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Sample records for glutamine binding protein

  1. Low-Cost Optical Lifetime Assisted Ratiometric Glutamine Sensor Based on Glutamine Binding Protein

    PubMed Central

    Lam, Hung; Kostov, Yordan; Rao, Govind; Tolosa, Leah

    2008-01-01

    Here we report a reagentless fluorescence sensing technique for glutamine in the submicromolar range based on the glutamine binding protein (QBP). The S179C mutant is labeled with the short-lived acrylodan (lifetime<5ns) and the long-lived tris(dibenzoylmethane) mono(5-amino-1,10-phenanthroline)europium(III) (lifetime>300µs) at the –SH and the N-terminal positions, respectively. In the presence of glutamine the fluorescence of acrylodan is quenched, while the fluorescence of europium complex remains constant. In this report we describe an innovative technique, the so called lifetime assisted ratiometric sensing to discriminate the two fluorescence signals using minimal optics and power requirements. This method exploits the large difference between the fluorescence lifetimes of the two fluorophores to isolate the individual fluorescence from each other by alternating the modulation frequency of the excitation light between 300 Hz and 10 kHz. The result is a ratiometric optical method that does not require expensive and highly attenuating band pass filters for each of the dyes, but only one long pass filter for both. Thus, the signal to noise ratio is enhanced, and at the same time, the optical setup is simplified. The end product is a simple sensing device suitable for low-cost applications such as point-of-care diagnostics or in-the-field analysis. PMID:18786501

  2. Dual-emitting biosensors for glucose and glutamine from genertically engineered E. coli binding proteins

    NASA Astrophysics Data System (ADS)

    Tolosa, Leah; Ge, Xudong; Kostov, Yordan; Lakowicz, Joseph R.; Rao, Govind

    2003-07-01

    Glucose is the major source of carbon, and glutamine is the major source of nitrogen in cell culture media. Thus, glucose and glutamine monitoring are important in maintaining optimal conditions in industrial bioprocesses. Here we report reagentless glucose and glutamine sensors using the E. coli glucose binding protein (GBP) and the glutamine binding protein (GlnBP). Both of these proteins are derived from the permease system of the gram-negative bacteria. The Q26C variant of GBP was labeled at the 26-position with anilino-naphthalene sulfonate (ANS), while the S179C variant of GlnBP was labeled at the 179-position with acrylodan. The ANS and acrylodan emissions are quenched in the presence of glucose and glutamine, respectively. The acrylodan-labeled GlnBP was labeled at the N-terminal with ruthenium bis-(2,2"-bipyridyl)-1,10-phenanthroline-9-isothiocyanate. The ruthenium acts as a non-responsive long-lived reference. The apparent binding constant, Kd", of 8.0 μM glucose was obtained from the decrease in intensity of ANS in GBP. The reliability of the method in monitoring glucose during yeast fermentation was determined by comparison with the YSI Biochemistry Analyzer. The apparent binding constant, Kd", of 0.72 μM glutamine was calculated from the ratio of emission intensities of acrylodan and ruthenium (I515/I610) in GlnBP. The presence of the long-lived ruthenium allowed for modulation sensing at lower frequencies (1-10 MHz) approaching an accuracy of +/- 0.02 μM. The conversion of the GBP into a similar ratiometric sensor was described.

  3. Dissecting the Binding between Glutamine Synthetase and Its Two Natively Unfolded Protein Inhibitors.

    PubMed

    Pantoja-Uceda, David; Neira, José L; Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel; Santoro, Jorge

    2016-06-21

    Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue (IF7) and the 149-residue one (IF17). In this work, we studied both IF7 and IF17 by nuclear magnetic resonance (NMR), and we described their binding to GS by using NMR and biolayer interferometry. We assigned the backbone nuclei of all residues of IF7. Analyses of chemical shifts and the (15)N-{(1)H} NOEs at two field strengths suggest that IF7 region Thr3-Arg13 and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The two-dimensional (1)H-(15)N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting first with IF7 were Gln6 and Ser27, belonging to those regions that appeared to be ordered in the isolated protein. We also determined the kon values and koff values for the binding of both IF7 and IF17 to GS, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model ([Formula: see text]), (ii) there is a strong electrostatic component in the determined kon, and (iii) the binding is not diffusion-limited. PMID:27232663

  4. Dissecting the Binding between Glutamine Synthetase and Its Two Natively Unfolded Protein Inhibitors.

    PubMed

    Pantoja-Uceda, David; Neira, José L; Saelices, Lorena; Robles-Rengel, Rocío; Florencio, Francisco J; Muro-Pastor, M Isabel; Santoro, Jorge

    2016-06-21

    Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue (IF7) and the 149-residue one (IF17). In this work, we studied both IF7 and IF17 by nuclear magnetic resonance (NMR), and we described their binding to GS by using NMR and biolayer interferometry. We assigned the backbone nuclei of all residues of IF7. Analyses of chemical shifts and the (15)N-{(1)H} NOEs at two field strengths suggest that IF7 region Thr3-Arg13 and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The two-dimensional (1)H-(15)N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting first with IF7 were Gln6 and Ser27, belonging to those regions that appeared to be ordered in the isolated protein. We also determined the kon values and koff values for the binding of both IF7 and IF17 to GS, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model ([Formula: see text]), (ii) there is a strong electrostatic component in the determined kon, and (iii) the binding is not diffusion-limited.

  5. The Rec protein of HERV-K(HML-2) upregulates androgen receptor activity by binding to the human small glutamine-rich tetratricopeptide repeat protein (hSGT).

    PubMed

    Hanke, Kirsten; Chudak, Claudia; Kurth, Reinhard; Bannert, Norbert

    2013-02-01

    The expression of endogenous retroviruses of the HERV-K(HML-2) family is strongly upregulated in germ cell tumors and several other cancers. Although the accessory Rec protein of HERV-K(HML-2) has been shown to induce carcinoma in situ in transgenic mice, to increase the activity of c-myc and to interact with the androgen receptor (AR), whether or not Rec expression is indeed implicated causally in the initiation or progression of any human malignancies remains unclear. We used the yeast two-hybrid system involving the Rec protein of a recently integrated HERV-K(HML-2) element in an effort to identify potential Rec-related oncogenic mechanisms. This revealed the human small glutamine-rich tetratricopeptide repeat (TPR)-containing protein (hSGT) to be a cellular binding partner. The interaction of Rec with this known negative regulator of the AR was confirmed by coimmunoprecipitation, pull-down assays and colocalization studies. The interaction involves the TPR motif of hSGT and takes place in the cytoplasm and in the nucleoli. Using an AR-responsive promoter and gene we could demonstrate that Rec interference with hSGT resulted in an up to five-fold increase in the activity of AR. Furthermore, in AR positive cells, Rec was shown to act as transactivator by enhancing AR-mediated activation of the HERV-K(HML-2) LTR promoter. This is in line with previous observations of elevated HERV-K(HML-2) expression in steroid-regulated tissues. On the basis of our findings we propose a "vicious cycle" model of Rec-driven hyperactivation of the AR leading to increased cell proliferation, inhibition of apoptosis and eventually to tumor induction or promotion.

  6. Resolution of fluorescence intensity decays of the two tryptophan residues in glutamine-binding protein from Escherichia coli using single tryptophan mutants.

    PubMed Central

    Axelsen, P H; Bajzer, Z; Prendergast, F G; Cottam, P F; Ho, C

    1991-01-01

    Time correlated single photon counting measurements of tryptophan (Trp) fluorescence intensity decay and other spectroscopic studies were performed on glutamine-binding protein (GlnBP) from Escherichia coli. Using site-specifically mutated forms of the protein in which tyrosine (Tyr) and phenylalanine (Phe) substitute for the Trp residues at positions 32 and 220, we have examined whether wild-type (Wtyp) intensity decay components may be assigned to specific Trp residues. Results indicate that: (a) two exponential intensity decay components are recovered from the Wtyp protein (6.16 ns, 0.46 ns); (b) the long decay component arises from Trp-220 and comprises greater than 90% of the total fluorescence emission; (c) the short component arises from Trp-32 and is highly quenched; (d) all four single-Trp mutants exhibit multiexponential intensity decays, yet equimolar mixtures of two single-Trp mutants yield only two decay components which are virtually indistinguishable from the Wtyp protein; (e) the recovery of additional components in protein mixtures is obscured by statistical noise inherent in the technique of photon counting; (f) various spectroscopic measurements suggest that Trp-Trp interactions occur in the Wtyp protein, but the Wtyp intensity decay may be closely approximated by a linear combination of intensity decays from single-Trp mutants; and (g) inferences derived independently from fluorescence and NMR spectroscopy which pertain to the presence of Trp-Trp interactions and the relative solvent exposure of the two Trp residues are in agreement. PMID:1932553

  7. Systematic Underutilization of Glutamine In Thermophile Proteins

    NASA Technical Reports Server (NTRS)

    Liang, Shoudan; Weber, Arthur; Biegel, Bryan A. (Technical Monitor)

    2002-01-01

    Rapid racemization above 100 C of L-amino acids to Domino acids, as well as deamidation, is probably a hazard for high temperature life. For example, the half-life of some asparaginyl peptides can be as short as 10 minutes at 100 C. High temperature organisms could protect themselves by reducing usage of amino acids that are easily racemized/deamidazed, by having a rapid rate of protein turnover which requires energy, or by adapting special cis-peptide conformations. We have searched eight completely sequenced thermophile genomes, and compare them to mesophile genomes, in order to identify underutilized amino acids. To our surprise, asparagine, the most unstable amino acid to deamidation, is used at about the same level in thermophile proteins in comparison to mesophiles whereas it is the second most unstable amino acid, glutamine, that is underutilized in all of eight thermophile species. Glutamines are present at 2% level in a typical thermophile protein, instead of 4% in mesophile. We argue that it is easier to protect asparagines from deamidation by cis-peptide conformations. We discuss statistical as well as structural evidence in support of our conclusions.

  8. Ancestral Protein Reconstruction Yields Insights into Adaptive Evolution of Binding Specificity in Solute-Binding Proteins.

    PubMed

    Clifton, Ben E; Jackson, Colin J

    2016-02-18

    The promiscuous functions of proteins are an important reservoir of functional novelty in protein evolution, but the molecular basis for binding promiscuity remains elusive. We used ancestral protein reconstruction to experimentally characterize evolutionary intermediates in the functional expansion of the polar amino acid-binding protein family, which has evolved to bind a variety of amino acids with high affinity and specificity. High-resolution crystal structures of an ancestral arginine-binding protein in complex with l-arginine and l-glutamine show that the promiscuous binding of l-glutamine is enabled by multi-scale conformational plasticity, water-mediated interactions, and selection of an alternative conformational substate productive for l-glutamine binding. Evolution of specialized glutamine-binding proteins from this ancestral protein was achieved by displacement of water molecules from the protein-ligand interface, reducing the entropic penalty associated with the promiscuous interaction. These results provide a structural and thermodynamic basis for the co-option of a promiscuous interaction in the evolution of binding specificity.

  9. Glutamine supplementation.

    PubMed

    Wernerman, Jan

    2011-07-18

    Intravenous glutamine supplementation is standard care when parenteral nutrition is given for critical illness. There are data of a reduced mortality when glutamine supplementation is given. In addition, standard commercial products for parenteral nutrition do not contain any glutamine due to glutamine instability in aqueous solutions. For the majority of critical ill patients who are fed enterally, the available evidence is insufficient to recommend glutamine supplementation. Standard formulation of enteral nutrition contains some glutamine: 2-4 g/L. However, this dose is insufficient to normalize glutamine plasma concentration.Plasma concentration of glutamine is low in many patients with critical illness and a low level is an independent risk factor for mortality. A low plasma glutamine concentration is the best indicator of glutamine depletion. Data are emerging about how the endogenous production of glutamine is regulated. We know that skeletal muscle is the major producer of glutamine and that a part of the profound depletion of skeletal muscle seen in critical illness is a reflection of the need to produce glutamine.Glutamine is utilized in rapidly dividing cells in the splanchnic area. Quantitatively most glutamine is oxidized, but the availability of glutamine in surplus is important for the de novo synthesis of nucleotides and necessary for cell division and protein synthesis. More knowledge about the regulation of the endogenous production of glutamine is needed to outline better guidelines for glutamine supplementation in the future.

  10. Targeting glutamine metabolism in multiple myeloma enhances BIM binding to BCL-2 eliciting synthetic lethality to venetoclax

    PubMed Central

    Bajpai, R; Matulis, SM; Wei, C; Nooka, AK; Von Hollen, HE; Lonial, S; Boise, LH; Shanmugam, M

    2016-01-01

    Multiple myeloma (MM) is a plasma cell malignancy that is largely incurable due to development of resistance to therapy-elicited cell death. Nutrients are intricately connected to maintenance of cellular viability in part by inhibition of apoptosis. We were interested to determine if examination of metabolic regulation of BCL-2 proteins may provide insight on alternative routes to engage apoptosis. MM cells are reliant on glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. We and others have demonstrated that glucose maintains levels of key resistance-promoting BCL-2 family member, myeloid cell leukemic factor 1 (MCL-1). Cells continuing to survive in the absence of glucose or glutamine were found to maintain expression of MCL-1 but importantly induce pro-apoptotic BIM expression. One potential mechanism for continued survival despite induction of BIM could be due to binding and sequestration of BIM to alternate pro-survival BCL-2 members. Our investigation revealed that cells surviving glutamine withdrawal in particular, enhance expression and binding of BIM to BCL-2, consequently sensitizing these cells to the BH3 mimetic venetoclax. Glutamine deprivation-driven sensitization to venetoclax can be reversed by metabolic supplementation with TCA cycle intermediate α-ketoglutarate. Inhibition of glucose metabolism with the GLUT4 inhibitor ritonavir elicits variable cytotoxicity in MM that is marginally enhanced with venetoclax treatment, however, targeting glutamine metabolism with 6-diazo-5-oxo-l-norleucine uniformly sensitized MM cell lines and relapse/refractory patient samples to venetoclax. Our studies reveal a potent therapeutic strategy of metabolically driven synthetic lethality involving targeting glutamine metabolism for sensitization to venetoclax in MM. PMID:26640142

  11. Targeting glutamine metabolism in multiple myeloma enhances BIM binding to BCL-2 eliciting synthetic lethality to venetoclax.

    PubMed

    Bajpai, R; Matulis, S M; Wei, C; Nooka, A K; Von Hollen, H E; Lonial, S; Boise, L H; Shanmugam, M

    2016-07-28

    Multiple myeloma (MM) is a plasma cell malignancy that is largely incurable due to development of resistance to therapy-elicited cell death. Nutrients are intricately connected to maintenance of cellular viability in part by inhibition of apoptosis. We were interested to determine if examination of metabolic regulation of BCL-2 proteins may provide insight on alternative routes to engage apoptosis. MM cells are reliant on glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. We and others have demonstrated that glucose maintains levels of key resistance-promoting BCL-2 family member, myeloid cell leukemic factor 1 (MCL-1). Cells continuing to survive in the absence of glucose or glutamine were found to maintain expression of MCL-1 but importantly induce pro-apoptotic BIM expression. One potential mechanism for continued survival despite induction of BIM could be due to binding and sequestration of BIM to alternate pro-survival BCL-2 members. Our investigation revealed that cells surviving glutamine withdrawal in particular, enhance expression and binding of BIM to BCL-2, consequently sensitizing these cells to the BH3 mimetic venetoclax. Glutamine deprivation-driven sensitization to venetoclax can be reversed by metabolic supplementation with TCA cycle intermediate α-ketoglutarate. Inhibition of glucose metabolism with the GLUT4 inhibitor ritonavir elicits variable cytotoxicity in MM that is marginally enhanced with venetoclax treatment, however, targeting glutamine metabolism with 6-diazo-5-oxo-l-norleucine uniformly sensitized MM cell lines and relapse/refractory patient samples to venetoclax. Our studies reveal a potent therapeutic strategy of metabolically driven synthetic lethality involving targeting glutamine metabolism for sensitization to venetoclax in MM.

  12. Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates.

    PubMed

    Kusevic, Denis; Kudithipudi, Srikanth; Jeltsch, Albert

    2016-03-18

    Bacterial HEMK2 homologs initially had been proposed to be involved in heme biogenesis or to function as adenine DNA methyltransferase. Later it was shown that this family of enzymes has protein glutamine methyltransferase activity, and they methylate the glutamine residue in the GGQ motif of ribosomal translation termination factors. The murine HEMK2 enzyme methylates Gln(185) of the eukaryotic translation termination factor eRF1. We have employed peptide array libraries to investigate the peptide sequence recognition specificity of murine HEMK2. Our data show that HEMK2 requires a GQX3R motif for methylation activity. In addition, amino acid preferences were observed between the -3 and +7 positions of the peptide substrate (considering the target glutamine as 0), including a preference for Ser, Arg, and Gly at the +1 and a preference for Arg at the +7 position. Based on our specificity profile, we identified several human proteins that contain putative HEMK2 methylation sites and show that HEMK2 methylates 58 novel peptide substrates. After cloning, expression, and purification of the corresponding protein domains, we confirmed methylation for 11 of them at the protein level. Transfected CHD5 (chromodomain helicase DNA-binding protein 5) and NUT (nuclear protein in testis) were also demonstrated to be methylated by HEMK2 in human HEK293 cells. Our data expand the range of proteins potentially subjected to glutamine methylation significantly, but further investigation will be required to understand the function of HEMK2-mediated methylation in proteins other than eRF1. PMID:26797129

  13. Structural and Functional Insights into Small, Glutamine-Rich, Tetratricopeptide Repeat Protein Alpha

    PubMed Central

    Roberts, Joanna D.; Thapaliya, Arjun; Martínez-Lumbreras, Santiago; Krysztofinska, Ewelina M.; Isaacson, Rivka L.

    2015-01-01

    The small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA) is an emerging player in the quality control of secretory and membrane proteins mislocalized to the cytosol, with established roles in tail-anchored (TA) membrane protein biogenesis. SGTA consists of three structural domains with individual functions, an N-terminal dimerization domain that assists protein sorting pathways, a central tetratricopeptide repeat (TPR) domain that mediates interactions with heat-shock proteins, proteasomal, and hormonal receptors, and viral proteins, and a C-terminal glutamine rich region that binds hydrophobic substrates. SGTA has been linked to viral lifecycles and hormone receptor signaling, with implications in the pathogenesis of various disease states. Thus far, a range of biophysical techniques have been employed to characterize SGTA structure in some detail, and to investigate its interactions with binding partners in different biological contexts. A complete description of SGTA structure, together with further investigation into its function as a co-chaperone involved quality control, could provide us with useful insights into its role in maintaining cellular proteostasis, and broaden our understanding of mechanisms underlying associated pathologies. This review describes how some structural features of SGTA have been elucidated, and what this has uncovered about its cellular functions. A brief background on the structure and function of SGTA is given, highlighting its importance to biomedicine and related fields. The current level of knowledge and what remains to be understood about the structure and function of SGTA is summarized, discussing the potential direction of future research. PMID:26734616

  14. Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes.

    PubMed

    Fulks, R M; Stadtman, E R

    1985-12-13

    When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged by the incorporation of [14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incorporation of [14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogenously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe2+ (or Ni2+) together with ATP (or other nucleoside di- and triphosphates. After 40-60 h dialysis Fe3+ together with NADH (but

  15. Free and protein-bound glutamine have identical splanchnic extraction in healthy human volunteers.

    PubMed

    Boza, J J; Dangin, M; Moënnoz, D; Montigon, F; Vuichoud, J; Jarret, A; Pouteau, E; Gremaud, G; Oguey-Araymon, S; Courtois, D; Woupeyi, A; Finot, P A; Ballèvre, O

    2001-07-01

    The objectives of the present study were to determine the splanchnic extraction of glutamine after ingestion of glutamine-rich protein ((15)N-labeled oat proteins) and to compare it with that of free glutamine and to determine de novo glutamine synthesis before and after glutamine consumption. Eight healthy adults were infused intravenously in the postabsorptive state with L-[1-(13)C]glutamine (3 micromol x kg(-1) x h(-1)) and L-[1-(13)C]lysine (1.5 micromol x kg(-1) x h(-1)) for 8 h. Four hours after the beginning of the infusion, subjects consumed (every 20 min) a liquid formula providing either 2.5 g of protein from (15)N-labeled oat proteins or a mixture of free amino acids that mimicked the oat-amino acid profile and contained L-[2,5-(15)N(2)]glutamine and L-[2-(15)N]lysine. Splanchnic extraction of glutamine reached 62.5 +/- 5.0% and 66.7 +/- 3.9% after administration of (15)N-labeled oat proteins and the mixture of free amino acids, respectively. Lysine splanchnic extraction was also not different (40.9 +/- 11.9% and 34.9 +/- 10.6% for (15)N-labeled oat proteins and free amino acids, respectively). The main conclusion of the present study is that glutamine is equally bioavailable when given enterally as a free amino acid and when protein bound. Therefore, and taking into consideration the drawbacks of free glutamine supplementation of ready-to-use formulas for enteral nutrition, protein sources naturally rich in this amino acid are the best option for providing stable glutamine.

  16. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  17. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  18. Controlling the prion propensity of glutamine/asparagine-rich proteins

    PubMed Central

    Paul, Kacy R; Ross, Eric D

    2015-01-01

    ABSTRACT The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve. PMID:26555096

  19. Controlling the prion propensity of glutamine/asparagine-rich proteins.

    PubMed

    Paul, Kacy R; Ross, Eric D

    2015-01-01

    The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve. PMID:26555096

  20. Controlling the prion propensity of glutamine/asparagine-rich proteins.

    PubMed

    Paul, Kacy R; Ross, Eric D

    2015-01-01

    The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve.

  1. Structural Characterization and Comparative Phylogenetic Analysis of Escherichia coli HemK, a Protein (N5)-glutamine Methyltransferase

    PubMed Central

    Yang, Zhe; Shipman, Lance; Zhang, Meng; Anton, Brian P.; Roberts, Richard J.; Cheng, Xiaodong

    2009-01-01

    Protein glutamine methylation at GGQ sites of protein chain release factors plays a pivotal role in the termination of translation. We report here the crystal structure of the Escherichia coli HemK protein (N5)-glutamine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-l-homocysteine (AdoHcy). HemK contains two domains: a putative substrate binding domain at the N terminus consisting of a five helix bundle and a seven-stranded catalytic domain at the C terminus that harbors the binding site for AdoHcy. The two domains are linked by a β-hairpin. Structure-guided sequence analysis of the HemK family revealed 11 invariant residues functioning in methyl-donor binding and catalysis of methyl transfer. The putative substrate-binding domains of HemK from E. coli and Thermotoga maritima are structurally similar, despite the fact that they share very little sequence similarity. When the two proteins are aligned structurally, the helical N-terminal domain is subject to approximately 10° of hinge movement relative to the C-terminal domain. The apparent hinge mobility of the two domains may reflect functional importance during the reaction cycle. Comparative phylogenetic analysis of the hemK gene and its frequent neighbor gene, prfA, which encodes a major substrate, provides evidence for several examples of lateral gene transfer. PMID:15223314

  2. The folate binding proteins.

    PubMed

    Corrocher, R; Olivieri, O; Pacor, M L

    1991-01-01

    Folates are essential molecules for cell life and, not surprisingly, their transport in biological fluids and their transfer to cells are finely regulated. Folate binding proteins play a major role in this regulation. This paper will review our knowledge on these proteins and examine the most recent advances in this field. PMID:1820987

  3. Glutamine supplementation does not improve protein synthesis rate by the jejunal mucosa of the malnourished rat.

    PubMed

    Tannus, Andrea Ferreira S; Darmaun, Dominique; Ribas, Durval F; Oliveira, José Eduardo D; Marchini, Julio Sergio

    2009-08-01

    It has been demonstrated that glutamine, a conditionally essential amino acid, improves nitrogen balance, acts as a stimulant of protein synthesis, and decreases proteolysis in myopathic children. In contrast, other studies have shown no beneficial effect of glutamine supplementation on burn victims or critically ill patients. Nonetheless, we hypothesized that glutamine supplementation would increase the fractional protein synthesis rate (FSR) in the jejunal mucosa of malnourished male Wistar rats. Thus, the objective of the present study was to test the effect of daily oral glutamine supplementation (0.42 g kg(-1) d(-1) for 14 days) on the FSR of the jejunal mucosa of healthy and malnourished rats. A 4-hour kinetic study with l-[1-(13)C]leucine was subsequently performed, and jejunal biopsies were obtained 1.5 cm from the Treitz angle and analyzed. Malnourished rats showed a 25% weight loss and increased urinary nitrogen excretion. Plasma amino acid concentration did not differ between groups. (13)C enrichment in plasma and jejunal cells was higher in the malnourished groups than in the healthy group. The FSR (percent per hour) was similar for the control and experimental groups (P > .05), with a mean range of 22%/h to 27%/h. Oral glutamine supplementation alone did not induce higher protein incorporation by the jejunal mucosa in malnourished rats, regardless of total food intake or the presence or absence of glutamine supplementation.

  4. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  5. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  6. Oral supplementations with free and dipeptide forms of L-glutamine in endotoxemic mice: effects on muscle glutamine-glutathione axis and heat shock proteins.

    PubMed

    Cruzat, Vinicius F; Pantaleão, Lucas C; Donato, José; de Bittencourt, Paulo Ivo Homem; Tirapegui, Julio

    2014-03-01

    Sepsis is a leading cause of death in intensive care units worldwide. Low availability of glutamine contributes to the catabolic state of sepsis. L-Glutamine supplementation has antioxidant properties and modulates the expression of heat shock proteins (HSPs). This study investigated the effects of oral supplementation with L-glutamine plus L-alanine (GLN+ALA), both in the free form and L-alanyl-L-glutamine dipeptide (DIP), on glutamine-glutathione (GSH) axis and HSPs expression in endotoxemic mice. B6.129F2/J mice were subjected to endotoxemia (lipopolysaccharides from Escherichia coli, 5 mg.kg(-1), LPS group) and orally supplemented for 48 h with either L-glutamine (1 g.kg(-1)) plus L-alanine (0.61 g.kg(-1)) (GLN+ALA-LPS group) or 1.49 g.kg(-1) of DIP (DIP-LPS group). Endotoxemia reduced plasma and muscle glutamine concentrations [relative to CTRL group] which were restored in both GLN+ALA-LPS and DIP-LPS groups (P<.05). In supplemented groups were re-established GSH content and intracellular redox status (GSSG/GSH ratio) in circulating erythrocytes and muscle. Thiobarbituric acid reactive substance was 4-fold in LPS treated mice relative to the untreated CTRL group, and plasma TNF-α and IL-1β levels were attenuated by the supplements. Heat shock proteins 27, 70 and 90 (protein and mRNA) were elevated in the LPS group and were returned to basal levels (relative to CTRL group) in both GLN+ALA-LPS and DIP-LPS groups. Supplementations to endotoxemic mice resulted in up-regulation of GSH reductase, GSH peroxidase and glutamate cysteine ligase mRNA expression in muscle. In conclusion, oral supplementations with GLN+ALA or DIP are effective in reversing the conditions of LPS-induced deleterious impact on glutamine-GSH axis in mice under endotoxemia.

  7. Amino acids, glutamine, and protein metabolism in very low birth weight infants.

    PubMed

    Parimi, Prabhu S; Kadrofske, Mark M; Gruca, Lourdes L; Hanson, Richard W; Kalhan, Satish C

    2005-12-01

    Glutamine has been proposed to be conditionally essential for premature infants, and the currently used parenteral nutrient mixtures do not contain glutamine. De novo glutamine synthesis (DGln) is linked to inflow of carbon into and out of the tricarboxylic acid (TCA) cycle. We hypothesized that a higher supply of parenteral amino acids by increasing the influx of amino acid carbon into the TCA cycle will enhance the rate of DGln. Very low birth weight infants were randomized to receive parenteral amino acids either 1.5 g/kg/d for 20 h followed by 3.0 g/kg/d for 5 h (AA1.5) or 3.0 g/kg/d for 20 h followed by 1.5 g/kg/d for 5 h (AA3.0). A third group of babies received amino acids 1.5 g/kg/d for 20 h followed by 3.0 g/kg/d for 20 h (AA-Ext). Glutamine and protein/nitrogen kinetics were examined using [5-(15)N]glutamine, [2H5]phenylalanine, [1-(13)C,15N]leucine, and [15N2]urea tracers. An acute increase in parenteral amino acid infusion for 5 h (AA1.5) resulted in decrease in rate of appearance (Ra) of phenylalanine and urea, but had no effect on glutamine Ra. Infusion of amino acids at 3.0 g/kg/d for 20 h resulted in increase in DGln, leucine transamination, and urea synthesis, but had no effect on Ra phenylalanine (AA-Ext). These data show an acute increase in parenteral amino acid-suppressed proteolysis, however, such an effect was not seen when amino acids were infused for 20 h and resulted in an increase in glutamine synthesis.

  8. Effects in vivo of decreased plasma and intracellular muscle glutamine concentration on whole-body and hindquarter protein kinetics in rats.

    PubMed

    Olde Damink, S W; de Blaauw, I; Deutz, N E; Soeters, P B

    1999-06-01

    Glutamine is considered to be a 'conditionally' essential amino acid. During situations of severe stress like sepsis or after trauma there is a fall in plasma glutamine levels, enhanced glutamine turnover and intracellular muscle glutamine depletion. Under these conditions, decreased intramuscular glutamine concentration correlates with reduced rates of protein synthesis. It has therefore been hypothesized that intracellular muscle glutamine levels have a regulatory role in muscle protein turnover rates. Administration of the glutamine synthetase inhibitor methionine sulphoximine (MSO) was used to decrease glutamine levels in male Wistar rats. Immediately after the MSO treatment (t=0 h), and at t=6 h and t=12 h, rats received intraperitoneal injections (10 ml/100 g body weight) with glutamine (200 mM) to test whether this attenuated the fall in plasma and intracellular muscle glutamine. Control animals received alanine and saline after MSO treatment, while saline was also given to a group of normal rats. At t=18 h rats received a primed constant infusion of L-[2,6-3H]phenylalanine. A three-pool compartment tracer model was used to measure whole-body protein turnover and muscle protein kinetics. Administration of MSO resulted in a 40% decrease in plasma glutamine and a 60% decrease in intracellular muscle glutamine, both of which were successfully attenuated by glutamine infusions. The decreased intracellular muscle glutamine levels had no effect on whole-body protein turnover or muscle protein kinetics. Also, glutamine supplementation did not alter these parameters. Alanine supplementation increased both hindquarter protein synthesis and breakdown but the net balance of phenylalanine remained unchanged. In conclusion, our results show that decreased plasma and muscle glutamine levels have no effect on whole-body protein turnover or muscle protein kinetics. Therefore, it is unlikely that, in vivo, the intracellular muscle concentration of glutamine is a major

  9. Tumor necrosis factor-α suppresses the protein fractional synthesis rate of the small intestine stimulated by glutamine in rats

    PubMed Central

    ZHOU, JIHONG; FAN, SHENGXIAN; CAO, YACHENG; ZHU, MINGFANG; HAN, YONG; CAO, XUEYING; LI, YOUSHENG

    2015-01-01

    The objective of this study was to examine whether and how TNF-α affects glutamine-enhanced protein synthesis and the expression of the amino acid transporter ASCT2 in the small intestine at the mRNA and protein levels. A total of 30 male Sprague-Dawley rats were randomly assigned into three groups, namely the total parenteral nutrition (TPN; control), glutamine-treated (Gln), and glutamine- and tumor necrosis factor-α (TNF-α)-treated (TNF-α) groups. At 30 min prior to examination, all rats were mainlined with [L-15N]leucine. The concentration of TNF-α in plasma and of glutamine in plasma and the small intestine was measured. The fractional synthesis rate (FSR) of protein and the mRNA and protein expression levels of ASCT2 in the small intestine were assessed. The level of TNF-α was highest in the TNF-α group and the glutamine concentration was elevated to a greater extent in the TNF-α group than in the other two groups. However, the FSR of protein in the small intestine was significantly higher in the Gln group compared with that in the TNF-α group. The mRNA and protein expression levels of ASCT2 in the experimental groups were significantly higher that those in the control group, but did not differ significantly between the Gln and TNF-α groups. These results indicate that TNF-α may attenuate glutamine-stimulated protein synthesis in the small intestine in the early stage of sepsis in rats. The mechanism may be that TNF-α inhibits the function of the glutamine transporter in the uptake the glutamine into target cells for protein synthesis. This inhibition may occur at or following protein translation. PMID:25574232

  10. Identification of the reactive cysteinyl residue and ATP binding site in Bacillus cereus glutamine synthetase by chemical modification.

    PubMed

    Nakano, Y; Itoh, M; Tanaka, E; Kimura, K

    1990-02-01

    Bacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2(+)-dependent activity and activated Mn2(+)-dependent activity. FSBA inactivated only Mn2(+)-dependent activity. Mg2+ plus Mn2(+)-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.

  11. The freshwater Amazonian stingray, Potamotrygon motoro, up-regulates glutamine synthetase activity and protein abundance, and accumulates glutamine when exposed to brackish (15 per thousand) water.

    PubMed

    Ip, Y K; Loong, A M; Ching, B; Tham, G H Y; Wong, W P; Chew, S F

    2009-12-01

    This study aimed to examine whether the stenohaline freshwater stingray, Potamotrygon motoro, which lacks a functional ornithine-urea cycle, would up-regulate glutamine synthetase (GS) activity and protein abundance, and accumulate glutamine during a progressive transfer from freshwater to brackish (15 per thousand) water with daily feeding. Our results revealed that, similar to other freshwater teleosts, P. motoro performed hyperosmotic regulation, with very low urea concentrations in plasma and tissues, in freshwater. In 15 per thousand water, it was non-ureotelic and non-ureoosmotic, acting mainly as an osmoconformer with its plasma osmolality, [Na+] and [Cl-] comparable to those of the external medium. There were significant increases in the content of several free amino acids (FAAs), including glutamate, glutamine and glycine, in muscle and liver, but not in plasma, indicating that FAAs could contribute in part to cell volume regulation. Furthermore, exposure of P. motoro to 15 per thousand water led to up-regulation of GS activity and protein abundance in both liver and muscle. Thus, our results indicate for the first time that, despite the inability to synthesize urea and the lack of functional carbamoyl phosphate synthetase III (CPS III) which uses glutamine as a substrate, P. motoro retained the capacity to up-regulate the activity and protein expression of GS in response to salinity stress. Potamotrygon motoro was not nitrogen (N) limited when exposed to 15 per thousand water with feeding, and there were no significant changes in the amination and deamination activities of hepatic glutamate dehydrogenase. In contrast, P. motoro became N limited when exposed to 10 per thousand water with fasting and could not survive well in 15 per thousand water without food.

  12. When is protein binding important?

    PubMed

    Heuberger, Jules; Schmidt, Stephan; Derendorf, Hartmut

    2013-09-01

    The present paper is an ode to a classic citation by Benet and Hoener (2002. Clin Pharm Ther 71(3):115-121). The now classic paper had a huge impact on drug development and the way the issue of protein binding is perceived and interpreted. Although the authors very clearly pointed out the limitations and underlying assumptions for their delineations, these are too often overlooked and the classic paper's message is misinterpreted by broadening to cases that were not intended. Some members of the scientific community concluded from the paper that protein binding is not important. This was clearly not intended by the authors, as they finished their paper with a paragraph entitled: "When is protein binding important?" Misinterpretation of the underlying assumptions in the classic work can result in major pitfalls in drug development. Therefore, we revisit the topic of protein binding with the intention of clarifying when clinically relevant changes should be considered during drug development.

  13. Function of a glutamine synthetase-like protein in bacterial aniline oxidation via γ-glutamylanilide.

    PubMed

    Takeo, Masahiro; Ohara, Akira; Sakae, Shinji; Okamoto, Yasuhiro; Kitamura, Chitoshi; Kato, Dai-ichiro; Negoro, Seiji

    2013-10-01

    Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl2. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.

  14. N-acetyl-L-glutamine, a liquid-stable source of glutamine, partially prevents changes in body weight and on intestinal immunity induced by protein energy malnutrition in pigs.

    PubMed

    López-Pedrosa, José M; Manzano, Manuel; Baxter, Jeffrey H; Rueda, Ricardo

    2007-03-01

    The goal of this study was to evaluate the preventive effect of free glutamine versus N-acetyl-L-glutamine, a liquid-stable source of glutamine, on gut damage induced by protein energy malnutrition in pigs. Healthy pigs (n = 6) were fed a liquid formula for 30 days. Three subgroups of malnourished pigs (n = 6) received daily 20% of the food intake recorded in control group, supplemented with calcium caseinate, glutamine, or N-acetyl-L-glutamine. Body weight was recorded, and small intestinal samples were evaluated for biochemical and immunologic parameters. Suppression in body weight gain was significantly lower in pigs fed with N-acetyl-L-glutamine than in the rest of malnourished pigs. Total number of lymphocytes, CD21+ B cells and CD4+ T cells in ileal Peyer patches were not significantly different in malnourished pigs fed with N-acetyl-L-glutamine and in healthy pigs. In conclusion, N-acetyl-L-glutamine has a moderate protective effect, partially preventing changes induced by protein energy malnutrition.

  15. DNA binding, BSA interaction and SOD activity of two new nickel(II) complexes with glutamine Schiff base ligands.

    PubMed

    Wei, Qiang; Dong, Jianfang; Zhao, Peiran; Li, Manman; Cheng, Fengling; Kong, Jinming; Li, Lianzhi

    2016-08-01

    Two hexacoordinated octahedral nickel(II) complexes, [Ni(o-van-gln)(phen)(H2O)](1) and [Ni(sal-gln)(phen)(H2O)](2) [o-van-gln=a Schiff base derived from o-vanillin and glutamine, sal-gln=a Schiff base derived from salicylaldehyde and glutamine, phen=1,10-phenanthroline], have been synthesized and characterized by elemental analysis, IR spectra and single crystal X-ray diffraction. X-ray studies showed that nickel atoms of both 1 and 2 exhibit distorted NiN3O3 octahedral geometry. In each crystal, intermolecular hydrogen bonds form a two-dimensional network structure. DNA-binding properties of these two nickel(II) complexes were investigated by using UV-Vis absorption, fluorescence, circular dichroism (CD) spectroscopies and viscosity measurements. Results indicated that the two complexes can bind to calf thymus DNA (CT-DNA) via an intercalative mode, and complex 1 exhibits higher interaction with CT-DNA than complex 2. Furthermore, the interactions between the nickel(II) complexes with bovine serum albumin (BSA) have been studied by spectroscopies. The results indicated that both complexes could quench the intrinsic fluorescence of BSA in a static quenching process. The binding constants (Kb) and the numbers of binding sites (n) obtained are 1.10×10(5)M(-1) and 1.05 for complex 1 and 5.05×10(4)M(-1) and 0.997 for complex 2, respectively. Site-selective competitive binding investigation indicated that the binding sites of both the complexes are located in site I of sub-domains IIA of BSA. Assay of superoxide dismutase (SOD) activity of the nickel(II) complexes revealed that they exhibit significant superoxide scavenging activity with IC50=3.4×10(-5)M for complex 1 and 4.3×10(-5)M for complex 2, respectively.

  16. DNA binding, BSA interaction and SOD activity of two new nickel(II) complexes with glutamine Schiff base ligands.

    PubMed

    Wei, Qiang; Dong, Jianfang; Zhao, Peiran; Li, Manman; Cheng, Fengling; Kong, Jinming; Li, Lianzhi

    2016-08-01

    Two hexacoordinated octahedral nickel(II) complexes, [Ni(o-van-gln)(phen)(H2O)](1) and [Ni(sal-gln)(phen)(H2O)](2) [o-van-gln=a Schiff base derived from o-vanillin and glutamine, sal-gln=a Schiff base derived from salicylaldehyde and glutamine, phen=1,10-phenanthroline], have been synthesized and characterized by elemental analysis, IR spectra and single crystal X-ray diffraction. X-ray studies showed that nickel atoms of both 1 and 2 exhibit distorted NiN3O3 octahedral geometry. In each crystal, intermolecular hydrogen bonds form a two-dimensional network structure. DNA-binding properties of these two nickel(II) complexes were investigated by using UV-Vis absorption, fluorescence, circular dichroism (CD) spectroscopies and viscosity measurements. Results indicated that the two complexes can bind to calf thymus DNA (CT-DNA) via an intercalative mode, and complex 1 exhibits higher interaction with CT-DNA than complex 2. Furthermore, the interactions between the nickel(II) complexes with bovine serum albumin (BSA) have been studied by spectroscopies. The results indicated that both complexes could quench the intrinsic fluorescence of BSA in a static quenching process. The binding constants (Kb) and the numbers of binding sites (n) obtained are 1.10×10(5)M(-1) and 1.05 for complex 1 and 5.05×10(4)M(-1) and 0.997 for complex 2, respectively. Site-selective competitive binding investigation indicated that the binding sites of both the complexes are located in site I of sub-domains IIA of BSA. Assay of superoxide dismutase (SOD) activity of the nickel(II) complexes revealed that they exhibit significant superoxide scavenging activity with IC50=3.4×10(-5)M for complex 1 and 4.3×10(-5)M for complex 2, respectively. PMID:27295415

  17. Distribution of glutamine and asparagine residues and their near neighbors in peptides and proteins.

    PubMed Central

    Robinson, A B; Robinson, L R

    1991-01-01

    In a statistical study of neighboring residues in 1465 peptides and proteins comprising 450,431 residues, it was found that the preferences for residues neighboring to glutamine and asparagine residues are consistent with the hypothesis that the rates of deamidation of these residues are of biological significance. Some dipeptide and tripeptide structures have special usefulness and some are especially undesirable. More such structures exist for amide residues than for other residues, and their specific types are those most relevant to the deamidation of amide residues under biological conditions. PMID:1924347

  18. Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box.

    PubMed Central

    Kiledjian, M; Dreyfuss, G

    1992-01-01

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to influence the structure of hnRNA and participate in the processing of hnRNA to mRNA. The hnRNP U protein is an abundant nucleoplasmic phosphoprotein that is the largest of the major hnRNP proteins (120 kDa by SDS-PAGE). HnRNP U binds pre-mRNA in vivo and binds both RNA and ssDNA in vitro. Here we describe the cloning and sequencing of a cDNA encoding the hnRNP U protein, the determination of its amino acid sequence and the delineation of a region in this protein that confers RNA binding. The predicted amino acid sequence of hnRNP U contains 806 amino acids (88,939 Daltons), and shows no extensive homology to any known proteins. The N-terminus is rich in acidic residues and the C-terminus is glycine-rich. In addition, a glutamine-rich stretch, a putative NTP binding site and a putative nuclear localization signal are present. It could not be defined from the sequence what segment of the protein confers its RNA binding activity. We identified an RNA binding activity within the C-terminal glycine-rich 112 amino acids. This region, designated U protein glycine-rich RNA binding region (U-gly), can by itself bind RNA. Furthermore, fusion of U-gly to a heterologous bacterial protein (maltose binding protein) converts this fusion protein into an RNA binding protein. A 26 amino acid peptide within U-gly is necessary for the RNA binding activity of the U protein. Interestingly, this peptide contains a cluster of RGG repeats with characteristic spacing and this motif is found also in several other RNA binding proteins. We have termed this region the RGG box and propose that it is an RNA binding motif and a predictor of RNA binding activity. Images PMID:1628625

  19. Site-selective dual modification of periplasmic binding proteins for sensing applications.

    PubMed

    Crochet, Amanda P; Kabir, Mohiuddin M; Francis, Matthew B; Paavola, Chad D

    2010-09-15

    We have developed three sensitive and specific amino acid sensors based on bacterial periplasmic solute binding proteins. A site-specific amino-terminal transamination reaction provides a useful complement to cysteine chemistry for the covalent modification of biomolecules in this application. We demonstrate this combination to attach two different chromophores to a single biomolecule in two locations. The periplasmic glutamine binding protein from E. coli was modified with a pair of dyes suitable for fluorescence resonance energy transfer, and this conjugate exhibited an l-glutamine dependent optical response. Two periplasmic binding proteins from the thermophilic organism Thermotoga maritima, for arginine and aliphatic amino acids, were modified and evaluated similarly. All three conjugates manifested signal changes mediated by resonant energy transfer upon binding their respective ligands, with nanomolar dissociation constants and stereochemical specificity. This represents a readily generalizable method for construction of reagentless biosensors. The double-labeling strategy was also exploited for the surface attachment of a dye-labeled glutamine binding protein via a biotin-streptavidin interaction.

  20. Effects of exercise on leukocyte death: prevention by hydrolyzed whey protein enriched with glutamine dipeptide.

    PubMed

    Cury-Boaventura, Maria Fernanda; Levada-Pires, Adriana C; Folador, Alessandra; Gorjão, Renata; Alba-Loureiro, Tatiana C; Hirabara, Sandro M; Peres, Fabiano P; Silva, Paulo R S; Curi, Rui; Pithon-Curi, Tania C

    2008-06-01

    Lymphocyte and neutrophil death induced by exercise and the role of hydrolyzed whey protein enriched with glutamine dipeptide (Gln) supplementation was investigated. Nine triathletes performed two exhaustive exercise trials with a 1-week interval in a randomized, double blind, crossover protocol. Thirty minutes before treadmill exhaustive exercise at variable speeds in an inclination of 1% the subjects ingested 50 g of maltodextrin (placebo) or 50 g of maltodextrin plus 4 tablets of 700 mg of hydrolyzed whey protein enriched with 175 mg of glutamine dipeptide dissolved in 250 mL water. Cell viability, DNA fragmentation, mitochondrial transmembrane potential and production of reactive oxygen species (ROS) were determined in lymphocytes and neutrophils. Exhaustive exercise decreased viable lymphocytes but had no effect on neutrophils. A 2.2-fold increase in the proportion of lymphocytes and neutrophils with depolarized mitochondria was observed after exhaustive exercise. Supplementation of maltodextrin plus Gln (MGln) prevented the loss of lymphocyte membrane integrity and the mitochondrial membrane depolarization induced by exercise. Exercise caused an increase in ROS production by neutrophils, whereas supplementation of MGln had no additional effect. MGln supplementation partially prevented lymphocyte apoptosis induced by exhaustive exercise possibly by a protective effect on mitochondrial function. PMID:18320208

  1. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

    PubMed

    Nguyen, T Van; Lee, J Eugene; Sweredoski, Michael J; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J

    2016-03-17

    Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation. PMID:26990986

  2. Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

    PubMed

    Nguyen, T Van; Lee, J Eugene; Sweredoski, Michael J; Yang, Seung-Joo; Jeon, Seung-Je; Harrison, Joseph S; Yim, Jung-Hyuk; Lee, Sang Ghil; Handa, Hiroshi; Kuhlman, Brian; Jeong, Ji-Seon; Reitsma, Justin M; Park, Chul-Seung; Hess, Sonja; Deshaies, Raymond J

    2016-03-17

    Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.

  3. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, O.; Yosef, K.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1998-02-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  4. Cellulose binding domain fusion proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  5. Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase.

    PubMed

    Fiebig, David; Schmelz, Stefan; Zindel, Stephan; Ehret, Vera; Beck, Jan; Ebenig, Aileen; Ehret, Marina; Fröls, Sabrina; Pfeifer, Felicitas; Kolmar, Harald; Fuchsbauer, Hans-Lothar; Scrima, Andrea

    2016-09-23

    Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency. PMID:27493205

  6. Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase.

    PubMed

    Fiebig, David; Schmelz, Stefan; Zindel, Stephan; Ehret, Vera; Beck, Jan; Ebenig, Aileen; Ehret, Marina; Fröls, Sabrina; Pfeifer, Felicitas; Kolmar, Harald; Fuchsbauer, Hans-Lothar; Scrima, Andrea

    2016-09-23

    Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed β-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

  7. Distribution of immunoreactive glutamine synthetase in the adult human and mouse brain. Qualitative and quantitative observations with special emphasis on extra-astroglial protein localization.

    PubMed

    Bernstein, Hans-Gert; Bannier, Jana; Meyer-Lotz, Gabriela; Steiner, Johann; Keilhoff, Gerburg; Dobrowolny, Henrik; Walter, Martin; Bogerts, Bernhard

    2014-11-01

    Glutamine synthetase catalyzes the ATP-dependent condensation of ammonia and glutamate to form glutamine, thus playing a pivotal role in glutamate and glutamine homoeostasis. Despite a plethora of studies on this enzyme, knowledge about the regional and cellular distribution of this enzyme in human brain is still fragmentary. Therefore, we mapped fourteen post-mortem brains of psychically healthy individuals for the distribution of the glutamine synthetase immunoreactive protein. It was found that glutamine synthetase immunoreactivity is expressed in multiple gray and white matter astrocytes, but also in oligodendrocytes, ependymal cells and certain neurons. Since a possible extra-astrocytic expression of glutamine synthetase is highly controversial, we paid special attention to its appearance in oligodendrocytes and neurons. By double immunolabeling of mouse brain slices and cultured mouse brain cells for glutamine synthetase and cell-type-specific markers we provide evidence that besides astrocytes subpopulations of oligodendrocytes, microglial cells and neurons express glutamine synthetase. Moreover, we show that glutamine synthetase-immunopositive neurons are not randomly distributed throughout human and mouse brain, but represent a subpopulation of nitrergic (i.e. neuronal nitric oxide synthase expressing) neurons. Possible functional implications of an extra-astrocytic localization of glutamine synthetase are discussed.

  8. H1TF2A, the large subunit of a heterodimeric, glutamine-rich CCAAT-binding transcription factor involved in histone H1 cell cycle regulation.

