 1% of the UniRef100 sequences sampled uniformly at random. To the best of our knowledge, FastRNABindR is the only protein-RNA interface residue prediction online server that requires generation of PSSM profiles for query sequences and accepts hundreds of protein sequences per submission. Our approach for determining the optimal BLAST database for a protein-RNA interface residue classification task has the potential of substantially speeding up, and hence increasing the practical utility of, other amino acid sequence based predictors of protein-protein and protein

  14. Intramolecular cyclization of aspartic acid residues assisted by three water molecules: a density functional theory study

    NASA Astrophysics Data System (ADS)

    Takahashi, Ohgi; Kirikoshi, Ryota

    2014-01-01

    Aspartic acid (Asp) residues in peptides and proteins (l-Asp) are known to undergo spontaneous nonenzymatic reactions to form l-β-Asp, d-Asp, and d-β-Asp residues. The formation of these abnormal Asp residues in proteins may affect their three-dimensional structures and hence their properties and functions. Indeed, the reactions have been thought to contribute to aging and pathologies. Most of the above reactions of the l-Asp residues proceed via a cyclic succinimide intermediate. In this paper, a novel three-water-assisted mechanism is proposed for cyclization of an Asp residue (forming a gem-diol precursor of the succinimide) by the B3LYP/6-31 + G(d,p) density functional theory calculations carried out for an Asp-containing model compound (Ace-Asp-Nme, where Ace = acetyl and Nme = NHCH3). The three water molecules act as catalysts by mediating ‘long-range’ proton transfers. In the proposed mechanism, the amide group on the C-terminal side of the Asp residue is first converted to the tautomeric iminol form (iminolization). Then, reorientation of a water molecule and a conformational change occur successively, followed by the nucleophilic attack of the iminol nitrogen on the carboxyl carbon of the Asp side chain to form the gem-diol species. A satisfactory agreement was obtained between the calculated and experimental energetics.

  15. Residue network in protein native structure belongs to the universality class of a three-dimensional critical percolation cluster.

    PubMed

    Morita, Hidetoshi; Takano, Mitsunori

    2009-02-01

    Single protein molecules are regarded as contact networks of amino-acid residues. Relationships between the shortest path lengths and the numbers of residues within single molecules in the native structures are examined for various sized proteins. A universal scaling among proteins is obtained, which shows that the residue networks are fractal networks. This universal fractal network is characterized with three kinds of dimensions: the network topological dimension D{c} approximately 1.9 , the fractal dimension D{f} approximately 2.5 , and the spectral dimension D{s} approximately 1.3 . These values are in surprisingly good coincidence with those of the three-dimensional critical percolation cluster. Hence the residue contact networks in the protein native structures belong to the universality class of the three-dimensional percolation cluster. The criticality is relevant to the ambivalence in the protein native structures, the coexistence of stability and instability, both of which are necessary for protein functions.

  16. Residue network in protein native structure belongs to the universality class of a three-dimensional critical percolation cluster

    NASA Astrophysics Data System (ADS)

    Morita, Hidetoshi; Takano, Mitsunori

    2009-02-01

    Single protein molecules are regarded as contact networks of amino-acid residues. Relationships between the shortest path lengths and the numbers of residues within single molecules in the native structures are examined for various sized proteins. A universal scaling among proteins is obtained, which shows that the residue networks are fractal networks. This universal fractal network is characterized with three kinds of dimensions: the network topological dimension Dc≈1.9 , the fractal dimension Df≈2.5 , and the spectral dimension Ds≈1.3 . These values are in surprisingly good coincidence with those of the three-dimensional critical percolation cluster. Hence the residue contact networks in the protein native structures belong to the universality class of the three-dimensional percolation cluster. The criticality is relevant to the ambivalence in the protein native structures, the coexistence of stability and instability, both of which are necessary for protein functions.

  17. Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein.

    PubMed Central

    Zhang, Dang-Quan; Liu, Bing; Feng, Dong-Ru; He, Yan-Ming; Wang, Shu-Qi; Wang, Hong-Bin; Wang, Jin-Fa

    2004-01-01

    The approximately 24-amino-acid leucine-rich tandem repeat motif (PXXXXXLXXLXXLXLSXNXLXGXI) of carrot antifreeze protein comprises most of the processed protein and should contribute at least partly to the ice-binding site. Structural predictions using publicly available online sources indicated that the theoretical three-dimensional model of this plant protein includes a 10-loop beta-helix containing the approximately 24-amino-acid tandem repeat. This theoretical model indicated that conservative asparagine residues create putative ice-binding sites with surface complementarity to the 1010 prism plane of ice. We used site-specific mutagenesis to test the importance of these residues, and observed a distinct loss of thermal hysteresis activity when conservative asparagines were replaced with valine or glutamine, whereas a large increase in thermal hysteresis was observed when phenylalanine or threonine residues were replaced with asparagine, putatively resulting in the formation of an ice-binding site. These results confirmed that the ice-binding site of carrot antifreeze protein consists of conservative asparagine residues in each beta-loop. We also found that its thermal hysteresis activity is directly correlated with the length of its asparagine-rich binding site, and hence with the size of its ice-binding face. PMID:14531728

  18. Methodology for detecting residual phosphoric acid in polybenzoxazole fibers.

    PubMed

    Park, Eun Su; Sieber, John; Guttman, Charles; Rice, Kirk; Flynn, Kathleen; Watson, Stephanie; Holmes, Gale

    2009-12-01

    Because of the premature failure of in-service soft-body armor containing the ballistic fiber poly[(benzo-[1,2-d:5,4-d']-benzoxazole-2,6-diyl)-1,4-phenylene] (PBO), the Office of Law Enforcement Standards (OLES) at the National Institute of Standards and Technology (NIST) initiated a research program to investigate the reasons for this failure and to develop testing methodologies and protocols to ensure that these types of failures do not reoccur. In a report that focused on the stability of the benzoxazole ring that is characteristic of PBO fibers, Holmes, G. A.; Rice, K.; Snyder, C. R. J. Mater. Sci. 2006, 41, 4105-4116, showed that the benzoxazole ring was susceptible to hydrolytic degradation under acid conditions. Because of the processing conditions for the fibers, it is suspected by many researchers that residual phosphoric acid may cause degradation of the benzoxazole ring resulting in a reduction of ballistic performance. Prior to this work, no definitive data have indicated the presence of phosphoric acid since the residual phosphorus is not easily extracted and the processed fibers are known to incorporate phosphorus containing processing aids. Methods to efficiently extract phosphorus from PBO are described in this article. Further, characterization determined that the majority of the extractable phosphorus in PBO was attributed to the octyldecyl phosphate processing aid with some phosphoric acid being detected. Analysis by matrix assisted laser desorption ionization of model PBO oligomers indicates that the nonextractable phosphorus is attached to the PBO polymer chain as a monoaryl phosphate ester. The response of model aryl phosphates to NaOH exposure indicates that monoaryl phosphate ester is stable to NaOH washes used in the manufacturing process to neutralize the phosphoric acid reaction medium and to extract residual phosphorus impurities.

  19. svmPRAT: SVM-based Protein Residue Annotation Toolkit

    PubMed Central

    2009-01-01

    Background Over the last decade several prediction methods have been developed for determining the structural and functional properties of individual protein residues using sequence and sequence-derived information. Most of these methods are based on support vector machines as they provide accurate and generalizable prediction models. Results We present a general purpose protein residue annotation toolkit (svmPRAT) to allow biologists to formulate residue-wise prediction problems. svmPRAT formulates the annotation problem as a classification or regression problem using support vector machines. One of the key features of svmPRAT is its ease of use in incorporating any user-provided information in the form of feature matrices. For every residue svmPRAT captures local information around the reside to create fixed length feature vectors. svmPRAT implements accurate and fast kernel functions, and also introduces a flexible window-based encoding scheme that accurately captures signals and pattern for training effective predictive models. Conclusions In this work we evaluate svmPRAT on several classification and regression problems including disorder prediction, residue-wise contact order estimation, DNA-binding site prediction, and local structure alphabet prediction. svmPRAT has also been used for the development of state-of-the-art transmembrane helix prediction method called TOPTMH, and secondary structure prediction method called YASSPP. This toolkit developed provides practitioners an efficient and easy-to-use tool for a wide variety of annotation problems. Availability: http://www.cs.gmu.edu/~mlbio/svmprat PMID:20028521

  20. Utilization of protein-rich residues in biotechnological processes.

    PubMed

    Pleissner, Daniel; Venus, Joachim

    2016-03-01

    A drawback of biotechnological processes, where microorganisms convert biomass constituents, such as starch, cellulose, hemicelluloses, lipids, and proteins, into wanted products, is the economic feasibility. Particularly the cost of nitrogen sources in biotechnological processes can make up a large fraction of total process expenses. To further develop the bioeconomy, it is of considerable interest to substitute cost-intensive by inexpensive nitrogen sources. The aim of this mini-review was to provide a comprehensive insight of utilization methods of protein-rich residues, such as fish waste, green biomass, hairs, and food waste. The methods described include (i) production of enzymes, (ii) recovery of bioactive compounds, and/or (iii) usage as nitrogen source for microorganisms in biotechnological processes. In this aspect, the utilization of protein-rich residues, which are conventionally considered as waste, allows the development of value-adding processes for the production of bioactive compounds, biomolecules, chemicals, and materials.

  1. Residue Geometry Networks: A Rigidity-Based Approach to the Amino Acid Network and Evolutionary Rate Analysis

    PubMed Central

    Fokas, Alexander S.; Cole, Daniel J.; Ahnert, Sebastian E.; Chin, Alex W.

    2016-01-01

    Amino acid networks (AANs) abstract the protein structure by recording the amino acid contacts and can provide insight into protein function. Herein, we describe a novel AAN construction technique that employs the rigidity analysis tool, FIRST, to build the AAN, which we refer to as the residue geometry network (RGN). We show that this new construction can be combined with network theory methods to include the effects of allowed conformal motions and local chemical environments. Importantly, this is done without costly molecular dynamics simulations required by other AAN-related methods, which allows us to analyse large proteins and/or data sets. We have calculated the centrality of the residues belonging to 795 proteins. The results display a strong, negative correlation between residue centrality and the evolutionary rate. Furthermore, among residues with high closeness, those with low degree were particularly strongly conserved. Random walk simulations using the RGN were also successful in identifying allosteric residues in proteins involved in GPCR signalling. The dynamic function of these residues largely remain hidden in the traditional distance-cutoff construction technique. Despite being constructed from only the crystal structure, the results in this paper suggests that the RGN can identify residues that fulfil a dynamical function. PMID:27623708

  2. Residue Geometry Networks: A Rigidity-Based Approach to the Amino Acid Network and Evolutionary Rate Analysis

    NASA Astrophysics Data System (ADS)

    Fokas, Alexander S.; Cole, Daniel J.; Ahnert, Sebastian E.; Chin, Alex W.

    2016-09-01

    Amino acid networks (AANs) abstract the protein structure by recording the amino acid contacts and can provide insight into protein function. Herein, we describe a novel AAN construction technique that employs the rigidity analysis tool, FIRST, to build the AAN, which we refer to as the residue geometry network (RGN). We show that this new construction can be combined with network theory methods to include the effects of allowed conformal motions and local chemical environments. Importantly, this is done without costly molecular dynamics simulations required by other AAN-related methods, which allows us to analyse large proteins and/or data sets. We have calculated the centrality of the residues belonging to 795 proteins. The results display a strong, negative correlation between residue centrality and the evolutionary rate. Furthermore, among residues with high closeness, those with low degree were particularly strongly conserved. Random walk simulations using the RGN were also successful in identifying allosteric residues in proteins involved in GPCR signalling. The dynamic function of these residues largely remain hidden in the traditional distance-cutoff construction technique. Despite being constructed from only the crystal structure, the results in this paper suggests that the RGN can identify residues that fulfil a dynamical function.

  3. Fast and accurate multivariate Gaussian modeling of protein families: predicting residue contacts and protein-interaction partners.

    PubMed

    Baldassi, Carlo; Zamparo, Marco; Feinauer, Christoph; Procaccini, Andrea; Zecchina, Riccardo; Weigt, Martin; Pagnani, Andrea

    2014-01-01

    In the course of evolution, proteins show a remarkable conservation of their three-dimensional structure and their biological function, leading to strong evolutionary constraints on the sequence variability between homologous proteins. Our method aims at extracting such constraints from rapidly accumulating sequence data, and thereby at inferring protein structure and function from sequence information alone. Recently, global statistical inference methods (e.g. direct-coupling analysis, sparse inverse covariance estimation) have achieved a breakthrough towards this aim, and their predictions have been successfully implemented into tertiary and quaternary protein structure prediction methods. However, due to the discrete nature of the underlying variable (amino-acids), exact inference requires exponential time in the protein length, and efficient approximations are needed for practical applicability. Here we propose a very efficient multivariate Gaussian modeling approach as a variant of direct-coupling analysis: the discrete amino-acid variables are replaced by continuous Gaussian random variables. The resulting statistical inference problem is efficiently and exactly solvable. We show that the quality of inference is comparable or superior to the one achieved by mean-field approximations to inference with discrete variables, as done by direct-coupling analysis. This is true for (i) the prediction of residue-residue contacts in proteins, and (ii) the identification of protein-protein interaction partner in bacterial signal transduction. An implementation of our multivariate Gaussian approach is available at the website http://areeweb.polito.it/ricerca/cmp/code.

  4. Enhancing interacting residue prediction with integrated contact matrix prediction in protein-protein interaction.

    PubMed

    Du, Tianchuan; Liao, Li; Wu, Cathy H

    2016-12-01

    Identifying the residues in a protein that are involved in protein-protein interaction and identifying the contact matrix for a pair of interacting proteins are two computational tasks at different levels of an in-depth analysis of protein-protein interaction. Various methods for solving these two problems have been reported in the literature. However, the interacting residue prediction and contact matrix prediction were handled by and large independently in those existing methods, though intuitively good prediction of interacting residues will help with predicting the contact matrix. In this work, we developed a novel protein interacting residue prediction system, contact matrix-interaction profile hidden Markov model (CM-ipHMM), with the integration of contact matrix prediction and the ipHMM interaction residue prediction. We propose to leverage what is learned from the contact matrix prediction and utilize the predicted contact matrix as "feedback" to enhance the interaction residue prediction. The CM-ipHMM model showed significant improvement over the previous method that uses the ipHMM for predicting interaction residues only. It indicates that the downstream contact matrix prediction could help the interaction site prediction.

  5. 77 FR 65834 - Residues of Fatty Acids, Tall-Oil, Ethoxylated Propoxylated; Tolerance Exemption

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-31

    ... AGENCY 40 CFR Part 180 Residues of Fatty Acids, Tall-Oil, Ethoxylated Propoxylated; Tolerance Exemption... an exemption from the requirement of a tolerance for residues of fatty acids, tall-oil, ethoxylated... residues of fatty ] acids, tall-oil, ethoxylated propoxylated on food or feed commodities. DATES:...

  6. Quantifying protein by bicinchoninic Acid.

    PubMed

    Simpson, Richard J

    2008-08-01

    INTRODUCTIONThis protocol describes a method of quantifying protein that is a variation of the Lowry assay. It uses bicinchoninic acid (BCA) to enhance the detection of Cu(+) generated under alkaline conditions at sites of complexes between Cu(2+) and protein. The resulting chromophore absorbs at 562 nm. This technique is divided into three parts: Standard Procedure, Microprocedure, and 96-Well Microtiter Plate Procedure. For each procedure, test samples are assayed in parallel with protein standards that are used to generate a calibration curve, and the exact concentration of protein in the test samples is interpolated. The standard BCA assay uses large volumes of both reagents and samples and cannot easily be automated. If these issues are important, the Microprocedure is recommended. This in turn can be adapted for use with a microplate reader in the 96-Well Microtiter Plate Procedure. If the microplate reader is interfaced with a computer, more than 1000 samples can be read per hour.

  7. XPS and STEM studies of Allende acid insoluble residues

    NASA Technical Reports Server (NTRS)

    Housley, R. M.; Clarke, D. R.

    1980-01-01

    Data on Allende acid residues obtained both before and after etching with hot HNO3 are presented. X-ray photoelectron spectra show predominantly carbonaceous material plus Fe-deficient chromite in both cases. The HNO3 oxidizes the carbonaceous material to some extent. The small chromites in these residues have a wide range of compositions somewhat paralleling those observed in larger Allende chromites and in Murchison chromites, especially in the high Al contents; however, they are deficient in divalent cations, which makes them metastable and indicates that they must have formed at relatively low temperatures. It is suggested that they formed by precipitation of Cr(3+) and Fe(3+) from olivine at low temperature or during rapid cooling.

  8. Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study.

    PubMed

    Albani, J R; Sillen, A; Plancke, Y D; Coddeville, B; Engelborghs, Y

    2000-07-24

    Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Carbohydr. Res., 318 (1999) 193-200]. Alpha1-acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to alpha1-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated alpha1-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation (one Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of alpha1-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor

  9. An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

    PubMed

    Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

    2014-05-20

    Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies.

  10. Development of a Machine Learning Method to Predict Membrane Protein-Ligand Binding Residues Using Basic Sequence Information

    PubMed Central

    Suresh, M. Xavier; Gromiha, M. Michael; Suwa, Makiko

    2015-01-01

    Locating ligand binding sites and finding the functionally important residues from protein sequences as well as structures became one of the challenges in understanding their function. Hence a Naïve Bayes classifier has been trained to predict whether a given amino acid residue in membrane protein sequence is a ligand binding residue or not using only sequence based information. The input to the classifier consists of the features of the target residue and two sequence neighbors on each side of the target residue. The classifier is trained and evaluated on a nonredundant set of 42 sequences (chains with at least one transmembrane domain) from 31 alpha-helical membrane proteins. The classifier achieves an overall accuracy of 70.7% with 72.5% specificity and 61.1% sensitivity in identifying ligand binding residues from sequence. The classifier performs better when the sequence is encoded by psi-blast generated PSSM profiles. Assessment of the predictions in the context of three-dimensional structures of proteins reveals the effectiveness of this method in identifying ligand binding sites from sequence information. In 83.3% (35 out of 42) of the proteins, the classifier identifies the ligand binding sites by correctly recognizing more than half of the binding residues. This will be useful to protein engineers in exploiting potential residues for functional assessment. PMID:25802517

  11. Chemical and isotopic compositions in acid residues from various meteorites

    NASA Technical Reports Server (NTRS)

    Kano, N.; Yamakoshi, K.; Matsuzaki, H.; Nogami, K.

    1993-01-01

    We are planning to carry out systematic isotopic investigations of Ru, Mg, etc., in primordial samples. The investigations will be pursued in the context of a study of the pre-history of the solar system. It is hoped that the study will yield direct evidence for processes of nucleosynthesis in the pre-solar stage and detection of extinct radioactive nuclides. In this paper, we present the results of chemical compositions of acid residues obtained from three types of meteorites: Canyon Diablo (IA), Allende (CV3), and Nuevo Mercuro (H5); and the preliminary results of Ru isotopic compositions.

  12. 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.

  13. Tripping Up Trp: Modification of Protein Tryptophan Residues by Reactive Oxygen Species, Modes of Detection, and Biological Consequences

    PubMed Central

    Ehrenshaft, Marilyn; Deterding, Leesa J.; Mason, Ronald P.

    2015-01-01

    Proteins comprise a majority of the dry weight of a cell, rendering them a major target for oxidative modification. Oxidation of proteins can result in significant alterations in protein molecular mass such as breakage of the polypeptide backbone, and/or polymerization of monomers into dimers, multimers and sometimes into insoluble aggregates. Protein oxidation can also result in structural changes to amino acid residue side chains, conversions which have only a modest effect on protein size but can have widespread consequences for protein function. There are a wide range of rate constants for amino acid reactivity, with cysteine, methionine, tyrosine, phenylalanine and tryptophan having the highest rate constants with commonly encountered biological oxidants. Free tryptophan and tryptophan protein residues react at a diffusion limited rate with hydroxyl radical, and also have high rate constants for reactions with singlet oxygen and ozone. Although oxidation of proteins in general and tryptophan residues specifically can have effects detrimental to the health of cells and organisms, some modifications are neutral while others contribute to the function of the protein in question or may act as a signal that damaged proteins need to be replaced. This review provides a brief overview of the chemical mechanisms by which tryptophan residues become oxidized, presents both the strengths and weaknesses of some of the techniques used to detect these oxidative interactions and discusses selected examples of the biological consequences of tryptophan oxidation in proteins from animals, plants and microbes. PMID:26393422

  14. Natural populations of lactic acid bacteria isolated from vegetable residues and silage fermentation.

    PubMed

    Yang, J; Cao, Y; Cai, Y; Terada, F

    2010-07-01

    Natural populations of lactic acid bacteria (LAB) and silage fermentation of vegetable residues were studied. Fifty-two strains of LAB isolated from cabbage, Chinese cabbage, and lettuce residues were identified and characterized. The LAB strains were gram-positive and catalase-negative bacteria, which were divided into 6 groups (A to F) according to morphological and biochemical characteristics. The strains in group A were rods that did not produce gas from glucose and formed the d and l isomers of lactate. Groups B and C were homofermentative cocci that formed l-lactic acid. Groups D, E, and F were heterofermentative cocci that formed d-lactic acid. Based on 16S rDNA gene sequence analysis, group A to F strains were identified as Lactobacillus plantarum, Lactococcus piscium, Lactococcus lactis, Leuconostoc citreum, Weissella soli and Leuconostoc gelidum, respectively. The prevalent LAB, predominantly homofermentative lactobacilli, consisted of Lactobacillus plantarum (34.6%), Weissella soli (19.2%), Leuconostoc gelidum (15.4%), Leuconostoc citreum (13.5%), Lactococcus lactis (9.6%), and Lactococcus piscium (7.7%). Lactobacillus plantarum was the dominant member of the LAB population in 3 types of vegetable residues. These vegetable residues contained a high level of crude protein (20.2 to 28.4% of dry matter). These silages prepared by using a small-scale fermentation system were well preserved, with low pH and a relatively high content of lactate. This study suggests that the vegetable residues contain abundant LAB species and nutrients, and that they could be well preserved by making silage, which is a potentially good vegetable protein source for livestock diets.

  15. Development of a sugar-binding residue prediction system from protein sequences using support vector machine.

    PubMed

    Banno, Masaki; Komiyama, Yusuke; Cao, Wei; Oku, Yuya; Ueki, Kokoro; Sumikoshi, Kazuya; Nakamura, Shugo; Terada, Tohru; Shimizu, Kentaro

    2017-02-01

    Several methods have been proposed for protein-sugar binding site prediction using machine learning algorithms. However, they are not effective to learn various properties of binding site residues caused by various interactions between proteins and sugars. In this study, we classified sugars into acidic and nonacidic sugars and showed that their binding sites have different amino acid occurrence frequencies. By using this result, we developed sugar-binding residue predictors dedicated to the two classes of sugars: an acid sugar binding predictor and a nonacidic sugar binding predictor. We also developed a combination predictor which combines the results of the two predictors. We showed that when a sugar is known to be an acidic sugar, the acidic sugar binding predictor achieves the best performance, and showed that when a sugar is known to be a nonacidic sugar or is not known to be either of the two classes, the combination predictor achieves the best performance. Our method uses only amino acid sequences for prediction. Support vector machine was used as a machine learning algorithm and the position-specific scoring matrix created by the position-specific iterative basic local alignment search tool was used as the feature vector. We evaluated the performance of the predictors using five-fold cross-validation. We have launched our system, as an open source freeware tool on the GitHub repository (https://doi.org/10.5281/zenodo.61513).

  16. Camphorquinone-10-sulfonic acid and derivatives: convenient reagents for reversible modification of arginine residues

    SciTech Connect

    Pande, C.S.; Pelzig, M.; Glass, J.D.

    1980-02-01

    Camphorquinone-10-sulfonic acid hydrate was prepared by the action of selenous acid on camphor-10-sulfonic acid. Camphorquinone-10-sulfonylnorleucine was prepared either from the sulfonic acid via the sulfonyl chloride or by selenous acid oxidation of camphor-10-sulfonylnorleucine. These reagents are useful for specific, reversible modification of the guanidino groups of arginine residues. Camphorquinonsulfonic acid is a crystalline water-soluble reagent that is especially suitable for use with small arginine-containing molecules, because the sulfonic acid group of the reagent is a convenient handle for analytical and preparative separation of products. Camphorquinonesulfonylnorleucine is more useful for work with large polypeptides and proteins, because hydrolysates of modified proteins may be analyzed for norleucine to determine the extent of arginine modification. The adducts of the camphorquinone derivatives with the guanidino group are stable to 0.5 M hydroxylamine solutions at pH 7, the recommended conditions for cleavage of the corresponding cyclohexanedione adducts. At pH 8-9 the adducts of the camphorquinone derivatives with the guanidino group are cleaved by o-phenylenediamine. The modification and regeneration of arginine, of the dipeptide arginylaspartic acid, of ribonuclease S-peptide, and of soybean trypsin inhibitor are presented as demonstrations of the use of the reagents.The use of camphorquinonesulfonyl chloride to prepare polymers containing arginine-specific ligands is discussed.

  17. Being a binding site: characterizing residue composition of binding sites on proteins.

    PubMed

    Iván, Gábor; Szabadka, Zoltán; Grolmusz, Vince

    2007-12-30

    The Protein Data Bank contains the description of more than 45,000 three-dimensional protein and nucleic-acid structures today. Started to exist as the computer-readable depository of crystallographic data complementing printed articles, the proper interpretation of the content of the individual files in the PDB still frequently needs the detailed information found in the citing publication. This fact implies that the fully automatic processing of the whole PDB is a very hard task. We first cleaned and re-structured the PDB data, then analyzed the residue composition of the binding sites in the whole PDB for frequency and for hidden association rules. Main results of the paper: (i) the cleaning and repairing algorithm (ii) redundancy elimination from the data (iii) application of association rule mining to the cleaned non-redundant data set. We have found numerous significant relations of the residue-composition of the ligand binding sites on protein surfaces, summarized in two figures. One of the classical data-mining methods for exploring implication-rules, the association-rule mining, is capable to find previously unknown residue-set preferences of bind ligands on protein surfaces. Since protein-ligand binding is a key step in enzymatic mechanisms and in drug discovery, these uncovered preferences in the study of more than 19,500 binding sites may help in identifying new binding protein-ligand pairs.

  18. Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein.

    PubMed

    Katsuma, Susumu; Sugano, Yudai; Kiuchi, Takashi; Shimada, Toru

    2015-10-23

    We have recently discovered that the Masculinizer (Masc) gene encodes a CCCH tandem zinc finger protein, which controls both masculinization and dosage compensation in the silkworm Bombyx mori. In this study, we attempted to identify functional regions or residues that are required for the masculinizing activity of the Masc protein. We constructed a series of plasmids that expressed the Masc derivatives and transfected them into a B. mori ovary-derived cell line, BmN-4. To assess the masculinizing activity of the Masc derivatives, we investigated the splicing patterns of B. mori doublesex (Bmdsx) and the expression levels of B. mori IGF-II mRNA-binding protein, a splicing regulator of Bmdsx, in Masc cDNA-transfected BmN-4 cells. We found that two zinc finger domains are not required for the masculinizing activity. We also identified that the C-terminal 288 amino acid residues are sufficient for the masculinizing activity of the Masc protein. Further detailed analyses revealed that two cysteine residues, Cys-301 and Cys-304, in the highly conserved region among lepidopteran Masc proteins are essential for the masculinizing activity in BmN-4 cells. Finally, we showed that Masc is a nuclear protein, but its nuclear localization is not tightly associated with the masculinizing activity.

  19. Comparison of the effects of ozone on the modification of amino acid residues in glutamine synthetase and bovine serum albumin.

    PubMed

    Berlett, B S; Levine, R L; Stadtman, E R

    1996-02-23

    During exposure to ozone, the methionine and aromatic amino acid residues of Escherichia coli glutamine synthetase (GS) and bovine serum albumin (BSA) are oxidized rapidly in the order Met > Trp > Tyr approximately His > Phe. The loss of His is matched by a nearly equivalent formation of aspartate or of a derivative that is converted to aspartic acid upon acid hydrolysis. Conversion of His to aspartate was confirmed by showing that the oxidation of E. coli protein in which all His residues were uniformly labeled with 14C gave rise to 14C-labeled aspartic acid in 80% yield and also by the demonstration that His residues in the tripeptides Ala-His-Ala or Ala-Ala-His gave rise to nearly stoichiometric amounts of aspartic acid whereas oxidation of His-Ala-Ala yielded only 36% aspartate. The oxidation of BSA and GS led to formation, respectively, of 11 and 3.3 eq of carbonyl groups and 0.5 and 0.3 eq of quinoprotein per subunit. Although BSA and GS contain nearly identical amounts of each kind of aromatic amino acid residues, oxidation of these residues in BSA was about 1.5-2.0 times faster than in GS indicating that the susceptibility to oxidation is dependent on the primary, secondary, tertiary, and quaternary structure of the protein.

  20. Acetic Acid Can Catalyze Succinimide Formation from Aspartic Acid Residues by a Concerted Bond Reorganization Mechanism: A Computational Study

    PubMed Central

    Takahashi, Ohgi; Kirikoshi, Ryota; Manabe, Noriyoshi

    2015-01-01

    Succinimide formation from aspartic acid (Asp) residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe) as a model compound, we propose the possibility that acetic acid (AA), which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition) to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds) occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism. PMID:25588215

  1. Diverse amino acid residues function within the type 1 peroxisomal targeting signal. Implications for the role of accessory residues upstream of the type 1 peroxisomal targeting signal.

    PubMed

    Mullen, R T; Lee, M S; Flynn, C R; Trelease, R N

    1997-11-01

    The purpose of this study was to determine whether the plant type 1 peroxisomal targeting signal (PTS1) utilizes amino acid residues that do not strictly adhere to the serine-lysine-leucine (SKL) motif (small-basic-hydrophobic residues). Selected residues were appended to the C terminus of chloramphenicol acetyltransferase (CAT) and were tested for their ability to target CAT fusion proteins to glyoxysomes in tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 suspension-cultured cells. CAT was redirected from the cytosol into glyoxysomes by a wide range of residues, i.e. A/C/G/S/T-H/K/ L/N/R-I/L/M/Y. Although L and N at the -2 position (-SLL, -ANL) do not conform to the SKL motif, both functioned, but in a temporally less-efficient manner. Other SKL divergent residues, however, did not target CAT to glyoxysomes, i.e. F or P at the -3 position (-FKL, -PKL), S or T at the -2 position (-SSI, STL), or D at the -1 position (-SKD). The targeting inefficiency of CAT-ANL could be ameliorated when K was included at the -4 position (-KANL). In summary, the plant PTS1 mostly conforms to the SKL motif. For those PTS1s that possess nonconforming residue(s), other residues upstream of the PTS1 appear to function as accessory sequences that enhance the temporal efficiency of peroxisomal targeting.

  2. Chemical modification of amino acid residues in glycerinated Vorticella stalk and Ca(2+)-induced contractility.

    PubMed

    Kono, R; Ochiai, T; Asai, H

    1997-01-01

    The glycerinated stalk of the peritrich ciliate Vorticella, was treated with various reagents to chemically modify the amino acid residues. The influences of these modifcations on spasmoneme contractility were investigated. First, it was confirmed that the spasmoneme contraction is not inhibited by alteration of SH groups. It was also demonstrated that chemical modification of methionine and tryptophan residues abolishes spasmoneme contractility. The reagents used for chemical modification were N-bromosuccinimide (NBS), chloramine T, and 2-hydroxy-5-nitrobenzyl bromide (HNBB), which abolished spasmoneme contractility at concentrations of 40-50 microM, 200-300 microM, and 4 mM, respectively. These results suggest that, along with Ca2+ binding proteins, there are other as yet to be identified proteins involved in contractility.

  3. Conservation of cysteine residues in fungal histidine acid phytases.

    PubMed

    Mullaney, Edward J; Ullah, Abul H J

    2005-03-11

    Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides. An additional disulfide bridge formed by two cysteines at the N-terminus of all the filamentous ascomycete phytases was also observed. Disulfide bridges are known to increase both stability and heat tolerance in proteins. It is therefore plausible that this extra disulfide bridge contributes to the higher stability found in phytase from some Aspergillus species. To engineer an enhanced phytase for the feed industry, it is imperative that the role of disulfide bridges be taken into cognizance and possibly be increased in number to further elevate stability in this enzyme.

  4. Removal of coagulant aluminum from water treatment residuals by acid.

    PubMed

    Okuda, Tetsuji; Nishijima, Wataru; Sugimoto, Mayo; Saka, Naoyuki; Nakai, Satoshi; Tanabe, Kazuyasu; Ito, Junki; Takenaka, Kenji; Okada, Mitsumasa

    2014-09-01

    Sediment sludge during coagulation and sedimentation in drinking water treatment is called "water treatment residuals (WTR)". Polyaluminum chloride (PAC) is mainly used as a coagulant in Japan. The recycling of WTR has been desired; one method for its reuse is as plowed soil. However, WTR reuse in this way is inhibited by the aluminum from the added PAC, because of its high adsorption capacity for phosphate and other fertilizer components. The removal of such aluminum from WTR would therefore be advantageous for its reuse as plowed soil; this research clarified the effect of acid washing on aluminum removal from WTR and on plant growth in the treated soil. The percentage of aluminum removal from raw WTR by sulphuric acid solution was around 90% at pH 3, the percentage decreasing to 40% in the case of a sun-dried sample. The maximum phosphate adsorption capacity was decreased and the available phosphorus was increased by acid washing, with 90% of aluminum removal. The enhancement of Japanese mustard spinach growth and the increased in plant uptake of phosphates following acid washing were observed.

  5. Extension of UNRES force field to treat polypeptide chains with D-amino-acid residues

    PubMed Central

    Sieradzan, Adam K.; Hansmann, Ulrich H.E.; Scheraga, Harold A.; Liwo, Adam

    2013-01-01

    Coarse-grained force fields for protein simulations are usually designed and parameterized to treat proteins composed of natural L-amino-acid residues. However, D-amino-acid residues occur in bacterial, fungal (e.g., gramicidins), as well as human-designed proteins. For this reason, we have extended the UNRES coarse-grained force field developed in our laboratory to treat systems with D-amino-acid residues. We developed the respective virtual-bond-torsional and double-torsional potentials for rotation about the Cα · · · Cα virtual-bond axis and two consecutive Cα · · · Cα virtual-bond axes, respectively, as functions of virtual-bond-dihedral angles γ. In turn, these were calculated as potentials of mean force (PMFs) from the diabatic energy surfaces of terminally-blocked model compounds for glycine, alanine, and proline. The potential-energy surfaces were calculated by using the ab initio method of molecular quantum mechanics at the Møller-Plesset (MP2) level of theory and the 6-31G(d,p) basis set, with the rotation angles of the peptide groups about Ci-1α⋯Ciα(λ(1)) and Ciα⋯Ci+1α(λ(2)) used as variables, and the energy was minimized with respect to the remaining degrees of freedom. The PMFs were calculated by numerical integration for all pairs and triplets with all possible combinations of types (glycine, alanine, and proline) and chirality (D or L); however, symmetry relations reduce the number of non-equivalent torsional potentials to 13 and the number of double-torsional potentials to 63 for a given C-terminal blocking group. Subsequently, one- (for torsional) and two-dimensional (for double-torsional potentials) Fourier series were fitted to the PMFs to obtain analytical expressions. It was found that the torsional potentials of the x-Y and X-y types, where X and Y are Ala or Pro, respectively, and a lowercase letter denotes D-chirality, have global minima for small absolute values of γ, accounting for the double-helical structure of

  6. Standard test method for acidity of distillation residues or hydrocarbon liquids

    SciTech Connect

    Not Available

    1980-01-01

    This method covers the qualitative determination of the acidity of the distillation residue from a gasoline. The sample of distillation residue or hydrocarbon liquid is shaken with water and the aqueous layer tested for acidity to methyl orange. Some petroleum products are treated with mineral acid as part of the refining procedure. Obviously, any residual mineral acid in a petroleum product is undesirable. The absence of a positive indication in the test for acidity of the distillation residue or aqueous extract of a hydrocarbon liquid is an assurance of the care used in refining the fuel or solvent.

  7. Effect of protein on the detection of fluoroquinolone residues in fish meat.

    PubMed

    Li, Huifang; Yin, Jungang; Liu, Yongming; Shang, Jian

    2012-02-22

    Using fish serum albumin (FSA) as the model protein, molecular fluorescence spectrometry and high-performance liquid chromatography (HPLC) were applied to study the effect of protein on the extraction of fluoroquinolone (FQ) residues in fish meat. There was a strong interaction between FQs and protein through hydrogen bonds, which could be broken as protein degenerated with 60-100% (v/v) acetonitrile acid solution, and FQs bound with protein were released in various degrees. On the basis of the results, a novel sample preparation procedure loosely based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) methodology was developed for the determination of FQ residues in fish muscle samples, using 90% (v/v) acetonitrile acid solution as the extractant, combined with a dispersive solid-phase extraction (DSPE) cleanup step. Mean recoveries of four FQs from spiked samples at a concentration range of 50-200 ng g(-1) were 73.3-95.9% with relative standard deviations (RSD) lower than 10.7%.

  8. C-H…pi interactions in proteins: prevalence, pattern of occurrence, residue propensities, location, and contribution to protein stability.

    PubMed

    Kumar, Manjeet; Balaji, Petety V

    2014-02-01

    C-H…pi interactions are a class of non-covalent interactions found in different molecular systems including organic crystals, proteins and nucleic acids. High-resolution protein structures have been analyzed in the present study to delineate various aspects of C-H…pi interactions. Additionally, to determine the extent to which redundancy of a database biases the outcome, two datasets differing from each other in the level of redundancy have been analyzed. On average, only one out of six {with C-H(Aro) group} or eight {with C-H(Ali) group} residues in a protein participate as C-H group donors. Neither the frequency of occurrence in proteins nor the number of C-H groups present in it is correlated to the propensity of an amino acid to participate in C-H…pi interactions. Most of the residues that participate in C-H…pi interactions are solvent-shielded. Solvent shielded nature of most of the C-H…pi interactions and prevalence of intra- as well as inter-secondary structural element C-H…pi interactions suggest that the contribution of these interactions to the enthalpy of folded form will be significant. The separation in the primary structure between donor and acceptor residues is found to be correlated to secondary structure type. Other insights obtained from this study include the presence of networks of C-H…pi interactions spanning multiple secondary structural elements. To our knowledge this has not been reported so far. A substantial number of residues involved in C-H…pi interactions are found in catalytic and ligand binding sites suggesting their possible role in maintaining active site geometry. No significant differences of C-H…pi interactions in the two datasets are found for any of the parameters/features analyzed.

  9. 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.

  10. Differentiating amino acid residues and side chain orientations in peptides using scanning tunneling microscopy.

    PubMed

    Claridge, Shelley A; Thomas, John C; Silverman, Miles A; Schwartz, Jeffrey J; Yang, Yanlian; Wang, Chen; Weiss, Paul S

    2013-12-11

    Single-molecule measurements of complex biological structures such as proteins are an attractive route for determining structures of the large number of important biomolecules that have proved refractory to analysis through standard techniques such as X-ray crystallography and nuclear magnetic resonance. We use a custom-built low-current scanning tunneling microscope to image peptide structures at the single-molecule scale in a model peptide that forms β sheets, a structural motif common in protein misfolding diseases. We successfully differentiate between histidine and alanine amino acid residues, and further differentiate side chain orientations in individual histidine residues, by correlating features in scanning tunneling microscope images with those in energy-optimized models. Beta sheets containing histidine residues are used as a model system due to the role histidine plays in transition metal binding associated with amyloid oligomerization in Alzheimer's and other diseases. Such measurements are a first step toward analyzing peptide and protein structures at the single-molecule level.

  11. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  12. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  13. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  14. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  15. 40 CFR 180.202 - p-Chlorophenoxyacetic acid; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false p-Chlorophenoxyacetic acid; tolerances... Tolerances § 180.202 p-Chlorophenoxyacetic acid; tolerances for residues. (a) General. A tolerance is established for the combined residues of the plant regulator p-chlorophenoxyacetic acid and its metabolite...

  16. 40 CFR 180.325 - 2-(m-Chlorophenoxy) propionic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false 2-(m-Chlorophenoxy) propionic acid... Tolerances § 180.325 2-(m-Chlorophenoxy) propionic acid; tolerances for residues. (a) General. A tolerance is established for negligible residues of the plant regulator 2-(m-chlorophenoxy) propionic acid from...

  17. Differential contribution of basic residues to HIV-1 nucleocapsid protein’s nucleic acid chaperone function and retroviral replication

    PubMed Central

    Wu, Hao; Mitra, Mithun; Naufer, M. Nabuan; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC’s NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC’s aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity. PMID:24293648

  18. Characterization of Proteins in Filtrate from Biodegradation of Crop Residue

    NASA Technical Reports Server (NTRS)

    Horton, Wileatha; Trotman, A. A.

    1997-01-01

    Biodegradation of plant biomass is a feasible path for transformation of crop residue and recycling of nutrients for crop growth. The need to model the effects of factors associated with recycling of plant biomass resulting from hydroponic sweet potato production has led to investigation of natural soil isolates with the capacity for starch hydrolysis. This study sought to use nondenaturing gel electrophoresis to characterize the proteins present in filtered effluent from bioreactors seeded with starch hydrolyzing bacterial culture used in the biodegradation of senesced sweet potato biomass. The study determined the relative molecular weight of proteins in sampled effluent and the protein banding pattern was characterized. The protein profiles of effluent were similar for samples taken from independent runs under similar conditions of starch hydrolysis. The method can be used as a quality control tool for confirmation of starch hydrolysis of crop biomass. In addition, this method will allow monitoring for presence of contaminants within the system-protein profiles indicative of new enzymes in the bioreactors.

  19. Spatio-temporal coordination among functional residues in protein

    PubMed Central

    Dutta, Sutapa; Ghosh, Mahua; Chakrabarti, J.

    2017-01-01

    The microscopic basis of communication among the functional sites in bio-macromolecules is a fundamental challenge in uncovering their functions. We study the communication through temporal cross-correlation among the binding sites. We illustrate via Molecular Dynamics simulations the properties of the temporal cross-correlation between the dihedrals of a small protein, ubiquitin which participates in protein degradation in eukaryotes. We show that the dihedral angles of the residues possess non-trivial temporal cross-correlations with asymmetry with respect to exchange of the dihedrals, having peaks at low frequencies with time scales in nano-seconds and an algebraic tail with a universal exponent for large frequencies. We show the existence of path for temporally correlated degrees of freedom among the functional residues. We explain the qualitative features of the cross-correlations through a general mathematical model. The generality of our analysis suggests that temporal cross-correlation functions may provide convenient theoretical framework to understand bio-molecular functions on microscopic basis. PMID:28091537

  20. Spatio-temporal coordination among functional residues in protein

    NASA Astrophysics Data System (ADS)

    Dutta, Sutapa; Ghosh, Mahua; Chakrabarti, J.

    2017-01-01

    The microscopic basis of communication among the functional sites in bio-macromolecules is a fundamental challenge in uncovering their functions. We study the communication through temporal cross-correlation among the binding sites. We illustrate via Molecular Dynamics simulations the properties of the temporal cross-correlation between the dihedrals of a small protein, ubiquitin which participates in protein degradation in eukaryotes. We show that the dihedral angles of the residues possess non-trivial temporal cross-correlations with asymmetry with respect to exchange of the dihedrals, having peaks at low frequencies with time scales in nano-seconds and an algebraic tail with a universal exponent for large frequencies. We show the existence of path for temporally correlated degrees of freedom among the functional residues. We explain the qualitative features of the cross-correlations through a general mathematical model. The generality of our analysis suggests that temporal cross-correlation functions may provide convenient theoretical framework to understand bio-molecular functions on microscopic basis.

  1. Spatio-temporal coordination among functional residues in protein.

    PubMed

    Dutta, Sutapa; Ghosh, Mahua; Chakrabarti, J

    2017-01-16

    The microscopic basis of communication among the functional sites in bio-macromolecules is a fundamental challenge in uncovering their functions. We study the communication through temporal cross-correlation among the binding sites. We illustrate via Molecular Dynamics simulations the properties of the temporal cross-correlation between the dihedrals of a small protein, ubiquitin which participates in protein degradation in eukaryotes. We show that the dihedral angles of the residues possess non-trivial temporal cross-correlations with asymmetry with respect to exchange of the dihedrals, having peaks at low frequencies with time scales in nano-seconds and an algebraic tail with a universal exponent for large frequencies. We show the existence of path for temporally correlated degrees of freedom among the functional residues. We explain the qualitative features of the cross-correlations through a general mathematical model. The generality of our analysis suggests that temporal cross-correlation functions may provide convenient theoretical framework to understand bio-molecular functions on microscopic basis.

  2. Cyst(e)ine residues of bovine white-matter proteolipid proteins. Role of disulphides in proteolipid conformation.

    PubMed Central

    Oteiza, P I; Adamo, A M; Aloise, P A; Paladini, A C; Paladini, A A; Soto, E F

    1987-01-01

    Cyst(e)ine residues of bovine white-matter proteolipid proteins were characterized in a highly purified preparation. From a total of 10.6 cyst(e)ine residues/molecule of protein, as determined by performic acid oxidation, 2.5-3 thiol groups were freely accessible to iodoacetamide, iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), when the proteins were solubilized in chloroform/methanol (C/M) (2:1, v/v). The presence of lipids had no effect on thiol-group exposure. One thiol group available to DTNB in C/M could not be detected when proteolipids were solubilized in the more polar solvent n-butanol. In a C/M solution of purified proteolipid proteins, SDS did not increase the number of reactive thiol groups, but the cleavage of one disulphide bridge made it possible to alkylate six more groups. C.d. and fluorescence studies showed that rupture of this disulphide bond changed the protein conformation, which was reflected in partial loss of helical structure and in a greater exposure to the solvent of at least one tryptophan residue. Cyst(e)ine residues were also characterized in the different components [PLP (principal proteolipid protein), DM20 and LMW (low-Mr proteins)] of the proteolipid preparation. Although the numbers of cyst(e)ine residues in PLP and DM20 were similar, in LMW fewer residues were alkylated under four different experimental conditions. The differences, however, are not simply related to differences in Mr. PMID:3663175

  3. Nucleic acid (cDNA) and amino acid sequences of the maize endosperm protein glutelin-2.

    PubMed Central

    Prat, S; Cortadas, J; Puigdomènech, P; Palau, J

    1985-01-01

    The cDNA coding for a glutelin-2 protein from maize endosperm has been cloned and the complete amino acid sequence of the protein derived for the first time. An immature maize endosperm cDNA bank was screened for the expression of a beta-lactamase:glutelin-2 (G2) fusion polypeptide by using antibodies against the purified 28 kd G2 protein. A clone corresponding to the 28 kd G2 protein was sequenced and the primary structure of this protein was derived. Five regions can be defined in the protein sequence: an 11 residue N-terminal part, a repeated region formed by eight units of the sequence Pro-Pro-Pro-Val-His-Leu, an alternating Pro-X stretch 21 residues long, a Cys rich domain and a C-terminal part rich in Gln. The protein sequence is preceded by 19 residues which have the characteristics of the signal peptide found in secreted proteins. Unlike zeins, the main maize storage proteins, 28 kd glutelin-2 has several homologous sequences in common with other cereal storage proteins. Images PMID:3839076

  4. Location of the serine residue involved in the linkage between the terminal protein and the DNA of phage phi 29.

    PubMed Central

    Hermoso, J M; Méndez, E; Soriano, F; Salas, M

    1985-01-01

    B. subtilis phage phi 29 has a terminal protein, p3, covalently linked to the 5' ends of the DNA through a phosphodiester bond between a serine residue and 5'-dAMP. This protein acts as a primer in DNA replication by forming an initiation complex with the 5'-terminal nucleotide dAMP. The amino acid sequence of the terminal protein, deduced from the nucleotide sequence of gene 3, showed the presence of 18 serine residues in a total of 266 amino acids. In this paper we have identified the serine involved in the linkage with the DNA as the residue 232, located close to the C-terminus of the molecule. This result was obtained by amino acid analysis of the peptide that remains linked to the DNA after proteinase K digestion of the terminal protein-phi 29 DNA complex and automated Edman degradation of the corresponding [125I]-labeled tryptic peptide. Prediction of the secondary structure of the terminal protein suggested that the serine residue involved in the linkage with the DNA is placed in a beta-turn, probably located on the external part of the molecule, as indicated by hydropathic values. Images PMID:3934646

  5. Mutation of aspartic acid residues in the fructosyltransferase of Streptococcus salivarius ATCC 25975.

    PubMed Central

    Song, D D; Jacques, N A

    1999-01-01

    The site-directed mutated fructosyltransferases (Ftfs) of Streptococcus salivarius ATCC 25975, D312E, D312S, D312N and D312K were all active at 37 degrees C, indicating that Asp-312 present in the 'sucrose box' was not the nucleophilic Asp residue responsible for the formation of a covalent fructosyl-enzyme intermediate required for enzyme activity. Analysis of the kinetic constants of the purified mutated forms of the enzyme showed that Asp-312 was most likely an essential amino acid involved in determining acceptor recognition and/or stabilizing a beta-turn in the protein. In contrast, when the Asp-397 of the Ftf present in the conserved triplet RDP motif of all 60 bacterial and plant family-32 glycosylhydrolases was mutated to a Ser residue, both sucrose hydrolysis and polymerization ceased. Tryptophan emission spectra confirmed that this mutation did not alter protein structure. Comparison of published data from other site-directed mutated enzymes implicated the Asp residue in the RDP motif as the one that may form a transient covalent fructosyl intermediate during the catalysis of sucrose by the Ftf of S. salivarius. PMID:10548559

  6. 40 CFR 180.297 - N-1-Naphthyl phthalamic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...; tolerances for residues. 180.297 Section 180.297 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.297 N-1-Naphthyl phthalamic acid; tolerances for residues. (a) General. Tolerances...

  7. 40 CFR 180.297 - N-1-Naphthyl phthalamic acid; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...; tolerances for residues. 180.297 Section 180.297 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.297 N-1-Naphthyl phthalamic acid; tolerances for residues. (a) General. Tolerances...

  8. 40 CFR 180.297 - N-1-Naphthyl phthalamic acid; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...; tolerances for residues. 180.297 Section 180.297 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.297 N-1-Naphthyl phthalamic acid; tolerances for residues. (a) General. Tolerances...

  9. 40 CFR 180.297 - N-1-Naphthyl phthalamic acid; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...; tolerances for residues. 180.297 Section 180.297 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.297 N-1-Naphthyl phthalamic acid; tolerances for residues. (a) General. Tolerances...

  10. 40 CFR 180.297 - N-1-Naphthyl phthalamic acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...; tolerances for residues. 180.297 Section 180.297 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances § 180.297 N-1-Naphthyl phthalamic acid; tolerances for residues. (a) General. Tolerances...

  11. Effect of additives on the tensile performance and protein solubility of industrial oilseed residual based plastics.

    PubMed

    Newson, William R; Kuktaite, Ramune; Hedenqvist, Mikael S; Gällstedt, Mikael; Johansson, Eva

    2014-07-16

    Ten chemical additives were selected from the literature for their proposed modifying activity in protein-protein interactions. These consisted of acids, bases, reducing agents, and denaturants and were added to residual deoiled meals of Crambe abyssinica (crambe) and Brassica carinata (carinata) to modify the properties of plastics produced through hot compression molding at 130 °C. The films produced were examined for tensile properties, protein solubility, molecular weight distribution, and water absorption. Of the additives tested, NaOH had the greatest positive effect on tensile properties, with increases of 105% in maximum stress and 200% in strain at maximum stress for crambe and a 70% increase in strain at maximum stress for carinata. Stiffness was not increased by any of the applied additives. Changes in tensile strength and elongation for crambe and elongation for carinata were related to changes in protein solubility. Increased pH was the most successful in improving the protein aggregation and mechanical properties within the complex chemistry of residual oilseed meals.

  12. Schematic representation of residue-based protein context-dependent data: an application to transmembrane proteins.

    PubMed

    Campagne, F; Weinstein, H

    1999-01-01

    An algorithmic method for drawing residue-based schematic diagrams of proteins on a 2D page is presented and illustrated. The method allows the creation of rendering engines dedicated to a given family of sequences, or fold. The initial implementation provides an engine that can produce a 2D diagram representing secondary structure for any transmembrane protein sequence. We present the details of the strategy for automating the drawing of these diagrams. The most important part of this strategy is the development of an algorithm for laying out residues of a loop that connects to arbitrary points of a 2D plane. As implemented, this algorithm is suitable for real-time modification of the loop layout. This work is of interest for the representation and analysis of data from (1) protein databases, (2) mutagenesis results, or (3) various kinds of protein context-dependent annotations or data.

  13. Functional role of polar amino acid residues in Na+/H+ exchangers.

    PubMed Central

    Wiebe, C A; Dibattista, E R; Fliegel, L

    2001-01-01

    Na(+)/H(+) exchangers are a family of ubiquitous membrane proteins. In higher eukaryotes they regulate cytosolic pH by removing an intracellular H(+) in exchange for an extracellular Na(+). In yeast and Escherichia coli, Na(+)/H(+) exchangers function in the opposite direction to remove intracellular Na(+) in exchange for extracellular H(+). Na(+)/H(+) exchangers display an internal pH-sensitivity that varies with the different antiporter types. Only recently have investigations examined the amino acids involved in pH-sensitivity and in cation binding and transport. Histidine residues are good candidates for H(+)-sensing amino acids, since they can ionize within the physiological pH range. Histidine residues have been shown to be important in the function of the E. coli Na(+)/H(+) exchanger NhaA and in the yeast Na(+)/H(+) exchanger sod2. In E. coli, His(225) of NhaA may function to interact with, or regulate, the pH-sensory region of NhaA. In sod2, His(367) is also critical to transport and may be a functional analogue of His(225) of NhaA. Histidine residues are not critical for the function of the mammalian Na(+)/H(+) exchanger, although an unusual histidine-rich sequence of the C-terminal tail has some influence on activity. Other amino acids involved in cation binding and transport by Na(+)/H(+) exchangers are only beginning to be studied. Amino acids with polar side chains such as aspartate and glutamate have been implicated in transport activity of NhaA and sod2, but have not been studied in the mammalian Na(+)/H(+) exchanger. Further studies are needed to elucidate the mechanisms involved in pH-sensitivity and cation binding and transport by Na(+)/H(+) exchangers. PMID:11415429

  14. Volatile fatty acids distribution during acidogenesis of algal residues with pH control.

    PubMed

    Li, Yan; Hua, Dongliang; Zhang, Jie; Zhao, Yuxiao; Xu, Haipeng; Liang, Xiaohui; Zhang, Xiaodong

    2013-06-01

    The anaerobic acidification of protein-rich algal residues with pH control (4, 6, 8, 10) was studied in batch reactors, which was operated at mesophilic(35 °C) condition. The distribution of major volatile fatty acids (VFAs) during acidogenesis was emphasized in this paper. The results showed that the acidification efficiency and VFAs distribution in the acid reactor strongly depended on the pH. The main product for all the runs involved acetic acid except that the proportion of butyric acid acidified at pH 6 was relatively higher. The other organic acids remained at lower levels. The VFAs yield reached the maximum value with about 0.6 g VFAs/g volatile solid (VS) added as pH was 8, and also the content of total ammonia nitrogen (TAN) reached the highest values of 9,629 mg/l. Low acidification degrees were obtained under the conditions at pH 4 and 10, which was not suitable for the metabolism of acidogens. Hydralic retention time (HRT) required for different conditions varied. As a consequence, it was indicated that pH was crucial to the acidification efficiency and products distribution. The investigation of acidogenesis process, which was producing the major substrates, short-chain fatty acids, would play the primary role in the efficient operation of methanogenesis.

  15. Oxidation in Acidic Medium of Lignins from Agricultural Residues

    NASA Astrophysics Data System (ADS)

    Labat, Gisele Aparecida Amaral; Gonçalves, Adilson Roberto

    Agricultural residues as sugarcane straw and bagasse are burned in boilers for generation of energy in sugar and alcohol industries. However, excess of those by-products could be used to obtain products with higher value. Pulping process generates cellulosic pulps and lignin. The lignin could be oxidized and applied in effluent treatments for heavy metal removal. Oxidized lignin presents very strong chelating properties. Lignins from sugarcane straw and bagasse were obtained by ethanol-water pulping. Oxidation of lignins was carried out using acetic acid and Co/Mn/Br catalytical system at 50, 80, and 115 °C for 5 h. Kinetics of the reaction was accomplished by measuring the UV-visible region. Activation energy was calculated for lignins from sugarcane straw and bagasse (34.2 and 23.4 kJ mol-1, respectively). The first value indicates higher cross-linked formation. Fourier-transformed infrared spectroscopy data of samples collected during oxidation are very similar. Principal component analysis applied to spectra shows only slight structure modifications in lignins after oxidation reaction.

  16. Pyrolytic characteristics of biomass acid hydrolysis residue rich in lignin.

    PubMed

    Huang, Yanqin; Wei, Zhiguo; Yin, Xiuli; Wu, Chuangzhi

    2012-01-01

    Pyrolytic characteristics of acid hydrolysis residue (AHR) of corncob and pinewood (CAHR, WAHR) were investigated using a thermo-gravimetric analyzer (TGA) and a self-designed pyrolysis apparatus. Gasification reactivity of CAHR char was then examined using TGA and X-ray diffractometer. Result of TGA showed that thermal degradation curves of AHR descended smoothly along with temperature increasing from 150 °C to 850 °C, while a "sharp mass loss stage" for original biomass feedstock (OBF) was observed. Char yield from AHR (42.64-30.35 wt.%) was found to be much greater than that from OBF (26.4-19.15 wt.%). In addition, gasification reactivity of CAHR char was lower than that of corncob char, and there was big difference in micro-crystallite structure. It was also found that CAHR char reactivity decreased with pyrolysis temperature, but increased with pyrolysis heating rate and gasification temperature at 850-950 °C. Furthermore, CAHR char reactivity performed better under steam atmosphere than under CO2 atmosphere.

  17. Residue-level resolution of alphavirus envelope protein interactions in pH-dependent fusion.

    PubMed

    Zeng, Xiancheng; Mukhopadhyay, Suchetana; Brooks, Charles L

    2015-02-17

    Alphavirus envelope proteins, organized as trimers of E2-E1 heterodimers on the surface of the pathogenic alphavirus, mediate the low pH-triggered fusion of viral and endosomal membranes in human cells. The lack of specific treatment for alphaviral infections motivates our exploration of potential antiviral approaches by inhibiting one or more fusion steps in the common endocytic viral entry pathway. In this work, we performed constant pH molecular dynamics based on an atomic model of the alphavirus envelope with icosahedral symmetry. We have identified pH-sensitive residues that cause the largest shifts in thermodynamic driving forces under neutral and acidic pH conditions for various fusion steps. A series of conserved interdomain His residues is identified to be responsible for the pH-dependent conformational changes in the fusion process, and ligand binding sites in their vicinity are anticipated to be potential drug targets aimed at inhibiting viral infections.

  18. Residue-level resolution of alphavirus envelope protein interactions in pH-dependent fusion

    PubMed Central

    Zeng, Xiancheng; Mukhopadhyay, Suchetana; Brooks, Charles L.

    2015-01-01

    Alphavirus envelope proteins, organized as trimers of E2–E1 heterodimers on the surface of the pathogenic alphavirus, mediate the low pH-triggered fusion of viral and endosomal membranes in human cells. The lack of specific treatment for alphaviral infections motivates our exploration of potential antiviral approaches by inhibiting one or more fusion steps in the common endocytic viral entry pathway. In this work, we performed constant pH molecular dynamics based on an atomic model of the alphavirus envelope with icosahedral symmetry. We have identified pH-sensitive residues that cause the largest shifts in thermodynamic driving forces under neutral and acidic pH conditions for various fusion steps. A series of conserved interdomain His residues is identified to be responsible for the pH-dependent conformational changes in the fusion process, and ligand binding sites in their vicinity are anticipated to be potential drug targets aimed at inhibiting viral infections. PMID:25646410

  19. 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

  20. Protein Crosslinking by Genetically Encoded Noncanonical Amino Acids with Reactive Aryl Carbamate Side Chains.

    PubMed

    Xuan, Weimin; Shao, Sida; Schultz, Peter G

    2017-04-03

    The use of genetically encoded noncanonical amino acids (ncAAs) to construct crosslinks within or between proteins has emerged as a useful method to enhance protein stability, investigate protein-protein interactions, and improve the pharmacological properties of proteins. We report ncAAs with aryl carbamate side chains (PheK and FPheK) that can react with proximal nucleophilic residues to form intra- or intermolecular protein crosslinks. We evolved a pyrrolysyl-tRNA synthetase that incorporates site-specifically PheK and FPheK into proteins in both E. coli and mammalian cells. PheK and FPheK when incorporated into proteins showed good stability during protein expression and purification. FPheK reacted with adjacent Lys, Cys, and Tyr residues in thioredoxin in high yields. In addition, crosslinks could be formed between FPheK and Lys residue of two interacting proteins, including the heavy chain and light chain of an antibody Fab.

  1. Amino Acid Substitutions in Cold-Adapted Proteins from Halorubrum lacusprofundi, an Extremely Halophilic Microbe from Antarctica

    PubMed Central

    DasSarma, Shiladitya; Capes, Melinda D.; Karan, Ram; DasSarma, Priya

    2013-01-01

    The halophilic Archaeon Halorubrum lacusprofundi, isolated from the perennially cold and hypersaline Deep Lake in Antarctica, was recently sequenced and compared to 12 Haloarchaea from temperate climates by comparative genomics. Amino acid substitutions for 604 H. lacusprofundi proteins belonging to conserved haloarchaeal orthologous groups (cHOGs) were determined and found to occur at 7.85% of positions invariant in proteins from mesophilic Haloarchaea. The following substitutions were observed most frequently: (a) glutamic acid with aspartic acid or alanine; (b) small polar residues with other small polar or non-polar amino acids; (c) small non-polar residues with other small non-polar residues; (d) aromatic residues, especially tryptophan, with other aromatic residues; and (e) some larger polar residues with other similar residues. Amino acid substitutions for a cold-active H. lacusprofundi β-galactosidase were then examined in the context of a homology modeled structure at residues invariant in homologous enzymes from mesophilic Haloarchaea. Similar substitutions were observed as in the genome-wide approach, with the surface accessible regions of β-galactosidase displaying reduced acidity and increased hydrophobicity, and internal regions displaying mainly subtle changes among smaller non-polar and polar residues. These findings are consistent with H. lacusprofundi proteins displaying amino acid substitutions that increase structural flexibility and protein function at low temperature. We discuss the likely mechanisms of protein adaptation to a cold, hypersaline environment on Earth, with possible relevance to life elsewhere. PMID:23536799

  2. Real-time measurements of amino acid and protein hydroperoxides using coumarin boronic acid.

    PubMed

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

    2014-08-08

    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 × 10(3) 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.

  3. Functional domains of the fatty acid transport proteins: studies using protein chimeras.

    PubMed

    DiRusso, Concetta C; Darwis, Dina; Obermeyer, Thomas; Black, Paul N

    2008-03-01

    Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1-FATP4 and FATP6-FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C(20:4)) and lignocerate (C(24:0)), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function.

  4. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...)butanoic acid, and its metabolite MCPA, (4-chloro-2-methylphenoxy)acetic acid, in or on the following food... acid; tolerance for residues. 180.318 Section 180.318 Protection of Environment ENVIRONMENTAL... FOOD Specific Tolerances § 180.318 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for...

  5. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...)butanoic acid, and its metabolite MCPA, (4-chloro-2-methylphenoxy)acetic acid, in or on the following food... acid; tolerance for residues. 180.318 Section 180.318 Protection of Environment ENVIRONMENTAL... FOOD Specific Tolerances § 180.318 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for...

  6. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...)butanoic acid, and its metabolite MCPA, (4-chloro-2-methylphenoxy)acetic acid, in or on the following food... acid; tolerance for residues. 180.318 Section 180.318 Protection of Environment ENVIRONMENTAL... FOOD Specific Tolerances § 180.318 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for...

  7. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...)butanoic acid, and its metabolite MCPA, (4-chloro-2-methylphenoxy)acetic acid, in or on the following food... acid; tolerance for residues. 180.318 Section 180.318 Protection of Environment ENVIRONMENTAL... FOOD Specific Tolerances § 180.318 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for...

  8. 40 CFR 180.318 - 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...)butanoic acid, and its metabolite MCPA, (4-chloro-2-methylphenoxy)acetic acid, in or on the following food... acid; tolerance for residues. 180.318 Section 180.318 Protection of Environment ENVIRONMENTAL... FOOD Specific Tolerances § 180.318 4-(2-Methyl-4-chlorophenoxy) butyric acid; tolerance for...

  9. Essential Amino Acids of the Hantaan Virus N Protein in Its Interaction with RNA

    PubMed Central

    Severson, William; Xu, Xiaolin; Kuhn, Michaela; Senutovitch, Nina; Thokala, Mercy; Ferron, François; Longhi, Sonia; Canard, Bruno; Jonsson, Colleen B.

    2005-01-01

    The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding. PMID:16014963

  10. Influence of glutamic acid residues and pH on the properties of transmembrane helices.

    PubMed

    Rajagopalan, Venkatesan; Greathouse, Denise V; Koeppe, Roger E

    2017-03-01

    Negatively charged side chains are important for the function of particular ion channels and certain other membrane proteins. To investigate the influence of single glutamic acid side chains on helices that span lipid-bilayer membranes, we have employed GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-amide) as a favorable host peptide framework. We substituted individual Leu residues with Glu residues (L12E or L14E or L16E) and incorporated specific (2)H-labeled alanine residues within the core helical region or near the ends of the sequence. Solid-state (2)H NMR spectra reveal little change for the core labels in GWALP23-E12, -E14 and -E16 over a pH range of 4 to 12.5, with the spectra being broader for samples in DOPC compared to DLPC bilayers. The spectra for samples with deuterium labels near the helix ends on alanines 3 and 21 show modest pH-dependent changes in the extent of unwinding of the helix terminals in DLPC and DOPC bilayers. The combined results indicate minor overall responses of these transmembrane helices to changes in pH, with the most buried residue E12 showing no pH dependence. While the Glu residues E14 and E16 may have high pKa values in the lipid bilayer environment, it is also possible that a paucity of helix response is masking the pKa values. Interestingly, when E16 is present, spectral changes at high pH report significant local unwinding of the core helix. Our results are consistent with the expectation that buried carboxyl groups aggressively hold their protons and/or waters of hydration.

  11. Properties of nanocellulose isolated from corncob residue using sulfuric acid, formic acid, oxidative and mechanical methods.

    PubMed

    Liu, Chao; Li, Bin; Du, Haishun; Lv, Dong; Zhang, Yuedong; Yu, Guang; Mu, Xindong; Peng, Hui

    2016-10-20

    In this work, nanocellulose was extracted from bleached corncob residue (CCR), an underutilized lignocellulose waste from furfural industry, using four different methods (i.e. sulfuric acid hydrolysis, formic acid (FA) hydrolysis, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-mediated oxidation, and pulp refining, respectively). The self-assembled structure, morphology, dimension, crystallinity, chemical structure and thermal stability of prepared nanocellulose were investigated. FA hydrolysis produced longer cellulose nanocrystals (CNCs) than the one obtained by sulfuric acid hydrolysis, and resulted in high crystallinity and thermal stability due to its preferential degradation of amorphous cellulose and lignin. The cellulose nanofibrils (CNFs) with fine and individualized structure could be isolated by TEMPO-mediated oxidation. In comparison with other nanocellulose products, the intensive pulp refining led to the CNFs with the longest length and the thickest diameter. This comparative study can help to provide an insight into the utilization of CCR as a potential source for nanocellulose production.

  12. Canine Distemper Virus Envelope Protein Interactions Modulated by Hydrophobic Residues in the Fusion Protein Globular Head

    PubMed Central

    Avila, Mislay; Khosravi, Mojtaba; Alves, Lisa; Ader-Ebert, Nadine; Bringolf, Fanny; Zurbriggen, Andreas; Plemper, Richard K.

    2014-01-01

    Membrane fusion for morbillivirus cell entry relies on critical interactions between the viral fusion (F) and attachment (H) envelope glycoproteins. Through extensive mutagenesis of an F cavity recently proposed to contribute to F's interaction with the H protein, we identified two neighboring hydrophobic residues responsible for severe F-to-H binding and fusion-triggering deficiencies when they were mutated in combination. Since both residues reside on one side of the F cavity, the data suggest that H binds the F globular head domain sideways. PMID:25355896

  13. Protein-borne methionine residues as structural antioxidants in mitochondria.

    PubMed

    Schindeldecker, Mario; Moosmann, Bernd

    2015-07-01

    Methionine is an oxidant-labile amino acid whose major oxidation products, methionine sulfoxides, can be readily repaired by various NADPH-dependent methionine sulfoxide reductases. Formally, the methionine oxidation-reduction circuit could act as a cellular antioxidant system, by providing a safe sink for oxidants that might cause much more damage if reacting otherwise. This concept is supported by focal experimental evidence; however, the global importance, scope and biochemical role of protein-borne methionine as an inbuilt macromolecular antioxidant have remained incompletely defined. In analyzing proteomic methionine usage on different levels of comparison, we find that protein methionine (i) is primarily an antioxidant of mitochondria, especially of the inner mitochondrial membrane, (ii) responds strongly to respiratory demands on an evolutionary timescale, (iii) acts locally, by selectively protecting its carrier protein, and (iv) might be utilized as a molecular predictor of aerobic metabolic rate in animals, to complement traditional markers like the presence of a respiratory pigment. Our data support the idea that proteins in need of a long lifespan or acting in dangerous environments may acquire massive structural alterations aimed at increasing their resistance to oxidation. Counterintuitively though, they sometimes do so by accumulating particularly labile rather than particularly stable building blocks, illustrating that the technical concept of cathodic protection is also employed by the animate nature.

  14. Conformational analysis of amyloid precursor protein fragment containing amino acids 667-676, and the effect of D-Asp and iso-Asp substitution at Asp₆₇₂ residue.

    PubMed

    Shanmugam, Ganesh; Polavarapu, Prasad L; Láng, Emma; Majer, Zsuzsa

    2012-03-01

    Amyloid precursor protein (APP) fragment containing amino acids 667-676, (APP₆₆₇₋₆₇₆), is a substrate for β-secretase which is responsible for generating amyloid β peptides. Conformational analysis of APP₆₆₇₋₆₇₆ peptide [Ac-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH₂] and the effect of substitution of Asp₆₇₂ with D-Asp and iso-L-Asp, studied for the first time, demonstrate that the peptide backbone of APP₆₆₇₋₆₇₆ is flexible and adopts different conformations in different solvent environments (water, trifluoroethanol and dimethylsulfoxide). A major conformational difference was observed in trifluoroethanol solvent when Asp₆₇₂ is substituted with D-Asp and iso-Asp. These conformational changes involved in APP₆₆₇₋₆₇₆ may assist in understanding the interactions between β-secretase and APP₆₆₇₋₆₇₆, with relevance to Alzheimer's disease.

  15. Modification of amino acid residues in carious dentin matrix.

    PubMed

    Kleter, G A; Damen, J J; Buijs, M J; Ten Cate, J M

    1998-03-01

    The Maillard reaction between sugar and protein has been postulated as the cause for the browning and arrestment of caries lesions. This reaction has been implicated as the cause for decreased degradability of collagen in vivo. The aim of the present study was to verify the occurrence of the reaction in vivo. Carious and sound dentin samples were taken from extracted human teeth and analyzed for the fluorescence characteristic of the Maillard reaction and oxidation and, by HPLC, for Maillard products. In addition, physiological cross-links were analyzed by HPLC. Oxidation- and Maillard reaction-related fluorescence increased in collagenase digests from carious dentin. Advanced Maillard products (carboxymethyllysine and pentosidine) increased, whereas furosine, a marker for the initial reaction, was not observed consistently. This implies no direct addition of sugars to protein, but rather the addi-tion of smaller metabolites and glycoxidation products. In addition, the physiological cross-links hydroxylysinonorleucine and dihydroxylysinonorleucine decreased in carious dentin. Also for hydroxylysylpyridinoline, a decrease was observed, but not consistently. In conclusion, the caries process modifies amino acids in dentin collagen, which can lead to increased resistance against proteolysis and ultimately to caries arrestment.

  16. Amino acid residues of bitter taste receptor TAS2R16 that determine sensitivity in primates to β-glycosides

    PubMed Central

    Imai, Hiroo; Suzuki-Hashido, Nami; Ishimaru, Yoshiro; Sakurai, Takanobu; Yin, Lijie; Pan, Wenshi; Ishiguro, Masaji; Masuda, Katsuyoshi; Abe, Keiko; Misaka, Takumi; Hirai, Hirohisa

    2016-01-01

    In mammals, bitter taste is mediated by TAS2Rs, which belong to the family of seven transmembrane G protein-coupled receptors. Since TAS2Rs are directly involved in the interaction between mammals and their dietary sources, it is likely that these genes evolved to reflect species-specific diets during mammalian evolution. Here, we analyzed the amino acids responsible for the difference in sensitivities of TAS2R16s of various primates using a cultured cell expression system. We found that the sensitivity of TAS2R16 varied due to several amino acid residues. Mutation of amino acid residues at E86T, L247M, and V260F in human and langur TAS2R16 for mimicking the macaque TAS2R16 decreased the sensitivity of the receptor in an additive manner, which suggests its contribution to the potency of salicin, possibly via direct interaction. However, mutation of amino acid residues 125 and 133 in human TAS2R16, which are situated in helix 4, to the macaque sequence increased the sensitivity of the receptor. These results suggest the possibility that bitter taste sensitivities evolved independently by replacing specific amino acid residues of TAS2Rs in different primate species to adapt to species-specific food. PMID:27924271

  17. Chlorine residuals and haloacetic acid reduction in rapid sand filtration.

    PubMed

    Chuang, Yi-Hsueh; Wang, Gen-Shuch; Tung, Hsin-hsin

    2011-11-01

    It is quite rare to find biodegradation in rapid sand filtration for drinking water treatment. This might be due to frequent backwashes and low substrate levels. High chlorine concentrations may inhibit biofilm development, especially for plants with pre-chlorination. However, in tropical or subtropical regions, bioactivity on the sand surface may be quite significant due to high biofilm development--a result of year-round high temperature. The objective of this study is to explore the correlation between biodegradation and chlorine concentration in rapid sand filters, especially for the water treatment plants that practise pre-chlorination. In this study, haloacetic acid (HAA) biodegradation was found in conventional rapid sand filters practising pre-chlorination. Laboratory column studies and field investigations were conducted to explore the association between the biodegradation of HAAs and chlorine concentrations. The results showed that chlorine residual was an important factor that alters bioactivity development. A model based on filter influent and effluent chlorine was developed for determining threshold chlorine for biodegradation. From the model, a temperature independent chlorine concentration threshold (Cl(threshold)) for biodegradation was estimated at 0.46-0.5mgL(-1). The results imply that conventional filters with adequate control could be conducive to bioactivity, resulting in lower HAA concentrations. Optimizing biodegradable disinfection by-product removal in conventional rapid sand filter could be achieved with minor variation and a lower-than-Cl(threshold) influent chlorine concentration. Bacteria isolation was also carried out, successfully identifying several HAA degraders. These degraders are very commonly seen in drinking water systems and can be speculated as the main contributor of HAA loss.

  18. Sequence-based discrimination of protein-RNA interacting residues using a probabilistic approach.

    PubMed

    Pai, Priyadarshini P; Dash, Tirtharaj; Mondal, Sukanta

    2017-04-07

    Protein interactions with ribonucleic acids (RNA) are well-known to be crucial for a wide range of cellular processes such as transcriptional regulation, protein synthesis or translation, and post-translational modifications. Identification of the RNA-interacting residues can provide insights into these processes and aid in relevant biotechnological manipulations. Owing to their eventual potential in combating diseases and industrial production, several computational attempts have been made over years using sequence- and structure-based information. Recent comparative studies suggest that despite these developments, many problems are faced with respect to the usability, prerequisites, and accessibility of various tools, thereby calling for an alternative approach and perspective supplementation in the prediction scenario. With this motivation, in this paper, we propose the use of a simple-yet-efficient conditional probabilistic approach based on the application of local occurrence of amino acids in the interacting region in a non-numeric sequence feature space, for discriminating between RNA interacting and non-interacting residues. The proposed method has been meticulously tested for robustness using a cross-estimation method showing MCC of 0.341 and F- measure of 66.84%. Upon exploring large scale applications using benchmark datasets available to date, this approach showed an encouraging performance comparable with the state-of-art. The software is available at https://github.com/ABCgrp/DORAEMON.

  19. Proteasomal degradation of ubiquitinated Insig proteins is determined by serine residues flanking ubiquitinated lysines

    PubMed Central

    Lee, Joon No; Gong, Yi; Zhang, Xiangyu; Ye, Jin

    2006-01-01

    Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum that play crucial roles in cholesterol homeostasis by inhibiting excessive cholesterol synthesis and uptake. In sterol-depleted cells Insig-1 is degraded at least 15 times more rapidly than Insig-2, owing to ubiquitination of Lys-156 and Lys-158 in Insig-1. In this study, we use domain-swapping methods to localize amino acid residues responsible for this differential degradation. In the case of Insig-2, Glu-214 stabilizes the protein by preventing ubiquitination. When Glu-214 is changed to alanine, Insig-2 becomes ubiquitinated, but it is still not degraded as rapidly as ubiquitinated Insig-1. The difference in the degradation rates is traced to two amino acids: Ser-149 in Insig-1 and Ser-106 in Insig-2. Ser-149, which lies NH2-terminal to the ubiquitination sites, accelerates the degradation of ubiquitinated Insig-1. Ser-106, which is COOH-terminal to the ubiquitination sites, retards the degradation of ubiquitinated Insig-2. The current studies indicate that the degradation of ubiquitinated Insigs is controlled by serine residues flanking the sites of ubiquitination. PMID:16549805

  20. Phosphorous acid residues in apples after foliar fertilization: results of field trials.

    PubMed

    Malusà, E; Tosi, L

    2005-06-01

    The levels of phosphorous acid residues in apples after foliar fertilization with P fertilizers and after treatment with a phosphonate fungicide (Fosetyl-Al) were determined and compared. Two field trials and a glasshouse experiment, using different genotypes and plants of different age, were carried out and monitored over a three-year period. Phosphorous acid residues were found in apples after application of foliar P fertilizers. Concentrations of the residues ranged between 0.02 and 14 mg kg(-1) depending on the phosphorous acid content in the fertilizer used and the plant size and yield. The treatments induced an accumulation of the residue in the course of the experiments, which in some cases reached a level exceeding the maximum limit set by EU legislation. Residues were also detected in other plant organs, i.e., roots and buds. Plants treated with Fosetyl-Al contained phosphorous acid residues in their fruits and buds two years after the suspension of the treatment, suggesting a long-term persistence of the substance in plant storage organs. A second experiment, involving treatment of trees with seven foliar fertilizers of different composition, also induced accumulation of phosphorous acid residues in fruits. It is concluded that a wide array of foliar products containing phosphorous acid, even as a minor component, could mimic the residue effect of phosphonate fungicide treatments.

  1. Analysis of proteins with the 'hot dog' fold: Prediction of function and identification of catalytic residues of hypothetical proteins

    PubMed Central

    Pidugu, Lakshmi S; Maity, Koustav; Ramaswamy, Karthikeyan; Surolia, Namita; Suguna, Kaza

    2009-01-01

    Background The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. Results We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. Conclusion The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects. PMID:19473548

  2. Residual on column host cell protein analysis during lifetime studies of protein A chromatography.

    PubMed

    Lintern, Katherine; Pathak, Mili; Smales, C Mark; Howland, Kevin; Rathore, Anurag; Bracewell, Daniel G

    2016-08-26

    Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC-MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell proteins (HCPs) by LC-MS/MS present on the resin is shown to increase 10-fold between 50 and 100 cycles. During this same period the functional class of the predominant HCPs associated with the resin increased in diversity, with number of proteins identified increasing 5-fold. This ageing is observed in the context of the product quality of the eluate HCP and protein A leachate concentration remaining constant with cycle number.

  3. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization.

    PubMed

    Shiheido, Hirokazu; Shimizu, Jun

    2015-02-20

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND356-58, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3.