    PubMed Central

    Martinelli, R; Heintz, N

    1994-01-01

    H1TF2 is a CCAAT transcription factor that binds to the histone H1 subtype-specific consensus sequence, which has previously been shown to be necessary for temporal regulation of histone H1 transcription during the cell cycle (F. La Bella, P. Gallinari, J. McKinney, and N. Heintz, Genes Dev. 3:1982-1990, 1989). In this study, we report that H1TF2 is a heteromeric CCAAT-binding protein composed of two polypeptide doublets of 33 and 34 kDa and 43 and 44 kDa that are not antigenically related. The 33- and 34-kDa species were not detected in our previous studies (P. Gallinari, F. La Bella, and N. Heintz, Mol. Cell. Biol. 9:1566-1575, 1989) because of technical problems in detection of these heavily glycosylated subunits. The cloning of H1TF2A, the large subunit of this factor, reveals it to be a glutamine-rich protein with extremely limited similarity to previously cloned CCAAT-binding proteins. A monospecific antiserum produced against bacterially synthesized H1TF2A was used to establish that HeLa cell H1TF2A is phosphorylated in vivo and that, in contrast to the H2b transcription factor Oct1 (S. B. Roberts, N. Segil, and N. Heintz, Science 253:1022-1026, 1991; N. Segil, S. B. Roberts, and N. Heintz, Cold Spring Harbor Symp. Quant. Biol. 56:285-292, 1991), no gross change in H1TF2A phosphorylation is evident during the cell cycle. Further immunoprecipitation studies demonstrated that H1TF2 is heterodimeric in the absence of DNA in vivo and identified several H1TF2-interacting proteins that may play a role in H1TF2 function in vivo. Images PMID:7969168

  9. Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy

    PubMed Central

    Swamy, Mahima; Pathak, Shalini; Grzes, Katarzyna M.; Damerow, Sebastian; Sinclair, Linda V.; van Aalten, Daan M. F.; Cantrell, Doreen A.

    2016-01-01

    Sustained glucose and glutamine transport are essential for activated T lymphocytes to support ATP and macromolecule biosynthesis. We now show that glutamine and glucose also fuel an indispensible dynamic regulation of intracellular protein O-GlcNAcylation at key stages of T cell development, transformation and differentiation. Glucose and glutamine are precursors of UDP-GlcNAc, a substrate for cellular glycosyltransferases. Immune activated T cells contained higher concentrations of UDP-GlcNAc and increased intracellular protein O-GlcNAcylation controlled by the enzyme O-GlcNAc glycosyltransferase as compared to naïve cells. We identified Notch, the T cell antigen receptor and c-Myc as key controllers of T cell protein O-GlcNAcylation, via regulation of glucose and glutamine transport. Loss of O-GlcNAc transferase blocked T cell progenitor renewal, malignant transformation, and peripheral T cell clonal expansion. Nutrient-dependent signaling pathways regulated by O-GlcNAc glycosyltransferase are thus fundamental for T cell biology. PMID:27111141

  10. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, G.K.

    1997-04-29

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

  11. Erythropoietin binding protein from mammalian serum

    DOEpatents

    Clemons, Gisela K.

    1997-01-01

    Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

  12. Effects of dietary glutamine supplementation on the body composition and protein status of early-weaned mice inoculated with Mycobacterium bovis Bacillus Calmette-Guerin.

    PubMed

    Rogero, Marcelo Macedo; Borges, Maria Carolina; de Castro, Inar Alves; Pires, Ivanir S O; Borelli, Primavera; Tirapegui, Julio

    2011-09-01

    Glutamine, one of the most abundant amino acids found in maternal milk, favors protein anabolism. Early-weaned babies are deprived of this source of glutamine, in a period during which endogenous biosynthesis may be insufficient for tissue needs in states of metabolic stress, mainly during infections. The objective of this study was to verify the effects of dietary glutamine supplementation on the body composition and visceral protein status of early-weaned mice inoculated with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). Mice were weaned early on their 14th day of life and seperated into two groups, one of which was fed a glutamine-free diet (n = 16) and the other a glutamine-supplemented diet (40 g/kg diet) (n = 16). At 21 days of age, some mice were intraperitoneally injected with BCG. Euthanasia was performed at the 28th day of age. BCG inoculation significantly reduced body weight (P < 0.001), lean mass (P = 0.002), water (P = 0.006), protein (P = 0.007) and lipid content (P = 0.001) in the carcass. Dietary glutamine supplementation resulted in a significant increase in serum IGF-1 (P = 0.019) and albumin (P = 0.025) concentration, muscle protein concentration (P = 0.035) and lipid content (P = 0.002) in the carcass. In conclusion, dietary glutamine supplementation had a positive influence on visceral protein status but did not affect body composition in early-weaned mice inoculated with BCG.

  13. Designing ligands to bind proteins.

    PubMed

    Whitesides, George M; Krishnamurthy, Vijay M

    2005-11-01

    The ability to design drugs (so-called 'rational drug design') has been one of the long-term objectives of chemistry for 50 years. It is an exceptionally difficult problem, and many of its parts lie outside the expertise of chemistry. The much more limited problem - how to design tight-binding ligands (rational ligand design) - would seem to be one that chemistry could solve, but has also proved remarkably recalcitrant. The question is 'Why is it so difficult?' and the answer is 'We still don't entirely know'. This perspective discusses some of the technical issues - potential functions, protein plasticity, enthalpy/entropy compensation, and others - that contribute, and suggests areas where fundamental understanding of protein-ligand interactions falls short of what is needed. It surveys recent technological developments (in particular, isothermal titration calorimetry) that will, hopefully, make now the time for serious progress in this area. It concludes with the calorimetric examination of the association of a series of systematically varied ligands with a model protein. The counterintuitive thermodynamic results observed serve to illustrate that, even in relatively simple systems, understanding protein-ligand association is challenging.

  14. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

    PubMed

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L; Paradiso, Annalisa; Gu, Yong Q; Blanco, Antonio; de Pinto, Maria C; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes.

  15. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content.

    PubMed

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L; Paradiso, Annalisa; Gu, Yong Q; Blanco, Antonio; de Pinto, Maria C; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes. PMID:27468287

  16. Glutamine synthetase in Durum Wheat: Genotypic Variation and Relationship with Grain Protein Content

    PubMed Central

    Nigro, Domenica; Fortunato, Stefania; Giove, Stefania L.; Paradiso, Annalisa; Gu, Yong Q.; Blanco, Antonio; de Pinto, Maria C.; Gadaleta, Agata

    2016-01-01

    Grain protein content (GPC), is one of the most important trait in wheat and its characterized by a very complex genetic control. The identification of wheat varieties with high GPC (HGPC), as well as the characterization of central enzymes involved in these processes, are important for more sustainable agricultural practices. In this study, we focused on Glutamine synthetase (GS) as a candidate to study GPC in wheat. We analyzed GS expression and its enzymatic activity in different tissues and phenological stages in 10 durum wheat genotypes with different GPC. Although each genotype performed quite differently from the others, both because their genetic variability and their adaptability to specific environmental conditions, the highest GS activity and expression were found in genotypes with HGPC and vice versa the lowest ones in genotypes with low GPC (LGPC). Moreover, in genotypes contrasting in GPC bred at different nitrogen regimes (0, 60, 140 N Unit/ha) GS behaved differently in diverse organs. Nitrogen supplement increased GS expression and activity in roots of all genotypes, highlighting the key role of this enzyme in nitrogen assimilation and ammonium detoxification in roots. Otherwise, nitrogen treatments decreased GS expression and activity in the leaves of HGPC genotypes and did not affect GS in the leaves of LGPC genotypes. Finally, no changes in GS and soluble protein content occurred at the filling stage in the caryopses of all analyzed genotypes. PMID:27468287

  17. Calcium-binding proteins and development

    NASA Technical Reports Server (NTRS)

    Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

  18. The detection of DNA-binding proteins by protein blotting.

    PubMed Central

    Bowen, B; Steinberg, J; Laemmli, U K; Weintraub, H

    1980-01-01

    A method, called "protein blotting," for the detection of DNA-binding proteins is described. Proteins are separated on an SDA-polyacrylamide gel. The gel is sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replica is used to detect DNA-binding proteins, RNA-binding proteins or histone-binding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed. Images PMID:6243775

  19. Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy.

    PubMed Central

    Reynaldo, L. P.; Villafranca, J. J.; Horrocks, W. D.

    1996-01-01

    Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation

  20. Calmodulin Binding Proteins and Alzheimer's Disease.

    PubMed

    O'Day, Danton H; Eshak, Kristeen; Myre, Michael A

    2015-01-01

    The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer's disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer's disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer's disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  1. Computational Prediction of RNA-Binding Proteins and Binding Sites.

    PubMed

    Si, Jingna; Cui, Jing; Cheng, Jin; Wu, Rongling

    2015-01-01

    Proteins and RNA interaction have vital roles in many cellular processes such as protein synthesis, sequence encoding, RNA transfer, and gene regulation at the transcriptional and post-transcriptional levels. Approximately 6%-8% of all proteins are RNA-binding proteins (RBPs). Distinguishing these RBPs or their binding residues is a major aim of structural biology. Previously, a number of experimental methods were developed for the determination of protein-RNA interactions. However, these experimental methods are expensive, time-consuming, and labor-intensive. Alternatively, researchers have developed many computational approaches to predict RBPs and protein-RNA binding sites, by combining various machine learning methods and abundant sequence and/or structural features. There are three kinds of computational approaches, which are prediction from protein sequence, prediction from protein structure, and protein-RNA docking. In this paper, we review all existing studies of predictions of RNA-binding sites and RBPs and complexes, including data sets used in different approaches, sequence and structural features used in several predictors, prediction method classifications, performance comparisons, evaluation methods, and future directions.

  2. The Cochaperone SGTA (Small Glutamine-rich Tetratricopeptide Repeat-containing Protein Alpha) Demonstrates Regulatory Specificity for the Androgen, Glucocorticoid, and Progesterone Receptors*

    PubMed Central

    Paul, Atanu; Garcia, Yenni A.; Zierer, Bettina; Patwardhan, Chaitanya; Gutierrez, Omar; Hildenbrand, Zacariah; Harris, Diondra C.; Balsiger, Heather A.; Sivils, Jeffrey C.; Johnson, Jill L.; Buchner, Johannes; Chadli, Ahmed; Cox, Marc B.

    2014-01-01

    Steroid hormone receptors are ligand-dependent transcription factors that require the ordered assembly of multichaperone complexes for transcriptional activity. Although heat shock protein (Hsp) 90 and Hsp70 are key players in this process, multiple Hsp70- and Hsp90-associated cochaperones associate with receptor-chaperone complexes to regulate receptor folding and activation. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was recently characterized as an Hsp70 and Hsp90-associated cochaperone that specifically regulates androgen receptor activity. However, the specificity of SGTA for additional members of the steroid hormone receptor superfamily and the mechanism by which SGTA regulates receptor activity remain unclear. Here we report that SGTA associates with and specifically regulates the androgen, glucocorticoid, and progesterone receptors and has no effect on the mineralocorticoid and estrogen receptors in both yeast and mammalian cell-based reporter assays. In both systems, SGTA knockdown/deletion enhances receptor activity, whereas SGTA overexpression suppresses receptor activity. We demonstrate that SGTA binds directly to Hsp70 and Hsp90 in vitro with similar affinities yet predominately precipitates with Hsp70 from cell lysates, suggesting a role for SGTA in early, Hsp70-mediated folding. Furthermore, SGTA expression completely abrogates the regulation of receptor function by FKBP52 (52-kDa FK506-binding protein), which acts at a later stage of the chaperone cycle. Taken together, our data suggest a role for SGTA at distinct steps in the chaperone-dependent modulation of androgen, glucocorticoid, and progesterone receptor activity. PMID:24753260

  3. Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets.

    PubMed

    Endrizzi, James A; Kim, Hanseong; Anderson, Paul M; Baldwin, Enoch P

    2004-06-01

    Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics. PMID:15157079

  4. Mercury-binding proteins of Mytilus edulis

    SciTech Connect

    Roesijadi, G.; Morris, J. E.; Calabrese, A.

    1981-11-01

    Mytilus edulis possesses low molecular weight, mercury-binding proteins. The predominant protein isolated from gill tissue is enriched in cysteinyl residues (8%) and possesses an amino acid composition similar to cadmium-binding proteins of mussels and oysters. Continuous exposure of mussels to 5 ..mu..g/l mercury results in spillover of mercury from these proteins to high molecular weight proteins. Antibodies to these proteins have been isolated, and development of immunoassays is presently underway. Preliminary studies to determine whether exposure of adult mussels to mercury will result in induction of mercury-binding proteins in offspring suggest that such proteins occur in larvae although additional studies are indicated for a conclusive demonstration.

  5. Why are proteins with glutamine- and asparagine-rich regions associated with protein misfolding diseases?

    NASA Astrophysics Data System (ADS)

    Cruzeiro, Leonor

    2005-12-01

    The possibility that vibrational excited states (VESs) are the drivers of protein folding and function (the VES hypothesis) is explored to explain the reason why Gln- and Asn-rich proteins are associated with degenerative diseases. The Davydov/Scott model is extended to describe energy transfer from the water solution to the protein and vice versa. Computer simulations show that, on average, Gln and Asn residues lead to an initial larger absorption of energy from the environment to the protein, something that can explain the greater structural instability of prions. The sporadic, inherited and infectious character of prion diseases is discussed in the light of the VES hypothesis. An alternative treatment for prion diseases is suggested.

  6. Chemokine binding proteins encoded by pathogens.

    PubMed

    Alcami, Antonio; Saraiva, Margarida

    2009-01-01

    Chemokines are chemoattractant cytokines that play an important role in immunity. The role of chemokines against invading pathogens is emphasized by the expression of chemokine inhibitors by many pathogens. A mechanims employed by poxviruses and herpesviruses is the secretion of chemokine bindingproteins unrelated to host receptors that bind chemokines with high affinity and block their activity. Soluble chemokine binding proteins have also been identified in the human parasite Schistosoma mansoni and in ticks. The binding specificity of these inhibitors of cell migration point at chemokines that contribute to host defense mechanisms against various pathogens. Chemokine binding proteins modulate the immune response and may lead to new therapeutic approaches to treat inflamatory diseases.

  7. PUB1: a major yeast poly(A)+ RNA-binding protein.

    PubMed Central

    Matunis, M J; Matunis, E L; Dreyfuss, G

    1993-01-01

    The expression of RNA polymerase II transcripts can be regulated at the posttranscriptional level by RNA-binding proteins. Although extensively characterized in metazoans, relatively few RNA-binding proteins have been characterized in the yeast Saccharomyces cerevisiae. Three major proteins are cross-linked by UV light to poly(A)+ RNA in living S. cerevisiae cells. These are the 72-kDa poly(A)-binding protein and proteins of 60 and 50 kDa (S.A. Adam, T.Y. Nakagawa, M.S. Swanson, T. Woodruff, and G. Dreyfuss, Mol. Cell. Biol. 6:2932-2943, 1986). Here, we describe the 60-kDa protein, one of the major poly(A)+ RNA-binding proteins in S. cerevisiae. This protein, PUB1 [for poly(U)-binding protein 1], was purified by affinity chromatography on immobilized poly(rU), and specific monoclonal antibodies to it were produced. UV cross-linking demonstrated that PUB1 is bound to poly(A)+ RNA (mRNA or pre-mRNA) in living cells, and it was detected primarily in the cytoplasm by indirect immunofluorescence. The gene for PUB1 was cloned and sequenced, and the sequence was found to predict a 51-kDa protein with three ribonucleoprotein consensus RNA-binding domains and three glutamine- and asparagine-rich auxiliary domains. This overall structure is remarkably similar to the structures of the Drosophila melanogaster elav gene product, the human neuronal antigen HuD, and the cytolytic lymphocyte protein TIA-1. Each of these proteins has an important role in development and differentiation, potentially by affecting RNA processing. PUB1 was found to be nonessential in S. cerevisiae by gene replacement; however, further genetic analysis should reveal important features of this class of RNA-binding proteins. Images PMID:8413213

  8. Effects of oral glutamine supplementation on exercise-induced gastrointestinal permeability and tight junction protein expression.

    PubMed

    Zuhl, Micah N; Lanphere, Kathryn R; Kravitz, Len; Mermier, Christine M; Schneider, Suzanne; Dokladny, Karol; Moseley, Pope L

    2014-01-15

    The objectives of this study are threefold: 1) to assess whether 7 days of oral glutamine (GLN) supplementation reduces exercise-induced intestinal permeability; 2) whether supplementation prevents the proinflammatory response; and 3) whether these changes are associated with upregulation of the heat shock response. On separate occasions, eight human subjects participated in baseline testing and in GLN and placebo (PLA) supplementation trials, followed by a 60-min treadmill run. Intestinal permeability was higher in the PLA trial compared with baseline and GLN trials (0.0604 ± 0.047 vs. 0.0218 ± 0.008 and 0.0272 ± 0.007, respectively; P < 0.05). IκBα expression in peripheral blood mononuclear cells was higher 240 min after exercise in the GLN trial compared with the PLA trial (1.411 ± 0.523 vs. 0.9839 ± 0.343, respectively; P < 0.05). In vitro using the intestinal epithelial cell line Caco-2, we measured effects of GLN supplementation (0, 4, and 6 mM) on heat-induced (37° or 41.8°C) heat shock protein 70 (HSP70), heat shock factor-1 (HSF-1), and occludin expression. HSF-1 and HSP70 levels increased in 6 mM supplementation at 41°C compared with 0 mM at 41°C (1.785 ± 0.495 vs. 0.6681 ± 0.290, and 1.973 ± 0.325 vs. 1.133 ± 0.129, respectively; P < 0.05). Occludin levels increased after 4 mM supplementation at 41°C and 6 mM at 41°C compared with 0 mM at 41°C (1.236 ± 0.219 and 1.849 ± 0.564 vs. 0.7434 ± 0.027, respectively; P < 0.001). GLN supplementation prevented exercise-induced permeability, possibly through HSF-1 activation. PMID:24285149

  9. Quantitative proteomics reveals dynamic interaction of c-Jun N-terminal kinase (JNK) with RNA transport granule proteins splicing factor proline- and glutamine-rich (Sfpq) and non-POU domain-containing octamer-binding protein (Nono) during neuronal differentiation.

    PubMed

    Sury, Matthias D; McShane, Erik; Hernandez-Miranda, Luis Rodrigo; Birchmeier, Carmen; Selbach, Matthias

    2015-01-01

    The c-Jun N-terminal kinase (JNK) is an important mediator of physiological and pathophysiological processes in the central nervous system. Importantly, JNK not only is involved in neuronal cell death, but also plays a significant role in neuronal differentiation and regeneration. For example, nerve growth factor induces JNK-dependent neuronal differentiation in several model systems. The mechanism by which JNK mediates neuronal differentiation is not well understood. Here, we employed a proteomic strategy to better characterize the function of JNK during neuronal differentiation. We used SILAC-based quantitative proteomics to identify proteins that interact with JNK in PC12 cells in a nerve growth factor-dependent manner. Intriguingly, we found that JNK interacted with neuronal transport granule proteins such as Sfpq and Nono upon NGF treatment. We validated the specificity of these interactions by showing that they were disrupted by a specific peptide inhibitor that blocks the interaction of JNK with its substrates. Immunoprecipitation and Western blotting experiments confirmed the interaction of JNK1 with Sfpq/Nono and demonstrated that it was RNA dependent. Confocal microscopy indicated that JNK1 associated with neuronal granule proteins in the cytosol of PC12 cells, primary cortical neurons, and P19 neuronal cells. Finally, siRNA experiments confirmed that Sfpq was necessary for neurite outgrowth in PC12 cells and that it most likely acted in the same pathway as JNK. In summary, our data indicate that the interaction of JNK1 with transport granule proteins in the cytosol of differentiating neurons plays an important role during neuronal development.

  10. Molecular beacons for detecting DNA binding proteins.

    PubMed

    Heyduk, Tomasz; Heyduk, Ewa

    2002-02-01

    We report here a simple, rapid, homogeneous fluorescence assay, the molecular beacon assay, for the detection and quantification of sequence-specific DNA-binding proteins. The central feature of the assay is the protein-dependent association of two DNA fragments each containing about half of a DNA sequence defining a protein-binding site. Protein-dependent association of DNA fragments can be detected by any proximity-based spectroscopic signal, such as fluorescence resonance energy transfer (FRET) between fluorochromes introduced into these DNA molecules. The assay is fully homogeneous and requires no manipulations aside from mixing of the sample and the test solution. It offers flexibility with respect to the mode of signal detection and the fluorescence probe, and is compatible with multicolor simultaneous detection of several proteins. The assay can be used in research and medical diagnosis and for high-throughput screening of drugs targeted to DNA-binding proteins.

  11. Phage display of engineered binding proteins.

    PubMed

    Levisson, Mark; Spruijt, Ruud B; Winkel, Ingrid Nolla; Kengen, Servé W M; van der Oost, John

    2014-01-01

    In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.

  12. II. Glutamine and glutamate.

    PubMed

    Tapiero, H; Mathé, G; Couvreur, P; Tew, K D

    2002-11-01

    Glutamine and glutamate with proline, histidine, arginine and ornithine, comprise 25% of the dietary amino acid intake and constitute the "glutamate family" of amino acids, which are disposed of through conversion to glutamate. Although glutamine has been classified as a nonessential amino acid, in major trauma, major surgery, sepsis, bone marrow transplantation, intense chemotherapy and radiotherapy, when its consumption exceeds its synthesis, it becomes a conditionally essential amino acid. In mammals the physiological levels of glutamine is 650 micromol/l and it is one of the most important substrate for ammoniagenesis in the gut and in the kidney due to its important role in the regulation of acid-base homeostasis. In cells, glutamine is a key link between carbon metabolism of carbohydrates and proteins and plays an important role in the growth of fibroblasts, lymphocytes and enterocytes. It improves nitrogen balance and preserves the concentration of glutamine in skeletal muscle. Deamidation of glutamine via glutaminase produces glutamate a precursor of gamma-amino butyric acid, a neurotransmission inhibitor. L-Glutamic acid is a ubiquitous amino acid present in many foods either in free form or in peptides and proteins. Animal protein may contain from 11 to 22% and plants protein as much as 40% glutamate by weight. The sodium salt of glutamic acid is added to several foods to enhance flavor. L-Glutamate is the most abundant free amino acid in brain and it is the major excitatory neurotransmitter of the vertebrate central nervous system. Most free L-glutamic acid in brain is derived from local synthesis from L-glutamine and Kreb's cycle intermediates. It clearly plays an important role in neuronal differentiation, migration and survival in the developing brain via facilitated Ca++ transport. Glutamate also plays a critical role in synaptic maintenance and plasticity. It contributes to learning and memory through use-dependent changes in synaptic efficacy and

  13. Lipid binding proteins from parasitic platyhelminthes.

    PubMed

    Alvite, Gabriela; Esteves, Adriana

    2012-01-01

    TWO MAIN FAMILIES OF LIPID BINDING PROTEINS HAVE BEEN IDENTIFIED IN PARASITIC PLATYHELMINTHES: hydrophobic ligand binding proteins (HLBPs) and fatty acid binding proteins (FABPs). Members of the former family of proteins are specific to the Cestoda class, while FABPs are conserved across a wide range of animal species. Because Platyhelminthes are unable to synthesize their own lipids, these lipid-binding proteins are important molecules in these organisms. HLBPs are a high molecular mass complex of proteins and lipids. They are composed of subunits of low molecular mass proteins and a wide array of lipid molecules ranging from CoA esters to cholesterol. These proteins are excretory-secretory molecules and are key serological tools for diagnosis of diseases caused by cestodes. FABPs are mainly intracellular proteins of low molecular weight. They are also vaccine candidates. Despite that the knowledge of their function is scarce, the differences in their molecular organization, ligand preferences, intra/extracellular localization, evolution, and phylogenetic distribution, suggest that platyhelminths HLBPs and FABPs should play different functions. FABPs might be involved in the removal of fatty acids from the inner surface of the cell membrane and in their subsequent targeting to specific cellular destinations. In contrast, HLBPs might be involved in fatty acid uptake from the host environment.

  14. Copper binding in the prion protein.

    PubMed

    Millhauser, Glenn L

    2004-02-01

    A conformational change of the prion protein is responsible for a class of neurodegenerative diseases called the transmissible spongiform encephalopathies that include mad cow disease and the human afflictions kuru and Creutzfeldt-Jakob disease. Despite the attention given to these diseases, the normal function of the prion protein in healthy tissue is unknown. Research over the past few years, however, demonstrates that the prion protein is a copper binding protein with high selectivity for Cu(2+). The structural features of the Cu(2+) binding sites have now been characterized and are providing important clues about the normal function of the prion protein and perhaps how metals or loss of protein function play a role in disease. The link between prion protein and copper may provide insight into the general, and recently appreciated, role of metals in neurodegenerative disease. PMID:14967054

  15. The binding domain structure of retinoblastoma-binding proteins.

    PubMed Central

    Figge, J.; Breese, K.; Vajda, S.; Zhu, Q. L.; Eisele, L.; Andersen, T. T.; MacColl, R.; Friedrich, T.; Smith, T. F.

    1993-01-01

    The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu-X-Cys-X-Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb-binding protein to another. In this report, we present a diagnostic computer pattern for a high-affinity Rb-binding domain featuring the three conserved residues as well as the conserved physico-chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico-chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding. PMID:8382993

  16. Folding funnels, binding funnels, and protein function.

    PubMed Central

    Tsai, C. J.; Kumar, S.; Ma, B.; Nussinov, R.

    1999-01-01

    Folding funnels have been the focus of considerable attention during the last few years. These have mostly been discussed in the general context of the theory of protein folding. Here we extend the utility of the concept of folding funnels, relating them to biological mechanisms and function. In particular, here we describe the shape of the funnels in light of protein synthesis and folding; flexibility, conformational diversity, and binding mechanisms; and the associated binding funnels, illustrating the multiple routes and the range of complexed conformers. Specifically, the walls of the folding funnels, their crevices, and bumps are related to the complexity of protein folding, and hence to sequential vs. nonsequential folding. Whereas the former is more frequently observed in eukaryotic proteins, where the rate of protein synthesis is slower, the latter is more frequent in prokaryotes, with faster translation rates. The bottoms of the funnels reflect the extent of the flexibility of the proteins. Rugged floors imply a range of conformational isomers, which may be close on the energy landscape. Rather than undergoing an induced fit binding mechanism, the conformational ensembles around the rugged bottoms argue that the conformers, which are most complementary to the ligand, will bind to it with the equilibrium shifting in their favor. Furthermore, depending on the extent of the ruggedness, or of the smoothness with only a few minima, we may infer nonspecific, broad range vs. specific binding. In particular, folding and binding are similar processes, with similar underlying principles. Hence, the shape of the folding funnel of the monomer enables making reasonable guesses regarding the shape of the corresponding binding funnel. Proteins having a broad range of binding, such as proteolytic enzymes or relatively nonspecific endonucleases, may be expected to have not only rugged floors in their folding funnels, but their binding funnels will also behave similarly

  17. Diagnostic utility and limitations of glutamine synthetase and serum amyloid-associated protein immunohistochemistry in the distinction of focal nodular hyperplasia and inflammatory hepatocellular adenoma.

    PubMed

    Joseph, Nancy M; Ferrell, Linda D; Jain, Dhanpat; Torbenson, Michael S; Wu, Tsung-Teh; Yeh, Matthew M; Kakar, Sanjay

    2014-01-01

    Inflammatory hepatocellular adenoma can show overlapping histological features with focal nodular hyperplasia, including inflammation, fibrous stroma, and ductular reaction. Expression of serum amyloid-associated protein in inflammatory hepatocellular adenoma and map-like pattern of glutamine synthetase in focal nodular hyperplasia can be helpful in this distinction, but the pitfalls and limitations of these markers have not been established. Morphology and immunohistochemistry were analyzed in 54 inflammatory hepatocellular adenomas, 40 focal nodular hyperplasia, and 3 indeterminate lesions. Morphological analysis demonstrated that nodularity, fibrous stroma, dystrophic blood vessels, and ductular reaction were more common in focal nodular hyperplasia, while telangiectasia, hemorrhage, and steatosis were more common in inflammatory hepatocellular adenoma, but there was frequent overlap of morphological features. The majority of inflammatory hepatocellular adenomas demonstrated perivascular and/or patchy glutamine synthetase staining (73.6%), while the remaining cases had diffuse (7.5%), negative (3.8%), or patchy pattern of staining (15%) that showed subtle differences from the classic map-like staining pattern and was designated as pseudo map-like staining. Positive staining for serum amyloid-associated protein was seen in the majority of inflammatory hepatocellular adenomas (92.6%) and in the minority of focal nodular hyperplasia (17.5%). The glutamine synthetase staining pattern was map-like in 90% of focal nodular hyperplasia cases, with the remaining 10% of cases showing pseudo map-like staining. Three cases were labeled as indeterminate and showed focal nodular hyperplasia-like morphology but lacked map-like glutamine synthetase staining pattern; these cases demonstrated a patchy pseudo map-like glutamine synthetase pattern along with the expression of serum amyloid-associated protein. Our results highlight the diagnostic errors that can be caused by variant

  18. Regulation of glutamine carrier proteins by RNF5 determines breast cancer response to ER stress-inducing chemotherapies.

    PubMed

    Jeon, Young Joo; Khelifa, Sihem; Ratnikov, Boris; Scott, David A; Feng, Yongmei; Parisi, Fabio; Ruller, Chelsea; Lau, Eric; Kim, Hyungsoo; Brill, Laurence M; Jiang, Tingting; Rimm, David L; Cardiff, Robert D; Mills, Gordon B; Smith, Jeffrey W; Osterman, Andrei L; Kluger, Yuval; Ronai, Ze'ev A

    2015-03-01

    Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination, and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling, and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5(hi)/SLC1A5/38A2(lo) expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies. PMID:25759021

  19. Regulation of Glutamine Carrier Proteins by RNF5 Determines Breast Cancer Response to ER Stress-inducing Chemotherapies

    PubMed Central

    Jeon, Young Joo; Khelifa, Sihem; Ratnikov, Boris; Scott, David A.; Feng, Yongmei; Parisi, Fabio; Ruller, Chelsea; Lau, Eric; Kim, Hyungsoo; Brill, Laurence M.; Jiang, Tingting; Rimm, David; Cardiff, Robert D.; Mills, Gordon B.; Smith, Jeffrey W.; Osterman, Andrei L.; Kluger, Yuval; Ronai, Ze'ev A.

    2015-01-01

    Summary Many tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5HI/SLC1A5/38A2LO expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies. PMID:25759021

  20. A Detour for Yeast Oxysterol Binding Proteins*

    PubMed Central

    Beh, Christopher T.; McMaster, Christopher R.; Kozminski, Keith G.; Menon, Anant K.

    2012-01-01

    Oxysterol binding protein-related proteins, including the yeast proteins encoded by the OSH gene family (OSH1–OSH7), are implicated in the non-vesicular transfer of sterols between intracellular membranes and the plasma membrane. In light of recent studies, we revisited the proposal that Osh proteins are sterol transfer proteins and present new models consistent with known Osh protein functions. These models focus on the role of Osh proteins as sterol-dependent regulators of phosphoinositide and sphingolipid pathways. In contrast to their posited role as non-vesicular sterol transfer proteins, we propose that Osh proteins coordinate lipid signaling and membrane reorganization with the assembly of tethering complexes to promote molecular exchanges at membrane contact sites. PMID:22334669

  1. Plasma protein binding of zomepirac sodium.

    PubMed

    O'Neill, P J

    1981-07-01

    The plasma protein binding of zomepirac, a new nonnarcotic analgesic, was studied using equilibrium dialysis. Experiments were performed using human plasma and plasma from mice, rats, and rhesus monkeys, all species of pharmacological or toxicological interest. At concentrations approximating those achieved in vivo, the binding was fairly constant at 98-99% in all species except the rhesus monkey, where binding was decreased from 98 to approximately 96% at higher concentrations (greater then 50 microgram/ml). Zomepirac (10 microgram/ml) did not appear to displace or to be displaced by warfarin (10 microgram/ml) caused a concentration-dependent decrease in zomepirac (10 microgram/ml) binding. Zomepirac did not affect salicylate binding.

  2. Aspects of Protein, Chemistry, Part II: Oxygen-Binding Proteins

    ERIC Educational Resources Information Center

    Nixon, J. E.

    1977-01-01

    Compares differences in function and behavior of two oxygen-binding proteins, myoglobin found in muscle and hemoglobin found in blood. Describes the mechanism of oxygen-binding and allosteric effect in hemoglobin; also describes the effect of pH on the affinity of hemoglobin for oxygen. (CS)

  3. Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 72 and 82 of the cyclic nucleotide binding pocket.

    PubMed Central

    Belduz, A O; Lee, E J; Harman, J G

    1993-01-01

    The 3', 5' cyclic adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute leucine, glutamine, or aspartate for glutamate 72; and lysine, histidine, leucine, isoleucine, or glutamine for arginine 82. Substitutions were made in wild-type CRP and in a CRP*, or cAMP-independent, form of the protein to assess the effects of the amino acid substitutions on CRP structure. Cells containing the binding pocket residue-substituted forms of CRP were characterized through beta-galactosidase activity and by measurement of cAMP binding activity. This study confirms a role for both glutamate 72 and arginine 82 in cAMP binding and activation of CRP. Glutamine or leucine substitution of glutamate 72 produced forms of CRP having low affinity for the cAMP and unresponsive to the nucleotide. Aspartate substituted for glutamate 72 produced a low affinity cAMP-responsive form of CRP. CRP has a stringent requirement for the positioning of the position 72 glutamate carboxyl group within the cyclic nucleotide binding pocket. Results of this study also indicate that there are differences in the binding requirements of cAMP and cGMP, a competitive inhibitor of cAMP binding to CRP. PMID:8388097

  4. Protein-protein binding site identification by enumerating the configurations

    PubMed Central

    2012-01-01

    Background The ability to predict protein-protein binding sites has a wide range of applications, including signal transduction studies, de novo drug design, structure identification and comparison of functional sites. The interface in a complex involves two structurally matched protein subunits, and the binding sites can be predicted by identifying structural matches at protein surfaces. Results We propose a method which enumerates “all” the configurations (or poses) between two proteins (3D coordinates of the two subunits in a complex) and evaluates each configuration by the interaction between its components using the Atomic Contact Energy function. The enumeration is achieved efficiently by exploring a set of rigid transformations. Our approach incorporates a surface identification technique and a method for avoiding clashes of two subunits when computing rigid transformations. When the optimal transformations according to the Atomic Contact Energy function are identified, the corresponding binding sites are given as predictions. Our results show that this approach consistently performs better than other methods in binding site identification. Conclusions Our method achieved a success rate higher than other methods, with the prediction quality improved in terms of both accuracy and coverage. Moreover, our method is being able to predict the configurations of two binding proteins, where most of other methods predict only the binding sites. The software package is available at http://sites.google.com/site/guofeics/dobi for non-commercial use. PMID:22768846

  5. Ice-Binding Proteins and Their Function.

    PubMed

    Bar Dolev, Maya; Braslavsky, Ido; Davies, Peter L

    2016-06-01

    Ice-binding proteins (IBPs) are a diverse class of proteins that assist organism survival in the presence of ice in cold climates. They have different origins in many organisms, including bacteria, fungi, algae, diatoms, plants, insects, and fish. This review covers the gamut of IBP structures and functions and the common features they use to bind ice. We discuss mechanisms by which IBPs adsorb to ice and interfere with its growth, evidence for their irreversible association with ice, and methods for enhancing the activity of IBPs. The applications of IBPs in the food industry, in cryopreservation, and in other technologies are vast, and we chart out some possibilities. PMID:27145844

  6. A microscopic insight from conformational thermodynamics to functional ligand binding in proteins.

    PubMed

    Sikdar, Samapan; Chakrabarti, J; Ghosh, Mahua

    2014-12-01

    We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for β-1,4-galactosyltransferase (β4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock β4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.

  7. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    PubMed

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

  8. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia*

    PubMed Central

    Dong, Qian; Ernst, Sarah E.; Ostedgaard, Lynda S.; Shah, Viral S.; Ver Heul, Amanda R.; Welsh, Michael J.; Randak, Christoph O.

    2015-01-01

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

  9. Calcium-binding proteins: an overview.

    PubMed

    Weinman, S

    1991-03-01

    In order to understand the mechanism of the various responses evoked by calcium in the cell, the identification and characterization of a number of calcium receptors were undertaken within the past two decades. Advances in amino acid sequence and protein three-dimensional structure led to the description of two families of calcium-binding proteins, the EF-hand homolog family and the annexin family. The EF-hand motif consists of two alpha helices, "E" and "F", joined by a Ca(2+)-binding loop. EF-hands have been identified in numerous Ca(2+)-binding proteins by similarity of amino acid sequence and confirmed in some crystal structures. Functional EF-hands seem always to occur in pairs. To date, the EF-hand homolog family contains more than 160 different Ca(2+)-modulated proteins which have a broad range of functions. Among them, are the calmodulin, the troponin C, the myosin regulatory light chain, the parvalbumin, the S-100 proteins and the calbindins 9- and 28 kDa. The most striking feature of the EF-hand family is the ability to modulate the activity of a number of enzymes. Several groups have identified proteins from various tissues that show calcium-dependent binding to membranes. These proteins, termed annexins have a molecular weight of 35- or 67 kDa. The amino acid sequences of the members of the annexin family show that each protein contains conserved internal repeats of about 70 amino acids each. The 35 kDa annexins contain four repeats, which show a high degree of homology with each other and with the repeat sequences of the other proteins. These repeats correspond to structural domains with a similar fold.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1864864

  10. Information flow through calcium binding proteins

    NASA Astrophysics Data System (ADS)

    Bak, Ji Hyun; Bialek, William

    2013-03-01

    Calcium signaling is a ubiquitous mode of biological communication, which regulates a great variety of vital processes in living systems. Such a signal typically begins with an elementary event, in which calcium ions bind to a protein, inducing a change in the protein's structure. Information can only be lost, from what was conveyed through this initial event, as the signal is further transduced through the downstream networks. In the present work we analyze and optimize the information flow in the calcium binding process. We explicitly calculate the mutual information between the calcium concentration and the states of the protein, using a simple model for allosteric regulation in a dimeric protein. The optimal solution depends on the dynamic range of the input as well as on the timescale of signal integration. According to our result, the optimizing strategy involves allowing the calcium-binding protein to be ``activated'' by a partial occupation of its sites, and tuning independently the strengths of cooperative interactions in the binding and unbinding processes.

  11. Cadmium-binding protein (metallothionein) in carp

    SciTech Connect

    Kito, H.; Ose, Y.; Sato, T.

    1986-03-01

    When carp (Cyprinus carpio) were exposed to 5 and 30 ppm Cd in the water, the contents of Cd-binding protein, which has low molecular weight, increased in the hepatopancreas, kidney, gills and gastrointestinal tract with duration of exposure. This Cd-binding protein was purified from hepatopancreas, kidney, gills, and spleen of carp administered 2 mg/kg Cd (as CdCl/sub 2/), intraperitoneally for 6 days. Two Cd-binding proteins were separated by DEAE-Sephadex A-25 column chromatography. These proteins had Cd-mercaptide bond, high cysteine contents (ca. 29-34%), but no aromatic amino acids or histidine. From these characteristics the Cd-binding proteins were identified as metallothionein. By using antiserum obtained from a rabbit to which carp hepatopancreas MT-II had been administered, immunological characteristics between hepatopancreas MT-I, II and kidney MT-II were studied, and a slight difference in antigenic determinant was observed among them. By immunological staining techniques with horseradish peroxidase, the localization of metallothionein was investigated. Carp were bred in 1 ppm Cd, 5 ppm Zn solution, and tap water for 14 days, following transfer to 15 ppm Cd solution, respectively. The survival ratio was the highest in the Zn group followed by Cd-treated and control groups.

  12. Antibodies against the calcium-binding protein

    SciTech Connect

    Chou, Mei; Jensen, K.G.; Sjolund, R.D. ); Krause, K.H.; Campbell, K.P. )

    1989-12-01

    Plant microsomes contain a protein clearly related to a calcium-binding protein, calsequestrin, originally found in the sarcoplasmic reticulum of muscle cells, responsible for the rapid release and uptake of Ca{sup 2+} within the cells. The location and role of calsequestrin in plant cells is unknown. To generate monoclonal antibodies specific to plant calsequestrin, mice were immunized with a microsomal fraction from cultured cells of Streptanthus tortuosus (Brassicaceae). Two clones cross-reacted with one protein band with a molecular weight equal to that of calsequestrin (57 kilodaltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. This band is able to bind {sup 45}Ca{sup 2+} and can be recognized by a polyclonal antibody against the canine cardiac muscle calsequestrin. Rabbit skeletal muscle calsequestrin cross-reacted with the plant monoclonal antibodies. The plant monoclonal antibodies generated here are specific to calsequestrin protein.

  13. AUXIN BINDING PROTEIN1: The Outsider

    PubMed Central

    Sauer, Michael; Kleine-Vehn, Jürgen

    2011-01-01

    AUXIN BINDING PROTEIN1 (ABP1) is one of the first characterized proteins that bind auxin and has been implied as a receptor for a number of auxin responses. Early studies characterized its auxin binding properties and focused on rapid electrophysiological and cell expansion responses, while subsequent work indicated a role in cell cycle and cell division control. Very recently, ABP1 has been ascribed a role in modulating endocytic events at the plasma membrane and RHO OF PLANTS-mediated cytoskeletal rearrangements during asymmetric cell expansion. The exact molecular function of ABP1 is still unresolved, but its main activity apparently lies in influencing events at the plasma membrane. This review aims to connect the novel findings with the more classical literature on ABP1 and to point out the many open questions that still separate us from a comprehensive model of ABP1 action, almost 40 years after the first reports of its existence. PMID:21719690

  14. Protein Binding Studies with Zero Mode Waveguides

    NASA Astrophysics Data System (ADS)

    Samiee, K.; Foquet, M.; Cox, E. C.; Craighead, H. G.

    2004-03-01

    Single protein molecules binding to their DNA operator site are observed using zero mode waveguides, novel quasi one-dimensional optical nanostructures. The subwavelength features of the waveguides allow the formation of a focal volume smaller than those allowed by classical diffraction limited optics. The small observation volume allows the use of fluorescence correlation spectroscopy to measure diffusion constants at fluorophore concentrations as high as10uM. Binding is observed between a DNA oligomer containing OR1, an operator site on the Lambda genome, and CI, the repressor protein that inhibits the bacteriophage's lytic growth cycle. The dimensions of the waveguide should allow a single DNA fragment to be fixed at the bottom where its binding dynamics can be characterized on a single molecule basis.

  15. Effect of intravenous amino acids on glutamine and protein kinetics in low-birth-weight preterm infants during the immediate neonatal period.

    PubMed

    Kadrofske, Mark M; Parimi, Prabhu S; Gruca, Lourdes L; Kalhan, Satish C

    2006-04-01

    Glutamine may be a conditionally essential amino acid in low-birth-weight (LBW) preterm neonates. Exogenously administered amino acids, by providing anaplerotic carbon into the tricarboxylic acid cycle, could result in greater cataplerotic efflux and glutamine de novo synthesis. The effect of dose and duration of amino acid infusion on glutamine and nitrogen (N) kinetics was examined in LBW infants in the period immediately after birth. Preterm neonates (<32 weeks gestation, birth weights 809-1,755 g) were randomized to initially receive either 480 or 960 micromol x kg(-1) x h(-1) of an intravenous amino acid solution for 19-24 hours, followed by a higher or lower amino acid load for either 5 h or 24 h. Glutamine de novo synthesis, leucine N, phenylalanine, and urea kinetics were determined using stable isotopic tracers. An increase in amino acid infusion from 480 to 960 micromol x kg(-1) x h(-1) for 5 h resulted in decreased glutamine de novo synthesis in every neonate (384.4 +/- 38.0 to 368.9 +/- 38.2 micromol x kg(-1) x h(-1), P < 0.01) and a lower whole body rate of proteolysis (P < 0.001) and urea synthesis (P < 0.001). However, when the increased amino acid infusion was extended for 24 h, glutamine de novo synthesis increased (369.7 +/- 92.6 to 483.4 +/- 97.5 micromol x kg(-1) x h(-1), P < 0.001), whole body rate of proteolysis did not change, and urea production increased. Decreasing the amino acid load resulted in a decrease in glutamine rate of appearance (R(a)) and leucine N R(a), but had no effect on phenylalanine R(a). Acutely stressed LBW infants responded to an increase in amino acid load by transiently suppressing whole body rate of glutamine synthesis, proteolysis, and oxidation of protein. The mechanisms of this transient effect on whole body protein/nitrogen metabolism remain unknown.

  16. Exogenous glutamine: the clinical evidence.

    PubMed

    Bongers, Thomas; Griffiths, Richard D; McArdle, Anne

    2007-09-01

    We know that critically ill patients suffering from undernutrition with a limited nutritional reserve have a poorer outcome. Furthermore, having a low body mass index has been shown to be an independent predictor of excess mortality in multiple organ failure. Therefore, nutritional support has gained increasing interest in critical illness with the hope of preventing or attenuating the effects of malnutrition. A negative nitrogen balance is the characteristic metabolic feature in critical illness, with the major protein loss derived from skeletal muscle. In particular, glutamine concentrations are rapidly reduced in plasma and muscle. Over the last 20 yrs or so, increasing evidence is emerging to support the use of glutamine supplementation in critical illness. Clinical trials have found a mortality and morbidity advantage with glutamine supplementation. The advantage appears to be greater the more glutamine is given and greater again when given parenterally. Various modes of action have been postulated. Glutamine seems to have an effect on the immune system, antioxidant status, glucose metabolism, and heat shock protein response. However, the benefit of exogenous glutamine on morbidity and mortality is not universally accepted. This review critically appraises the current clinical evidence regarding glutamine supplementation in critical illness.

  17. The human small glutamine-rich TPR-containing protein is required for progress through cell division.

    PubMed

    Winnefeld, Marc; Rommelaere, Jean; Cziepluch, Celina

    2004-02-01

    Eukaryotic organisms from yeast to human harbor genes encoding the small glutamine-rich tetratricopeptide repeat-containing (SGT) protein. Work presented here demonstrated the presence of human SGT (hSGT) protein in a panel of human cell lines and throughout the cell cycle. To identify cellular processes in which hSGT is involved, knock down populations were analyzed which were generated through transfection of hsgt-specific small interfering RNA. Most strikingly, depletion of hSGT led to reduced proliferation of the affected cell populations while the mitotic index was increased. Time-lapse video microscopy revealed that cells from hSGT-depleted populations were unable to complete cell division due to mitotic arrest which was frequently followed by cell death. Further evidence for a role in cell division was given by the accumulation of hSGT in the midzone and the midbody, and by a mitosis-specific migration pattern of hSGT as detected by Western blotting after SDS-PAGE or two-dimensional gel electrophoresis. In conclusion, results obtained in this study demonstrate that hSGT protein is a constitutive component of all human cell lines tested and that this protein is essential for successful completion of cell division. PMID:14729056

  18. Evolution of Protein-binding DNA Sequences through Competitive Binding

    NASA Astrophysics Data System (ADS)

    Peng, Weiqun; Gerland, Ulrich; Hwa, Terence; Levine, Herbert

    2002-03-01

    The dynamics of in vitro DNA evolution controlled via competitive binding of DNA sequences to proteins has been explored in a recent serial transfer experiment footnote B. Dubertret, S.Liu, Q. Ouyang, A. Libchaber, Phys. Rev. Lett. 86, 6022 (2001).. Motivated by the experiment, we investigate a continuum model for this evolution process in various parameter regimes. We establish a self-consistent mean-field evolution equation, determine its dynamical properties and finite population size corrections. In addition, we discuss the experimental implications of our results.

  19. Quantifying drug-protein binding in vivo.

    SciTech Connect

    Buchholz, B; Bench, G; Keating III, G; Palmblad, M; Vogel, J; Grant, P G; Hillegonds, D

    2004-02-17

    Accelerator mass spectrometry (AMS) provides precise quantitation of isotope labeled compounds that are bound to biological macromolecules such as DNA or proteins. The sensitivity is high enough to allow for sub-pharmacological (''micro-'') dosing to determine macromolecular targets without inducing toxicities or altering the system under study, whether it is healthy or diseased. We demonstrated an application of AMS in quantifying the physiologic effects of one dosed chemical compound upon the binding level of another compound in vivo at sub-toxic doses [4].We are using tissues left from this study to develop protocols for quantifying specific binding to isolated and identified proteins. We also developed a new technique to quantify nanogram to milligram amounts of isolated protein at precisions that are comparable to those for quantifying the bound compound by AMS.

  20. The dynamics of ligands binding to proteins

    NASA Astrophysics Data System (ADS)

    Callender, Robert

    2001-03-01

    The static structures of many proteins have been solved, and this has revealed much about how they function. On the other hand, although the importance of atomic motion to how proteins function has been conjectured for several decades, the characterization of protein dynamics on multiple time scales is scant. This is because of severe experimental and theoretical difficulties, particularly characterizing the nanosecond to millisecond time scales. Recently, several new techniques have been introduced that make it possible to initiate chemical reactions on fast time scales. We have applied advanced laser induced temperature jump relaxation spectroscopy with nanosecond resolution to examine the binding kinetics of ligands to several enzymes. The observed kinetics take place over multiple time scales. The results reveal the dynamical nature of the binding process and show that there are substantial populations of many structures that are in a constant dynamic equilibrium in some cases. Some of these structures lie quite far from the static structure defined in crystallographic studies, which suggest that the conventional thermodynamical picture of binding (an equilibrium between ligand free in solution and bound) is far off the mark. Moreover, the results suggest that the dynamics can certainly play a crucial role in kinetic control of protein function as in, for example, affecting the rates of enzymatic catalysis. This work is a collaborative project with Hong Deng and Nick Zhadin, also at Albert Einstein. Work supported by the NSF and NIH.

  1. Sterol carrier protein-2: binding protein for endocannabinoids.

    PubMed

    Liedhegner, Elizabeth Sabens; Vogt, Caleb D; Sem, Daniel S; Cunningham, Christopher W; Hillard, Cecilia J

    2014-08-01

    The endocannabinoid (eCB) system, consisting of eCB ligands and the type 1 cannabinoid receptor (CB1R), subserves retrograde, activity-dependent synaptic plasticity in the brain. eCB signaling occurs "on-demand," thus the processes regulating synthesis, mobilization and degradation of eCBs are also primary mechanisms for the regulation of CB1R activity. The eCBs, N-arachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are poorly soluble in water. We hypothesize that their aqueous solubility, and, therefore, their intracellular and transcellular distribution, are facilitated by protein binding. Using in silico docking studies, we have identified the nonspecific lipid binding protein, sterol carrier protein 2 (SCP-2), as a potential AEA binding protein. The docking studies predict that AEA and AM404 associate with SCP-2 at a putative cholesterol binding pocket with ∆G values of -3.6 and -4.6 kcal/mol, respectively. These values are considerably higher than cholesterol (-6.62 kcal/mol) but consistent with a favorable binding interaction. In support of the docking studies, SCP-2-mediated transfer of cholesterol in vitro is inhibited by micromolar concentrations of AEA; and heterologous expression of SCP-2 in HEK 293 cells increases time-related accumulation of AEA in a temperature-dependent fashion. These results suggest that SCP-2 facilitates cellular uptake of AEA. However, there is no effect of SCP-2 transfection on the cellular accumulation of AEA determined at equilibrium or the IC50 values for AEA, AM404 or 2-AG to inhibit steady state accumulation of radiolabelled AEA. We conclude that SCP-2 is a low affinity binding protein for AEA that can facilitate its cellular uptake but does not contribute significantly to intracellular sequestration of AEA.

  2. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  3. Polynucleotides encoding TRF1 binding proteins

    DOEpatents

    Campisi, Judith; Kim, Sahn-Ho

    2002-01-01

    The present invention provides a novel telomere associated protein (Trf1-interacting nuclear protein 2 "Tin2") that hinders the binding of Trf1 to its specific telomere repeat sequence and mediates the formation of a Tin2-Trf1-telomeric DNA complex that limits telomerase access to the telomere. Also included are the corresponding nucleic acids that encode the Tin2 of the present invention, as well as mutants of Tin2. Methods of making, purifying and using Tin2 of the present invention are described. In addition, drug screening assays to identify drugs that mimic and/or complement the effect of Tin2 are presented.

  4. EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression.

    PubMed

    Lee, Nara; Yario, Therese A; Gao, Jessica S; Steitz, Joan A

    2016-03-22

    Epstein-Barr virus (EBV) produces a highly abundant noncoding RNA called EBV-encoded RNA 2 (EBER2) that interacts indirectly with the host transcription factor paired box protein 5 (PAX5) to regulate viral latent membrane protein 1/2 (LMP1/2) gene expression as well as EBV lytic replication. To identify intermediary proteins, we isolated EBER2-PAX5-containing complexes and analyzed the protein components by mass spectrometry. The top candidates include three host proteins splicing factor proline and glutamine rich (SFPQ), non-POU domain-containing octamer-binding protein (NONO), and RNA binding motif protein 14 (RBM14), all reported to be components of nuclear bodies called paraspeckles. In vivo RNA-protein crosslinking indicates that SFPQ and RBM14 contact EBER2 directly. Binding studies using recombinant proteins demonstrate that SFPQ and NONO associate with PAX5, potentially bridging its interaction with EBER2. Similar to EBER2 or PAX5 depletion, knockdown of any of the three host RNA-binding proteins results in the up-regulation of viral LMP2A mRNA levels, supporting a physiologically relevant interaction of these newly identified factors with EBER2 and PAX5. Identification of these EBER2-interacting proteins enables the search for cellular noncoding RNAs that regulate host gene expression in a manner similar to EBER2. PMID:26951683

  5. EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression.

    PubMed

    Lee, Nara; Yario, Therese A; Gao, Jessica S; Steitz, Joan A

    2016-03-22

    Epstein-Barr virus (EBV) produces a highly abundant noncoding RNA called EBV-encoded RNA 2 (EBER2) that interacts indirectly with the host transcription factor paired box protein 5 (PAX5) to regulate viral latent membrane protein 1/2 (LMP1/2) gene expression as well as EBV lytic replication. To identify intermediary proteins, we isolated EBER2-PAX5-containing complexes and analyzed the protein components by mass spectrometry. The top candidates include three host proteins splicing factor proline and glutamine rich (SFPQ), non-POU domain-containing octamer-binding protein (NONO), and RNA binding motif protein 14 (RBM14), all reported to be components of nuclear bodies called paraspeckles. In vivo RNA-protein crosslinking indicates that SFPQ and RBM14 contact EBER2 directly. Binding studies using recombinant proteins demonstrate that SFPQ and NONO associate with PAX5, potentially bridging its interaction with EBER2. Similar to EBER2 or PAX5 depletion, knockdown of any of the three host RNA-binding proteins results in the up-regulation of viral LMP2A mRNA levels, supporting a physiologically relevant interaction of these newly identified factors with EBER2 and PAX5. Identification of these EBER2-interacting proteins enables the search for cellular noncoding RNAs that regulate host gene expression in a manner similar to EBER2.

  6. Small Glutamine-Rich Tetratricopeptide Repeat-Containing Protein Alpha (SGTA) Ablation Limits Offspring Viability and Growth in Mice

    PubMed Central

    Philp, Lisa K.; Day, Tanya K.; Butler, Miriam S.; Laven-Law, Geraldine; Jindal, Shalini; Hickey, Theresa E.; Scher, Howard I.; Butler, Lisa M.; Tilley, Wayne D.

    2016-01-01

    Small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) has been implicated as a co-chaperone and regulator of androgen and growth hormone receptor (AR, GHR) signalling. We investigated the functional consequences of partial and full Sgta ablation in vivo using Cre-lox Sgta-null mice. Sgta+/− breeders generated viable Sgta−/− offspring, but at less than Mendelian expectancy. Sgta−/− breeders were subfertile with small litters and higher neonatal death (P < 0.02). Body size was significantly and proportionately smaller in male and female Sgta−/− (vs WT, Sgta+/− P < 0.001) from d19. Serum IGF-1 levels were genotype- and sex-dependent. Food intake, muscle and bone mass and adiposity were unchanged in Sgta−/−. Vital and sex organs had normal relative weight, morphology and histology, although certain androgen-sensitive measures such as penis and preputial size, and testis descent, were greater in Sgta−/−. Expression of AR and its targets remained largely unchanged, although AR localisation was genotype- and tissue-dependent. Generally expression of other TPR-containing proteins was unchanged. In conclusion, this thorough investigation of SGTA-null mutation reports a mild phenotype of reduced body size. The model’s full potential likely will be realised by genetic crosses with other models to interrogate the role of SGTA in the many diseases in which it has been implicated. PMID:27358191

  7. Small Glutamine-Rich Tetratricopeptide Repeat-Containing Protein Alpha (SGTA) Ablation Limits Offspring Viability and Growth in Mice.

    PubMed

    Philp, Lisa K; Day, Tanya K; Butler, Miriam S; Laven-Law, Geraldine; Jindal, Shalini; Hickey, Theresa E; Scher, Howard I; Butler, Lisa M; Tilley, Wayne D

    2016-01-01

    Small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) has been implicated as a co-chaperone and regulator of androgen and growth hormone receptor (AR, GHR) signalling. We investigated the functional consequences of partial and full Sgta ablation in vivo using Cre-lox Sgta-null mice. Sgta(+/-) breeders generated viable Sgta(-/-) offspring, but at less than Mendelian expectancy. Sgta(-/-) breeders were subfertile with small litters and higher neonatal death (P < 0.02). Body size was significantly and proportionately smaller in male and female Sgta(-/-) (vs WT, Sgta(+/-) P < 0.001) from d19. Serum IGF-1 levels were genotype- and sex-dependent. Food intake, muscle and bone mass and adiposity were unchanged in Sgta(-/-). Vital and sex organs had normal relative weight, morphology and histology, although certain androgen-sensitive measures such as penis and preputial size, and testis descent, were greater in Sgta(-/-). Expression of AR and its targets remained largely unchanged, although AR localisation was genotype- and tissue-dependent. Generally expression of other TPR-containing proteins was unchanged. In conclusion, this thorough investigation of SGTA-null mutation reports a mild phenotype of reduced body size. The model's full potential likely will be realised by genetic crosses with other models to interrogate the role of SGTA in the many diseases in which it has been implicated. PMID:27358191

  8. Novel stereospecificity of the L-arabinose-binding protein

    NASA Astrophysics Data System (ADS)

    Quiocho, Florante A.; Vyas, Nand K.

    1984-08-01

    Tertiary structure refinement at 1.7 Å resolution of the liganded form of L-arabinose-binding protein from Escherichia coli has revealed a novel binding site geometry which accommodates both α- and β-anomers of L-arabinose. This detailed structure analysis provides new understanding of protein-sugar interaction, the process by which the binding protein minimizes the difference in the stability of the two bound sugar anomers, and the roles of periplasmic binding proteins in active transport

  9. Systematic discovery of Xist RNA binding proteins.

    PubMed

    Chu, Ci; Zhang, Qiangfeng Cliff; da Rocha, Simão Teixeira; Flynn, Ryan A; Bharadwaj, Maheetha; Calabrese, J Mauro; Magnuson, Terry; Heard, Edith; Chang, Howard Y

    2015-04-01

    Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA-protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3' RNA processing machinery. Xist, an essential lncRNA for X chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK, which participates in Xist-mediated gene silencing and histone modifications but not Xist localization, and Drosophila Split ends homolog Spen, which interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing.

  10. Systematic discovery of Xist RNA binding proteins

    PubMed Central

    Chu, Ci; Zhang, Qiangfeng Cliff; da Rocha, Simão Teixeira; Flynn, Ryan A.; Bharadwaj, Maheetha; Calabrese, J. Mauro; Magnuson, Terry; Heard, Edith; Chang, Howard Y.

    2015-01-01

    Summary Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA- protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3′ RNA processing machinery. Xist, an essential lncRNA for X-chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK that participates in Xist-mediated gene silencing and histone modifications, but not Xist localization and Drosophila Split ends homolog Spen that interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing. PMID:25843628

  11. Engineering a uranyl specific binding protein from NikR.

    SciTech Connect

    Wegner, S. V.; Boyaci, H.; Chen, H.; Jensen, M. P.; He, C.

    2009-03-16

    The first uranyl-selective DNA-binding protein is designed using the E. coli nickel(II)-responsive protein NikR as the template. The resulting NikR? protein binds uranyl (see picture) with a dissociation constant Kd=53?nM and selectively binds to DNA in the presence of uranyl.

  12. Copper-binding protein in Mimulus guttatus

    SciTech Connect

    Robinson, N.J.; Thurman, D.A.

    1985-01-01

    A Cu-binding protein has been purified from the roots of Mimulus guttatus using gel permeation chromatography on Sephadex G-75 and anion exchange chromatography on DEAE Biogel A. The protein has similar properties to putative metallothioneins (MTS) purified from other angiosperms. Putative MT was estimated by measuring the relative percentage incorporation of (/sup 35/S) into fractions containing the protein after HPLC on SW 3000-gel. In the roots of both Cu-tolerant and non tolerant plants synthesis of putative MT is induced by increased Cu concentration in the nutrient solution. The relative percentage incorporation of (/sup 35/S) into putative MT is significantly higher in extracts from the roots of Cu-tolerant than non tolerant M. guttatus after growth in 1 ..mu..M Cu suggesting involvement in the mechanism of tolerance. 22 refs., 2 figs., 1 tab.

  13. A Crayfish Insulin-like-binding Protein

    PubMed Central

    Rosen, Ohad; Weil, Simy; Manor, Rivka; Roth, Ziv; Khalaila, Isam; Sagi, Amir

    2013-01-01

    Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development. PMID:23775079

  14. Identification of DNA-binding and protein-binding proteins using enhanced graph wavelet features.

    PubMed

    Zhu, Yuan; Zhou, Weiqiang; Dai, Dao-Qing; Yan, Hong

    2013-01-01

    Interactions between biomolecules play an essential role in various biological processes. For predicting DNA-binding or protein-binding proteins, many machine-learning-based techniques have used various types of features to represent the interface of the complexes, but they only deal with the properties of a single atom in the interface and do not take into account the information of neighborhood atoms directly. This paper proposes a new feature representation method for biomolecular interfaces based on the theory of graph wavelet. The enhanced graph wavelet features (EGWF) provides an effective way to characterize interface feature through adding physicochemical features and exploiting a graph wavelet formulation. Particularly, graph wavelet condenses the information around the center atom, and thus enhances the discrimination of features of biomolecule binding proteins in the feature space. Experiment results show that EGWF performs effectively for predicting DNA-binding and protein-binding proteins in terms of Matthew's correlation coefficient (MCC) score and the area value under the receiver operating characteristic curve (AUC). PMID:24334394

  15. Competitive protein binding assay for piritrexim

    SciTech Connect

    Woolley, J.L. Jr.; Ringstad, J.L.; Sigel, C.W. )

    1989-09-01

    A competitive protein binding assay for piritrexim (PTX, 1) that makes use of a commercially available radioassay kit for methotrexate has been developed. After it is selectively extracted from plasma, PTX competes with ({sup 125}I)methotrexate for binding to dihydrofolate reductase isolated from Lactobacillus casei. Free drug is separated from bound drug by adsorption to dextran-coated charcoal. Piritrexim is measurable over a range of 0.01 to 10.0 micrograms/mL in plasma with a coefficient of variation less than 15%. The limit of sensitivity of the assay is approximately 2 ng/mL. An excellent correlation between this assay and a previously published HPLC method was found.

  16. [Platelet cytochrome c-oxidase and glutamine synthetase-like protein in patients with mild cognitive impairment].

    PubMed

    Burbaeva, G Sh; Boksha, I S; Savushkina, O K; Turishcheva, M S; Tereshkina, E B; Starodubtseva, L I; Gavrilova, S I; Fedorova, Ia B; Zhuravin, I A

    2012-01-01

    The study aimed to develop pre-clinical diagnosis of Alzheimer's disease (AD) and - in future - preventive therapy in patients with mild cognitive impairment (MCI). The MCI group (n=44) and AD group (n=42, including 18 patients with soft dementia and 24 patients with mild dementia) were studied. The groups were matched for age (median 70 and 69 years for MCI and AD groups, respectively). The control group comprised 24 mentally healthy relatives of the patients. Correlations between the activity/amounts of platelet enzymes: cytochrome c-oxidase (COX), glutamine synthetase-like protein (GSLP) and the extent of cognitive impairment were studied. The COX activity in MCI and AD groups was significantly lower than in the control group (Kruskal-Wallis test p=0.0001, χ²=11.6, p=0.003). These tests showed significant differences in GSLP amount between three groups (p=0.04 and χ²=9.38, p=0.01, respectively). Significant reverse correlation (Spearman R= -0.43, p=0.007) was found between GSLP amount and MMSE scores for MCI+AD group, i.e., the lower MMSE scores, the higher platelet GSLP level. Platelet COX and GSLP may be considered as early markers of cognitive impairment.

  17. Glutamine supplementation in the newborn infant.

    PubMed

    Parimi, Prabhu S; Kalhan, Satish C

    2007-02-01

    Glutamine is a non-essential amino acid that can be synthesized de novo from glutamate. This synthesis can be increased by intravenous infusion of carbon precursors (alpha-ketoglutarate or amino acids) in adults and in infants. The metabolism of glutamine is highly compartmentalized between the splanchnic tissues and the periphery, so that orally administered glutamine is completely metabolized in the splanchnic compartment. Data from studies in adults and children show that plasma levels of glutamine decline during acute stress and illness. Because of its importance in several physiological functions (the demonstrated benefits of supplemental glutamine in adult burns and trauma patients and the inhibitory effect on proteolysis in the skeletal muscle in vitro), it has been suggested that during 'acute stress' the demands of glutamine outweigh its de novo synthesis, resulting in a fall in plasma glutamine levels. As a consequence, glutamine has been considered a 'conditionally essential' amino acid. Because of its instability in solution, glutamine is not routinely added to the parenteral amino acid mixtures. A number of clinical trials of parenteral and enteral supplementation of glutamine have been performed. The outcome measures examined have varied between acute effects and long-term complex clinical events such as mortality and risk of infections. Although acute studies in LBW babies have shown some beneficial effects such as changes in protein metabolism and activation of immune system, these have not been translated into prolonged advantages such as reduction in mortality or in nosocomial infection. The reasons for these differences are discussed.

  18. Mechanical unfolding of ribose binding protein and its comparison with other periplasmic binding proteins.

    PubMed

    Kotamarthi, Hema Chandra; Narayan, Satya; Ainavarapu, Sri Rama Koti

    2014-10-01

    Folding and unfolding studies on large, multidomain proteins are still rare despite their high abundance in genomes of prokaryotes and eukaryotes. Here, we investigate the unfolding properties of a 271 residue, two-domain ribose binding protein (RBP) from the bacterial periplasm using single-molecule force spectroscopy. We observe that RBP predominately unfolds via a two-state pathway with an unfolding force of ∼80 pN and an unfolding contour length of ∼95 nm. Only a small population (∼15%) of RBP follows three-state pathways. The ligand binding neither increases the mechanical stability nor influences the unfolding flux of RBP through different pathways. The kinetic partitioning between two-state and three-state pathways, which has been reported earlier for other periplasmic proteins, is also observed in RBP, albeit to a lesser extent. These results provide important insights into the mechanical stability and unfolding processes of large two-domain proteins and highlight the contrasting features upon ligand binding. Protein structural topology diagrams are used to explain the differences in the mechanical unfolding behavior of RBP with other periplasmic binding proteins.

  19. Human complement protein C99 is a calcium binding protein

    SciTech Connect

    Thielens, N.M.; Lohner, K.; Esser, A.F.

    1988-05-15

    Human complement protein C9 is shown to be a metalloprotein that binds 1 mol of Ca/sup 2 +//mol of C9 with a dissociation constant of 3 ..mu..m as measured by equilibrium dialysis. Incubation with EDTA removes the bound calcium, resulting in a apoprotein with decreased thermal stability. This loss in stability leads to aggregation and, therefore, to loss of hemolytic activity upon heating to a few degrees above the physiological temperature. Heat-induced aggregation of apoC9 can be prevented by salts that stabilize proteins according to the Hofmeister series of lyotropic ions, suggesting that the ion in native C9 may ligand with more than one structural element of domain of the protein. Ligand blotting indicates that the calcium binding site is located in the amino-terminal half of the protein. Removal of calcium by inclusion of EDTA in assay mixtures has no effect on the hemolytic activity of C9, and its capacity to bind to C8 in solution, or to small unilamellar lipid vesicles at temperatures at or below the physiological range. Although the precise structural and functional role of the bound calcium is not know, it is clear that it provides thermal stability to C9 and it may have a function in regulation of membrane insertion.

  20. Glutamine supplementation in the critically ill: friend or foe?

    PubMed

    Oudemans-van Straaten, Heleen M; van Zanten, Arthur R H

    2014-05-19

    In the previous issue of Critical Care, Mori and colleagues demonstrate that glutamine supplementation in mechanically ventilated patients as part of parenteral nutrition increases plasma glutamine concentration and glutamine utilization, but does not mitigate protein degradation and even increases de novo glutamine production. Studies suggest that protein degradation is regulated by the degree of inflammation. Immune cells utilize large amounts of glutamine and derive their glutamine requirements from muscle protein degradation. We hypothesize that the effects of glutamine supplementation depend on the degree of inflammation. Infusing large amounts of exogenous glutamine into patients with inflammatory conditions like sepsis and multiple organ failure may not only enhance immune competence, but may potentially augment the inflammatory response and thereby negatively influence outcome.

  1. Landscape of protein-small ligand binding modes.

    PubMed

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them. PMID:27327045

  2. Landscape of protein-small ligand binding modes.

    PubMed

    Kasahara, Kota; Kinoshita, Kengo

    2016-09-01

    Elucidating the mechanisms of specific small-molecule (ligand) recognition by proteins is a long-standing conundrum. While the structures of these molecules, proteins and ligands, have been extensively studied, protein-ligand interactions, or binding modes, have not been comprehensively analyzed. Although methods for assessing similarities of binding site structures have been extensively developed, the methods for the computational treatment of binding modes have not been well established. Here, we developed a computational method for encoding the information about binding modes as graphs, and assessing their similarities. An all-against-all comparison of 20,040 protein-ligand complexes provided the landscape of the protein-ligand binding modes and its relationships with protein- and chemical spaces. While similar proteins in the same SCOP Family tend to bind relatively similar ligands with similar binding modes, the correlation between ligand and binding similarities was not very high (R(2)  = 0.443). We found many pairs with novel relationships, in which two evolutionally distant proteins recognize dissimilar ligands by similar binding modes (757,474 pairs out of 200,790,780 pairs were categorized into this relationship, in our dataset). In addition, there were an abundance of pairs of homologous proteins binding to similar ligands with different binding modes (68,217 pairs). Our results showed that many interesting relationships between protein-ligand complexes are still hidden in the structure database, and our new method for assessing binding mode similarities is effective to find them.

  3. Regulation of Pluripotency by RNA Binding Proteins

    PubMed Central

    Ye, Julia; Blelloch, Robert

    2015-01-01

    Establishment, maintenance, and exit from pluripotency require precise coordination of a cell’s molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells. PMID:25192462

  4. Gene encoding herbicide safener binding protein

    DOEpatents

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

  5. Computational Design of DNA-Binding Proteins.

    PubMed

    Thyme, Summer; Song, Yifan

    2016-01-01

    Predicting the outcome of engineered and naturally occurring sequence perturbations to protein-DNA interfaces requires accurate computational modeling technologies. It has been well established that computational design to accommodate small numbers of DNA target site substitutions is possible. This chapter details the basic method of design used in the Rosetta macromolecular modeling program that has been successfully used to modulate the specificity of DNA-binding proteins. More recently, combining computational design and directed evolution has become a common approach for increasing the success rate of protein engineering projects. The power of such high-throughput screening depends on computational methods producing multiple potential solutions. Therefore, this chapter describes several protocols for increasing the diversity of designed output. Lastly, we describe an approach for building comparative models of protein-DNA complexes in order to utilize information from homologous sequences. These models can be used to explore how nature modulates specificity of protein-DNA interfaces and potentially can even be used as starting templates for further engineering. PMID:27094297

  6. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    ERIC Educational Resources Information Center

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  7. Two hypervariable minisatellite DNA binding proteins.

    PubMed

    Wahls, W P; Swenson, G; Moore, P D

    1991-06-25

    Hypervariable minisatellite DNA sequences are short, tandemly repeated sequences present at numerous loci in eukaryotes. They stimulate intermolecular homologous recombination up to 13-fold in human cells in culture and may be specific sites for the initiation of recombination in the eukaryotic genome (Wahls, W.P., Wallace, L.J., & Moore, P.D. (1990) Cell 60, 95-103). Reported here is the detection and partial purification of two hypervariable minisatellite DNA binding proteins, called Msbp-2 and Msbp-3, present in the nuclear extracts of human HeLa cells. The proteins elute from a gel filtration column with a native mass of 200-250 kDa and have sizes of 77 kDa and 115 kDa respectively. PMID:2062643

  8. Alternative polyadenylation and RNA-binding proteins.

    PubMed

    Erson-Bensan, Ayse Elif

    2016-08-01

    Our understanding of the extent of microRNA-based gene regulation has expanded in an impressive pace over the past decade. Now, we are beginning to better appreciate the role of 3'-UTR (untranslated region) cis-elements which harbor not only microRNA but also RNA-binding protein (RBP) binding sites that have significant effect on the stability and translational rate of mRNAs. To add further complexity, alternative polyadenylation (APA) emerges as a widespread mechanism to regulate gene expression by producing shorter or longer mRNA isoforms that differ in the length of their 3'-UTRs or even coding sequences. Resulting shorter mRNA isoforms generally lack cis-elements where trans-acting factors bind, and hence are differentially regulated compared with the longer isoforms. This review focuses on the RBPs involved in APA regulation and their action mechanisms on APA-generated isoforms. A better understanding of the complex interactions between APA and RBPs is promising for mechanistic and clinical implications including biomarker discovery and new therapeutic approaches. PMID:27208003

  9. Butachlor impact on protein, free amino acid and glutamine contents, and on activity levels of aminotransferases, glutamate dehydrogenase and glutamine synthetase in the fresh water snail, Pila globosa (Swainson).

    PubMed

    Rajyalakshmi, T; Srinivas, T; Swamy, K V; Mohan, P M

    1996-08-01

    Biochemical changes followed in the freshwater snail Pila globosa (Swainson) during exposure to sublethal concentrations of the herbicide butachlor (26.6 ppm) in the ambient medium, at 3,6,12,24 and 48 h intervals, were marked by a significant decrease in total and soluble proteins, and an increase in free amino acids in foot and hepatopancreas up to 12 h before gradually recovering. Aminotransferase activities and glutamine content decreased during the early periods of exposure, while glutamate dehydrogenase activity increased. After an initial elevation, glutamate synthetase activity decreased at later intervals. Maximum effect of butachlor on the enzymes was seen after 12 h exposure. The extent of increase or decrease in different parameters examined varied between the two tissues studied. These changes are discussed in relation to the toxic stress of butachlor.

  10. Comparison of the Folding Mechanism of Highly Homologous Proteins in the Lipid-binding Protein Family

    EPA Science Inventory

    The folding mechanism of two closely related proteins in the intracellular lipid binding protein family, human bile acid binding protein (hBABP) and rat bile acid binding protein (rBABP) were examined. These proteins are 77% identical (93% similar) in sequence Both of these singl...

  11. Helical Defects in MicroRNA Influence Protein Binding by TAR RNA Binding Protein

    PubMed Central

    Acevedo, Roderico; Orench-Rivera, Nichole; Quarles, Kaycee A.; Showalter, Scott A.

    2015-01-01

    Background MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression. Their precursors have a globally A-form helical geometry, which prevents most proteins from identifying their nucleotide sequence. This suggests the hypothesis that local structural features (e.g., bulges, internal loops) play a central role in specific double-stranded RNA (dsRNA) selection from cellular RNA pools by dsRNA binding domain (dsRBD) containing proteins. Furthermore, the processing enzymes in the miRNA maturation pathway require tandem-dsRBD cofactor proteins for optimal function, suggesting that dsRBDs play a key role in the molecular mechanism for precise positioning of the RNA within these multi-protein complexes. Here, we focus on the tandem-dsRBDs of TRBP, which have been shown to bind dsRNA tightly. Methodology/Principal Findings We present a combination of dsRNA binding assays demonstrating that TRBP binds dsRNA in an RNA-length dependent manner. Moreover, circular dichroism data shows that the number of dsRBD moieties bound to RNA at saturation is different for a tandem-dsRBD construct than for constructs with only one dsRBD per polypeptide, revealing another reason for the selective pressure to maintain multiple domains within a polypeptide chain. Finally, we show that helical defects in precursor miRNA alter the apparent dsRNA size, demonstrating that imperfections in RNA structure influence the strength of TRBP binding. Conclusion/Significance We conclude that TRBP is responsible for recognizing structural imperfections in miRNA precursors, in the sense that TRBP is unable to bind imperfections efficiently and thus is positioned around them. We propose that once positioned around structural defects, TRBP assists Dicer and the rest of the RNA-induced silencing complex (RISC) in providing efficient and homogenous conversion of substrate precursor miRNA into mature miRNA downstream. PMID:25608000

  12. Glutamine synthetase in durum wheat: Genotypic variation and relationship with grain protein content

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrogen Use Efficiency (NUE), one of the most valuable indicators for nitrogen use in crops, both in terms of yield and final grain protein content (GPC), is a very complex trait. The identification of wheat varieties with high NUE, as well as the characterization of central enzymes involved in th...

  13. Roles for RNA-binding proteins in development and disease.

    PubMed

    Brinegar, Amy E; Cooper, Thomas A

    2016-09-15

    RNA-binding protein activities are highly regulated through protein levels, intracellular localization, and post-translation modifications. During development, mRNA processing of specific gene sets is regulated through manipulation of functional RNA-binding protein activities. The impact of altered RNA-binding protein activities also affects human diseases in which there are either a gain-of-function or loss-of-function causes pathogenesis. We will discuss RNA-binding proteins and their normal developmental RNA metabolism and contrast how their function is disrupted in disease. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease.

  14. Glutamine and cancer.

    PubMed Central

    Souba, W W

    1993-01-01

    OBJECTIVE: This overview on glutamine and cancer discusses the importance of glutamine for tumor growth, summarizes the alterations in interorgan glutamine metabolism that develop in the tumor-bearing host, and reviews the potential benefits of glutamine nutrition in the patient with cancer. SUMMARY BACKGROUND DATA: Glutamine is the most abundant amino acid in the blood and tissues. It is essential for tumor growth and marked changes in organ glutamine metabolism are characteristic of the host with cancer. Because host glutamine depletion has adverse effects, it is important to study the regulation of glutamine metabolism in cancer and to evaluate the impact of glutamine nutrition in the tumor-bearing state. METHODS: Data from a variety of investigations on glutamine metabolism and nutrition related to the host with cancer were compiled and summarized. RESULTS: Numerous studies on glutamine metabolism in cancer indicate that many tumors are avid glutamine consumers in vivo and in vitro. As a consequence of progressive tumor growth, host glutamine depletion develops and becomes a hallmark. This glutamine depletion occurs in part because the tumor behaves as a "glutamine trap" but also because of cytokine-mediated alterations in glutamine metabolism in host tissues. Animal and human studies that have investigated the use of glutamine-supplemented nutrition in the host with cancer suggest that pharmacologic doses of dietary glutamine may be beneficial. CONCLUSIONS: Understanding the control of glutamine metabolism in the tumor-bearing host not only improves the knowledge of metabolic regulation in the patient with cancer but also will lead to improved nutritional support regimens targeted to benefit the host. PMID:8257221

  15. Calcyclin Binding Protein/Siah-1 Interacting Protein Is a Hsp90 Binding Chaperone

    PubMed Central

    Góral, Agnieszka; Bieganowski, Paweł; Prus, Wiktor; Krzemień-Ojak, Łucja; Kądziołka, Beata; Fabczak, Hanna; Filipek, Anna

    2016-01-01

    The Hsp90 chaperone activity is tightly regulated by interaction with many co-chaperones. Since CacyBP/SIP shares some sequence homology with a known Hsp90 co-chaperone, Sgt1, in this work we performed a set of experiments in order to verify whether CacyBP/SIP can interact with Hsp90. By applying the immunoprecipitation assay we have found that CacyBP/SIP binds to Hsp90 and that the middle (M) domain of Hsp90 is responsible for this binding. Furthermore, the proximity ligation assay (PLA) performed on HEp-2 cells has shown that the CacyBP/SIP-Hsp90 complexes are mainly localized in the cytoplasm of these cells. Using purified proteins and applying an ELISA we have shown that Hsp90 interacts directly with CacyBP/SIP and that the latter protein does not compete with Sgt1 for the binding to Hsp90. Moreover, inhibitors of Hsp90 do not perturb CacyBP/SIP-Hsp90 binding. Luciferase renaturation assay and citrate synthase aggregation assay with the use of recombinant proteins have revealed that CacyBP/SIP exhibits chaperone properties. Also, CacyBP/SIP-3xFLAG expression in HEp-2 cells results in the appearance of more basic Hsp90 forms in 2D electrophoresis, which may indicate that CacyBP/SIP dephosphorylates Hsp90. Altogether, the obtained results suggest that CacyBP/SIP is involved in regulation of the Hsp90 chaperone machinery. PMID:27249023

  16. Latent TGF-β-binding proteins

    PubMed Central

    Robertson, Ian B.; Horiguchi, Masahito; Zilberberg, Lior; Dabovic, Branka; Hadjiolova, Krassimira; Rifkin, Daniel B.

    2016-01-01

    The LTBPs (or latent transforming growth factor β binding proteins) are important components of the extracellular matrix (ECM) that interact with fibrillin microfibrils and have a number of different roles in microfibril biology. There are four LTBPs isoforms in the human genome (LTBP-1, -2, -3, and -4), all of which appear to associate with fibrillin and the biology of each isoform is reviewed here. The LTBPs were first identified as forming latent complexes with TGFβ by covalently binding the TGFβ propeptide (LAP) via disulfide bonds in the endoplasmic reticulum. LAP in turn is cleaved from the mature TGFβ precursor in the trans golgi network but LAP and TGFβ remain strongly bound through non-covalent interactions. LAP, TGFβ, and LTBP together form the large latent complex (LLC). LTBPs were originally thought to primarily play a role in maintaining TGFβ latency and targeting the latent growth factor to the extracellular matrix (ECM), but it has also been shown that LTBP-1 participates in TGFβ activation by integrins and may also regulate activation by proteases and other factors. LTBP-3 appears to have a role in skeletal formation including tooth development. As well as having important functions in TGFβ regulation, TGFβ-independent activities have recently been identified for LTBP-2 and LTBP-4 in stabilizing microfibril bundles and regulating elastic fiber assembly. PMID:25960419

  17. Characterizing the morphology of protein binding patches.

    PubMed

    Malod-Dognin, Noël; Bansal, Achin; Cazals, Frédéric

    2012-12-01

    Let the patch of a partner in a protein complex be the collection of atoms accounting for the interaction. To improve our understanding of the structure-function relationship, we present a patch model decoupling the topological and geometric properties. While the geometry is classically encoded by the atomic positions, the topology is recorded in a graph encoding the relative position of concentric shells partitioning the interface atoms. The topological-geometric duality provides the basis of a generic dynamic programming-based algorithm comparing patches at the shell level, which may favor topological or geometric features. On the biological side, we address four questions, using 249 cocrystallized heterodimers organized in biological families. First, we dissect the morphology of binding patches and show that Nature enjoyed the topological and geometric degrees of freedom independently while retaining a finite set of qualitatively distinct topological signatures. Second, we argue that our shell-based comparison is effective to perform atomic-level comparisons and show that topological similarity is a less stringent than geometric similarity. We also use the topological versus geometric duality to exhibit topo-rigid patches, whose topology (but not geometry) remains stable upon docking. Third, we use our comparison algorithms to infer specificity-related information amidst a database of complexes. Finally, we exhibit a descriptor outperforming its contenders to predict the binding affinities of the affinity benchmark. The softwares developed with this article are availablefrom http://team.inria.fr/abs/vorpatch_compatch/.

  18. Liver Fatty Acid Binding Protein and Obesity

    PubMed Central

    Atshaves, B.P.; Martin, G.G.; Hostetler, H.A.; McIntosh, A.L.; Kier, A.B.; Schroeder, F.

    2010-01-01

    While low levels of unesterified long chain fatty acids (LCFAs) are normal metabolic intermediates of dietary and endogenous fat, LCFAs are also potent regulators of key receptors/enzymes, and at high levels become toxic detergents within the cell. Elevated levels of LCFAs are associated with diabetes, obesity, and metabolic syndrome. Consequently, mammals evolved fatty acid binding proteins (FABPs) that bind/sequester these potentially toxic free fatty acids in the cytosol and present them for rapid removal in oxidative (mitochondria, peroxisomes) or storage (endoplasmic reticulum, lipid droplets) organelles. Mammals have a large (15 member) family of FABPs with multiple members occurring within a single cell type. The first described FABP, liver-FABP (L-FABP, or FABP1), is expressed in very high levels (2-5% of cytosolic protein) in liver as well as intestine and kidney. Since L-FABP facilitates uptake and metabolism of LCFAs in vitro and in cultured cells, it was expected that abnormal function or loss of L-FABP would reduce hepatic LCFA uptake/oxidation and thereby increase LCFAs available for oxidation in muscle and/or storage in adipose. This prediction was confirmed in vitro with isolated liver slices and cultured primary hepatocytes from L-FABP gene-ablated mice. Despite unaltered food consumption when fed a control diet ad libitum, the L-FABP null mice exhibited age- and sex-dependent weight gain and increased fat tissue mass. The obese phenotype was exacerbated in L-FABP null mice pair-fed a high fat diet. Taken together with other findings, these data suggest that L-FABP could have an important role in preventing age- or diet-induced obesity. PMID:20537520

  19. Protein function annotation by local binding site surface similarity.

    PubMed

    Spitzer, Russell; Cleves, Ann E; Varela, Rocco; Jain, Ajay N

    2014-04-01

    Hundreds of protein crystal structures exist for proteins whose function cannot be confidently determined from sequence similarity. Surflex-PSIM, a previously reported surface-based protein similarity algorithm, provides an alternative method for hypothesizing function for such proteins. The method now supports fully automatic binding site detection and is fast enough to screen comprehensive databases of protein binding sites. The binding site detection methodology was validated on apo/holo cognate protein pairs, correctly identifying 91% of ligand binding sites in holo structures and 88% in apo structures where corresponding sites existed. For correctly detected apo binding sites, the cognate holo site was the most similar binding site 87% of the time. PSIM was used to screen a set of proteins that had poorly characterized functions at the time of crystallization, but were later biochemically annotated. Using a fully automated protocol, this set of 8 proteins was screened against ∼60,000 ligand binding sites from the PDB. PSIM correctly identified functional matches that predated query protein biochemical annotation for five out of the eight query proteins. A panel of 12 currently unannotated proteins was also screened, resulting in a large number of statistically significant binding site matches, some of which suggest likely functions for the poorly characterized proteins.

  20. Detection of secondary binding sites in proteins using fragment screening

    PubMed Central

    Ludlow, R. Frederick; Verdonk, Marcel L.; Saini, Harpreet K.; Tickle, Ian J.; Jhoti, Harren

    2015-01-01

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets. PMID:26655740

  1. Detection of secondary binding sites in proteins using fragment screening.

    PubMed

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  2. Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

    PubMed

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

    The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids. PMID:27094290

  3. Minimalistic predictor of protein binding energy: contribution of solvation factor to protein binding.

    PubMed

    Choi, Jeong-Mo; Serohijos, Adrian W R; Murphy, Sean; Lucarelli, Dennis; Lofranco, Leo L; Feldman, Andrew; Shakhnovich, Eugene I

    2015-02-17

    It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: http://faculty.chemistry.harvard.edu/shakhnovich/software.

  4. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins.

    PubMed

    Figueroa-Angulo, Elisa E; Calla-Choque, Jaeson S; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-11-26

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis.

  5. Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

    PubMed

    Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

    2014-10-01

    Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets.

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

    SciTech Connect

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

    2014-07-01

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

  7. Topological Analyses of Protein-Ligand Binding: a Network Approach.

    PubMed

    Costanzi, Stefano

    2016-01-01

    Proteins can be conveniently represented as networks of interacting residues, thus allowing the study of several network parameters that can shed light onto several of their structural and functional aspects. With respect to the binding of ligands, which are central for the function of many proteins, network analysis may constitute a possible route to assist the identification of binding sites. As the bulk of this review illustrates, this has generally been easier for enzymes than for non-enzyme proteins, perhaps due to the different topological nature of the binding sites of the former over those of the latter. The article also illustrates how network representations of binding sites can be used to search PDB structures in order to identify proteins that bind similar molecules and, lastly, how codifying proteins as networks can assist the analysis of the conformational changes consequent to ligand binding.

  8. Minisatellite binding protein Msbp-1 is a sequence-specific single-stranded DNA-binding protein.

    PubMed Central

    Collick, A; Dunn, M G; Jeffreys, A J

    1991-01-01

    Msbp-1 is a minisatellite-specific DNA-binding protein. Using synthetic binding substrates, we now show that Msbp-1 binds not to double-stranded DNA, but exclusively to single-stranded DNA. Binding is specific to the guanine-rich strand of the minisatellite duplex, interactions with the cytosine-rich strand being undetectable by southwestern analysis. Furthermore, the binding site required for successful DNA-protein interactions appears to be two or more minisatellite repeat units. We have also isolated, by whole-genome PCR and cloning, one Msbp-1 binding site from the human genome. Again, the binding strand of this molecule contains a repetitive G-rich structure equivalent to that of a small minisatellite. These observations are discussed with respect to other single-stranded DNA-binding proteins known to play a role in recombination processes. Images PMID:1754375

  9. Conformity of RNAs that interact with tetranucleotide loop binding proteins.

    PubMed Central

    Zwieb, C

    1992-01-01

    A group of RNA binding proteins, termed tetraloop binding proteins, includes ribosomal protein S15 and protein SRP19 of signal recognition particle. They are primary RNA binding proteins, recognize RNA tetranucleotide loops with a GNAR consensus motif, and require a helical region located adjacent to the tetraloop. Closely related RNA structures that fit these criteria appear in helix 6 of SRP RNA, in helices 22 and 23A of 16 S ribosomal RNA, and, as a pseudoknot, in the regulatory region of the rpsO gene. Images PMID:1329024

  10. Imbalanced expression of glutamate-glutamine cycle enzymes induced by human T-cell lymphotropic virus type 1 Tax protein in cultivated astrocytes.

    PubMed Central

    Akaoka, H; Hardin-Pouzet, H; Bernard, A; Verrier, B; Belin, M F; Giraudon, P

    1996-01-01

    Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent involved in the disease HTLV-1-associated myelopathy, or tropical spastic paraparesis (HAM/TSP). The pathogenesis of HAM/TSP is poorly understood, but it is probable that viral infection has an indirect, deleterious effect on neural function. In this regard, dysfunction in astrocytes may be severely detrimental, as they supply neurons with metabolic precursors, control the extracellular levels of ion and excitatory neurotransmitters, and are electrically coupled with oligodendrocytes. In a model in vitro, we demonstrate that HTLV-1 induces an imbalance in the expression of two astrocyte enzymes, at both the transcriptional and translational levels. In both human astrocyte precursors and rat glial cells, the levels of expression of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were increased and decreased, respectively, after coculture with HTLV-1 T cells. The enhancement of GS expression may result from the action of the protein Tax, which is demonstrated to transactivate the GS gene promoter, while the decreased expression of GDH seems to reflect some compensatory mechanism in response to GS induction. GS and GDH are involved in the conversion of glutamate into glutamine or alpha-ketoglutarate, which then acts as a precursor for glutamatergic and gamma-aminobutyric acid (GABA)-ergic neurons. Metabolism in astrocytes altered by Tax protein may lead to deleterious effects if it modifies the extracellular levels of glutamine, glutamate, and GABA and thus modulates neuronal excitability and osmotic equilibrium in the central nervous system of HTLV-1-infected patients. PMID:8971000

  11. A β-hairpin-binding protein for three different disease-related amyloidogenic proteins.

    PubMed

    Shaykhalishahi, Hamed; Mirecka, Ewa A; Gauhar, Aziz; Grüning, Clara S R; Willbold, Dieter; Härd, Torleif; Stoldt, Matthias; Hoyer, Wolfgang

    2015-02-01

    Amyloidogenic proteins share a propensity to convert to the β-structure-rich amyloid state that is associated with the progression of several protein-misfolding disorders. Here we show that a single engineered β-hairpin-binding protein, the β-wrapin AS10, binds monomers of three different amyloidogenic proteins, that is, amyloid-β peptide, α-synuclein, and islet amyloid polypeptide, with sub-micromolar affinity. AS10 binding inhibits the aggregation and toxicity of all three proteins. The results demonstrate common conformational preferences and related binding sites in a subset of the amyloidogenic proteins. These commonalities enable the generation of multispecific monomer-binding agents.

  12. Calmodulin Binding Proteins and Alzheimer’s Disease

    PubMed Central

    O’Day, Danton H.; Eshak, Kristeen; Myre, Michael A.

    2015-01-01

    Abstract The small, calcium-sensor protein, calmodulin, is ubiquitously expressed and central to cell function in all cell types. Here the literature linking calmodulin to Alzheimer’s disease is reviewed. Several experimentally-verified calmodulin-binding proteins are involved in the formation of amyloid-β plaques including amyloid-β protein precursor, β-secretase, presenilin-1, and ADAM10. Many others possess potential calmodulin-binding domains that remain to be verified. Three calmodulin binding proteins are associated with the formation of neurofibrillary tangles: two kinases (CaMKII, CDK5) and one protein phosphatase (PP2B or calcineurin). Many of the genes recently identified by genome wide association studies and other studies encode proteins that contain putative calmodulin-binding domains but only a couple (e.g., APOE, BIN1) have been experimentally confirmed as calmodulin binding proteins. At least two receptors involved in calcium metabolism and linked to Alzheimer’s disease (mAchR; NMDAR) have also been identified as calmodulin-binding proteins. In addition to this, many proteins that are involved in other cellular events intimately associated with Alzheimer’s disease including calcium channel function, cholesterol metabolism, neuroinflammation, endocytosis, cell cycle events, and apoptosis have been tentatively or experimentally verified as calmodulin binding proteins. The use of calmodulin as a potential biomarker and as a therapeutic target is discussed. PMID:25812852

  13. Protein Binding: Do We Ever Learn?▿

    PubMed Central

    Zeitlinger, Markus A.; Derendorf, Hartmut; Mouton, Johan W.; Cars, Otto; Craig, William A.; Andes, David; Theuretzbacher, Ursula

    2011-01-01

    Although the influence of protein binding (PB) on antibacterial activity has been reported for many antibiotics and over many years, there is currently no standardization for pharmacodynamic models that account for the impact of protein binding of antimicrobial agents in vitro. This might explain the somewhat contradictory results obtained from different studies. Simple in vitro models which compare the MIC obtained in protein-free standard medium versus a protein-rich medium are prone to methodological pitfalls and may lead to flawed conclusions. Within in vitro test systems, a range of test conditions, including source of protein, concentration of the tested antibiotic, temperature, pH, electrolytes, and supplements may influence the impact of protein binding. As new antibiotics with a high degree of protein binding are in clinical development, attention and action directed toward the optimization and standardization of testing the impact of protein binding on the activity of antibiotics in vitro become even more urgent. In addition, the quantitative relationship between the effects of protein binding in vitro and in vivo needs to be established, since the physiological conditions differ. General recommendations for testing the impact of protein binding in vitro are suggested. PMID:21537013

  14. Actin binding proteins, spermatid transport and spermiation.

    PubMed

    Qian, Xiaojing; Mruk, Dolores D; Cheng, Yan-Ho; Tang, Elizabeth I; Han, Daishu; Lee, Will M; Wong, Elissa W P; Cheng, C Yan

    2014-06-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby arriving the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium beyond stage VIII of the epithelial cycle will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come.

  15. Odorant-Binding Protein: Localization to Nasal Glands and Secretions

    NASA Astrophysics Data System (ADS)

    Pevsner, Jonathan; Sklar, Pamela B.; Snyder, Solomon H.

    1986-07-01

    An odorant-binding protein (OBP) was isolated from bovine olfactory and respiratory mucosa. We have produced polyclonal antisera to this protein and report its immunohistochemical localization to mucus-secreting glands of the olfactory and respiratory mucosa. Although OBP was originally isolated as a pyrazine binding protein, both rat and bovine OBP also bind the odorants [3H]methyldihydrojasmonate and 3,7-dimethyl-octan-1-ol as well as 2-isobutyl-3-[3H]methoxypyrazine. We detect substantial odorant-binding activity attributable to OBP in secreted rat nasal mucus and tears but not in saliva, suggesting a role for OBP in transporting or concentrating odorants.

  16. Therapeutic and analytical applications of arsenic binding to proteins.

    PubMed

    Chen, Beibei; Liu, Qingqing; Popowich, Aleksandra; Shen, Shengwen; Yan, Xiaowen; Zhang, Qi; Li, Xing-Fang; Weinfeld, Michael; Cullen, William R; Le, X Chris

    2015-01-01

    Arsenic binding to proteins plays a pivotal role in the health effects of arsenic. Further knowledge of arsenic binding to proteins will advance the development of bioanalytical techniques and therapeutic drugs. This review summarizes recent work on arsenic-based drugs, imaging of cellular events, capture and purification of arsenic-binding proteins, and biosensing of arsenic. Binding of arsenic to the promyelocytic leukemia fusion oncoprotein (PML-RARα) is a plausible mode of action leading to the successful treatment of acute promyelocytic leukemia (APL). Identification of other oncoproteins critical to other cancers and the development of various arsenicals and targeted delivery systems are promising approaches to the treatment of other types of cancers. Techniques for capture, purification, and identification of arsenic-binding proteins make use of specific binding between trivalent arsenicals and the thiols in proteins. Biarsenical probes, such as FlAsH-EDT2 and ReAsH-EDT2, coupled with tetracysteine tags that are genetically incorporated into the target proteins, are used for site-specific fluorescence labelling and imaging of the target proteins in living cells. These allow protein dynamics and protein-protein interactions to be studied. Arsenic affinity chromatography is useful for purification of thiol-containing proteins, and its combination with mass spectrometry provides a targeted proteomic approach for studying the interactions between arsenicals and proteins in cells. Arsenic biosensors evolved from the knowledge of arsenic resistance and arsenic binding to proteins in bacteria, and have now been developed into analytical techniques that are suitable for the detection of arsenic in the field. Examples in the four areas, arsenic-based drugs, imaging of cellular events, purification of specific proteins, and arsenic biosensors, demonstrate important therapeutic and analytical applications of arsenic protein binding. PMID:25356501

  17. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen P.

    2006-10-17

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  18. RNA binding protein and binding site useful for expression of recombinant molecules

    DOEpatents

    Mayfield, Stephen

    2000-01-01

    The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

  19. Immobilized purified folate-binding protein: binding characteristics and use for quantifying folate in erythrocytes

    SciTech Connect

    Hansen, S.I.; Holm, J.; Nexo, E.

    1987-08-01

    Purified folate-binding protein from cow's milk was immobilized on monodisperse polymer particles (Dynospheres) activated by rho-toluenesulfonyl chloride. Leakage from the spheres was less than 0.1%, and the binding properties were similar to those of the soluble protein with regard to dissociation, pH optimum for binding pteroylglutamic acid, and specificity for binding various folate derivatives. We used the immobilized folate-binding protein as binding protein in an isotope-dilution assay for quantifying folate in erythrocytes. The detection limit was 50 nmol/L and the CV over a six-month period was 2.3% (means = 1.25 mumol/L, n = 15). The reference interval, for folate measured in erythrocytes of 43 blood donors, was 0.4-1.5 mumol/L.