  4. Glycolic acid-catalyzed deamidation of asparagine residues in degrading PLGA matrices: a computational study.

    PubMed

    Manabe, Noriyoshi; Kirikoshi, Ryota; Takahashi, Ohgi

    2015-03-31

    Poly(lactic-co-glycolic acid) (PLGA) is a strong candidate for being a drug carrier in drug delivery systems because of its biocompatibility and biodegradability. However, in degrading PLGA matrices, the encapsulated peptide and protein drugs can undergo various degradation reactions, including deamidation at asparagine (Asn) residues to give a succinimide species, which may affect their potency and/or safety. Here, we show computationally that glycolic acid (GA) in its undissociated form, which can exist in high concentration in degrading PLGA matrices, can catalyze the succinimide formation from Asn residues by acting as a proton-transfer mediator. A two-step mechanism was studied by quantum-chemical calculations using Ace-Asn-Nme (Ace = acetyl, Nme = NHCH3) as a model compound. The first step is cyclization (intramolecular addition) to form a tetrahedral intermediate, and the second step is elimination of ammonia from the intermediate. Both steps involve an extensive bond reorganization mediated by a GA molecule, and the first step was predicted to be rate-determining. The present findings are expected to be useful in the design of more effective and safe PLGA devices.

  5. An Amino Acid Code for Irregular and Mixed Protein Packing

    PubMed Central

    Joo, Hyun; Chavan, Archana; Fraga, Keith; Tsai, Jerry

    2015-01-01

    To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of 2 motifs: a 3 residue socket for packing within 2° structure and a 4 residue knob-socket for 3° packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. Irregular secondary structure involves 3 residue cliques of consecutive contacting residues or XYZ sockets. In irregular sockets, Gly, Pro, Asp and Ser are favored, while Cys, His, Met and Trp are not. For irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly, while Cys, His, Met and Trp are not. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helices/β-sheets identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map. PMID:26370334

  6. Identification of Coevolving Residues and Coevolution Potentials Emphasizing Structure, Bond Formation and Catalytic Coordination in Protein Evolution

    PubMed Central

    Little, Daniel Y.; Chen, Lu

    2009-01-01

    The structure and function of a protein is dependent on coordinated interactions between its residues. The selective pressures associated with a mutation at one site should therefore depend on the amino acid identity of interacting sites. Mutual information has previously been applied to multiple sequence alignments as a means of detecting coevolutionary interactions. Here, we introduce a refinement of the mutual information method that: 1) removes a significant, non-coevolutionary bias and 2) accounts for heteroscedasticity. Using a large, non-overlapping database of protein alignments, we demonstrate that predicted coevolving residue-pairs tend to lie in close physical proximity. We introduce coevolution potentials as a novel measure of the propensity for the 20 amino acids to pair amongst predicted coevolutionary interactions. Ionic, hydrogen, and disulfide bond-forming pairs exhibited the highest potentials. Finally, we demonstrate that pairs of catalytic residues have a significantly increased likelihood to be identified as coevolving. These correlations to distinct protein features verify the accuracy of our algorithm and are consistent with a model of coevolution in which selective pressures towards preserving residue interactions act to shape the mutational landscape of a protein by restricting the set of admissible neutral mutations. PMID:19274093

  7. 40 CFR 180.311 - Cacodylic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) PESTICIDE PROGRAMS TOLERANCES AND EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Specific Tolerances... million Expiration/Revocation Date Cotton, undelinted seed 2.8 1/1/12 (b) Section 18 emergency...

  8. Conversion of Undaria pinnatifida residue to glycolic acid with recyclable methylamine in low temperature hydrothermal liquefaction.

    PubMed

    Chen, Yongxing; Ren, Xiulian; Wei, Qifeng

    2017-03-01

    The conversion of Undaria pinnatifida residue to glycolic acid was carried out using methylamine as catalyst by hydrothermal method at relatively low temperature. GC-MS and HPLC were used to identify the composition of bio-oil and liquid products which provide the knowledge of the chemical reaction pathways of the hydrothermal liquefaction. The main liquid product was organic acid which contained glycolic acid, lactic acid, formic acid and acetic acid. And the major organic acid was glycolic acid with the highest yield of 46.52% or 33.98% of dry biomass. Methylamine promoted the dissolution of cellulose from Undaria pinnatifida residue, and significantly improved the yield of glycolic acid. The mechanism of HTL was investigated and the results show that the carbocation C3 was attacked by methylamine molecule which led to the high yield of glycolic acid. In addition, the recovery of methylamine was studied and the highest recovery rate reached 99.28%.

  9. SLC27 fatty acid transport proteins.

    PubMed

    Anderson, Courtney M; Stahl, Andreas

    2013-01-01

    The uptake and metabolism of long chain fatty acids (LCFA) are critical to many physiological and cellular processes. Aberrant accumulation or depletion of LCFA underlie the pathology of numerous metabolic diseases. Protein-mediated transport of LCFA has been proposed as the major mode of LCFA uptake and activation. Several proteins have been identified to be involved in LCFA uptake. This review focuses on the SLC27 family of fatty acid transport proteins, also known as FATPs, with an emphasis on the gain- and loss-of-function animal models that elucidate the functions of FATPs in vivo and how these transport proteins play a role in physiological and pathological situations.

  10. 40 CFR 180.331 - 4-(2,4-Dichlorophenoxy) butyric acid; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... and conjugated, determined as the acid, in or on food commodities, as follows: Commodity Parts per... 40 Protection of Environment 24 2014-07-01 2014-07-01 false 4-(2,4-Dichlorophenoxy) butyric acid... Tolerances § 180.331 4-(2,4-Dichlorophenoxy) butyric acid; tolerances for residues. (a) General....

  11. 40 CFR 180.331 - 4-(2,4-Dichlorophenoxy) butyric acid; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... and conjugated, determined as the acid, in or on food commodities, as follows: Commodity Parts per... 40 Protection of Environment 25 2012-07-01 2012-07-01 false 4-(2,4-Dichlorophenoxy) butyric acid... Tolerances § 180.331 4-(2,4-Dichlorophenoxy) butyric acid; tolerances for residues. (a) General....

  12. 40 CFR 180.331 - 4-(2,4-Dichlorophenoxy) butyric acid; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... and conjugated, determined as the acid, in or on food commodities, as follows: Commodity Parts per... 40 Protection of Environment 25 2013-07-01 2013-07-01 false 4-(2,4-Dichlorophenoxy) butyric acid... Tolerances § 180.331 4-(2,4-Dichlorophenoxy) butyric acid; tolerances for residues. (a) General....

  13. 40 CFR 180.331 - 4-(2,4-Dichlorophenoxy) butyric acid; tolerances for residues.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... and conjugated, determined as the acid, in or on food commodities, as follows: Commodity Parts per... 40 Protection of Environment 23 2010-07-01 2010-07-01 false 4-(2,4-Dichlorophenoxy) butyric acid... Tolerances § 180.331 4-(2,4-Dichlorophenoxy) butyric acid; tolerances for residues. (a) General....

  14. Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation.

    PubMed

    Aguilera, Paulina; Marcoleta, Andrés; Lobos-Ruiz, Pablo; Arranz, Rocío; Valpuesta, José M; Monasterio, Octavio; Lagos, Rosalba

    2016-01-01

    Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril's morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4'-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54-63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with

  15. Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation

    PubMed Central

    Aguilera, Paulina; Marcoleta, Andrés; Lobos-Ruiz, Pablo; Arranz, Rocío; Valpuesta, José M.; Monasterio, Octavio; Lagos, Rosalba

    2016-01-01

    Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4′-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54–63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54–63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although

  16. Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro.

    PubMed Central

    Engelman, A; Craigie, R

    1992-01-01

    We have probed the structural organization of the human immunodeficiency virus type 1 integrase protein by limited proteolysis and the functional organization by site-directed mutagenesis of selected amino acid residues. A central region of the protein was relatively resistant to proteolysis. Proteins with altered amino acids in this region, or in the N-terminal part of the protein that includes a putative zinc-binding motif, were purified and assayed for 3' processing, DNA strand transfer, and disintegration activities in vitro. In general, these mutations had parallel effects on 3' processing and DNA strand transfer, suggesting that integrase may utilize a single active site for both reactions. The only proteins that were completely inactive in all three assays contained mutations at conserved amino acids in the central region, suggesting that this part of the protein may be involved in catalysis. In contrast, none of the mutations in the N-terminal region resulted in a protein that was inactive in all three assays, suggesting that this part of integrase may not be essential for catalysis. The disintegration reaction was particularly insensitive to these amino acid substitutions, indicating that some function that is important for 3' processing and DNA strand transfer may be dispensable for disintegration. Images PMID:1404595

  17. Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis.

    PubMed

    Pedersen, Anja K; Guo, Xiao-Ling; Møller, Karin B; Peters, Günther H; Andersen, Henrik S; Kastrup, Jette S; Mortensen, Steen B; Iversen, Lars F; Zhang, Zhong-Yin; Møller, Niels Peter H

    2004-03-01

    Previous enzyme kinetic and structural studies have revealed a critical role for Asp181 (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp181 functions as a general acid, while in the E-P hydrolysis step it acts as a general base. Most of our understanding of the role of Asp181 is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and the related PTPa and PTPe. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes and a glutamine in Yersinia PTP. Surprisingly, little attention has been paid to the fact that this residue is a histidine in most other mammalian PTPs. Using a reciprocal single-point mutational approach with introduction of His182 in PTP1B and Phe182 in PTPH1, we demonstrate here that His182-PTPs, in comparison with Phe182-PTPs, have significantly decreased kcat values, and to a lesser degree, decreased kcat/Km values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His182 is due to interactions with Asp181 and with Gln262. We conclude that residue 182 can modulate the functionality of both Asp181 and Gln262 and therefore affect the E-P hydrolysis step of PTP-mediated catalysis.

  18. Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins.

    PubMed

    Pradhan, Mohan R; Pal, Arumay; Hu, Zhongqiao; Kannan, Srinivasaraghavan; Chee Keong, Kwoh; Lane, David P; Verma, Chandra S

    2016-02-01

    Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using "Dehydrons." "Dehydrons" are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein-protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non-conserved dehydron-residues) from other interaction interfaces (conserved dehydron-residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation.

  19. CAB-Align: A Flexible Protein Structure Alignment Method Based on the Residue-Residue Contact Area.

    PubMed

    Terashi, Genki; Takeda-Shitaka, Mayuko

    2015-01-01

    Proteins are flexible, and this flexibility has an essential functional role. Flexibility can be observed in loop regions, rearrangements between secondary structure elements, and conformational changes between entire domains. However, most protein structure alignment methods treat protein structures as rigid bodies. Thus, these methods fail to identify the equivalences of residue pairs in regions with flexibility. In this study, we considered that the evolutionary relationship between proteins corresponds directly to the residue-residue physical contacts rather than the three-dimensional (3D) coordinates of proteins. Thus, we developed a new protein structure alignment method, contact area-based alignment (CAB-align), which uses the residue-residue contact area to identify regions of similarity. The main purpose of CAB-align is to identify homologous relationships at the residue level between related protein structures. The CAB-align procedure comprises two main steps: First, a rigid-body alignment method based on local and global 3D structure superposition is employed to generate a sufficient number of initial alignments. Then, iterative dynamic programming is executed to find the optimal alignment. We evaluated the performance and advantages of CAB-align based on four main points: (1) agreement with the gold standard alignment, (2) alignment quality based on an evolutionary relationship without 3D coordinate superposition, (3) consistency of the multiple alignments, and (4) classification agreement with the gold standard classification. Comparisons of CAB-align with other state-of-the-art protein structure alignment methods (TM-align, FATCAT, and DaliLite) using our benchmark dataset showed that CAB-align performed robustly in obtaining high-quality alignments and generating consistent multiple alignments with high coverage and accuracy rates, and it performed extremely well when discriminating between homologous and nonhomologous pairs of proteins in both

  20. SAS-Pro: simultaneous residue assignment and structure superposition for protein structure alignment.

    PubMed

    Shah, Shweta B; Sahinidis, Nikolaos V

    2012-01-01

    Protein structure alignment is the problem of determining an assignment between the amino-acid residues of two given proteins in a way that maximizes a measure of similarity between the two superimposed protein structures. By identifying geometric similarities, structure alignment algorithms provide critical insights into protein functional similarities. Existing structure alignment tools adopt a two-stage approach to structure alignment by decoupling and iterating between the assignment evaluation and structure superposition problems. We introduce a novel approach, SAS-Pro, which addresses the assignment evaluation and structure superposition simultaneously by formulating the alignment problem as a single bilevel optimization problem. The new formulation does not require the sequentiality constraints, thus generalizing the scope of the alignment methodology to include non-sequential protein alignments. We employ derivative-free optimization methodologies for searching for the global optimum of the highly nonlinear and non-differentiable RMSD function encountered in the proposed model. Alignments obtained with SAS-Pro have better RMSD values and larger lengths than those obtained from other alignment tools. For non-sequential alignment problems, SAS-Pro leads to alignments with high degree of similarity with known reference alignments. The source code of SAS-Pro is available for download at http://eudoxus.cheme.cmu.edu/saspro/SAS-Pro.html.

  1. Teichuronic acid reducing terminal N-acetylglucosamine residue linked by phosphodiester to peptidoglycan of Micrococcus luteus

    SciTech Connect

    Gassner, G.T.; Dickie, J.P.; Hamerski, D.A.; Magnuson, J.K.; Anderson, J.S. )

    1990-05-01

    Teichuronic acid-peptidoglycan complex isolated from Micrococcus luteus cells by lysozyme digestion in osmotically stabilized medium was treated with mild acid to cleave the linkage joining teichuronic acid to peptidoglycan. This labile linkage was shown to be the phosphodiester which joins N-acetylglucosamine, the residue located at the reducing end of the teichuronic acid, through its anomeric hydroxyl group to a 6-phosphomuramic acid, a residue of the glycan strand of peptidoglycan. {sup 31}P nuclear magnetic resonance spectroscopy of the lysozyme digest of cell walls demonstrated the presence of a phosphodiester which was converted to a phosphomonoester by the conditions which released teichuronic acid from cell walls. Reduction of acid-liberated reducing end groups by NaB{sup 3}H{sub 4} followed by complete acid hydrolysis yielded ({sup 3}H) glucosaminitol from the true reducing end residue of teichuronic acid and ({sup 3}H)glucitol from the sites of fragmentation of teichuronic acid. The amount of N-acetylglucosamine detected was approximately stoichiometric with the amount of phosphate in the complex. Partial fragmentation of teichuronic acid provides an explanation of the previous erroneous identification of the reducing end residue.

  2. 40 CFR 180.155 - 1-Naphthaleneacetic acid; tolerances for residues.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... established for the combined residues of the plant growth regulator 1-naphthaleneacetic acid and its... ammonium, sodium, or potassium salts, ethyl ester, and acetamide in or on food commodities as...

  3. Environment-specific amino acid substitution tables: tertiary templates and prediction of protein folds.

    PubMed Central

    Overington, J.; Donnelly, D.; Johnson, M. S.; Sali, A.; Blundell, T. L.

    1992-01-01

    The local environment of an amino acid in a folded protein determines the acceptability of mutations at that position. In order to characterize and quantify these structural constraints, we have made a comparative analysis of families of homologous proteins. Residues in each structure are classified according to amino acid type, secondary structure, accessibility of the side chain, and existence of hydrogen bonds from the side chains. Analysis of the pattern of observed substitutions as a function of local environment shows that there are distinct patterns, especially for buried polar residues. The substitution data tables are available on diskette with Protein Science. Given the fold of a protein, one is able to predict sequences compatible with the fold (profiles or templates) and potentially to discriminate between a correctly folded and misfolded protein. Conversely, analysis of residue variation across a family of aligned sequences in terms of substitution profiles can allow prediction of secondary structure or tertiary environment. PMID:1304904

  4. Two amino acid residues confer type specificity to a neutralizing, conformationally dependent epitope on human papillomavirus type 11.

    PubMed Central

    Ludmerer, S W; Benincasa, D; Mark, G E

    1996-01-01

    Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism. PMID:8676509

  5. The amino acid sequence of protein AA from a burro (Equus asinus).

    PubMed

    Sletten, Knut; Johnson, Kenneth H; Westermark, Per

    2003-09-01

    The primary structure of amyloid fibril protein AA of a burro has been determined by Edman degradation. The 80 amino acid residue long protein shows strong resemblance to that of other mammalian AA-proteins and differs from equine protein AA at 5 positions: Burro/horse positions 20 (Q/N), 44 (R,Q, K/K,Q), 59 (G,L/G,A), 61 (Q/E) and 65 (N/R).

  6. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    NASA Astrophysics Data System (ADS)

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-09-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct.

  7. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    PubMed Central

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-01-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct. PMID:27681031

  8. Are tyrosine residues involved in the photoconversion of the water-soluble chlorophyll-binding protein of Chenopodium album?

    PubMed

    Takahashi, S; Seki, Y; Uchida, A; Nakayama, K; Satoh, H

    2015-05-01

    Non-photosynthetic and hydrophilic chlorophyll (Chl) proteins, called water-soluble Chl-binding proteins (WSCPs), are distributed in various species of Chenopodiaceae, Amaranthaceae, Polygonaceae and Brassicaceae. Based on their photoconvertibility, WSCPs are categorised into two classes: Class I (photoconvertible) and Class II (non-photoconvertible). Chenopodium album WSCP (CaWSCP; Class I) is able to convert the chlorin skeleton of Chl a into a bacteriochlorin-like skeleton under light in the presence of molecular oxygen. Potassium iodide (KI) is a strong inhibitor of the photoconversion. Because KI attacks tyrosine residues in proteins, tyrosine residues in CaWSCP are considered to be important amino acid residues for the photoconversion. Recently, we identified the gene encoding CaWSCP and found that the mature region of CaWSCP contained four tyrosine residues: Tyr13, Tyr14, Tyr87 and Tyr134. To gain insight into the effect of the tyrosine residues on the photoconversion, we constructed 15 mutant proteins (Y13A, Y14A, Y87A, Y134A, Y13-14A, Y13-87A, Y13-134A, Y14-87A, Y14-134A, Y87-134A, Y13-14-87A, Y13-14-134A, Y13-87-134A, Y14-87-134A and Y13-14-87-134A) using site-directed mutagenesis. Amazingly, all the mutant proteins retained not only chlorophyll-binding activity, but also photoconvertibility. Furthermore, we found that KI strongly inhibited the photoconversion of Y13-14-87-134A. These findings indicated that the four tyrosine residues are not essential for the photoconversion.

  9. Structural consequences of chromophore formation and exploration of conserved lid residues amongst naturally occurring fluorescent proteins

    NASA Astrophysics Data System (ADS)

    Zimmer, Matthew H.; Li, Binsen; Shahid, Ramza; Peshkepija, Paola; Zimmer, Marc

    2014-01-01

    Computational methods were used to generate the lowest energy conformations of the immature precyclized forms of the 28 naturally occurring GFP-like proteins deposited in the pdb. In all 28 GFP-like proteins, the beta-barrel contracts upon chromophore formation and becomes more rigid. Our prior analysis of over 260 distinct naturally occurring GFP-like proteins revealed that most of the conserved residues are located in the top and bottom of the barrel in the turns between the β-sheets (Ong et al. 2011) [1]. Structural analyses, molecular dynamics simulations and the Anisotropic Network Model were used to explore the role of these conserved lid residues as possible folding nuclei. Our results are internally consistent and show that the conserved residues in the top and bottom lids undergo relatively less translational movement than other lid residues, and a number of these residues may play an important role as hinges or folding nuclei in the fluorescent proteins.

  10. SDR: a database of predicted specificity-determining residues in proteins.

    PubMed

    Donald, Jason E; Shakhnovich, Eugene I

    2009-01-01

    The specificity-determining residue database (SDR database) presents residue positions where mutations are predicted to have changed protein function in large protein families. Because the database pre-calculates predictions on existing protein sequence alignments, users can quickly find the predictions by selecting the appropriate protein family or searching by protein sequence. Predictions can be used to guide mutagenesis or to gain a better understanding of specificity changes in a protein family. The database is available on the web at http://paradox.harvard.edu/sdr.

  11. Effect of temperature on iron leaching from bauxite residue by sulfuric acid.

    PubMed

    Liu, Zhi-Rong; Zeng, Kai; Zhao, Wei; Li, Ying

    2009-01-01

    Bauxite residue, as solid waste from alumina production, contains mainly hematite [Fe2O3]. Kinetic study of iron leaching of bauxite residue by diluted sulfuric acid at atmospheric pressure has been investigated. The results have been obtained as following: (i) Temperature play an important role in iron leaching from bauxite residue. Higher temperature is favor of Fe(III) leaching from bauxite residue. (ii) The leaching process is applicable to the intra-particle diffusion model and the apparent activation energy of model of leaching is found to be 17.32 kJ/mol.

  12. SucStruct: Prediction of succinylated lysine residues by using structural properties of amino acids.

    PubMed

    López, Yosvany; Dehzangi, Abdollah; Lal, Sunil Pranit; Taherzadeh, Ghazaleh; Michaelson, Jacob; Sattar, Abdul; Tsunoda, Tatsuhiko; Sharma, Alok

    2017-03-28

    Post-Translational Modification (PTM) is a biological reaction which contributes to diversify the proteome. Despite many modifications with important roles in the cellular activity, lysine succinylation has recently emerged as an important PTM mark. It alters the chemical structure of lysines, leading to remarkable changes in the structure and function of proteins. Given the huge amount of proteins being sequenced in the post-genome era, the experimental detection of succinylated residues remains expensive, inefficient and time-consuming. Therefore, the development of computational tools for accurately predicting succinylated lysines is an urgent necessity. To date, several approaches have been proposed but their sensitivity has been reportedly poor. In this paper, we propose an approach that utilizes structural features of amino acids to improve lysine succinylation prediction. Succinylated and non-succinylated lysines were first retrieved from 670 proteins and characteristics such as accessible surface area, backbone torsion angles, and local structure conformations were incorporated. We used the k-nearest neighbors cleaning for dealing with class imbalance and designed a pruned decision tree for classification. Our predictor, referred as SucStruct (Succinylation using Structural features), proved to significantly improve performance when compared to previous predictors, with sensitivity, accuracy and Mathew's correlation coefficient equal to 0.7334-0.7946, 0.7444-0.7608 and 0.4884-0.5240, respectively.

  13. Allosteric Inhibition of Phosphoenolpyruvate Carboxylases is Determined by a Single Amino Acid Residue in Cyanobacteria

    PubMed Central

    Takeya, Masahiro; Hirai, Masami Yokota; Osanai, Takashi

    2017-01-01

    Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme for CO2 fixation and primary metabolism in photosynthetic organisms including cyanobacteria. The kinetics and allosteric regulation of PEPCs have been studied in many organisms, but the biochemical properties of PEPC in the unicellular, non-nitrogen-fixing cyanobacterium Synechocystis sp. PCC 6803 have not been clarified. In this study, biochemical analysis revealed that the optimum pH and temperature of Synechocystis 6803 PEPC proteins were 7.3 and 30 °C, respectively. Synechocystis 6803 PEPC was found to be tolerant to allosteric inhibition by several metabolic effectors such as malate, aspartate, and fumarate compared with other cyanobacterial PEPCs. Comparative sequence and biochemical analysis showed that substitution of the glutamate residue at position 954 with lysine altered the enzyme so that it was inhibited by malate, aspartate, and fumarate. PEPC of the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 was purified, and its activity was inhibited in the presence of malate. Substitution of the lysine at position 946 (equivalent to position 954 in Synechocystis 6803) with glutamate made Anabaena 7120 PEPC tolerant to malate. These results demonstrate that the allosteric regulation of PEPC in cyanobacteria is determined by a single amino acid residue, a characteristic that is conserved in different orders. PMID:28117365

  14. Effects of advanced oxidation pretreatment on residual aluminum control in high humic acid water purification.

    PubMed

    Wang, Wendong; Li, Hua; Ding, Zhenzhen; Wang, Xiaochang

    2011-01-01

    Due to the formation of disinfection by-products and high concentrations of Al residue in drinking water purification, humic substances are a major component of organic matter in natural waters and have therefore received a great deal of attention in recent years. We investigated the effects of advanced oxidation pretreatment methods usually applied for removing dissolved organic matters on residual Al control. Results showed that the presence of humic acid increased residual Al concentration notably. With 15 mg/L of humic acid in raw water, the concentrations of soluble aluminum and total aluminum in the treated water were close to the quantity of Al addition. After increasing coagulant dosage from 12 to 120 mg/L, the total-Al in the treated water was controlled to below 0.2 mg/L. Purification systems with ozonation, chlorination, or potassium permanganate oxidation pretreatment units had little effects on residual Al control; while UV radiation decreased Al concentration notably. Combined with ozonation, the effects of UV radiation were enhanced. Optimal dosages were 0.5 mg O3/mg C and 3 hr for raw water with 15 mg/L of humic acid. Under UV light radiation, the combined forces or bonds that existed among humic acid molecules were destroyed; adsorption sites increased positively with radiation time, which promoted adsorption of humic acid onto polymeric aluminum and Al(OH)3(s). This work provides a new solution for humic acid coagulation and residual Al control for raw water with humic acid purification.

  15. Minimal genome encoding proteins with constrained amino acid repertoire

    PubMed Central

    Tsoy, Olga; Yurieva, Marina; Kucharavy, Andrey; O'Reilly, Mary; Mushegian, Arcady

    2013-01-01

    Minimal bacterial gene set comprises the genetic elements needed for survival of engineered bacterium on a rich medium. This set is estimated to include 300–350 protein-coding genes. One way of simplifying an organism with such a minimal genome even further is to constrain the amino acid content of its proteins. In this study, comparative genomics approaches and the results of gene knockout experiments were used to extrapolate the minimal gene set of mollicutes, and bioinformatics combined with the knowledge-based analysis of the structure-function relationships in these proteins and their orthologs, paralogs and analogs was applied to examine the challenges of completely replacing the rarest residue, cysteine. Among several known functions of cysteine residues, their roles in the active centers of the enzymes responsible for deoxyribonucleoside synthesis and transfer RNA modification appear to be crucial, as no alternative chemistry is known for these reactions. Thus, drastic reduction of the content of the rarest amino acid in a minimal proteome appears to be possible, but its complete elimination is challenging. PMID:23873957

  16. Photo-CIDNP NMR spectroscopy of amino acids and proteins.

    PubMed

    Kuhn, Lars T

    2013-01-01

    Photo-chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance (NMR) phenomenon which, among other things, is exploited to extract information on biomolecular structure via probing solvent-accessibilities of tryptophan (Trp), tyrosine (Tyr), and histidine (His) amino acid side chains both in polypeptides and proteins in solution. The effect, normally triggered by a (laser) light-induced photochemical reaction in situ, yields both positive and/or negative signal enhancements in the resulting NMR spectra which reflect the solvent exposure of these residues both in equilibrium and during structural transformations in "real time". As such, the method can offer - qualitatively and, to a certain extent, quantitatively - residue-specific structural and kinetic information on both the native and, in particular, the non-native states of proteins which, often, is not readily available from more routine NMR techniques. In this review, basic experimental procedures of the photo-CIDNP technique as applied to amino acids and proteins are discussed, recent improvements to the method highlighted, and future perspectives presented. First, the basic principles of the phenomenon based on the theory of the radical pair mechanism (RPM) are outlined. Second, a description of standard photo-CIDNP applications is given and it is shown how the effect can be exploited to extract residue-specific structural information on the conformational space sampled by unfolded or partially folded proteins on their "path" to the natively folded form. Last, recent methodological advances in the field are highlighted, modern applications of photo-CIDNP in the context of biological NMR evaluated, and an outlook into future perspectives of the method is given.

  17. Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein.

    PubMed Central

    Montalto, G; Bonicel, J; Multigner, L; Rovery, M; Sarles, H; De Caro, A

    1986-01-01

    Pancreatic stone protein (PSP) is the major organic component of human pancreatic stones. With the use of monoclonal antibody immunoadsorbents, five immunoreactive forms (PSP-S) with close Mr values (14,000-19,000) were isolated from normal pancreatic juice. By CM-Trisacryl M chromatography the lowest-Mr form (PSP-S1) was separated from the others and some of its molecular characteristics were investigated. The Mr of the PSP-S1 polypeptide chain calculated from the amino acid composition was about 16,100. The N-terminal sequences (40 residues) of PSP and PSP-S1 are identical, which suggests that the peptide backbone is the same for both of these polypeptides. The PSP-S1 sequence was determined up to residue 65 and was found to be different from all other known protein sequences. Images Fig. 1. PMID:3541906

  18. 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.

  19. 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.

  20. Estimation of Position Specific Energy as a Feature of Protein Residues from Sequence Alone for Structural Classification

    PubMed Central

    Iqbal, Sumaiya; Hoque, Md Tamjidul

    2016-01-01

    A set of features computed from the primary amino acid sequence of proteins, is crucial in the process of inducing a machine learning model that is capable of accurately predicting three-dimensional protein structures. Solutions for existing protein structure prediction problems are in need of features that can capture the complexity of molecular level interactions. With a view to this, we propose a novel approach to estimate position specific estimated energy (PSEE) of a residue using contact energy and predicted relative solvent accessibility (RSA). Furthermore, we demonstrate PSEE can be reasonably estimated based on sequence information alone. PSEE is useful in identifying the structured as well as unstructured or, intrinsically disordered region of a protein by computing favorable and unfavorable energy respectively, characterized by appropriate threshold. The most intriguing finding, verified empirically, is the indication that the PSEE feature can effectively classify disorder versus ordered residues and can segregate different secondary structure type residues by computing the constituent energies. PSEE values for each amino acid strongly correlate with the hydrophobicity value of the corresponding amino acid. Further, PSEE can be used to detect the existence of critical binding regions that essentially undergo disorder-to-order transitions to perform crucial biological functions. Towards an application of disorder prediction using the PSEE feature, we have rigorously tested and found that a support vector machine model informed by a set of features including PSEE consistently outperforms a model with an identical set of features with PSEE removed. In addition, the new disorder predictor, DisPredict2, shows competitive performance in predicting protein disorder when compared with six existing disordered protein predictors. PMID:27588752

  1. How Does Alkali Aid Protein Extraction in Green Tea Leaf Residue: A Basis for Integrated Biorefinery of Leaves

    PubMed Central

    Zhang, Chen; Sanders, Johan P. M.; Xiao, Ting T.; Bruins, Marieke E.

    2015-01-01

    Leaf protein can be obtained cost-efficiently by alkaline extraction, but overuse of chemicals and low quality of (denatured) protein limits its application. The research objective was to investigate how alkali aids protein extraction of green tea leaf residue, and use these results for further improvements in alkaline protein biorefinery. Protein extraction yield was studied for correlation to morphology of leaf tissue structure, protein solubility and hydrolysis degree, and yields of non-protein components obtained at various conditions. Alkaline protein extraction was not facilitated by increased solubility or hydrolysis of protein, but positively correlated to leaf tissue disruption. HG pectin, RGII pectin, and organic acids were extracted before protein extraction, which was followed by the extraction of cellulose and hemi-cellulose. RGI pectin and lignin were both linear to protein yield. The yields of these two components were 80% and 25% respectively when 95% protein was extracted, which indicated that RGI pectin is more likely to be the key limitation to leaf protein extraction. An integrated biorefinery was designed based on these results. PMID:26200774

  2. CAB-Align: A Flexible Protein Structure Alignment Method Based on the Residue-Residue Contact Area

    PubMed Central

    Terashi, Genki; Takeda-Shitaka, Mayuko

    2015-01-01

    Proteins are flexible, and this flexibility has an essential functional role. Flexibility can be observed in loop regions, rearrangements between secondary structure elements, and conformational changes between entire domains. However, most protein structure alignment methods treat protein structures as rigid bodies. Thus, these methods fail to identify the equivalences of residue pairs in regions with flexibility. In this study, we considered that the evolutionary relationship between proteins corresponds directly to the residue–residue physical contacts rather than the three-dimensional (3D) coordinates of proteins. Thus, we developed a new protein structure alignment method, contact area-based alignment (CAB-align), which uses the residue–residue contact area to identify regions of similarity. The main purpose of CAB-align is to identify homologous relationships at the residue level between related protein structures. The CAB-align procedure comprises two main steps: First, a rigid-body alignment method based on local and global 3D structure superposition is employed to generate a sufficient number of initial alignments. Then, iterative dynamic programming is executed to find the optimal alignment. We evaluated the performance and advantages of CAB-align based on four main points: (1) agreement with the gold standard alignment, (2) alignment quality based on an evolutionary relationship without 3D coordinate superposition, (3) consistency of the multiple alignments, and (4) classification agreement with the gold standard classification. Comparisons of CAB-align with other state-of-the-art protein structure alignment methods (TM-align, FATCAT, and DaliLite) using our benchmark dataset showed that CAB-align performed robustly in obtaining high-quality alignments and generating consistent multiple alignments with high coverage and accuracy rates, and it performed extremely well when discriminating between homologous and nonhomologous pairs of proteins in both

  3. Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

    PubMed Central

    Du, Yushen; Wu, Nicholas C.; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting

    2016-01-01

    ABSTRACT Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. PMID:27803181

  4. Statistical analysis of protein structures suggests that buried ionizable residues in proteins are hydrogen bonded or form salt bridges.

    PubMed

    Bush, Jeffrey; Makhatadze, George I

    2011-07-01

    It is well known that nonpolar residues are largely buried in the interior of proteins, whereas polar and ionizable residues tend to be more localized on the protein surface where they are solvent exposed. Such a distribution of residues between surface and interior is well understood from a thermodynamic point: nonpolar side chains are excluded from the contact with the solvent water, whereas polar and ionizable groups have favorable interactions with the water and thus are preferred at the protein surface. However, there is an increasing amount of information suggesting that polar and ionizable residues do occur in the protein core, including at positions that have no known functional importance. This is inconsistent with the observations that dehydration of polar and in particular ionizable groups is very energetically unfavorable. To resolve this, we performed a detailed analysis of the distribution of fractional burial of polar and ionizable residues using a large set of ˜2600 nonhomologous protein structures. We show that when ionizable residues are fully buried, the vast majority of them form hydrogen bonds and/or salt bridges with other polar/ionizable groups. This observation resolves an apparent contradiction: the energetic penalty of dehydration of polar/ionizable groups is paid off by favorable energy of hydrogen bonding and/or salt bridge formation in the protein interior. Our conclusion agrees well with the previous findings based on the continuum models for electrostatic interactions in proteins.

  5. 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.

  6. Detection of outlier residues for improving interface prediction in protein heterocomplexes.

    PubMed

    Chen, Peng; Wong, Limsoon; Li, Jinyan

    2012-01-01

    Sequence-based understanding and identification of protein binding interfaces is a challenging research topic due to the complexity in protein systems and the imbalanced distribution between interface and noninterface residues. This paper presents an outlier detection idea to address the redundancy problem in protein interaction data. The cleaned training data are then used for improving the prediction performance. We use three novel measures to describe the extent a residue is considered as an outlier in comparison to the other residues: the distance of a residue instance from the center instance of all residue instances of the same class label (Dist), the probability of the class label of the residue instance (PCL), and the importance of within-class and between-class (IWB) residue instances. Outlier scores are computed by integrating the three factors; instances with a sufficiently large score are treated as outliers and removed. The data sets without outliers are taken as input for a support vector machine (SVM) ensemble. The proposed SVM ensemble trained on input data without outliers performs better than that with outliers. Our method is also more accurate than many literature methods on benchmark data sets. From our empirical studies, we found that some outlier interface residues are truly near to noninterface regions, and some outlier noninterface residues are close to interface regions.

  7. OMPcontact: An Outer Membrane Protein Inter-Barrel Residue Contact Prediction Method.

    PubMed

    Zhang, Li; Wang, Han; Yan, Lun; Su, Lingtao; Xu, Dong

    2017-03-01

    In the two transmembrane protein types, outer membrane proteins (OMPs) perform diverse important biochemical functions, including substrate transport and passive nutrient uptake and intake. Hence their 3D structures are expected to reveal these functions. Because experimental structures are scarce, predicted 3D structures are more adapted to OMP research instead, and the inter-barrel residue contact is becoming one of the most remarkable features, improving prediction accuracy by describing the structural information of OMPs. To predict OMP structures accurately, we explored an OMP inter-barrel residue contact prediction method: OMPcontact. Multiple OMP-specific features were integrated in the method, including residue evolutionary covariation, topology-based transmembrane segment relative residue position, OMP lipid layer accessibility, and residue evolution conservation. These features describe the properties of a residue pair in different respects: sequential, structural, evolutionary, and biochemical. Within a 3-residues slide window, a Support Vector Machine (SVM) could accurately determinate the inter-barrel contact residue pair using above features. A 5-fold cross-valuation process was applied in testing the OMPcontact performance against a non-redundant OMP set with 75 samples inside. The tests compared four evolutionary covariation methods and screen analyzed the adaptive ones for inter-barrel contact prediction. The results showed our method not only efficiently realized the prediction, but also scored the possibility for residue pairs reliably. This is expected to improve OMP tertiary structure prediction. Therefore, OMPcontact will be helpful in compiling a structural census of outer membrane protein.

  8. The contrasting properties of conservation and correlated phylogeny in protein functional residue prediction

    PubMed Central

    Manning, Jonathan R; Jefferson, Emily R; Barton, Geoffrey J

    2008-01-01

    Background Amino acids responsible for structure, core function or specificity may be inferred from multiple protein sequence alignments where a limited set of residue types are tolerated. The rise in available protein sequences continues to increase the power of techniques based on this principle. Results A new algorithm, SMERFS, for predicting protein functional sites from multiple sequences alignments was compared to 14 conservation measures and to the MINER algorithm. Validation was performed on an automatically generated dataset of 1457 families derived from the protein interactions database SNAPPI-DB, and a smaller manually curated set of 148 families. The best performing measure overall was Williamson property entropy, with ROC0.1 scores of 0.0087 and 0.0114 for domain and small molecule contact prediction, respectively. The Lancet method performed worse than random on protein-protein interaction site prediction (ROC0.1 score of 0.0008). The SMERFS algorithm gave similar accuracy to the phylogenetic tree-based MINER algorithm but was superior to Williamson in prediction of non-catalytic transient complex interfaces. SMERFS predicts sites that are significantly more solvent accessible compared to Williamson. Conclusion Williamson property entropy is the the best performing of 14 conservation measures examined. The difference in performance of SMERFS relative to Williamson in manually defined complexes was dependent on complex type. The best choice of analysis method is therefore dependent on the system of interest. Additional computation employed by Miner in calculation of phylogenetic trees did not produce improved results over SMERFS. SMERFS performance was improved by use of windows over alignment columns, illustrating the necessity of considering the local environment of positions when assessing their functional significance. PMID:18221517

  9. Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization

    SciTech Connect

    Shiheido, Hirokazu Shimizu, Jun

    2015-02-20

    BEN domain-containing protein 3 (BEND3) has recently been reported to function as a heterochromatin-associated protein in transcriptional repression in the nucleus. BEND3 should have nuclear localization signals (NLSs) to localize to the nucleus in light of its molecular weight, which is higher than that allowed to pass through nuclear pore complexes. We here analyzed the subcellular localization of deletion/site-directed mutants of human BEND3 by an immunofluorescence assay in an attempt to identify the amino acids essential for its nuclear localization. We found that three basic amino acid residues located in the N-terminal region of BEND3 (BEND3{sub 56–58}, KRK) are essential, suggesting that these residues play a role as a functional NLS. These results provide valuable information for progressing research on BEND3. - Highlights: • BEND3 localizes to the nucleus. • The N-terminal 60 amino acids region of BEND3 contains NLS. • Amino acids located between 56 and 58 of BEND3 (KRK) are part of NLS. • KRK motif is highly conserved among BEND3 homologs.

  10. A noncanonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins.

    PubMed

    Bellucci, Joseph J; Bhattacharyya, Jayanta; Chilkoti, Ashutosh

    2015-01-07

    We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full-length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε-amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site-specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site-specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox.

  11. 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.

  12. Identification of essential histidine residues involved in heme binding and Hemozoin formation in heme detoxification protein from Plasmodium falciparum.