  20. Affinity Purification of Sequence-Specific DNA Binding Proteins

    NASA Astrophysics Data System (ADS)

    Kadonaga, James T.; Tjian, Robert

    1986-08-01

    We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed through the DNA-Sepharose resin. The desired sequence-specific DNA binding protein is purified because it preferentially binds to the recognition sites in the affinity resin rather than to the nonspecific competitor DNA in solution. For example, a protein fraction that is enriched for transcription factor Sp1 can be further purified 500- to 1000-fold by two sequential affinity chromatography steps to give Sp1 of an estimated 90% homogeneity with 30% yield. In addition, the use of tandem affinity columns containing different protein binding sites allows the simultaneous purification of multiple DNA binding proteins from the same extract. This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor.

  1. Characterization of the DNA binding properties of polyomavirus capsid protein

    NASA Technical Reports Server (NTRS)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  2. Clinical relevance of drug binding to plasma proteins

    NASA Astrophysics Data System (ADS)

    Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

    2014-12-01

    Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

  3. Global discovery of protein kinases and other nucleotide-binding proteins by mass spectrometry.

    PubMed

    Xiao, Yongsheng; Wang, Yinsheng

    2016-09-01

    Nucleotide-binding proteins, such as protein kinases, ATPases and GTP-binding proteins, are among the most important families of proteins that are involved in a number of pivotal cellular processes. However, global study of the structure, function, and expression level of nucleotide-binding proteins as well as protein-nucleotide interactions can hardly be achieved with the use of conventional approaches owing to enormous diversity of the nucleotide-binding protein family. Recent advances in mass spectrometry (MS) instrumentation, coupled with a variety of nucleotide-binding protein enrichment methods, rendered MS-based proteomics a powerful tool for the comprehensive characterizations of the nucleotide-binding proteome, especially the kinome. Here, we review the recent developments in the use of mass spectrometry, together with general and widely used affinity enrichment approaches, for the proteome-wide capture, identification and quantification of nucleotide-binding proteins, including protein kinases, ATPases, GTPases, and other nucleotide-binding proteins. The working principles, advantages, and limitations of each enrichment platform in identifying nucleotide-binding proteins as well as profiling protein-nucleotide interactions are summarized. The perspectives in developing novel MS-based nucleotide-binding protein detection platform are also discussed. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:601-619, 2016.

  4. Poly-A binding protein-1 localization to a subset of TAR DNA-binding protein of 43 kDa inclusions in amyotrophic lateral sclerosis occurs more frequently in patients harboring an expansion in C9orf72

    PubMed Central

    McGurk, Leeanne; Lee, Virginia, M.; Trojanowksi, John Q.; Van Deerlin, Vivianna M.; Lee, Edward B.; Bonini, Nancy M.

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease in which the loss of spinal cord motor neurons leads to paralysis and death within a few years of clinical disease onset. In almost all cases of ALS, TAR DNA binding protein of 43 kDa (TDP-43) forms cytoplasmic neuronal inclusions. A second causative gene for a subset of ALS is fused in sarcoma (FUS), an RNA binding protein that also forms cytoplasmic inclusions in spinal cord motor neurons. Poly A binding protein 1 (PABP-1) is a marker of stress granules, i.e. accumulations of proteins and RNA indicative of translational arrest in cells under stress. We report on the colocalization PABP-1 to both TDP-43 and FUS inclusions in 4 patient cohorts: ALS without a mutation, ALS with an intermediate poly glutamine repeat expansion in ATXN2, ALS with a GGGGCC-hexanucleotide repeat expansion in C9orf72, and ALS with basophilic inclusion body disease. Notably, PABP-1 colocalization to TDP-43 was twice as frequent in ALS with C9orf72 expansions compared to ALS with no mutation. This study highlights PABP-1 as a protein important to the pathology of ALS and indicates that the proteomic profile of TDP-43 inclusions in ALS may be different depending on the causative genetic mutation. PMID:25111021

  5. The 3′ untranslated region of a soybean cytosolic glutamine synthetase (GS1) affects transcript stability and protein accumulation in transgenic alfalfa

    PubMed Central

    Ortega, Jose L.; Moguel-Esponda, Salvador; Potenza, Carol; Conklin, Cristina F.; Quintana, Anita; Sengupta-Gopalan, Champa

    2013-01-01

    Summary Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). Glutamine synthetase 1 is regulated in different plants at the transcriptional level and there are some reports of regulation at the level of protein stability. Here we present data that clearly establish that GS1 in plants is also regulated at the level of transcript turnover and at the translational level. Using a Glycine max (soybean) GS1 transgene, with and without its 3′ untranslated region (UTR), driven by the constitutive CaMV 35S promoter in Medicago sativa (alfalfa) and Nicotiana tabacum (tobacco), we show that the 3′ UTR plays a major role in both transcript turnover and translation repression in both the leaves and the nodules. Our data suggest that the 3′ UTR mediated turnover of the transcript is regulated by a nitrogen metabolite or carbon/nitrogen ratios. We also show that the 3′ UTR of the gene for the soybean GS1 confers post-transcriptional regulation on a reporter gene. Our dissection of post-transcriptional and translational levels of regulation of GS in plants shows that the situation in plants strongly resembles that in other organisms where GS is regulated at almost all levels. Multistep regulation of GS shows the high priority given by organisms to regulating and ensuring optimal control of nitrogen substrates and preventing overproduction of glutamine and drainage of the glutamate pool. PMID:16460515

  6. The 3' untranslated region of a soybean cytosolic glutamine synthetase (GS1) affects transcript stability and protein accumulation in transgenic alfalfa.

    PubMed

    Ortega, Jose L; Moguel-Esponda, Salvador; Potenza, Carol; Conklin, Cristina F; Quintana, Anita; Sengupta-Gopalan, Champa

    2006-03-01

    Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). Glutamine synthetase 1 is regulated in different plants at the transcriptional level and there are some reports of regulation at the level of protein stability. Here we present data that clearly establish that GS1 in plants is also regulated at the level of transcript turnover and at the translational level. Using a Glycine max (soybean) GS1 transgene, with and without its 3' untranslated region (UTR), driven by the constitutive CaMV 35S promoter in Medicago sativa (alfalfa) and Nicotiana tabacum (tobacco), we show that the 3' UTR plays a major role in both transcript turnover and translation repression in both the leaves and the nodules. Our data suggest that the 3' UTR mediated turnover of the transcript is regulated by a nitrogen metabolite or carbon/nitrogen ratios. We also show that the 3' UTR of the gene for the soybean GS1 confers post-transcriptional regulation on a reporter gene. Our dissection of post-transcriptional and translational levels of regulation of GS in plants shows that the situation in plants strongly resembles that in other organisms where GS is regulated at almost all levels. Multistep regulation of GS shows the high priority given by organisms to regulating and ensuring optimal control of nitrogen substrates and preventing overproduction of glutamine and drainage of the glutamate pool.

  7. Search for Amyloid-Binding Proteins by Affinity Chromatography

    PubMed Central

    Calero, Miguel; Rostagno, Agueda; Ghiso, Jorge

    2013-01-01

    ‘Amyloid binging proteins’ is a generic term used to designate proteins that interact with different forms of amyloidogenic peptides or proteins and that, as a result, may modulate their physiological and pathological functions by altering solubility, transport, clearance, degradation, and fibril formation. We describe a simple affinity chromatography protocol to isolate and characterize amyloid-binding proteins based on the use of sequential elution steps that may provide further information on the type of binding interaction. As an example, we depict the application of this protocol to the study of Alzheimer’s amyloid β (Aβ) peptide-binding proteins derived from human plasma. Biochemical analysis of the proteins eluted under different conditions identified serum amyloid P component (SAP) and apolipoprotein J (clusterin) as the main plasma Aβ-binding proteins while various apolipoproteins (apoA-IV, apoE, and apoA-I), as well as albumin (HSA) and fibulin were identified as minor contributors. PMID:22528093

  8. Phosphorylation of platelet actin-binding protein during platelet activation

    SciTech Connect

    Carroll, R.C.; Gerrard, J.M.

    1982-03-01

    In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide-sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2 to 3-fold labeling increases in actin-binding protein 30 to 60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P-labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

  9. Concentration-dependent Cu(II) binding to prion protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry

    2008-03-01

    The prion protein plays a causative role in several neurodegenerative diseases, including mad cow disease in cattle and Creutzfeldt-Jakob disease in humans. The normal function of the prion protein is unknown, but it has been linked to its ability to bind copper ions. Experimental evidence suggests that copper can be bound in three distinct modes depending on its concentration, but only one of those binding modes has been fully characterized experimentally. Using a newly developed hybrid DFT/DFT method [1], which combines Kohn-Sham DFT with orbital-free DFT, we have examined all the binding modes and obtained their detailed binding geometries and copper ion binding energies. Our results also provide explanation for experiments, which have found that when the copper concentration increases the copper binding mode changes, surprisingly, from a stronger to a weaker one. Overall, our results indicate that prion protein can function as a copper buffer. 1. Hodak, Lu, Bernholc, JCP, in press.

  10. Cooperative binding modes of Cu(II) in prion protein

    NASA Astrophysics Data System (ADS)

    Hodak, Miroslav; Chisnell, Robin; Lu, Wenchang; Bernholc, Jerry

    2007-03-01

    The misfolding of the prion protein, PrP, is responsible for a group of neurodegenerative diseases including mad cow disease and Creutzfeldt-Jakob disease. It is known that the PrP can efficiently bind copper ions; four high-affinity binding sites located in the octarepeat region of PrP are now well known. Recent experiments suggest that at low copper concentrations new binding modes, in which one copper ion is shared between two or more binding sites, are possible. Using our hybrid Thomas-Fermi/DFT computational scheme, which is well suited for simulations of biomolecules in solution, we investigate the geometries and energetics of two, three and four binding sites cooperatively binding one copper ion. These geometries are then used as inputs for classical molecular dynamics simulations. We find that copper binding affects the secondary structure of the PrP and that it stabilizes the unstructured (unfolded) part of the protein.

  11. Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.

    PubMed

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

    2014-06-01

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

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

    PubMed Central

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

    2014-01-01

    Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states. PMID:25004958

  13. Stereoselective binding of chiral drugs to plasma proteins

    PubMed Central

    Shen, Qi; Wang, Lu; Zhou, Hui; Jiang, Hui-di; Yu, Lu-shan; Zeng, Su

    2013-01-01

    Chiral drugs show distinct biochemical and pharmacological behaviors in the human body. The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity, which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles. In this review, the stereoselective binding of chiral drugs to human serum albumin (HSA), α1-acid glycoprotein (AGP) and lipoprotein, three most important proteins in human plasma, are detailed. Furthermore, the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed. Apart from the stereoselectivity of enantiomer-protein binding, enantiomer-enantiomer interactions that may induce allosteric effects are also described. Additionally, the techniques and methods used to determine drug-protein binding parameters are briefly reviewed. PMID:23852086

  14. The RNA-binding protein Gemin5 binds directly to the ribosome and regulates global translation

    PubMed Central

    Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Ramajo, Jorge; Martinez-Salas, Encarnación

    2016-01-01

    RNA-binding proteins (RBPs) play crucial roles in all organisms. The protein Gemin5 harbors two functional domains. The N-terminal domain binds to snRNAs targeting them for snRNPs assembly, while the C-terminal domain binds to IRES elements through a non-canonical RNA-binding site. Here we report a comprehensive view of the Gemin5 interactome; most partners copurified with the N-terminal domain via RNA bridges. Notably, Gemin5 sediments with the subcellular ribosome fraction, and His-Gemin5 binds to ribosome particles via its N-terminal domain. The interaction with the ribosome was lost in F381A and Y474A Gemin5 mutants, but not in W14A and Y15A. Moreover, the ribosomal proteins L3 and L4 bind directly with Gemin5, and conversely, Gemin5 mutants impairing the binding to the ribosome are defective in the interaction with L3 and L4. The overall polysome profile was affected by Gemin5 depletion or overexpression, concomitant to an increase or a decrease, respectively, of global protein synthesis. Gemin5, and G5-Nter as well, were detected on the polysome fractions. These results reveal the ribosome-binding capacity of the N-ter moiety, enabling Gemin5 to control global protein synthesis. Our study uncovers a crosstalk between this protein and the ribosome, and provides support for the view that Gemin5 may control translation elongation. PMID:27507887

  15. The effects of acute oral glutamine supplementation on exercise-induced gastrointestinal permeability and heat shock protein expression in peripheral blood mononuclear cells.

    PubMed

    Zuhl, Micah; Dokladny, Karol; Mermier, Christine; Schneider, Suzanne; Salgado, Roy; Moseley, Pope

    2015-01-01

    Chronic glutamine supplementation reduces exercise-induced intestinal permeability and inhibits the NF-κB pro-inflammatory pathway in human peripheral blood mononuclear cells. These effects were correlated with activation of HSP70. The purpose of this paper is to test if an acute dose of oral glutamine prior to exercise reduces intestinal permeability along with activation of the heat shock response leading to inhibition of pro-inflammatory markers. Physically active subjects (N = 7) completed baseline and exercise intestinal permeability tests, determined by the percent ratio of urinary lactulose (5 g) to rhamnose (2 g). Exercise included two 60-min treadmill runs at 70 % of VO2max at 30 °C after ingestion of glutamine (Gln) or placebo (Pla). Plasma levels of endotoxin and TNF-α, along with peripheral blood mononuclear cell (PBMC) protein expression of HSP70 and IκBα, were measured pre- and post-exercise and 2 and 4 h post-exercise. Permeability increased in the Pla trial compared to that at rest (0.06 ± 0.01 vs. 0.02 ± 0.018) and did not increase in the Gln trial. Plasma endotoxin was lower at the 4-h time point in the Gln vs. 4 h in the Pla (6.715 ± 0.046 pg/ml vs. 7.952 ± 1.11 pg/ml). TNF-α was lower 4 h post-exercise in the Gln vs. Pla (1.64 ± 0.09 pg/ml vs. 1.87 ± 0.12 pg/ml). PBMC expression of IkBα was higher 4 h post-exercise in the Gln vs. 4 h in the Pla (1.29 ± 0.43 vs. 0.8892 ± 0.040). HSP70 was higher pre-exercise and 2 h post-exercise in the Gln vs. Pla (1.35 ± 0.21 vs. 1.000 ± 0.000 and 1.65 ± 0.21 vs. 1.27 ± 0.40). Acute oral glutamine supplementation prevents an exercise-induced rise in intestinal permeability and suppresses NF-κB activation in peripheral blood mononuclear cells. PMID:25062931

  16. The effects of acute oral glutamine supplementation on exercise-induced gastrointestinal permeability and heat shock protein expression in peripheral blood mononuclear cells.

    PubMed

    Zuhl, Micah; Dokladny, Karol; Mermier, Christine; Schneider, Suzanne; Salgado, Roy; Moseley, Pope

    2015-01-01

    Chronic glutamine supplementation reduces exercise-induced intestinal permeability and inhibits the NF-κB pro-inflammatory pathway in human peripheral blood mononuclear cells. These effects were correlated with activation of HSP70. The purpose of this paper is to test if an acute dose of oral glutamine prior to exercise reduces intestinal permeability along with activation of the heat shock response leading to inhibition of pro-inflammatory markers. Physically active subjects (N = 7) completed baseline and exercise intestinal permeability tests, determined by the percent ratio of urinary lactulose (5 g) to rhamnose (2 g). Exercise included two 60-min treadmill runs at 70 % of VO2max at 30 °C after ingestion of glutamine (Gln) or placebo (Pla). Plasma levels of endotoxin and TNF-α, along with peripheral blood mononuclear cell (PBMC) protein expression of HSP70 and IκBα, were measured pre- and post-exercise and 2 and 4 h post-exercise. Permeability increased in the Pla trial compared to that at rest (0.06 ± 0.01 vs. 0.02 ± 0.018) and did not increase in the Gln trial. Plasma endotoxin was lower at the 4-h time point in the Gln vs. 4 h in the Pla (6.715 ± 0.046 pg/ml vs. 7.952 ± 1.11 pg/ml). TNF-α was lower 4 h post-exercise in the Gln vs. Pla (1.64 ± 0.09 pg/ml vs. 1.87 ± 0.12 pg/ml). PBMC expression of IkBα was higher 4 h post-exercise in the Gln vs. 4 h in the Pla (1.29 ± 0.43 vs. 0.8892 ± 0.040). HSP70 was higher pre-exercise and 2 h post-exercise in the Gln vs. Pla (1.35 ± 0.21 vs. 1.000 ± 0.000 and 1.65 ± 0.21 vs. 1.27 ± 0.40). Acute oral glutamine supplementation prevents an exercise-induced rise in intestinal permeability and suppresses NF-κB activation in peripheral blood mononuclear cells.

  17. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    NASA Technical Reports Server (NTRS)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  18. Guardian of Genetic Messenger-RNA-Binding Proteins

    PubMed Central

    Anji, Antje; Kumari, Meena

    2016-01-01

    RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins. PMID:26751491

  19. Constitutive overexpression of cytosolic glutamine synthetase (GS1) gene in transgenic alfalfa demonstrates that GS1 may be regulated at the level of RNA stability and protein turnover.

    PubMed

    Ortega, J L; Temple, S J; Sengupta-Gopalan, C

    2001-05-01

    Glutamine synthetase (GS) catalyzes the ATP-dependent condensation of NH4+ with glutanate to yield glutamine. Gene constructs consisting of the cauliflower mosaic virus (CaMV) 35S promoter driving a cytosolic isoform of GS (GS1) gene have been introduced into alfalfa (Medicago sativa). Although transcripts for the transgene were shown to accumulate to high levels in the leaves, they were undetectable in the nodules. However, significant amounts of beta-glucuronidase activity could be detected in nodules of plants containing the CaMV 35S promoter-beta-glucuronidase gene construct, suggesting that the transcript for the GS1 transgene is not stable in the root nodules. Leaves of alfalfa plants with the CaMV 35S promoter-GS1 gene showed high levels of accumulation of the transcript for the transgene when grown under low-nitrogen conditions and showed a significant drop in the level of GS1 transcripts when fed with high levels of NO3-. However, no increase in GS activity or polypeptide level was detected in the leaves of transgenic plants. The results suggest that GS1 is regulated at the level of RNA stability and protein turnover.

  20. Identification of AOSC-binding proteins in neurons

    NASA Astrophysics Data System (ADS)

    Liu, Ming; Nie, Qin; Xin, Xianliang; Geng, Meiyu

    2008-11-01

    Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer’s Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.

  1. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    PubMed

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

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

  2. HTLV-1 Tax Protein Stimulation of DNA Binding of bZIP Proteins by Enhancing Dimerization

    NASA Astrophysics Data System (ADS)

    Wagner, Susanne; Green, Michael R.

    1993-10-01

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

  3. Protein surface-distribution and protein-protein interactions in the binding of peripheral proteins to charged lipid membranes.

    PubMed Central

    Heimburg, T; Marsh, D

    1995-01-01

    The binding of native cytochrome c to negatively charged lipid dispersions of dioleoyl phosphatidylglycerol has been studied over a wide range of ionic strengths. Not only is the strength of protein binding found to decrease rapidly with increasing ionic strength, but also the binding curves reach an apparent saturation level that decreases rapidly with increasing ionic strength. Analysis of the binding isotherms with a general statistical thermodynamic model that takes into account not only the free energy of the electrostatic double layer, but also the free energy of the surface distribution of the protein, demonstrates that the apparent saturation effects could arise from a competition between the out-of-plane binding reaction and the lateral in-plane interactions between proteins at the surface. It is found that association with nonlocalized sites results in binding isotherms that display the apparent saturation effect to a much more pronounced extent than does the Langmuir adsorption isotherm for binding to localized sites. With the model for nonlocalized sites, the binding isotherms of native cytochrome c can be described adequately by taking into account only the entropy of the surface distribution of the protein, without appreciable enthalpic interactions between the bound proteins. The binding of cytochrome c to dioleoyl phosphatidylglycerol dispersions at a temperature at which the bound protein is denatured on the lipid surface, but is nondenatured when free in solution, has also been studied. The binding curves for the surface-denatured protein differ from those for the native protein in that the apparent saturation at high ionic strength is less pronounced. This indicates the tendency of the denatured protein to aggregate on the lipid surface, and can be described by the binding isotherms for nonlocalized sites only if attractive interactions between the surface-bound proteins are included in addition to the distributional entropic terms. Additionally

  4. DNA sequence analysis of a 10 624 bp fragment of the left arm of chromosome XV from Saccharomyces cerevisiae reveals a RNA binding protein, a mitochondrial protein, two ribosomal proteins and two new open reading frames.

    PubMed

    Lafuente, M J; Gamo, F J; Gancedo, C

    1996-09-01

    We have determined the sequence of a 10624 bp DNA segment located in the left arm of chromosome XV of Saccharomyces cerevisiae. The sequence contains eight open reading frames (ORFs) longer than 100 amino acids. Two of them do not present significant homology with sequences found in the databases. The product of ORF o0553 is identical to the protein encoded by the gene SMF1. Internal to it there is another ORF, o0555 that is apparently expressed. The proteins encoded by ORFs o0559 and o0565 are identical to ribosomal proteins S19.e and L18 respectively. ORF o0550 encodes a protein with an RNA binding signature including RNP motifs and stretches rich in asparagine, glutamine and arginine.

  5. Fibronectin-binding protein of Streptococcus equi subsp. zooepidemicus.

    PubMed Central

    Lindmark, H; Jacobsson, K; Frykberg, L; Guss, B

    1996-01-01

    By screening a genomic lambda library of Streptococcus equi subsp. zooepidemicus, we have cloned and sequenced a gene, termed fnz, encoding a fibronectin (Fn)-binding protein called FNZ. On the basis of the deduced amino acid sequence of FNZ, the mature protein has a molecular mass of approximately 61 kDa. Analysis of FNZ reveals a structural organization similar to that of other cell surface proteins from streptococci and staphylococci. The Fn-binding activity is localized to two domains in the C-terminal part of FNZ. One domain is composed of five repeats, which contain a motif similar to what has earlier been found in other Fn-binding proteins in streptococci and staphylococci. The first and second repeats are separated by a short stretch of amino acids, including the motif LAGESGET, which is an important part of the second Fn-binding domain. This motif is also present in an Fn-binding domain (UR) in protein F of Streptococcus pyogenes. A fusion protein covering the Fn-binding domain of FNZ inhibits the binding of the 29-kDa N-terminal fragment of Fn to cells of various streptococcal species as well as to Staphylococcus aureus. PMID:8926060

  6. Paramagnetic Ligand Tagging To Identify Protein Binding Sites

    PubMed Central

    2015-01-01

    Transient biomolecular interactions are the cornerstones of the cellular machinery. The identification of the binding sites for low affinity molecular encounters is essential for the development of high affinity pharmaceuticals from weakly binding leads but is hindered by the lack of robust methodologies for characterization of weakly binding complexes. We introduce a paramagnetic ligand tagging approach that enables localization of low affinity protein–ligand binding clefts by detection and analysis of intermolecular protein NMR pseudocontact shifts, which are invoked by the covalent attachment of a paramagnetic lanthanoid chelating tag to the ligand of interest. The methodology is corroborated by identification of the low millimolar volatile anesthetic interaction site of the calcium sensor protein calmodulin. It presents an efficient route to binding site localization for low affinity complexes and is applicable to rapid screening of protein–ligand systems with varying binding affinity. PMID:26289584

  7. Niobium Uptake and Release by Bacterial Ferric Ion Binding Protein

    PubMed Central

    Shi, Yanbo; Harvey, Ian; Campopiano, Dominic; Sadler, Peter J.

    2010-01-01

    Ferric ion binding proteins (Fbps) transport FeIII across the periplasm and are vital for the virulence of many Gram negative bacteria. Iron(III) is tightly bound in a hinged binding cleft with octahedral coordination geometry involving binding to protein side chains (including tyrosinate residues) together with a synergistic anion such as phosphate. Niobium compounds are of interest for their potential biological activity, which has been little explored. We have studied the binding of cyclopentadienyl and nitrilotriacetato NbV complexes to the Fbp from Neisseria gonorrhoeae by UV-vis spectroscopy, chromatography, ICP-OES, mass spectrometry, and Nb K-edge X-ray absorption spectroscopy. These data suggest that NbV binds strongly to Fbp and that a dinuclear NbV centre can be readily accommodated in the interdomain binding cleft. The possibility of designing niobium-based antibiotics which block iron uptake by pathogenic bacteria is discussed. PMID:20445753

  8. Exchange Kinetics of a Hydrophobic Ligand Binding Protein

    NASA Astrophysics Data System (ADS)

    Vaughn, Jeff; Stone, Martin

    2002-03-01

    Conformational fluctuations of proteins are thought to be important for determining the functional roles in biological activity. In some cases, the rates of these conformational changes may be directly correlated to, for example, the rates of catalysis or ligand binding. We are studying the role of conformational fluctuations in the binding of small volatile hydrophobic pheromones by the mouse major urinary proteins (MUPs). Communication among mice occurs, in part, with the MUP-1 protein. This urinary protein binds pheromones as a way to increase the longevity of the pheromone in an extracellular environment. Of interest is that the crystal structure of MUP-1 with a pheromone ligand shows the ligand to be completely occluded from the solvent with no obvious pathway to enter or exit. This suggests that conformational exchange of the protein may be required for ligand binding and release to occur. We hypothesize that the rate of conformational exchange may be a limiting factor determining the rate of ligand association and dissociation. By careful measurement of the on- and off-rates of ligand binding and the rates of conformational changes of the protein, a more defined picture of the interplay between protein structure and function can be obtained. To this end, heteronuclear saturation transfer, ^15N-exchange and ^15N dynamics experiments have been employed to probe the kinetics of ligand binding to MUP-1.

  9. Estradiol binding to nuclear matrix protein of pig adrenal cortex

    SciTech Connect

    Ungar, F.; Johnson, S.R.; Johnston, J.A.

    1987-05-01

    Binding of TH-estradiol can be shown in vitro after incubation with purified washed nuclei of sow adrenal cortex or with the insoluble nuclear matrix protein isolated from nuclei. The procedure modified after Berezney and Coffey treated washed nuclei sequentially with 1% Triton-X100, DNase, RNase and 2M NaCl to give an insoluble nuclear matrix protein preparation in which most of the phospholipid, DNA, RNA and protein was removed. Reagents were added to 10 mM Tris buffer containing 1 mM phenylmethyl sulfonyl fluoride, dithiothreitol and 0.2 mM or 5.0 mM MgCl2. Each treatment and washes were centrifuged at 4C. Suspensions of nuclei and nuclear matrix protein were incubated at 4C for 24 hrs. with 0.25 to 3.0 ng of TH-estradiol in 0.5 ml 10 mM Tris buffer with 5 mM MgCl2. Scatchard analysis of binding in duplicate or triplicate tubes with or without excess unlabeled estradiol gave specific binding for sow adrenal nuclei and for nuclear matrix protein. Total binding sites varied between 780 to 1380 fmoles/mg protein. Estradiol binding was not shown in the fetal adrenal matrix nor in mitochondria. Noncompetitive controls included progesterone and pregnenolone. Nuclear matrix protein binding of estradiol may have significance in functional or morphological changes of the adrenal cortex in fetal, neonatal, or pubertal development.

  10. General RNA binding proteins render translation cap dependent.

    PubMed Central

    Svitkin, Y V; Ovchinnikov, L P; Dreyfuss, G; Sonenberg, N

    1996-01-01

    Translation in rabbit reticulocyte lysate is relatively independent of the presence of the mRNA m7G cap structure and the cap binding protein, eIF-4E. In addition, initiation occurs frequently at spurious internal sites. Here we show that a critical parameter which contributes to cap-dependent translation is the amount of general RNA binding proteins in the extract. Addition of several general RNA binding proteins, such as hnRNP A1, La autoantigen, pyrimidine tract binding protein (hnRNP I/PTB) and the major core protein of cytoplasmic mRNP (p50), rendered translation in a rabbit reticulocyte lysate cap dependent. These proteins drastically inhibited the translation of an uncapped mRNA, but had no effect on translation of a capped mRNA. Based on these and other results, we suggest that one function of general mRNA binding proteins in the cytoplasm is to promote ribosome binding by a 5' end, cap-mediated mechanism, and prevent spurious initiations at aberrant translation start sites. Images PMID:9003790

  11. Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms.

    PubMed

    Chou, Shan-Ho; Galperin, Michael Y

    2016-01-01

    Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms.

  12. Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

    PubMed Central

    2015-01-01

    ABSTRACT Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms. PMID:26055114

  13. Analysis of Arf GTP-binding Protein Function in Cells

    PubMed Central

    Cohen, Lee Ann; Donaldson, Julie G.

    2010-01-01

    This unit describes techniques and approaches that can be used to study the functions of the ADP-ribosylation factor (Arf) GTP-binding proteins in cells. There are 6 mammalian Arfs and many more Arf-like proteins (Arls) and these proteins are conserved in eukaryotes from yeast to man. Like all GTPases, Arfs cycle between GDP-bound, inactive and GTP-bound active conformations, facilitated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that catalyze GTP binding and hydrolysis respectively. Here we describe approaches that can be taken to examine the localization and function of Arf and Arl proteins in cells (Protocol 1). We also provide a simple protocol for measuring activation (GTP-binding) of specific Arf proteins in cells using a pull-down assay (Protocol 2). We then discuss approaches that can be taken to assess function of GEFs and GAPs in cells (Protocol 3). PMID:20853342

  14. Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations.

    PubMed

    Hunter, S A; Cochran, J R

    2016-01-01

    Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein-protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities-time to equilibrium and ligand depletion-will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented. PMID:27586327

  15. A new aspect of serum protein binding of tolbutamide.

    PubMed

    Ayanoğlu, G; Uihlein, M; Grigoleit, H G

    1986-02-01

    Tolbutamide is known to bind highly to serum proteins. Quite different values have, however, been reported for binding, ranging from 80 to 99 percent. In this study, in vivo and in vitro binding of increasing concentrations of tolbutamide to human serum proteins were evaluated. In vitro studies were done serum from three healthy males and for in vivo studies serum samples from eight healthy males who had received 1,000 mg tolbutamide were used. Protein binding was determined by equilibrium dialysis, using DIANORM system. Tolbutamide concentrations were determined by HPLC method of Uihlein and Hack. The results suggest that there is an increase in percent tolbutamide bound with increasing concentrations of tolbutamide. Generally, an inverse relationship between the total concentration of a drug in serum and its bound fraction is observed. Our findings seem to be contrary to this, at least within the concentration range studied. There exist at least two binding sites on albumin with different affinities for tolbutamide and most probably, at low concentrations, the drug binds mainly to the high affinity sites, whereas at higher concentrations additional drug will bind to the lower affinity sites leading to the observed increase in fraction bound with concentration. In conclusion it may be said that serum protein binding is a much more complicated phenomenon than generally stated and that the normal observations are only true for some ideal compounds where only one site of adsorption has to be taken into account.

  16. Conformational thermodynamics of metal-ion binding to a protein

    NASA Astrophysics Data System (ADS)

    Das, Amit; Chakrabarti, J.; Ghosh, Mahua

    2013-08-01

    Conformational changes in proteins induced by metal-ions play extremely important role in various cellular processes and technological applications. Dihedral angles are suitable conformational variables to describe microscopic conformations of a biomacromolecule. Here, we use the histograms of the dihedral angles to study the thermodynamics of conformational changes of a protein upon metal-ion binding. Our method applied to Ca2+ ion binding to an important metalloprotein, Calmodulin, reveals different thermodynamic changes in different metal-binding sites. The ligands coordinating to Ca2+ ions also play different roles in stabilizing the metal-ion coordinated protein-structure. Metal-ion binding induce remarkable thermodynamic changes in distant part of the protein via modification of secondary structural elements.

  17. SIENA: Efficient Compilation of Selective Protein Binding Site Ensembles.

    PubMed

    Bietz, Stefan; Rarey, Matthias

    2016-01-25

    Structural flexibility of proteins has an important influence on molecular recognition and enzymatic function. In modeling, structure ensembles are therefore often applied as a valuable source of alternative protein conformations. However, their usage is often complicated by structural artifacts and inconsistent data annotation. Here, we present SIENA, a new computational approach for the automated assembly and preprocessing of protein binding site ensembles. Starting with an arbitrarily defined binding site in a single protein structure, SIENA searches for alternative conformations of the same or sequentially closely related binding sites. The method is based on an indexed database for identifying perfect k-mer matches and a recently published algorithm for the alignment of protein binding site conformations. Furthermore, SIENA provides a new algorithm for the interaction-based selection of binding site conformations which aims at covering all known ligand-binding geometries. Various experiments highlight that SIENA is able to generate comprehensive and well selected binding site ensembles improving the compatibility to both known and unconsidered ligand molecules. Starting with the whole PDB as data source, the computation time of the whole ensemble generation takes only a few seconds. SIENA is available via a Web service at www.zbh.uni-hamburg.de/siena .

  18. Subcellular distribution of small GTP binding proteins in pancreas: Identification of small GTP binding proteins in the rough endoplasmic reticulum

    SciTech Connect

    Nigam, S.K. )

    1990-02-01

    Subfractionation of a canine pancreatic homogenate was performed by several differential centrifugation steps, which gave rise to fractions with distinct marker profiles. Specific binding of guanosine 5{prime}-({gamma}-({sup 35}S)thio)triphosphate (GTP({gamma}-{sup 35}S)) was assayed in each fraction. Enrichment of GTP({gamma}-{sup 35}S) binding was greatest in the interfacial smooth microsomal fraction, expected to contain Golgi and other smooth vesicles. There was also marked enrichment in the rough microsomal fraction. Electron microscopy and marker protein analysis revealed the rough microsomes (RMs) to be highly purified rough endoplasmic reticulum (RER). The distribution of small (low molecular weight) GTP binding proteins was examined by a ({alpha}-{sup 32}P)GTP blot-overlay assay. Several apparent GTP binding proteins of molecular masses 22-25 kDa were detected in various subcellular fractions. In particular, at least two such proteins were found in the Golgi-enriched and RM fractions, suggesting that these small GTP binding proteins were localized to the Golgi and RER. To more precisely localize these proteins to the RER, native RMs and RMs stripped of ribosomes by puromycin/high salt were subjected to isopycnic centrifugation. The total GTP({gamma}-{sup 35}S) binding, as well as the small GTP binding proteins detected by the ({alpha}-{sup 32}P)GTP blot overlay, distributed into fractions of high sucrose density, as did the RER marker ribophorin I. Consistent with a RER localization, when the RMS were stripped of ribosomes and subjected to isopycnic centrifugation, the total GTP({gamma}-{sup 35}S) binding and the small GTP binding proteins detected in the blot-overlay assay shifted to fractions of lighter sucrose density along with the RER marker.

  19. Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals.

    PubMed

    tom Dieck, S; Sanmartí-Vila, L; Langnaese, K; Richter, K; Kindler, S; Soyke, A; Wex, H; Smalla, K H; Kämpf, U; Fränzer, J T; Stumm, M; Garner, C C; Gundelfinger, E D

    1998-07-27

    The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 Mr, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.

  20. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  1. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  2. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  3. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  4. 21 CFR 866.5765 - Retinol-binding protein immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement...

  5. Protein-DNA binding in high-resolution

    PubMed Central

    Mahony, Shaun; Pugh, B. Franklin

    2015-01-01

    Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein-DNA binding events. For example, the ChIP-exo protocol precisely characterizes protein-DNA crosslinking patterns by combining chromatin immunoprecipitation (ChIP) with 5′ → 3′ exonuclease digestion. Similarly, deeply sequenced chromatin accessibility assays (e.g. DNase-seq and ATACseq) enable the detection of protected footprints at protein-DNA binding sites. With these techniques and others, we have the potential to characterize the individual nucleotides that interact with transcription factors, nucleosomes, RNA polymerases, and other regulatory proteins in a particular cellular context. In this review, we explain the experimental assays and computational analysis methods that enable high-resolution profiling of protein-DNA binding events. We discuss the challenges and opportunities associated with such approaches. PMID:26038153

  6. Discodermolide interferes with the binding of tau protein to microtubules.

    PubMed

    Kar, Santwana; Florence, Gordon J; Paterson, Ian; Amos, Linda A

    2003-03-27

    We investigated whether discodermolide, a novel antimitotic agent, affects the binding to microtubules of tau protein repeat motifs. Like taxol, the new drug reduces the proportion of tau that pellets with microtubules. Despite their differing structures, discodermolide, taxol and tau repeats all bind to a site on beta-tubulin that lies within the microtubule lumen and is crucial in controlling microtubule assembly. Low concentrations of tau still bind strongly to the outer surfaces of preformed microtubules when the acidic C-terminal regions of at least six tubulin dimers are available for interaction with each tau molecule; otherwise binding is very weak.

  7. Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine

    SciTech Connect

    Kuroki, Y.; Akino, T. )

    1991-02-15

    Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of reducing the surface tension at the air-liquid interface. Surfactant protein A (SP-A) with a reduced denatured molecular mass of 26-38 kDa, characterized by a collagen-like structure and N-linked glycosylation, interacts strongly with a mixture of surfactant-like phospholipids. In the present study the direct binding of SP-A to phospholipids on a thin layer chromatogram was visualized using 125I-SP-A as a probe, so that the phospholipid specificities of SP-A binding and the structural requirements of SP-A and phospholipids for the binding could be examined. Although 125I-SP-A bound phosphatidylcholine and sphingomyeline, it was especially strong in binding dipalmitoylphosphatidylcholine, but failed to bind phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. Labeled SP-A also exhibited strong binding to distearoylphosphatidylcholine, but weak binding to dimyristoyl-, 1-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. Unlabeled SP-A readily competed with labeled SP-A for phospholipid binding. SP-A strongly bound dipalmitoylglycerol produced by phospholipase C treatment of dipalmitoylphosphatidylcholine, but not palmitic acid. This protein also failed to bind lysophosphatidylcholine produced by phospholipase A2 treatment of dipalmitoylphosphatidylcholine. 125I-SP-A shows almost no binding to dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine. The addition of 10 mM EGTA into the binding buffer reduced much of the 125I-SP-A binding to phospholipids. Excess deglycosylated SP-A competed with labeled SP-A for binding to dipalmitoylphosphatidylcholine, but the excess collagenase-resistant fragment of SP-A failed.

  8. Wild-Type p53 Binds to the TATA-Binding Protein and Represses Transcription

    NASA Astrophysics Data System (ADS)

    Seto, Edward; Usheva, Anny; Zambetti, Gerard P.; Momand, Jamil; Horikoshi, Nobuo; Weinmann, Roberto; Levine, Arnold J.; Shenk, Thomas

    1992-12-01

    p53 activates transcription of genes with a p53 response element, and it can repress genes lacking the element. Here we demonstrate that wild-type but not mutant p53 inhibits transcription in a HeLa nuclear extract from minimal promoters. Wild-type but not mutant p53 binds to human TATA-binding protein (TBP). p53 does not bind to yeast TBP, and it cannot inhibit transcription in a HeLa extract where yeast TBP substitutes for human TBP. These results suggest a model in which p53 binds to TBP and interferes with transcriptional initiation.

  9. Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment.

    PubMed

    Mollica, Luca; Bessa, Luiza M; Hanoulle, Xavier; Jensen, Malene Ringkjøbing; Blackledge, Martin; Schneider, Robert

    2016-01-01

    In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs). In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an associated function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiological partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed, e.g., in the "fly-casting" hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit), are subjects of intense debate. Experimentally, these questions remain difficult to address. Here, we review experimental studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theoretical concepts and numerical methods that can be applied to describe these interactions at the atomic level. The available literature suggests that the kinetic and thermodynamic parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes observed experimentally. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context.

  10. Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment

    PubMed Central

    Mollica, Luca; Bessa, Luiza M.; Hanoulle, Xavier; Jensen, Malene Ringkjøbing; Blackledge, Martin; Schneider, Robert

    2016-01-01

    In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs). In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an associated function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiological partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed, e.g., in the “fly-casting” hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit), are subjects of intense debate. Experimentally, these questions remain difficult to address. Here, we review experimental studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theoretical concepts and numerical methods that can be applied to describe these interactions at the atomic level. The available literature suggests that the kinetic and thermodynamic parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes observed experimentally. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context. PMID:27668217

  11. Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment.

    PubMed

    Mollica, Luca; Bessa, Luiza M; Hanoulle, Xavier; Jensen, Malene Ringkjøbing; Blackledge, Martin; Schneider, Robert

    2016-01-01

    In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs). In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an associated function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiological partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed, e.g., in the "fly-casting" hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit), are subjects of intense debate. Experimentally, these questions remain difficult to address. Here, we review experimental studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theoretical concepts and numerical methods that can be applied to describe these interactions at the atomic level. The available literature suggests that the kinetic and thermodynamic parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes observed experimentally. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context. PMID:27668217

  12. Binding Mechanisms of Intrinsically Disordered Proteins: Theory, Simulation, and Experiment

    PubMed Central

    Mollica, Luca; Bessa, Luiza M.; Hanoulle, Xavier; Jensen, Malene Ringkjøbing; Blackledge, Martin; Schneider, Robert

    2016-01-01

    In recent years, protein science has been revolutionized by the discovery of intrinsically disordered proteins (IDPs). In contrast to the classical paradigm that a given protein sequence corresponds to a defined structure and an associated function, we now know that proteins can be functional in the absence of a stable three-dimensional structure. In many cases, disordered proteins or protein regions become structured, at least locally, upon interacting with their physiological partners. Many, sometimes conflicting, hypotheses have been put forward regarding the interaction mechanisms of IDPs and the potential advantages of disorder for protein-protein interactions. Whether disorder may increase, as proposed, e.g., in the “fly-casting” hypothesis, or decrease binding rates, increase or decrease binding specificity, or what role pre-formed structure might play in interactions involving IDPs (conformational selection vs. induced fit), are subjects of intense debate. Experimentally, these questions remain difficult to address. Here, we review experimental studies of binding mechanisms of IDPs using NMR spectroscopy and transient kinetic techniques, as well as the underlying theoretical concepts and numerical methods that can be applied to describe these interactions at the atomic level. The available literature suggests that the kinetic and thermodynamic parameters characterizing interactions involving IDPs can vary widely and that there may be no single common mechanism that can explain the different binding modes observed experimentally. Rather, disordered proteins appear to make combined use of features such as pre-formed structure and flexibility, depending on the individual system and the functional context.

  13. GTP-binding proteins in rat liver nuclear envelopes.

    PubMed Central

    Rubins, J B; Benditt, J O; Dickey, B F; Riedel, N

    1990-01-01

    Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membrane vesicles required for reassembly of the nucleus after mitosis. Images PMID:2119502

  14. Structural Perspectives on the Evolutionary Expansion of Unique Protein-Protein Binding Sites.

    PubMed

    Goncearenco, Alexander; Shaytan, Alexey K; Shoemaker, Benjamin A; Panchenko, Anna R

    2015-09-15

    Structures of protein complexes provide atomistic insights into protein interactions. Human proteins represent a quarter of all structures in the Protein Data Bank; however, available protein complexes cover less than 10% of the human proteome. Although it is theoretically possible to infer interactions in human proteins based on structures of homologous protein complexes, it is still unclear to what extent protein interactions and binding sites are conserved, and whether protein complexes from remotely related species can be used to infer interactions and binding sites. We considered biological units of protein complexes and clustered protein-protein binding sites into similarity groups based on their structure and sequence, which allowed us to identify unique binding sites. We showed that the growth rate of the number of unique binding sites in the Protein Data Bank was much slower than the growth rate of the number of structural complexes. Next, we investigated the evolutionary roots of unique binding sites and identified the major phyletic branches with the largest expansion in the number of novel binding sites. We found that many binding sites could be traced to the universal common ancestor of all cellular organisms, whereas relatively few binding sites emerged at the major evolutionary branching points. We analyzed the physicochemical properties of unique binding sites and found that the most ancient sites were the largest in size, involved many salt bridges, and were the most compact and least planar. In contrast, binding sites that appeared more recently in the evolution of eukaryotes were characterized by a larger fraction of polar and aromatic residues, and were less compact and more planar, possibly due to their more transient nature and roles in signaling processes.

  15. Metal-binding proteins as metal pollution indicators

    SciTech Connect

    Hennig, H.F.

    1986-03-01

    The fact that metal-binding proteins are a consequence of elevated metal concentration in organisms is well known. What has been overlooked is that the presence of these proteins provides a unique opportunity to reformulate the criteria of metal pollution. The detoxification effect of metal-binding proteins in animals from polluted areas has been cited, but there have been only very few studies relating metal-binding proteins to pollution. This lack is due partly to the design of most experiments, which were aimed at isolation of metal-binding proteins and hence were of too short duration to allow for correlation to adverse physiological effects on the organism. In this study metal-binding proteins were isolated and characterized from five different marine animals (rock lobster, Jasus lalandii; hermit crab, Diogenes brevirostris; sandshrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis; and limpet, Patella granularis). These animals were kept under identical metal-enriched conditions, hence eliminating differences in method and seasons. The study animals belonged to different phyla; varied in size, mass, age, behavior, food requirements and life stages; and accumulated metals at different rates. It is possible to link unseasonal moulting in crustacea, a known physiological effect due to a metal-enriched environment, to the production of the metal-binding protein without evidence of obvious metal body burden. Thus a new concept of pollution is defined: the presence of metal-binding proteins confirms toxic metal pollution. This concept was then tested under field conditions in the whelk Bullia digitalis and in metal-enriched grass.

  16. Review: the liver bile acid-binding proteins.

    PubMed

    Monaco, Hugo L

    2009-12-01

    The liver bile acid-binding proteins, L-BABPs, formerly called the liver "basic" fatty acid-binding proteins, are a subfamily of the fatty acid-binding proteins, FABPs. All the members of this protein group share the same fold: a 10 stranded beta barrel in which two short helices are inserted in between the first and the second strand of antiparallel beta sheet. The barrel encloses the ligand binding cavity of the protein while the two helices are believed to be involved in ligand accessibility to the binding site. The L-BABP subfamily has been found to be present in the liver of several vertebrates: fish, amphibians, reptiles, and birds but not in mammals. The members of the FABP family present in mammals that appear to be more closely related to the L-BABPs are the liver FABPs and the ileal BABPs, both very extensively studied. Several L-BABP X-ray structures are available and chicken L-BABP has also been studied using NMR spectroscopy. The stoichiometry of ligand binding for bile acids, first determined by X-ray crystallography for the chicken liver protein, is of two cholates per protein molecule with the only exception of zebrafish L-BABP which, due to the presence of a disulfide bridge, has a stoichiometry of 1:1. The stoichiometry of ligand binding for fatty acids, determined with several different techniques, is 1:1. An unanswered question of great relevance is the identity of the protein that in mammals performs the function that in other vertebrates is carried out by the L-BABPS.

  17. Using evolutionary and structural information to predict DNA-binding sites on DNA-binding proteins.

    PubMed

    Kuznetsov, Igor B; Gou, Zhenkun; Li, Run; Hwang, Seungwoo

    2006-07-01

    Proteins that interact with DNA are involved in a number of fundamental biological activities such as DNA replication, transcription, and repair. A reliable identification of DNA-binding sites in DNA-binding proteins is important for functional annotation, site-directed mutagenesis, and modeling protein-DNA interactions. We apply Support Vector Machine (SVM), a supervised pattern recognition method, to predict DNA-binding sites in DNA-binding proteins using the following features: amino acid sequence, profile of evolutionary conservation of sequence positions, and low-resolution structural information. We use a rigorous statistical approach to study the performance of predictors that utilize different combinations of features and how this performance is affected by structural and sequence properties of proteins. Our results indicate that an SVM predictor based on a properly scaled profile of evolutionary conservation in the form of a position specific scoring matrix (PSSM) significantly outperforms a PSSM-based neural network predictor. The highest accuracy is achieved by SVM predictor that combines the profile of evolutionary conservation with low-resolution structural information. Our results also show that knowledge-based predictors of DNA-binding sites perform significantly better on proteins from mainly-alpha structural class and that the performance of these predictors is significantly correlated with certain structural and sequence properties of proteins. These observations suggest that it may be possible to assign a reliability index to the overall accuracy of the prediction of DNA-binding sites in any given protein using its sequence and structural properties. A web-server implementation of the predictors is freely available online at http://lcg.rit.albany.edu/dp-bind/.

  18. Detecting O2 binding sites in protein cavities

    PubMed Central

    Kitahara, Ryo; Yoshimura, Yuichi; Xue, Mengjun; Kameda, Tomoshi; Mulder, Frans A. A.