    PubMed

    Nakatani, Keisuke; Ishikawa, Haruto; Aono, Shigetoshi; Mizutani, Yasuhisa

    2014-08-20

    Malaria parasites digest hemoglobin within a food vacuole to supply amino acids, releasing the toxic product heme. During the detoxification, toxic free heme is converted into an insoluble crystalline form called hemozoin (Hz). Heme detoxification protein (HDP) in Plasmodium falciparum is one of the most potent of the hemozoin-producing enzymes. However, the reaction mechanisms of HDP are poorly understood. We identified the active site residues in HDP using a combination of Hz formation assay and spectroscopic characterization of mutant proteins. Replacement of the critical histidine residues His122, His172, His175, and His197 resulted in a reduction in the Hz formation activity to approximately 50% of the wild-type protein. Spectroscopic characterization of histidine-substituted mutants revealed that His122 binds heme and that His172 and His175 form a part of another heme-binding site. Our results show that the histidine residues could be present in the individual active sites and could be ligated to each heme. The interaction between heme and the histidine residues would serve as a molecular tether, allowing the proper positioning of two hemes to enable heme dimer formation. The heme dimer would act as a seed for the crystal growth of Hz in P. falciparum.

  13. Optimization of acid hydrolysis from the hemicellulosic fraction of Eucalyptus grandis residue using response surface methodology.

    PubMed

    Canettieri, Eliana Vieira; de Moraes Rocha, George Jackson; de Carvalho, João Andrade; de Almeida e Silva, João Batista

    2007-01-01

    Biotechnological conversion of biomass into fuels and chemicals requires hydrolysis of the polysaccharide fraction into monomeric sugars. Hydrolysis can be performed enzymatically and with dilute or concentrate mineral acids. The present study used dilute sulfuric acid as a catalyst for hydrolysis of Eucalyptus grandis residue. The purpose of this paper was to optimize the hydrolysis process in a 1.4 l pilot-scale reactor and investigate the effects of the acid concentration, temperature and residue/acid solution ratio on the hemicellulose removal and consequently on the production of sugars (xylose, glucose and arabinose) as well as on the formation of by-products (furfural, 5-hydroxymethylfurfural and acetic acid). This study was based on a model composition corresponding to a 2(3) orthogonal factorial design and employed the response surface methodology (RSM) to optimize the hydrolysis conditions, aiming to attain maximum xylose extraction from hemicellulose of residue. The considered optimum conditions were: H(2)SO(4) concentration of 0.65%, temperature of 157 degrees C and residue/acid solution ratio of 1/8.6 with a reaction time of 20 min. Under these conditions, 79.6% of the total xylose was removed and the hydrolysate contained 1.65 g/l glucose, 13.65 g/l xylose, 1.55 g/l arabinose, 3.10 g/l acetic acid, 1.23 g/l furfural and 0.20 g/l 5-hydroxymethylfurfural.

  14. Entropy Transfer between Residue Pairs and Allostery in Proteins: Quantifying Allosteric Communication in Ubiquitin

    PubMed Central

    2017-01-01

    It has recently been proposed by Gunasakaran et al. that allostery may be an intrinsic property of all proteins. Here, we develop a computational method that can determine and quantify allosteric activity in any given protein. Based on Schreiber's transfer entropy formulation, our approach leads to an information transfer landscape for the protein that shows the presence of entropy sinks and sources and explains how pairs of residues communicate with each other using entropy transfer. The model can identify the residues that drive the fluctuations of others. We apply the model to Ubiquitin, whose allosteric activity has not been emphasized until recently, and show that there are indeed systematic pathways of entropy and information transfer between residues that correlate well with the activities of the protein. We use 600 nanosecond molecular dynamics trajectories for Ubiquitin and its complex with human polymerase iota and evaluate entropy transfer between all pairs of residues of Ubiquitin and quantify the binding susceptibility changes upon complex formation. We explain the complex formation propensities of Ubiquitin in terms of entropy transfer. Important residues taking part in allosteric communication in Ubiquitin predicted by our approach are in agreement with results of NMR relaxation dispersion experiments. Finally, we show that time delayed correlation of fluctuations of two interacting residues possesses an intrinsic causality that tells which residue controls the interaction and which one is controlled. Our work shows that time delayed correlations, entropy transfer and causality are the required new concepts for explaining allosteric communication in proteins. PMID:28095404

  15. Requirement of the N-terminal residues of human cytomegalovirus UL112-113 proteins for viral growth and oriLyt-dependent DNA replication.

    PubMed

    Kim, Young-Eui; Park, Mi Young; Kang, Kyeong Jin; Han, Tae Hee; Lee, Chan Hee; Ahn, Jin-Hyun

    2015-08-01

    The UL112-113 region of the human cytomegalovirus (HCMV) genome encodes four phosphoproteins of 34, 43, 50, and 84 kDa that promote viral DNA replication. Co-transfection assays have demonstrated that self-interaction of these proteins via the shared N-termini is necessary for their intranuclear distribution as foci and for the efficient relocation of a viral DNA polymerase processivity factor (UL44) to the viral replication sites. However, the requirement of UL112-113 N-terminal residues for viral growth and DNA replication has not been fully elucidated. Here, we investigated the effect of deletion of the N-terminal regions of UL112-113 proteins on viral growth and oriLyt-dependent DNA replication. A deletion of the entire UL112 region or the region encoding the 25 N-terminal amino-acid residues from the HCMV (Towne strain) bacmid impaired viral growth in bacmid-transfected human fibroblast cells, indicating their requirement for viral growth. In co-immunoprecipitation assays using the genomic gene expressing the four UL112-113 proteins together, the 25 N-terminal amino-acid residues were found to be necessary for stable expression of UL112-113 proteins and their self-interaction. These residues were also required for efficient binding to and relocation of UL44, but not for interaction with IE2, an origin-binding transcription factor. In co-transfection/replication assays, replication of the oriLyt-containing plasmid was promoted by expression of intact UL112-113 proteins, but not by the expression of 25-amino-acid residue-deleted proteins. Our results demonstrate that the 25 N-terminal amino-acid residues of UL112-113 proteins that mediate self-interaction contribute to viral growth by promoting their binding to UL44 and the initiation of oriLyt-dependent DNA replication.

  16. Characterization of protein and carbohydrate mid-IR spectral features in crop residues

    NASA Astrophysics Data System (ADS)

    Xin, Hangshu; Zhang, Yonggen; Wang, Mingjun; Li, Zhongyu; Wang, Zhibo; Yu, Peiqiang

    2014-08-01

    To the best of our knowledge, a few studies have been conducted on inherent structure spectral traits related to biopolymers of crop residues. The objective of this study was to characterize protein and carbohydrate structure spectral features of three field crop residues (rice straw, wheat straw and millet straw) in comparison with two crop vines (peanut vine and pea vine) by using Fourier transform infrared spectroscopy (FTIR) technique with attenuated total reflectance (ATR). Also, multivariate analyses were performed on spectral data sets within the regions mainly related to protein and carbohydrate in this study. The results showed that spectral differences existed in mid-IR peak intensities that are mainly related to protein and carbohydrate among these crop residue samples. With regard to protein spectral profile, peanut vine showed the greatest mid-IR band intensities that are related to protein amide and protein secondary structures, followed by pea vine and the rest three field crop straws. The crop vines had 48-134% higher spectral band intensity than the grain straws in spectral features associated with protein. Similar trends were also found in the bands that are mainly related to structural carbohydrates (such as cellulosic compounds). However, the field crop residues had higher peak intensity in total carbohydrates region than the crop vines. Furthermore, spectral ratios varied among the residue samples, indicating that these five crop residues had different internal structural conformation. However, multivariate spectral analyses showed that structural similarities still exhibited among crop residues in the regions associated with protein biopolymers and carbohydrate. Further study is needed to find out whether there is any relationship between spectroscopic information and nutrition supply in various kinds of crop residue when fed to animals.

  17. Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues.

    PubMed

    Coïc, Yves-Marie; Vincent, Michel; Gallay, Jacques; Baleux, Françoise; Mousson, Florence; Beswick, Veronica; Neumann, Jean-Michel; de Foresta, Béatrice

    2005-12-01

    Membrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H(+)-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H(+)-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185-97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (lambda(max)), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k(q)) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We

  18. NIAS-Server: Neighbors Influence of Amino acids and Secondary Structures in Proteins.

    PubMed

    Borguesan, Bruno; Inostroza-Ponta, Mario; Dorn, Márcio

    2017-03-01

    The exponential growth in the number of experimentally determined three-dimensional protein structures provide a new and relevant knowledge about the conformation of amino acids in proteins. Only a few of probability densities of amino acids are publicly available for use in structure validation and prediction methods. NIAS (Neighbors Influence of Amino acids and Secondary structures) is a web-based tool used to extract information about conformational preferences of amino acid residues and secondary structures in experimental-determined protein templates. This information is useful, for example, to characterize folds and local motifs in proteins, molecular folding, and can help the solution of complex problems such as protein structure prediction, protein design, among others. The NIAS-Server and supplementary data are available at http://sbcb.inf.ufrgs.br/nias .

  19. DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues

    PubMed Central

    Guo, Jing; Sun, Xiao

    2016-01-01

    DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/. PMID:27907159

  20. Functional analysis of amino acid residues essential for activity in the Na+/H+ exchanger of fission yeast.

    PubMed

    Dibrov, P; Young, P G; Fliegel, L

    1998-06-09

    We identified amino acid residues important for activity of sod2, the Na+/H+ antiporter of Schizosaccharomyces pombe. We mutated all eight His residues of sod2 into Arg. Only His367-->Arg affected function and resulted in complete inability of sod2 to allow growth of S. pombe in LiCl-containing medium. Mutant S. pombe (H367R) could not expel sodium in acidic (pH 4.0) medium and were defective in their ability to alkalinize external medium. When His367 was replaced by Asp, sodium export of S. pombe was suppressed at acidic pH while the sodium-dependent proton influx at pH 6.1 was increased compared to wild type. We also mutated three residues conserved in putative membrane regions of various eukaryotic and prokaryotic Na+/H+ exchangers. S. pombe containing Asp241-->Asn and Asp266, 267-->Asn mutations had greatly impaired growth in LiCl-containing medium. In addition, sodium-dependent proton influx at external pH 6. 1 was impaired. Sodium export from S. pombe cells at external pH 4.0 was also almost completely abolished by the D266,267N mutation; however, the D241N mutant protein retained almost normal Na+ export. The results demonstrate that His367, Asp241, and Asp266,267 are important in the function of the eukaryotic Na+/H+ exchanger sod2.

  1. Sequence of the canine herpesvirus thymidine kinase gene: taxon-preferred amino acid residues in the alphaherpesviral thymidine kinases.

    PubMed

    Rémond, M; Sheldrick, P; Lebreton, F; Foulon, T

    1995-12-01

    Multiple sequence alignments of evolutionarily related proteins are finding increasing use as indicators of critical amino acid residues necessary for structural stability or involved in functional domains responsible for catalytic activities. In the past, a number of alignments have provided such information for the herpesviral thymidine kinases, for which three-dimensional structures are not yet available. We have sequenced the thymidine kinase gene of a canine herpesvirus, and with a multiple alignment have identified amino acids preferentially conserved in either of two taxons, the genera Varicellovirus and Simplexvirus, of the subfamily Alphaherpesvirinae. Since some regions of the thymidine kinases show otherwise elevated levels of substitutional tolerance, these conserved amino acids are candidates for critical residues which have become fixed through selection during the evolutionary divergence of these enzymes. Several pairs with distinctive patterns of distribution among the various viruses occur in or near highly conserved sequence motifs previously proposed to form the catalytic site, and we speculate that they may represent interacting, co-ordinately variable residues.

  2. 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.

  3. Measurement of the individual pKa values of acidic residues of hen and turkey lysozymes by two-dimensional 1H NMR.

    PubMed Central

    Bartik, K; Redfield, C; Dobson, C M

    1994-01-01

    The pH dependence of the two-dimensional 1H nuclear magnetic resonance spectra of hen and turkey egg-white lysozymes has been recorded over the pH range 1-7. By monitoring the chemical shifts of the resonances of the various protons of ionizable residues, individual pKa values for the acidic residues have been determined for both proteins. The pKa values are displaced, with the exception of those of the residues in the active site cleft, by an average of 1 unit to low pH compared to model compounds. PMID:8038389

  4. A conserved proline residue in Dothideomycete Avr4 effector proteins is required to trigger a Cf-4-dependent hypersensitive response.

    PubMed

    Mesarich, Carl H; Stergiopoulos, Ioannis; Beenen, Henriek G; Cordovez, Viviane; Guo, Yanan; Karimi Jashni, Mansoor; Bradshaw, Rosie E; de Wit, Pierre J G M

    2016-01-01

    CfAvr4, a chitin-binding effector protein produced by the Dothideomycete tomato pathogen Cladosporium fulvum, protects the cell wall of this fungus against hydrolysis by secreted host chitinases during infection. However, in the presence of the Cf-4 immune receptor of tomato, CfAvr4 triggers a hypersensitive response (HR), which renders the pathogen avirulent. Recently, several orthologues of CfAvr4 have been identified from phylogenetically closely related species of Dothideomycete fungi. Of these, DsAvr4 from Dothistroma septosporum also triggers a Cf-4-dependent HR, but CaAvr4 and CbAvr4 from Cercospora apii and Cercospora beticola, respectively, do not. All, however, bind chitin. To identify the region(s) and specific amino acid residue(s) of CfAvr4 and DsAvr4 required to trigger a Cf-4-dependent HR, chimeric and mutant proteins, in which specific protein regions or single amino acid residues, respectively, were exchanged between CfAvr4 and CaAvr4 or DsAvr4 and CbAvr4, were tested for their ability to trigger an HR in Nicotiana benthamiana plants transgenic for the Cf-4 immune receptor gene. Based on this approach, a single region common to CfAvr4 and DsAvr4 was determined to carry a conserved proline residue necessary for the elicitation of this HR. In support of this result, a Cf-4-dependent HR was triggered by mutant CaAvr4 and CbAvr4 proteins carrying an arginine-to-proline substitution at this position. This study provides the first step in deciphering how Avr4 orthologues from different Dothideomycete fungi trigger a Cf-4-dependent HR.

  5. An amino acid code for irregular and mixed protein packing.

    PubMed

    Joo, Hyun; Chavan, Archana G; Fraga, Keith J; Tsai, Jerry

    2015-12-01

    To advance our understanding of protein tertiary structure, the development of the knob-socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob-socket model simplifies packing based on repeated patterns of two motifs: a three-residue socket for packing within secondary (2°) structure and a four-residue knob-socket for tertiary (3°) packing. For coil and turn secondary structure, knob-sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α-helices to tertiary packing. In irregular sockets, Gly, Pro, Asp, and Ser are favored, while in irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly. Cys, His,Met, and Trp are not favored in either. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β-sheet socket may potentially function to inhibit β-sheet extension. In addition, analysis of the preferred crossing angles for strands within a β-sheet and mixed α-helice/β-sheet identifies canonical packing patterns useful in protein design. Lastly, the knob-socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map.

  6. Alimentary proteins, amino acids and cholesterolemia.

    PubMed

    Blachier, François; Lancha, Antonio H; Boutry, Claire; Tomé, Daniel

    2010-01-01

    Numerous data from both epidemiological and experimental origins indicate that some alimentary proteins and amino acids in supplements can modify the blood LDL cholesterol, HDL cholesterol and total cholesterol. After an initial approval of the health claim for soy protein consumption for the prevention of coronary heart disease, more recently it has been concluded from an overall analysis of literature that isolated soy protein with isoflavones only slightly decrease LDL and total cholesterol. Other plant extracts and also some proteins from animal origin have been reported to exert a lowering effect on blood cholesterol when compared with a reference protein (often casein). The underlying mechanisms are still little understood. Individual amino acids and mixture of amino acids have also been tested (mostly in animal studies) for their effects on cholesterol parameters and on cholesterol metabolism. Methionine, lysine, cystine, leucine, aspartate and glutamate have been tested individually and in combination in different models of either normo or hypercholesterolemic animals and found to be able to modify blood cholesterol and/or LDL cholesterol and/or HDL cholesterol. It is however not known if these results are relevant to human nutrition.

  7. Plasmodium falciparum Field Isolates Commonly Use Erythrocyte Invasion Pathways That Are Independent of Sialic Acid Residues of Glycophorin A

    PubMed Central

    Okoyeh, Jude Nnaemeka; Pillai, C. R.; Chitnis, Chetan E.

    1999-01-01

    Erythrocyte invasion by malaria parasites is mediated by specific molecular interactions. Sialic acid residues of glycophorin A are used as invasion receptors by Plasmodium falciparum. In vitro invasion studies have demonstrated that some cloned P. falciparum lines can use alternate receptors independent of sialic acid residues of glycophorin A. It is not known if invasion by alternate pathways occurs commonly in the field. In this study, we used in vitro growth assays and erythrocyte invasion assays to determine the invasion phenotypes of 15 P. falciparum field isolates. Of the 15 field isolates tested, 5 multiply in both neuraminidase and trypsin-treated erythrocytes, 3 multiply in neuraminidase-treated but not trypsin-treated erythrocytes, and 4 multiply in trypsin-treated but not neuraminidase-treated erythrocytes; 12 of the 15 field isolates tested use alternate invasion pathways that are not dependent on sialic acid residues of glycophorin A. Alternate invasion pathways are thus commonly used by P. falciparum field isolates. Typing based on two polymorphic markers, MSP-1 and MSP-2, and two microsatellite markers suggests that only 1 of the 15 field isolates tested contains multiple parasite genotypes. Individual P. falciparum lines can thus use multiple invasion pathways in the field. These observations have important implications for malaria vaccine development efforts based on EBA-175, the P. falciparum protein that binds sialic acid residues of glycophorin A during invasion. It may be necessary to target parasite ligands responsible for the alternate invasion pathways in addition to EBA-175 to effectively block erythrocyte invasion by P. falciparum. PMID:10531229

  8. 75 FR 1773 - Notice of Receipt of a Pesticide Petition Filed for Residues of Polymeric Polyhydroxy Acid in or...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-13

    ... AGENCY Notice of Receipt of a Pesticide Petition Filed for Residues of Polymeric Polyhydroxy Acid in or... establishment of a regulation for residues of the plant growth regulator, polymeric polyhydroxy acid, in or on... polymeric polyhydroxy acid in or on all food commodities. EPA has determined that the pesticide...

  9. Identification of critical amino acid residues and functional conservation of the Neurospora crassa and Rattus norvegicus orthologues of neuronal calcium sensor-1.

    PubMed

    Gohain, Dibakar; Deka, Rekha; Tamuli, Ranjan

    2016-12-01

    Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca(2+)) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca(2+) binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N. crassa strains possessing the ncs-1 mutant allele of these three amino acid residues showed impairment in functions ranging from growth, Ca(2+) stress tolerance, and ultraviolet survival. In addition, heterologous expression of the NCS-1 from Rattus norvegicus in N. crassa confirmed its interspecies functional conservation. Moreover, functions of glutamic acid at position 120, the first Ca(2+) binding residue among all the EF-hands of the R. norvegicus NCS-1 was found conserved. Thus, we identified three critical amino acid residues of N. crassa NCS-1, and demonstrated its functional conservation across species using the orthologue from R. norvegicus.

  10. Structural Reorganization Triggered by Charging of Lys Residues in the Hydrophobic Interior of a Protein

    SciTech Connect

    Chimenti M. S.; Heroux A.; Khangulov, V. S.; Robinson, A. C.; Majumdar, A.; Schlessman, J. L.; Garcia-Moreno, B.

    2012-06-06

    Structural consequences of ionization of residues buried in the hydrophobic interior of proteins were examined systematically in 25 proteins with internal Lys residues. Crystal structures showed that the ionizable groups are buried. NMR spectroscopy showed that in 2 of 25 cases studied, the ionization of an internal Lys unfolded the protein globally. In five cases, the internal charge triggered localized changes in structure and dynamics, and in three cases, it promoted partial or local unfolding. Remarkably, in 15 proteins, the ionization of the internal Lys had no detectable structural consequences. Highly stable proteins appear to be inherently capable of withstanding the presence of charge in their hydrophobic interior, without the need for specialized structural adaptations. The extent of structural reorganization paralleled loosely with global thermodynamic stability, suggesting that structure-based pK{sub a} calculations for buried residues could be improved by calculation of thermodynamic stability and by enhanced conformational sampling.

  11. Functional analyses of carnivorous plant-specific amino acid residues in S-like ribonucleases.

    PubMed

    Arai, Naoki; Nishimura, Emi; Kikuchi, Yo; Ohyama, Takashi

    2015-09-11

    Unlike plants with no carnivory, carnivorous plants seem to use S-like ribonucleases (RNases) as an enzyme for carnivory. Carnivorous plant-specific conserved amino acid residues are present at four positions around the conserved active site (CAS). The roles of these conserved amino acid residues in the enzymatic function were explored in the current study by preparing five recombinant variants of DA-I, the S-like RNase of Drosera adelae. The kcat and kcat/Km values of the enzymes revealed that among the four variants with a single mutation, the serine to glycine mutation at position 111 most negatively influenced the enzymatic activity. The change in the bulkiness of the amino acid residue side-chain seemed to be the major cause of the above effect. Modeling of the three dimensional (3D) structures strongly suggested that the S to G mutation at 111 greatly altered the overall enzyme conformation. The conserved four amino acid residues are likely to function in keeping the two histidine residues, which are essential for the cleavage of RNA strands, and the CAS in the most functional enzymatic conformation.

  12. Just three water molecules can trigger the undesired nonenzymatic reactions of aspartic acid residues: new insight from a quantum-chemical study

    NASA Astrophysics Data System (ADS)

    Takahashi, O.

    2014-03-01

    Aspartic acid (Asp) residues in peptides and proteins (L-Asp) can undergo spontaneous, nonenzymatic reactions under physiological conditions by which abnormal L-β-Asp, D-Asp, and/or D-β-Asp residues are formed. These altered Asp residues may affect the three-dimensional structures of the peptides and proteins and hence their properties and functions. In fact, the altered Asp residues are relevant to age-related diseases such as cataract and Alzheimer's disease. Most of the above reactions of the L-Asp residue proceed via a cyclic succinimide intermediate. In this paper, I propose a detailed mechanism of cyclization of an Asp residue (forming a precursor of the succinimide) by the B3LYP/6-31+G(d,p) density functional theory calculations carried out for a small Asp-containing model compound complexed with three water molecules which act as general acid-base catalysts in proton transfers. In the proposed mechanism, the amide group on the C-terminal side of the Asp residue is first converted to the tautomeric iminol form. Then, successive reorientation of a water molecule and conformational change occur followed by the nucleophilic attack of the iminol nitrogen atom on the carboxyl carbon atom of the Asp side chain to form a five-membered ring. A satisfactory agreement was obtained between the calculated and experimental energetics.

  13. A microalgae residue based carbon solid acid catalyst for biodiesel production.

    PubMed

    Fu, Xiaobo; Li, Dianhong; Chen, Jie; Zhang, Yuanming; Huang, Weiya; Zhu, Yi; Yang, Jun; Zhang, Chengwu

    2013-10-01

    Biodiesel production from microalgae is recognized as one of the best solutions to deal with the energy crisis issues. However, after the oil extraction from the microalgae, the microalgae residue was generally discarded or burned. Here a novel carbon-based solid acid catalyst derived from microalgae residue by in situ hydrothermal partially carbonization were synthesized. The obtained catalyst was characterized and subjected to both the esterification of oleic acid and transesterification of triglyceride to produce biodiesel. The catalyst showed high catalytic activity and can be regenerated while its activity can be well maintained after five cycles.

  14. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    PubMed Central

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea

    2015-01-01

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin. PMID:25724962

  15. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    DOE PAGES

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; ...

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

  16. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    SciTech Connect

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

  17. Identification of residues in a hydrophilic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen.

    PubMed Central

    Kakeda, K; Jordan, N D; Conner, A; Ride, J P; Franklin-Tong, V E; Franklin, F C

    1998-01-01

    The self-incompatibility response involves S allele-specific recognition between stigmatic S proteins and incompatible pollen. This response results in pollen inhibition. Defining the amino acid residues within the stigmatic S proteins that participate in S allele-specific inhibition of incompatible pollen is essential for the elucidation of the molecular basis of the self-incompatibility response. We have constructed mutant derivatives of the S1 protein from Papaver rhoeas by using site-directed mutagenesis and have tested their biological activity. This has enabled us to identify amino acid residues in the stigmatic S proteins of P. rhoeas that are required for S-specific inhibition of incompatible pollen. We report here the identification of several amino acid residues in the predicted hydrophilic loop 6 of the P. rhoeas stigmatic S1 protein that are involved in the inhibition of S1 pollen. Mutation of the only hypervariable amino acid, which is situated in this loop, resulted in the complete loss of ability of the S protein to inhibit S1 pollen. This clearly demonstrates that this residue plays a crucial role in pollen recognition and may also participate in defining allelic specificity. We have also established the importance of highly conserved amino acids adjacent to this hypervariable site. Our studies demonstrate that both variable and conserved amino acids in the region of the S protein corresponding to surface loop 6 are key elements that play a role in the recognition and inhibition of incompatible pollen in the pollen-pistil self-incompatibility reaction. PMID:9761798

  18. Conformations of amino acids in proteins.

    PubMed

    Hovmöller, Sven; Zhou, Tuping; Ohlson, Tomas

    2002-05-01

    The main-chain conformations of 237 384 amino acids in 1042 protein subunits from the PDB were analyzed with Ramachandran plots. The populated areas of the empirical Ramachandran plot differed markedly from the classical plot in all regions. All amino acids in alpha-helices are found within a very narrow range of phi, psi angles. As many as 40% of all amino acids are found in this most populated region, covering only 2% of the Ramachandran plot. The beta-sheet region is clearly subdivided into two distinct regions. These do not arise from the parallel and antiparallel beta-strands, which have quite similar conformations. One beta region is mainly from amino acids in random coil. The third and smallest populated area of the Ramachandran plot, often denoted left-handed alpha-helix, has a different position than that originally suggested by Ramachandran. Each of the 20 amino acids has its own very characteristic Ramachandran plot. Most of the glycines have conformations that were considered to be less favoured. These results may be useful for checking secondary-structure assignments in the PDB and for predicting protein folding.

  19. Relationship between residual feed intake and lymphocyte mitochondrial complex protein concentration and ratio in crossbred steers.

    PubMed

    Davis, M P; Brooks, M A; Kerley, M S

    2016-04-01

    Rate of oxygen uptake by muscle mitochondria and respiratory chain protein concentrations differed between high- and low-residual feed intake (RFI) animals. The hypothesis of this research was that complex I (CI), II (CII), and III (CIII) mitochondria protein concentrations in lymphocyte (blood) mitochondria were related to the RFI phenotype of beef steers. Daily feed intake (ADFI) was individually recorded for 92 Hereford-crossbreed steers over 63 d using GrowSafe individual feed intake system. Predicted ADFI was calculated as the regression of ADFI on ADG and midtest BW. Difference between ADFI and predicted ADFI was RFI. Lymphocytes were isolated from low-RFI (-1.32 ± 0.11 kg/d; = 10) and high-RFI (1.34 ± 0.18 kg/d; = 8) steers. Immunocapture of CI, CII, and CIII proteins from the lymphocyte was done using MitoProfile CI, CII, and CIII immunocapture kits (MitoSciences Inc., Eugene, OR). Protein concentrations of CI, CII, and CIII and total protein were quantified using bicinchoninic acid colorimetric procedures. Low-RFI steers consumed 30% less ( = 0.0004) feed and had a 40% improvement ( < 0.0001) in feed efficiency compared with high-RFI steers with similar growth ( = 0.78) and weight measurements ( > 0.65). High- and low-RFI steers did not differ in CI ( = 0.22), CII ( = 0.69), and CIII ( = 0.59) protein concentrations. The protein concentration ratios for CI to CII ( = 0.03) were 20% higher and the ratios of CI to CIII ( = 0.01) were 30% higher, but the ratios of CII to CIII ( = 0.89) did not differ when comparing low-RFI steers with high-RFI steers. The similar magnitude difference in feed intake, feed efficiency measurements, and CI-to-CIII ratio between RFI phenotypes provides a plausible explanation for differences between the phenotypes. We also concluded that mitochondria isolated from lymphocytes could be used to study respiratory chain differences among differing RFI phenotypes. Further research is needed to determine if lymphocyte mitochondrial

  20. Snake venoms. The amino-acid sequence of protein S5C4 from Dendroaspis jamesoni kaimosae (Jameson's mamba) venom.

    PubMed

    Joubert, F J; Strydom, A J; Taljaard, N

    1978-06-01

    A major component (S5C4) was purified from Jameson's mamba by gel filtration on Sephadex G-50 and by ion-exchange chromotography on CM-cellulose. Protein S5C4 contains 60 amino acid residues and is cross-linked by four intrachain disulphide bridges. The complete primary structure of the protein has been elucidated. The toxicities, the immunochemical properties, the sequence and the invariant amino acid residues of protein S5C4 resemble subgroup II of the angusticeps-type proteins.

  1. Measurement of protein using bicinchoninic acid.

    PubMed

    Smith, P K; Krohn, R I; Hermanson, G T; Mallia, A K; Gartner, F H; Provenzano, M D; Fujimoto, E K; Goeke, N M; Olson, B J; Klenk, D C

    1985-10-01

    Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.

  2. Hyperdimensional Analysis of Amino Acid Pair Distributions in Proteins

    PubMed Central

    Henriksen, Svend B.; 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

  3. Pulsed EPR Determination of Water Accessibility to Spin-Labeled Amino Acid Residues in LHCIIb

    PubMed Central

    Volkov, A.; Dockter, C.; Bund, T.; Paulsen, H.; Jeschke, G.

    2009-01-01

    Membrane proteins reside in a structured environment in which some of their residues are accessible to water, some are in contact with alkyl chains of lipid molecules, and some are buried in the protein. Water accessibility of residues may change during folding or function-related structural dynamics. Several techniques based on the combination of pulsed electron paramagnetic resonance (EPR) with site-directed spin labeling can be used to quantify such water accessibility. Accessibility parameters for different residues in major plant light-harvesting complex IIb are determined by electron spin echo envelope modulation spectroscopy in the presence of deuterated water, deuterium contrast in transversal relaxation rates, analysis of longitudinal relaxation rates, and line shape analysis of electron-spin-echo-detected EPR spectra as well as by the conventional techniques of measuring the maximum hyperfine splitting and progressive saturation in continuous-wave EPR. Systematic comparison of these parameters allows for a more detailed characterization of the environment of the spin-labeled residues. These techniques are applicable independently of protein size and require ∼10–20 nmol of singly spin-labeled protein per sample. For a residue close to the N-terminus, in a domain unresolved in the existing x-ray structures of light-harvesting complex IIb, all methods indicate high water accessibility. PMID:19186148

  4. Highly conserved residues in the helical domain of dengue virus type 1 precursor membrane protein are involved in assembly, precursor membrane (prM) protein cleavage, and entry.

    PubMed

    Hsieh, Szu-Chia; Wu, Yi-Chieh; Zou, Gang; Nerurkar, Vivek R; Shi, Pei-Yong; Wang, Wei-Kung

    2014-11-28

    The envelope and precursor membrane (prM) proteins of dengue virus (DENV) are present on the surface of immature virions. During maturation, prM protein is cleaved by furin protease into pr peptide and membrane (M) protein. Although previous studies mainly focusing on the pr region have identified several residues important for DENV replication, the functional role of M protein, particularly the α-helical domain (MH), which is predicted to undergo a large conformational change during maturation, remains largely unknown. In this study, we investigated the role of nine highly conserved MH domain residues in the replication cycle of DENV by site-directed mutagenesis in a DENV1 prME expression construct and found that alanine substitutions introduced to four highly conserved residues at the C terminus and one at the N terminus of the MH domain greatly affect the production of both virus-like particles and replicon particles. Eight of the nine alanine mutants affected the entry of replicon particles, which correlated with the impairment in prM cleavage. Moreover, seven mutants were found to have reduced prM-E interaction at low pH, which may inhibit the formation of smooth immature particles and exposure of prM cleavage site during maturation, thus contributing to inefficient prM cleavage. Taken together, these results are the first report showing that highly conserved MH domain residues, located at 20-38 amino acids downstream from the prM cleavage site, can modulate the prM cleavage, maturation of particles, and virus entry. The highly conserved nature of these residues suggests potential targets of antiviral strategy.

  5. Toward amino acid typing for proteins in FFLUX.

    PubMed

    Fletcher, Timothy L; Popelier, Paul L A

    2017-03-05

    Continuing the development of the FFLUX, a multipolar polarizable force field driven by machine learning, we present a modern approach to atom-typing and building transferable models for predicting atomic properties in proteins. Amino acid atomic charges in a peptide chain respond to the substitution of a neighboring residue and this response can be categorized in a manner similar to atom-typing. Using a machine learning method called kriging, we are able to build predictive models for an atom that is defined, not only by its local environment, but also by its neighboring residues, for a minimal additional computational cost. We found that prediction errors were up to 11 times lower when using a model specific to the correct group of neighboring residues, with a mean prediction of ∼0.0015 au. This finding suggests that atoms in a force field should be defined by more than just their immediate atomic neighbors. When comparing an atom in a single alanine to an analogous atom in a deca-alanine helix, the mean difference in charge is 0.026 au. Meanwhile, the same difference between a trialanine and a deca-alanine helix is only 0.012 au. When compared to deca-alanine models, the transferable models are up to 20 times faster to train, and require significantly less ab initio calculation, providing a practical route to modeling large biological systems. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

  6. Evaluation of microwave-accelerated residue-specific acid cleavage for proteomic applications.

    PubMed

    Swatkoski, Stephen; Gutierrez, Peter; Wynne, Colin; Petrov, Alexey; Dinman, Jonathan D; Edwards, Nathan; Fenselau, Catherine

    2008-02-01

    Microwave-accelerated proteolysis using acetic acid has been shown to occur specifically on either or both sides of aspartic acid residues. This chemical cleavage has been applied to ovalbumin and several model peptides to test the effect on some of the more common post-translational modifications. No oxidation of methionine or cysteine was observed; however, hydrolysis of phosphate groups proceeds at a detectable rate. Acid cleavage was also extended to the yeast ribosome model proteome, where it provided information on 74% of that proteome. Aspartic acid occurs across the proteome with approximately half the frequency of the combined occurrence of the trypsin residues lysine and arginine, and implications of this are considered.

  7. Evolutionary diversification of aminopeptidase N in Lepidoptera by conserved clade-specific amino acid residues.

    PubMed

    Hughes, Austin L

    2014-07-01

    Members of the aminopepidase N (APN) gene family of the insect order Lepidoptera (moths and butterflies) bind the naturally insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis. Phylogenetic analysis of amino acid sequences of seven lepidopteran APN classes provided strong support for the hypothesis that lepidopteran APN2 class arose by gene duplication prior to the most recent common ancestor of Lepidoptera and Diptera. The Cry toxin-binding region (BR) of lepidopteran and dipteran APNs was subject to stronger purifying selection within APN classes than was the remainder of the molecule, reflecting conservation of catalytic site and adjoining residues within the BR. Of lepidopteran APN classes, APN2, APN6, and APN8 showed the strongest evidence of functional specialization, both in expression patterns and in the occurrence of conserved derived amino acid residues. The latter three APN classes also shared a convergently evolved conserved residue close to the catalytic site. APN8 showed a particularly strong tendency towards class-specific conserved residues, including one of the catalytic site residues in the BR and ten others in close vicinity to the catalytic site residues. The occurrence of class-specific sequences along with the conservation of enzymatic function is consistent with the hypothesis that the presence of Cry toxins in the environment has been a factor shaping the evolution of this multi-gene family.

  8. Revised Backbone-Virtual-Bond-Angle Potentials to Treat the l- and d-Amino Acid Residues in the Coarse-Grained United Residue (UNRES) Force Field

    PubMed Central

    2015-01-01

    Continuing our effort to introduce d-amino-acid residues in the united residue (UNRES) force field developed in our laboratory, in this work the Cα ··· Cα ··· Cα backbone-virtual-bond-valence-angle (θ) potentials for systems containing d-amino-acid residues have been developed. The potentials were determined by integrating the combined energy surfaces of all possible triplets of terminally blocked glycine, alanine, and proline obtained with ab initio molecular quantum mechanics at the MP2/6-31G(d,p) level to calculate the corresponding potentials of mean force (PMFs). Subsequently, analytical expressions were fitted to the PMFs to give the virtual-bond-valence potentials to be used in UNRES. Alanine represented all types of amino-acid residues except glycine and proline. The blocking groups were either the N-acetyl and N′,N′-dimethyl or N-acetyl and pyrrolidyl group, depending on whether the residue next in sequence was an alanine-type or a proline residue. A total of 126 potentials (63 symmetry-unrelated potentials for each set of terminally blocking groups) were determined. Together with the torsional, double-torsional, and side-chain-rotamer potentials for polypeptide chains containing d-amino-acid residues determined in our earlier work (Sieradzan et al. J. Chem. Theory Comput., 2012, 8, 4746), the new virtual-bond-angle (θ) potentials now constitute the complete set of physics-based potentials with which to run coarse-grained simulations of systems containing d-amino-acid residues. The ability of the extended UNRES force field to reproduce thermodynamics of polypeptide systems with d-amino-acid residues was tested by comparing the experimentally measured and the calculated free energies of helix formation of model KLALKLALxxLKLALKLA peptides, where x denotes any d- or l- amino-acid residue. The obtained results demonstrate that the UNRES force field with the new potentials reproduce the changes of free energies of helix formation upon d

  9. The role of basic residues in the adsorption of blood proteins onto the graphene surface

    NASA Astrophysics Data System (ADS)

    Gu, Zonglin; Yang, Zaixing; Wang, Lingle; Zhou, Hong; Jimenez-Cruz, Camilo A.; Zhou, Ruhong

    2015-06-01

    With its many unique properties, graphene has shown great potential in various biomedical applications, while its biocompatibility has also attracted growing concerns. Previous studies have shown that the formation of protein-graphene corona could effectively reduce its cytotoxicity; however, the underlying molecular mechanism remains not well-understood. Herein, we use extensive molecular dynamics simulations to demonstrate that blood proteins such as bovine fibrinogen (BFG) can absorb onto the graphene surface quickly and tightly to form a corona complex. Aromatic residues contributed significantly during this adsorption process due to the strong π-π stacking interactions between their aromatic rings and the graphene sp2-carbons. Somewhat surprisingly, basic residues like arginine, also played an equally or even stronger role during this process. The strong dispersion interactions between the sidechains of these solvent-exposed basic residues and the graphene surface provide the driving force for a tight binding of these basic residues. To the best of our knowledge, this is the first study with blood proteins to show that, in addition to the aromatic residues, the basic residues also play an important role in the formation of protein-graphene corona complexes.

  10. Increasing protein stability by polar surface residues : domain-wide consequences of interactions within a loop.

    SciTech Connect

    Pokkuluri, P. R.; Raffen, R.; Dieckman, L.; Boogaard, C.; Stevens, F. J.; Schiffer, M.; Biosciences Division; C. Boogaard

    2002-01-01

    We have examined the influence of surface hydrogen bonds on the stability of proteins by studying the effects of mutations of human immunoglobulin light chain variable domain (V(L)). In addition to the variants Y27dD, N28F, and T94H of protein kappa IV Len that were previously described, we characterized mutants M4L, L27cN, L27cQ, and K39T, double mutant M4L/Y27dD, and triple mutant M4L/Y27dD/T94H. The triple mutant had an enhanced thermodynamic stability of 4.2 kcal/mol. We determined the structure of the triple mutant by x-ray diffraction and correlated the changes in stability due to the mutations with changes in the three-dimensional structure. Y27dD mutant had increased stability of Len by 2.7 kcal/mol, a large value for a single mutation. Asp27d present in CDR1 formed hydrogen bonds with the side-chain and main-chain atoms within the loop. In the case of the K39T mutant, which reduces stability by 2 kcal/mol, Lys39 in addition to forming a hydrogen bond with a carbonyl oxygen of a neighboring loop may also favorably influence the surface electrostatics of the molecule. We showed that hydrogen bonds between residues in surface loops can add to the overall stability of the V(L) domains. The contribution to stability is further increased if the surface residue makes more than one hydrogen bond or if it forms a hydrogen bond between neighboring turns or loops separated from each other in the amino acid sequence. Based on our experiments we suggest that stabilization of proteins might be systematically accomplished by introducing additional hydrogen bonds on the surface. These substitutions are more straightforward to predict than core-packing interactions and can be selected to avoid affecting the protein's function.