    2016-01-01

    Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O2) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O2 concentration to 8.9 mM, changes in 1H, 15N, and 13C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O2-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O2 binds specifically to the hydrophobic, and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O2 in the cavities, as well as the binding and egress of O2, suggesting that the channel consisting of helices D, E, G, H, and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O2 gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1%, and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins. PMID:26830762

  19. Binding profile of spiramycin to oviducal proteins of laying hens.

    PubMed

    Furusawa, N

    2000-12-01

    In vitro protein binding of spiramycin (SP) in the plasma and oviducts of laying hens was studied. The data for SP were compared with those for oxytetracycline (OTC), sulphadimidine (SDD), sulphamonomethoxine (SMM) and sulphaquinoxaline (SQ). The two oviduct segments, magnum (M) and isthmus plus shell gland (IS), were collected. The soluble (cell sap) fractions from the magnum (M-S9) and the isthmus plus shell gland (IS-S9) were used as samples. Plasma protein binding was highest for SQ (81.4%) (P < 0.01), and lowest for SDD (30.9%) (P < 0.01). No M-S9 protein binding of OTC was found. The IS-S9 protein binding of SP (60.4%) was very much higher than those of OTC (0.8%), SDD (4.1%), SMM (4.0%) and SQ (12.3%) (P < 0.01). Biological half-lives of these drugs in egg albumen were directly correlated to the extent of their binding to IS proteins. Of plasma, M-S9 and IS-S9, variation in SP concentration in the ranges from 1 to 20 micrograms/ml did not alter the binding properties of the drug. PMID:11199206

  20. Perturbation Approaches for Exploring Protein Binding Site Flexibility to Predict Transient Binding Pockets.

    PubMed

    Kokh, Daria B; Czodrowski, Paul; Rippmann, Friedrich; Wade, Rebecca C

    2016-08-01

    Simulations of the long-time scale motions of a ligand binding pocket in a protein may open up new perspectives for the design of compounds with steric or chemical properties differing from those of known binders. However, slow motions of proteins are difficult to access using standard molecular dynamics (MD) simulations and are thus usually neglected in computational drug design. Here, we introduce two nonequilibrium MD approaches to identify conformational changes of a binding site and detect transient pockets associated with these motions. The methods proposed are based on the rotamerically induced perturbation (RIP) MD approach, which employs perturbation of side-chain torsional motion for initiating large-scale protein movement. The first approach, Langevin-RIP (L-RIP), entails a series of short Langevin MD simulations, each starting with perturbation of one of the side-chains lining the binding site of interest. L-RIP provides extensive sampling of conformational changes of the binding site. In less than 1 ns of MD simulation with L-RIP, we observed distortions of the α-helix in the ATP binding site of HSP90 and flipping of the DFG loop in Src kinase. In the second approach, RIPlig, a perturbation is applied to a pseudoligand placed in different parts of a binding pocket, which enables flexible regions of the binding site to be identified in a small number of 10 ps MD simulations. The methods were evaluated for four test proteins displaying different types and degrees of binding site flexibility. Both methods reveal all transient pocket regions in less than a total of 10 ns of simulations, even though many of these regions remained closed in 100 ns conventional MD. The proposed methods provide computationally efficient tools to explore binding site flexibility and can aid in the functional characterization of protein pockets, and the identification of transient pockets for ligand design. PMID:27399277

  1. Perturbation Approaches for Exploring Protein Binding Site Flexibility to Predict Transient Binding Pockets.

    PubMed

    Kokh, Daria B; Czodrowski, Paul; Rippmann, Friedrich; Wade, Rebecca C

    2016-08-01

    Simulations of the long-time scale motions of a ligand binding pocket in a protein may open up new perspectives for the design of compounds with steric or chemical properties differing from those of known binders. However, slow motions of proteins are difficult to access using standard molecular dynamics (MD) simulations and are thus usually neglected in computational drug design. Here, we introduce two nonequilibrium MD approaches to identify conformational changes of a binding site and detect transient pockets associated with these motions. The methods proposed are based on the rotamerically induced perturbation (RIP) MD approach, which employs perturbation of side-chain torsional motion for initiating large-scale protein movement. The first approach, Langevin-RIP (L-RIP), entails a series of short Langevin MD simulations, each starting with perturbation of one of the side-chains lining the binding site of interest. L-RIP provides extensive sampling of conformational changes of the binding site. In less than 1 ns of MD simulation with L-RIP, we observed distortions of the α-helix in the ATP binding site of HSP90 and flipping of the DFG loop in Src kinase. In the second approach, RIPlig, a perturbation is applied to a pseudoligand placed in different parts of a binding pocket, which enables flexible regions of the binding site to be identified in a small number of 10 ps MD simulations. The methods were evaluated for four test proteins displaying different types and degrees of binding site flexibility. Both methods reveal all transient pocket regions in less than a total of 10 ns of simulations, even though many of these regions remained closed in 100 ns conventional MD. The proposed methods provide computationally efficient tools to explore binding site flexibility and can aid in the functional characterization of protein pockets, and the identification of transient pockets for ligand design.

  2. Assessing Energetic Contributions to Binding from a Disordered Region in a Protein-Protein Interaction

    SciTech Connect

    S Cho; C Swaminathan; D Bonsor; M Kerzic; R Guan; J Yang; C Kieke; P Anderson; D Kranz; et al.

    2011-12-31

    Many functional proteins are at least partially disordered prior to binding. Although the structural transitions upon binding of disordered protein regions can influence the affinity and specificity of protein complexes, their precise energetic contributions to binding are unknown. Here, we use a model protein-protein interaction system in which a locally disordered region has been modified by directed evolution to quantitatively assess the thermodynamic and structural contributions to binding of disorder-to-order transitions. Through X-ray structure determination of the protein binding partners before and after complex formation and isothermal titration calorimetry of the interactions, we observe a correlation between protein ordering and binding affinity for complexes along this affinity maturation pathway. Additionally, we show that discrepancies between observed and calculated heat capacities based on buried surface area changes in the protein complexes can be explained largely by heat capacity changes that would result solely from folding the locally disordered region. Previously developed algorithms for predicting binding energies of protein-protein interactions, however, are unable to correctly model the energetic contributions of the structural transitions in our model system. While this highlights the shortcomings of current computational methods in modeling conformational flexibility, it suggests that the experimental methods used here could provide training sets of molecular interactions for improving these algorithms and further rationalizing molecular recognition in protein-protein interactions.

  3. TALE proteins bind to both active and inactive chromatin.

    PubMed

    Scott, James N F; Kupinski, Adam P; Kirkham, Christopher M; Tuma, Roman; Boyes, Joan

    2014-02-15

    TALE (transcription activator-like effector) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. In the present study, we have systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites, but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only 2-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.

  4. Characterization of EhCaBP, a calcium-binding protein of Entamoeba histolytica and its binding proteins.

    PubMed

    Yadava, N; Chandok, M R; Prasad, J; Bhattacharya, S; Sopory, S K; Bhattacharya, A

    1997-01-01

    A novel calcium-binding protein (EhCaBP) has been recently identified and characterized from the protozoan parasite Entamoeba histolytica. In order to decipher the function of this protein, a few basic properties were investigated and compared with the ubiquitous Ca(2+)-signal transducing protein calmodulin (CaM). Indirect immunofluorescence and immunoprecipitation analyses using specific antibodies against EhCaBP suggest that it is a soluble cytoplasmic protein with no major post-translational modification. EhCaBP did not stimulate cAMP-phosphodiesterase activity, differentiating it from all known CaMs. Affinity chromatography of [35S]methionine-labelled proteins of E. histolytica trophozoites using EhCaBP-sepharose column showed Ca(2+)-dependent binding of a group of proteins. Radiolabelled proteins from the same extract also bound to CaM-sepharose. However, the proteins bound to the two columns were different as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At least one of the EhCaBP-binding proteins became phosphorylated as revealed by in vivo phosphorylation analysis. The binding-proteins could not be detected in E. invadens (a species that is pathogenic in reptiles) and E. moshkovskii (which is found in the human gut but is not pathogenic), two species in which EhCaBP-like protein has not been found. Two distinct Ca(2+)-dependent protein kinases, which get activated by EhCaBP and CaM respectively, were detected in E. histolytica. These kinases require different levels of Ca2+ for their maximal activities. Affinity chromatography also showed the binding of protein kinase(s) to EhCaBP in a Ca(2+)-dependent manner. Our data suggest that there may be novel Ca(2+)-signal transduction pathway in E. histolytica mediated by EhCaBP.

  5. Theoretical studies of binding of mannose-binding protein to monosaccharides

    NASA Astrophysics Data System (ADS)

    Aida-Hyugaji, Sachiko; Takano, Keiko; Takada, Toshikazu; Hosoya, Haruo; Kojima, Naoya; Mizuochi, Tsuguo; Inoue, Yasushi

    2004-11-01

    Binding properties of mannose-binding protein (MBP) to monosaccharides are discussed based on ab initio molecular orbital calculations for cluster models constructed. The calculated binding energies indicate that MBP has an affinity for N-acetyl- D-glucosamine, D-mannose, L-fucose, and D-glucose rather than D-galactose and N-acetyl- D-galactosamine, which is consistent with the biochemical experimental results. Electrostatic potential surfaces at the binding site of four monosaccharides having binding properties matched well with that of MBP. A vacant frontier orbital was found to be localized around the binding site of MBP, suggesting that MBP-monosaccharide interaction may occur through electrostatic and orbital interactions.

  6. Detection of GTP-binding proteins in barley aleurone protoplasts.

    PubMed

    Wang, M; Sedee, N J; Heidekamp, F; Snaar-Jagalska, B E

    1993-08-30

    We report the existence of several families of GTP-binding proteins in barley aleurone protoplasts. Partial purified plasma membrane proteins were separated by SDS-PAGE, transferred to a nitrocellulose filter and incubated with either antisera raised against a highly conserved animal G protein alpha subunit peptide/or Ras protein, or with [alpha-32P]GTP. Two sets of proteins of M(r) = 32-36 kDa and 22-24 kDa were strongly recognized by the antisera. Binding of [alpha-32P]GTP was detected on Western blots with proteins of M(r) = 22-24 kDa and 16 kDa. Binding was inhibited by 10(-7)-10(-6) M GTP gamma S, GTP or GDP; binding was not affected by 10(-6)-10(-5) M ATP gamma S or ADP. The kinetics, specificity and the effects of phytohormones in a [35S]GTP gamma S binding assay were also studied in isolated plasma membranes of barley aleurone protoplasts.

  7. Detergent activation of the binding protein in the folate radioassay

    SciTech Connect

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1982-01-01

    A minor cow's whey protein associated with ..beta..-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to ..beta..-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants (lipids/detergents).

  8. Architectural repertoire of ligand-binding pockets on protein surfaces.

    PubMed

    Weisel, Martin; Kriegl, Jan M; Schneider, Gisbert

    2010-03-01

    Knowledge of the three-dimensional structure of ligand binding sites in proteins provides valuable information for computer-assisted drug design. We present a method for the automated extraction and classification of ligand binding site topologies, in which protein surface cavities are represented as branched frameworks. The procedure employs a growing neural gas approach for pocket topology assignment and pocket framework generation. We assessed the structural diversity of 623 known ligand binding site topologies based on framework cluster analysis. At a resolution of 5 A only 23 structurally distinct topology groups were formed; this suggests an overall limited structural diversity of ligand-accommodating protein cavities. Higher resolution allowed for identification of protein-family specific pocket features. Pocket frameworks highlight potentially preferred modes of ligand-receptor interactions and will help facilitate the identification of druggable subpockets suitable for ligand affinity and selectivity optimization. PMID:20069621

  9. Liver takes up retinol-binding protein from plasma

    SciTech Connect

    Gjoen, T.; Bjerkelund, T.; Blomhoff, H.K.; Norum, K.R.; Berg, T.; Blomhoff, R.

    1987-08-15

    Retinol is transported in plasma bound to a specific transport protein, retinol-binding protein. We prepared /sup 125/I-tyramine cellobiose-labeled rat retinol-binding protein and studied its tissue uptake 1, 5, and 24 h after intravenous injection into rats. The liver was the organ containing most radioactivity at all time points studied. After 5 and 24 h, 30 and 22% of the injected dose were recovered in liver, respectively. After separating the liver into parenchymal and nonparenchymal cells in the 5-h group, we found that both cell fractions contained approximately the same amount of radioactivity (per gram of liver). Most of the retinol-binding protein radioactivity in the nonparenchymal cell fraction was in the stellate cells. The implication of these results for a possible transfer mechanism for retinol between parenchymal and stellate cells is discussed.

  10. Studies of Fibronectin-Binding Proteins of Streptococcus equi

    PubMed Central

    Lannergård, Jonas; Flock, Margareta; Johansson, Staffan; Flock, Jan-Ingmar; Guss, Bengt

    2005-01-01

    Streptococcus equi subsp. equi is the causative agent of strangles, a disease of the upper respiratory tract in horses. The initiation of S. equi subsp. equi infection is likely to involve cell surface-anchored molecules mediating bacterial adhesion to the epithelium of the host. The present study describes the cloning and characterization of FNEB, a fibronectin-binding protein with cell wall-anchoring motifs. FNEB can thus be predicted as cell surface located, contrary to the two previously characterized fibronectin-binding proteins in S. equi subsp. equi, FNE and SFS. Assays of antibody titers in horses and in experimentally infected mice indicate that the protein is immunogenic and expressed in vivo during S. equi subsp. equi infection. Using Western ligand blotting, it was shown that FNEB binds to the N-terminal 29-kDa fragment of fibronectin, while SFS and FNE both bind to the adjacent 40-kDa fragment. S. equi subsp. equi is known to bind fibronectin to a much lower degree than the closely related S. equi subsp. zooepidemicus, but the binding is primarily directed to the 29-kDa fragment. Inhibition studies using S. equi subsp. equi cells indicate that FNEB mediates cellular binding to fibronectin in this species. PMID:16239519

  11. CAG trinucleotide RNA repeats interact with RNA-binding proteins.

    PubMed Central

    McLaughlin, B. A.; Spencer, C.; Eberwine, J.

    1996-01-01

    Genes associated with several neurological diseases are characterized by the presence of an abnormally long trinucleotide repeat sequence. By way of example, Huntington's disease (HD), is characterized by selective neuronal degeneration associated with the expansion of a polyglutamine-encoding CAG tract. Normally, this CAG tract is comprised of 11-34 repeats, but in HD it is expanded to > 37 repeats in affected individuals. The mechanism by which CAG repeats cause neuronal degeneration is unknown, but it has been speculated that the expansion primarily causes abnormal protein functioning, which in turn causes HD pathology. Other mechanisms, however, have not been ruled out. Interactions between RNA and RNA-binding proteins have previously been shown to play a role in the expression of several eukaryotic genes. Herein, we report the association of cytoplasmic proteins with normal length and extended CAG repeats, using gel shift and UV crosslinking assays. Cytoplasmic protein extracts from several rat brain regions, including the striatum and cortex, sites of neuronal degeneration in HD, contain a 63-kD RNA-binding protein that specifically interacts with these CAG-repeat sequences. These protein-RNA interactions are dependent on the length of the CAG repeat, with longer repeats binding substantially more protein. Two CAG repeat-binding proteins are present in human cortex and striatum; one comigrates with the rat protein at 63 kD, while the other migrates at 49 kD. These data suggest mechanisms by which RNA-binding proteins may be involved in the pathological course of trinucleotide repeat-associated neurological diseases. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8751857

  12. Computational design of a PAK1 binding protein.

    PubMed

    Jha, Ramesh K; Leaver-Fay, Andrew; Yin, Shuangye; Wu, Yibing; Butterfoss, Glenn L; Szyperski, Thomas; Dokholyan, Nikolay V; Kuhlman, Brian

    2010-07-01

    We describe a computational protocol, called DDMI, for redesigning scaffold proteins to bind to a specified region on a target protein. The DDMI protocol is implemented within the Rosetta molecular modeling program and uses rigid-body docking, sequence design, and gradient-based minimization of backbone and side-chain torsion angles to design low-energy interfaces between the scaffold and target protein. Iterative rounds of sequence design and conformational optimization were needed to produce models that have calculated binding energies that are similar to binding energies calculated for native complexes. We also show that additional conformation sampling with molecular dynamics can be iterated with sequence design to further lower the computed energy of the designed complexes. To experimentally test the DDMI protocol, we redesigned the human hyperplastic discs protein to bind to the kinase domain of p21-activated kinase 1 (PAK1). Six designs were experimentally characterized. Two of the designs aggregated and were not characterized further. Of the remaining four designs, three bound to the PAK1 with affinities tighter than 350 muM. The tightest binding design, named Spider Roll, bound with an affinity of 100 muM. NMR-based structure prediction of Spider Roll based on backbone and (13)C(beta) chemical shifts using the program CS-ROSETTA indicated that the architecture of human hyperplastic discs protein is preserved. Mutagenesis studies confirmed that Spider Roll binds the target patch on PAK1. Additionally, Spider Roll binds to full-length PAK1 in its activated state but does not bind PAK1 when it forms an auto-inhibited conformation that blocks the Spider Roll target site. Subsequent NMR characterization of the binding of Spider Roll to PAK1 revealed a comparably small binding 'on-rate' constant (<10(5) M(-1) s(-1)). The ability to rationally design the site of novel protein-protein interactions is an important step towards creating new proteins that are useful

  13. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    PubMed

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  14. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins

    PubMed Central

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements. PMID:21087992

  15. Amidoligases with ATP-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins

    PubMed Central

    Iyer, Lakshminarayan M.; Abhiman, Saraswathi; Burroughs, A. Maxwell; Aravind, L.

    2011-01-01

    Recent studies have shown that the ubiquitin system had its origins in ancient cofactor/amino acid biosynthesis pathways. Preliminary studies also indicated that conjugation systems for other peptide tags on proteins, such as pupylation, have evolutionary links to cofactor/amino acid biosynthesis pathways. Following up on these observations, we systematically investigated the non-ribosomal amidoligases of the ATP-grasp, glutamine synthetase-like and acetyltransferase folds by classifying the known members and identifying novel versions. We then established their contextual connections using information from domain architectures and conserved gene neighborhoods. This showed remarkable, previously uncharacterized functional links between diverse peptide ligases, several peptidases of unrelated folds and enzymes involved in synthesis of modified amino acids. Using the network of contextual connections we were able to predict numerous novel pathways for peptide synthesis and modification, amine-utilization, secondary metabolite synthesis and potential peptide-tagging systems. One potential peptide-tagging system, which is widely distributed in bacteria, involves an ATP-grasp domain and a glutamine synthetase-like ligase, both of which are circularly permuted, an NTN hydrolase fold peptidase and a novel alpha helical domain. Our analysis also elucidates key steps in the biosynthesis of antibiotics such as friulimicin, butirosin and bacilysin and cell surface structures such as capsular polymers and teichuronopeptides. We also report the discovery of several novel ribosomally synthesized bacterial peptide metabolites that are cyclized via amide and lactone linkages formed by ATP-grasp enzymes. We present an evolutionary scenario for the multiple convergent origins of peptide ligases in various folds and clarify the bacterial origin of eukaryotic peptide-tagging enzymes of the TTL family. PMID:20023723

  16. Affinity labeling of GTP-binding proteins in cellular extracts.

    PubMed

    Löw, A; Faulhammer, H G; Sprinzl, M

    1992-05-25

    GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [alpha-32P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross-linking of [alpha-32P]GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. thermophilus and initiation factor 2 (IF2) from T. thermophilus. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins. PMID:1592117

  17. Binding-protein-dependent lactose transport in Agrobacterium radiobacter.

    PubMed

    Greenwood, J A; Cornish, A; Jones, C W

    1990-04-01

    Agrobacterium radiobacter NCIB 11883 was grown in lactose-limited continuous culture at a dilution rate of 0.045/h. Washed cells transported [14C]lactose and [methyl-14C]beta-D-thiogalactoside, a nonmetabolisable analog of lactose, at similar rates and with similar affinities (Km for transport, less than 1 microM). Transport was inhibited to various extents by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, by unlabeled beta-galactosides and D-galactose, and by osmotic shock. The accumulation ratio for methyl-beta-D-thiogalactoside was greater than or equal to 4,100. An abundant protein (molecular weight, 41,000) was purified from osmotic-shock fluid and shown by equilibrium dialysis to bind lactose and methyl-beta-D-thiogalactoside, the former with very high affinity (binding constant, 0.14 microM). The N-terminal amino acid sequence of this lactose-binding protein exhibited some homology with several other sugar-binding proteins from bacteria. Antiserum raised against the lactose-binding protein did not cross-react with two glucose-binding proteins from A. radiobacter or with extracts of other bacteria grown under lactose limitation. Lactose transport and beta-galactosidase were induced in batch cultures by lactose, melibiose [O-alpha-D-galactoside-(1----6)alpha-D-glucose], and isopropyl-beta-D-thiogalactoside and were subject to catabolite repression by glucose, galactose, and succinate which was not alleviated by cyclic AMP. We conclude that lactose is transported into A. radiobacter via a binding protein-dependent active transport system (in contrast to the H+ symport and phosphotransferase systems found in other bacteria) and that the expression of this transport system is closely linked to that of beta-galactosidase.

  18. PRBP: Prediction of RNA-Binding Proteins Using a Random Forest Algorithm Combined with an RNA-Binding Residue Predictor.

    PubMed

    Ma, Xin; Guo, Jing; Xiao, Ke; Sun, Xiao

    2015-01-01

    The prediction of RNA-binding proteins is an incredibly challenging problem in computational biology. Although great progress has been made using various machine learning approaches with numerous features, the problem is still far from being solved. In this study, we attempt to predict RNA-binding proteins directly from amino acid sequences. A novel approach, PRBP predicts RNA-binding proteins using the information of predicted RNA-binding residues in conjunction with a random forest based method. For a given protein, we first predict its RNA-binding residues and then judge whether the protein binds RNA or not based on information from that prediction. If the protein cannot be identified by the information associated with its predicted RNA-binding residues, then a novel random forest predictor is used to determine if the query protein is a RNA-binding protein. We incorporated features of evolutionary information combined with physicochemical features (EIPP) and amino acid composition feature to establish the random forest predictor. Feature analysis showed that EIPP contributed the most to the prediction of RNA-binding proteins. The results also showed that the information from the RNA-binding residue prediction improved the overall performance of our RNA-binding protein prediction. It is anticipated that the PRBP method will become a useful tool for identifying RNA-binding proteins. A PRBP Web server implementation is freely available at http://www.cbi.seu.edu.cn/PRBP/.

  19. Calmodulin and calmodulin binding proteins in amphibian rod outer segments

    SciTech Connect

    Nagao, S.; Yamazaki, A.; Bitensky, M.W.

    1987-03-24

    The calmodulin (CaM) content of fully intact frog rod outer segments (ROS) has been measured using radioimmunoassay. The molar ratio between rhodopsin and total CaM in ROS is 800:1. In the absence of Ca/sup 2 +/, the ROS membrane fraction contains only 4% of total ROS CaM. In contrast, in the presence of Ca/sup 2 +/, 15% of total ROS CaM is found in the membrane fraction. For half-maximal binding of CaM to CaM-depleted ROS membranes, 3 x 10/sup -7/ M Ca/sup 2 +/ is required. This CaM binding is inhibited by trifluoperazine. CaM binding proteins in the ROS membrane fraction are identified by using two different methods: the overlay method and the use of 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), a bifunctional cross-linking reagent. Ca/sup 2 +/-dependent CaM binding proteins with apparent molecular weights of 240,000, 140,000, 53,000, and 47,000 are detected in the ROS membrane fraction by the overlay method. Anomalous, Ca/sup 2 +/-independent CaM binding to rhodopsin is also detected with this method, and this CaM binding is inhibited by the presence of Ca/sup 2 +/. With the bifunctional cross-linking reagent, DTSSP, three discrete proteins with molecular weights of 240,000, 53,000, and 47,000 are detected in the native ROS membrane fraction. CaM binding to rhodopsin is not detected with this method. These data suggest that both the Ca/sup 2 +/-independent binding of CaM to rhodopsin and the Ca/sup 2 +/-dependent binding of CaM to the M/sub r/ 140,000 protein represent binding of CaM to a site(s) which is (are) exposed only after denaturation. Ca/sup 2 +/-dependent CaM binding in the cytoplasmic fraction is also evaluated with the overlay method. These data suggest that CaM and its binding proteins participate in the regulation of Ca/sup 2 +/-sensitive processes primarily on the ROS disk membranes.

  20. Specific binding of gibberellic acid by cytokinin-specific binding proteins: a new aspect of plant hormone-binding proteins with the PR-10 fold.

    PubMed

    Ruszkowski, Milosz; Sliwiak, Joanna; Ciesielska, Agnieszka; Barciszewski, Jakub; Sikorski, Michal; Jaskolski, Mariusz

    2014-07-01

    Pathogenesis-related proteins of class 10 (PR-10) are a family of plant proteins with the same fold characterized by a large hydrophobic cavity that allows them to bind various ligands, such as phytohormones. A subfamily with only ~20% sequence identity but with a conserved canonical PR-10 fold have previously been recognized as Cytokinin-Specific Binding Proteins (CSBPs), although structurally the binding mode of trans-zeatin (a cytokinin phytohormone) was found to be quite diversified. Here, it is shown that two CSBP orthologues from Medicago truncatula and Vigna radiata bind gibberellic acid (GA3), which is an entirely different phytohormone, in a conserved and highly specific manner. In both cases a single GA3 molecule is found in the internal cavity of the protein. The structural data derived from high-resolution crystal structures are corroborated by isothermal titration calorimetry (ITC), which reveals a much stronger interaction with GA3 than with trans-zeatin and pH dependence of the binding profile. As a conclusion, it is postulated that the CSBP subfamily of plant PR-10 proteins should be more properly linked with general phytohormone-binding properties and termed phytohormone-binding proteins (PhBP).

  1. A Simple Method for Discovering Druggable, Specific Glycosaminoglycan-Protein Systems. Elucidation of Key Principles from Heparin/Heparan Sulfate-Binding Proteins.

    PubMed

    Sarkar, Aurijit; Desai, Umesh R

    2015-01-01

    Glycosaminoglycans (GAGs) affect human physiology and pathology by modulating more than 500 proteins. GAG-protein interactions are generally assumed to be ionic and nonspecific, but specific interactions do exist. Here, we present a simple method to identify the GAG-binding site (GBS) on proteins that in turn helps predict high specific GAG-protein systems. Contrary to contemporary thinking, we found that the electrostatic potential at basic arginine and lysine residues neither identifies the GBS consistently, nor its specificity. GBSs are better identified by considering the potential at neutral hydrogen bond donors such as asparagine or glutamine sidechains. Our studies also reveal that an unusual constellation of ionic and non-ionic residues in the binding site leads to specificity. Nature engineers the local environment of Asn45 of antithrombin, Gln255 of 3-O-sulfotransferase 3, Gln163 and Asn167 of 3-O-sulfotransferase 1 and Asn27 of basic fibroblast growth factor in the respective GBSs to induce specificity. Such residues are distinct from other uncharged residues on the same protein structure in possessing a significantly higher electrostatic potential, resultant from the local topology. In contrast, uncharged residues on nonspecific GBSs such as thrombin and serum albumin possess a diffuse spread of electrostatic potential. Our findings also contradict the paradigm that GAG-binding sites are simply a collection of contiguous Arg/Lys residues. Our work demonstrates the basis for discovering specifically interacting and druggable GAG-protein systems based on the structure of protein alone, without requiring access to any structure-function relationship data.

  2. A DNA-binding protein factor recognizes two binding domains within the octopine synthase enhancer element.

    PubMed Central

    Tokuhisa, J G; Singh, K; Dennis, E S; Peacock, W J

    1990-01-01

    A protein that binds to the enhancing element of the octopine synthase gene has been identified in nuclear extracts from maize cell suspension cultures. Two protein-DNA complexes are distinguishable by electrophoretic mobility in gel retardation assays. Footprint analyses of these low and high molecular weight complexes show, respectively, half and complete protection of the ocs-element DNA from cleavage by methidiumpropyl-EDTA.FE(II). Two lines of evidence indicate that the element has two recognition sites, each of which can bind identical protein units. Elements that are mutated in one or the other half and form only the low molecular weight complex interfere with the formation of both the low and high molecular weight complexes by the wild-type element. Protein isolated from a complex with only one binding site occupied can bind to the wild-type ocs-element and generate complexes with protein occupying one or both binding sites. Occupation of both sites of the ocs-element is a prerequisite for transcriptional enhancement. PMID:2152113

  3. Lactation-induced cadmium-binding proteins

    SciTech Connect

    Bhattacharyya, M.H.; Solaiman, D.; Garvey, J.S.; Miyazaki, W.Y.

    1987-01-01

    Previously we have demonstrated an increase during midlactation in /sup 109/Cd adsorption and increased retention by the duodenum, kidney, and mammary tissue of mouse dams receiving environmental levels of cadmium//sup 109/Cd via drinking water, with little change in /sup 109/Cd retention in liver and jejunum compared to nonpregnant controls. Results are reported here of a study of cadmium deposition during midlactation as associated with induction of metallothionein (MT). A cadmium/hemoglobin (Cd/Hb) assay and radioimmunoassay for MT which measures heat-stable cadmium binding capacity in tissues was used to determine MT concentrations in fractions of kidney, liver, duodenum, and jejunum from female mice. Both assays demonstrated clear lactation-induced increases in MT concentrations in liver, kidney, and duodenum, with MT concentrations falling rapidly to control levels after weaning. 4 refs., 1 tab.

  4. Evaluation of silica nanoparticle binding to major human blood proteins

    NASA Astrophysics Data System (ADS)

    Hata, Katsutomo; Higashisaka, Kazuma; Nagano, Kazuya; Mukai, Yohei; Kamada, Haruhiko; Tsunoda, Shin-ichi; Yoshioka, Yasuo; Tsutsumi, Yasuo

    2014-12-01

    Nanomaterials are used for various biomedical applications because they are often more effective than conventional materials. Recently, however, it has become clear that the protein corona that forms on the surface of nanomaterials when they make contact with biological fluids, such as blood, influences the pharmacokinetics and biological responses induced by the nanomaterials. Therefore, when evaluating nanomaterial safety and efficacy, it is important to analyze the interaction between nanomaterials and proteins in biological fluids and to evaluate the effects of the protein corona. Here, we evaluated the interaction of silica nanoparticles, a commonly used nanomaterial, with the human blood proteins albumin, transferrin, fibrinogen, and IgG. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the amount of albumin, transferrin, and IgG binding to the silica particles increased as the particle size decreased under conditions where the silica particle mass remained the same. However, under conditions in which the specific surface area remained constant, there were no differences in the binding of human plasma proteins to the silica particles tested, suggesting that the binding of silica particles with human plasma proteins is dependent on the specific surface area of the silica particles. Furthermore, the amount of albumin, transferrin, and IgG binding to silica nanoparticles with a diameter of 70 nm (nSP70) and a functional amino group was lower than that with unmodified nSP70, although there was no difference in the binding between nSP70 with the surface modification of a carboxyl functional group and nSP70. These results suggest that the characteristics of nanomaterials are important for binding with human blood proteins; this information may contribute to the development of safe and effective nanomaterials.

  5. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    SciTech Connect

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-02-10

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the ..cap alpha.. subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single ..beta.. subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the ..cap alpha.. subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub s..cap alpha../ relative to G/sub ichemically bond/ and G/sub ochemically bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with (/sup 125/I)protein. Immunohistochemical studies using an antiserum against the ..beta.. subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the ..cap alpha.. subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium.

  6. Metal binding proteins, recombinant host cells and methods

    DOEpatents

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  7. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    PubMed

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein. PMID:24878641

  8. Organic solvents identify specific ligand binding sites on protein surfaces.

    PubMed

    Liepinsh, E; Otting, G

    1997-03-01

    Enzymes frequently recognize substrates and pharmaceutical drugs through specific binding interactions in deep pockets on the protein surface. We show how the specificity-determining substrate binding site of hen egg-white lysozyme (HEWL) can be readily identified in aqueous solution by nuclear magnetic resonance spectroscopy using small organic solvent molecules as detection probes. Exchange of magnetization between the 1H nuclei of the protein and the ligands through dipole-dipole interactions is observed which allows the modeling of their position and orientation at the binding site. Combined with site-specific binding constants measured by titration experiments with different organic solvents, the method can provide important information for rational drug design. In addition, the lifetime of nonspecific interactions of HEWL with organic solvents is shown to be in the sub-nanosecond time range. PMID:9062927

  9. RNA-binding region of Macrobrachium rosenbergii nodavirus capsid protein.

    PubMed

    Goh, Zee Hong; Mohd, Nur Azmina Syakirin; Tan, Soon Guan; Bhassu, Subha; Tan, Wen Siang

    2014-09-01

    White tail disease (WTD) kills prawn larvae and causes drastic losses to the freshwater prawn (Macrobrachium rosenbergii) industry. The main causative agent of WTD is Macrobrachium rosenbergii nodavirus (MrNV). The N-terminal end of the MrNV capsid protein is very rich in positively charged amino acids and is postulated to interact with RNA molecules. N-terminal and internal deletion mutagenesis revealed that the RNA-binding region is located at positions 20-29, where 80 % of amino acids are positively charged. Substitution of all these positively charged residues with alanine abolished the RNA binding. Mutants without the RNA-binding region still assembled into virus-like particles, suggesting that this region is not a part of the capsid assembly domain. This paper is, to the best of our knowledge, the first to report the specific RNA-binding region of MrNV capsid protein.

  10. The human mannose-binding protein functions as an opsonin

    PubMed Central

    1989-01-01

    The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains: a cysteine-rich NH2-terminal domain that stabilizes the alpha-helix of the second collagen-like domain, and a third COOH-terminal carbohydrate binding region. The function of MBP is unknown, although a role in host defense is suggested by its ability to bind yeast mannans. In this report we show that native and recombinant human MBP can serve in an opsonic role in serum and thereby enhance clearance of mannose rich pathogens by phagocytes. MBP binds to wild-type virulent Salmonella montevideo that express a mannose-rich O- polysaccharide. Interaction of MBP with these organisms results in attachment, uptake, and killing of the opsonized bacteria by phagocytes. These results demonstrate that MBP plays a role in first line host defense against certain pathogenic organisms. PMID:2469767

  11. Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

    PubMed Central

    Goldstein, M A; Takagi, M; Hashida, S; Shoseyov, O; Doi, R H; Segel, I H

    1993-01-01

    Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain. Images PMID:8376323

  12. A new zinc binding fold underlines the versatility of zinc binding modules in protein evolution.

    PubMed

    Sharpe, Belinda K; Matthews, Jacqueline M; Kwan, Ann H Y; Newton, Anthea; Gell, David A; Crossley, Merlin; Mackay, Joel P

    2002-05-01

    Many different zinc binding modules have been identified. Their abundance and variety suggests that the formation of zinc binding folds might be relatively common. We have determined the structure of CH1(1), a 27-residue peptide derived from the first cysteine/histidine-rich region (CH1) of CREB binding protein (CBP). This peptide forms a highly ordered zinc-dependent fold that is distinct from known folds. The structure differs from a subsequently determined structure of a larger region from the CH3 region of CBP, and the CH1(1) fold probably represents a nonphysiologically active form. Despite this, the fold is thermostable and tolerant to both multiple alanine mutations and changes in the zinc-ligand spacing. Our data support the idea that zinc binding domains may arise frequently. Additionally, such structures may prove useful as scaffolds for protein design, given their stability and robustness.

  13. Protein D of Haemophilus influenzae is not a universal immunoglobulin D-binding protein.

    PubMed Central

    Sasaki, K; Munson, R S

    1993-01-01

    Haemophilus influenzae type b and nontypeable H. influenzae have been reported to bind human immunoglobulin D (IgD). IgD myeloma sera from five patients were tested for the ability of IgD to bind to H. influenzae. Serotype b strains bound human IgD in four of the five sera tested. IgD in the fifth serum bound strongly to type b strain MinnA but poorly to other type b strains. Additionally, IgD binding was not observed when nontypeable strains were tested. The gene for protein D, the putative IgD-binding protein, was cloned from the IgD-binding H. influenzae type b strain MinnA and expressed in Escherichia coli. IgD binding to E. coli expressing protein D was not demonstrable. Recombinant protein D was purified, and antisera were generated in rabbits. Using these rabbit sera, we detected protein D in nontypeable as well as serotype b strains by Western blotting (immunoblotting). In contrast, IgD myeloma protein 4490, which was previously reported to bind to protein D by Ruan and coworkers (M. Ruan, M. Akkoyunlu, A. Grubb, and A. Forsgren, J. Immunol. 145:3379-3384), bound strongly to both type b and nontypeable H. influenzae as well as to E. coli expressing protein D. Thus, IgD binding is a general property of H. influenzae type b strains but not a general property of nontypeable strains, although both type b and nontypeable strains produce protein D. With the exception of IgD myeloma protein 4490 binding, we have no evidence for a role of protein D in IgD binding to H. influenzae. Images PMID:8514409

  14. PrionW: a server to identify proteins containing glutamine/asparagine rich prion-like domains and their amyloid cores.

    PubMed

    Zambrano, Rafael; Conchillo-Sole, Oscar; Iglesias, Valentin; Illa, Ricard; Rousseau, Frederic; Schymkowitz, Joost; Sabate, Raimon; Daura, Xavier; Ventura, Salvador

    2015-07-01

    Prions are a particular type of amyloids with the ability to self-perpetuate and propagate in vivo. Prion-like conversion underlies important biological processes but is also connected to human disease. Yeast prions are the best understood transmissible amyloids. In these proteins, prion formation from an initially soluble state involves a structural conversion, driven, in many cases, by specific domains enriched in glutamine/asparagine (Q/N) residues. Importantly, domains sharing this compositional bias are also present in the proteomes of higher organisms, thus suggesting that prion-like conversion might be an evolutionary conserved mechanism. We have recently shown that the identification and evaluation of the potency of amyloid nucleating sequences in putative prion domains allows discrimination of genuine prions. PrionW is a web application that exploits this principle to scan sequences in order to identify proteins containing Q/N enriched prion-like domains (PrLDs) in large datasets. When used to scan the complete yeast proteome, PrionW identifies previously experimentally validated prions with high accuracy. Users can analyze up to 10 000 sequences at a time, PrLD-containing proteins are identified and their putative PrLDs and amyloid nucleating cores visualized and scored. The output files can be downloaded for further analysis. PrionW server can be accessed at http://bioinf.uab.cat/prionw/.

  15. PrionW: a server to identify proteins containing glutamine/asparagine rich prion-like domains and their amyloid cores

    PubMed Central

    Zambrano, Rafael; Conchillo-Sole, Oscar; Iglesias, Valentin; Illa, Ricard; Rousseau, Frederic; Schymkowitz, Joost; Sabate, Raimon; Daura, Xavier; Ventura, Salvador

    2015-01-01

    Prions are a particular type of amyloids with the ability to self-perpetuate and propagate in vivo. Prion-like conversion underlies important biological processes but is also connected to human disease. Yeast prions are the best understood transmissible amyloids. In these proteins, prion formation from an initially soluble state involves a structural conversion, driven, in many cases, by specific domains enriched in glutamine/asparagine (Q/N) residues. Importantly, domains sharing this compositional bias are also present in the proteomes of higher organisms, thus suggesting that prion-like conversion might be an evolutionary conserved mechanism. We have recently shown that the identification and evaluation of the potency of amyloid nucleating sequences in putative prion domains allows discrimination of genuine prions. PrionW is a web application that exploits this principle to scan sequences in order to identify proteins containing Q/N enriched prion-like domains (PrLDs) in large datasets. When used to scan the complete yeast proteome, PrionW identifies previously experimentally validated prions with high accuracy. Users can analyze up to 10 000 sequences at a time, PrLD-containing proteins are identified and their putative PrLDs and amyloid nucleating cores visualized and scored. The output files can be downloaded for further analysis. PrionW server can be accessed at http://bioinf.uab.cat/prionw/. PMID:25977297

  16. Characterization of adenosine binding proteins in human placental membranes

    SciTech Connect

    Hutchison, K.A.

    1989-01-01

    We have characterized two adenosine binding proteins in human placenta. In membranes, one site is detected with ({sup 3}H) -N-ethylcarboxamidoadenosine (({sup 3}H)NECA). This site is similar to the adenosine A{sub 2} receptor. We call this site the adenosine A{sub 2}-like binding site. In detergent extracts, the second site is detected and has the characteristics of an adenosine A{sub 1} receptor. The soluble adenosine A{sub 2}-like binding site cannot be detected without a rapid assay. Binding to the adenosine A{sub 1} receptor with ({sup 3}H)-2-chloroadenosine and ({sup 3}H)NECA is time dependent, saturable, and reversible. Equilibrium displacement analysis with adenosine agonists reveals an A{sub 1} specificity: 2-chloroadenosine > R-phenylisopropyladenosine > 5{prime}-N-ethylcarboxamidoadenosine. The antagonist potency order is 1,3-diethyl-8-phenylxanthine > isobutylmethylxanthine > theophylline. Competition analysis of membranes with the A,-selective ligands ({sup 3}H)-cyclohexyladenosine ({sup 3}H) cylopentylxanthine revealed adenosine A{sub 1} agonist and antagonist potency orders. We have purified the adenosine A{sub 2}-like binding site. The adenosine A{sub 2}-like binding site is an ubiquitous major cellular protein. It is glycosylated, highly asymmetric, and acidic. The native protein is an homodimer with a subunit molecular mass of 98 kDa. The sedimentation coefficient and partial specific volume of the binding complex are 6.9 s and 0.698 ml/g, respectively. The Stokes' radius is 70 {Angstrom}. The native molecular mass of the detergent-protein complex is 230 kDa. The adenosine A{sub 2}-like binding site has an agonist potency order of 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine >> R-phenylisopropyladenosine and an antagonist potency order of isobutylmethylxanthine > theophylline >> 1,3-diethyl-8-phenylxanthine.

  17. Predicting the Binding Patterns of Hub Proteins: A Study Using Yeast Protein Interaction Networks

    PubMed Central

    Andorf, Carson M.; Honavar, Vasant; Sen, Taner Z.

    2013-01-01

    Background Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH) with one or two binding sites, or multiple-interface hubs (MIH) with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations) or party hubs (i.e., simultaneously interact with multiple partners). Methodology Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB) protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques. Conclusions Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions. Availability We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu. PMID:23431393

  18. Quantifying Aptamer-Protein Binding via Thermofluorimetric Analysis

    PubMed Central

    Hu, Juan; Kim, Joonyul; Easley, Christopher J.

    2015-01-01

    Effective aptamer-based protein assays require coupling to a quantitative reporter of aptamer-protein binding. Typically, this involves a direct optical or electrochemical readout of DNA hybridization or an amplification step coupled to the readout. However, method development is often hampered by the multiplicity of aptamer-target binding mechanisms, which can interfere with the hybridization step. As a simpler and more generalizable readout of aptamer-protein binding, we report that thermofluorimetric analysis (TFA) can be used to quantitatively assay protein levels. Sub-nanomolar detection (0.74 nM) of platelet-derived growth factor (PDGF) with its corresponding aptamer is shown as a test case. In the presence of various DNA intercalating dyes, protein-bound aptamers exhibit a change in fluorescence intensity compared to the intercalated, unbound aptamer. This allows thermal resolution of bound and unbound aptamers using fluorescence melting analysis (−dF/dT curves). Remarkably, the homogeneous optical method allows subtraction of autofluorescence in human serum, giving PDGF detection limits of 1.8 and 10.7 nM in serum diluted 1:7 and 1:3, respectively. We have thus demonstrated that bound and unbound aptamers can be thermally resolved in a homogeneous format using a simple qPCR instrument—even in human serum. The simplicity of this approach provides an important step toward a robust, generalizable readout of aptamer-protein binding. PMID:26366207

  19. Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

    PubMed

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-02-21

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10-100 μM; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.

  20. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    PubMed Central

    Villamor, Joji Grace; Kaschani, Farnusch; Colby, Tom; Oeljeklaus, Julian; Zhao, David; Kaiser, Markus; Patricelli, Matthew P.; van der Hoorn, Renier A. L.

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188. PMID:23722185

  1. A general approach to visualize protein binding and DNA conformation without protein labelling.

    PubMed

    Song, Dan; Graham, Thomas G W; Loparo, Joseph J

    2016-03-08

    Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein-DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein-DNA interactions.

  2. Flexible Linker Modulates Glycosaminoglycan Affinity of Decorin Binding Protein A.

    PubMed

    Morgan, Ashli; Sepuru, Krishna Mohan; Feng, Wei; Rajarathnam, Krishna; Wang, Xu

    2015-08-18

    Decorin binding protein A (DBPA) is a glycosaminoglycan (GAG)-binding adhesin found on the surface of the bacterium Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease. DBPA facilitates bacterial adherence to extracellular matrices of human tissues and is crucial during the early stage of the infection process. Interestingly, DBPA from different strains (B31, N40, and PBr) show significant differences in GAG affinities, but the structural basis for the differences is not clear. In this study, we show that GAG affinity of N40 DBPA is modulated in part by flexible segments that control access to the GAG binding site, such that shortening of the linker leads to higher GAG affinity when analyzed using ELISA, gel mobility shift assay, solution NMR, and isothermal titration calorimetry. Our observation that GAG affinity differences among different B. burgdorferi strains can be attributed to a flexible linker domain regulating access to the GAG-binding domain is novel. It also provides a rare example of how neutral amino acids and dynamic segments in GAG binding proteins can have a large influence on GAG affinity and provides insights into why the number of basic amino acids in the GAG-binding site may not be the only factor determining GAG affinity of proteins. PMID:26223367

  3. Escherchia coli ribose binding protein based bioreporters revisited

    PubMed Central

    Reimer, Artur; Yagur-Kroll, Sharon; Belkin, Shimshon; Roy, Shantanu; van der Meer, Jan Roelof

    2014-01-01

    Bioreporter bacteria, i.e., strains engineered to respond to chemical exposure by production of reporter proteins, have attracted wide interest because of their potential to offer cheap and simple alternative analytics for specified compounds or conditions. Bioreporter construction has mostly exploited the natural variation of sensory proteins, but it has been proposed that computational design of new substrate binding properties could lead to completely novel detection specificities at very low affinities. Here we reconstruct a bioreporter system based on the native Escherichia coli ribose binding protein RbsB and one of its computationally designed variants, reported to be capable of binding 2,4,6-trinitrotoluene (TNT). Our results show in vivo reporter induction at 50 nM ribose, and a 125 nM affinity constant for in vitro ribose binding to RbsB. In contrast, the purified published TNT-binding variant did not bind TNT nor did TNT cause induction of the E. coli reporter system. PMID:25005019

  4. Grafting odorant binding proteins on diamond bio-MEMS.

    PubMed

    Manai, R; Scorsone, E; Rousseau, L; Ghassemi, F; Possas Abreu, M; Lissorgues, G; Tremillon, N; Ginisty, H; Arnault, J-C; Tuccori, E; Bernabei, M; Cali, K; Persaud, K C; Bergonzo, P

    2014-10-15

    Odorant binding proteins (OBPs) are small soluble proteins found in olfactory systems that are capable of binding several types of odorant molecules. Cantilevers based on polycrystalline diamond surfaces are very promising as chemical transducers. Here two methods were investigated for chemically grafting porcine OBPs on polycrystalline diamond surfaces for biosensor development. The first approach resulted in random orientation of the immobilized proteins over the surface. The second approach based on complexing a histidine-tag located on the protein with nickel allowed control of the proteins' orientation. Evidence confirming protein grafting was obtained using electrochemical impedance spectroscopy, fluorescence imaging and X-ray photoelectron spectroscopy. The chemical sensing performances of these OBP modified transducers were assessed. The second grafting method led to typically 20% more sensitive sensors, as a result of better access of ligands to the proteins active sites and also perhaps a better yield of protein immobilization. This new grafting method appears to be highly promising for further investigation of the ligand binding properties of OBPs in general and for the development of arrays of non-specific biosensors for artificial olfaction applications.

  5. RNA binding proteins in neurodegeneration: Seq and you shall receive

    PubMed Central

    Nussbacher, Julia K.; Batra, Ranjan; Lagier-Tourenne, Clotilde; Yeo, Gene W.

    2015-01-01

    As critical players in gene regulation, RNA binding proteins are taking center stage in our understanding of cellular function and disease. In our era of bench-top sequencers and unprecedented computational power, biological questions can be addressed in a systematic, genome-wide manner. Development of high-throughput sequencing methodologies provides unparalleled potential to discover new mechanisms of disease-associated perturbations of RNA homeostasis. Complementary to candidate single-gene studies, these innovative technologies may elicit the discovery of unexpected mechanisms, and allow us to determine the widespread influence of the multifunctional RNA binding proteins on their targets. As disruption of RNA processing is increasingly implicated in neurological diseases, these approaches will continue to provide insights into the roles of RNA binding proteins in disease pathogenesis. PMID:25765321

  6. Differential plasma protein binding to metal oxide nanoparticles

    NASA Astrophysics Data System (ADS)

    Deng, Zhou J.; Mortimer, Gysell; Schiller, Tara; Musumeci, Anthony; Martin, Darren; Minchin, Rodney F.

    2009-11-01

    Nanoparticles rapidly interact with the proteins present in biological fluids, such as blood. The proteins that are adsorbed onto the surface potentially dictate the biokinetics of the nanomaterials and their fate in vivo. Using nanoparticles with different sizes and surface characteristics, studies have reported the effects of physicochemical properties on the composition of adsorbed plasma proteins. However, to date, few studies have been conducted focusing on the nanoparticles that are commonly exposed to the general public, such as the metal oxides. Using previously established ultracentrifugation approaches, two-dimensional gel electrophoresis and mass spectrometry, the current study investigated the binding of human plasma proteins to commercially available titanium dioxide, silicon dioxide and zinc oxide nanoparticles. We found that, despite these particles having similar surface charges in buffer, they bound different plasma proteins. For TiO2, the shape of the nanoparticles was also an important determinant of protein binding. Agglomeration in water was observed for all of the nanoparticles and both TiO2 and ZnO further agglomerated in biological media. This led to an increase in the amount and number of different proteins bound to these nanoparticles. Proteins with important biological functions were identified, including immunoglobulins, lipoproteins, acute-phase proteins and proteins involved in complement pathways and coagulation. These results provide important insights into which human plasma proteins bind to particular metal oxide nanoparticles. Because protein absorption to nanoparticles may determine their interaction with cells and tissues in vivo, understanding how and why plasma proteins are adsorbed to these particles may be important for understanding their biological responses.

  7. SVM based prediction of RNA-binding proteins using binding residues and evolutionary information.

    PubMed

    Kumar, Manish; Gromiha, M Michael; Raghava, Gajendra P S

    2011-01-01

    RNA-binding proteins (RBPs) play crucial role in transcription and gene-regulation. This paper describes a support vector machine (SVM) based method for discriminating and classifying RNA-binding and non-binding proteins using sequence features. With the threshold of 30% interacting residues, RNA-binding amino acid prediction method PPRINT achieved the Matthews correlation coefficient (MCC) of 0.32. BLAST and PSI-BLAST identified RBPs with the coverage of 32.63 and 33.16%, respectively, at the e-value of 1e-4. The SVM models developed with amino acid, dipeptide and four-part amino acid compositions showed the MCC of 0.60, 0.46, and 0.53, respectively. This is the first study in which evolutionary information in form of position specific scoring matrix (PSSM) profile has been successfully used for predicting RBPs. We achieved the maximum MCC of 0.62 using SVM model based on PSSM called PSSM-400. Finally, we developed different hybrid approaches and achieved maximum MCC of 0.66. We also developed a method for predicting three subclasses of RNA binding proteins (e.g., rRNA, tRNA, mRNA binding proteins). The performance of the method was also evaluated on an independent dataset of 69 RBPs and 100 non-RBPs (NBPs). An additional benchmarking was also performed using gene ontology (GO) based annotation. Based on the hybrid approach a web-server RNApred has been developed for predicting RNA binding proteins from amino acid sequences (http://www.imtech.res.in/raghava/rnapred/).

  8. BindUP: a web server for non-homology-based prediction of DNA and RNA binding proteins.

    PubMed

    Paz, Inbal; Kligun, Efrat; Bengad, Barak; Mandel-Gutfreund, Yael

    2016-07-01

    Gene expression is a multi-step process involving many layers of regulation. The main regulators of the pathway are DNA and RNA binding proteins. While over the years, a large number of DNA and RNA binding proteins have been identified and extensively studied, it is still expected that many other proteins, some with yet another known function, are awaiting to be discovered. Here we present a new web server, BindUP, freely accessible through the website http://bindup.technion.ac.il/, for predicting DNA and RNA binding proteins using a non-homology-based approach. Our method is based on the electrostatic features of the protein surface and other general properties of the protein. BindUP predicts nucleic acid binding function given the proteins three-dimensional structure or a structural model. Additionally, BindUP provides information on the largest electrostatic surface patches, visualized on the server. The server was tested on several datasets of DNA and RNA binding proteins, including proteins which do not possess DNA or RNA binding domains and have no similarity to known nucleic acid binding proteins, achieving very high accuracy. BindUP is applicable in either single or batch modes and can be applied for testing hundreds of proteins simultaneously in a highly efficient manner.

  9. BindUP: a web server for non-homology-based prediction of DNA and RNA binding proteins

    PubMed Central

    Paz, Inbal; Kligun, Efrat; Bengad, Barak; Mandel-Gutfreund, Yael

    2016-01-01

    Gene expression is a multi-step process involving many layers of regulation. The main regulators of the pathway are DNA and RNA binding proteins. While over the years, a large number of DNA and RNA binding proteins have been identified and extensively studied, it is still expected that many other proteins, some with yet another known function, are awaiting to be discovered. Here we present a new web server, BindUP, freely accessible through the website http://bindup.technion.ac.il/, for predicting DNA and RNA binding proteins using a non-homology-based approach. Our method is based on the electrostatic features of the protein surface and other general properties of the protein. BindUP predicts nucleic acid binding function given the proteins three-dimensional structure or a structural model. Additionally, BindUP provides information on the largest electrostatic surface patches, visualized on the server. The server was tested on several datasets of DNA and RNA binding proteins, including proteins which do not possess DNA or RNA binding domains and have no similarity to known nucleic acid binding proteins, achieving very high accuracy. BindUP is applicable in either single or batch modes and can be applied for testing hundreds of proteins simultaneously in a highly efficient manner. PMID:27198220

  10. Can cofactor-binding sites in proteins be flexible? Desulfovibrio desulfuricans flavodoxin binds FMN dimer.

    PubMed

    Muralidhara, B K; Wittung-Stafshede, Pernilla

    2003-11-11

    Flavodoxins catalyze redox reactions using the isoalloxazine moiety of the flavin mononucleotide (FMN) cofactor stacked between two aromatic residues located in two peptide loops. At high FMN concentrations that favor stacked FMN dimers in solution, isothermal titration calorimetric studies show that these dimers bind strongly to apo-flavodoxin from Desulfovibrio desulfuricans (30 degrees C, 20 mM Hepes, pH 7, K(D) = 5.8 microM). Upon increasing the temperature so the FMN dimers dissociate (as shown by (1)H NMR), only one-to-one (FMN-to-protein) binding is observed. Calorimetric titrations result in one-to-one binding also in the presence of phosphate or sulfate (30 degrees C, 13 mM anion, pH 7, K(D) = 0.4 microM). FMN remains dimeric in the presence of phosphate and sulfate, suggesting that specific binding of a divalent anion to the phosphate-binding site triggers ordering of the peptide loops so only one isoalloxazine can fit. Although the physiological relevance of FMN and other nucleotides as dimers has not been explored, our study shows that high-affinity binding to proteins of such dimers can occur in vitro. This emphasizes that the cofactor-binding site in flavodoxin is more flexible than previously expected. PMID:14596623

  11. Coenzyme Q10-Binding/Transfer Protein Saposin B also Binds gamma-Tocopherol.

    PubMed

    Jin, Guangzhi; Horinouchi, Ryo; Sagawa, Tomofumi; Orimo, Nobutsune; Kubo, Hiroshi; Yoshimura, Shinichi; Fujisawa, Akio; Kashiba, Misato; Yamamoto, Yorihiro

    2008-09-01

    gamma-Tocopherol, the major form of dietary vitamin E, is absorbed in the intestine and is secreted in chylomicrons, which are then transferred to liver lysosomes. Most gamma-tocopherol is transferred to liver microsomes and is catabolized by cytochrome p450. Due to the hydrophobicity of gamma-tocopherol, a binding and transfer protein is plausible, but none have yet been isolated and characterized. We recently found that a ubiquitous cytosolic protein, saposin B, binds and transfers coenzyme Q10 (CoQ10), which is an essential factor for ATP production and an important antioxidant. Here, we report that saposin B also binds gamma-tocopherol, but not alpha-tocopherol, as efficiently as CoQ10 at pH 7.4. At acidic pH, saposin B binds gamma-tocopherol preferentially to CoQ10 and alpha-tocopherol. Furthermore, we confirmed that saposin B selectively binds gamma-tocopherol instead of CoQ10 and alpha-tocopherol at every pH between 5.4 and 8.0 when all three lipids are competing for binding. We detected gamma-tocopherol in human saposin B monoclonal antibody-induced immunoprecipitates from human urine, although the amount of gamma-tocopherol was much smaller than that of CoQ10. These results suggest that saposin B binds and transports gamma-tocopherol in human cells.

  12. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    SciTech Connect

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  13. Solvation structure of ice-binding antifreeze proteins

    NASA Astrophysics Data System (ADS)

    Hansen-Goos, Hendrik; Wettlaufer, John

    2009-03-01

    Antifreeze proteins (AFPs) can be found in organisms which survive at subzero temperatures. They were first discovered in polar fishes since the 1950's [1] and have been isolated meanwhile also from insects, plants, and bacteria. While AFPs shift the freezing point of water below the bulk melting point and hence can prevent recrystallization; the effect is non-colligative and there is a pronounced hysteresis between freezing and melting. For many AFPs it is generally accepted that they function through an irreversible binding to the ice-water interface which leads to a piecewise convex growth front with a lower nonequilibrium freezing point due to the Kelvin effect. Recent molecular dynamics simulations of the AFP from Choristoneura fumiferana reveal that the solvation structures of water at ice-binding and non-ice-binding faces of the protein are crucial for understanding how the AFP binds to the ice surface and how it is protected from being overgrown [2]. We use density functional theory of classical fluids in order to assess the microscopic solvent structure in the vicinity of protein faces with different surface properties. With our method, binding energies of different protein faces to the water-ice-interface can be computed efficiently in a simplified model. [1] Y. Yeh and R.E. Feeney, Chem. Rev. 96, 601 (1996). [2] D.R. Nutt and J.C. Smith, J. Am. Chem. Soc. 130, 13066 (2008).

  14. Reprogramming cellular events by poly(ADP-ribose)-binding proteins

    PubMed Central

    Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

    2013-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

  15. MutaBind estimates and interprets the effects of sequence variants on protein-protein interactions.

    PubMed

    Li, Minghui; Simonetti, Franco L; Goncearenco, Alexander; Panchenko, Anna R

    2016-07-01

    Proteins engage in highly selective interactions with their macromolecular partners. Sequence variants that alter protein binding affinity may cause significant perturbations or complete abolishment of function, potentially leading to diseases. There exists a persistent need to develop a mechanistic understanding of impacts of variants on proteins. To address this need we introduce a new computational method MutaBind to evaluate the effects of sequence variants and disease mutations on protein interactions and calculate the quantitative changes in binding affinity. The MutaBind method uses molecular mechanics force fields, statistical potentials and fast side-chain optimization algorithms. The MutaBind server maps mutations on a structural protein complex, calculates the associated changes in binding affinity, determines the deleterious effect of a mutation, estimates the confidence of this prediction and produces a mutant structural model for download. MutaBind can be applied to a large number of problems, including determination of potential driver mutations in cancer and other diseases, elucidation of the effects of sequence variants on protein fitness in evolution and protein design. MutaBind is available at http://www.ncbi.nlm.nih.gov/projects/mutabind/. PMID:27150810

  16. Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

    PubMed

    Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

    2009-10-01

    Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes. PMID:19754879

  17. Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

    PubMed

    Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

    2009-10-01

    Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

  18. Behind the scenes of vitamin D binding protein: more than vitamin D binding.

    PubMed

    Delanghe, Joris R; Speeckaert, Reinhart; Speeckaert, Marijn M

    2015-10-01

    Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein. PMID:26522461

  19. Glutamine as an immunonutrient.

    PubMed

    Kim, Hyeyoung

    2011-11-01

    Dietary supplementation with nutrients enhancing immune function is beneficial in patients with surgical and critical illness. Malnutrition and immune dysfunction are common features in hospitalized patients. Specific nutrients with immunological and pharmacological effects, when consumed in amounts above the daily requirement, are referred to as immune-enhancing nutrients or immunonutrients. Supplementation of immunonutrients is important especially for patients with immunodeficiency, virus or overwhelming infections accompanied by a state of malnutrition. Representative immunonutrients are arginine, omega-3 fatty acids, glutamine, nucleotides, beta-carotene, and/or branched-chain amino acids. Glutamine is the most abundant amino acid and performs multiple roles in human body. However, glutamine is depleted from muscle stores during severe metabolic stress including sepsis and major surgery. Therefore it is considered conditionally essential under these conditions. This review discusses the physiological role of glutamine, mode and dose for glutamine administration, as well as improvement of certain disease state after glutamine supplementation. Even though immunonutrition has not been widely assimilated by clinicians other than nutritionists, immunonutrients including glutamine may exert beneficial influence on diverse patient populations.

  20. Quantitative analysis of pheromone-binding protein specificity

    PubMed Central

    Katti, S.; Lokhande, N.; González, D.; Cassill, A.; Renthal, R.

    2012-01-01

    Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ~100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ~200 nM for the silk moth pheromone bombykol and ~90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the pheromone receptor model proposed by Laughlin et al. (Cell 133: 1255–65, 2008) are discussed. PMID:23121132

  1. Binding of C-reactive protein to human lymphocytes. I. Requirement for a binding specificity.

    PubMed

    James, K; Hansen, B; Gewurz, H

    1981-12-01

    Our laboratory previously reported that C-reactive protein (CRP) binds selectively to T lymphocytes and inhibits certain of their reactivities in vitro. However, these findings could not be repeated using more highly purified CRP preparations even under a variety of experimental conditions. Purified CRP alone did not bind to peripheral blood lymphocytes (PBL); however, in the presence of a ligand such as pneumococcal C-polysaccharide (CPS), CRP binding was readily detectable both by immunofluorescence and by a radioassay established for this purpose. The optimal concentration of CRP, ratio of CRP:CPS, and time and temperature for reactivity were determined using both assays. A markedly enhanced rate of binding was observed after pre-equilibration of CRP with calcium. A small percentage (mean 3.0%; range 0.5 to 8.0%) of PBL bound complexed CRP, and saturation was reached with 200 microgram CRP/ml. Reactivity of CRP with a multimeric form of phosphocholine (PC) (KLH-PC44) led to binding comparable to that observed with CPS, whereas monomeric PC inhibited the binding. Thus, in the presence of a multimeric binding specificity, CRP binds to a small fraction of peripheral blood lymphocytes, which are characterized in the accompanying paper.

  2. Binding-regulated click ligation for selective detection of proteins.

    PubMed

    Cao, Ya; Han, Peng; Wang, Zhuxin; Chen, Weiwei; Shu, Yongqian; Xiang, Yang

    2016-04-15

    Herein, a binding-regulated click ligation (BRCL) strategy for endowing selective detection of proteins is developed with the incorporation of small-molecule ligand and clickable DNA probes. The fundamental principle underlying the strategy is the regulating capability of specific protein-ligand binding against the ligation between clickable DNA probes, which could efficiently combine the detection of particular protein with enormous DNA-based sensing technologies. In this work, the feasibly of the BRCL strategy is first verified through agarose gel electrophoresis and electrochemical impedance spectroscopy measurements, and then confirmed by transferring it to a nanomaterial-assisted fluorescence assay. Significantly, the BRCL strategy-based assay is able to respond to target protein with desirable selectivity, attributing to the specific recognition between small-molecule ligand and its target. Further experiments validate the general applicability of the sensing method by tailoring the ligand toward different proteins (i.e., avidin and folate receptor), and demonstrate its usability in complex biological samples. To our knowledge, this work pioneers the practice of click chemistry in probing specific small-molecule ligand-protein binding, and therefore may pave a new way for selective detection of proteins.

  3. Drug-drug plasma protein binding interactions of ivacaftor.

    PubMed

    Schneider, Elena K; Huang, Johnny X; Carbone, Vincenzo; Baker, Mark; Azad, Mohammad A K; Cooper, Matthew A; Li, Jian; Velkov, Tony

    2015-06-01

    Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR-protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co-administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co-administered CF drugs for human serum albumin (HSA) and α1 -acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site-selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug-drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug-drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug-drug interactions of ivacaftor with co-administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor. PMID:25707701

  4. Structural and functional analysis of fatty acid-binding proteins

    PubMed Central

    Storch, Judith; McDermott, Lindsay

    2009-01-01

    The mammalian FA-binding proteins (FABPs) bind long-chain FA with high affinity. The large number of FABP types is suggestive of distinct functions in specific tissues. Multiple experimental approaches have shown that individual FABPs possess both unique and overlapping functions, some of which are based on specific elements in the protein structure. Although FA binding affinities for all FABPs tend to correlate directly with FA hydrophobicity, structure-function studies indicate that subtle three-dimensional changes that occur upon ligand binding may promote specific protein-protein or protein-membrane interactions that ultimately determine the function of each FABP. The conformational changes are focused in the FABP helical/portal domain, a region that was identified by in vitro studies to be vital for the FA transport properties of the FABPs. Thus, the FABPs modulate intracellular lipid homeostasis by regulating FA transport in the nuclear and extra-nuclear compartments of the cell; in so doing, they also impact systemic energy homeostasis. PMID:19017610

  5. The RNA-binding protein repertoire of Arabidopsis thaliana

    PubMed Central

    Marondedze, Claudius; Thomas, Ludivine; Serrano, Natalia L.; Lilley, Kathryn S.; Gehring, Chris

    2016-01-01

    RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses. PMID:27405932

  6. Carotenoid-binding proteins; accessories to carotenoid function.

    PubMed

    Pilbrow, Jodi; Garama, Daniel; Carne, Alan

    2012-01-01

    Understanding of the widespread biological importance of carotenoids is increasing. Accompanying this is the developing recognition that the interaction of carotenoids with other molecules, such as proteins, is also essential. Here the significance of carotenoid-protein interactions with respect to biological function is reviewed for three well characterised carotenoprotein complexes; crustacyanin, the orange carotenoid protein and glutathione-S-transferase P1. In addition a preliminary report is made on the recent partial purification of an echinenone-binding protein extracted from a New Zealand sea urchin, Evechinus chloroticus. PMID:22428138

  7. Carotenoid-binding proteins; accessories to carotenoid function.

    PubMed

    Pilbrow, Jodi; Garama, Daniel; Carne, Alan

    2012-01-01

    Understanding of the widespread biological importance of carotenoids is increasing. Accompanying this is the developing recognition that the interaction of carotenoids with other molecules, such as proteins, is also essential. Here the significance of carotenoid-protein interactions with respect to biological function is reviewed for three well characterised carotenoprotein complexes; crustacyanin, the orange carotenoid protein and glutathione-S-transferase P1. In addition a preliminary report is made on the recent partial purification of an echinenone-binding protein extracted from a New Zealand sea urchin, Evechinus chloroticus.

  8. Binding of IKe gene 5 protein to polynucleotides. Fluorescence binding experiments of IKe gene 5 protein and mutual cooperativity of IKe and M13 gene 5 proteins.

    PubMed

    de Jong, E A; Harmsen, B J; Konings, R N; Hilbers, C W

    1987-04-01

    Fluorescence studies of the binding of IKe gene 5 protein to various polynucleotides were performed to obtain insight into the question as to what extent the binding characteristics of the gene 5 proteins of the IKe and M13 phages resemble and/or differ from each other. The fluorescence of IKe gene 5 protein is quenched 60% upon binding to most polynucleotides. At moderate salt concentrations, i.e., below 1 M salt, the binding stoichiometry is 4.0 +/- 0.5 nucleotides per IKe gene 5 protein monomer. The affinity of the protein for homopolynucleotides depends strongly on sugar and base type; in order of increasing affinities we find poly(rC) less than poly(dA) less than poly(rA) less than poly(dI) less than poly(rU) less than poly(dU) less than poly(dT). For most polynucleotides studied, the affinity depends linearly on the salt concentration: [d log (Kint omega)]/(d log [M+]) = -3. The binding is highly cooperative. The cooperativity parameter omega, as deduced from protein titration curves, is 300 +/- 150 and appears independent of the type of polynucleotide studied. Estimation of this binding parameter from salt titrations of gene 5 protein-polynucleotide complexes results in systematically higher values. A comparison of the binding data of the IKe and M13 gene 5 proteins shows that the fluorescence quenching, stoichiometry, order of binding affinities, and cooperativity in the binding are similar for both proteins. From this it is concluded that at least the DNA binding grooves of both proteins must show a close resemblance.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Fluorescence turn-on detection of a protein through the displaced single-stranded DNA binding protein binding to a molecular beacon.

    PubMed

    Tang, Dan; Liao, Dongli; Zhu, Qiankun; Wang, Fangyuan; Jiao, Huping; Zhang, Yujing; Yu, Cong

    2011-05-21

    A new approach has been developed for the highly sensitive and selective sensing of a protein. Lysozyme binding to its aptamer prevents SSB protein binding, and the subsequent binding of the free SSB protein to a molecular beacon results in a turn-on fluorescence signal, which can be used for lysozyme quantification.

  10. Thioredoxin-Interacting Protein (TXNIP) Suppresses Expression of Glutamine Synthetase by Inducing Oxidative Stress in Retinal Muller Glia Under Diabetic Conditions

    PubMed Central

    Zhou, Jia; Shen, Xi; Lu, Qiong; Zhang, Min

    2016-01-01

    Background Diabetic retinopathy (DR) is a progressive neurodegenerative disease with early-stage symptoms such as dysfunction of Muller cells, which leads to ganglion cell death. Its pathogenesis is probably associated with oxidative stress and a recently discovered protein, thioredoxin-interacting protein (TXNIP). Material/Methods To explore the role of TXNIP in DR, we cultured Muller cells under diabetic conditions, and then used immunohistochemistry, Western blot, and RT-PCR to detect the expression level of TXNIP under diabetic conditions. We demonstrated the expression level of glutamine synthetase (GS) when TXNIP was inhibited. To explore the potential pathway of TXNIP-induced cell damage in DR, we confirmed the role of IL-1β under diabetic conditions. Results Diabetes induces TXNIP expressions at mRNA levels, but shows the opposite effect on GS. IL-1β plays an important role in this pathway. Azaserine effectively increased the expression of GS via attenuating the expression of TXNIP. Conclusions This study demonstrates the role of TXNIP and its mechanism in DR, provides a possible treatment for DR, and lays a new theoretical foundation for the clinical treatment of DR and other diabetic microvascular changes. PMID:27131835

  11. Novel folate binding protein-1 interactions in embryonic orofacial tissue

    PubMed Central

    Pisano, M. Michele; Bhattacherjee, Vasker; Wong, Leeyean; Finnell, Richard H.; Greene, Robert M.

    2010-01-01

    Aim To identify proteins with which FolBp1 may interact within lipid rafts in tissue derived from embryonic orofacial tissue. Methods A yeast two-hybrid screen of a cDNA library, derived from orofacial tissue from gestational day 11 to 13 mouse embryos, was conducted. Key Findings Using the full-length FolBp1 protein as bait, two proteins that bind FolBp1 were identified, Bat2d, and a fibronectin type III-containing domain protein. Results were confirmed by glutathione S-transferase pull-down assays. Significance As a component of membrane lipid raft protein complexes, these binding proteins may represent “helper” or chaperone proteins that associate with FolBp1 in order to facilitate the transport of folate across the plasma membrane. The protein-protein interactions detected, while limited in number, may be critical in mediating the role of FolBp1 in folate transport, particularly in the developing embryo. PMID:20045418

  12. Use of native gels to measure protein binding to SSB.

    PubMed

    Inoue, Jin; Mikawa, Tsutomu

    2012-01-01

    We describe a procedure to detect protein binding to SSB by polyacrylamide gel electrophoresis under non-denaturing conditions. As an example, we show the interaction of Thermus thermophilus (Tth) SSB with its cognate RecO protein. The interaction is detected as decay of the band corresponding to SSB by addition of RecO. We also demonstrate analysis of the RecO-RecR interaction as another example of this method. PMID:22976186

  13. Tritium NMR spectroscopy of ligand binding to maltose-binding protein

    SciTech Connect

    Gehring, K.; Williams, P.G.; Pelton, J.G.; Morimoto, H.; Wemmer, D.E. )

    1991-06-04

    Tritium-labeled {alpha}- and {beta}-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR specctroscopy of the labeled sugars showed large upfield chamical shift changes upon binding and strong anomeric specficity. At 10{degrees}C, MBP bound {alpha}-maltose with 2.7 {plus minus} 0.5-fold higher affinity than {beta}-maltose, and, for longer maltodextrins, the ratio of affinities was even larger. The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound {alpha}-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound {beta}-maltotriose resonances in rapid exchange. The authors interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the {beta}-maltodextrin is bound by its reducing end, and, in the other complex, the {beta}-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins.

  14. The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative disease.

    PubMed

    King, Oliver D; Gitler, Aaron D; Shorter, James

    2012-06-26

    Prions are self-templating protein conformers that are naturally transmitted between individuals and promote phenotypic change. In yeast, prion-encoded phenotypes can be beneficial, neutral or deleterious depending upon genetic background and environmental conditions. A distinctive and portable 'prion domain' enriched in asparagine, glutamine, tyrosine and glycine residues unifies the majority of yeast prion proteins. Deletion of this domain precludes prionogenesis and appending this domain to reporter proteins can confer prionogenicity. An algorithm designed to detect prion domains has successfully identified 19 domains that can confer prion behavior. Scouring the human genome with this algorithm enriches a select group of RNA-binding proteins harboring a canonical RNA recognition motif (RRM) and a putative prion domain. Indeed, of 210 human RRM-bearing proteins, 29 have a putative prion domain, and 12 of these are in the top 60 prion candidates in the entire genome. Startlingly, these RNA-binding prion candidates are inexorably emerging, one by one, in the pathology and genetics of devastating neurodegenerative disorders, including: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), Alzheimer's disease and Huntington's disease. For example, FUS and TDP-43, which rank 1st and 10th among RRM-bearing prion candidates, form cytoplasmic inclusions in the degenerating motor neurons of ALS patients and mutations in TDP-43 and FUS cause familial ALS. Recently, perturbed RNA-binding proteostasis of TAF15, which is the 2nd ranked RRM-bearing prion candidate, has been connected with ALS and FTLD-U. We strongly suspect that we have now merely reached the tip of the iceberg. We predict that additional RNA-binding prion candidates identified by our algorithm will soon surface as genetic modifiers or causes of diverse neurodegenerative conditions. Indeed, simple prion-like transfer mechanisms involving the prion

  15. Capacitance-modulated transistor detects odorant binding protein chiral interactions.

    PubMed

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters. PMID:25591754

  16. RNA–protein binding kinetics in an automated microfluidic reactor

    PubMed Central

    Ridgeway, William K.; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R.

    2009-01-01

    Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome. PMID:19759214

  17. Methods of use of cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1997-09-23

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  18. RNA-protein binding kinetics in an automated microfluidic reactor.

    PubMed

    Ridgeway, William K; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R

    2009-11-01

    Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA-protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic 'Riboreactor' has been designed and constructed to facilitate the study of kinetics of RNA-protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA-protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.

  19. Methods of use of cellulose binding domain proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1997-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  20. Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A.

    PubMed Central

    Shoseyov, O; Takagi, M; Goldstein, M A; Doi, R H

    1992-01-01

    The cbpA gene for the Clostridium cellulovorans cellulose binding protein (CbpA), which is part of the multisubunit cellulase complex, has been cloned and sequenced. When cbpA was expressed in Escherichia coli, proteins capable of binding to crystalline cellulose and of interacting with anti-CbpA were observed. The cbpA gene consists of 5544 base pairs and encodes a protein containing 1848 amino acids with a molecular mass of 189,036 Da. The open reading frame is preceded by a Gram-positive-type ribosome binding site. A signal peptide sequence of 28 amino acids is present at its N terminus. The encoded protein is highly hydrophobic with extremely high levels of threonine and valine residues. There are two types of putative cellulose binding domains of approximately 100 amino acids that are slightly hydrophilic and eight conserved, highly hydrophobic beta-sheet regions of approximately 140 amino acids. These latter hydrophobic regions may be the CbpA domains that interact with the different enzymatic subunits of the cellulase complex. Images PMID:1565642

  1. Capacitance-modulated transistor detects odorant binding protein chiral interactions

    NASA Astrophysics Data System (ADS)

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.

  2. Structural analysis of ibuprofen binding to human adipocyte fatty-acid binding protein (FABP4)

    PubMed Central

    González, Javier M.; Fisher, S. Zoë

    2015-01-01

    Inhibition of human adipocyte fatty-acid binding protein (FABP4) has been proposed as a treatment for type 2 diabetes, fatty liver disease and atherosclerosis. However, FABP4 displays a naturally low selectivity towards hydrophobic ligands, leading to the possibility of side effects arising from cross-inhibition of other FABP isoforms. In a search for structural determinants of ligand-binding selectivity, the binding of FABP4 towards a group of small molecules structurally related to the nonsteroidal anti-inflammatory drug ibuprofen was analyzed through X-ray crystallography. Several specific hydrophobic interactions are shown to enhance the binding affinities of these compounds, whereas an aromatic edge-to-face interaction is proposed to determine the conformation of bound ligands, highlighting the importance of aromatic interactions in hydrophobic environments. PMID:25664790

  3. Structural analysis of ibuprofen binding to human adipocyte fatty-acid binding protein (FABP4).

    PubMed

    González, Javier M; Fisher, S Zoë

    2015-02-01

    Inhibition of human adipocyte fatty-acid binding protein (FABP4) has been proposed as a treatment for type 2 diabetes, fatty liver disease and atherosclerosis. However, FABP4 displays a naturally low selectivity towards hydrophobic ligands, leading to the possibility of side effects arising from cross-inhibition of other FABP isoforms. In a search for structural determinants of ligand-binding selectivity, the binding of FABP4 towards a group of small molecules structurally related to the nonsteroidal anti-inflammatory drug ibuprofen was analyzed through X-ray crystallography. Several specific hydrophobic interactions are shown to enhance the binding affinities of these compounds, whereas an aromatic edge-to-face interaction is proposed to determine the conformation of bound ligands, highlighting the importance of aromatic interactions in hydrophobic environments.

  4. The EGF receptor is an actin-binding protein

    PubMed Central

    1992-01-01

    In a number of recent studies it has been shown that in vivo part of the EGF receptor (EGFR) population is associated to the actin filament system. In this paper we demonstrate that the purified EGFR can be cosedimented with purified filamentous actin (F-actin) indicating a direct association between EGFR and actin. A truncated EGFR, previously shown not to be associated to the cytoskeleton, was used as a control and this receptor did not cosediment with actin filaments. Determination of the actin-binding domain of the EGFR was done by measuring competition of either a polyclonal antibody or synthetic peptides on EGFR cosedimentation with F-actin. A synthetic peptide was made homologous to amino acid residues 984-996 (HL-33) of the EGFR which shows high homology with the actin-binding domain of Acanthamoeba profilin. A polyclonal antibody raised against HL-33 was found to prevent cosedimentation of EGFR with F-actin. This peptide HL-33 was shown to bind directly to actin in contrast with a synthetic peptide homologous to residues 1001-1013 (HL-34). During cosedimentation, HL-33 competed for actin binding of the EGFR and HL-34 did not, indicating that the EGFR contains one actin-binding site. These results demonstrate that the EGFR is an actin-binding protein which binds to actin via a domain containing amino acids residues 984-996. PMID:1383230

  5. A general approach to visualize protein binding and DNA conformation without protein labelling

    PubMed Central

    Song, Dan; Graham, Thomas G. W.; Loparo, Joseph J.

    2016-01-01

    Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein–DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein–DNA interactions. PMID:26952553

  6. Monomeric Yeast Frataxin is an Iron-Binding Protein

    SciTech Connect

    Cook,J.; Bencze, K.; Jankovic, A.; Crater, A.; Busch, C.; Bradley, P.; Stemmler, A.; Spaller, M.; Stemmler, T.

    2006-01-01

    Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50 000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.

  7. The Archaeal Lsm Protein Binds to Small RNAs*

    PubMed Central

    Fischer, Susan; Benz, Juliane; Späth, Bettina; Maier, Lisa-Katharina; Straub, Julia; Granzow, Michaela; Raabe, Monika; Urlaub, Henning; Hoffmann, Jan; Brutschy, Bernd; Allers, Thorsten; Soppa, Jörg; Marchfelder, Anita

    2010-01-01

    Proteins of the Lsm family, including eukaryotic Sm proteins and bacterial Hfq, are key players in RNA metabolism. Little is known about the archaeal homologues of these proteins. Therefore, we characterized the Lsm protein from the haloarchaeon Haloferax volcanii using in vitro and in vivo approaches. H. volcanii encodes a single Lsm protein, which belongs to the Lsm1 subfamily. The lsm gene is co-transcribed and overlaps with the gene for the ribosomal protein L37e. Northern blot analysis shows that the lsm gene is differentially transcribed. The Lsm protein forms homoheptameric complexes and has a copy number of 4000 molecules/cell. In vitro analyses using electrophoretic mobility shift assays and ultrasoft mass spectrometry (laser-induced liquid bead ion desorption) showed a complex formation of the recombinant Lsm protein with oligo(U)-RNA, tRNAs, and an small RNA. Co-immunoprecipitation with a FLAG-tagged Lsm protein produced in vivo confirmed that the protein binds to small RNAs. Furthermore, the co-immunoprecipitation revealed several protein interaction partners, suggesting its involvement in different cellular pathways. The deletion of the lsm gene is viable, resulting in a pleiotropic phenotype, indicating that the haloarchaeal Lsm is involved in many cellular processes, which is in congruence with the number of protein interaction partners. PMID:20826804

  8. The neuronal calcium sensor family of Ca2+-binding proteins.

    PubMed Central

    Burgoyne, R D; Weiss, J L

    2001-01-01

    Ca(2+) plays a central role in the function of neurons as the trigger for neurotransmitter release, and many aspects of neuronal activity, from rapid modulation to changes in gene expression, are controlled by Ca(2+). These actions of Ca(2+) must be mediated by Ca(2+)-binding proteins, including calmodulin, which is involved in Ca(2+) regulation, not only in neurons, but in most other cell types. A large number of other EF-hand-containing Ca(2+)-binding proteins are known. One family of these, the neuronal calcium sensor (NCS) proteins, has a restricted expression in retinal photoreceptors or neurons and neuroendocrine cells, suggesting that they have specialized roles in these cell types. Two members of the family (recoverin and guanylate cyclase-activating protein) have established roles in the regulation of phototransduction. Despite close sequence similarities, the NCS proteins have distinct neuronal distributions, suggesting that they have different functions. Recent work has begun to demonstrate the physiological roles of members of this protein family. These include roles in the modulation of neurotransmitter release, control of cyclic nucleotide metabolism, biosynthesis of polyphosphoinositides, regulation of gene expression and in the direct regulation of ion channels. In the present review we describe the known sequences and structures of the NCS proteins, information on their interactions with target proteins and current knowledge about their cellular and physiological functions. PMID:11115393

  9. Glutamine supplementation in bone marrow transplantation.

    PubMed

    Ziegler, Thomas R

    2002-01-01

    An increasing number of clinical investigations have focused on supplementation of specialized enteral and parenteral nutrition with the amino acid glutamine. This interest derives from strong evidence in animal models and emerging clinical data on the efficacy of glutamine administration following chemotherapy, trauma, sepsis and other catabolic conditions. Glutamine has protein-anabolic effects in stressed patients and, among many key metabolic functions, is used as a major fuel/substrate by cells of the gastrointestinal epithelium and the immune system. These effects may be particularly advantageous in patients undergoing bone marrow transplantation (BMT), who exhibit post-transplant body protein wasting, gut mucosal injury and immunodeficiency. Studies to date indicate that enteral and parenteral glutamine supplementation is well tolerated and potentially efficacious after high-dose chemotherapy or BMT for cancer treatment. Although not all studies demonstrate benefits, sufficient positive data have been published to suggest that this nutrient should be considered as adjunctive metabolic support of some individuals undergoing marrow transplant. However, BMT is a rapidly evolving clinical procedure with regard to the conditioning and supportive protocols utilized. Thus, additional randomized, double-blind, controlled clinical trials are indicated to define the efficacy of glutamine with current BMT regimens.

  10. Glutamine Modulates Macrophage Lipotoxicity

    PubMed Central

    He, Li; Weber, Kassandra J.; Schilling, Joel D.

    2016-01-01

    Obesity and diabetes are associated with excessive inflammation and impaired wound healing. Increasing evidence suggests that macrophage dysfunction is responsible for these inflammatory defects. In the setting of excess nutrients, particularly dietary saturated fatty acids (SFAs), activated macrophages develop lysosome dysfunction, which triggers activation of the NLRP3 inflammasome and cell death. The molecular pathways that connect lipid stress to lysosome pathology are not well understood, but may represent a viable target for therapy. Glutamine uptake is increased in activated macrophages leading us to hypothesize that in the context of excess lipids glutamine metabolism could overwhelm the mitochondria and promote the accumulation of toxic metabolites. To investigate this question we assessed macrophage lipotoxicity in the absence of glutamine using LPS-activated peritoneal macrophages exposed to the SFA palmitate. We found that glutamine deficiency reduced lipid induced lysosome dysfunction, inflammasome activation, and cell death. Under glutamine deficient conditions mTOR activation was decreased and autophagy was enhanced; however, autophagy was dispensable for the rescue phenotype. Rather, glutamine deficiency prevented the suppressive effect of the SFA palmitate on mitochondrial respiration and this phenotype was associated with protection from macrophage cell death. Together, these findings reveal that crosstalk between activation-induced metabolic reprogramming and the nutrient microenvironment can dramatically alter macrophage responses to inflammatory stimuli. PMID:27077881

  11. Nuclear protein accumulation by facilitated transport and intranuclear binding.

    PubMed

    Paine, P L

    1993-10-01

    Nuclear proteins are transported from the cytoplasm into the nucleus via nuclear envelope pore complexes (NPCs). At the molecular level, the mechanisms responsible for this transport remain obscure. However, it is known that, for many proteins, the process requires ATP and proceeds against formidable nucleocytoplasmic concentration gradients. Therefore, the NPC is often thought of as an active transport site. In this article, Philip Paine presents the alternative hypothesis that, on current evidence, protein translocation across the nuclear envelope and accumulation in the nucleus can equally well be explained by facilitated transport through the NPC and subsequent intranuclear binding.

  12. Ligand-binding study of Anopheles gambiae chemosensory proteins.

    PubMed

    Iovinella, Immacolata; Bozza, Francesco; Caputo, Beniamino; Della Torre, Alessandra; Pelosi, Paolo

    2013-06-01

    Chemosensory proteins (CSPs) are a class of small proteins expressed only in arthropods and endowed with heterogeneous functions. Some of them are involved in chemical communications, others in development or other physiological roles. The numbers of CSPs in different species of insects range from 4 in Drosophila to at least 70 in locusts, whereas in other arthropods such as crustaceans and millipedes, only 2-3 very similar sequences have been reported in each species. We have expressed, in a bacterial system, 5 of the 8 CSPs predicted by the genome of the malaria mosquito Anopheles gambiae, 4 identified at the protein level (SAP1, SAP2, SAP3, and CSP3) and a fifth annotated as part of this work, obtaining the proteins with high yields and in their soluble forms. Purified CSPs have been used to study their ligand-binding properties, both using competitive binding assays and quenching of intrinsic tryptophan fluorescence, in order to get insights into their physiological functions. The agreement between the 2 sets of data supports the assumptions that the ligands, including the fluorescent reporter, bind within the core of the proteins. Their different affinities toward a set of pure chemicals suggest specific roles in chemical communication.

  13. Goodpasture Antigen-binding Protein (GPBP) Directs Myofibril Formation

    PubMed Central

    Revert-Ros, Francisco; López-Pascual, Ernesto; Granero-Moltó, Froilán; Macías, Jesús; Breyer, Richard; Zent, Roy; Hudson, Billy G.; Saadeddin, Anas; Revert, Fernando; Blasco, Raül; Navarro, Carmen; Burks, Deborah; Saus, Juan

    2011-01-01

    Goodpasture antigen-binding protein-1 (GPBP-1) is an exportable non-conventional Ser/Thr kinase that regulates glomerular basement membrane collagen organization. Here we provide evidence that GPBP-1 accumulates in the cytoplasm of differentiating mouse myoblasts prior to myosin synthesis. Myoblasts deficient in GPBP-1 display defective myofibril formation, whereas myofibrils assemble with enhanced efficiency in those overexpressing GPBP-1. We also show that GPBP-1 targets the previously unidentified GIP130 (GPBP-interacting protein of 130 kDa), which binds to myosin and promotes its myofibrillar assembly. This report reveals that GPBP-1 directs myofibril formation, an observation that expands its reported role in supramolecular organization of structural proteins to the intracellular compartment. PMID:21832087

  14. Cytosolic insulin-binding proteins of mouse liver cells.

    PubMed

    Lokhov, Petr G; Moshkovskii, Sergei A; Ipatova, Olga M; Prozorovskii, Vladimir N

    2004-02-01

    It has been recently shown that insulin retains its biological activity after receptor-directed internalization and it may affect the cell metabolism by interaction with cytosolic insulin-binding proteins (CIBPs). Using affinity chromatography combined with SDS-PAGE and MALDI-TOF mass-spectrometry we have identified 7 proteins from mouse liver cells that specifically bind to the insulin, including adenylate kinase 2 (25.6 kD), kinesin superfamily protein 20B (26.0 kD), hepatic arginase 1 (34.8 kD), fructose-bisphosphate aldolase B (39.5 kD), 4-hydroxyphenylpyruvate dioxygenase (45.1 kD), betaine-homocysteine methyl-transferase (45.0 kD) and KRIT1 (83.4 kD).

  15. Light aerobic physical exercise in combination with leucine and/or glutamine-rich diet can improve the body composition and muscle protein metabolism in young tumor-bearing rats.

    PubMed

    Salomão, Emilianne Miguel; Gomes-Marcondes, Maria Cristina Cintra

    2012-12-01

    Nutritional supplementation with some amino acids may influence host's responses and also certain mechanism involved in tumor progression. It is known that exercise influences body weight and muscle composition. Previous findings from our group have shown that leucine has beneficial effects on protein composition in cachectic rat model as the Walker 256 tumor. The main purpose of this study was to analyze the effects of light exercise and leucine and/or glutamine-rich diet in body composition and skeletal muscle protein synthesis and degradation in young tumor-bearing rats. Walker tumor-bearing rats were subjected to light aerobic exercise (swimming 30 min/day) and fed a leucine-rich (3%) and/or glutamine-rich (4%) diet for 10 days and compared to healthy young rats. The carcasses were analyzed as total water and fat body content and lean body mass. The gastrocnemious muscles were isolated and used for determination of total protein synthesis and degradation. The chemical body composition changed with tumor growth, increasing body water and reducing body fat content and total body nitrogen. After tumor growth, the muscle protein metabolism was impaired, showing that the muscle protein synthesis was also reduced and the protein degradation process was increased in the gastrocnemius muscle of exercised rats. Although short-term exercise (10 days) alone did not produce beneficial effects that would reduce tumor damage, host protein metabolism was improved when exercise was combined with a leucine-rich diet. Only total carcass nitrogen and protein were recovered by a glutamine-rich diet. Exercise, in combination with an amino acid-rich diet, in particular, leucine, had effects beyond reducing tumoral weight such as improving protein turnover and carcass nitrogen content in the tumor-bearing host. PMID:22460363

  16. RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome.

    PubMed

    Silverman, Ian M; Li, Fan; Alexander, Anissa; Goff, Loyal; Trapnell, Cole; Rinn, John L; Gregory, Brian D

    2014-01-07

    Although numerous approaches have been developed to map RNA-binding sites of individual RNA-binding proteins (RBPs), few methods exist that allow assessment of global RBP-RNA interactions. Here, we describe PIP-seq, a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply PIP-seq to the HeLa transcriptome and compare binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP-binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites.

  17. Glutamine as an immunoenhancing nutrient.

    PubMed

    Saito, H; Furukawa, S; Matsuda, T

    1999-01-01

    New strategies for immunonutritional support include administration of special nutrients such as glutamine. Glutamine is important in several key metabolic processes of immune cells and enterocytes. Exogenous glutamine augments the functions of lymphocytes and macrophages. Neutrophils also reportedly utilize glutamine at a significant rate. Our recent studies demonstrated that glutamine enhances neutrophil function. This article focuses on the effects of glutamine on neutrophil function in surgical stress. Enteral glutamine administration enhanced peritoneal and hepatic bacterial clearance in our rat peritonitis model. Furthermore, IV glutamine supplementation improved the outcome of animals with severe surgical stress. Our in vitro study revealed that supplemental glutamine augmented the bacterial killing function of neutrophils from postoperative patients. Glutamine increased phagocytosis of the neutrophils. In addition, glutamine dose-dependently increased production of reactive oxygen intermediates (ROI) by neutrophils. Thus, our studies suggest that glutamine supplementation may improve bactericidal function of neutrophils by increasing both phagocytosis and ROI production. In conclusion, glutamine plays an important role in neutrophil function. Glutamine may be useful for the prevention, and treatment, of severe infection in critical illness and trauma.

  18. Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

    PubMed

    Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

    2016-12-15

    In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins. PMID:27451201

  19. Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

    PubMed

    Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

    2016-12-15

    In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins.

  20. Protein interactors of acyl-CoA-binding protein ACBP2 mediate cadmium tolerance in Arabidopsis.

    PubMed

    Gao, Wei; Li, Hong-Ye; Xiao, Shi; Chye, Mee-Len

    2010-08-01

    In our recent paper in the Plant Journal, we reported that Arabidopsis thaliana lysophospholipase 2 (lysoPL2) binds acyl-CoA-binding protein 2 (ACBP2) to mediate cadmium [Cd(II)] tolerance in transgenic Arabidopsis. ACBP2 contains ankyrin repeats that have been previously shown to mediate protein-protein interactions with an ethylene-responsive element binding protein (AtEBP) and a farnesylated protein 6 (AtFP6). Transgenic Arabidopsis ACBP2-overexpressors, lysoPL2-overexpressors and AtFP6-overexpressors all display enhanced Cd(II) tolerance, in comparison to wild type, suggesting that ACBP2 and its protein partners work together to mediate Cd(II) tolerance. Given that recombinant ACBP2 and AtFP6 can independently bind Cd(II) in vitro, they may be able to participate in Cd(II) translocation. The binding of recombinant ACBP2 to [(14)C]linoleoyl-CoA and [(14)C]linolenoyl-CoA implies its role in phospholipid repair. In conclusion, ACBP2 can mediate tolerance to Cd(II)-induced oxidative stress by interacting with two protein partners, AtFP6 and lysoPL2. Observations that ACBP2 also binds lysophosphatidylcholine (lysoPC) in vitro and that recombinant lysoPL2 degrades lysoPC, further confirm an interactive role for ACBP2 and lysoPL2 in overcoming Cd(II)-induced stress.

  1. Polyamine binding to proteins in oat and Petunia protoplasts

    NASA Technical Reports Server (NTRS)

    Mizrahi, Y.; Applewhite, P. B.; Galston, A. W.

    1989-01-01

    Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

  2. Neisseria meningitis GNA1030 is a ubiquinone-8 binding protein.

    PubMed

    Donnarumma, Danilo; Golfieri, Giacomo; Brier, Sébastien; Castagnini, Marta; Veggi, Daniele; Bottomley, Matthew James; Delany, Isabel; Norais, Nathalie

    2015-06-01

    Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is composed of 3 main recombinant proteins and an outer membrane vesicle component. One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA), is present as a fusion protein with the accessory protein genome-derived neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene encoding for GNA1030 is present and highly conserved in all Neisseria strains, and although orthologs are present in numerous species, its biologic function is unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly involved in the electron transport chain and with antioxidant properties. Disc diffusion assays on the wild-type and knockout mutant of GNA1030, in the presence of various compounds, suggested that GNA1030 is not involved in oxidative stress or electron chain transport per se, although it contributes to constitutive refilling of the inner membrane with Ub8. These studies shed light on an accessory protein present in Bexsero and reveal functional insights into the family of related proteins. On the basis of our findings, we propose to name the protein neisseria ubiquinone binding protein (NUbp).

  3. A novel calcium-binding protein is associated with tau proteins in tauopathy

    PubMed Central

    Vega, Irving E.; Traverso, Edwin E.; Ferrer-Acosta, Yancy; Matos, Eduardo; Colon, Migdalisel; Gonzalez, John; Dickson, Dennis; Hutton, Michael; Lewis, Jada; Yen, Shu H.

    2013-01-01

    Tauopathies are a group of neurological disorders characterized by the presence of intraneuronal hyperphosphorylated and filamentous tau. Mutations in the tau gene have been found in kindred with tauopathy. The expression of the human tau mutant in transgenic mice induced neurodegeneration, indicating that tau plays a central pathological role. However, the molecular mechanism leading to tau-mediated neurodegeneration is poorly understood. To gain insights into the role that tau plays in neurodegeneration, human tau proteins were immunoprecipitated from brain lysates of the tauopathy mouse model JNPL3, which develops neurodegeneration in age-dependent manner. In the present work, a novel EF-hand domain-containing protein was found associated with tau proteins in brain lysate of 12-month-old JNPL3 mice. The association between tau proteins and the novel identified protein appears to be induced by the neurodegeneration process as these two proteins were not found associated in young JNPL3 mice. Consistently, the novel protein co-purified with the pathological sarkosyl insoluble tau in terminally ill JNPL3 mice. Calcium-binding assays demonstrated that this protein binds calcium effectively. Finally, the association between tau and the novel calcium-binding protein is conserved in human and enriched in Alzheimer's disease brain. Taken together, the identification of a novel calcium-binding protein associated with tau protein in terminally ill tauopathy mouse model and its confirmation in human brain lysate suggests that this association may play an important physiological and/or pathological role. PMID:18346207

  4. Finding the target sites of RNA-binding proteins

    PubMed Central

    Li, Xiao; Kazan, Hilal; Lipshitz, Howard D; Morris, Quaid D

    2014-01-01

    RNA–protein interactions differ from DNA–protein interactions because of the central role of RNA secondary structure. Some RNA-binding domains (RBDs) recognize their target sites mainly by their shape and geometry and others are sequence-specific but are sensitive to secondary structure context. A number of small- and large-scale experimental approaches have been developed to measure RNAs associated in vitro and in vivo with RNA-binding proteins (RBPs). Generalizing outside of the experimental conditions tested by these assays requires computational motif finding. Often RBP motif finding is done by adapting DNA motif finding methods; but modeling secondary structure context leads to better recovery of RBP-binding preferences. Genome-wide assessment of mRNA secondary structure has recently become possible, but these data must be combined with computational predictions of secondary structure before they add value in predicting in vivo binding. There are two main approaches to incorporating structural information into motif models: supplementing primary sequence motif models with preferred secondary structure contexts (e.g., MEMERIS and RNAcontext) and directly modeling secondary structure recognized by the RBP using stochastic context-free grammars (e.g., CMfinder and RNApromo). The former better reconstruct known binding preferences for sequence-specific RBPs but are not suitable for modeling RBPs that recognize shape and geometry of RNAs. Future work in RBP motif finding should incorporate interactions between multiple RBDs and multiple RBPs in binding to RNA. WIREs RNA 2014, 5:111–130. doi: 10.1002/wrna.1201 PMID:24217996

  5. Calcium binding proteins and calcium signaling in prokaryotes.

    PubMed

    Domínguez, Delfina C; Guragain, Manita; Patrauchan, Marianna

    2015-03-01

    With the continued increase of genomic information and computational analyses during the recent years, the number of newly discovered calcium binding proteins (CaBPs) in prokaryotic organisms has increased dramatically. These proteins contain sequences that closely resemble a variety of eukaryotic calcium (Ca(2+)) binding motifs including the canonical and pseudo EF-hand motifs, Ca(2+)-binding β-roll, Greek key motif and a novel putative Ca(2+)-binding domain, called the Big domain. Prokaryotic CaBPs have been implicated in diverse cellular activities such as division, development, motility, homeostasis, stress response, secretion, transport, signaling and host-pathogen interactions. However, the majority of these proteins are hypothetical, and only few of them have been studied functionally. The finding of many diverse CaBPs in prokaryotic genomes opens an exciting area of research to explore and define the role of Ca(2+) in organisms other than eukaryotes. This review presents the most recent developments in the field of CaBPs and novel advancements in the role of Ca(2+) in prokaryotes.