  11. Phospho-N-Acetyl-Muramyl-Pentapeptide Translocase from Escherichia coli: Catalytic Role of Conserved Aspartic Acid Residues

    PubMed Central

    Lloyd, Adrian J.; Brandish, Philip E.; Gilbey, Andrea M.; Bugg, Timothy D. H.

    2004-01-01

    Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His6) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His6 fusions. Enzymatic analysis established that all three mutations eliminated translocase 1 activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile. PMID:14996806

  12. Kinetic and mutagenic evidence for the role of histidine residues in the Lycopersicon esculentum 1-aminocyclopropane-1-carboxylic acid oxidase.

    PubMed

    Tayeh, M A; Howe, D L; Salleh, H M; Sheflyan, G Y; Son, J K; Woodard, R W

    1999-01-01

    The ACCO gene from Lycopersicon esculentum (tomato) has been cloned into the expression vector PT7-7. The highly expressed protein was recovered in the form of inclusion bodies. ACCO is inactivated by diethyl pyrocarbonate (DEPC) with a second-order rate constant of 170 M(-1) min(-1). The pH-inactivation rate data imply the involvement of an amino acid residue with a pK value of 6.05. The difference UV spectrum of the the DEPC-inactivated versus native ACCO showed a single peak at 242 nm indicating the modification of histidine residues. The inactivation was reversed by the addition of hydroxylamine to the DEPC-inactivated ACCO. Substrate/cofactor protection studies indicate that both iron and ACC bind near the active site, which contains histidine residues. Four histidines of ACCO were individually mutated to alanine and glycine. H39A is catalytically active, while H177A, H177G, H211A, H211G, H234A, and H234G are basically inactive. The results indicate that histidine residues 177, 211, and 234 may serve as ligands for the active-site iron of ACCO and/or may play some important structural or catalytic role.

  13. A single residue in the 126-kDa protein of pepper mild mottle virus controls the severity of symptoms on infected green bell pepper plants.

    PubMed

    Ichiki, T U; Nagaoka, E N; Hagiwara, K; Sasaya, T; Omura, T

    2009-01-01

    Infectious cDNA clones originally derived from a mild strain of Pepper mild mottle virus were constructed by replacing residue 649, a critical point for attenuation of this virus, with all possible amino acids. All clones were infectious to pepper plants and induced a variety of symptoms, including no visible symptoms. The results of this study showed that a single amino acid mutation at residue 649 could control the function of the 126- and 183-kDa proteins, replicases with multiple roles in the life cycle of this virus.

  14. 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

  15. Amino acid composition analysis of human secondary transport proteins and implications for reliable membrane topology prediction.

    PubMed

    Saidijam, Massoud; Azizpour, Sonia; Patching, Simon G

    2016-07-08

    Secondary transporters in humans are a large group of proteins that transport a wide range of ions, metals, organic and inorganic solutes involved in energy transduction, control of membrane potential and osmotic balance, metabolic processes and in the absorption or efflux of drugs and xenobiotics. They are also emerging as important targets for development of new drugs and as target sites for drug delivery to specific organs or tissues. We have performed amino acid composition (AAC) and phylogenetic analyses and membrane topology predictions for 336 human secondary transport proteins and used the results to confirm protein classification and to look for trends and correlations with structural domains and specific substrates and/or function. Some proteins showed statistically high contents of individual amino acids or of groups of amino acids with similar physicochemical properties. One recurring trend was a correlation between high contents of charged and/or polar residues with misleading results in predictions of membrane topology, which was especially prevalent in Mitochondrial Carrier family proteins. We demonstrate how charged or polar residues located in the middle of transmembrane helices can interfere with their identification by membrane topology tools resulting in missed helices in the prediction. Comparison of AAC in the human proteins with that in 235 secondary transport proteins from Escherichia coli revealed similar overall trends along with differences in average contents for some individual amino acids and groups of similar amino acids that are presumed to result from a greater number of functions and complexity in the higher organism.

  16. The role of aspartic acid residues 405 and 416 of the kidney isotype of sodium-bicarbonate cotransporter 1 in its targeting to the plasma membrane

    PubMed Central

    Kucher, Volodymyr; Li, Emily Y.; Conforti, Laura; Zahedi, Kamyar A.

    2012-01-01

    The NH2 terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH2-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH2 terminus of NBCe1A are important in its accurate targeting to the plasma membrane. PMID:22442137

  17. Amino Acid Residues in the Putative Transmembrane Domain 11 of Human Organic Anion Transporting Polypeptide 1B1 Dictate Transporter Substrate Binding, Stability, and Trafficking.

    PubMed

    Hong, Weifang; Wu, Zhixuan; Fang, Zihui; Huang, Jiujiu; Huang, Hong; Hong, Mei

    2015-12-07

    Organic anion transporting polypeptides (OATPs, gene symbol SLCO) are membrane proteins that mediate the sodium-independent transport of a wide range of endogenous and exogenous compounds. Due to their broad substrate specificity, wide tissue distribution, and involvement in drug-drug interactions, OATPs have been considered as key players in drug absorption, distribution, and excretion. Transmembrane domains (TMs) are crucial structural features involved in proper functions of many transporters. According to computer-based modeling and previous studies of our laboratory and others, TM11 of OATP1B1 may face the substrate interaction pocket and thus play an important role in the transport function of the protein. Alanine-scanning of the transmembrane domain identified seven critical amino acid residues within the region. Further analysis revealed that alanine substitution of these residues resulted in reduced protein stability, which led to significantly decreased protein expression on the plasma membrane. In addition, all mutants exhibited an altered Km for ES uptake (either high affinity or low affinity component, or both), though Km for taurocholate transport only changed in R580A, G584A, and F591A. These results suggested that critical residues in TM11 not only affect protein stability of the transporter, but its interaction with substrates as well. The identification of seven essential residues out of 21 TM amino acids highlighted the importance of this transmembrane domain in the proper function of OATP1B1.

  18. The F13 residue is critical for interaction among the coat protein subunits of papaya mosaic virus.

    PubMed

    Laliberté Gagné, M E; Lecours, K; Gagné, S; Leclerc, D

    2008-04-01

    Papaya mosaic virus (PapMV) coat protein (CP) in Escherichia coli was previously showed to self-assemble in nucleocapsid-like particles (NLPs) that were similar in shape and appearance to the native virus. We have also shown that a truncated CP missing the N-terminal 26 amino acids is monomeric and loses its ability to bind RNA. It is likely that the N-terminus of the CP is important for the interaction between the subunits in self-assembly into NLPs. In this work, through deletion and mutation analysis, we have shown that the deletion of 13 amino acids is sufficient to generate the monomeric form of the CP. Furthermore, we have shown that residue F13 is critical for self-assembly of the CP subunits into NLPs. The replacement of F13 with hydrophobic residues (L or Y) generated mutated forms of the CP that were able to self-assemble into NLPs. However, the replacement of F13 by A, G, R, E or S was detrimental to the self-assembly of the protein into NLPs. We concluded that a hydrophobic interaction at the N-terminus is important to ensure self-assembly of the protein into NLPs. We also discuss the importance of F13 for assembly of other members of the potexvirus family.

  19. Assessing side-chain perturbations of the protein backbone: a knowledge-based classification of residue Ramachandran space.

    PubMed

    Dahl, David B; Bohannan, Zach; Mo, Qianxing; Vannucci, Marina; Tsai, Jerry

    2008-05-02

    Grouping the 20 residues is a classic strategy to discover ordered patterns and insights about the fundamental nature of proteins, their structure, and how they fold. Usually, this categorization is based on the biophysical and/or structural properties of a residue's side-chain group. We extend this approach to understand the effects of side chains on backbone conformation and to perform a knowledge-based classification of amino acids by comparing their backbone phi, psi distributions in different types of secondary structure. At this finer, more specific resolution, torsion angle data are often sparse and discontinuous (especially for nonhelical classes) even though a comprehensive set of protein structures is used. To ensure the precision of Ramachandran plot comparisons, we applied a rigorous Bayesian density estimation method that produces continuous estimates of the backbone phi, psi distributions. Based on this statistical modeling, a robust hierarchical clustering was performed using a divergence score to measure the similarity between plots. There were seven general groups based on the clusters from the complete Ramachandran data: nonpolar/beta-branched (Ile and Val), AsX (Asn and Asp), long (Met, Gln, Arg, Glu, Lys, and Leu), aromatic (Phe, Tyr, His, and Cys), small (Ala and Ser), bulky (Thr and Trp), and, lastly, the singletons of Gly and Pro. At the level of secondary structure (helix, sheet, turn, and coil), these groups remain somewhat consistent, although there are a few significant variations. Besides the expected uniqueness of the Gly and Pro distributions, the nonpolar/beta-branched and AsX clusters were very consistent across all types of secondary structure. Effectively, this consistency across the secondary structure classes implies that side-chain steric effects strongly influence a residue's backbone torsion angle conformation. These results help to explain the plasticity of amino acid substitutions on protein structure and should help in

  20. Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies

    SciTech Connect

    Lakowiz, J.R.; Weber, G.

    1980-10-01

    Steady-state and lifetime-resolved fluorescence anisotropy measurements of protein fluorescence were used to investigate the depolarizing motions of tryptophan residues in proteins. Lifetime resolution was achieved by oxygen quenching. The proteins investigated were carbonic anhydrase, carboxypeptidase A, ..cap alpha..-chymotrypsin, trypsin, pepsin, and bovine and human serum albumin. When corrected for overall protein rotation, the steady state anisotropies indicate that, on the average, the tryptophan residues in these proteins rotate 29/sup 0/ +- 6/sup 0/ during the unquenched excited state lifetimes of these proteins, which range from 1.7 to 6.1 ns. The lifetime-resolved anisotropies reveal correlation times for these displacements ranging from 1 to 12 ns. On the average these correlation times are tenfold shorter than that expected for overall protein rotation. We conclude that the tryptophan residues in these proteins display remarkable freedom of motion within the protein matrix, which implies that these matrices are highly flexible on the nanosecond time scale.

  1. Inadequacy of prebiotic synthesis as origin of proteinous amino acids.

    PubMed

    Wong, J T; Bronskill, P M

    1979-07-18

    The production of some nonproteinous, and lack of production of other proteinous, amino acids in model prebiotic synthesis, along with the instability of glutamine and asparagine, suggest that not all of the 20 present day proteinous amino acids gained entry into proteins directly from the primordial soup. Instead, a process of active co-evolution of the genetic code and its constituent amino acids would have to precede the final selection of these proteinous amono acids.

  2. Packing of coat protein amphipathic and transmembrane helices in filamentous bacteriophage M13: role of small residues in protein oligomerization.

    PubMed

    Williams, K A; Glibowicka, M; Li, Z; Li, H; Khan, A R; Chen, Y M; Wang, J; Marvin, D A; Deber, C M

    1995-09-08

    Filamentous bacteriophage M13, an important cloning and phage display vector, is encapsulated by ca 2700 copies of its 50-residue major coat protein (gene 8). This protein occurs as a membrane protein while stably inserted into its E. coli host inner membrane, and as a coat protein upon assembly and packing onto phage DNA in the lipid-free virion. To examine the specific protein-protein interactions underlying these processes, we used a combination of randomized and saturation mutagenesis of the entire gene 8 to assess the susceptibility of each position to mutation. In the resulting library of ca 100 viable M13 mutants, "small" residues (Ala,Gly,Ser), which constitute the non-polar face of the N-terminal amphipathic helical segment, and a face of the hydrophobic (effective transmembrane) helical segment, were found to be highly conserved. These results support a model in which coat protein packing is stabilized by the presence within each protein subunit of two "oligomerization segments", i.e. specific helical regions with faces rich in small residues which function to promote the close approach of alpha-helices.

  3. Amino acid sequences of two nonspecific lipid-transfer proteins from germinated castor bean.

    PubMed

    Takishima, K; Watanabe, S; Yamada, M; Suga, T; Mamiya, G

    1988-11-01

    The amino acid sequence of two nonspecific lipid-transfer proteins (nsLTP) B and C from germinated castor bean seeds have been determined. Both the proteins consist of 92 residues, as for nsLTP previously reported, and their calculated Mr values are 9847 and 9593 for nsLTP-B and nsLTP-C, respectively. The sequences of nsLTP-B and nsLTP-C, compared to the known sequence of nsLTP-A from the same source, are 68% and 35% similar, respectively. No variation was found at the positions of the cysteine residues, indicating that they might be involved in disulfide bridges.

  4. 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.

  5. Residual mitochondrial transmembrane potential decreases unsaturated fatty acid level in sake yeast during alcoholic fermentation.

    PubMed

    Sawada, Kazutaka; Kitagaki, Hiroshi

    2016-01-01

    Oxygen, a key nutrient in alcoholic fermentation, is rapidly depleted during this process. Several pathways of oxygen utilization have been reported in the yeast Saccharomyces cerevisiae during alcoholic fermentation, namely synthesis of unsaturated fatty acid, sterols and heme, and the mitochondrial electron transport chain. However, the interaction between these pathways has not been investigated. In this study, we showed that the major proportion of unsaturated fatty acids of ester-linked lipids in sake fermentation mash is derived from the sake yeast rather than from rice or koji (rice fermented with Aspergillus). Additionally, during alcoholic fermentation, inhibition of the residual mitochondrial activity of sake yeast increases the levels of unsaturated fatty acids of ester-linked lipids. These findings indicate that the residual activity of the mitochondrial electron transport chain reduces molecular oxygen levels and decreases the synthesis of unsaturated fatty acids, thereby increasing the synthesis of estery flavors by sake yeast. This is the first report of a novel link between residual mitochondrial transmembrane potential and the synthesis of unsaturated fatty acids by the brewery yeast during alcoholic fermentation.

  6. Nonnatural protein–protein interaction-pair design by key residues grafting

    PubMed Central

    Liu, Sen; Liu, Shiyong; Zhu, Xiaolei; Liang, Huanhuan; Cao, Aoneng; Chang, Zhijie; Lai, Luhua

    2007-01-01

    Protein–protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein–protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein–protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat PLCδ1-PH (pleckstrin homology domain of phospholipase C-δ1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat PLCδ1-PH were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of PLCδ1-PH (ERPH1) was found to bind EPOR with high affinity (KD of 24 nM and an IC50 of 5.7 μM) both in vitro and in a cell-based assay, respectively, although the WT PLCδ1-PH did not show any detectable binding under the assay conditions. The in vitro binding affinities of the PLCδ1-PH mutants correlate qualitatively to the computational binding affinities, validating the design and the protein–protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting protein–protein interfaces. PMID:17372228

  7. 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

  8. Complete amino acid sequence and structure characterization of the taste-modifying protein, miraculin.

    PubMed

    Theerasilp, S; Hitotsuya, H; Nakajo, S; Nakaya, K; Nakamura, Y; Kurihara, Y

    1989-04-25

    The taste-modifying protein, miraculin, has the unusual property of modifying sour taste into sweet taste. The complete amino acid sequence of miraculin purified from miracle fruits by a newly developed method (Theerasilp, S., and Kurihara, Y. (1988) J. Biol. Chem. 263, 11536-11539) was determined by an automatic Edman degradation method. Miraculin was a single polypeptide with 191 amino acid residues. The calculated molecular weight based on the amino acid sequence and the carbohydrate content (13.9%) was 24,600. Asn-42 and Asn-186 were linked N-glycosidically to carbohydrate chains. High homology was found between the amino acid sequences of miraculin and soybean trypsin inhibitor.

  9. Structural evidence for solvent-stabilisation by aspartic acid as a mechanism for halophilic protein stability in high salt concentrations.

    PubMed

    Lenton, Samuel; Walsh, Danielle L; Rhys, Natasha H; Soper, Alan K; Dougan, Lorna

    2016-07-21

    Halophilic organisms have adapted to survive in high salt environments, where mesophilic organisms would perish. One of the biggest challenges faced by halophilic proteins is the ability to maintain both the structure and function at molar concentrations of salt. A distinct adaptation of halophilic proteins, compared to mesophilic homologues, is the abundance of aspartic acid on the protein surface. Mutagenesis and crystallographic studies of halophilic proteins suggest an important role for solvent interactions with the surface aspartic acid residues. This interaction, between the regions of the acidic protein surface and the solvent, is thought to maintain a hydration layer around the protein at molar salt concentrations thereby allowing halophilic proteins to retain their functional state. Here we present neutron diffraction data of the monomeric zwitterionic form of aspartic acid solutions at physiological pH in 0.25 M and 2.5 M concentration of potassium chloride, to mimic mesophilic and halophilic-like environmental conditions. We have used isotopic substitution in combination with empirical potential structure refinement to extract atomic-scale information from the data. Our study provides structural insights that support the hypothesis that carboxyl groups on acidic residues bind water more tightly under high salt conditions, in support of the residue-ion interaction model of halophilic protein stabilisation. Furthermore our data show that in the presence of high salt the self-association between the zwitterionic form of aspartic acid molecules is reduced, suggesting a possible mechanism through which protein aggregation is prevented.

  10. Residue-specific immobilization of protein molecules by size-selected clusters

    PubMed Central

    Prisco, Umberto; Leung, Carl; Xirouchaki, Chrisa; Jones, Celine H; Heath, John K; Palmer, Richard E

    2005-01-01

    The atomic force microscope (AFM), operating in contact mode, has been employed in buffer solution to study two proteins; (i) green fluorescent protein (GFP), from the hydromedusan jellyfish Aequorea victoria; and (ii) human oncostatin M (OSM), in the presence of size-selected gold nanoclusters pinned on to a highly oriented pyrolytic graphite substrate. The AFM images have revealed immobilization of single molecules of OSM, which are strongly bound to the gold nanoclusters. Conversely, no strong immobilization has been observed for the GFP, as these molecules were easily displaced by the scanning tip. The contrasting behaviour of the two proteins can be explained by the exposed molecular surface area of their cysteine residues as modelled on the basis of their respective X-ray crystallographic data structures. GFP contains two cysteine residues, but neither is readily available to chemisorb on the gold clusters, because the cysteines are largely inaccessible from the surface of the protein. In contrast, OSM has a total of five cysteine residues, with different degrees of accessibility, which make the protein amenable to anchoring on the nanoclusters. Statistical analysis of the height of the OSM molecules bound to the nanoclusters is in accordance with crystallographic data, and suggests various configurations of the proteins on the clusters, associated with the presence of different cysteine anchoring sites. These results suggest that the three-dimensional conformation of protein molecules is preserved when they are chemisorbed to size-selected gold clusters, thus opening a new route towards oriented immobilization of individual protein molecules. PMID:16849177

  11. Identification of the amino acid residues responsible for stable nucleosome formation by histone H3.Y.

    PubMed

    Kujirai, Tomoya; Horikoshi, Naoki; Xie, Yan; Taguchi, Hiroyuki; Kurumizaka, Hitoshi

    2017-01-24

    Histone H3.Y is conserved among primates. We previously reported that exogenously produced H3.Y accumulates around transcription start sites, suggesting that it may play a role in transcription regulation. The H3.Y nucleosome forms a relaxed chromatin conformation with flexible DNA ends. The H3.Y-specific Lys42 residue is partly responsible for enhancing the flexibility of the nucleosomal DNA. To our surprise, we found that H3.Y stably associates with chromatin and nucleosomes in vivo and in vitro. However, the H3.Y residues responsible for its stable nucleosome incorporation have not been identified yet. In the present study, we performed comprehensive mutational analyses of H3.Y, and determined that the H3.Y C-terminal region including amino acid residues 124-135 is responsible for its stable association with DNA. Among the H3.Y C-terminal residues, the H3.Y Met124 residue significantly contributed to the stable DNA association with the H3.Y-H4 tetramer. The H3.Y M124I mutation substantially reduced the H3.Y-H4 association in the nucleosome. In contrast, the H3.Y K42R mutation affected the nucleosome stability less, although it contributes to the flexible DNA ends of the nucleosome. Therefore, these H3.Y-specific residues, Lys42 and Met124, play different and specific roles in nucleosomal DNA relaxation and stable nucleosome formation, respectively, in chromatin.

  12. A biotin enrichment strategy identifies novel carbonylated amino acids in proteins from human plasma.

    PubMed

    Havelund, Jesper F; Wojdyla, Katarzyna; Davies, Michael J; Jensen, Ole N; Møller, Ian Max; Rogowska-Wrzesinska, Adelina

    2017-03-06

    Protein carbonylation is an irreversible protein oxidation correlated with oxidative stress, various diseases and ageing. Here we describe a peptide-centric approach for identification and characterisation of up to 14 different types of carbonylated amino acids in proteins. The modified residues are derivatised with biotin-hydrazide, enriched and characterised by tandem mass spectrometry. The strength of the method lies in an improved elution of biotinylated peptides from monomeric avidin resin using hot water (95°C) and increased sensitivity achieved by reduction of analyte losses during sample preparation and chromatography. For the first time MS/MS data analysis utilising diagnostic biotin fragment ions is used to pinpoint sites of biotin labelling and improve the confidence of carbonyl peptide assignments. We identified a total of 125 carbonylated residues in bovine serum albumin after extensive in vitro metal ion-catalysed oxidation. Furthermore, we assigned 133 carbonylated sites in 36 proteins in native human plasma protein samples. The optimised workflow enabled detection of 10 hitherto undetected types of carbonylated amino acids in proteins: aldehyde and ketone modifications of leucine, valine, alanine, isoleucine, glutamine, lysine and glutamic acid (+14Da), an oxidised form of methionine - aspartate semialdehyde (-32Da) - and decarboxylated glutamic acid and aspartic acid (-30Da).

  13. Do cysteine residues regulate transient receptor potential canonical type 6 channel protein expression?

    PubMed

    Thilo, Florian; Liu, Ying; Krueger, Katharina; Förste, Nora; Wittstock, Antje; Scholze, Alexandra; Tepel, Martin

    2012-03-01

    The regulation of calcium influx through transient receptor potential canonical type 6 (TRPC6) channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine (HC) or acetylcysteine (ACC) affect TRPC6 expression in human monocytes. We observed that patients with chronic renal failure had significantly elevated HC levels and TRPC6 mRNA expression levels in monocytes compared with control subjects. We further observed that administration of HC or ACC significantly increased TRPC6 channel protein expression compared with control conditions. We, therefore, hypothesize that cysteine residues increase TRPC6 channel protein expression in humans.

  14. Residue-specific force field based on protein coil library. RSFF2: modification of AMBER ff99SB.

    PubMed

    Zhou, Chen-Yang; Jiang, Fan; Wu, Yun-Dong

    2015-01-22

    Recently, we developed a residue-specific force field (RSFF1) based on conformational free-energy distributions of the 20 amino acid residues from a protein coil library. Most parameters in RSFF1 were adopted from the OPLS-AA/L force field, but some van der Waals and torsional parameters that effectively affect local conformational preferences were introduced specifically for individual residues to fit the coil library distributions. Here a similar strategy has been applied to modify the Amber ff99SB force field, and a new force field named RSFF2 is developed. It can successfully fold α-helical structures such as polyalanine peptides, Trp-cage miniprotein, and villin headpiece subdomain and β-sheet structures such as Trpzip-2, GB1 β-hairpins, and the WW domain, simultaneously. The properties of various popular force fields in balancing between α-helix and β-sheet are analyzed based on their descriptions of local conformational features of various residues, and the analysis reveals the importance of accurate local free-energy distributions. Unlike the RSFF1, which overestimates the stability of both α-helix and β-sheet, RSFF2 gives melting curves of α-helical peptides and Trp-cage in good agreement with experimental data. Fitting to the two-state model, RSFF2 gives folding enthalpies and entropies in reasonably good agreement with available experimental results.

  15. Thiostrepton Variants Containing a Contracted Quinaldic Acid Macrocycle Result from Mutagenesis of the Second Residue

    PubMed Central

    Zhang, Feifei; Li, Chaoxuan

    2016-01-01

    The thiopeptides are a family of ribosomally synthesized and posttranslationally modified peptide metabolites, and the vast majority of thiopeptides characterized to date possess one highly modified macrocycle. A few members, including thiostrepton A, harbor a second macrocycle that incorporates a quinaldic acid moiety and the four N-terminal residues of the peptide. The antibacterial properties of thiostrepton A are well established, and its recently discovered ability to inhibit the proteasome has additional implications for the development of antimalarial and anticancer therapeutics. We have conducted the saturation mutagenesis of Ala2 in the precursor peptide, TsrA, to examine which variants can be transformed into a mature thiostrepton analogue. Although the thiostrepton biosynthetic system is somewhat restrictive towards substitutions at the second residue, eight thiostrepton Ala2 analogues were isolated. The TsrA Ala2Ile and Ala2Val variants were largely channeled through an alternate processing pathway wherein the first residue of the core peptide, Ile1, is removed and the resulting thiostrepton analogues bear quinaldic acid macrocycles abridged by one residue. This is the first report revealing that quinaldic acid loop size is amenable to alteration during the course of thiostrepton biosynthesis. Both the antibacterial and proteasome inhibitory properties of the thiostrepton Ala2 analogues were examined. While the identity of the residue at the second position of the core peptide influences thiostrepton biosynthesis, our report suggests it may not be crucial for antibacterial and proteasome inhibitory properties of the full-length variants. In contrast, the contracted quinaldic acid loop can, to differing degrees, affect both types of biological activity. PMID:26630475

  16. A Hexasaccharide Containing Rare 2‐O‐Sulfate‐Glucuronic Acid Residues Selectively Activates Heparin Cofactor II

    PubMed Central

    Sankarayanarayanan, Nehru Viji; Strebel, Tamara R.; Boothello, Rio S.; Sheerin, Kevin; Raghuraman, Arjun; Sallas, Florence; Mosier, Philip D.; Watermeyer, Nicholas D.

    2017-01-01

    Abstract Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2‐O‐sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250‐fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG‐binding protein(s), which may lead to chemical biology or drug discovery tools. PMID:28124818

  17. beta-Hydroxyaspartic acid in vitamin K-dependent protein C.

    PubMed

    Drakenberg, T; Fernlund, P; Roepstorff, P; Stenflo, J

    1983-04-01

    Previous work has shown that the light chain of protein C, an anticoagulant plasma protein, contains an unusual amino acid [Fernlund, P. & Stenflo, J. (1982) J. Biol. Chem. 257, 12170-12179]. To determine the structure of this amino acid a heptapeptide, CMCys-Ile-X-Gly-Leu-Gly-Gly (residues 69-75 in the light chain), was isolated from enzymatic digests of the light chain. According to automatic Edman sequence analysis, 1H NMR spectroscopy, and mass spectrometry the heptapeptide had beta-hydroxyaspartic acid in its third position, which corresponds to position 71 in the light chain of protein C. Analysis of acid and aminopeptidase M hydrolysates of the heptapeptide showed the beta-hydroxyaspartic acid to be the erythro form. Acid hydrolysis of protein C released approximately equal to 1 mol of beta-hydroxyaspartic acid per mol of protein. The function of this amino acid, which, to the best of our knowledge, has not been found previously in proteins, is unknown.

  18. A novel sono-assisted acid pretreatment of chili post harvest residue for bioethanol production.

    PubMed

    Sindhu, Raveendran; Binod, Parameswaran; Pandey, Ashok

    2016-08-01

    The objective of the present study was to develop a sono-assisted acid pretreatment strategy for the effective removal of lignin and hemicelluloses and to improve the sugar yield from chili post harvest residue. Operational parameters that affect the pretreatment efficiency were studied and optimized. Inhibitor analysis of the hydrolyzate revealed that major fermentation inhibitors like furfural, hydroxymethyl furfural and organic acids like citric acid, succinic acid and propionic acid were absent. Changes in structural properties of the biomass were studied in relation to the pretreatment process using scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier Transform Infrared (FTIR) analysis, and the changes in chemical composition was also monitored. The biomass pretreated with the optimized novel method yielded 0.465g/g of reducing sugars on enzymatic hydrolysis. Fermentation of the non-detoxified hydrolysate yielded 2.14% of bioethanol with a fermentation efficiency of 71.03%.

  19. A Multifeatures Fusion and Discrete Firefly Optimization Method for Prediction of Protein Tyrosine Sulfation Residues

    PubMed Central

    Liu, Chunhua; Zhou, Peng; Li, Yanling

    2016-01-01

    Tyrosine sulfation is one of the ubiquitous protein posttranslational modifications, where some sulfate groups are added to the tyrosine residues. It plays significant roles in various physiological processes in eukaryotic cells. To explore the molecular mechanism of tyrosine sulfation, one of the prerequisites is to correctly identify possible protein tyrosine sulfation residues. In this paper, a novel method was presented to predict protein tyrosine sulfation residues from primary sequences. By means of informative feature construction and elaborate feature selection and parameter optimization scheme, the proposed predictor achieved promising results and outperformed many other state-of-the-art predictors. Using the optimal features subset, the proposed method achieved mean MCC of 94.41% on the benchmark dataset, and a MCC of 90.09% on the independent dataset. The experimental performance indicated that our new proposed method could be effective in identifying the important protein posttranslational modifications and the feature selection scheme would be powerful in protein functional residues prediction research fields. PMID:27034949

  20. SigniSite: Identification of residue-level genotype-phenotype correlations in protein multiple sequence alignments

    PubMed Central

    Jessen, Leon Eyrich; Hoof, Ilka; Lund, Ole; Nielsen, Morten

    2013-01-01

    Identifying which mutation(s) within a given genotype is responsible for an observable phenotype is important in many aspects of molecular biology. Here, we present SigniSite, an online application for subgroup-free residue-level genotype–phenotype correlation. In contrast to similar methods, SigniSite does not require any pre-definition of subgroups or binary classification. Input is a set of protein sequences where each sequence has an associated real number, quantifying a given phenotype. SigniSite will then identify which amino acid residues are significantly associated with the data set phenotype. As output, SigniSite displays a sequence logo, depicting the strength of the phenotype association of each residue and a heat-map identifying ‘hot’ or ‘cold’ regions. SigniSite was benchmarked against SPEER, a state-of-the-art method for the prediction of specificity determining positions (SDP) using a set of human immunodeficiency virus protease-inhibitor genotype–phenotype data and corresponding resistance mutation scores from the Stanford University HIV Drug Resistance Database, and a data set of protein families with experimentally annotated SDPs. For both data sets, SigniSite was found to outperform SPEER. SigniSite is available at: http://www.cbs.dtu.dk/services/SigniSite/. PMID:23761454

  1. Protein and Amino Acid Profiles of Different Whey Protein Supplements.

    PubMed

    Almeida, Cristine C; Alvares, Thiago S; Costa, Marion P; Conte-Junior, Carlos A

    2016-01-01

    Whey protein (WP) supplements have received increasing attention by consumers due to the high nutritional value of the proteins and amino acids they provide. However, some WP supplements may not contain the disclosed amounts of the ingredients listed on the label, compromising the nutritional quality and the effectiveness of these supplements. The aim of this study was to evaluate and compare the contents of total protein (TP), α-lactalbumin (α-LA), β-lactoglobulin (β-LG), free essential amino acids (free EAA), and free branched-chain amino acids (free BCAA), amongst different WP supplements produced by U.S. and Brazilian companies. Twenty commercial brands of WP supplements were selected, ten manufactured in U.S. (WP-USA) and ten in Brazil (WP-BRA). The TP was analyzed using the Kjeldahl method, while α-LA, β-LG, free EAA, and free BCAA were analyzed using HPLC system. There were higher (p < 0.05) concentrations of TP, α-LA, β-LG, and free BCAA in WP-USA supplements, as compared to the WP-BRA supplements; however, there was no difference (p > 0.05) in the content of free EAA between WP-USA and WP-BRA. Amongst the 20 brands evaluated, four WP-USA and seven WP-BRA had lower (p < 0.05) values of TP than those specified on the label. In conclusion, the WP-USA supplements exhibited better nutritional quality, evaluated by TP, α-LA, β-LG, and free BCAA when compared to WP-BRA.

  2. Conjugated fatty acid synthesis: residues 111 and 115 influence product partitioning of Momordica charantia conjugase.

    PubMed

    Rawat, Richa; Yu, Xiao-Hong; Sweet, Marie; Shanklin, John

    2012-05-11

    Conjugated linolenic acids (CLNs), 18:3 Δ(9,11,13), lack the methylene groups found between the double bonds of linolenic acid (18:3 Δ(9,12,15)). CLNs are produced by conjugase enzymes that are homologs of the oleate desaturases FAD2. The goal of this study was to map the domain(s) within the Momordica charantia conjugase (FADX) responsible for CLN formation. To achieve this, a series of Momordica FADX-Arabidopsis FAD2 chimeras were expressed in the Arabidopsis fad3fae1 mutant, and the transformed seeds were analyzed for the accumulation of CLN. These experiments identified helix 2 and the first histidine box as a determinant of conjugase product partitioning into punicic acid (18:3 Δ(9cis,11trans,13cis)) or α-eleostearic acid (18:3 Δ(9cis,11trans,13trans)). This was confirmed by analysis of a FADX mutant containing six substitutions in which the sequence of helix 2 and first histidine box was converted to that of FAD2. Each of the six FAD2 substitutions was individually converted back to the FADX equivalent identifying residues 111 and 115, adjacent to the first histidine box, as key determinants of conjugase product partitioning. Additionally, expression of FADX G111V and FADX G111V/D115E resulted in an approximate doubling of eleostearic acid accumulation to 20.4% and 21.2%, respectively, compared with 9.9% upon expression of the native Momordica FADX. Like the Momordica conjugase, FADX G111V and FADX D115E produced predominantly α-eleostearic acid and little punicic acid, but the FADX G111V/D115E double mutant produced approximately equal amounts of α-eleostearic acid and its isomer, punicic acid, implicating an interactive effect of residues 111 and 115 in punicic acid formation.

  3. Nitrogen dioxide reaction with proteins: Evidence for peptide bond cleavage at lysine residues

    SciTech Connect

    Hood, D.B.

    1991-01-01

    Nitrogen dioxide (NO{sub 2}), an air pollutant produced by burning fossil fuels and a component of cigarette smoke, is thought to contribute to the pathogenesis of pulmonary diseases, such as emphysema. To gain information on the mechanism by which NO{sub 2} damages the lung, in vitro exposures of {alpha}{sub 1}-proteinase inhibitor ({alpha}{sub 1}-PI), elastin, bovine serum albumin (BSA), human serum albumin (HSA) and synthetic poly-L-lysine were performed. A genetic deficiency of {alpha}{sup 1}-PI predisposes humans to emphysema and NO{sub 2} has been hypothesized to damage {alpha}{sub 1}-PI, which would leave proteases such as human neutrophil elastase, (HNE) free to attack lung structural proteins. The ability of {alpha}{sub 1}-PI to inhibit HNE declined with exposure to 50% of the control value at molar ratios of NO{sub 2}:{alpha}{sub 1}-PI of 400:1 and greater. Exposure of {alpha}{sub 1}-PI to NO{sub 2} resulted in a 50% lose of immunoreactivity with either monoclonal or polyclonal antibodies in an enzyme-linked immunosorbent assay at molar ratios of NO{sub 2}:{alpha}{sub 1}-PI of essentially 100:1 and greater. The mechanisms of these effects were investigated via ultraviolet-visible spectroscopy and amino acid analysis. The remaining target molecules were labeled by reductive methylation of amino groups with {sup 3}H-HCHO prior to treatment with NO{sub 2} in aqueous solutions at physiological pH. Time course exposure of 5 mg {sup 3}H-insoluble bovine ligamentum nuchae elastin suspensions with up to 120 {mu}moles of NO{sub 2} resulted in 90% solubilization of the label. Amino acid analysis of the soluble and insoluble fractions from these exposures confirmed that 80% of the {sup 3}H-dimethyllysine residues were in the soluble fraction.

  4. Hypochlorous and peracetic acid induced oxidation of dairy proteins.

    PubMed

    Kerkaert, Barbara; Mestdagh, Frédéric; Cucu, Tatiana; Aedo, Philip Roger; Ling, Shen Yan; De Meulenaer, Bruno

    2011-02-09

    Hypochlorous and peracetic acids, both known disinfectants in the food industry, were compared for their oxidative capacity toward dairy proteins. Whey proteins and caseins were oxidized under well controlled conditions at pH 8 as a function of the sanitizing concentration. Different markers for protein oxidation were monitored. The results established that the protein carbonyl content was a rather unspecific marker for protein oxidation, which did not allow one to differentiate the oxidant used especially at the lower concentrations. Cysteine, tryptophan, and methionine were proven to be the most vulnerable amino acids for degradation upon hypochlorous and peracetic acid treatment, while tyrosine was only prone to degradation in the presence of hypochlorous acid. Hypochlorous acid induced oxidation gave rise to protein aggregation, while during peracetic acid induced oxidation, no high molecular weight aggregates were observed. Protein aggregation upon hypochlorous acid oxidation could primarily be linked to tryptophan and tyrosine degradation.

  5. Residue-specific force field based on the protein coil library. RSFF1: modification of OPLS-AA/L.

    PubMed

    Jiang, Fan; Zhou, Chen-Yang; Wu, Yun-Dong

    2014-06-26

    Traditional protein force fields use one set of parameters for most of the 20 amino acids (AAs), allowing transferability of the parameters. However, a significant shortcoming is the difficulty to fit the Ramachandran plots of all AA residues simultaneously, affecting the accuracy of the force field. In this Feature Article, we report a new strategy for protein force field parametrization. Backbone and side-chain conformational distributions of all 20 AA residues obtained from protein coil library were used as the target data. The dihedral angle (torsion) potentials and some local nonbonded (1-4/1-5/1-6) interactions in OPLS-AA/L force field were modified such that the target data can be excellently reproduced by molecular dynamics simulations of dipeptides (blocked AAs) in explicit water, resulting in a new force field with AA-specific parameters, RSFF1. An efficient free energy decomposition approach was developed to separate the corrections on ϕ and ψ from the two-dimensional Ramachandran plots. RSFF1 is shown to reproduce the experimental NMR (3)J-coupling constants of AA dipeptides better than other force fields. It has a good balance between α-helical and β-sheet secondary structures. It can successfully fold a set of α-helix proteins (Trp-cage and Homeodomain) and β-hairpins (Trpzip-2, GB1 hairpin), which cannot be consistently stabilized by other state-of-the-art force fields. Interestingly, the RSFF1 force field systematically overestimates the melting temperature (and the stability of native state) of these peptides/proteins. It has a potential application in the simulation of protein folding and protein structure refinement.

  6. COMSAT: Residue contact prediction of transmembrane proteins based on support vector machines and mixed integer linear programming.

    PubMed

    Zhang, Huiling; Huang, Qingsheng; Bei, Zhendong; Wei, Yanjie; Floudas, Christodoulos A

    2016-03-01

    In this article, we present COMSAT, a hybrid framework for residue contact prediction of transmembrane (TM) proteins, integrating a support vector machine (SVM) method and a mixed integer linear programming (MILP) method. COMSAT consists of two modules: COMSAT_SVM which is trained mainly on position-specific scoring matrix features, and COMSAT_MILP which is an ab initio method based on optimization models. Contacts predicted by the SVM model are ranked by SVM confidence scores, and a threshold is trained to improve the reliability of the predicted contacts. For TM proteins with no contacts above the threshold, COMSAT_MILP is used. The proposed hybrid contact prediction scheme was tested on two independent TM protein sets based on the contact definition of 14 Å between Cα-Cα atoms. First, using a rigorous leave-one-protein-out cross validation on the training set of 90 TM proteins, an accuracy of 66.8%, a coverage of 12.3%, a specificity of 99.3% and a Matthews' correlation coefficient (MCC) of 0.184 were obtained for residue pairs that are at least six amino acids apart. Second, when tested on a test set of 87 TM proteins, the proposed method showed a prediction accuracy of 64.5%, a coverage of 5.3%, a specificity of 99.4% and a MCC of 0.106. COMSAT shows satisfactory results when compared with 12 other state-of-the-art predictors, and is more robust in terms of prediction accuracy as the length and complexity of TM protein increase. COMSAT is freely accessible at http://hpcc.siat.ac.cn/COMSAT/.

  7. Mutagenesis of bacteriophage IKe major coat protein transmembrane domain: role of an interfacial proline residue.

    PubMed

    Williams, K A; Deber, C M

    1993-10-15

    The transmembrane (TM) domain of the 53-residue major coat protein of the M13-related bacteriophage IKe (residues 24-42: LISQTWPVVTTVVVAGVLI) has been subjected to randomized mutagenesis to probe the conformation and stability of the TM domain, as well as the effect of structurally-important residues such as proline. TM mutants were obtained by the Eckstein method of site-directed mutagenesis using the IKe genome as template so as to eliminate the need for subcloning. Over 40 single- and double-site viable mutants of bacteriophage IKe were isolated. Every residue in the TM segment, except the highly conserved Trp29, could be mutated to at least one other residue; polar and charged mutations occurred in the TM segment adjacent to the N-terminal domain (residues 24-28), while non-polar substitutions predominated in the C-terminal portion (residues 30-42). The Pro30 locus tolerated four mutations-Ala, Gly, Cys, and Ser- which represent the four side chains of least volume. Mutant coat proteins obtained directly from the phage in milligram quantities were studied by circular dichroism spectroscopy and SDS-PAGE gels. Wild type IKe coat protein solubilized in sodium deoxycholate micelles was found to occur as an alpha-helical, monomeric species which is stable at 95 degrees C, whereas the mutant Pro30-->Gly undergoes an irreversible conformational transition at ca. 90 degrees C to an aggregated beta-sheet structure. The result that Pro30 stabilizes the TM helix in the micellar membrane suggests a sterically-restricted location for the wild type Pro pyrrolidine side chain in the bulky Trp-Pro-Val triad, where it may be positioned to direct the initiation of the subsequent TM core domain helix.

  8. Shark myelin basic protein: amino acid sequence, secondary structure, and self-association.

    PubMed

    Milne, T J; Atkins, A R; Warren, J A; Auton, W P; Smith, R

    1990-09-01

    Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self-association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure.

  9. Nucleic acid binding proteins in highly purified Creutzfeldt-Jakob disease preparations.

    PubMed Central

    Sklaviadis, T; Akowitz, A; Manuelidis, E E; Manuelidis, L

    1993-01-01

    The nature of the infectious agent causing human Creutzfeldt-Jakob disease (CJD), a slowly progressive dementia, is controversial. As in scrapie, no agent-specific proteins or nucleic acids have been identified. However, biological features of exponential replication and agent strain variation, as well as physical size and density data, are most consistent with a viral structure--i.e., a nucleic acid-protein complex. It is often assumed that nuclease treatment, which does not reduce infectious titer, leaves no nucleic acids of > 50 bp. However, nucleic acids of 500-6000 bp can be extracted from highly purified infectious complexes with a mass of approximately 1.5 x 10(7) daltons. It was therefore germane to search for nucleic acid binding proteins that might protect an agent genome. We here use Northwestern blotting to show that there are low levels of nonhistone nucleic acid binding proteins in highly purified infectious 120S gradient fractions. Several nucleic acid binding proteins were clearly host encoded, whereas others were apparent only in CJD, but not in parallel preparations from uninfected brain. Small amounts of residual host Gp34 (prion protein) did not bind any 32P-labeled nucleic acid probes. Most of the minor "CJD-specific" proteins had an acidic pI, a characteristic of many viral core proteins. Such proteins deserve further study, as they probably contribute to unique properties of resistance described for these agents. It remains to be seen if any of these proteins are agent encoded. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8516321

  10. Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

    SciTech Connect

    Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan; Bae, Brian; Mackie, Roderick I.; Nair, Satish K.; Cann, Isaac K.O.

    2010-11-22

    Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.

  11. Comparison between liquid and solid acids catalysts on reducing sugars conversion from furfural residues via pretreatments.

    PubMed

    Lin, Keying; Ma, Baojun; Sun, Yuan; Liu, Wanyi

    2014-09-01

    Liquid sulphuric acid is adopted and compared with carbon-based sulfonated solid acids (coal tar-based and active carbon-based) for furfural residues conversion into reducing sugars. The optimum hydrolysis conditions of liquid acid are at 4% of sulphuric acid, 25:1 of liquid and solid ratio, 175°C of reaction temperature and 120 min of reaction time. The reducing sugar yields are reached over 60% on liquid acid via NaOH/H2O2, NaOH/microwave and NaOH/ultrasonic pretreatments, whereas only over 30% on solid acids. The TOFs (turnover number frequency) via NaOH/H2O2 pretreatments are 0.093, 0.020 and 0.023 h(-1) for liquid sulphuric acid, coal tar-based and active carbon-based solid acids catalysts, respectively. Considering the efficiency, cost and environment factors, the liquid and solid acids have their own advantages of potential commercial application values.

  12. Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase.

    PubMed Central

    Tamura, H; Tameishi, K; Yamada, A; Tomita, M; Matsuo, Y; Nishikawa, K; Ikezawa, H

    1995-01-01

    Four aspartic acid residues (Asp126, Asp156, Asp233 and Asp295) of Bacillus cereus sphingomyelinase (SMase) in the conservative regions were changed to glycine by in vitro mutagenesis, and the mutant SMases [D126G (Asp126-->Gly etc.), D156G, D233G and D295G] were produced in Bacillus brevis 47, a protein-producing strain. The sphingomyelin (SM)-hydrolysing activity of D295G was completely abolished and those of D126G and D156G were reduced by more than 80%, whereas that of D233G was not so profoundly affected. Two mutant enzymes (D126G and D156G) were purified and characterized further. The hydrolytic activities of D126G and D156G toward four phosphocholine-containing substrates with different hydrophobicities, SM, 2-hexadecanoylamino-4-nitrophenylphosphocholine(HNP), lysophosphatidylcholine (lysoPC) and p-nitro-phenylphosphocholine (p-NPPC), were compared with those of the wild-type. The activity of D126G toward water-soluble p-NPPC was comparable with that of the wild-type. On the other hand, D156G catalysed the hydrolysis of hydrophilic substrates such as HNP and p-NPPC more efficiently (> 4-fold) than the wild-type. These results suggested that Asp126 and Asp156, located in the highly conserved region, may well be involved in a substrate recognition process rather than catalytic action. Haemolytic activities of the mutant enzymes were found to be parallel with their SM-hydrolysing activities. Two regions, including the C-terminal region containing Asp295, were found to show considerable sequence identity with the corresponding regions of bovine pancreatic DNase I. Structural predictions indicated structural similarity between SMase and DNase I. An evolutionary relationship based on the catalytic function was suggested between the structures of these two phosphodiesterases. Images Figure 2 Figure 3 Figure 4 Figure 6 PMID:7639690

  13. Coiled-coil interaction of N-terminal 36 residues of cyclase-associated protein with adenylyl cyclase is sufficient for its function in Saccharomyces cerevisiae ras pathway.

    PubMed

    Nishida, Y; Shima, F; Sen, H; Tanaka, Y; Yanagihara, C; Yamawaki-Kataoka, Y; Kariya, K; Kataoka, T

    1998-10-23

    In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to confer RAS2(Val-19)-dependent heat shock sensitivity to yeast cells. The CAP-binding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif alphaXXalphaXXX (where alpha represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAP-adenylyl cyclase interaction.

  14. Histaminylation of glutamine residues is a novel posttranslational modification implicated in G-protein signaling.

    PubMed

    Vowinckel, Jakob; Stahlberg, Silke; Paulmann, Nils; Bluemlein, Katharina; Grohmann, Maik; Ralser, Markus; Walther, Diego J

    2012-11-02

    Posttranslational modifications (PTM) have been shown to be essential for protein function and signaling. Here we report the identification of a novel modification, protein transfer of histamine, and provide evidence for its function in G protein signaling. Histamine, known as neurotransmitter and mediator of the inflammatory response, was found incorporated into mastocytoma proteins. Histaminylation was dependent on transglutaminase II. Mass spectrometry confirmed histamine modification of the small and heterotrimeric G proteins Cdc42, Gαo1 and Gαq. The modification was specific for glutamine residues in the catalytic core, and triggered their constitutive activation. TGM2-mediated histaminylation is thus a novel PTM that functions in G protein signaling. Protein αmonoaminylations, thus including histaminylation, serotonylation, dopaminylation and norepinephrinylation, hence emerge as a novel class of regulatory PTMs.

  15. Analysis and Ranking of Protein-Protein Docking Models Using Inter-Residue Contacts and Inter-Molecular Contact Maps.

    PubMed

    Oliva, Romina; Chermak, Edrisse; Cavallo, Luigi

    2015-07-01

    In view of the increasing interest both in inhibitors of protein-protein interactions and in protein drugs themselves, analysis of the three-dimensional structure of protein-protein complexes is assuming greater relevance in drug design. In the many cases where an experimental structure is not available, protein-protein docking becomes the method of choice for predicting the arrangement of the complex. However, reliably scoring protein-protein docking poses is still an unsolved problem. As a consequence, the screening of many docking models is usually required in the analysis step, to possibly single out the correct ones. Here, making use of exemplary cases, we review our recently introduced methods for the analysis of protein complex structures and for the scoring of protein docking poses, based on the use of inter-residue contacts and their visualization in inter-molecular contact maps. We also show that the ensemble of tools we developed can be used in the context of rational drug design targeting protein-protein interactions.

  16. Proteomic analysis of residual proteins in blades and petioles of fallen leaves of Brassica napus.

    PubMed

    Desclos-Théveniau, M; Coquet, L; Jouenne, T; Etienne, P

    2015-03-01

    Brassica napus L. is an important crop plant, characterised by high nitrogen (N) levels in fallen leaves, leading to a significant restitution of this element to the soil, with important consequences at the economic and environmental levels. It is now well established that the N in fallen leaves is due to weak N remobilisation that is especially related to incomplete degradation of foliar proteins during leaf senescence. Identification of residual proteins in a fallen leaf (i.e. incompletely degraded in the last step of the N remobilisation process) constitutes important information for improving nutrient use efficiency. Proteome analysis of the vascular system (petioles) and blades from fallen leaves of Brassica napus was performed, and the 30 most abundant residual proteins in each tissue were identified. Among them, several proteins involved in N recycling remain in the leaf after abscission. Moreover, this study reveals that some residual proteins are associated with energy metabolism, protection against oxidative stress, and more surprisingly, photosynthesis. Finally, comparison of blade and petiole proteomes show that, despite their different physiological roles in the non-senescing leaf, both organs redirect their metabolism in order to ensure catabolic reactions. Taken together, the results suggest that a better degradation of these leaf proteins during the senescence process could enable improvements in the N use efficiency of Brassica napus.

  17. Delivery of a foreign epitope by sharing amino acid residues with the carrier matrix.

    PubMed

    Cheong, Wan-Shoo; Drummer, Heidi Edelgard; Netter, Hans-Jürgen

    2009-06-01

    A broad range of structural viral proteins has the ability to assemble into virus-like particles (VLPs). Under the condition that modified subunits are still competent to assemble into VLPs, they are epitope delivery platforms suitable for vaccination purposes. The insertion of foreign sequences can be detrimental for the formation of chimeric VLPs as a result of misfolded subunit proteins. Hence, a strategy was adopted to screen for locations allowing the use of shared residues between the wildtype subunit sequence and the foreign insert. The insertion of a cysteine-containing sequence of hepatitis C virus (HCV) envelope protein 2 (E2) without adding an additional cysteine residue retained the ability of recombinant small hepatitis B surface antigen (HBsAg-S) to form secretion competent VLPs. A cysteine residue shared by the insert and the template protein avoided the formation of non-native disulfide bonds, and allowed the formation of VLPs. The chimeric HBsAg-S VLPs were similar to wildtype VLPs in density exposing the inserted foreign epitope and being immunogenic. Overall, the use of shared sequences between the insert and the subunit will facilitate the design of chimeric VLPs carrying multiple epitopes.

  18. A Highly Conserved Residue in HIV-1 Nef Alpha Helix 2 Modulates Protein Expression

    PubMed Central

    Johnson, Aaron L.; Dirk, Brennan S.; Coutu, Mathieu; Haeryfar, S. M. Mansour; Arts, Eric J.; Finzi, Andrés

    2016-01-01

    ABSTRACT Extensive genetic diversity is a defining characteristic of human immunodeficiency virus type 1 (HIV-1) and poses a significant barrier to the development of an effective vaccine. To better understand the impact of this genetic diversity on the HIV-1 pathogenic factor Nef, we compiled a panel of reference strains from the NIH Los Alamos HIV Database. Initial sequence analysis identified point mutations at Nef residues 13, 84, and 92 in subtype C reference strain C.BR92025 from Brazil. Functional analysis revealed impaired major histocompatibility complex class I and CD4 downregulation of strain C.BR92025 Nef, which corresponded to decreased protein expression. Metabolic labeling demonstrated that strain C.BR92025 Nef has a greater rate of protein turnover than subtype B reference strain B.JRFL that, on the basis of mutational analysis, is related to Nef residue A84. An alanine-to-valine substitution at position 84, located in alpha helix 2 of Nef, was sufficient to alter the rate of turnover of an otherwise highly expressed Nef protein. In conclusion, these findings highlight HIV-1 Nef residue A84 as a major determinant of protein expression that may offer an additional avenue to disrupt or mediate the effects of this key HIV-1 pathogenic factor. IMPORTANCE The HIV-1 Nef protein has been established as a key pathogenic determinant of HIV/AIDS, but there is little knowledge of how the extensive genetic diversity of HIV-1 affects Nef function. Upon compiling a set of subtype-specific reference strains, we identified a subtype C reference strain, C.BR92025, that contained natural polymorphisms at otherwise highly conserved residues 13, 84, and 92. Interestingly, strain C.BR92025 Nef displayed impaired Nef function and had decreased protein expression. We have demonstrated that strain C.BR92025 Nef has a higher rate of protein turnover than highly expressed Nef proteins and that this higher rate of protein turnover is due to an alanine-to-valine substitution

  19. 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.

  20. Halquinol modulated growth, physiology, and protein profile and halquinol residue withdrawal study in the Indian major carp Catla catla (Hamilton).

    PubMed

    Mushigeri, S B; Saha, S; Somashekar, B N; Nischal, K; Radhakrishna, P M

    2008-01-01

    The aim of the present study was to evaluate the effect of halquinol, an antimicrobial used as a growth promotor in poultry, on the fresh water fish Catla catla in terms of growth promotion, protein profile, and physiology as the rate of oxygen consumption. A synergic increment in the free amino acid level and total protein concentration suggested enhanced anabolic metabolism resulting in weight gain. When compared with an untreated control group, fishes treated with 0.1% halquinol (T1) showed a higher weight gain than those treated with 0.2% halquinol (T2). Variations in the rate of oxygen consumption among the three groups (control, T1, T2) expressed the physiological response of the animals toward the chemical along the time factor. After 7 days of treatment, the absence of halquinol revealed by post-withdrawal residual HPLC studies suggests its biosafety.

  1. Important amino acid residues of hexachlorocyclohexane dehydrochlorinases (LinA) for enantioselective transformation of hexachlorocyclohexane isomers.

    PubMed

    Shrivastava, Nidhi; Macwan, Ankit S; Kohler, Hans-Peter E; Kumar, Ashwani

    2017-03-01

    LinA-type1 and LinA-type2 are two well-characterized variants of the enzyme 'hexachlorocyclohexane (HCH)-dehydrochlorinase'. They differ from each other at ten amino acid positions and exhibit differing enantioselectivity for the transformation of the (-) and (+) enantiomers of α-HCH. Amino acids responsible for this enantioselectivity, however, are not known. An in silico docking analysis identified four amino acids (K20, L96, A131, and T133) in LinA-type1 that could be involved in selective binding of the substrates. Experimental studies with constructed mutant enzymes revealed that a combined presence of three amino acid changes in LinA-type1, i.e. K20Q, L96C, and A131G, caused a reversal in its preference from the (-) to the (+) enantiomer of α-HCH. This preference was enhanced by the additional amino acid change T133 M. Presence of these four changes also caused the reversal of enantioselectivity of LinA-type1 for δ-HCH, and β-, γ-, and δ-pentachlorocyclohexens. Thus, the residues K20, L96, A131, and T133 in LinA-type1 and the residues Q20, C96, G131, and M133 in LinA-type 2 appear to be important determinants for the enantioselectivity of LinA enzymes.

  2. Critical amino acid residues of maurocalcine involved in pharmacology, lipid interaction and cell penetration.

    PubMed

    Mabrouk, Kamel; Ram, Narendra; Boisseau, Sylvie; Strappazzon, Flavie; Rehaim, Amel; Sadoul, Rémy; Darbon, Hervé; Ronjat, Michel; De Waard, Michel

    2007-10-01

    Maurocalcine (MCa) is a 33-amino acid residue peptide that was initially identified in the Tunisian scorpion Scorpio maurus palmatus. This peptide triggers interest for three main reasons. First, it helps unravelling the mechanistic basis of Ca(2+) mobilization from the sarcoplasmic reticulum because of its sequence homology with a calcium channel domain involved in excitation-contraction coupling. Second, it shows potent pharmacological properties because of its ability to activate the ryanodine receptor. Finally, it is of technological value because of its ability to carry cell-impermeable compounds across the plasma membrane. Herein, we characterized the molecular determinants that underlie the pharmacological and cell-penetrating properties of maurocalcine. We identify several key amino acid residues of the peptide that will help the design of cell-penetrating analogues devoid of pharmacological activity and cell toxicity. Close examination of the determinants underlying cell penetration of maurocalcine reveals that basic amino acid residues are required for an interaction with negatively charged lipids of the plasma membrane. Maurocalcine analogues that penetrate better have also stronger interaction with negatively charged lipids. Conversely, less effective analogues present a diminished ability to interact with these lipids. These findings will also help the design of still more potent cell penetrating analogues of maurocalcine.

  3. Proline and gamma-carboxylated glutamate residues in matrix Gla protein are critical for binding of bone morphogenetic protein-4.

    PubMed

    Yao, Yucheng; Shahbazian, Ani; Boström, Kristina I

    2008-05-09

    Arterial calcification is ubiquitous in vascular disease and is, in part, prevented by matrix Gla protein (MGP). MGP binds calcium ions through gamma-carboxylated glutamates (Gla residues) and inhibits bone morphogenetic protein (BMP)-2/-4. We hypothesized that a conserved proline (Pro)64 is essential for BMP inhibition. We further hypothesized that calcium binding by the Gla residues is a prerequisite for BMP inhibition. Site-directed mutagenesis was used to modify Pro64 and the Gla residues, and the effect on BMP-4 activity, and binding of BMP-4 and calcium was tested using luciferase reporter gene assays, coimmunoprecipitation, crosslinking, and calcium quantification. The results showed that Pro64 was critical for binding and inhibition of BMP-4 but not for calcium binding. The Gla residues were also required for BMP-4 binding but flexibility existed. As long as 1 Gla residue remained on each side of Pro64, the ability to bind and inhibit BMP-4 was preserved. Chelation of calcium ions by EDTA or warfarin treatment of cells led to loss of ability of MGP to bind BMP-4. Our results also showed that phenylalanine could replace Pro64 without loss of function and that zebrafish MGP, which lacks upstream Gla residues, did not function as a BMP inhibitor. The effect of MGP mutagenesis on vascular calcification was determined in calcifying vascular cells. Only MGP proteins with preserved ability to bind and inhibit BMP-4 prevented osteogenic differentiation and calcification. Together, our results suggest that BMP and calcium binding in MGP are independent but functionally intertwined processes and that the BMP binding is essential for prevention of vascular calcification.

  4. The Loss and Gain of Functional Amino Acid Residues Is a Common Mechanism Causing Human Inherited Disease

    PubMed Central

    Lugo-Martinez, Jose; Pejaver, Vikas; Pagel, Kymberleigh A.; Mort, Matthew; Cooper, David N.; Mooney, Sean D.; Radivojac, Predrag

    2016-01-01

    Elucidating the precise molecular events altered by disease-causing genetic variants represents a major challenge in translational bioinformatics. To this end, many studies have investigated the structural and functional impact of amino acid substitutions. Most of these studies were however limited in scope to either individual molecular functions or were concerned with functional effects (e.g. deleterious vs. neutral) without specifically considering possible molecular alterations. The recent growth of structural, molecular and genetic data presents an opportunity for more comprehensive studies to consider the structural environment of a residue of interest, to hypothesize specific molecular effects of sequence variants and to statistically associate these effects with genetic disease. In this study, we analyzed data sets of disease-causing and putatively neutral human variants mapped to protein 3D structures as part of a systematic study of the loss and gain of various types of functional attribute potentially underlying pathogenic molecular alterations. We first propose a formal model to assess probabilistically function-impacting variants. We then develop an array of structure-based functional residue predictors, evaluate their performance, and use them to quantify the impact of disease-causing amino acid substitutions on catalytic activity, metal binding, macromolecular binding, ligand binding, allosteric regulation and post-translational modifications. We show that our methodology generates actionable biological hypotheses for up to 41% of disease-causing genetic variants mapped to protein structures suggesting that it can be reliably used to guide experimental validation. Our results suggest that a significant fraction of disease-causing human variants mapping to protein structures are function-altering both in the presence and absence of stability disruption. PMID:27564311

  5. 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.

  6. Energetics of galactose- and glucose-aromatic amino acid interactions: implications for binding in galactose-specific proteins.

    PubMed

    Sujatha, Mannargudi S; Sasidhar, Yellamraju U; Balaji, Petety V

    2004-09-01

    An aromatic amino acid is present in the binding site of a number of sugar binding proteins. The interaction of the saccharide with the aromatic residue is determined by their relative position as well as orientation. The position-orientation of the saccharide relative to the aromatic residue was found to vary in different sugar-binding proteins. In the present study, interaction energies of the complexes of galactose (Gal) and of glucose (Glc) with aromatic residue analogs have been calculated by ab initio density functional (U-B3LYP/ 6-31G**) theory. The position-orientations of the saccharide with respect to the aromatic residue observed in various Gal-, Glc-, and mannose-protein complexes were chosen for the interaction energy calculations. The results of these calculations show that galactose can interact with the aromatic residue with similar interaction energies in a number of position-orientations. The interaction energy of Gal-aromatic residue analog complex in position-orientations observed for the bound saccharide in Glc/Man-protein complexes is comparable to the Glc-aromatic residue analog complex in the same position-orientation. In contrast, there is a large variation in interaction energies of complexes of Glc- and of Gal- with the aromatic residue analog in position-orientations observed in Gal-protein complexes. Furthermore, the conformation wherein the O6 atom is away from the aromatic residue is preferred for the exocyclic -CH2OH group in Gal-aromatic residue analog complexes. The implications of these results for saccharide binding in Gal-specific proteins and the possible role of the aromatic amino acid to ensure proper positioning and orientation of galactose in the binding site have been discussed.

  7. Microwave-assisted 18O-labeling of proteins catalyzed by formic acid.

    PubMed

    Liu, Ning; Wu, Hanzhi; Liu, Hongxia; Chen, Guonan; Cai, Zongwei

    2010-11-01

    Oxygen exchange may occur at carboxyl groups catalyzed by acid. The reaction, however, takes at least several days at room temperature. The long-time exchanging reaction often prevents its application from protein analysis. In this study, an (18)O-labeling method utilizing microwave-assisted acid hydrolysis was developed. After being dissolved in (16)O/(18)O (1:1) water containing 2.5% formic acid, protein samples were exposed to microwave irradiation. LC-MS/MS analysis of the resulted peptide mixtures indicated that oxygen in the carboxyl groups from glutamic acid, aspartic acid, and the C-terminal residues could be efficiently exchanged with (18)O within less than 15 min. The rate of back exchange was so slow that no detectable back exchange could be found during the HPLC run.

  8. Moving Iron through Ferritin Protein Nanocages Depends on Residues throughout Each Four α-Helix Bundle Subunit*

    PubMed Central

    Haldar, Suranjana; Bevers, Loes E.; Tosha, Takehiko; Theil, Elizabeth C.

    2011-01-01

    Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe2+/O2 oxidoreduction and formation of [Fe3+O]n multimers within the protein cage, en route to the cavity, at sites distributed over ∼50 Å. Recent NMR and Co2+-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe2+ ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe3+O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A650 nm) and on mineral growth (Fe3+O-A350 nm), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe2+ access/selectivity to the active sites (Glu130), (ii) distribution of Fe2+ to each of the three active sites near each ion channel (Asp127), (iii) product (diferric oxo) release into the Fe3+O nucleation channels (Ala26), and (iv) [Fe3+O]n transit through subunits (Val42, Thr149). Synthesis of ferritin biominerals depends on residues along the entire length of H subunits from Fe2+ substrate entry at 3-fold cage axes at one subunit end through active sites and nucleation channels, at the other subunit end, inside the cage at 4-fold cage axes. Ferritin subunit-subunit geometry contributes to mineral order and explains the physiological impact of ferritin H and L subunits. PMID:21592958

  9. The 73 kilodalton heat shock cognate protein purified from rat brain contains nonesterified palmitic and stearic acids.

    PubMed

    Guidon, P T; Hightower, L E

    1986-08-01

    A protein related to the 71 kilodalton inducible rat heat shock protein was purified to electrophoretic homogeneity in milligram amounts from brain tissue of nonheat-stressed rats. The protein has been designated as a stress cognate protein based on previous studies and data presented herein that this protein cross-reacted with a monoclonal antibody originally raised against the Drosophila 70 kilodalton heat shock protein. The purified protein had an apparent molecular mass of 73 kilodaltons when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and an apparent mass of 150 kilodaltons as determined by nondissociative gel chromatography, suggesting that the purified protein is a homodimer. The purified protein had isoelectric points of 5.0 under nondissociative conditions and 5.6 when exposed to protein denaturants, suggesting loss of bound anionic molecules and/or net exposure of basic residues upon denaturation. Chloroform/methanol extraction of the purified protein and subsequent analyses by thin layer and gas-liquid chromatography resulted in the identification of palmitic and stearic acids noncovalently bound to the protein. Approximately four molecules of fatty acids were bound per dimer with palmitic and stearic acids present in a one-to-one ratio. The purified protein did not bind exogenously added radioactive palmitate, indicating that the fatty acid-binding sites of the cognate protein were fully occupied and that the associated fatty acids were too tightly bound to exchange readily. The possible significance of the fatty acids associated with the 73 kilodalton stress cognate protein is discussed.

  10. Amino acid residues Leu135 and Tyr236 are required for RNA binding activity of CFIm25 in Entamoeba histolytica.

    PubMed

    Ospina-Villa, Juan David; Zamorano-Carrillo, Absalom; Lopez-Camarillo, Cesar; Castañon-Sanchez, Carlos A; Soto-Sanchez, Jacqueline; Ramirez-Moreno, Esther; Marchat, Laurence A

    2015-08-01

    Pre-mRNA 3' end processing in the nucleus is essential for mRNA stability, efficient nuclear transport, and translation in eukaryotic cells. In Human, the cleavage/polyadenylation machinery contains the 25 kDa subunit of the Cleavage Factor Im (CFIm25), which specifically recognizes two UGUA elements and regulates the assembly of polyadenylation factors, poly(A) site selection and polyadenylation. In Entamoeba histolytica, the protozoan parasite responsible for human amoebiasis, EhCFIm25 has been reported as a RNA binding protein that interacts with the Poly(A) Polymerase. Here, we follow-up with the study of EhCFIm25 to characterize its interaction with RNA. Using in silico strategy, we identified Leu135 and Tyr236 in EhCFIm25 as conserved amino acids among CFIm25 homologues. We therefore generated mutant EhCFIm25 proteins to investigate the role of these residues for RNA interaction. Results showed that RNA binding activity was totally abrogated when Leu135 and Tyr236 were replaced with Ala residue, and Tyr236 was changed for Phe. In contrast, RNA binding activity was less affected when Leu135 was substituted by Thr. Our data revealed for the first time -until we know-the functional relevance of the conserved Leu135 and Tyr236 in EhCFIm25 for RNA binding activity. They also gave some insights about the possible chemical groups that could be interacting with the RNA molecule.

  11. Removal of copper from acid wastewater of bioleaching by adsorption onto ramie residue and uptake by Trichoderma viride.

    PubMed

    Wang, Buyun; Wang, Kai

    2013-05-01

    A continuous batch bioleaching was built to realize the bioleaching of sewage sludge in large scale. In the treatment, heavy metal in acid wastewater of bioleaching was removed by adsorption onto ramie residue. Then, acid wastewater was reused in next bioleaching batch. In this way, most time and water of bioleaching was saved and leaching efficiency of copper, lead and chromium kept at a high level in continuous batch bioleaching. It was found that residual heavy metal in sewage sludge is highly related to that in acid wastewater after bioleaching. To get a high leaching efficiency, concentration of heavy metal in acid wastewater should be low. Adsorption of copper from acid wastewater onto ramie residue can be described by pseudo first-order kinetics equation and Freundlich isotherm model. Trichoderma viride has the potential to be used for the concentration and recovery of heavy metal adsorbed onto ramie residue.

  12. Fidelity of the protein structure reconstruction from inter-residue proximity constraints.

    PubMed

    Chen, Yiwen; Ding, Feng; Dokholyan, Nikolay V

    2007-06-28

    Inter-residue proximity constraints obtained in such experiments as cross-linking/mass spectrometry are important sources of information for protein structure determination. A central question in structure determination using these constraints is, What is the minimal number of inter-residue constraints needed to determine the fold of a protein? It is also unknown how the different structural aspects of constraints differentiate their ability in determining the native fold and whether there is a rational strategy for selecting constraints that feature higher fidelity in structure determination. To shed light on these questions, we study the fidelity of protein fold determination using theoretical inter-residue proximity constraints derived from protein native structures and the effect of various subsets of such constraints on fold determination. We show that approximately 70% randomly selected constraints are sufficient for determining the fold of a domain (with an average root-mean-square deviation of protein structure determination relies on a composite of global and local structural information.

  13. Protein design by sampling an undirected graphical model of residue constraints.

    PubMed

    Thomas, John; Ramakrishnan, Naren; Bailey-Kellogg, Chris

    2009-01-01

    This paper develops an approach for designing protein variants by sampling sequences that satisfy residue constraints encoded in an undirected probabilistic graphical model. Due to evolutionary pressures on proteins to maintain structure and function, the sequence record of a protein family contains valuable information regarding position-specific residue conservation and coupling (or covariation) constraints. Representing these constraints with a graphical model provides two key benefits for protein design: a probabilistic semantics enabling evaluation of possible sequences for consistency with the constraints, and an explicit factorization of residue dependence and independence supporting efficient exploration of the constrained sequence space. We leverage these benefits in developing two complementary MCMC algorithms for protein design: constrained shuffling mixes wild-type sequences positionwise and evaluates graphical model likelihood, while component sampling directly generates sequences by sampling clique values and propagating to other cliques. We apply our methods to design WW domains. We demonstrate that likelihood under a model of wild-type WWs is highly predictive of foldedness of new WWs. We then show both theoretical and rapid empirical convergence of our algorithms in generating high-likelihood, diverse new sequences. We further show that these sequences capture the original sequence constraints, yielding a model as predictive of foldedness as the original one.

  14. An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from Penicillium purpurogenum

    NASA Astrophysics Data System (ADS)

    Colombres, Marcela; Garate, José A.; Lagos, Carlos F.; Araya-Secchi, Raúl; Norambuena, Patricia; Quiroz, Soledad; Larrondo, Luis; Pérez-Acle, Tomas; Eyzaguirre, Jaime

    2008-01-01

    The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 Å resolution (PDB 1G66). The enzyme possesses the α/β hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three-dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a comparative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop's residues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters.

  15. ERAD of proteins containing aberrant transmembrane domains requires ubiquitylation of cytoplasmic lysine residues

    PubMed Central

    Briant, Kit; Koay, Yee-Hui; Otsuka, Yuka; Swanton, Eileithyia

    2015-01-01

    ABSTRACT Clearance of misfolded proteins from the endoplasmic reticulum (ER) is mediated by the ubiquitin-proteasome system in a process known as ER-associated degradation (ERAD). The mechanisms through which proteins containing aberrant transmembrane domains are degraded by ERAD are poorly understood. To address this question, we generated model ERAD substrates based on CD8 with either a non-native transmembrane domain but a folded ER luminal domain (CD8TMD*), or the native transmembrane domain but a misfolded luminal domain (CD8LUM*). Although both chimeras were degraded by ERAD, we found that the location of the folding defect determined the initial site of ubiquitylation. Ubiquitylation of cytoplasmic lysine residues was required for the extraction of CD8TMD* from the ER membrane during ERAD, whereas CD8LUM* continued to be degraded in the absence of cytoplasmic lysine residues. Cytoplasmic lysine residues were also required for degradation of an additional ERAD substrate containing an unassembled transmembrane domain and when a non-native transmembrane domain was introduced into CD8LUM*. Our results suggest that proteins with defective transmembrane domains are removed from the ER through a specific ERAD mechanism that depends upon ubiquitylation of cytoplasmic lysine residues. PMID:26446255

  16. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    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.

  17. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    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.

  18. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G [La Jolla, CA; Wang, Lei [San Diego, CA

    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.

  19. 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.

  20. 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.

  1. Beta-methylthio-aspartic acid: identification of a novel posttranslational modification in ribosomal protein S12 from Escherichia coli.

    PubMed Central

    Kowalak, J. A.; Walsh, K. A.

    1996-01-01

    Utilizing microscale chemical derivatization reactions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we have identified a novel posttranslational modification of aspartic acid, beta-methylthio-aspartic acid. The modified residue is located at position 88 in ribosomal protein S12 from Escherichia coli, a phylogenetically conserved protein that has been implicated in maintaining translational accuracy of the ribosome. PMID:8844851

  2. Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster.

    PubMed

    Menger, Katja E; James, Andrew M; Cochemé, Helena M; Harbour, Michael E; Chouchani, Edward T; Ding, Shujing; Fearnley, Ian M; Partridge, Linda; Murphy, Michael P

    2015-06-30

    Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. Although aging and fasting alter many redox processes, the role of cysteine residues is uncertain. To address this, we used a redox proteomic technique, oxidative isotope-coded affinity tags (OxICAT), to assess cysteine-residue redox changes in Drosophila melanogaster during aging and fasting. This approach enabled us to simultaneously identify and quantify the redox state of several hundred cysteine residues in vivo. Cysteine residues within young flies had a bimodal distribution with peaks at ∼10% and ∼85% reversibly oxidized. Surprisingly, these cysteine residues did not become more oxidized with age. In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized. We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster.

  3. Conserved Cysteine Residue in the DNA-Binding Domain of the Bovine Papillomavirus Type 1 E2 Protein Confers Redox Regulation of the DNA- Binding Activity in Vitro

    NASA Astrophysics Data System (ADS)

    McBride, Alison A.; Klausner, Richard D.; Howley, Peter M.

    1992-08-01

    The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfydryl group(s) may be protected by DNA binding. Sensitivity to sulfydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.

  4. Mobilization of Cr(VI) from chromite ore processing residue through acid treatment.

    PubMed

    Tinjum, James M; Benson, Craig H; Edil, Tuncer B

    2008-02-25

    Batch leaching studies on chromite ore processing residue (COPR) were performed using acids to investigate leaching of hexavalent chromium, Cr(VI), with respect to particle size, reaction time, and type of acid (HNO(3) and H(2)SO(4)). Aqueous Cr(VI) is maximized at approximately 0.04 mol Cr(VI) per kg of dry COPR at pH 7.6-8.1. Cr(VI) mobilized more slowly for larger particles, and the pH increased with time and increased more rapidly for smaller particles, suggesting that rate limitations occur in the solid phase. With H(2)SO(4), the pH stabilized at a higher value (8.8 for H(2)SO(4) vs. 8.0 for HNO(3)) and more rapidly (16 h vs. 30 h), and the differences in pH for different particle sizes were smaller. The acid neutralization capacity (ANC) of COPR is very large (8 mol HNO(3) per kg of dry COPR for a stable eluate pH of 7.5). Changes to the elemental and mineralogical composition and distribution in COPR particles after mixing with acid indicate that Cr(VI)-bearing solids dissolved. However, concentrations of Cr(VI) >2800 mg kg(-1) (>50% of the pre-treatment concentration) were still found after mixing with acid, regardless of the particle size, reaction time, or type of acid used. The residual Cr(VI) appears to be partially associated with poorly-ordered Fe and Al oxyhydroxides that precipitated in the interstitial areas of COPR particles. Remediation strategies that use HNO(3) or H(2)SO(4) to neutralize COPR or to maximize Cr(VI) in solution are likely to require extensive amounts of acid, may not mobilize all of the Cr(VI), and may require extended contact time, even under well-mixed conditions.

  5. Novel fatty acid acylation of lens integral membrane protein aquaporin-0.

    PubMed

    Schey, Kevin L; Gutierrez, Danielle B; Wang, Zhen; Wei, Junhua; Grey, Angus C

    2010-11-16

    Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0. LC/MS/MS proteomic analysis of hydrophobic tryptic peptides from lens membrane proteins revealed both N-terminal and C-terminal peptides modified by 238 and 264 Da which were subsequently assigned by accurate mass measurement as palmitoylation and oleoylation, respectively. Specific sites of modification were the N-terminal methionine residue and lysine 238 revealing, for the first time, an oleic acid modification via an amide linkage to a lysine residue. The specific fatty acids involved reflect their abundance in the lens fiber cell plasma membrane. Imaging mass spectrometry indicated abundant acylated AQP0 in the inner cortical region of both bovine and human lenses and acylated truncation products in the lens nucleus. Additional analyses revealed that the lipid-modified forms partitioned exclusively to a detergent-resistant membrane fraction, suggesting a role in membrane domain targeting.

  6. Structure of the ordered hydration of amino acids in proteins: analysis of crystal structures.

    PubMed

    Biedermannová, Lada; Schneider, Bohdan

    2015-11-01

    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.

  7. Structure of the ordered hydration of amino acids in proteins: analysis of crystal structures

    PubMed Central

    Biedermannová, Lada; Schneider, Bohdan

    2015-01-01

    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. PMID:26527137

  8. Generation of pseudocontact shifts in proteins with lanthanides using small "clickable" nitrilotriacetic acid and iminodiacetic acid tags.

    PubMed

    Loh, Choy-Theng; Graham, Bim; Abdelkader, Elwy H; Tuck, Kellie L; Otting, Gottfried

    2015-03-23

    Pseudocontact shifts (PCS) induced by paramagnetic lanthanide ions provide unique long-range structural information in nuclear magnetic resonance (NMR) spectra, but the site-specific attachment of lanthanide tags to proteins remains a challenge. Here we incorporated p-azido-phenylalanine (AzF) site-specifically into the proteins ubiquitin and GB1, and ligated the AzF residue with alkyne derivatives of small nitrilotriacetic acid and iminodiacetic acid tags using the Cu(I) -catalysed "click" reaction. These tags form lanthanide complexes with no or only a small net charge and produced sizeable PCSs with paramagnetic lanthanide ions in all mutants tested. The PCSs were readily fitted by single magnetic susceptibility anisotropy tensors. Protein precipitation during the click reaction was greatly alleviated by the presence of 150 mM NaCl.

  9. Intrinsic propensities of amino acid residues in GxG peptides inferred from amide I' band profiles and NMR scalar coupling constants.

    PubMed

    Hagarman, Andrew; Measey, Thomas J; Mathieu, Daniel; Schwalbe, Harald; Schweitzer-Stenner, Reinhard

    2010-01-20

    A reliable intrinsic propensity scale of amino acid residues is indispensable for an assessment of how local conformational distributions in the unfolded state can affect the folding of peptides and proteins. Short host-guest peptides, such as GxG tripeptides, are suitable tools for probing such propensities. To explore the conformational distributions sampled by the central amino acid residue in these motifs, we combined vibrational (IR, Raman, and VCD) with NMR spectroscopy. The data were analyzed in terms of a superposition of two-dimensional Gaussian distribution functions in the Ramachandran space pertaining to subensembles of polyproline II, beta-strand, right- and left-handed helical, and gamma-turn-like conformations. The intrinsic propensities of eight amino acid residues (x = A, V, F, L, S, E, K, and M) in GxG peptides were determined as mole fractions of these subensembles. Our results show that alanine adopts primarily (approximately 80%) a PPII-like conformation, while valine and phenylalanine were found to sample PPII and beta-strand-like conformations equally. The centers of the respective beta-strand distributions generally do not coincide with canonical values of dihedral angles of residues in parallel or antiparallel beta-strands. In fact, the distributions for most residues found in the beta-region significantly overlap the PPII-region. A comparison with earlier reported results for trivaline reveals that the terminal valines increase the beta-strand propensity of the central valine residue even further. Of the remaining investigated amino acids, methionine preferred PPII the most (0.64), and E, S, L, and K exhibit moderate (0.56-0.45) PPII propensities. Residues V, F, S, E, and L sample, to a significant extent, a region between the canonical PPII and (antiparallel) beta-strand conformations. This region coincides with the sampling reported for L and V using theoretical predictions (Tran et al. Biochemistry 2005, 44, 11369). The distributions of

  10. Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis.