  6. Capacitance-modulated transistor detects odorant binding protein chiral interactions

    PubMed Central

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand–protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein–ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters. PMID:25591754

  7. [Glutamine synthetase-like protein, glutamate dehydrogenase, and cytochrome c-oxidase in platelets of patients with the first episode psychosis in the course of treatment].

    PubMed

    Burbaeva, G Sh; Boksha, I S; Kaleda, V G; Barkhatova, A N; Turishcheva, M S; Omel'chenko, M A; Tereshkina, E B; Savushkina, O K; Starodubtseva, L I; Prokhorova, T A; Vorob'eva, E A

    2011-01-01

    The authors searched for correlations between amounts of platelet proteins and results of psychometric tests in patients with the first episode psychosis (schizophrenia, schizoaffective psychosis) in the course of their combined antipsychotic treatment with haloperidol and clozapine. Psychometric evaluations (PANSS, BPRS) and analyses of platelet enzymes - glutamine synthetase-like protein (GSLP), glutamate dehydrogenase (GDH), and cytochrome c-oxidase (COX) - were carried out before, during, and after the treatment. These proteins were also analyzed in matched controls. All the parameters comprised a database, followed by statistical data processing using Statistica 6.0 (StatSoft) software, nonparametric statistics module. The patients before the treatment, when compared with controls, demonstrated significantly decreased COX activity (p=0,0000001) and increased GSLP amount (p=0,006) with a positive correlation between GSLP amount and PANSSneg (R=0,34, p<0,01). Those patients who displayed initially low COX activity (below median) demonstrated significant increase in COX activity after the treatment. Negative correlations were revealed between COX activity and PANSS, PANSSpsy scores during the treatment, i.e. the lower was COX activity, the more severe syndromes were observed. Negative correlations were found between the initial COX activity and PANSS, PANSSpsy, BPRS scores after the treatment, i.e., the higher was COX before the treatment, the less prominent syndromes were observed after the treatment. Significantly more "non-responders" by PANSSneg were found among the patients with low GSLP level (below median) than their calculated expected amount. The COX activity measured before the treatment was significantly lower in patients with schizophrenia than in patients with schizoaffective disorder (SAD) (p=0,038). In SAD patients, the initial COX activity was negatively correlated with PANSSpsy and BPRS scores after the treatment (R=-0,5, p=0,02), i.e. the lower

  8. Inhibitory serpins from wheat grain with reactive centers resembling glutamine-rich repeats of prolamin storage proteins. Cloning and characterization of five major molecular forms.

    PubMed

    Ostergaard, H; Rasmussen, S K; Roberts, T H; Hejgaard, J

    2000-10-27

    Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P(1)-P(1)' Gln-Gln, and WSZ2b at P(2)-P(1) Leu-Arg-one of two overlapping reactive centers. WSZ1c with P(1)-P(1)' Leu-Gln was the fastest inhibitor of chymotrypsin (k(a) = 1.3 x 10(6) m(-1) s(-1)). WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k(a) approximately 10(5) m(-1) s(-1)), which has P(1)-P(1)' Leu-Ser-a reactive center common in animal serpins. WSZ2b inhibited plasmin at P(1)-P(1)' Arg-Gln (k(a) approximately 10(3) m(-1) s(-1)). None of the five serpins inhibited Bacillus subtilisin A, Fusarium trypsin, or two subtilisin-like plant serine proteinases, hordolisin from barley green malt and cucumisin D from honeydew melon. Possible functions involving interactions with endogenous or exogenous proteinases adapted to prolamin degradation are discussed.

  9. Protein-folding location can regulate manganese-binding versus copper- or zinc-binding.

    PubMed

    Tottey, Steve; Waldron, Kevin J; Firbank, Susan J; Reale, Brian; Bessant, Conrad; Sato, Katsuko; Cheek, Timothy R; Gray, Joe; Banfield, Mark J; Dennison, Christopher; Robinson, Nigel J

    2008-10-23

    Metals are needed by at least one-quarter of all proteins. Although metallochaperones insert the correct metal into some proteins, they have not been found for the vast majority, and the view is that most metalloproteins acquire their metals directly from cellular pools. However, some metals form more stable complexes with proteins than do others. For instance, as described in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage metal acquisition by most nascent proteins. To investigate this question, we identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of the cyanobacterium Synechocystis PCC 6803. Each of these newly identified proteins binds its respective metal via identical ligands within a cupin fold. Consistent with the Irving-Williams series, MncA only binds Mn(2+) after folding in solutions containing at least a 10(4) times molar excess of Mn(2+) over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal does not exchange with Cu(2+). MncA and CucA have signal peptides for different export pathways into the periplasm, Tat and Sec respectively. Export by the Tat pathway allows MncA to fold in the cytoplasm, which contains only tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal a mechanism whereby the compartment in which a protein folds overrides its binding preference to control its metal content. They explain why the cytoplasm must contain only tightly bound and buffered copper and Zn(2+). PMID:18948958

  10. Calmodulin binds to maize lipid transfer protein and modulates its lipids binding ability.

    PubMed

    Li, Cuifeng; Xie, Wanqin; Bai, Wenyan; Li, Zhenpeng; Zhao, Yulong; Liu, Hua

    2008-11-01

    Although plant non-specific lipid transfer proteins (ns-LTPs) are characterized by their ability to bind and transfer a broad range of hydrophobic ligands in vitro, their biological functions in vivo remain unclear. Recently, it has been proposed that ns-LTPs may play a key role in plant defense mechanisms, particularly during the induction of systemic acquired resistance, however, very little is known about the regulation in this process. We report that the binding of maize non-specific lipid transfer protein (Zm-LTP) to calmodulin (CaM) is in a calcium-independent manner. To better understand the interaction mechanism between Zm-LTP and CaM, the CaM-binding site of Zm-LTP was mapped to the region of amino acids 46-60. Point mutations indicate that four amino acid residues, R46, R47, K54 and R58, in this region are crucial for binding. Furthermore, we tested the effects of CaM on the lipid-binding activity of Zm-LTP in the presence of Ca(2+), EGTA, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide and trifluoperazine respectively. We also investigated the structural features of CaM-binding motifs in LTPs from different species and strong differences were observed. Taken together, our results suggest that the interaction with CaM could be a common feature of plant LTPs. The identification and characterization of CaM-binding domain of LTPs should provide new insights into the mechanism by which the physiological functions of LTPs are regulated.

  11. Copper binding to the prion protein: Structural implications of four identical cooperative binding sites

    PubMed Central

    Viles, John H.; Cohen, Fred E.; Prusiner, Stanley B.; Goodin, David B.; Wright, Peter E.; Dyson, H. Jane

    1999-01-01

    Evidence is growing to support a functional role for the prion protein (PrP) in copper metabolism. Copper ions appear to bind to the protein in a highly conserved octapeptide repeat region (sequence PHGGGWGQ) near the N terminus. To delineate the site and mode of binding of Cu(II) to the PrP, the copper-binding properties of peptides of varying lengths corresponding to 2-, 3-, and 4-octarepeat sequences have been probed by using various spectroscopic techniques. A two-octarepeat peptide binds a single Cu(II) ion with Kd ≈ 6 μM whereas a four-octarepeat peptide cooperatively binds four Cu(II) ions. Circular dichroism spectra indicate a distinctive structuring of the octarepeat region on Cu(II) binding. Visible absorption, visible circular dichroism, and electron spin resonance spectra suggest that the coordination sphere of the copper is identical for 2, 3, or 4 octarepeats, consisting of a square-planar geometry with three nitrogen ligands and one oxygen ligand. Consistent with the pH dependence of Cu(II) binding, proton NMR spectroscopy indicates that the histidine residues in each octarepeat are coordinated to the Cu(II) ion. Our working model for the structure of the complex shows the histidine residues in successive octarepeats bridged between two copper ions, with both the Nɛ2 and Nδ1 imidazole nitrogen of each histidine residue coordinated and the remaining coordination sites occupied by a backbone amide nitrogen and a water molecule. This arrangement accounts for the cooperative nature of complex formation and for the apparent evolutionary requirement for four octarepeats in the PrP. PMID:10051591

  12. Transport effects on the kinetics of protein-surface binding.

    PubMed Central

    Balgi, G; Leckband, D E; Nitsche, J M

    1995-01-01

    A detailed model is presented for protein binding to active surfaces, with application to the binding of avidin molecules to a biotin-functionalized fiber optic sensor in experiments reported by S. Zhao and W. M. Reichert (American Chemical Society Symposium Series 493, 1992). Kinetic data for binding in solution are used to assign an intrinsic catalytic rate coefficient k to the biotin-avidin pair, deconvoluted from transport and electrostatic factors via application of coagulation theory. This intrinsic chemical constant is built into a reaction-diffusion analysis of surface binding where activity is restricted to localized sites (representing immobilized biotin molecules). The analysis leads to an effective catalytic rate coefficient keff characterizing the active surface. Thereafter, solution of the transport problem describing absorption of avidin molecules by the macroscopic sensor surface leads to predictions of the avidin flux, which are found to be in good agreement with the experimental data. The analysis suggests the following conclusions. 1) Translational diffusion limitations are negligible for avidin-biotin binding in solution owing to the small (kinetically limiting) value k = 0.00045 m/s. 2) The sparse distribution of biotin molecules and the presence of a repulsive hydration force produce an effective surface-average catalytic rate coefficient keff of order 10(-7) m/s, much smaller than k. 3) Avidin binding to the fiber optic sensor occurs in an intermediate regime where the rate is influenced by both kinetics and diffusion. Images FIGURE 1 FIGURE 3 PMID:7647232

  13. Using protein binding site prediction to improve protein docking.

    PubMed

    Huang, Bingding; Schroeder, Michael

    2008-10-01

    Predicting protein interaction interfaces and protein complexes are two important related problems. For interface prediction, there are a number of tools, such as PPI-Pred, PPISP, PINUP, Promate, and SPPIDER, which predict enzyme-inhibitor interfaces with success rates of 23% to 55% and other interfaces with 10% to 28% on a benchmark dataset of 62 complexes. Here, we develop, metaPPI, a meta server for interface prediction. It significantly improves prediction success rates to 70% for enzyme-inhibitor and 44% for other interfaces. As shown with Promate, predicted interfaces can be used to improve protein docking. Here, we follow this idea using the meta server instead of individual predictions. We confirm that filtering with predicted interfaces significantly improves candidate generation in rigid-body docking based on shape complementarity. Finally, we show that the initial ranking of candidate solutions in rigid-body docking can be further improved for the class of enzyme-inhibitor complexes by a geometrical scoring which rewards deep pockets. A web server of metaPPI is available at scoppi.tu-dresden.de/metappi. The source code of our docking algorithm BDOCK is also available at www.biotec.tu-dresden.de /approximately bhuang/bdock.

  14. Part I. Cobalt thiolate complexes modeling the active site of cobalt nitrile hydratase. Part II. Formation of inorganic nanoparticles on protein scaffolding in Escherichia coli glutamine synthetase

    NASA Astrophysics Data System (ADS)

    Kung, Irene Yuk Man

    Part I. A series of novel cobalt dithiolate complexes with mixed imine/amine ligand systems is presented here as electronic and structural models for the active site in the bacterial enzyme class, nitrile hydratase (NHase). Pentadentate cobalt(II) complexes with S2N 3 ligand environments are first studied as precursors to the more relevant cobalt(III) complexes. Adjustment of the backbone length by removal of a methylene group increases the reactivity of the system; whereas reduction of the two backbone imine bonds to allow free rotation about those bonds may decrease reactivity. Reactivity change due to the replacement of the backbone amine proton with a more sterically challenging methyl group is not yet clear. Upon oxidation, the monocationic pentadentate cobalt(III) complex, 1b, shows promising reactivity similar to that of NHase. The metal's open coordination site allows reversible binding of the endogenous, monoanionic ligands, N 3- and NCS-. Oxygenation of the thiolate sulfur atoms by exposure to O2 and H2O 2 produces sulfenate and sulfinate ligands in complex 8, which resembles the crystal structure of "deactivated" Fe NHase. However, its lack of reactivity argues against the oxygenated enzyme structure as the active form. Six-coordinate cobalt(III) complexes with S2N4 amine/amine ligand systems are also presented as analogues of previously reported iron(III) compounds, which mimic the spectroscopic properties of Fe NHase. The cobalt complexes do not seem to similarly model Co NHase. However, the S = 0 cobalt(III) center can be spectroscopically silent and difficult to detect, making comparison with synthetic models using common techniques hard. Part II. Dodecameric Escherichia coli glutamine synthetase mutant, E165C, stacks along its six-fold axis to produce tubular nanostructures in the presence of some divalent metal ions, as does the wild type enzyme. The centrally located, engineered Cys-165 residues appear to bind to various species and may serve as

  15. Schistosoma mansoni secretes a chemokine binding protein with antiinflammatory activity.

    PubMed

    Smith, Philip; Fallon, Rosie E; Mangan, Niamh E; Walsh, Caitriona M; Saraiva, Margarida; Sayers, Jon R; McKenzie, Andrew N J; Alcami, Antonio; Fallon, Padraic G

    2005-11-21

    The coevolution of humans and infectious agents has exerted selective pressure on the immune system to control potentially lethal infections. Correspondingly, pathogens have evolved with various strategies to modulate and circumvent the host's innate and adaptive immune response. Schistosoma species are helminth parasites with genes that have been selected to modulate the host to tolerate chronic worm infections, often for decades, without overt morbidity. The modulation of immunity by schistosomes has been shown to prevent a range of immune-mediated diseases, including allergies and autoimmunity. Individual immune-modulating schistosome molecules have, therefore, therapeutic potential as selective manipulators of the immune system to prevent unrelated diseases. Here we show that S. mansoni eggs secrete a protein into host tissues that binds certain chemokines and inhibits their interaction with host chemokine receptors and their biological activity. The purified recombinant S. mansoni chemokine binding protein (smCKBP) suppressed inflammation in several disease models. smCKBP is unrelated to host proteins and is the first described chemokine binding protein encoded by a pathogenic human parasite and may have potential as an antiinflammatory agent.

  16. Why are hyperactive ice-binding-proteins so active?

    NASA Astrophysics Data System (ADS)

    Braslavsky, Ido; Celik, Yeliz; Pertaya, Natalya; Eun Choi, Young; Bar, Maya; Davies, Peter L.

    2008-03-01

    Ice binding proteins (IBPs), also called `antifreeze proteins' or `ice structuring proteins', are a class of proteins that protect organisms from freezing injury. These proteins have many applications in medicine and agriculture, and as a platform for future biotechnology applications. One of the interesting questions in this field focuses on the hyperactivity of some IBPs. Ice binding proteins can be classified in two groups: moderate ones that can depress the freezing point up to ˜1.0 ^oC and hyperactive ones that can depress the freezing point several-fold further even at lower concentrations. It has been suggested that the hyperactivity of IBPs stem from the fact that they block growth out of specific ice surfaces, more specifically the basal planes of ice. Here we show experimental results based on fluorescence microscopy, highlighting the differences between moderate IBPs and hyperactive IBPs. These include direct evidence for basal plane affinity of hyperactive IBPs, the effects of IBPs on growth-melt behavior of ice and the dynamics of their interaction with ice.

  17. Sensitive NMR Approach for Determining the Binding Mode of Tightly Binding Ligand Molecules to Protein Targets.

    PubMed

    Chen, Wan-Na; Nitsche, Christoph; Pilla, Kala Bharath; Graham, Bim; Huber, Thomas; Klein, Christian D; Otting, Gottfried

    2016-04-01

    Structure-guided drug design relies on detailed structural knowledge of protein-ligand complexes, but crystallization of cocomplexes is not always possible. Here we present a sensitive nuclear magnetic resonance (NMR) approach to determine the binding mode of tightly binding lead compounds in complex with difficult target proteins. In contrast to established NMR methods, it does not depend on rapid exchange between bound and free ligand or on stable isotope labeling, relying instead on a tert-butyl group as a chemical label. tert-Butyl groups are found in numerous protein ligands and deliver an exceptionally narrow and tall (1)H NMR signal. We show that a tert-butyl group also produces outstandingly intense intra- and intermolecular NOESY cross-peaks. These enable measurements of pseudocontact shifts generated by lanthanide tags attached to the protein, which in turn allows positioning of the ligand on the protein. Once the ligand has been located, assignments of intermolecular NOEs become possible even without prior resonance assignments of protein side chains. The approach is demonstrated with the dengue virus NS2B-NS3 protease in complex with a high-affinity ligand containing a tert-butyl group. PMID:26974502

  18. Crystal Structure of Human Retinoblastoma Binding Protein 9

    SciTech Connect

    Vorobiev, S.; Su, M; Seetharaman, J; Huang, Y; Chen, C; Maglaqui, M; Janjua, H; Montelione, G; Tong, L; et. al.

    2009-01-01

    As a step towards better integrating protein three-dimensional (3D) structural information in cancer systems biology, the Northeast Structural Genomics Consortium (NESG) (www.nesg.org) has constructed a Human Cancer Pathway Protein Interaction Network (HCPIN) by analysis of several classical cancer-associated signaling pathways and their physical protein-protein interactions. Many well-known cancer-associated proteins play central roles as hubs or bottlenecks in the HCPIN (http://nmr.cabm.rutgers.edu/hcpin). NESG has selected more than 1000 human proteins and protein domains from the HCPIN for sample production and 3D structure determination. The long-range goal of this effort is to provide a comprehensive 3D structure-function database for human cancer-associated proteins and protein complexes, in the context of their interaction networks. Human retinoblastoma binding protein 9 (RBBP9) is one of the HCPIN proteins targeted by NESG. RBBP9 was initially identified as the product of a new gene, Bog (for B5T over-expressed gene), in several transformed rat liver epithelial cell lines resistant to the growth-inhibitory effect of TGF-1 as well as in primary human liver tumors. RBBP9 contains the retinoblastoma (Rb) binding motif LxCxE in its sequence, and was shown to interact with Rb by yeast two-hybrid and coimmunoprecipitation experiments. Mutation of the Leu residue in this motif to Gln blocked the binding to Rb. RBBP9 can displace E2F1 from E2F1-Rb complexes, and over expression of RBBP9 overcomes TGF-1 induced growth arrest and results in transformation of rat liver epithelial cells leading to hepatoblastoma-like tumors in nude mice. RBBP9 may also play a role in cellular responses to chronic low dose radiation. A close homolog of RBBP9, sharing 93% amino acid sequence identity and also known as RBBP10, interacts with a protein with sua5-yciO-yrdC domains.

  19. Purification of penicillin-binding protein 2 of Escherichia coli.

    PubMed Central

    Curtis, S J; Strominger, J L

    1981-01-01

    Penicillin-binding protein 2 (PBP-2) of Escherichia coli K-12 was purified by covalent affinity chromatography using 6-aminopenicillanic acid covalently coupled to carboxymethyl-Sepharose (6-APA-CM-Sepharose). Purification of PBP-2 was accomplished by prebinding the methoxy cephalosporin, cefoxitin, to the Triton X-100-solubilized PBPs of E. coli and then incubating the PBPs with 6-APA-CM-Sepharose. Cefoxitin readily binds to all the E. coli PBPs except PBP-2 and, thus, in the presence of cefoxitin, only PBP-2 could bind to the 6-APA-CM-Sepharose. The purification of a mixture of all of the PBPs of E. coli by affinity chromatography is also described. Images PMID:7007320

  20. Glutamate regulates kainate-binding protein expression in cultured chick Bergmann glia through an activator protein-1 binding site.

    PubMed

    Aguirre, A; López, T; López-Bayghen, E; Ortega, A

    2000-12-15

    The expression of the chick kainate-binding protein, a member of the ionotropic glutamate receptor family, is restricted to the cerebellum, specifically to Bergmann glia. Glutamate induces a membrane to nuclei signaling involved in gene expression regulation. Exposure of cultured chick Bergmann glia cells to glutamate leads to an increase in kainate binding protein and mRNA levels, suggesting a transcriptional level of regulation. The 5' proximal region of the chick kainate binding gene was cloned and transfected 4into Bergmann glia cells. Three main regulatory regions could be defined, a minimal promoter region, a negative regulatory region, and interestingly, a glutamate-responsive element. Deletion of this element abolishes the agonist effect. Moreover, electrophoretic mobility shift assays, cotransfection experiments, and site-directed mutagenesis clearly suggest that the glutamate effect is mediated through an AP-1 site by a Fos/Jun heterodimer. The present results favor the notion of a functional role of kainate-binding protein in glutamatergic cerebellar neurotransmission.

  1. Ice-binding mechanism of winter flounder antifreeze proteins.

    PubMed Central

    Cheng, A; Merz, K M

    1997-01-01

    We have studied the winter flounder antifreeze protein (AFP) and two of its mutants using molecular dynamics simulation techniques. The simulations were performed under four conditions: in the gas phase, solvated by water, adsorbed on the ice (2021) crystal plane in the gas phase and in aqueous solution. This study provided details of the ice-binding pattern of the winter flounder AFP. Simulation results indicated that the Asp, Asn, and Thr residues in the AFP are important in ice binding and that Asn and Thr as a group bind cooperatively to the ice surface. These ice-binding residues can be collected into four distinct ice-binding regions: Asp-1/Thr-2/Asp-5, Thr-13/Asn-16, Thr-24/Asn-27, and Thr-35/Arg-37. These four regions are 11 residues apart and the repeat distance between them matches the ice lattice constant along the (1102) direction. This match is crucial to ensure that all four groups can interact with the ice surface simultaneously, thereby, enhancing ice binding. These Asx (x = p or n)/Thr regions each form 5-6 hydrogen bonds with the ice surface: Asn forms about three hydrogen bonds with ice molecules located in the step region while Thr forms one to two hydrogen bonds with the ice molecules in the ridge of the (2021) crystal plane. Both the distance between Thr and Asn and the ordering of the two residues are crucial for effective ice binding. The proper sequence is necessary to generate a binding surface that is compatible with the ice surface topology, thus providing a perfect "host/guest" interaction that simultaneously satisfies both hydrogen bonding and van der Waals interactions. The results also show the relation among binding energy, the number of hydrogen bonds, and the activity. The activity is correlated to the binding energy, and in the case of the mutants we have studied the number of hydrogen bonds. The greater the number of the hydrogen bonds the greater the antifreeze activity. The roles van der Waals interactions and the hydrophobic

  2. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    NASA Astrophysics Data System (ADS)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  3. Heparin-binding mechanism of the IGF2/IGF-binding protein 2 complex.

    PubMed

    Lund, Jacob; Søndergaard, Mads T; Conover, Cheryl A; Overgaard, Michael T

    2014-06-01

    IGF1 and IGF2 are potent stimulators of diverse cellular activities such as differentiation and mitosis. Six IGF-binding proteins (IGFBP1-IGFBP6) are primary regulators of IGF half-life and receptor availability. Generally, the binding of IGFBPs inhibits IGF receptor activation. However, it has been shown that IGFBP2 in complex with IGF2 (IGF2/IGFBP2) stimulates osteoblast function in vitro and increases skeletal mass in vivo. IGF2 binding to IGFBP2 greatly increases the affinity for 2- or 3-carbon O-sulfated glycosaminoglycans (GAGs), e.g. heparin and heparan sulfate, which is hypothesized to preferentially and specifically target the IGF2/IGFBP2 complex to the bone matrix. In order to obtain a more detailed understanding of the interactions between the IGF2/IGFBP2 complex and GAGs, we investigated heparin-binding properties of IGFBP2 and the IGF2/IGFBP2 complex in a quantitative manner. For this study, we mutated key positively charged residues within the two heparin-binding domains (HBDs) in IGFBP2 and in one potential HBD in IGF2. Using heparin affinity chromatography, we demonstrate that the two IGFBP2 HBDs contribute differentially to GAG binding in free IGFBP2 and the IGF2/IGFBP2 protein complex. Moreover, we identify a significant contribution from the HBD in IGF2 to the increased IGF2/IGFBP2 heparin affinity. Using molecular modeling, we present a novel model for the IGF2/IGFBP2 interaction with heparin where all three proposed HBDs constitute a positively charged and surface-exposed area that would serve to promote the increased heparin affinity of the complex compared with free intact IGFBP2.

  4. Nucleic acid binding affinity of fd gene 5 protein in the cooperative binding mode.

    PubMed

    Bobst, A M; Ireland, J C; Bobst, E V

    1984-02-25

    A sensitive ESR method which allows a direct quantitative determination of nucleic acid binding affinities of proteins under physiologically relevant conditions has been applied to the gene 5 protein of bacteriophage fd. This was achieved with two spin-labeled nucleic acids, (ldT, dT)n and (lA,A)n, which served as macro-molecular spin probes in ESR competition experiments. With the two different macromolecular spin probes, it was possible to determine the relative apparent affinity constants, Kapp, over a large affinity domain. In 20 mM Tris X HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton, and 125 mM NaCl, the following affinity relationship was observed: K(dT)napp = 10(3) KfdDNAapp = 2 X 10(4) K(A)napp = 6.6 X 10(4) KrRNAapp = 1.5 X 10(5) KR17RNAapp. Increasing the [NaCl] from 125 to 200 mM caused considerably less tight binding of gene 5 protein to (lA,A)n, and a typical cooperative binding isotherm was observed, whereas at the lower [NaCl] used for the competition experiments, the binding was essentially stoichiometric. A computer fit of the experimental titration data at 200 mM NaCl gave an intrinsic binding constant, Kint, of 1300 M-1 and a cooperativity factor, omega, of 60 (Kint omega = Kapp) for (lA,A)n.

  5. Role of fatty acid binding protein on hepatic palmitate uptake.

    PubMed

    Burczynski, F J; Zhang, M N; Pavletic, P; Wang, G Q

    1997-12-01

    Expression of hepatic fatty acid binding protein (FABP) mRNA is regulated by growth hormone. In the absence of growth hormone, there is a 60% reduction in FABP mRNA levels (S.A. Berry, J.-B Yoon, U. List, and S. Seelig. J. Am. Coll. Nutr. 12:638-642. 1995). Previous work in our laboratory focused on the role of extracellular binding proteins in the hepatic uptake of long chain fatty acids. In the present study we were interested to determine the role of FABP in the transmembrane flux of long chain fatty acids. Using hepatocyte monolayers from control (n = 9) and hypophysectomized (n = 6) rats, we investigated the uptake of [3H]palmitate in the presence and absence of albumin. In the absence of albumin, total hepatocyte [3H]palmitate clearance rates from control (17.2 +/- 1.5 microL.mg-1 protein.s-1; mean +/- SEM; n = 9) and hypophysectomized (15.5 +/- 2.1 microL.mg-1 protein.s-1; n = 6) animals were similar (p > 0.05). In the presence of 2 microM albumin the total [3H]palmitate clearance rate from control hepatocytes (1.63 +/- 0.11 microL.mg-1 protein.s-1; n = 9) was significantly larger (40%) than from hepatocytes obtained from hypophysectomized (0.97 +/- 0.15 microL.mg-1 protein.s-1; n = 6; p < 0.01) animals. SDS-PAGE electrophoresis revealed that plasma membrane FABP levels from control and hypophysectomized animals were similar. However, there was a 49% decrease in the cytosolic FABP levels of hepatocytes isolated from hypophysectomized as compared with control animals. The decreased cytosolic FABB levels paralleled the decrease in palmitate uptake. We conclude that in the absence of extracellular binding proteins the rate-limiting step in the overall uptake of long chain fatty acids is diffusion to the cell surface. However, in the presence of albumin, the rate of palmitate uptake is determined primarily by cytosolic FABP levels.

  6. Structural and binding studies of SAP-1 protein with heparin.

    PubMed

    Yadav, Vikash K; Mandal, Rahul S; Puniya, Bhanwar L; Kumar, Rahul; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

    2015-03-01

    SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (KD = 158 nm). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity.

  7. Pyruvate kinase M2 is a phosphotyrosine-binding protein

    SciTech Connect

    Christofk, H.R.; Vander Heiden, M.G.; Wu, N.; Asara, J.M.; Cantley, L.C.

    2008-06-03

    Growth factors stimulate cells to take up excess nutrients and to use them for anabolic processes. The biochemical mechanism by which this is accomplished is not fully understood but it is initiated by phosphorylation of signalling proteins on tyrosine residues. Using a novel proteomic screen for phosphotyrosine-binding proteins, we have made the observation that an enzyme involved in glycolysis, the human M2 (fetal) isoform of pyruvate kinase (PKM2), binds directly and selectively to tyrosine-phosphorylated peptides. We show that binding of phosphotyrosine peptides to PKM2 results in release of the allosteric activator fructose-1,6-bisphosphate, leading to inhibition of PKM2 enzymatic activity. We also provide evidence that this regulation of PKM2 by phosphotyrosine signalling diverts glucose metabolites from energy production to anabolic processes when cells are stimulated by certain growth factors. Collectively, our results indicate that expression of this phosphotyrosine-binding form of pyruvate kinase is critical for rapid growth in cancer cells.

  8. Immunochemical similarity of GTP-binding proteins from different systems

    SciTech Connect

    Kalinina, S.N.

    1986-06-20

    It was found that antibodies against the GTP-binding proteins of bovine retinal photoreceptor membranes blocked the inhibitory effect of estradiol on phosphodiesterase from rat and human uterine cytosol and prevented the cumulative effect of catecholamines and guanylyl-5'-imidodiphosphate on rat skeletal muscle adenylate cyclase. It was established by means of double radial immunodiffusion that these antibodies form a precipitating complex with purified bovine brain tubulin as well as with retinal preparations obtained from eyes of the bull, pig, rat, frog, some species of fish, and one reptile species. Bands of precipitation were not observed with these antibodies when retinal preparations from invertebrates (squid and octopus) were used as the antigens. The antibodies obtained interacted with the ..cap alpha..- and ..beta..-subunits of GTP-binding proteins from bovine retinal photoreceptor membranes.

  9. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    PubMed

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B; Patel, Trushar R; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems. PMID:27029048

  10. Human neutrophil calmodulin-binding proteins: identification of the calmodulin-dependent protein phosphatase

    SciTech Connect

    Blackburn, W.D.; Tallant, E.A.; Wallace, R.W.

    1986-05-01

    The molecular events in linking neutrophil activation and ligand binding to specific membrane receptors are mediated in part by an increase in intracellular Ca/sup 2 +/. One mechanism by which Ca/sup 2 +/ may trigger neutrophil activation is through Ca/sup 2 +//calmodulin (CaM)-regulated proteins and enzymes. To determine which Ca/sup 2 +//CaM-regulated enzymes may be present in the neutrophil, they have used Western blotting techniques and /sup 125/I-CaM to identify neutrophil CaM-binding proteins. Eleven proteins with molecular weights ranging from 230K to 13.5K bound /sup 125/I-CaM in a Ca/sup 2 +/-dependent manner. One predominant region of /sup 125/I-Cam binding was to a 59K protein; a protein with an identical mobility was labeled by an antisera against brain CaM-dependent phosphatase. Ca/sup 2 +/-dependent phosphatase activity, which was inhibited by the CaM antagonist trifluoperazine, was detected in a neutrophil extract; a radioimmunoassay for the phosphatase indicated that it was present in the extract at approximately 0.2 ..mu..g/mg protein. Most of the CaM-binding proteins, including the 59K protein, were rapidly degraded upon lysis of the neutrophil. There was a close correlation between the degradation of the 59K protein and the loss of Ca/sup 2 +/-dependent phosphatase activity in the neutrophil extract. Thus, human neutrophils contain numerous CaM-binding proteins which are presumably Ca/sup 2 +//calmodulin-regulated enzymes and proteins; the 59K protein is a CaM-dependent phosphatase.

  11. Mammalian synthetic circuits with RNA binding proteins delivered by RNA

    PubMed Central

    Wroblewska, Liliana; Kitada, Tasuku; Endo, Kei; Siciliano, Velia; Stillo, Breanna; Saito, Hirohide; Weiss, Ron

    2015-01-01

    Synthetic regulatory circuits encoded on RNA rather than DNA could provide a means to control cell behavior while avoiding potentially harmful genomic integration in therapeutic applications. We create post-transcriptional circuits using RNA-binding proteins, which can be wired in a plug-and-play fashion to create networks of higher complexity. We show that the circuits function in mammalian cells when encoded on modified mRNA or self-replicating RNA. PMID:26237515

  12. Liposolubility and protein binding of oxycodone in vitro.

    PubMed

    Pöyhiä, R; Seppälä, T

    1994-01-01

    The liposolubility and protein-binding of oxycodone were studied in vitro and compared with other opioids. Liposolubility was assessed by three different methods: 1) the shake-flask method with n-octanol at pH 4-9, 2) measuring the retention time in reversed-phase high-performance liquid chromatography (RP-HPLC) with a LiChrosorb RP-18 and 3) studying the solubility in human epidural and subcutaneous fat. Human fat was obtained from patients undergoing surgery for herniated intervertebral disc. After incubation, pieces of fatty tissue immersed in a buffer solution containing oxycodone, morphine, pethidine or fentanyl for 10-40 min.; tissue pieces were homogenated, opioids extracted and opioid concentrations measured by gas- and high-performance liquid chromatography. The binding of oxycodone, morphine and fentanyl in plasma proteins was studied by ultrafiltration (Amicon-kit). The mean apparent partition coefficients Papp of oxycodone, morphine, pethidine and fentanyl in n-octanol at pH 7 were 0.7, 0.5, 10.5 and 399, respectively. The retention times in RP-HPLC for oxycodone, morphine, pethidine, fentanyl and buprenorphine were 0.6 min., 0.2 min., 2.4 min., 2.3 min. and 10.5 min., respectively. Only buprenorphine and fentanyl appeared to be highly lipophilic in the human fat tissue experiments; no difference was found between epidural or subcutaneous fat in this respect. The in vitro protein binding of oxycodone was 38%, of morphine 31% and of fentanyl 87% in average. It is concluded that, in terms of physiochemical properties, liposolubility and protein-binding, oxycodone resembles morphine more than it does fentanyl.

  13. Analysis of zinc binding sites in protein crystal structures.

    PubMed Central

    Alberts, I. L.; Nadassy, K.; Wodak, S. J.

    1998-01-01

    The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations. PMID:10082367

  14. Anchored clathrate waters bind antifreeze proteins to ice

    PubMed Central

    Garnham, Christopher P.; Campbell, Robert L.; Davies, Peter L.

    2011-01-01

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca2+-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFP∶ice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the “anchored clathrate” mechanism of AFP action. PMID:21482800

  15. Anchored Clathrate Waters Bind Antifreeze Proteins to Ice

    SciTech Connect

    C Garnham; R Campbell; P Davies

    2011-12-31

    The mechanism by which antifreeze proteins (AFPs) irreversibly bind to ice has not yet been resolved. The ice-binding site of an AFP is relatively hydrophobic, but also contains many potential hydrogen bond donors/acceptors. The extent to which hydrogen bonding and the hydrophobic effect contribute to ice binding has been debated for over 30 years. Here we have elucidated the ice-binding mechanism through solving the first crystal structure of an Antarctic bacterial AFP. This 34-kDa domain, the largest AFP structure determined to date, folds as a Ca{sup 2+}-bound parallel beta-helix with an extensive array of ice-like surface waters that are anchored via hydrogen bonds directly to the polypeptide backbone and adjacent side chains. These bound waters make an excellent three-dimensional match to both the primary prism and basal planes of ice and in effect provide an extensive X-ray crystallographic picture of the AFP{vert_ellipsis}ice interaction. This unobstructed view, free from crystal-packing artefacts, shows the contributions of both the hydrophobic effect and hydrogen bonding during AFP adsorption to ice. We term this mode of binding the 'anchored clathrate' mechanism of AFP action.

  16. Fatty acid binding protein in the intestine of the chicken.

    PubMed

    Katongole, J B; March, B E

    1979-03-01

    The mucosa of the mesenteric intestine of the chicken has been found to contain a fatty acid binding protein (FABP) with a molecular weight of less than 12,400. The protein is present in the newly hatched chick before ingestion of feed and in the adult bird. When a low-fat diet is fed, the concentration of the FABP is highest in the proximal portion of the intestine and decreases posteriorly. When a high-fat diet is fed, an increase occurs in the amount of FABP in the lower section of the intestine.

  17. Fluctuations in Mass-Action Equilibrium of Protein Binding Networks

    NASA Astrophysics Data System (ADS)

    Yan, Koon-Kiu; Walker, Dylan; Maslov, Sergei

    2008-12-01

    We consider two types of fluctuations in the mass-action equilibrium in protein binding networks. The first type is driven by slow changes in total concentrations of interacting proteins. The second type (spontaneous) is caused by quickly decaying thermodynamic deviations away from equilibrium. We investigate the effects of network connectivity on fluctuations by comparing them to scenarios in which the interacting pair is isolated from the network and analytically derives bounds on fluctuations. Collective effects are shown to sometimes lead to large amplification of spontaneous fluctuations. The strength of both types of fluctuations is positively correlated with the complex connectivity and negatively correlated with complex concentration. Our general findings are illustrated using a curated network of protein interactions and multiprotein complexes in baker’s yeast, with empirical protein concentrations.

  18. Characterization of auxin-binding proteins from zucchini plasma membrane

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  19. Mycobacteriophage cell binding proteins for the capture of mycobacteria

    PubMed Central

    Arutyunov, Denis; Singh, Upasana; El-Hawiet, Amr; Seckler, Henrique dos Santos; Nikjah, Sanaz; Joe, Maju; Bai, Yu; Lowary, Todd L; Klassen, John S; Evoy, Stephane; Szymanski, Christine M

    2014-01-01

    Slow growing Mycobacterium avium subsp. paratuberculosis (MAP) causes a deadly condition in cattle known as Johne's disease where asymptomatic carriers are the major source of disease transmission. MAP was also shown to be associated with chronic Crohn's disease in humans. Mycobacterium smegmatis is a model mycobacterium that can cause opportunistic infections in a number of human tissues and, rarely, a respiratory disease. Currently, there are no rapid, culture-independent, reliable and inexpensive tests for the diagnostics of MAP or M. smegmatis infections. Bacteriophages are viruses producing a number of proteins that effectively and specifically recognize the cell envelopes of their bacterial hosts. We demonstrate that the mycobacterial phage L5 minor tail protein Gp6 and lysin Gp10 are useful tools for the rapid capture of mycobacteria. Immobilized Gp10 was able to bind both MAP and M. smegmatis cells whereas Gp6 was M. smegmatis specific. Neither of the 2 proteins was able to capture E. coli, salmonella, campylobacter or Mycobacterium marinum cells. Gp6 was detected previously as a component of the phage particle and shows no homology to proteins with known function. Therefore, electrospray ionization mass spectrometry was used to determine whether recombinant Gp6 could bind to a number of chemically synthesized fragments of mycobacterial surface glycans. These findings demonstrate that mycobacteriophage proteins could be used as a pathogen capturing platform that can potentially improve the effectiveness of existing diagnostic methods. PMID:26713219

  20. Odorant binding characteristics of three recombinant odorant binding proteins in Microplitis mediator (Hymenoptera: Braconidae).

    PubMed

    Li, Keming; Wang, Shanning; Zhang, Kang; Ren, Liyan; Ali, Abid; Zhang, Yongjun; Zhou, Jingjiang; Guo, Yuyuan

    2014-06-01

    Odorant binding proteins (OBPs) are believed to be important for transporting semiochemicals through the aqueous sensillar lymph to the olfactory receptor cells within the insect antennal sensilla. In this study, three new putative OBP genes, MmedOBP8-10, were identified from a Microplitis mediator (Hymenoptera: Braconidae) antennal cDNA library. Quantitative real-time PCR (qRT-PCR) analysis revealed that all three of the OBP genes were expressed mainly in the antennae of adult wasps. The three OBPs were recombinantly expressed in Escherichia coli and purified by Ni ion affinity chromatography. Fluorescence competitive binding assays were performed using N-phenyl-naphthylamine as a fluorescent probe and 45 small organic compounds as competitors. These assays demonstrated that the three M. mediator OBPs can bind a broad range of odorant molecules with different binding affinities. They can bind the following ligands: nonane, farnesol, nerolidol, nonanal, β-ionone, acetic ether, and farnesene. In a Y-tube assay with these ligands as odor stimuli and paraffin oil as a control, all ligands, except nerolidol and acetic ether, were able to elicit behavioral responses in adult M. mediator. The wasps were significantly attracted to β-ionone, nonanal, and farnesene and repelled by nonane and farnesol. The results of this work provide insight into the chemosensory functions of the OBPs in M. mediator.

  1. Determinants of affinity and specificity in RNA-binding proteins.

    PubMed

    Helder, Stephanie; Blythe, Amanda J; Bond, Charles S; Mackay, Joel P

    2016-06-01

    Emerging data suggest that the mechanisms by which RNA-binding proteins (RBPs) interact with RNA and the rules governing specificity might be substantially more complex than those underlying their DNA-binding counterparts. Even our knowledge of what constitutes the RNA-bound proteome is contentious; recent studies suggest that 10-30% of RBPs contain no known RNA-binding domain. Adding to this situation is a growing disconnect between the avalanche of identified interactions between proteins and long noncoding RNAs and the absence of biophysical data on these interactions. RNA-protein interactions are also at the centre of what might emerge as one of the biggest shifts in thinking about cell and molecular biology this century, following from recent reports of ribonucleoprotein complexes that drive reversible membrane-free phase separation events within the cell. Unexpectedly, low-complexity motifs are important in the formation of these structures. Here we briefly survey recent advances in our understanding of the specificity of RBPs. PMID:27315040

  2. Are odorant-binding proteins involved in odorant discrimination?

    PubMed

    Steinbrecht, R A

    1996-12-01

    Pheromone-sensitive sensilla trichodea of nine moth species belonging to six families and three superfamilies of Lepidoptera were immunolabelled with an antiserum against the pheromone-binding protein of Antheraea polyphemus. Strong immunolabelling of the sensillum lymph was observed in all long sensilla trichodea of A. polyphemus, A. pernyi (Saturniidae), Bombyx mori (Bombycidae) and Manduca sexta (Sphingidae). Very weak labelling was found with all sensilla trichodea of Dendrolimus kikuchii (Lasiocampidae) and Lymantria dispar (Lymantriidae). In three noctuid species, some long sensilla trichodea were labelled strongly, some only weakly and some were not labelled at all. The fraction of long sensilla trichodea that were strongly labelled was large in Helicoverpa armigera, but small in Spodoptera littoralis and Autographa gamma. The observed cross-reactivity was not correlated with taxonomic relatedness of the species but rather with chemical relatedness of the pheromones used by these species, as a high labelling density was consistently observed in sensilla tuned to pheromones with an alcyl chain of 16 carbon atoms. The highly divergent specificity of pheromone-receptor cells in Noctuidae appears to be mirrored by a similar diversity of the pheromone-binding proteins in the sensilla trichodea. These data support the notion that pheromone-binding proteins participate in odorant discrimination.

  3. Proteins as binding targets of isothiocyanates in cancer prevention

    PubMed Central

    Mi, Lixin; Di Pasqua, Anthony J.

    2011-01-01

    Isothiocyanates are versatile cancer-preventive compounds. Evidence from animal studies indicates that the anticarcinogenic activities of ITCs involve all the major stages of tumor growth: initiation, promotion and progression. Epidemiological studies have also shown that dietary intake of ITCs is associated with reduced risk of certain human cancers. A number of mechanisms have been proposed for the chemopreventive activities of ITCs. To identify the molecular targets of ITCs is a first step to understand the molecular mechanisms of ITCs. Studies in recent years have shown that the covalent binding to certain protein targets by ITCs seems to play an important role in ITC-induced apoptosis and cell growth inhibition and other cellular effects. The knowledge gained from these studies may be used to guide future design and screen of better and more efficacious compounds. In this review, we intend to cover all potential protein targets of ITCs so far studied and summarize what are known about their binding sites and the potential biological consequences. In the end, we also offer discussions to shed light onto the relationship between protein binding and reactive oxygen species generation by ITCs. PMID:21665889

  4. Are odorant-binding proteins involved in odorant discrimination?

    PubMed

    Steinbrecht, R A

    1996-12-01

    Pheromone-sensitive sensilla trichodea of nine moth species belonging to six families and three superfamilies of Lepidoptera were immunolabelled with an antiserum against the pheromone-binding protein of Antheraea polyphemus. Strong immunolabelling of the sensillum lymph was observed in all long sensilla trichodea of A. polyphemus, A. pernyi (Saturniidae), Bombyx mori (Bombycidae) and Manduca sexta (Sphingidae). Very weak labelling was found with all sensilla trichodea of Dendrolimus kikuchii (Lasiocampidae) and Lymantria dispar (Lymantriidae). In three noctuid species, some long sensilla trichodea were labelled strongly, some only weakly and some were not labelled at all. The fraction of long sensilla trichodea that were strongly labelled was large in Helicoverpa armigera, but small in Spodoptera littoralis and Autographa gamma. The observed cross-reactivity was not correlated with taxonomic relatedness of the species but rather with chemical relatedness of the pheromones used by these species, as a high labelling density was consistently observed in sensilla tuned to pheromones with an alcyl chain of 16 carbon atoms. The highly divergent specificity of pheromone-receptor cells in Noctuidae appears to be mirrored by a similar diversity of the pheromone-binding proteins in the sensilla trichodea. These data support the notion that pheromone-binding proteins participate in odorant discrimination. PMID:8985600

  5. PSCDB: a database for protein structural change upon ligand binding.

    PubMed

    Amemiya, Takayuki; Koike, Ryotaro; Kidera, Akinori; Ota, Motonori

    2012-01-01

    Proteins are flexible molecules that undergo structural changes to function. The Protein Data Bank contains multiple entries for identical proteins determined under different conditions, e.g. with and without a ligand molecule, which provides important information for understanding the structural changes related to protein functions. We gathered 839 protein structural pairs of ligand-free and ligand-bound states from monomeric or homo-dimeric proteins, and constructed the Protein Structural Change DataBase (PSCDB). In the database, we focused on whether the motions were coupled with ligand binding. As a result, the protein structural changes were classified into seven classes, i.e. coupled domain motion (59 structural changes), independent domain motion (70), coupled local motion (125), independent local motion (135), burying ligand motion (104), no significant motion (311) and other type motion (35). PSCDB provides lists of each class. On each entry page, users can view detailed information about the motion, accompanied by a morphing animation of the structural changes. PSCDB is available at http://idp1.force.cs.is.nagoya-u.ac.jp/pscdb/. PMID:22080505

  6. Identification of Immunogenic and Serum Binding Proteins of Staphylococcus epidermidis

    PubMed Central

    Sellman, Bret R.; Howell, Alan P.; Kelly-Boyd, Cari; Baker, Steve M.

    2005-01-01

    Staphylococcus epidermidis is a commensal of human skin and a leading cause of nosocomial bloodstream infections. Limited information is available about S. epidermidis proteins that are expressed upon transition to the bloodstream or those involved in host-pathogen interactions. A cell surface fraction from S. epidermidis 0-47 grown in rabbit serum to mimic environmental signals encountered during a bloodstream infection was separated by two-dimensional (2D) gel electrophoresis. Following 2D separation, the proteins were transferred to nitrocellulose and detected with either pooled sera generated in rabbits immunized with live S. epidermidis 0-47 or with biotin-labeled serum proteins eluted from the surface of bacteria grown in rabbit serum. Twenty-nine immunoreactive or serum binding proteins of S. epidermidis were identified by mass spectrometry. Twenty-seven of the corresponding genes were expressed in Escherichia coli, and the purified recombinant proteins were used to immunize mice. In a preliminary screen, 12 of the 27 recombinant proteins induced a response that reduced the number of bacteria recovered from the spleen or bloodstream of infected mice. In subsequent vaccination studies, 5 of the 12 proteins resulted in a statistically significant reduction in the number of bacteria. The identification of five candidate vaccine antigens from the initial screen of only 29 proteins demonstrates the utility of this approach. PMID:16177335

  7. Spatial analysis and quantification of the thermodynamic driving forces in protein-ligand binding: binding site variability.

    PubMed

    Raman, E Prabhu; MacKerell, Alexander D

    2015-02-25

    The thermodynamic driving forces behind small molecule-protein binding are still not well-understood, including the variability of those forces associated with different types of ligands in different binding pockets. To better understand these phenomena we calculate spatially resolved thermodynamic contributions of the different molecular degrees of freedom for the binding of propane and methanol to multiple pockets on the proteins Factor Xa and p38 MAP kinase. Binding thermodynamics are computed using a statistical thermodynamics based end-point method applied on a canonical ensemble comprising the protein-ligand complexes and the corresponding free states in an explicit solvent environment. Energetic and entropic contributions of water and ligand degrees of freedom computed from the configurational ensemble provide an unprecedented level of detail into the mechanisms of binding. Direct protein-ligand interaction energies play a significant role in both nonpolar and polar binding, which is comparable to water reorganization energy. Loss of interactions with water upon binding strongly compensates these contributions leading to relatively small binding enthalpies. For both solutes, the entropy of water reorganization is found to favor binding in agreement with the classical view of the "hydrophobic effect". Depending on the specifics of the binding pocket, both energy-entropy compensation and reinforcement mechanisms are observed. It is notable to have the ability to visualize the spatial distribution of the thermodynamic contributions to binding at atomic resolution showing significant differences in the thermodynamic contributions of water to the binding of propane versus methanol. PMID:25625202

  8. Hierarchical mechanisms build the DNA-binding specificity of FUSE binding protein.

    PubMed

    Benjamin, Lawrence R; Chung, Hye-Jung; Sanford, Suzanne; Kouzine, Fedor; Liu, Juhong; Levens, David

    2008-11-25

    The far upstream element (FUSE) binding protein (FBP), a single-stranded nucleic acid binding protein, is recruited to the c-myc promoter after melting of FUSE by transcriptionally generated dynamic supercoils. Via interactions with TFIIH and FBP-interacting repressor (FIR), FBP modulates c-myc transcription. Here, we investigate the contributions of FBP's 4 K Homology (KH) domains to sequence selectivity. EMSA and missing contact point analysis revealed that FBP contacts 4 separate patches spanning a large segment of FUSE. A SELEX procedure using paired KH-domains defined the preferred subsequences for each KH domain. Unexpectedly, there was also a strong selection for the noncontacted residues between these subsequences, showing that the contact points must be optimally presented in a backbone that minimizes secondary structure. Strategic mutation of contact points defined in this study disabled FUSE activity in vivo. Because the biological specificity of FBP is tuned at several layers: (i) accessibility of the site; (ii) supercoil-driven melting; (iii) presentation of unhindered bases for recognition; and (iv) modular interaction of KH-domains with cognate bases, the FBP-FIR system and sequence-specific, single-strand DNA binding proteins in general are likely to prove versatile tools for adjusting gene expression.

  9. Immunological characterization of honey proteins and identification of MRJP 1 as an IgE-binding protein.

    PubMed

    Hayashi, Takeshi; Takamatsu, Nobue; Nakashima, Takashi; Arita, Takashi

    2011-01-01

    We encountered a fourth case of honey allergy in Japan. We characterized and identified the IgE-binding proteins in honey using the serum of a honey-allergenic patient. Immunoblot analysis revealed that IgE in the patient serum specifically bound to four proteins in each honey sample. At least three of these IgE-binding proteins were N-linked glycoproteins. To identify the 60-kDa IgE-binding protein in dandelion honey, the N-terminal sequences of the fragmented protein were analyzed, revealing the protein to be major royal jelly protein 1 (MRJP 1). Three IgE-binding proteins removed of N-linked oligosaccharide showed a large reduction in IgE-binding activity as compared with the intact protein. This suggests that the carbohydrates in the IgE-binding proteins are a major epitope for patient IgE.

  10. THE ROLE OF PROTEIN BINDING OF TRIVALENT ARSENICALS IN ARSENIC CARCINOGENESIS AND TOXICITY

    EPA Science Inventory

    Three of the most plausible biological theories of arsenic carcinogenesis are protein binding, oxidative stress and altered DNA methylation. This review presents the role of trivalent arsenicals binding to proteins in arsenic carcinogenesis. Using vacuum filtration based receptor...

  11. Glutamine deprivation initiates reversible assembly of mammalian rods and rings.

    PubMed

    Calise, S John; Carcamo, Wendy C; Krueger, Claire; Yin, Joyce D; Purich, Daniel L; Chan, Edward K L

    2014-08-01

    Rods and rings (RR) are protein assemblies composed of cytidine triphosphate synthetase type 1 (CTPS1) and inosine monophosphate dehydrogenase type 2 (IMPDH2), key enzymes in CTP and GTP biosynthesis. Small-molecule inhibitors of CTPS1 or IMPDH2 induce RR assembly in various cancer cell lines within 15 min to hours. Since glutamine is an essential amide nitrogen donor in these nucleotide biosynthetic pathways, glutamine deprivation was examined to determine whether it leads to RR formation. HeLa cells cultured in normal conditions did not show RR, but after culturing in media lacking glutamine, short rods (<2 μm) assembled after 24 h, and longer rods (>5 μm) formed after 48 h. Upon supplementation with glutamine or guanosine, these RR underwent almost complete disassembly within 15 min. Inhibition of glutamine synthetase with methionine sulfoximine also increased RR assembly in cells deprived of glutamine. Taken together, our data support the hypothesis that CTP/GTP biosynthetic enzymes polymerize to form RR in response to a decreased intracellular level of glutamine. We speculate that rod and ring formation is an adaptive metabolic response linked to disruption of glutamine homeostasis.

  12. Dosing and efficacy of glutamine supplementation in human exercise and sport training.

    PubMed

    Gleeson, Michael

    2008-10-01

    Some athletes can have high intakes of l-glutamine because of their high energy and protein intakes and also because they consume protein supplements, protein hydrolysates, and free amino acids. Prolonged exercise and periods of heavy training are associated with a decrease in the plasma glutamine concentration and this has been suggested to be a potential cause of the exercise-induced immune impairment and increased susceptibility to infection in athletes. However, several recent glutamine feeding intervention studies indicate that although the plasma glutamine concentration can be kept constant during and after prolonged strenuous exercise, the glutamine supplementation does not prevent the postexercise changes in several aspects of immune function. Although glutamine is essential for lymphocyte proliferation, the plasma glutamine concentration does not fall sufficiently low after exercise to compromise the rate of proliferation. Acute intakes of glutamine of approximately 20-30 g seem to be without ill effect in healthy adult humans and no harm was reported in 1 study in which athletes consumed 28 g glutamine every day for 14 d. Doses of up to 0.65 g/kg body mass of glutamine (in solution or as a suspension) have been reported to be tolerated by patients and did not result in abnormal plasma ammonia levels. However, the suggested reasons for taking glutamine supplements (support for immune system, increased glycogen synthesis, anticatabolic effect) have received little support from well-controlled scientific studies in healthy, well-nourished humans.

  13. Dosing and efficacy of glutamine supplementation in human exercise and sport training.

    PubMed

    Gleeson, Michael

    2008-10-01

    Some athletes can have high intakes of l-glutamine because of their high energy and protein intakes and also because they consume protein supplements, protein hydrolysates, and free amino acids. Prolonged exercise and periods of heavy training are associated with a decrease in the plasma glutamine concentration and this has been suggested to be a potential cause of the exercise-induced immune impairment and increased susceptibility to infection in athletes. However, several recent glutamine feeding intervention studies indicate that although the plasma glutamine concentration can be kept constant during and after prolonged strenuous exercise, the glutamine supplementation does not prevent the postexercise changes in several aspects of immune function. Although glutamine is essential for lymphocyte proliferation, the plasma glutamine concentration does not fall sufficiently low after exercise to compromise the rate of proliferation. Acute intakes of glutamine of approximately 20-30 g seem to be without ill effect in healthy adult humans and no harm was reported in 1 study in which athletes consumed 28 g glutamine every day for 14 d. Doses of up to 0.65 g/kg body mass of glutamine (in solution or as a suspension) have been reported to be tolerated by patients and did not result in abnormal plasma ammonia levels. However, the suggested reasons for taking glutamine supplements (support for immune system, increased glycogen synthesis, anticatabolic effect) have received little support from well-controlled scientific studies in healthy, well-nourished humans. PMID:18806122

  14. Top-down HPLC-ESI-MS characterization of rat gliadoralin A, a new member of the family of rat submandibular gland glutamine-rich proteins and potential substrate of transglutaminase.

    PubMed

    Cabras, Tiziana; Iavarone, Federica; Pirolli, Davide; De Rosa, Maria Cristina; Vitali, Alberto; Faa, Gavino; Cordaro, Massimo; Messana, Irene; Ekström, Jörgen; Castagnola, Massimo

    2013-09-01

    During HPLC-ESI-MS/MS analysis of rat submandibular saliva secreted under isoprenaline stimulation, a protein with an experimental [M+H](1+) = 10,544.24 m/z was detected (17.5 ± 0.7 min). The MS/MS fragmentation pattern, manually investigated, allowed establishing an internal sequence in agreement with a DNA-derived sequence of an unknown rat protein coded D3Z9M3 (Swiss-Prot). To match the experimental MS/MS fragmentation pattern and protein mass with theoretical data, the removal from the N terminus of the signal peptide and from the C terminus of three amino acid (a.a.) residues (Arg-Ala-Val) and the cyclization of the N-terminal glutamine in pyroglutamic had to be supposed, resulting in a mature protein of 90 a.a. HPLC-ESI-MS/MS of the trypsin digest ensured 100% sequence coverage. For the high glutamine content (34/90 = 37.8%) we propose to name this protein rat gliadoralin A 1-90. Low amounts of five different isoforms were sporadically detected, which did not significantly change their relative amounts after stimulation. Gliadoralin A is substrate for transglutaminase-2, having Lys 60 and different Gln residues as major determinants for enzyme recognition. In silico investigation of superior structures evidenced that a small part of the protein adopts an α-helical fold, whereas large segments are unfolded, suggesting an unordered conformation.

  15. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

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

    PubMed

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

    2015-04-01

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

  17. Copper–zinc cross-modulation in prion protein binding

    PubMed Central

    Stellato, Francesco; Minicozzi, Velia; Millhauser, Glenn L.; Pascucci, Marco; Proux, Olivier; Rossi, Giancarlo C.; Spevacek, Ann

    2016-01-01

    In this paper we report a systematic XAS study of a set of samples in which Cu(II) was progressively added to complexes in which Zn(II) was bound to the tetra-octarepeat portion of the prion protein. This work extends previous EPR and XAS analysis in which, in contrast, the effect of adding Zn(II) to Cu(II)–tetra-octarepeat complexes was investigated. Detailed structural analysis of the XAS spectra taken at both the Cu and Zn K-edge when the two metals are present at different relative concentrations revealed that Zn(II) and Cu(II) ions compete for binding to the tetra-octarepeat peptide by cross-regulating their relative binding modes. We show that the specific metal–peptide coordination mode depends not only, as expected, on the relative metal concentrations, but also on whether Zn(II) or Cu(II) was first bound to the peptide. In particular, it seems that the Zn(II) binding mode in the absence of Cu(II) is able to promote the formation of small peptide clusters in which triplets of tetra-octarepeats are bridged by pairs of Zn ions. When Cu(II) is added, it starts competing with Zn(II) for binding, disrupting the existing peptide cluster arrangement, despite the fact that Cu(II) is unable to completely displace Zn(II). These results may have a bearing on our understanding of peptide-aggregation processes and, with the delicate cross-regulation balancing we have revealed, seem to suggest the existence of an interesting, finely tuned interplay among metal ions affecting protein binding, capable of providing a mechanism for regulation of metal concentration in cells. PMID:25395329

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

    PubMed

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

    2015-04-01

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

  19. Expression profile and ligand-binding characterization of odorant-binding protein 2 in Batocera horsfieldi (Hope)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Odorant-binding proteins (OBPs) are important components in insect olfactory systems that transport semiochemicals through the aqueous sensillum lymph to surface of olfactory receptor neurons. In this study, we cloned the cDNA of odorant-binding protein 2 (BhorOBP2) in Batocera horsfieldi (Hope) and...

  20. A Venom Gland Extracellular Chitin-Binding-Like Protein from Pupal Endoparasitoid Wasps, Pteromalus Puparum, Selectively Binds Chitin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chitin-binding proteins (CBPs) existed in various species and involved in different biology processes. In the present study, we cloned a full length cDNA of chitin-binding protein-like (PpCBP-like) from Pteromalus puparum, a pupal endoparasitoid of Pieris rapae. PpCBP-like encoded a 96 putative amin...

  1. Immunomodulation. Part IV: Glutamine.

    PubMed

    Bell, Susan Givens

    2006-01-01

    Glutamine, a nonessential amino acid that appears to be conditionally essential during periods of physiologic stress, plays important physiologic roles in the immune system. However, neither enteral nor parenteral glutamine supplementation makes a difference in the rate of systemic infection or of NEC in very low birth weight infants. Thus, the search for agents to enhance the neonate's immune system and to serve as safe and effective adjuvants to antibiotics continues. Part V, the final article in this immunomodulation series, will explore the use of probiotics to support the neonatal immune system.

  2. Differential induction of PPAR-gamma by luminal glutamine and iNOS by luminal arginine in the rodent postischemic small bowel.

    PubMed

    Sato, N; Moore, F A; Kone, B C; Zou, L; Smith, M A; Childs, M A; Moore-Olufemi, S; Schultz, S G; Kozar, R A

    2006-04-01

    Using a rodent model of gut ischemia-reperfusion (I/R), we have previously shown that the induction of inducible nitric oxide synthase (iNOS) is harmful, whereas the induction of heme oxygenase 1 (HO-1) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is protective. In the present study, we hypothesized that the luminal nutrients arginine and glutamine differentially modulate these molecular events in the postischemic gut. Jejunal sacs were created in rats at laparotomy, filled with either 60 mM glutamine, arginine, or magnesium sulfate (osmotic control) followed by 60 min of superior mesenteric artery occlusion and 6 h of reperfusion, and compared with shams. The jejunum was harvested for histology or myeloperoxidase (MPO) activity (inflammation). Heat shock proteins and iNOS were quantitated by Western blot analysis and PPAR-gamma by DNA binding activity. In some experiments, rats were pretreated with the PPAR-gamma inhibitor G9662 or with the iNOS inhibitor N-[3(aminomethyl)benzyl]acetamidine (1400W). iNOS was significantly increased by arginine but not by glutamine following gut I/R and was associated with increased MPO activity and mucosal injury. On the other hand, PPAR-gamma was significantly increased by glutamine but decreased by arginine, whereas heat shock proteins were similarly increased in all experimental groups. The PPAR-gamma inhibitor G9662 abrogated the protective effects of glutamine, whereas the iNOS inhibitor 1400W attenuated the injurious effects of arginine. We concluded that luminal arginine and glutamine differentially modulate the molecular events that regulate injurious I/R-mediated gut inflammation and injury. The induction of PPAR-gamma by luminal glutamine is a novel protective mechanism, whereas luminal arginine appears harmful to the postischemic gut due to enhanced expression of iNOS.

  3. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue.

    PubMed

    Chen, Wentao; Dong, Jiajia; Plate, Lars; Mortenson, David E; Brighty, Gabriel J; Li, Suhua; Liu, Yu; Galmozzi, Andrea; Lee, Peter S; Hulce, Jonathan J; Cravatt, Benjamin F; Saez, Enrique; Powers, Evan T; Wilson, Ian A; Sharpless, K Barry; Kelly, Jeffery W

    2016-06-15

    Arylfluorosulfates have appeared only rarely in the literature and have not been explored as probes for covalent conjugation to proteins, possibly because they were assumed to possess high reactivity, as with other sulfur(VI) halides. However, we find that arylfluorosulfates become reactive only under certain circumstances, e.g., when fluoride displacement by a nucleophile is facilitated. Herein, we explore the reactivity of structurally simple arylfluorosulfates toward the proteome of human cells. We demonstrate that the protein reactivity of arylfluorosulfates is lower than that of the corresponding aryl sulfonyl fluorides, which are better characterized with regard to proteome reactivity. We discovered that simple hydrophobic arylfluorosulfates selectively react with a few members of the intracellular lipid binding protein (iLBP) family. A central function of iLBPs is to deliver small-molecule ligands to nuclear hormone receptors. Arylfluorosulfate probe 1 reacts with a conserved tyrosine residue in the ligand-binding site of a subset of iLBPs. Arylfluorosulfate probes 3 and 4, featuring a biphenyl core, very selectively and efficiently modify cellular retinoic acid binding protein 2 (CRABP2), both in vitro and in living cells. The X-ray crystal structure of the CRABP2-4 conjugate, when considered together with binding site mutagenesis experiments, provides insight into how CRABP2 might activate arylfluorosulfates toward site-specific reaction. Treatment of breast cancer cells with probe 4 attenuates nuclear hormone receptor activity mediated by retinoic acid, an endogenous client lipid of CRABP2. Our findings demonstrate that arylfluorosulfates can selectively target single iLBPs, making them useful for understanding iLBP function. PMID:27191344

  4. Efficient determination of protein-protein standard binding free energies from first principles.

    PubMed

    Gumbart, James C; Roux, Benoît; Chipot, Christophe

    2013-08-13

    Characterizing protein-protein association quantitatively has been a longstanding challenge for computer simulations. Here, a theoretical framework is put forth that addresses this challenge on the basis of detailed all-atom molecular dynamics simulations with explicit solvent. The proposed methodology relies upon independent potential of mean force (PMF) free-energy calculations carried out sequentially, wherein the biological objects are restrained in the conformation, position and orientation of the bound state, using adequately chosen biasing potentials. These restraints systematically narrow down the configurational entropy available to the system and effectively guarantee that the relevant network of interactions is properly sampled as the two proteins reversibly associate. Decomposition of the binding process into consecutive, well-delineated stages, for both the protein complex and the individual, unbound partners, offers a rigorous definition of the standard state, from which the absolute binding free energy can be determined. The method is applied to the difficult case of the extracellular ribonuclease barnase binding to its intracellular inhibitor barstar. The calculated binding free energy is -21.0 ± 1.4 kcal/mol, which compares well with the experimental value of -19.0 ± 0.2 kcal/mol. The relatively small statistical error reflects the precision and convergence afforded by the PMF-based simulation methodology. In addition to providing an accurate reproduction of the standard binding free energy, the proposed strategy offers a detailed picture of the protein-protein interface, illuminating the thermodynamic forces that underlie reversible association. The application of the present formal framework to barnase:barstar binding provides a foundation for tackling nearly any protein-protein complex.

  5. Preferential binding of an unfolded protein to DsbA.

    PubMed Central

    Frech, C; Wunderlich, M; Glockshuber, R; Schmid, F X

    1996-01-01

    The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate. During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA. To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1. We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol. This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions. The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds. Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold. PMID:8617214

  6. Identification of prolactin and growth hormone binding proteins in rabbit milk.

    PubMed Central

    Postel-Vinay, M C; Belair, L; Kayser, C; Kelly, P A; Djiane, J

    1991-01-01

    Two distinct soluble proteins that specifically bind 125I-labeled human growth hormone (GH) are identified in the supernatant of ultracentrifuged rabbit milk, using HPLC gel filtration. The higher molecular weight proteins is GH specific, whereas the other one is specific for prolactin (PRL). The PRL-binding protein has a very high affinity for the hormone, almost 10 times higher than the affinity of the mammary gland membrane receptor. The PRL-binding protein is immunoprecipitated by a monoclonal antibody against the PRL receptor; another monoclonal antibody, which inhibits the PRL binding to mammary gland membranes, is a poor competitor for the PRL binding to the milk protein. These findings suggest that the milk PRL-binding protein corresponds to the binding domain of the receptor, but also that the conformation of the receptor and of the binding protein might differ. The milk and the plasma GH-binding proteins have a similar binding affinity. In cross-linking experiments using 125I-labeled human GH, the Mr of the GH-binding protein and of the PRL-binding protein were estimated to be 51,000 and 33,000, respectively. The binding proteins identified in the present work are probably responsible for the transport of their specific ligands in the milk. It is also conceivable that they have a role in the effects of GH and PRL in the mammary gland and/or the intestine of the young. Images PMID:1862093

  7. Prediction of Protein-DNA binding by Monte Carlo method

    NASA Astrophysics Data System (ADS)

    Deng, Yuefan; Eisenberg, Moises; Korobka, Alex

    1997-08-01

    We present an analysis and prediction of protein-DNA binding specificity based on the hydrogen bonding between DNA, protein, and auxillary clusters of water molecules. Zif268, glucocorticoid receptor, λ-repressor mutant, HIN-recombinase, and tramtrack protein-DNA complexes are studied. Hydrogen bonds are approximated by the Lennard-Jones potential with a cutoff distance between the hydrogen and the acceptor atoms set to 3.2 Åand an angular component based on a dipole-dipole interaction. We use a three-stage docking algorithm: geometric hashing that matches pairs of hydrogen bonding sites; (2) least-squares minimization of pairwise distances to filter out insignificant matches; and (3) Monte Carlo stochastic search to minimize the energy of the system. More information can be obtained from our first paper on this subject [Y.Deng et all, J.Computational Chemistry (1995)]. Results show that the biologically correct base pair is selected preferentially when there are two or more strong hydrogen bonds (with LJ potential lower than -0.20) that bind it to the protein. Predicted sequences are less stable in the case of weaker bonding sites. In general the inclusion of water bridges does increase the number of base pairs for which correct specificity is predicted.

  8. Identification of lectin binding proteins in human tears.

    PubMed

    Kuizenga, A; van Haeringen, N J; Kijlstra, A

    1991-12-01

    The identity of glycoproteins in stimulated normal human tears was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of tears onto minigels, blotting, and subsequent incubation with different biotinylated lectins (concanavalin A [Con A], peanut agglutinin [PNA], glycine max agglutinin [SBA], Phaseolus vulgaris agglutinin, wheat germ agglutinin [WGA, native form], Artocarpus integrifolia agglutinin [Jacalin], and Pisum sativum agglutinin). Control proteins included purified secretory immunoglobulin A (sIgA) from human colostrum, human milk lactoferrin, and chicken-egg lysozyme. All samples were prepared in a denaturing (SDS) buffer under nonreducing and reducing conditions. The sIgA in tears and IgA (alpha) heavy chain fragments (reduced sample) were identified with most of the lectins tested. A particular high molecular weight (greater than 200 kD) protein fraction in tears that just entered the separation gel on SDS-PAGE was detected with WGA and Jacalin. This fraction stain poorly with silver. Tear lactoferrin was identified with all lectins used, although binding was low with SBA. Purified milk lactoferrin showed a poor reaction with Jacalin, but a protein in tears of similar mobility bound this lectin (nonreduced samples). Under both nonreducing and reducing conditions, tear-specific prealbumin in tears did not bind any of the lectins tested. Tear lysozyme only reacted with lectin after reduction. The techniques described may provide additional valuable information in addition to commonly used methods for tear protein analysis and further knowledge concerning the role of glycoproteins on the ocular surface.

  9. Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

    PubMed Central

    Barros, Marilia; Nanda, Hirsh

    2016-01-01

    ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

  10. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    NASA Astrophysics Data System (ADS)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-11-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp ( Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  11. Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

    NASA Astrophysics Data System (ADS)

    Ma, Cuiyan; Gao, Qiang; Liang, Yan; Li, Chen; Liu, Chao; Huang, Jie

    2016-03-01

    Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp (Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

  12. Interplay between binding affinity and kinetics in protein-protein interactions.

    PubMed

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. PMID:27018856

  13. Interplay between binding affinity and kinetics in protein-protein interactions.

    PubMed

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc.

  14. Protein Binding in Patients With Late-Life Depression

    PubMed Central

    Kumar, Anand; Kepe, Vladimir; Barrio, Jorge R.; Siddarth, Prabha; Manoukian, Vicki; Elderkin-Thompson, Virginia; Small, Gary W.

    2013-01-01

    Context Depression has been identified as a risk factor and a prodrome of dementia. Common neurobiological mechanisms may underlie this clinical and phenomenologic overlap. Objective To examine and compare protein (amyloid and tau) binding in critical brain regions in patients diagnosed as having late-life major depressive disorder (MDD) and healthy control individuals using 2-(1-{6-[(2-[18F]fluoroethyl) (methyl)-amino]-2-naphthyl}ethylidene) malononitrile ([18F]FDDNP) positron emission tomography. Design A cross-section neuroimaging study using positron emission tomography. Setting University of California, Los Angeles. Patients Our samples comprised 20 patients diagnosed as having MDD and 19 healthy control individuals of comparable age, sex, and educational level. Main Outcome Measure Relative distribution volume in regions of interest was used as the measure of [18F]FDDNP binding in all study participants. Results When compared with controls, [18F]FDDNP binding was significantly higher overall and in the posterior cingulate and lateral temporal regions in the MDD group. Conclusions These findings suggest that neuronal injury associated with higher protein load in critical brain regions might provide a mechanism in the pathophysiologic manifestation of MDD in late life and have implications for the therapeutics of depression in elderly individuals. PMID:22065530

  15. Streptomyces coelicolor SCO4226 is a nickel binding protein.

    PubMed

    Lu, Mo; Jiang, Yong-Liang; Wang, Shu; Jin, Hua; Zhang, Rong-Guang; Virolle, Marie-Joelle; Chen, Yuxing; Zhou, Cong-Zhao

    2014-01-01

    The open reading frame SCO4226 of Streptomyces coelicolor A3(2) encodes an 82-residue hypothetical protein. Biochemical assays revealed that each SCO4226 dimer binds four nickel ions. To decipher the molecular function, we solved the crystal structures of SCO4226 in both apo- and nickel-bound (Ni-SCO4226) forms at 1.30 and 2.04 Å resolution, respectively. Each subunit of SCO4226 dimer adopts a canonical ferredoxin-like fold with five β-strands flanked by two α-helices. In the structure of Ni-SCO4226, four nickel ions are coordinated at the surface of the dimer. Further biochemical assays suggested that the binding of Ni2+ triggers the self-aggregation of SCO4226 in vitro. In addition, RT-qPCR assays demonstrated that the expression of SCO4226 gene in S. coelicolor is specifically up-regulated by the addition of Ni2+, but not other divalent ions such as Cu2+, Mn2+ or Co2+. All these results suggested that SCO4226 acts as a nickel binding protein, probably required for nickel sequestration and/or detoxification.

  16. Cloud computing for protein-ligand binding site comparison.

    PubMed

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

  17. Flies expand the repertoire of protein structures that bind ice.

    PubMed

    Basu, Koli; Graham, Laurie A; Campbell, Robert L; Davies, Peter L

    2015-01-20

    An antifreeze protein (AFP) with no known homologs has been identified in Lake Ontario midges (Chironomidae). The midge AFP is expressed as a family of isoforms at low levels in adults, which emerge from fresh water in spring before the threat of freezing temperatures has passed. The 9.1-kDa major isoform derived from a preproprotein precursor is glycosylated and has a 10-residue tandem repeating sequence xxCxGxYCxG, with regularly spaced cysteines, glycines, and tyrosines comprising one-half its 79 residues. Modeling and molecular dynamics predict a tightly wound left-handed solenoid fold in which the cysteines form a disulfide core to brace each of the eight 10-residue coils. The solenoid is reinforced by intrachain hydrogen bonds, side-chain salt bridges, and a row of seven stacked tyrosines on the hydrophobic side that forms the putative ice-binding site. A disulfide core is also a feature of the similar-sized beetle AFP that is a β-helix with seven 12-residue coils and a comparable circular dichroism spectrum. The midge and beetle AFPs are not homologous and their ice-binding sites are radically different, with the latter comprising two parallel arrays of outward-pointing threonines. However, their structural similarities is an amazing example of convergent evolution in different orders of insects to cope with change to a colder climate and provide confirmation about the physical features needed for a protein to bind ice.

  18. Cloud computing for protein-ligand binding site comparison.

    PubMed

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  19. Flies expand the repertoire of protein structures that bind ice

    PubMed Central

    Basu, Koli; Graham, Laurie A.; Campbell, Robert L.; Davies, Peter L.

    2015-01-01

    An antifreeze protein (AFP) with no known homologs has been identified in Lake Ontario midges (Chironomidae). The midge AFP is expressed as a family of isoforms at low levels in adults, which emerge from fresh water in spring before the threat of freezing temperatures has passed. The 9.1-kDa major isoform derived from a preproprotein precursor is glycosylated and has a 10-residue tandem repeating sequence xxCxGxYCxG, with regularly spaced cysteines, glycines, and tyrosines comprising one-half its 79 residues. Modeling and molecular dynamics predict a tightly wound left-handed solenoid fold in which the cysteines form a disulfide core to brace each of the eight 10-residue coils. The solenoid is reinforced by intrachain hydrogen bonds, side-chain salt bridges, and a row of seven stacked tyrosines on the hydrophobic side that forms the putative ice-binding site. A disulfide core is also a feature of the similar-sized beetle AFP that is a β-helix with seven 12-residue coils and a comparable circular dichroism spectrum. The midge and beetle AFPs are not homologous and their ice-binding sites are radically different, with the latter comprising two parallel arrays of outward-pointing threonines. However, their structural similarities is an amazing example of convergent evolution in different orders of insects to cope with change to a colder climate and provide confirmation about the physical features needed for a protein to bind ice. PMID:25561557

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

    PubMed

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

    1982-01-01

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

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

    PubMed

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

    1982-01-01

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

  2. Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein

    NASA Technical Reports Server (NTRS)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis. Copyright 2000 Academic Press.

  3. Genes encoding calmodulin-binding proteins in the Arabidopsis genome

    NASA Technical Reports Server (NTRS)

    Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

    2002-01-01

    Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

  4. Identification of zinc-binding sites of proteins: zinc binds to the amino-terminal region of tubulin

    SciTech Connect

    Serrano, L.; Dominguez, J.E.; Avila, J.

    1988-07-01

    The discovery that certain proteins may require zinc for their activity, and the fact that several of them cannot be purified in large amounts, has led us to develop a rapid, sensitive method to detect these proteins in samples. This method is based on the fractionation of the proteins by gel electrophoresis, blotting onto nitrocellulose paper, and overlaying with /sup 65/Zn. We have tested the procedure with well-characterized zinc-binding proteins. In the case of tubulin, we have used this method to localize its zinc-binding site. It was found that zinc binds to the first 150 amino acids of both alpha- and beta-tubulin subunits.

  5. Multiple Binding Poses in the Hydrophobic Cavity of Bee Odorant Binding Protein AmelOBP14

    PubMed Central

    2015-01-01

    In the first step of olfaction, odorants are bound and solubilized by small globular odorant binding proteins (OBPs) which shuttle them to the membrane of a sensory neuron. Low ligand affinity and selectivity at this step enable the recognition of a wide range of chemicals. Honey bee Apis mellifera’s OBP14 (AmelOBP14) binds different plant odorants in a largely hydrophobic cavity. In long molecular dynamics simulations in the presence and absence of ligand eugenol, we observe a highly dynamic C-terminal region which forms one side of the ligand-binding cavity, and the ligand drifts away from its crystallized orientation. Hamiltonian replica exchange simulations, allowing exchanges of conformations sampled by the real ligand with those sampled by a noninteracting dummy molecule and several intermediates, suggest an alternative, quite different ligand pose which is adopted immediately and which is stable in long simulations. Thermodynamic integration yields binding free energies which are in reasonable agreement with experimental data. PMID:26633245

  6. Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP-binding Protein Superfamily†,‡

    PubMed Central

    Morar, Mariya; Anand, Ruchi; Hoskins, Aaron A.; Stubbe, JoAnne; Ealick, Steven E.

    2008-01-01

    Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two types of PurL have been detected. The first type, found in eukaryotes and Gram-negative bacteria, consists of a single 140 kDa polypeptide chain and is designated large PurL (lgPurL). The second type, small PurL (smPurL), is found in archaea and Gram-positive bacteria and consists of an 80 kDa polypeptide chain. Small PurL requires two additional gene products, PurQ and PurS, for activity. PurL is a member of a protein superfamily that contains a novel ATP-binding domain. Structures of several members of this superfamily are available in the apo form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analog, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. Availability of a ternary complex enabled mapping of the active site thus identifying potential residues involved in catalysis. The complexes show a conformational change in the active site compared to the unliganded structure. A surprising discovery, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculations about the regulatory role of the auxiliary site in PurLSQ complex formation as well as the evolutionary relationship of PurL's from different organisms. PMID:17154526

  7. Time-resolved fluorescence of bacteriophage Pf1 DNA-binding protein. Determination of oligonucleotide and polynucleotide binding parameters.

    PubMed

    Kneale, G G; Wijnaendts van Resandt, R W

    1985-05-15

    The binding of oligonucleotides and polynucleotides to the Pf1 DNA-binding protein was followed by fluorescence spectral shift and lifetime measurements, which gave an anomalous value for the stoichiometry of binding. The anomaly was investigated in detail using fluorescence depolarisation to measure the aggregation during the titration and showed that all the fluorescence parameters are related to the specific aggregation of dimers on ligand binding. At saturation, complexes of the protein with the octanucleotide d(GCGTTGCG) and the hexadecanucleotide (dT)16 have rotational correlation times, phi, of 50 ns and 85 ns, corresponding to protein tetramers and octamers, respectively. In the presence of the tetranucleotide d(CGCA) the protein remains as the native dimer (phi = 19 ns). The titration curves could be analysed in terms of two non-equivalent binding sites, with binding constants K1 and K2. Comparison of K1 values for oligonucleotide binding leads to an estimated (single-site) intrinsic binding constant Kint approximately equal to 3 X 10(4) M-1 and a cooperativity parameter omega approximately equal to 100, in agreement with the apparent binding constant Kapp approximately equal to 3 X 10(6) M-1 for polynucleotides. Binding to the second site on the protein dimer is greatly reduced and cannot be determined accurately. The results suggest that the protein dimers bind cooperatively by lateral association along the DNA and that occupation of only one of the two DNA-binding sites of the protein dimers is sufficient to stabilize the nucleoprotein complexes.

  8. Functional analysis of glucan binding protein B from Streptococcus mutans.

    PubMed

    Mattos-Graner, Renata O; Porter, Kristen A; Smith, Daniel J; Hosogi, Yumiko; Duncan, Margaret J

    2006-06-01

    Mutans streptococci are major etiological agents of dental caries, and several of their secreted products contribute to bacterial accumulation on teeth. Of these, Streptococcus mutans glucan binding protein B (GbpB) is a novel, immunologically dominant protein. Its biological function is unclear, although GbpB shares homology with a putative peptidoglycan hydrolase from S. agalactiae and S. pneumoniae, indicative of a role in murein biosynthesis. To determine the cellular function of GbpB, we used several approaches to inactivate the gene, analyze its expression, and identify interacting proteins. None of the transformants analyzed were true gbpB mutants, since they all contained both disrupted and wild-type gene copies, and expression of functional GbpB was always conserved. Thus, the inability to obtain viable gbpB null mutants supports the notion that gbpB is an essential gene. Northern blot and real-time PCR analyses suggested that induction of gbpB expression in response to stress was a strain-dependent phenomenon. Proteins that interacted with GbpB were identified in pull-down and coimmunoprecipitation assays, and these data suggest that GbpB interacts with ribosomal protein L7/L12, possibly as part of a protein complex involved in peptidoglycan synthesis and cell division. PMID:16707674

  9. Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe. Binding properties of the protein and nucleotide sequence of the gene.

    PubMed

    Peeters, B P; Konings, R N; Schoenmakers, J G

    1983-09-01

    A DNA binding protein encoded by the filamentous single-stranded DNA phage IKe has been isolated from IKe-infected Escherichia coli cells. Fluorescence and in vitro binding studies have shown that the protein binds co-operatively and with a high specificity to single-stranded but not to double-stranded DNA. From titration of the protein to poly(dA) it has been calculated that approximately four bases of the DNA are covered by one monomer of protein. These binding characteristics closely resemble those of gene V protein encoded by the F-specific filamentous phages M13 and fd. The nucleotide sequence of the gene specifying the IKe DNA binding protein has been established. When compared to the nucleotide sequence of gene V of phage M13 it shows an homology of 58%, indicating that these two phages are evolutionarily related. The IKe DNA binding protein is 88 amino acids long which is one amino acid residue larger than the gene V protein sequence. When the IKe DNA binding protein sequence is compared with that of gene V protein it was found that 39 amino acid residues have identical positions in both proteins. The positions of all five tyrosine residues, a number of which are known to be involved in DNA binding, are conserved. Secondary structure predictions indicate that the two proteins contain similar structural domains. It is proposed that the tyrosine residues which are involved in DNA binding are the ones in or next to a beta-turn, at positions 26, 41 and 56 in gene V protein and at positions 27, 42 and 57 in the IKe DNA binding protein.

  10. The oxysterol-binding protein superfamily: new concepts and old proteins

    PubMed Central

    Villasmil, Michelle L.; Bankaitis, Vytas A.; Mousley, Carl J.

    2013-01-01

    The Kes1 OSBP (oxysterol-binding protein) is a key regulator of membrane trafficking through the TGN (trans-Golgi network) and endosomal membranes. We demonstrated recently that Kes1 acts as a sterol-regulated rheostat for TGN/endosomal phosphatidylinositol 4-phosphate signalling. Kes1 utilizes its dual lipid-binding activities to integrate endosomal lipid metabolism with TORC1 (target of rapamycin complex 1)-dependent proliferative pathways and transcriptional control of nutrient signalling. PMID:22435832

  11. TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein.

    PubMed Central

    Hateboer, G; Timmers, H T; Rustgi, A K; Billaud, M; van 't Veer, L J; Bernards, R

    1993-01-01

    Using a protein binding assay, we show that the amino-terminal 204 amino acids of the c-Myc protein interact directly with a key component of the basal transcription factor TFIID, the TATA box-binding protein (TBP). Essentially the same region of the c-Myc protein also binds the product of the retinoblastoma gene, the RB protein. c-Myc protein coimmunoprecipitates with TBP in lysates of mammalian cells, demonstrating that the proteins are also complexed in vivo. A short peptide that spans the RB binding site of the E7 protein of human papilloma virus type 16 interferes with the binding of c-Myc to TBP. The same peptide also blocks binding of adenovirus E1A protein to TBP, suggesting that c-Myc and E1A bind to RB and TBP through overlapping epitopes. Furthermore, we show that binding of RB to E1A prevents association of E1A with TBP. Our data suggest that one of the functions of RB and RB-like proteins is to prevent interaction of viral and cellular oncoproteins, such as c-Myc and E1A, with TBP. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7690963

  12. Factor H-binding protein, a unique meningococcal vaccine antigen.

    PubMed

    Pizza, Mariagrazia; Donnelly, John; Rappuoli, Rino

    2008-12-30

    GNA1870, also named factor H-binding protein (fHbp) or rLP-2086, is a genome-derived antigen and one of the components of a rationally designed vaccine against Neisseria meningitidis serogroup B, which has entered phase III clinical trials. It has been classified into three main non-cross-protective variant groups. GNA1870 has also been termed fHbp because of its ability to bind factor H, a key regulatory component of the alternative complement pathway. fHbp is important for survival in human blood, human sera, and in presence of antimicrobial peptides, independently of its expression level. All these properties make fHbp a unique vaccine antigen.

  13. Specific mutagenesis of a chlorophyll-binding protein. Progress report.

    SciTech Connect

    Eaton-Rye, Dr., Julian; Shen, Gaozhong

    1990-01-01

    During the first phase of the project regarding specific mutagenesis of the chlorophyll-binding protein CP47 in photosystem II (PS II) most of the time has been devoted to (1) establishment of an optimal procedure for the reintroduction of psbB (the gene encoding CP47) carrying a site-directed mutation into the experimental organism, the cyanobacterium Synechocystis sp. PCC 6803, (2) preparations for site-directed mutagenesis, and (3) creation and analysis of chimaeric spinach/cyanobacterial CP47 mutants of Synechocystis. In the coming year, psbB constructs with site-directed mutations in potential chlorophyll-binding regions of CP47 will be introduced into the Synechocystis genome, and site-directed mutants will be characterized according to procedures described in the original project description. In addition, analysis of chimaeric CP47 mutants will be continued.

  14. Identification of FUSE-binding proteins as interacting partners of TIA proteins

    SciTech Connect

    Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique . E-mail: vkruys@ulb.ac.be

    2006-04-28

    TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm.

  15. Glutamine alimentation in catabolic state.

    PubMed

    Boelens, P G; Nijveldt, R J; Houdijk, A P; Meijer, S; van Leeuwen, P A

    2001-09-01

    Glutamine should be reclassified as a conditionally essential amino acid in the catabolic state because the body's glutamine expenditures exceed synthesis and low glutamine levels in plasma are associated with poor clinical outcome. After severe stress, several amino acids are mobilized from muscle tissue to supply energy and substrate to the host. Glutamine is one of the most important amino acids that provide this function. Glutamine acts as the preferred respiratory fuel for lymphocytes, hepatocytes and intestinal mucosal cells and is metabolized in the gut to citrulline, ammonium and other amino acids. Low concentrations of glutamine in plasma reflect reduced stores in muscle and this reduced availability of glutamine in the catabolic state seems to correlate with increased morbidity and mortality. Adding glutamine to the nutrition of clinical patients, enterally or parenterally, may reduce morbidity. Several excellent clinical trials have been performed to prove efficacy and feasibility of the use of glutamine supplementation in parenteral and enteral nutrition. The increased intake of glutamine has resulted in lower septic morbidity in certain critically ill patient populations. This review will focus on the efficacy and the importance of glutamine supplementation in diverse catabolic states.

  16. Biologically active protein fragments containing specific binding regions of serum albumin or related proteins

    NASA Technical Reports Server (NTRS)

    Carter, Daniel C. (Inventor)

    1998-01-01

    In accordance with the present invention, biologically active protein fragments can be constructed which contain only those specific portions of the serum albumin family of proteins such as regions known as subdomains IIA and IIIA which are primarily responsible for the binding properties of the serum albumins. The artificial serums that can be prepared from these biologically active protein fragments are advantageous in that they can be produced much more easily than serums containing the whole albumin, yet still retain all or most of the original binding potential of the full albumin proteins. In addition, since the protein fragment serums of the present invention can be made from non-natural sources using conventional recombinant DNA techniques, they are far safer than serums containing natural albumin because they do not carry the potentially harmful viruses and other contaminants that will be found in the natural substances.

  17. Eukaryotic damaged DNA-binding proteins: DNA repair proteins or transcription factors?

    SciTech Connect

    Protic, M.

    1994-12-31

    Recognition and removal of structural defects in the genome, caused by diverse physical and chemical agents, are among the most important cell functions. Proteins that recognize and bind to modified DNA, and thereby initiate damage-induced recovery processes, have been identified in prokaryotic and eukaryotic cells. Damaged DNA-binding (DDB) proteins from prokaryotes are either DNA repair enzymes or noncatalytic subunits of larger DNA repair complexes that participate in excision repair, or in recombinational repair and SOS-mutagenesis. Although the methods employed may not have allowed detection of all eukaryotic DDB proteins and identification of their functions, it appears that during evolution cells have developed a wide array of DDB proteins that can discriminate among the diversity of DNA conformations found in the eukaryotic nucleus, as well as a gene-sharing feature found in DDB proteins that also act as transcription factors.

  18. DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication.

    PubMed

    Salas, Margarita; Holguera, Isabel; Redrejo-Rodríguez, Modesto; de Vega, Miguel

    2016-01-01

    Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5' ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3'-5' exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the

  19. DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

    PubMed Central

    Salas, Margarita; Holguera, Isabel; Redrejo-Rodríguez, Modesto; de Vega, Miguel

    2016-01-01

    Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5′ ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3′–5′ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and

  20. DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication.

    PubMed

    Salas, Margarita; Holguera, Isabel; Redrejo-Rodríguez, Modesto; de Vega, Miguel

    2016-01-01

    Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5' ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3'-5' exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the

  1. Proteins and Their Interacting Partners: An Introduction to Protein-Ligand Binding Site Prediction Methods.

    PubMed

    Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

    2015-12-15

    Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein-ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein-ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein-ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.

  2. Localization of StarD5 cholesterol binding protein.

    PubMed

    Rodriguez-Agudo, Daniel; Ren, Shunlin; Hylemon, Phillip B; Montañez, Raul; Redford, Kaye; Natarajan, Ramesh; Medina, Miguel Angel; Gil, Gregorio; Pandak, William M

    2006-06-01

    Human StarD5 belongs to the StarD4 subfamily of START (for steroidogenic acute regulatory lipid transfer) domain proteins. We previously reported that StarD5 is located in the cytosolic fraction of human liver and binds cholesterol and 25-hydroxycholesterol. After overexpression of the gene encoding StarD5 in primary rat hepatocytes, free cholesterol accumulated in intracellular membranes. These findings suggested StarD5 to be a directional cytosolic sterol transporter. The objective of this study was to determine the localization of StarD5 in human liver. Western blot analysis confirmed StarD5's presence in the liver but not in human hepatocytes. Immunohistochemistry studies showed StarD5 localized within sinusoidal lining cells in the human liver and colocalized with CD68, a marker for Kupffer cells. Western blot analyses identified the presence of StarD5 in monocytes and macrophages as well as mast cells, basophils, and promyelocytic cells, but not in human hepatocytes, endothelial cells, fibroblasts, osteocytes, astrocytes, or brain tissue. Cell fractionation and immunocytochemistry studies on THP-1 macrophages localized StarD5 to the cytosol and supported an association with the Golgi. The presence of this cholesterol/25-hydroxycholesterol-binding protein in cells related to inflammatory processes provides new clues to the role of this protein in free sterol transport in the cells and in lipid-mediated atherogenesis.

  3. Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

    PubMed

    Tahiri, Abdelghani; Delporte, Fabienne; Muhovski, Yordan; Ongena, Marc; Thonart, Philippe; Druart, Philippe

    2016-01-01

    Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization.

  4. Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

    PubMed

    Tahiri, Abdelghani; Delporte, Fabienne; Muhovski, Yordan; Ongena, Marc; Thonart, Philippe; Druart, Philippe

    2016-01-01

    Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization. PMID:26595095

  5. Identification of novel PTEN-binding partners: PTEN interaction with fatty acid binding protein FABP4.

    PubMed

    Gorbenko, O; Panayotou, G; Zhyvoloup, A; Volkova, D; Gout, I; Filonenko, V

    2010-04-01

    PTEN is a tumor suppressor with dual protein and lipid-phosphatase activity, which is frequently deleted or mutated in many human advanced cancers. Recent studies have also demonstrated that PTEN is a promising target in type II diabetes and obesity treatment. Using C-terminal PTEN sequence in pEG202-NLS as bait, yeast two-hybrid screening on Mouse Embryo, Colon Cancer, and HeLa cDNA libraries was carried out. Isolated positive clones were validated by mating assay and identified through automated DNA sequencing and BLAST database searches. Sequence analysis revealed a number of PTEN-binding proteins linking this phosphatase to a number of different signaling cascades, suggesting that PTEN may perform other functions besides tumor-suppressing activity in different cell types. In particular, the interplay between PTEN function and adipocyte-specific fatty-acid-binding protein FABP4 is of notable interest. The demonstrable tautology of PTEN to FABP4 suggested a role for this phosphatase in the regulation of lipid metabolism and adipocyte differentiation. This interaction was further studied using coimmunoprecipitation and gel-filtration assays. Finally, based on Biacore assay, we have calculated the K(D) of PTEN-FABP4 complex, which is around 2.8 microM.

  6. DnaT is a PriC-binding protein.

    PubMed

    Huang, Chien-Chih; Huang, Cheng-Yang

    2016-09-01

    DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT-PriC changed from red to purple, which indicated the protein-protein interactions due to gold nanoparticle aggregate. In addition, this DnaT-PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10(-8) M. These results constitute a pioneering study of the DnaT-PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC-SSB-DNA tricomplex and regulates the PriC-dependent replication restart.

  7. Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

    PubMed

    Jongerius, Ilse; Lavender, Hayley; Tan, Lionel; Ruivo, Nicola; Exley, Rachel M; Caesar, Joseph J E; Lea, Susan M; Johnson, Steven; Tang, Christoph M

    2013-01-01

    Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.

  8. Distinct binding and immunogenic properties of the gonococcal homologue of meningococcal factor h binding protein.

    PubMed

    Jongerius, Ilse; Lavender, Hayley; Tan, Lionel; Ruivo, Nicola; Exley, Rachel M; Caesar, Joseph J E; Lea, Susan M; Johnson, Steven; Tang, Christoph M

    2013-01-01

    Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups. PMID:23935503

  9. Clinical use of glutamine supplementation.

    PubMed

    Wernerman, Jan

    2008-10-01

    Endogenous production of glutamine may become insufficient during critical illness. The shortage of glutamine is reflected as a decrease in plasma concentration, which is a prognostic factor for poor outcome in sepsis. Because glutamine is a precursor for nucleotide synthesis, rapidly dividing cells are most likely to suffer from a shortage. Therefore, exogenous glutamine supplementation is necessary. In particular, when i.v. nutrition is given, extra glutamine supplementation becomes critical, because most present formulations for i.v. use do not contain any glutamine for technical reasons. The major part of endogenously produced glutamine comes from skeletal muscle. For patients staying a long time in the intensive care unit (ICU), the muscle mass decreases rapidly, which leaves a tissue of diminishing size to maintain the export of glutamine. The metabolic and nutritional adaptation in long-staying ICU patients is poorly studied and is one of the fields that needs more scientific evidence for clinical recommendations. To date, there is evidence to support the clinical use of glutamine supplementation in critically ill patients, in hematology patients, and in oncology patients. Strong evidence is presently available for i.v. glutamine supplementation to critically ill patients on parenteral nutrition. This must be regarded as the standard of care. For patients on enteral nutrition, more evidence is needed. To guide administration of glutamine, there are good arguments to use measurement of plasma glutamine concentration for guidance. This will give an indication for treatment as well as proper dosing. Most patients will have a normalized plasma glutamine concentration by adding 20-25 g/24 h. Furthermore, there are no reported adverse or negative effects attributable to glutamine supplementation.

  10. Direct DNA Methylation Profiling Using Methyl Binding Domain Proteins

    PubMed Central

    Yu, Yinni; Blair, Steve; Gillespie, David; Jensen, Randy; Myszka, David G.; Badran, Ahmed H.; Ghosh, Indraneel; Chagovetz, Alexander

    2010-01-01

    Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring methyl binding domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG), and subsequently recruit other chromatin-modifying proteins to suppress transcription. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay. We focus on proof-of-concept studies for a direct microarray-based assay using surface-bound methylated probes. The recombinant protein 1xMBD-GFP recognizes hemi-methylation and symmetric methylation of the CpG sequence of hybridized dsDNA, while displaying greater affinity for the symmetric methylation motif, as evaluated by SPR. From these studies, for symmetric mCpG, the KD for 1xMBD-GFP ranged from 106 nM to 870 nM, depending upon the proximity of the methylation site to the sensor surface. The KD values for non-symmetrical methylation motifs were consistently greater (> 2 µM), but the binding selectivity between symmetric and hemi-methylation motifs ranged from 4 to 30, with reduced selectivity for sites close to the surface or multiple sites in proximity, which we attribute to steric effects. Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density. PMID:20507169

  11. Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids.

    PubMed Central

    Folmer, R H; Nilges, M; Konings, R N; Hilbers, C W

    1995-01-01

    The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics. NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate. The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude. The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics. The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e. the cold shock domain) of proteins belonging to the Y-box family. Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins. Images PMID:7556054

  12. Structural analysis of the wheat genes encoding NADH-dependent glutamine-2-oxoglutarate amidotransferases genes and correlation with grain protein content

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nitrogen uptake and the efficient absorption and metabolism of nitrogen are essential elements in attempts to breed improved cereal cultivars for grain or silage production. One of the enzymes related to nitrogen metabolism is glutamine-2-oxoglutarate amidotransferase (GOGAT). Together with glutami...

  13. RNA-binding proteins