    PubMed

    Praefcke, Gerrit J K; Kloep, Stephan; Benscheid, Utz; Lilie, Hauke; Prakash, Balaji; Herrmann, Christian

    2004-11-12

    The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP. In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies.

  11. Evidence that the amino acid residue Cys117 of chloroplastic monodehydroascorbate reductase is involved in its activity and structural stability.

    PubMed

    Li, Feng; Wu, Qing-Yun; Sun, Yan-Li; Ma, Na-Na; Wang, Xiao-Yun; Meng, Qing-Wei

    2010-04-01

    Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) is crucial for AsA regeneration and essential for maintaining the reduced pool of AsA. And the amino acid residue C117 of chloroplastic MDAR is the conserved cysteine residue in MDAR isoforms. A series mutation of conserved amino acid residue cysteine117 (C117) was constructed to investigate its role in MDAR structural stability and activity. Our study revealed that mutation in this conserved residue could cause pronounced loss of activity and conformational changes. Spectroscopic experiments indicated that these mutations influenced transition from the molten globule intermediate to the native state in folding process. These results suggested that amino acid residue C117 played a relatively important role in keeping MDAR structural stability and activity.

  12. FKBP12.6 activates RyR1: investigating the amino acid residues critical for channel modulation.

    PubMed

    Venturi, Elisa; Galfré, Elena; O'Brien, Fiona; Pitt, Samantha J; Bellamy, Stuart; Sessions, Richard B; Sitsapesan, Rebecca

    2014-02-18

    We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12E31Q/D32N/W59F) where the residues Glu(31), Asp(32), and Trp(59) were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12E31Q/D32N/W59F mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12E31Q/D32N/W59F activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu(31), Asp(32), and Trp(59) in FKBP12 and Gln(31), Asn(32), and Phe(59) in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.

  13. Leaching of lead from zinc leach residue in acidic calcium chloride aqueous solution

    NASA Astrophysics Data System (ADS)

    Wang, Le; Mu, Wen-ning; Shen, Hong-tao; Liu, Shao-ming; Zhai, Yu-chun

    2015-05-01

    A process with potentially reduced environmental impacts and occupational hazards of lead-bearing zinc plant residue was studied to achieve a higher recovery of lead via a cost-effective and environmentally friendly process. This paper describes an optimization study on the leaching of lead from zinc leach residue using acidic calcium chloride aqueous solution. Six main process conditions, i.e., the solution pH value, stirring rate, concentration of CaCl2 aqueous solution, liquid-to-solid (L/S) ratio, leaching temperature, and leaching time, were investigated. The microstructure and components of the residue and tailing were characterized using scanning electron microscopy (SEM) and X-ray diffraction (XRD). On the basis of experimental results, the optimum reaction conditions were determined to be a solution pH value of 1, a stirring rate of 500 r·min-1, a CaCl2 aqueous solution concentration of 400 g·L-1, a liquid-to-solid mass ratio of 7:1, a leaching temperature of 80°C, and a leaching time of 45 min. The leaching rate of lead under these conditions reached 93.79%, with an iron dissolution rate of 19.28%. Silica did not take part in the chemical reaction during the leaching process and was accumulated in the residue.

  14. Radionuclide Leaching from Residual Solids Remaining after Acid Dissolution of Composite K East Canister Sludge

    SciTech Connect

    Delegard, C.H.; Rinehart, D.E.; Soderquist, C.Z.; Fadeff, S.K.

    1999-04-02

    Laboratory tests were performed to examine mixed nitric/hydrofluoric acid leach treatments for decontaminating dissolver residual solids (KECDVSR24H-2) produced during a 20- to 24-hr dissolution of a composite K East (KE) Basin canister sludge in 95 C 6 M nitric acid (HNO{sub 3}). The scope of this testing has been described in Section 4.5 of ''Testing Strategy to Support the Development of K Basin Sludge Treatment Process'' (Flament 1998). Radionuclides sorbed or associated with the residual solids generated in the K Basin sludge treatment process can restrict disposal of this solid to the Environmental Restoration Disposal Facility (ERDF). The starting dissolver residual solid for this testing, KECDVSR24H-2, contains radionuclides at concentrations which exceed the ERDF Waste Acceptance Criteria for TRU by about a factor of 70, for {sup 239}Pu by a factor of 200, and for {sup 241}Am by a factor of 50. The solids also exceed the ERDF criterion for {sup 137}Cs by a factor of 2 and uranium by a factor of 5. Therefore, the radionuclides of greatest interest in this leaching study are first {sup 239}Pu and {sup 241}Am (both components of TRU) and then uranium and {sup 137}Cs.

  15. Immunohistochemical localization of D-β-aspartic acid-containing proteins in pterygium.

    PubMed

    Kaji, Yuichi; Oshika, Tetsuro; Nejima, Ryouhei; Mori, Saiyo; Miyata, Kazunori; Fujii, Noriko

    2015-12-10

    Biologically uncommon D-β-aspartic acid (D-β-Asp) residues have been reported to accumulate in organs affected by age-related disorders. In the present study, we investigated the localization of D-β-Asp-containing proteins in cases of pterygium, one of the most prominent age-related ocular conditions. Immunohistochemical localization of D-β-Asp-containing proteins was investigated in surgical specimens of pterygium from 20 patients and control specimens from 10 patients. Strong immunoreactivity to D-β-Asp-containing proteins was observed in subepithelial elastotic lesions and surrounding collagenous lesions from all surgical specimens with pterygia. In contrast, no immunoreactivity to D-β-Asp-containing proteins was seen in pterygium-free specimens. D-β-Asp-containing proteins are produced in organs as they are affected by the aging process. In addition, conversion of L- to D-aspartyl residues is accelerated by ultraviolet (UV) irradiation. Since pterygia can form due to aging or UV exposure, it is reasonable to find D-β-Asp-containing proteins in specimens with pterygia. Furthermore, since D-β-Asp is a non-native amino acid, D-β-Asp-containing proteins may be recognized as allogeneic antigens. Therefore, D-β-Asp-containing proteins in pterygia may responsible for the fibrovascular changes seen in the disorder.

  16. Conformational characterization of the 1-aminocyclobutane-1-carboxylic acid residue in model peptides.

    PubMed

    Gatos, M; Formaggio, F; Crisma, M; Toniolo, C; Bonora, G M; Benedetti, Z; Di Blasio, B; Iacovino, R; Santini, A; Saviano, M; Kamphuis, J

    1997-01-01

    A series of N- and C-protected, monodispersed homo-oligopeptides (to the dodecamer level) from the small-ring alicyclic C alpha, alpha-dialkylated glycine 1-aminocyclobutane-1-carboxylic acid (Ac4c) and two Ala/Ac4c tripeptides were synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and 1H-NMR. The molecular structures of the amino acid derivatives Z-Ac4c-OH and Z2-Ac4c-OH, the tripeptides Z-(Ac4c)3-OtBu, Z-Ac4c-(L-Ala)2-OMe and Z-L-Ala-Ac4c-L-Ala-OMe, and the tetrapeptide Z-(Ac4c)4-OtBu were determined in the crystal state by X-ray diffraction. The average geometry of the cyclobutyl moiety of the Ac4c residue was assessed and the tau(N-C alpha-C') bond angle was found to be significantly expanded from the regular tetrahedral value. The conformational data are strongly in favour of the conclusion that the Ac4c residue is an effective beta-turn and helix former. A comparison with the structural propensities of alpha-aminoisobutyric acid, the prototype of C alpha, alpha-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc, with n = 3, 5-8) is made and the implications for the use of the Ac4c residue in conformationally constrained peptide analogues are briefly examined.

  17. A Novel Treatment for Acid Mine Drainage Utilizing Reclaimed Limestone Residual

    SciTech Connect

    Horace K. Moo-Young; Charles E. Ochola

    2004-08-31

    The viability of utilizing Reclaimed Limestone Residual (RLR) to remediate Acid Mine Drainage (AMD) was investigated. Physical and chemical characterization of RLR showed that it is composed of various minerals that contain significant quantities of limestone or calcium bearing compounds that can be exploited for acid neutralization. Acid Neutralization Potential (ANP) test results showed that RLR has a neutralization potential of approximately 83% as calcium carbonate (CaCO{sub 3}). Neutralization tests with most of the heavy metals associated with AMD showed removal efficiencies of over 99%. An unexpected benefit of utilizing RLR was the removal of hexavalent chromium Cr (VI) from the aqueous phase. Due to an elevation in pH by RLR most AMD heavy metals are removed from solution by precipitation as their metal hydroxides. Cr (VI) however is not removed by pH elevation and therefore subsequent ongoing tests to elucidate the mechanism responsible for this reaction were conducted.

  18. Computational prediction of methylation types of covalently modified lysine and arginine residues in proteins.

    PubMed

    Deng, Wankun; Wang, Yongbo; Ma, Lili; Zhang, Ying; Ullah, Shahid; Xue, Yu

    2016-05-30

    Protein methylation is an essential posttranslational modification (PTM) mostly occurs at lysine and arginine residues, and regulates a variety of cellular processes. Owing to the rapid progresses in the large-scale identification of methylation sites, the available data set was dramatically expanded, and more attention has been paid on the identification of specific methylation types of modification residues. Here, we briefly summarized the current progresses in computational prediction of methylation sites, which provided an accurate, rapid and efficient approach in contrast with labor-intensive experiments. We collected 5421 methyllysines and methylarginines in 2592 proteins from the literature, and classified most of the sites into different types. Data analyses demonstrated that different types of methylated proteins were preferentially involved in different biological processes and pathways, whereas a unique sequence preference was observed for each type of methylation sites. Thus, we developed a predictor of GPS-MSP, which can predict mono-, di- and tri-methylation types for specific lysines, and mono-, symmetric di- and asymmetrical di-methylation types for specific arginines. We critically evaluated the performance of GPS-MSP, and compared it with other existing tools. The satisfying results exhibited that the classification of methylation sites into different types for training can considerably improve the prediction accuracy. Taken together, we anticipate that our study provides a new lead for future computational analysis of protein methylation, and the prediction of methylation types of covalently modified lysine and arginine residues can generate more useful information for further experimental manipulation.

  19. Interaction between dimer interface residues of native and mutated SOD1 protein: a theoretical study.

    PubMed

    Keerthana, S P; Kolandaivel, P

    2015-04-01

    Cu-Zn superoxide dismutase 1 (SOD1) is a highly conserved bimetallic protein enzyme, used for the scavenging the superoxide radicals (O2 (-)) produced due to aerobic metabolism in the mitochondrial respiratory chain. Over 100 mutations have been identified and found to be in the homodimeric structure of SOD1. The enzyme has to be maintained in its dimeric state for the structural stability and enzymatic activity. From our investigation, we found that the mutations apart from the dimer interface residues are found to affect the dimer stability of protein and hence enhancing the aggregation and misfolding tendency of mutated protein. The homodimeric state of SOD1 is found to be held together by the non-covalent interactions. The molecular dynamics simulation has been used to study the hydrogen bond interactions between the dimer interface residues of the monomers in native and mutated forms of SOD1 in apo- and holo-states. The results obtained by this analysis reveal the fact that the loss of hydrogen bond interactions between the monomers of the dimer is responsible for the reduced stability of the apo- and holo-mutant forms of SOD1. The conformers with dimer interface residues in native and mutated protein obtained by the molecular dynamics simulation is subjected to quantum mechanical study using M052X/6-31G(d) level of theory. The charge transfer between N-H···O interactions in the dimer interface residues were studied. The weak interaction between the monomers of the dimer accounts for the reduced dimerization and enhanced deformation energy in the mutated SOD1 protein.

  20. Acid hydrolysis of Curcuma longa residue for ethanol and lactic acid fermentation.

    PubMed

    Nguyen, Cuong Mai; Nguyen, Thanh Ngoc; Choi, Gyung Ja; Choi, Yong Ho; Jang, Kyoung Soo; Park, Youn-Je; Kim, Jin-Cheol

    2014-01-01

    This research examines the acid hydrolysis of Curcuma longa waste, to obtain the hydrolysate containing lactic acid and ethanol fermentative sugars. A central composite design for describing regression equations of variables was used. The selected optimum condition was 4.91% sulphuric acid, 122.68°C and 50 min using the desirability function under the following conditions: the maximum reducing sugar (RS) yield is within the limited range of the 5-hydroxymethylfurfural (HMF) and furfural concentrations. Under the condition, the obtained solution contained 144 g RS/L, 0.79 g furfural/L and 2.59 g HMF/L and was directly fermented without a detoxification step. The maximum product concentration, average productivity, RS conversion and product yield were 115.36 g/L, 2.88 g/L/h, 89.43% and 64% for L-lactic acid; 113.92 g/L, 2.59 g/L/h, 88.31% and 63.29% for D-lactic acid; and 55.03 g/L, 1.38 g/L/h, 42.66 and 30.57%, respectively, for ethanol using a 7-L jar fermenter.

  1. Key residues for the oligomerization of A{beta}42 protein in Alzheimer's disease

    SciTech Connect

    Ngo, Sam; Guo, Zhefeng

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer A{beta} oligomers are neurotoxins and likely the causing agents for Alzheimer's disease. Black-Right-Pointing-Pointer A{beta}42 fusion protein form globular oligomers. Black-Right-Pointing-Pointer A{beta}42 fusion protein oligomers contain SDS-resistant tetramers and hexamers. Black-Right-Pointing-Pointer Cysteine substitutions at residues 31, 32, 34, 39-41 disrupt A{beta}42 oligomerization. -- Abstract: Deposition of amyloid fibrils consisting of amyloid {beta} (A{beta}) protein as senile plaques in the brain is a pathological hallmark of Alzheimer's disease. However, a growing body of evidence shows that soluble A{beta} oligomers correlate better with dementia than fibrils, suggesting that A{beta} oligomers may be the primary toxic species. The structure and oligomerization mechanism of these A{beta} oligomers are crucial for developing effective therapeutics. Here we investigated the oligomerization of A{beta}42 in the context of a fusion protein containing GroES and ubiquitin fused to the N-terminus of A{beta} sequence. The presence of fusion protein partners, in combination with a denaturing buffer containing 8 M urea at pH 10, is unfavorable for A{beta}42 aggregation, thus allowing only the most stable structures to be observed. Transmission electron microscopy showed that A{beta}42 fusion protein formed globular oligomers, which bound weakly to thioflavin T and Congo red. SDS-PAGE shows that A{beta}42 fusion protein formed SDS-resistant hexamers and tetramers. In contrast, A{beta}40 fusion protein remained as monomers on SDS gel, suggesting that the oligomerization of A{beta}42 fusion protein is not due to the fusion protein partners. Cysteine scanning mutagenesis at 22 residue positions further revealed that single cysteine substitutions of the C-terminal hydrophobic residues (I31, I32, L34, V39, V40, and I41) led to disruption of hexamer and tetramer formation, suggesting that hydrophobic interactions between these

  2. [Nitrate nitrogen leaching and residue of humic acid fertilizer in field soil].

    PubMed

    Liu, Fang-chun; Xing, Shang-jun; Duan, Chun-hua; Du, Zhen-yu; Ma, Hai-lin; Ma, Bing-yao

    2010-07-01

    To elucidate the potential influence of humic acidfertilizer on groundwater and soil quality in clay soil (CS) and sandy soil (SS), nitrate nitrogen leaching and residue of different fertilizers in field soil were studied using a self-made leaching field device. Nitrate nitrogen concentration in leaching water of fertilizer treatments was 28.1%-222.2% higher than that of non-nitrogen treatment in different times, but humic acid fertilizer could prevent nitrate nitrogen leaching both in CS and SS, especially in CS. Nitrate nitrogen concentration of leaching water in CS was 41.2%-59.1% less than that in SS and the inhibiting effect in CS was greater than that in SS. Nitrate nitrogen could be accumulated in soil profile by fertilizer application. The residue of nitrate nitrogen retained in 0-40 cm soil layer of humic acid fertilizer treatment was 59.8% and 54.4% respectively, higher than that of urea and compound fertilizer treatments. Nitrate nitrogen amount of humic acid, urea and compound fertilizer treatments in SS was significantly less than that in CS, being 81.7%, 81.1% and 47.6% respectively. Compared with the conventional fertilizer, humic acid fertilizer treatment improved the contents of organic matter, available nitrogen, phosphorus, and potassium of upper layer soil as well as cation exchange capacity. Besides, total amount of water-soluble salts in humic acid fertilizer treatment was decreased by 24.8% and 22.5% in comparison to urea and compound fertilizer treatments in CS, respectively. In summary, the application of humic acid fertilizer could improve physical and chemical properties of upper layer soil and reduce the risk of potential pollution to groundwater.

  3. Computational prediction of the tolerance to amino-acid deletion in green-fluorescent protein

    PubMed Central

    Jackson, Eleisha L.; Spielman, Stephanie J.

    2017-01-01

    Proteins evolve through two primary mechanisms: substitution, where mutations alter a protein’s amino-acid sequence, and insertions and deletions (indels), where amino acids are either added to or removed from the sequence. Protein structure has been shown to influence the rate at which substitutions accumulate across sites in proteins, but whether structure similarly constrains the occurrence of indels has not been rigorously studied. Here, we investigate the extent to which structural properties known to covary with protein evolutionary rates might also predict protein tolerance to indels. Specifically, we analyze a publicly available dataset of single—amino-acid deletion mutations in enhanced green fluorescent protein (eGFP) to assess how well the functional effect of deletions can be predicted from protein structure. We find that weighted contact number (WCN), which measures how densely packed a residue is within the protein’s three-dimensional structure, provides the best single predictor for whether eGFP will tolerate a given deletion. We additionally find that using protein design to explicitly model deletions results in improved predictions of functional status when combined with other structural predictors. Our work suggests that structure plays fundamental role in constraining deletions at sites in proteins, and further that similar biophysical constraints influence both substitutions and deletions. This study therefore provides a solid foundation for future work to examine how protein structure influences tolerance of more complex indel events, such as insertions or large deletions. PMID:28369116

  4. Method for identification of rigid domains and hinge residues in proteins based on exhaustive enumeration.

    PubMed

    Sim, Jaehyun; Sim, Jun; Park, Eunsung; Lee, Julian

    2015-06-01

    Many proteins undergo large-scale motions where relatively rigid domains move against each other. The identification of rigid domains, as well as the hinge residues important for their relative movements, is important for various applications including flexible docking simulations. In this work, we develop a method for protein rigid domain identification based on an exhaustive enumeration of maximal rigid domains, the rigid domains not fully contained within other domains. The computation is performed by mapping the problem to that of finding maximal cliques in a graph. A minimal set of rigid domains are then selected, which cover most of the protein with minimal overlap. In contrast to the results of existing methods that partition a protein into non-overlapping domains using approximate algorithms, the rigid domains obtained from exact enumeration naturally contain overlapping regions, which correspond to the hinges of the inter-domain bending motion. The performance of the algorithm is demonstrated on several proteins.

  5. Binding of a C-terminal fragment (residues 369 to 435) of vitamin D-binding protein to actin.

    PubMed

    Buch, Stefan; Gremm, Dagmar; Wegner, Albrecht; Mannherz, Hans Georg

    2002-10-01

    The vitamin D-binding protein (DBP) binds to monomeric actin with high affinity. The variation in DBP isoforms is due to genetic polymorphism and varying glycosylation. To obtain a homogeneous preparation, the cDNA for human DBP and truncations thereof were cloned and various systems were applied for heterologous bacterial and yeast expression. The full-length protein and the N- and C-terminal halves of DBP remained insoluble probably because the protein did not fold to its native three-dimensional structure due to formation of accidental intra- and inter-molecular disulfide bonds during expression in bacteria or yeast. This problem was overcome by cloning of a C-terminal fragment comprising residues 369 to 435 that did not contain disulfide bonds and was completely soluble. Binding of the C-terminal fragment to monomeric actin was demonstrated by comigration with actin during native polyacrylamide gel electrophoresis and surface plasmon resonance, however, at considerably lower affinity than full-length DBP. This suggests that in addition to the C-terminal amino acid sequence other parts (amino acid residues or sugar moieties) of DBP participate in actin binding. The C-terminal fragment was found to inhibit denaturation of actin and to decrease the rate of actin polymerisation both at the barbed and at the pointed end in a concentration-dependent manner. According to a quantitative analysis of the polymerisation kinetics, association of actin monomers to nucleate filaments was not prevented by binding of the C-terminal fragment to actin. These data suggest that the sites on the surface of actin that are involved in actin nucleation and elongation are different.

  6. Cellular Retinoic Acid Binding Protein and Breast Cancer

    DTIC Science & Technology

    2006-05-01

    fatty acid probe anilinonaphtalene-8- sulphonic acid (ANS) was measured. ANS readily associates with various FABPs and its fluorescence is highly...DAMD17-03-1-0249 TITLE: Cellular Retinoic Acid Binding Protein and Breast Cancer PRINCIPAL INVESTIGATOR: Leslie J. (Willmert) Donato...DATES COVERED (From - To) 14 Apr 03 – 13 Apr 06 5a. CONTRACT NUMBER Cellular Retinoic Acid Binding Protein and Breast Cancer 5b. GRANT NUMBER

  7. A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

    PubMed Central

    Salmon, Melissa; Thimmappa, Ramesha B.; Minto, Robert E.; Melton, Rachel E.; O’Maille, Paul E.; Hemmings, Andrew M.; Osbourn, Anne

    2016-01-01

    Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

  8. 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

  9. Basic residues within the ebolavirus VP35 protein are required for its viral polymerase cofactor function.

    PubMed

    Prins, Kathleen C; Binning, Jennifer M; Shabman, Reed S; Leung, Daisy W; Amarasinghe, Gaya K; Basler, Christopher F

    2010-10-01

    The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-α/β) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and a separate cluster of basic residues, called the first basic patch, were also identified. Functional analysis of alanine substitution mutants indicates that basic residues outside the central basic patch are not required for dsRNA binding or for IFN inhibition. However, minigenome assays, which assess viral RNA polymerase complex function, identified these other basic residues to be critical for viral RNA synthesis. Of these, a subset located within the first basic patch is important for VP35-nucleoprotein (NP) interaction, as evidenced by the inability of alanine substitution mutants to coimmunoprecipitate with NP. Therefore, first basic patch residues are likely critical for replication complex formation through interactions with NP. Coimmunoprecipitation studies further demonstrate that the VP35 IID is sufficient to interact with NP and that dsRNA can modulate VP35 IID interactions with NP. Other basic residue mutations that disrupt the VP35 polymerase cofactor function do not affect interaction with NP or with the amino terminus of the viral polymerase. Collectively, these results highlight the importance of conserved basic residues from the EBOV VP35 C-terminal IID and validate the VP35 IID as a potential therapeutic target.

  10. Leucine residues in conserved region of 33K protein of bovine adenovirus - 3 are important for binding to major late promoter and activation of late gene expression.

    PubMed

    Kulshreshtha, Vikas; Islam, Azharul; Ayalew, Lisanework E; Tikoo, Suresh K

    2015-09-01

    The L6 region of bovine adenovirus 3 (BAdV-3) encode 33K (spliced) and 22K (unspliced) proteins. Earlier, anti-33K serum detected five major and three minor proteins in BAdV-3 infected cells. Here, we demonstrate that anti-sera raised against L6-22K protein detected two proteins of 42 and 37 kDa in BAdV-3 infected cells and one protein of 42 kDa in transfected cells expressing splice-site variant 22K protein (pC.22K containing substituted splice acceptor/donor sequence). Unlike 22K, 33K stimulated the transcription from the major late promoter (MLP) by binding to the downstream sequence elements (DE). Analysis of the variant proteins demonstrated that amino acids 201-240 of the conserved C-terminus of 33K containing the potential leucine zipper and RS repeat are required for the activation of MLP. Furthermore, amino acid substitution analysis demonstrated that unlike arginine residues of RS repeat, the leucine residues (217, 224, 232 and 240) of the conserved leucine zipper appear required for the binding of 33K to the MLP.

  11. 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.

  12. 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

  13. Microbial Physiology of the Conversion of Residual Oil to Methane: A Protein Prospective

    NASA Astrophysics Data System (ADS)

    Morris, Brandon E. L.; Bastida-Lopez, Felipe; von Bergen, Martin; Richnow, Hans-Hermann; Suflita, Joseph M.

    2010-05-01

    Traditional petroleum recovery techniques are unable to extract the majority of oil in most petroliferous deposits. The recovery of even a fraction of residual hydrocarbon in conventional reserves could represent a substantive energy supply. To this end, the microbial conversion of residual oil to methane has gained increasing relevance in recent years [1,2]. Worldwide demand for methane is expected to increase through 2030 [3], as it is a cleaner-burning alternative to traditional fuels [4]. To investigate the microbial physiology of hydrocarbon-decomposition and ultimate methanogenesis, we initiated a two-pronged approach. First, a model alkane-degrading sulfate-reducing bacterium, Desulfoglaeba alkanexedens, was used to interrogate the predominant metabolic pathway(s) differentially expressed during growth on either n-decane or butyrate. A total of 81 proteins were differentially expressed during bacterial growth on butyrate, while 100 proteins were unique to the alkane-grown condition. Proteins related to alkylsuccinate synthase, or the homologous 1-methyl alkylsuccinate synthase, were identified only in the presence of the hydrocarbon. Secondly, we used a newly developed stable isotope probing technique [5] targeted towards proteins to monitor the flux of carbon through a residual oil-degrading bacterial consortium enriched from a gas-condensate contaminated aquifer [1]. Combined carbon and hydrogen stable isotope fractionation identified acetoclastic methanogenesis as the dominant process in this system. Such findings agree with the previous clone library characterization of the consortium. Furthermore, hydrocarbon activation was determined to be the rate-limiting process during the net conversion of residual oil to methane. References 1. Gieg, L.M., K.E. Duncan, and J.M. Suflita, Bioenegy production via microbial conversion of residual oil to natural gas. Appl Environ Micro, 2008. 74(10): p. 3022-3029. 2. Jones, D.M., et al., Crude-oil biodegradation via

  14. Dynamics of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and emission anisotropy studies of Calcofluor White.

    PubMed

    Albani, J R; Sillen, A; Coddeville, B; Plancke, Y D; Engelborghs, Y

    1999-11-23

    Dynamics studies on Calcofluor White bound to the carbohydrate residues of sialylated and asialylated alpha 1-acid glycoprotein (orosomucoid) have been performed. The interaction between the fluorophore and the protein was found to occur preferentially with the glycan residues with a dependence on their spatial conformation. In the presence of sialylated alpha 1-acid glycoprotein, excitation at the red edge of the absorption spectrum of calcofluor does not lead to a shift in the fluorescence emission maximum (440 nm) of the fluorophore. Thus, the emission of calcofluor occurs from a relaxed state. This is confirmed by anisotropy studies as a function of temperature (Perrin plot). In the presence of asialylated alpha 1-acid glycoprotein, red-edge excitation spectra show an important shift (8 nm) of the fluorescence emission maximum of the probe. This reveals that emission of calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that Calcofluor molecules are bound tightly to the carbohydrate residues, a result confirmed by anisotropy studies.

  15. Prediction algorithm for amino acid types with their secondary structure in proteins (PLATON) using chemical shifts.

    PubMed

    Labudde, D; Leitner, D; Krüger, M; Oschkinat, H

    2003-01-01

    The algorithm PLATON is able to assign sets of chemical shifts derived from a single residue to amino acid types with its secondary structure (amino acid species). A subsequent ranking procedure using optionally two different penalty functions yields predictions for possible amino acid species for the given set of chemical shifts. This was demonstrated in the case of the alpha-spectrin SH3 domain and applied to 9 further protein data sets taken from the BioMagRes database. A database consisting of reference chemical shift patterns (reference CSPs) was generated from assigned chemical shifts of proteins with known 3D-structure. This reference CSP database is used in our approach for extracting distributions of amino acid types with their most likely secondary structure elements (namely alpha-helix, beta-sheet, and coil) for single amino acids by comparison with query CSPs. Results obtained for the 10 investigated proteins indicates that the percentage of correct amino acid species in the first three positions in the ranking list, ranges from 71.4% to 93.2% for the more favorable penalty function. Where only the top result of the ranking list for these 10 proteins is considered, 36.5% to 83.1% of the amino acid species are correctly predicted. The main advantage of our approach, over other methods that rely on average chemical shift values is the ability to increase database content by incorporating newly derived CSPs, and therefore to improve PLATON's performance over time.

  16. Measuring membrane protein bond orientations in nanodiscs via residual dipolar couplings

    PubMed Central

    Bibow, Stefan; Carneiro, Marta G; Sabo, T Michael; Schwiegk, Claudia; Becker, Stefan; Riek, Roland; Lee, Donghan

    2014-01-01

    Membrane proteins are involved in numerous vital biological processes. To understand membrane protein functionality, accurate structural information is required. Usually, structure determination and dynamics of membrane proteins are studied in micelles using either solution state NMR or X-ray crystallography. Even though invaluable information has been obtained by this approach, micelles are known to be far from ideal mimics of biological membranes often causing the loss or decrease of membrane protein activity. Recently, nanodiscs, which are composed of a lipid bilayer surrounded by apolipoproteins, have been introduced as a more physiological alternative than micelles for NMR investigations on membrane proteins. Here, we show that membrane protein bond orientations in nanodiscs can be obtained by measuring residual dipolar couplings (RDCs) with the outer membrane protein OmpX embedded in nanodiscs using Pf1 phage as an alignment medium. The presented collection of membrane protein RDCs in nanodiscs represents an important step toward more comprehensive structural and dynamical NMR-based investigations of membrane proteins in a natural bilayer environment. PMID:24752984

  17. Highly sensitive rapid fluorescence detection of protein residues on surgical instruments

    NASA Astrophysics Data System (ADS)

    Kovalev, Valeri I.; Bartona, James S.; Richardson, Patricia R.; Jones, Anita C.

    2006-07-01

    There is a risk of contamination of surgical instruments by infectious protein residues, in particular, prions which are the agents for Creutzfeldt-Jakob Disease in humans. They are exceptionally resistant to conventional sterilization, therefore it is important to detect their presence as contaminants so that alternative cleaning procedures can be applied. We describe the development of an optimized detection system for fluorescently labelled protein, suitable for in-hospital use. We show that under optimum conditions the technique can detect ~10 attomole/cm2 with a scan speed of ~3-10 cm2/s of the test instrument's surface. A theoretical analysis and experimental measurements will be discussed.

  18. Conformation of dehydropentapeptides containing four achiral amino acid residues – controlling the role of L-valine

    PubMed Central

    Krzciuk-Gula, Joanna; Makowski, Maciej; Latajka, Rafał; Kafarski, Paweł

    2014-01-01

    Summary Structural studies of pentapeptides containing an achiral block, built from two dehydroamino acid residues (ΔZPhe and ΔAla) and two glycines, as well as one chiral L-Val residue were performed using NMR spectroscopy. The key role of the L-Val residue in the generation of the secondary structure of peptides is discussed. The obtained results suggest that the strongest influence on the conformation of peptides arises from a valine residue inserted at the C-terminal position. The most ordered conformation was found for peptide Boc-Gly-ΔAla-Gly-ΔZPhe-Val-OMe (3), which adopts a right-handed helical conformation. PMID:24778717

  19. Bacterial GRAS domain proteins throw new light on gibberellic acid response mechanisms

    PubMed Central

    Zhang, Dapeng; Iyer, Lakshminarayan M.; Aravind, L.

    2012-01-01

    Summary: Gibberellic acids (GAs) are key plant hormones, regulating various aspects of growth and development, which have been at the center of the ‘green revolution’. GRAS family proteins, the primary players in GA signaling pathways, remain poorly understood. Using sequence-profile searches, structural comparisons and phylogenetic analysis, we establish that the GRAS family first emerged in bacteria and belongs to the Rossmann fold methyltransferase superfamily. All bacterial and a subset of plant GRAS proteins are likely to function as small-molecule methylases. The remaining plant versions have lost one or more AdoMet (SAM)-binding residues while preserving their substrate-binding residues. We predict that GRAS proteins might either modify or bind small molecules such as GAs or their derivatives. Contact: aravind@ncbi.nlm.nih.gov Supplementary Information: Supplementary Material for this article is available at Bioinformatics online. PMID:22829623

  20. Amino acid sequence of band-3 protein from rainbow trout erythrocytes derived from cDNA.

    PubMed Central

    Hübner, S; Michel, F; Rudloff, V; Appelhans, H

    1992-01-01

    In this report we present the first complete band-3 cDNA sequence of a poikilothermic lower vertebrate. The primary structure of the anion-exchange protein band 3 (AE1) from rainbow trout erythrocytes was determined by nucleotide sequencing of cDNA clones. The overlapping clones have a total length of 3827 bp with a 5'-terminal untranslated region of 150 bp, a 2754 bp open reading frame and a 3'-untranslated region of 924 bp. Band-3 protein from trout erythrocytes consists of 918 amino acid residues with a calculated molecular mass of 101 827 Da. Comparison of its amino acid sequence revealed a 60-65% identity within the transmembrane spanning sequence of band-3 proteins published so far. An additional insertion of 24 amino acid residues within the membrane-associated domain of trout band-3 protein was identified, which until now was thought to be a general feature only of mammalian band-3-related proteins. PMID:1637296

  1. Identification of essential amino acid residues of an alpha-amylase inhibitor from Phaseolus vulgaris white kidney beans.

    PubMed

    Takahashi, T; Hiramoto, S; Wato, S; Nishimoto, T; Wada, Y; Nagai, K; Yamaguchi, H

    1999-11-01

    Kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors, which are bivalent inhibitors with the subunit stoichiometry of (alphabeta)(2) complex, have been inferred to contain unique arginine, tryptophan, and tyrosine residues essential for the inhibitory activity. To test the validity of this inference, an attempt was made to identify the essential amino acid residues of a white kidney bean (P. vulgaris) alpha-amylase inhibitor (PHA-I) by using the chemical modification technique combined with amino acid sequencing and mass spectrometry. Exhaustive modification of the arginine residues by phenylglyoxal did not lead to a marked loss of activity, suggesting that no arginine residue is directly associated with the inhibitory activity. N-Bromosuccinimide treatment of PHA-I in the presence or absence of a substrate alpha-amylase revealed the involvement of two tryptophan residues in alpha-amylase inhibition, and they were identified as Trp188 of the beta-subunit by amino acid sequencing and mass spectrometry of lysylendopeptidase peptides. Further, two tyrosine residues were preferentially modified either by N-acetylimidazole or by tetranitromethane, resulting in a concomitant loss of most of the PHA-I activity. Amino acid sequencing of the lysylendopeptidase peptides from a tetranitromethane-modified PHA-I identified Tyr186 of the beta-subunit as an essential residue.

  2. An Efficient Docking Algorithm Using Conserved Residue Information to Study Protein-Protein Interactions

    DTIC Science & Technology

    2004-12-01

    employ genetic algorithms . In principle, calculation of the free energy change upon binding of two proteins should allow determination of the... Genetic Algorithm Approach to Protein Docking in CAPRI round 1. Proteins 52: 10-14. Glaser, F., Pupko, T., Paz, I., Bell, R.E., Bechor-Shental, D... genetic algorithm and an empirical binding free energy function. Journal of Computational Chemistry 19: 1639-1662. Palma, P.N., Krippahl, L., Wampler

  3. Modification of cysteine residues by cyclopentenone prostaglandins: interplay with redox regulation of protein function.

    PubMed

    Oeste, Clara L; Pérez-Sala, Dolores

    2014-01-01

    Cyclopentenone prostaglandins (cyPG) are endogenous lipid mediators involved in the resolution of inflammation and the regulation of cell proliferation and cellular redox status. Upon exogenous administration they have shown beneficial effects in models of inflammation and tissue injury, as well as potential antitumoral actions, which have raised a considerable interest in their study for the development of therapeutic tools. Due to their electrophilic nature, the best-known mechanism of action of these mediators is the covalent modification of proteins at cysteine residues through Michael addition. Identification of cyPG targets through proteomic approaches, including MS/MS analysis to pinpoint the modified residues, is proving critical to characterize their mechanisms of action. Among the targets of cyPG are proinflammatory transcription factors, proteins involved in cell defense, such as the regulator of the antioxidant response Keap1 and detoxifying enzymes like GST, and key signaling proteins like Ras proteins. Moreover, cyPG may interact with redox-active small molecules, such as glutathione and hydrogen sulfide. Much has been learned about cyPG in the past few years and this knowledge has also contributed to clarify both pharmacological actions and signaling mechanisms of these and other electrophilic lipids. Given the fact that many cyPG targets are involved in or are targets for redox regulation, there is a complex interplay with redox-induced modifications. Here we address the modification of protein cysteine residues by cyPG elucidated by proteomic studies, paying special attention to the interplay with redox signaling.

  4. RBscore&NBench: a high-level web server for nucleic acid binding residues prediction with a large-scale benchmarking database.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2016-07-08

    RBscore&NBench combines a web server, RBscore and a database, NBench. RBscore predicts RNA-/DNA-binding residues in proteins and visualizes the prediction scores and features on protein structures. The scoring scheme of RBscore directly links feature values to nucleic acid binding probabilities and illustrates the nucleic acid binding energy funnel on the protein surface. To avoid dataset, binding site definition and assessment metric biases, we compared RBscore with 18 web servers and 3 stand-alone programs on 41 datasets, which demonstrated the high and stable accuracy of RBscore. A comprehensive comparison led us to develop a benchmark database named NBench. The web server is available on: http://ahsoka.u-strasbg.fr/rbscorenbench/.

  5. Optimization of thermal-dilute sulfuric acid pretreatment for enhancement of methane production from cassava residues.

    PubMed

    Zhang, Qinghua; Tang, Lei; Zhang, Jianhua; Mao, Zhonggui; Jiang, Li

    2011-02-01

    In this study, the pretreatment of cassava residues by thermal-dilute sulfuric acid (TDSA) hydrolysis was investigated by means of a statistically designed set of experiments. A three-factor central composite design (CCD) was employed to identify the optimum pretreatment condition of cassava residues for methane production. The individual and interactive effects of temperature, H(2)SO(4) concentration and reaction time on increase of methane yield (IMY) were evaluated by applying response surface methodology (RSM). After optimization, the resulting optimum pretreatment condition was 157.84°C, utilizing 2.99% (w/w TS) H(2)SO(4) for 20.15 min, where the maximum methane yield (248 mL/g VS) was 56.96% higher than the control (158 mL/g VS), which was very close to the predict value 56.53%. These results indicate the model obtained through RSM analysis is suit to predict the optimum pretreatment condition and there is great potential of using TDSA pretreatment of cassava residues to enhance methane yield.

  6. Identification of Structural and Catalytic Classes of Highly Conserved Amino Acid Residues in Lysine 2,3-Aminomutase †

    PubMed Central

    Chen, Dawei; Frey, Perry A.; Lepore, Bryan W.; Ringe, Dagmar; Ruzicka, Frank J.

    2008-01-01

    Lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4 catalyzes the interconversion of (S)-lysine and (S)-β-lysine by a radical mechanism involving coenzymatic actions of S-adenosylmethionine (SAM), a [4Fe-4S] cluster, and pyridoxal-5′-phosphate (PLP). The enzyme contains a number of conserved acidic residues and a cysteine and arginine-rich motif, that binds iron and sulfide in the [4Fe–4S] cluster. The results of activity and iron, sulfide, and PLP analysis of variants resulting from site-specific mutations of the conserved acidic residues and the arginine residues in the iron-sulfide binding motif indicate two classes of conserved residues of each type. Mutation of the conserved residues Arg134, Asp293, and Asp330 abolish all enzymatic activity. Based on the x-ray crystal structure, these residues bind the ε-aminium and α-carboxylate groups of (S)-lysine. However, among these residues only Asp293 appears to be important for stabilizing the [4Fe–4S] cluster. Members of a second group of conserved residues appear to stabilize the structure of LAM. Mutations of arginine residues 130, 135, and 136 and acidic residues Glu86, Asp165, Glu236, and Asp172 dramatically decrease iron and sulfide contents in the purified variants. Mutation of Asp96 significantly decreases iron and sulfide content. Variants in Arg130 or Asp172 display no detectable activity, whereas variants in the other positions display low to very low activities. Structural roles are assigned to this latter class of conserved amino acids. In particular, a network of hydrogen bonded interactions of Arg130, Glu86, Arg135 and the main chain carbonyl groups of Cys132 and Leu55 appears to stabilize the [4Fe–4S] cluster. PMID:17042481

  7. Multisite clickable modification of proteins using lipoic acid ligase.

    PubMed

    Plaks, Joseph G; Falatach, Rebecca; Kastantin, Mark; Berberich, Jason A; Kaar, Joel L

    2015-06-17

    Approaches that allow bioorthogonal and, in turn, site-specific chemical modification of proteins present considerable opportunities for modulating protein activity and stability. However, the development of such approaches that enable site-selective modification of proteins at multiple positions, including internal sites within a protein, has remained elusive. To overcome this void, we have developed an enzymatic approach for multisite clickable modification based on the incorporation of azide moieties in proteins using lipoic acid ligase (LplA). The ligation of azide moieties to the model protein, green fluorescent protein (GFP), at the N-terminus and two internal sites using lipoic acid ligase was shown to proceed efficiently with near-complete conversion. Modification of the ligated azide groups with poly(ethylene glycol) (PEG), α-d-mannopyranoside, and palmitic acid resulted in highly homogeneous populations of protein-polymer, protein-sugar, and protein-fatty acid conjugates. The homogeneity of the conjugates was confirmed by mass spectrometry (MALDI-TOF) and SDS-PAGE electrophoresis. In the case of PEG attachment, which involved the use of strain-promoted azide-alkyne click chemistry, the conjugation reaction resulted in highly homogeneous PEG-GFP conjugates in less than 30 min. As further demonstration of the utility of this approach, ligated GFP was also covalently immobilized on alkyne-terminated self-assembled monolayers. These results underscore the potential of this approach for, among other applications, site-specific multipoint protein PEGylation, glycosylation, fatty acid modification, and protein immobilization.

  8. Effects of the number of fatty acid residues on the phase behaviors of decaglycerol fatty acid esters.

    PubMed

    Ai, Sakiko; Ishitobi, Masahiko

    2006-04-15

    The effects of the number of fatty acid residues (n) in decaglycerol fatty acid esters, i.e., decaglycerol laurates (abbreviated to (C11)nG10), on the phase behaviors of three laurate esters, (C11)1.9G10, (C11)2.7G10, and (C11)3.4G10, were investigated. The unreacted decaglycerol remaining in each ester was removed by liquid extraction before use. (C11)1.9G10 formed hexagonal liquid crystals in aqueous solutions, while (C11)2.7G10 and (C11)3.4G10, which are more hydrophobic than (C11)1.9G10, formed lamellar liquid crystals. The cloud point in aqueous solution was measured for mixtures of these three esters. The cloud phenomenon was observed when the weight ratio of hydrophilic groups to the total surfactant (WH/WS) was around 0.6. The cloud point shifted to a markedly higher temperature, even with a slight increase in the WH/WS ratio. The solubilization abilities of (C11)nG10 for the oils m-xylene and (R)-(+)-limonene were also examined. When the WH/WS ratio was between 0.60 and 0.64, (C11)nG10 formed microemulsions and lyotropic liquid crystals in the presence of water and the oils. These self-organized structures were stable, even above 90 degrees C. It is concluded that the phase behavior of (C11)nG10 are insensitive to temperature, but strongly dependent on both the WH/WS ratio and the number of fatty acid residues (n).

  9. pH-sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability

    PubMed Central

    Law, Sean M; Zhang, Bin W; Brooks, Charles L

    2013-01-01

    Tombusviruses, such as Carnation Italian ringspot virus (CIRV), encode a protein homodimer called p19 that is capable of suppressing RNA silencing in their infected hosts by binding to and sequestering short-interfering RNA (siRNA) away from the RNA silencing pathway. P19 binding stability has been shown to be sensitive to changes in pH but the specific amino acid residues involved have remained unclear. Using constant pH molecular dynamics simulations, we have identified key pH-dependent residues that affect CIRV p19–siRNA binding stability at various pH ranges based on calculated changes in the free energy contribution from each titratable residue. At high pH, the deprotonation of Lys60, Lys67, Lys71, and Cys134 has the largest effect on the binding stability. Similarly, deprotonation of several acidic residues (Asp9, Glu12, Asp20, Glu35, and/or Glu41) at low pH results in a decrease in binding stability. At neutral pH, residues Glu17 and His132 provide a small increase in the binding stability and we find that the optimal pH range for siRNA binding is between 7.0 and 10.0. Overall, our findings further inform recent experiments and are in excellent agreement with data on the pH-dependent binding profile. PMID:23450521

  10. pH-sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability.

    PubMed

    Law, Sean M; Zhang, Bin W; Brooks, Charles L

    2013-05-01

    Tombusviruses, such as Carnation Italian ringspot virus (CIRV), encode a protein homodimer called p19 that is capable of suppressing RNA silencing in their infected hosts by binding to and sequestering short-interfering RNA (siRNA) away from the RNA silencing pathway. P19 binding stability has been shown to be sensitive to changes in pH but the specific amino acid residues involved have remained unclear. Using constant pH molecular dynamics simulations, we have identified key pH-dependent residues that affect CIRV p19-siRNA binding stability at various pH ranges based on calculated changes in the free energy contribution from each titratable residue. At high pH, the deprotonation of Lys60, Lys67, Lys71, and Cys134 has the largest effect on the binding stability. Similarly, deprotonation of several acidic residues (Asp9, Glu12, Asp20, Glu35, and/or Glu41) at low pH results in a decrease in binding stability. At neutral pH, residues Glu17 and His132 provide a small increase in the binding stability and we find that the optimal pH range for siRNA binding is between 7.0 and 10.0. Overall, our findings further inform recent experiments and are in excellent agreement with data on the pH-dependent binding profile.

  11. Differences in sialic acid residues among bone alkaline phosphatase isoforms: a physical, biochemical, and immunological characterization.

    PubMed

    Magnusson, P; Farley, J R

    2002-12-01

    High-performance liquid chromatography (HPLC) separates three human bone alkaline phosphatase (BALP) isoforms in serum; two major BALP isoforms, B1 and B2, and a minor fraction, B/I, which is composed on average of 70% bone and 30% intestinal ALP. The current studies were intended to identify an in vitro source of the BALP isoforms for physical, biochemical, and immunological characterizations. The three BALP isoforms were identified in extracts of human osteosarcoma (SaOS-2) cells, by HPLC, after separation by anion-exchange chromatography. All three BALP isoforms were similar with respect to freeze-thaw stability, solubility, heat inactivation, and inhibition by L-phenylalanine, L-homoarginine, and levamisole. The isoforms were also kinetically similar (i.e., maximal velocity and KM at pH 8.8 and pH 10.0). The isoforms differed, however, with respect to sensitivity to precipitation with wheat germ agglutinin (WGA), P < 0.001, but not Concanavalin A. At 3.0 mg/ml, WGA precipitated approximately 25% of B/I but more than 80% of B1 and B2. Molecular weights were estimated by native gradient gel electrophoresis: B/I, 126 kDa; B1, 136 kDa; and B2, 141 kDa. Desialylation with neuraminidase reduced the apparent sizes of B1 and B2 to 127 kDa (i.e., approximately to that of B/I). The total carbohydrate content was calculated to be 18 kDa, 28 kDa, and 33 kDa (i.e., 14%, 21%, and 23%) for the BALP isofonns, B/I, B1, and B2, respectively. The number of sialic acid residues was estimated to be 29 and 45, for each B1 and B2 homodimer, respectively. Apparent discrepancies between these estimates of molecular weight and estimates based on gel filtration chromatography were attributed to nonspecific interactions between carbohydrate residues and the gel filtration beads. All three BALP isoforms showed similar dose-dependent linearity in the commercial Alkphase-B and Tandem-MP Ostase immunoassays, r = 0.944 and r = 0.985, respectively (P < 0.001). In summary, our data indicate that

  12. Role of enthalpy-entropy compensation interactions in determining the conformational propensities of amino acid residues in unfolded peptides.

    PubMed

    Toal, Siobhan E; Verbaro, Daniel J; Schweitzer-Stenner, Reinhard

    2014-02-06

    The driving forces governing the unique and restricted conformational preferences of amino acid residues in the unfolded state are still not well understood. In this study, we experimentally determine the individual thermodynamic components underlying intrinsic conformational propensities of these residues. Thermodynamic analysis of ultraviolet-circular dichroism (UV-CD) and (1)H NMR data for a series of glycine capped amino acid residues (i.e., G-x-G peptides) reveals the existence of a nearly exact enthalpy-entropy compensation for the polyproline II-β strand equilibrium for all investigated residues. The respective ΔHβ, ΔSβ values exhibit a nearly perfect linear relationship with an apparent compensation temperature of 295 ± 2 K. Moreover, we identified iso-equilibrium points for two subsets of residues at 297 and 305 K. Thus, our data suggest that within this temperature regime, which is only slightly below physiological temperatures, the conformational ensembles of amino acid residues in the unfolded state differ solely with respect to their capability to adopt turn-like conformations. Such iso-equilibria are rarely observed, and their existence herein indicates a common physical origin behind conformational preferences, which we are able to assign to side-chain dependent backbone solvation. Conformational effects such as differences between the number of sterically allowed side chain rotamers can contribute to enthalpy and entropy but not to the Gibbs energy associated with conformational preferences. Interestingly, we found that alanine, aspartic acid, and threonine are the only residues which do not share these iso-equilbiria. The enthalpy-entropy compensation discovered as well as the iso-equilbrium and thermodynamics obtained for each amino acid residue provide a new and informative way of identifying the determinants of amino acid propensities in unfolded and disordered states.

  13. Quantitative solid state NMR analysis of residues from acid hydrolysis of loblolly pine wood.

    PubMed

    Sievers, Carsten; Marzialetti, Teresita; Hoskins, Travis J C; Valenzuela Olarte, Mariefel B; Agrawal, Pradeep K; Jones, Christopher W

    2009-10-01

    The composition of solid residues from hydrolysis reactions of loblolly pine wood with dilute mineral acids is analyzed by (13)C Cross Polarization Magic Angle Spinning (CP MAS) NMR spectroscopy. Using this method, the carbohydrate and lignin fractions are quantified in less than 3h as compared to over a day using wet chemical methods. In addition to the quantitative information, (13)C CP MAS NMR spectroscopy provides information on the formation of additional extractives and pseudo lignin from the carbohydrates. Being a non-destructive technique, NMR spectroscopy provides unambiguous evidence of the presence of side reactions and products, which is a clear advantage over the wet chemical analytical methods. Quantitative results from NMR spectroscopy and proximate analysis are compared for the residues from hydrolysis of loblolly pine wood under 13 different conditions; samples were treated either at 150 degrees C or 200 degrees C in the presence of various acids (HCl, H(2)SO(4), H(3)PO(4), HNO(3) and TFA) or water. The lignin content determined by both methods differed on averaged by 2.9 wt% resulting in a standard deviation of 3.5 wt%. It is shown that solid degradation products are formed from saccharide precursors under harsh reaction conditions. These degradation reactions limit the total possible yield of monosaccharides from any subsequent reaction.

  14. Theoretical exploration of the cooperative effect in NMF-NMF-amino acid residue hydrogen bonding system.

    PubMed

    Li, Xichen; Liu, Wenlan; Sun, Kening; Wang, Yan; Tan, Hongwei; Chen, Guangju

    2008-09-28

    This paper presents a theoretical study of the cooperative effect in sixteen linearly-arranged trimer systems consisting of N-methylformamide dimer and an extra amino acid residue. These trimer systems, NMF-NMF-AAR, in short, have been systematically investigated by full optimization at B3LYP/cc-pVTZ level and subsequent electronic energy calculations at PBE1PBE/cc-pVTZ, HF/cc-pVTZ and MP2/cc-pVTZ, respectively. Obvious spatial transformation due to energetic factors has been found in almost all the trimers. Systematic analysis in weak interaction energy components has shown that: (1) in these trimer systems, the bonding structure and the cooperative effect combine to determine the stability of both HB1 and HB2. For HB2, the structure of the constituent amino acid residue also plays a crucial role by interfering with the neighboring moieties; (2) the large contribution of the cooperative effect to the overall hydrogen bonding energy has claimed the importance of cooperativity in our systems; (3) the non-hydrogen bonding weak interaction components are found to be non-negligible in these trimer systems; (4) moreover, the cooperative effect between these non-hydrogen bonding components is always found to be positive. The good performances of PBE1PBE and PM6 have been established by comparisons between these methods.

  15. Accurate determination of residual acrylic acid in superabsorbent polymer of hygiene products by headspace gas chromatography.

    PubMed

    Zhang, Shu-Xin; Chai, Xin-Sheng; Jiang, Ran

    2017-02-17

    This work reports on a method for the determination of residual acrylic acid (AA) in the superabsorbent polymers for hygiene products by headspace analysis. It was based on water extraction for the polymer sample at a room temperature for 50min. Then, the AA in the extractant reacted with bicarbonate solution in a closed headspace sample vial, from which the carbon dioxide generated from the reaction (within 20min at 70°C) was detected by gas chromatography (GC). It was found that there is adsorption partition equilibrium of AA between solid-liquid phases. Therefore, an equation for calculating the total AA content in the original polymers sample was derived based on the above phase equilibrium. The results show that the HS-GC method has good precision (RSD<2.51%) and good accuracy (recoveries from 93 to 105%); the limit of quantification (LOQ) was 373mg/kg. The present method is rapid, accurate, and suitable for determining total residual acrylic acid in a wide variety of applications from processing of superabsorbent polymer to commercial products quality control.

  16. The Dual NOD1/NOD2 Agonism of Muropeptides Containing a Meso-Diaminopimelic Acid Residue

    PubMed Central

    Dagil, Yulia A.; Arbatsky, Nikolai P.; Alkhazova, Biana I.; L’vov, Vyacheslav L.; Mazurov, Dmitriy V.; Pashenkov, Mikhail V.

    2016-01-01

    Muropeptides are fragments of peptidoglycan that trigger innate immune responses by activating nucleotide-binding oligomerization domain (NOD) 1 and NOD2. Muropeptides from Gram-negative bacteria contain a meso-diaminopimelic acid (meso-DAP) residue in either a terminal or a non-terminal position. While the former ones are known to be recognized by NOD1, much less is known about recognition of muropeptides with non-terminal meso-DAP, which are most abundant moieties of Gram-negative peptidoglycans. Here, we developed a novel system to assess biological activity of muropeptides, based on CRISPR/Cas9-mediated knockout (KO) of NOD1 and NOD2 genes in modified HEK293T cells. Using NOD1/NOD2 knockout and overexpression systems, as well as human monocytes and macrophages, we refine the current view of muropeptide recognition. We show that NOD2 can recognize different natural muropeptides containing a meso-DAP residue (preferably in a non-terminal position), provided they are present at micromolar concentrations. NOD2 accepts muropeptides with long and branched peptide chains and requires an intact N-acetylmuramyl residue. Muropeptides with non-terminal meso-DAP can activate NOD1 as well, but, in this case, probably require peptidase pre-processing to expose the meso-DAP residue. Depending on NOD1/NOD2 ratio in specific cell types, meso-DAP-containing muropeptides can be recognized either primarily via NOD2 (in monocytes) or via NOD1 (in monocyte-derived macrophages and HEK293T-derived cells). The dual NOD1/NOD2 agonism of meso-DAP-containing muropeptides should be taken into account when assessing cellular responses to muropeptides and designing muropeptide immunostimulants and vaccine adjuvants. PMID:27513337

  17. Amino acid residues 196–225 of LcrV represent a plague protective epitope

    PubMed Central

    Quenee, Lauriane E.; Berube, Bryan J.; Segal, Joshua; Elli, Derek; Ciletti, Nancy A.; Anderson, Deborah; Schneewind, Olaf

    2010-01-01

    LcrV, a protein that resides at the tip of the type III secretion needles of Yersinia pestis, is the single most important plague protective antigen. Earlier work reported monoclonal antibody MAb 7.3, which binds a conformational epitope of LcrV and protects experimental animals against lethal plague challenge. By screening monoclonal antibodies directed against LcrV for their ability to protect immunized mice against bubonic plague challenge, we examined here the possibility of additional protective epitopes. MAb BA5 protected animals against plague, neutralized the Y. pestis type III secretion pathway and promoted opsonophagocytic clearance of bacteria in blood. LcrV residues 196–225 were necessary and sufficient for MAb-BA5 binding. Compared to full length LcrV, a variant lacking its residues 196–225 retained the ability of eliciting plague protection. These results identify LcrV residues 196–225 as a linear epitope that is recognized by the murine immune system to confer plague protection. PMID:20005318

  18. Benchmarking Deep Networks for Predicting Residue-Specific Quality of Individual Protein Models in CASP11.

    PubMed

    Liu, Tong; Wang, Yiheng; Eickholt, Jesse; Wang, Zheng

    2016-01-14

    Quality assessment of a protein model is to predict the absolute or relative quality of a protein model using computational methods before the native structure is available. Single-model methods only need one model as input and can predict the absolute residue-specific quality of an individual model. Here, we have developed four novel single-model methods (Wang_deep_1, Wang_deep_2, Wang_deep_3, and Wang_SVM) based on stacked denoising autoencoders (SdAs) and support vector machines (SVMs). We evaluated these four methods along with six other methods participating in CASP11 at the global and local levels using Pearson's correlation coefficients and ROC analysis. As for residue-specific quality assessment, our four methods achieved better performance than most of the six other CASP11 methods in distinguishing the reliably modeled residues from the unreliable measured by ROC analysis; and our SdA-based method Wang_deep_1 has achieved the highest accuracy, 0.77, compared to SVM-based methods and our ensemble of an SVM and SdAs. However, we found that Wang_deep_2 and Wang_deep_3, both based on an ensemble of multiple SdAs and an SVM, performed slightly better than Wang_deep_1 in terms of ROC analysis, indicating that integrating an SVM with deep networks works well in terms of certain measurements.

  19. Crystal structure of the tumor-promoter okadaic acid bound to protein phosphatase-1.

    PubMed

    Maynes, J T; Bateman, K S; Cherney, M M; Das, A K; Luu, H A; Holmes, C F; James, M N

    2001-11-23

    Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 A. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive beta12-beta13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and -2A but not PP-2B (calcineurin) may be reassessed in light of this study.

  20. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  1. Kinetics of sulfuric acid leaching of cadmium from Cd-Ni zinc plant residues.

    PubMed

    Safarzadeh, Mohammad Sadegh; Moradkhani, Davood; Ojaghi-Ilkhchi, Mehdi

    2009-04-30

    Cd-Ni filtercakes are produced continuously at the third purification step in the electrolytic production of zinc in the National Iranian Lead and Zinc Company (NILZ) in northwestern Iran. In this research, the dissolution kinetics of cadmium from Cd-Ni residues produced in NILZ plant has been investigated. Hence, the effects of temperature, sulfuric acid concentration, particle size and stirring speed on the kinetics of cadmium dissolution in sulfuric acid were studied. The dissolution kinetics at 25-55 degrees C and tacid concentration, solid/liquid ratio and particle size were also achieved. The rate of reaction at first 5 min based on diffusion-controlled process can be expressed by a semi-empirical equation as:It was determined that the dissolution rate increased with increasing sulfuric acid concentration and decreasing particle size.

  2. Identification of acid-base catalytic residues of high-Mr thioredoxin reductase from Plasmodium falciparum.

    PubMed

    McMillan, Paul J; Arscott, L David; Ballou, David P; Becker, Katja; Williams, Charles H; Müller, Sylke

    2006-11-03

    High-M(r) thioredoxin reductase from the malaria parasite Plasmodium falciparum (PfTrxR) contains three redox active centers (FAD, Cys-88/Cys-93, and Cys-535/Cys-540) that are in redox communication. The catalytic mechanism of PfTrxR, which involves dithiol-disulfide interchanges requiring acid-base catalysis, was studied by steady-state kinetics, spectral analyses of anaerobic static titrations, and rapid kinetics analysis of wild-type enzyme and variants involving the His-509-Glu-514 dyad as the presumed acid-base catalyst. The dyad is conserved in all members of the enzyme family. Substitution of His-509 with glutamine and Glu-514 with alanine led to TrxR with only 0.5 and 7% of wild type activity, respectively, thus demonstrating the crucial roles of these residues for enzymatic activity. The H509Q variant had rate constants in both the reductive and oxidative half-reactions that were dramatically less than those of wild-type enzyme, and no thiolateflavin charge-transfer complex was observed. Glu-514 was shown to be involved in dithiol-disulfide interchange between the Cys-88/Cys-93 and Cys-535/Cys-540 pairs. In addition, Glu-514 appears to greatly enhance the role of His-509 in acid-base catalysis. It can be concluded that the His-509-Glu-514 dyad, in analogy to those in related oxidoreductases, acts as the acid-base catalyst in PfTrxR.

  3. Acid-catalyzed hydrothermal severity on the fractionation of agricultural residues for xylose-rich hydrolyzates.

    PubMed

    Lee, Ji Ye; Ryu, Hyun Jin; Oh, Kyeong Keun

    2013-03-01

    The objective of this work was to investigate the feasibility of acid-catalyzed hydrothermal fractionation for maximum solubilization of the hemicellulosic portion of three agricultural residues. The fractionation conditions converted into combined severity factor (CS) in the range of 1.2-2.9. The highest hemicellulose yield of 87.88% was achieved when barley straw was fractionated at a CS of 2.19. However, the maximum glucose release of 15.29% was achieved for the case of rice straw. The maximum productions of various by-products were observed with the fractionation of rape straw: 0.88 g/L of 5-hydroxymethylfurfural (5-HMF), 2.16 g/L of furfural, 0.44 g/L of levulinic acid, 1.59 g/L of formic acid, and 3.06 g/L of acetic acid. The highest selectivities, a criterion for evaluating the fractionation of 21.55 for fractionated solid and 7.48 for liquid hydrolyzate were obtained from barley straw.

  4. Protein and sulfur amino acid requirements of broiler breeder hens.

    PubMed

    Harms, R H; Wilson, H R

    1980-02-01

    Two experiments were conducted with Cobb color-sexed broiler breeder hens to determine their protein and sulfur amino acid requirement. A daily intake between 400 and 478 mg of methionine and between 722 and 839 mg of total sulfur amino acids was necessary for maximum egg production, the latter in a diet of 13.07% protein. Slightly lower levels supported maximum body weights. Hens laying at the highest rate consumed 23.4 g of protein per day.

  5. Los Alamos sequence analysis package for nucleic acids and proteins.

    PubMed Central

    Kanehisa, M I

    1982-01-01

    An interactive system for computer analysis of nucleic acid and protein sequences has been developed for the Los Alamos DNA Sequence Database. It provides a convenient way to search or verify various sequence features, e.g., restriction enzyme sites, protein coding frames, and properties of coded proteins. Further, the comprehensive analysis package on a large-scale database can be used for comparative studies on sequence and structural homologies in order to find unnoted information stored in nucleic acid sequences. PMID:6174934

  6. Elicitin-membrane interaction is driven by a positive charge on the protein surface: role of Lys13 residue in lipids loading and resistance induction.

    PubMed

    Plešková, Veronika; Kašparovský, Tomáš; Obořil, Michal; Ptáčková, Nikola; Chaloupková, Radka; Ladislav, Dokládal; Damborský, Jiří; Lochman, Jan

    2011-03-01

    Elicitins are family of small proteins secreted by species of the pathogenic fungus Phytophthora inducing a defence reaction in plants. They contain a hydrophobic cavity capable of binding sterols and fatty acids, and on the basis of their pI they are classified as either α-elicitins or more necrotising β-elicitins. The residue Lys13 was previously identified as a key determinant of the necrotising activity of basic elicitins. In the present study we describe changes in the ability of cryptogein, a β-elicitin inducing a hypersensitive response in tobacco, to transfer sterols and fatty acids between micelles and liposomes upon Lys13Val mutation. We propose that the change in activity is influenced by the elimination of positive charge on the surface of cryptogein, which is significant for correct positioning of the protein during lipid loading, without adversely affecting the binding of sterol to the cavity of the protein. Compared to wild type cryptogein, mutation Lys13Val resulted in lowered expression of defence-related genes and compromised resistance to Phytophthora parasitica. Furthermore, resistance induced by Lys13Val mutant was similar to that induced by acidic elicitin capsicein containing at amino position 13 valine Determined results sustained a crucial role of positive lysine residues on the surface of basic elicitins and suggested their significant role in correct protein-membrane interaction and thus on their biological activity.

  7. Osmium isotope anomalies in chondrites: Results for acid residues and related leachates

    NASA Astrophysics Data System (ADS)

    Yokoyama, Tetsuya; O'D. Alexander, Conel M.; Walker, Richard J.

    2010-03-01

    We have investigated Os isotope anomalies in acid residues enriched in insoluble organic matter (IOM) extracted from ten primitive chondrites, acid leachates and residues of these fractions, as well as acid leachates of bulk chondrites. Osmium isotopic compositions of bulk carbonaceous, ordinary and enstatite chondrites are also reported. Consistent with prior results, bulk chondrites have homogeneous Os isotope compositions for s-, r-, and p-process nuclides that are indistinguishable from terrestrial, at the current level of resolution. In contrast, nearly all the IOM-rich residues are enriched in s-process Os, evidently due to the preferential incorporation of s-process enriched presolar grains (most likely presolar SiC). Presolar silicate grains that formed in red giant branch (RGB) or asymptotic giant branch (AGB) stars are also likely hosts of additional s-process Os in chondrites. Consistent with one prior study, Os released by weak acid leaching of bulk chondrites is slightly to strongly enriched in r-process nuclides, of which the carrier may be fine-grained presolar silicates formed in supernovae or unidentified solar phases. Collectively, the different, chemically concentrated components in these meteorites are variably enriched in s-, r-, and possibly p-process Os, of which the individual carriers must have been produced in multiple stellar environments. The lack of evidence for Os isotopic heterogeneity among bulk chondrites contrasts with evidence for isotopic heterogeneities for various other elements at approximately the same levels of resolution (e.g., Cr, Mo, Ru, Ba, Sm, and Nd). One possible explanation for this is that the heterogeneities for some elements in bulk materials reflect selective removal of some types of presolar grains as a result of nebular processes, and that because of the strong chemical differences between Os and the other elements, the Os was not significantly affected. Another possible explanation is that late-stage injection

  8. Non-random distribution of amino acids in the transmembrane segments of human type I single span membrane proteins.

    PubMed

    Landolt-Marticorena, C; Williams, K A; Deber, C M; Reithmeier, R A

    1993-02-05

    The distribution of amino acids in the transmembrane segments and flanking regions of 115 human type I single span (amino terminus extracellular and carboxyl terminus cytosolic) plasma membrane proteins was found to be non-random. In this sample, Ile was preferentially localized to the amino-terminal region of the hydrophobic transmembrane segments, followed by Val, while Leu predominated in the carboxyl-terminal half of the segment. Although Gly residues were preferentially located in the transmembrane segment, this residue was excluded from the carboxyl-terminal and adjacent boundary regions. Aromatic residues (Tyr, Trp and Phe) occurred preferentially at the cytoplasmic boundary, with Trp also favored at the extracellular boundary. The extracellular flanking sequence amino-terminal to the transmembrane segment was enriched in residues predicted to initiate helix formation (Pro, Asn and Ser), while Arg and Lys were enriched in the cytoplasmic flank where they may function as topological determinants. The positional preferences of these particular amino acids within the transmembrane segment and flanking regions suggests that, in addition to lipid-protein interactions, these residues may participate in specific protein-protein interactions. A consensus sequence motif for type I membrane proteins is proposed and its role in the biosynthesis, folding, assembly and function of these segments is discussed.

  9. Identification of residues in beta-lactamase critical for binding beta-lactamase inhibitory protein.

    PubMed

    Rudgers, G W; Palzkill, T

    1999-03-12

    beta-Lactamase inhibitory protein (BLIP) is a potent inhibitor of several beta-lactamases including TEM-1 beta-lactamase (Ki = 0.1 nM). The co-crystal structure of TEM-1 beta-lactamase and BLIP has been solved, revealing the contact residues involved in the interface between the enzyme and inhibitor. To determine which residues in TEM-1 beta-lactamase are critical for binding BLIP, the method of monovalent phage display was employed. Random mutants of TEM-1 beta-lactamase in the 99-114 loop-helix and 235-240 B3 beta-strand regions were displayed as fusion proteins on the surface of the M13 bacteriophage. Functional mutants were selected based on the ability to bind BLIP. After three rounds of enrichment, the sequences of a collection of functional beta-lactamase mutants revealed a consensus sequence for the binding of BLIP. Seven loop-helix residues including Asp-101, Leu-102, Val-103, Ser-106, Pro-107, Thr-109, and His-112 and three B3 beta-strand residues including Ser-235, Gly-236, and Gly-238 were found to be critical for tight binding of BLIP. In addition, the selected beta-lactamase mutants A113L/T114R and E240K were found to increase binding of BLIP by over 6- and 11-fold, respectively. Combining these substitutions resulted in 550-fold tighter binding between the enzyme and BLIP with a Ki of 0.40 pM. These results reveal that the binding between TEM-1 beta-lactamase and BLIP can be improved and that there are a large number of sequences consistent with tight binding between BLIP and beta-lactamase.

  10. Role of positively charged residues of the second transmembrane domain in the ion transport activity and conformation of human uncoupling protein-2.

    PubMed

    Hoang, Tuan; Matovic, Tijana; Parker, James; Smith, Matthew D; Jelokhani-Niaraki, Masoud

    2015-04-14

    Residing at the inner mitochondrial membrane, uncoupling protein-2 (UCP2) mediates proton transport from the intermembrane space (IMS) to the mitochondrial matrix and consequently reduces the rate of ATP synthesis in the mitochondria. The ubiquitous expression of UCP2 in humans can be attributed to the protein's multiple physiological roles in tissues, including its involvement in protective mechanisms against oxidative stress, as well as glucose and lipid metabolisms. Currently, the structural properties and ion transport mechanism of UCP2 and other UCP homologues remain poorly understood. UCP2-mediated proton transport is activated by fatty acids and inhibited by di- and triphosphate purine nucleotides. UCP2 also transports chloride and some other small anions. Identification of key amino acid residues of UCP2 in its ion transport pathway can shed light on the protein's ion transport function. On the basis of our previous studies, the second transmembrane helix segment (TM2) of UCP2 exhibited chloride channel activity. In addition, it was suggested that the positively charged residues on TM2 domains of UCPs 1 and 2 were important for their chloride transport activity. On this basis, to further understand the role of these positively charged residues on the ion transport activity of UCP2, we recombinantly expressed four TM2 mutants: R76Q, R88Q, R96Q, and K104Q. The wild type UCP2 and its mutants were purified and reconstituted into liposomes, and their conformation and ion (proton and chloride) transport activity were studied. TM2 Arg residues at the matrix interface of UCP2 proved to be crucial for the protein's anion transport function, and their absence resulted in highly diminished Cl(-) transport rates. On the other hand, the two other positively charged residues of TM2, located at the UCP2-IMS interface, could participate in the salt-bridge formation in the protein and promote the interhelical tight packing in the UCP2. Absence of these residues did not

  11. The use of unnatural amino acids to study and engineer protein function.

    PubMed

    Neumann-Staubitz, Petra; Neumann, Heinz

    2016-06-01

    The expansion of the genetic code for the incorporation of unnatural amino acids (UAAs) in proteins of bacteria, yeasts, mammalian cells or whole animals provides molecular and structural biologists with an amazing kit of novel tools. UAAs can be used to investigate the structure and dynamics of proteins, to study their interactions or to control their activity in living cells. Incorporation of UAAs with bioorthogonal reactivity facilitates the site-specific installation of labels for spectroscopy and microscopy. Light-activatable crosslinker UAAs can be used to trap interacting molecules in living cells with a precision almost at the structural level. Post-translational modifications such as lysine acetylation and serine phosphorylation can be directly encoded to analyse their impact on protein function, and caging groups can be installed on critical residues to create light-activatable proteins. In this review we highlight recent applications of this technology to investigate protein function.

  12. Kainic acid inhibits protein amino acid incorporation in select rat brain regions.

    PubMed

    Planas, A M; Soriano, M A; Ferrer, I; Rodríguez-Farré, E

    1994-11-21

    Regional incorporation of labelled methionine into proteins was studied with quantitative autoradiography in different regions of the rat brain 2.5 h following systemic kainic acid administration. Labelled protein concentration was found reduced to approximately 40% of control values in the pyramidal cell layer of hippocampus, piriform, entorhinal and perirhinal cortices, ventral lateral septum and mediodorsal thalamic nucleus. These regions showed increased levels of label not incorporated into proteins, indicating that free labelled methionine was available for protein synthesis. Reduction of protein amino acid incorporation in those brain regions selectively affected by kainic acid may be involved in subsequent tissue damage.

  13. Defining membrane spanning domains and crucial membrane-localized acidic amino acid residues for K⁺ transport of a Kup/HAK/KT-type Escherichia coli potassium transporter.

    PubMed

    Sato, Yoko; Nanatani, Kei; Hamamoto, Shin; Shimizu, Makoto; Takahashi, Miho; Tabuchi-Kobayashi, Mayumi; Mizutani, Akifumi; Schroeder, Julian I; Souma, Satoshi; Uozumi, Nobuyuki

    2014-05-01

    Potassium (K(+))-uptake transport proteins present in prokaryote and eukaryote cells are categorized into two classes; Trk/Ktr/HKT, K(+) channel, and Kdp belong to the same superfamily, whereas the remaining K(+)-uptake family, Kup/HAK/KT has no homology to the others, and neither its membrane topology nor crucial residues for K(+) uptake have been identified. We examined the topology of Kup from Escherichia coli. Results from the reporter fusion and cysteine labeling assays support a model with 12 membrane-spanning domains. A model for proton-coupled K(+) uptake mediated by Kup has been proposed. However, this study did not show any stimulation of Kup activity at low pH and any evidence of involvement of the three His in Kup-mediated K(+) uptake. Moreover, replacement of all four cysteines of Kup with serine did not abolish K(+) transport activity. To gain insight on crucial residues of Kup-mediated K(+) uptake activity, we focused on acidic residues in the predicted external and transmembrane regions, and identified four residues in the membrane regions required for K(+) uptake activity. This is different from no membrane-localized acidic residues essential for Trk/Ktr/HKTs, K(+) channels and Kdp. Taken together, these results demonstrate that Kup belongs to a distinct type of K(+) transport system.

  14. 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

  15. Anion-π interactions in complexes of proteins and halogen-containing amino acids.

    PubMed

    Borozan, Sunčica Z; Zlatović, Mario V; Stojanović, Srđan Đ

    2016-06-01

    We analyzed the potential influence of anion-π interactions on the stability of complexes of proteins and halogen-containing non-natural amino acids. Anion-π interactions are distance and orientation dependent and our ab initio calculations showed that their energy can be lower than -8 kcal mol(-1), while most of their interaction energies lie in the range from -1 to -4 kcal mol(-1). About 20 % of these interactions were found to be repulsive. We have observed that Tyr has the highest occurrence among the aromatic residues involved in anion-π interactions, while His made the least contribution. Furthermore, our study showed that 67 % of total interactions in the dataset are multiple anion-π interactions. Most of the amino acid residues involved in anion-π interactions tend to be buried in the solvent-excluded environment. The majority of the anion-π interacting residues are located in regions with helical secondary structure. Analysis of stabilization centers for these complexes showed that all of the six residues capable of anion-π interactions are important in locating one or more of such centers. We found that anion-π interacting residues are sometimes involved in simultaneous interactions with halogens as well. With all that in mind, we can conclude that the anion-π interactions can show significant influence on molecular organization and on the structural stability of the complexes of proteins and halogen-containing non-natural amino acids. Their influence should not be neglected in supramolecular chemistry and crystal engineering fields as well.

  16. Design of a Protein-Targeting System for Lactic Acid Bacteria

    PubMed Central

    Dieye, Y.; Usai, S.; Clier, F.; Gruss, A.; Piard, J.-C.

    2001-01-01

    We designed an expression and export system that enabled the targeting of a reporter protein (the staphylococcal nuclease Nuc) to specific locations in Lactococcus lactis cells, i.e., cytoplasm, cell wall, or medium. Optimization of protein secretion and of protein cell wall anchoring was performed with L. lactis cells by modifying the signals located at the N and C termini, respectively, of the reporter protein. Efficient translocation of precursor (∼95%) is obtained using the signal peptide from the lactococcal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interactions of Nuc with the cell envelope. Nuc could be covalently anchored to the peptidoglycan by using the cell wall anchor motif of the Streptococcus pyogenes M6 protein. However, the anchoring step proved to not be totally efficient in L. lactis, as considerable amounts of protein remained membrane associated. Our results may suggest that the defect is due to limiting sortase in the cell. The optimized expression and export vectors also allowed secretion and cell wall anchoring of Nuc in food-fermenting and commensal strains of Lactobacillus. In all strains tested, both secreted and cell wall-anchored Nuc was enzymatically active, suggesting proper enzyme folding in the different locations. These results provide the first report of a targeting system in lactic acid bacteria in which the final location of a protein is controlled and biological activity is maintained. PMID:11418555

  17. Microbial production of amino acid-modified spider dragline silk protein with intensively improved mechanical properties.

    PubMed

    Zhang, Haibo; Zhou, Fengli; Jiang, Xinglin; Cao, Mingle; Wang, Shilu; Zou, Huibin; Cao, Yujin; Xian, Mo; Liu, Huizhou

    2016-08-17

    Spider dragline silk is a remarkably strong fiber with impressive mechanical properties, which were thought to result from the specific structures of the underlying proteins and their molecular size. In this study, silk protein 11R26 from the dragline silk protein of Nephila clavipes was used to analyze the potential effects of the special amino acids on the function of 11R26. Three protein derivatives, ZF4, ZF5, and ZF6, were obtained by site-directed mutagenesis, based on the sequence of 11R26, and among these derivatives, serine was replaced with cysteine, isoleucine, and arginine, respectively. After these were expressed and purified, the mechanical performance of the fibers derived from the four proteins was tested. Both hardness and average elastic modulus of ZF4 fiber increased 2.2 times compared with those of 11R26. The number of disulfide bonds in ZF4 protein was 4.67 times that of 11R26, which implied that disulfide bonds outside the poly-Ala region affect the mechanical properties of spider silk more efficiently. The results indicated that the mechanical performances of spider silk proteins with small molecular size can be enhanced by modification of the amino acids residues. Our research not only has shown the feasibility of large-scale production of spider silk proteins but also provides valuable information for protein rational design.

  18. G4IPDB: A database for G-quadruplex structure forming nucleic acid interacting proteins

    PubMed Central

    Mishra, Subodh Kumar; Tawani, Arpita; Mishra, Amit; Kumar, Amit

    2016-01-01

    Nucleic acid G-quadruplex structure (G4) Interacting Proteins DataBase (G4IPDB) is an important database that contains detailed information about proteins interacting with nucleic acids that forms G-quadruplex structures. G4IPDB is the first database that provides comprehensive information about this interaction at a single platform. This database contains more than 200 entries with details of interaction such as interacting protein name and their synonyms, their UniProt-ID, source organism, target name and its sequences, ∆Tm, binding/dissociation constants, protein gene name, protein FASTA sequence, interacting residue in protein, related PDB entries, interaction ID, graphical view, PMID, author’s name and techniques that were used to detect their interactions. G4IPDB also provides an efficient web-based “G-quadruplex predictor tool” that searches putative G-quadruplex forming sequences simultaneously in both sense and anti-sense strands of the query nucleotide sequence and provides the predicted G score. Studying the interaction between proteins and nucleic acids forming G-quadruplex structures could be of therapeutic significance for various diseases including cancer and neurological disease, therefore, having detail information about their interactions on a single platform would be helpful for the discovery and development of novel therapeutics. G4IPDB can be routinely updated (twice in year) and freely available on http://bsbe.iiti.ac.in/bsbe/ipdb/index.php. PMID:27905517

  19. 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

  20. Regulation of translesion DNA synthesis: Posttranslational modification of lysine residues in key proteins.

    PubMed

    McIntyre, Justyna; Woodgate, Roger

    2015-05-01

    Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiqu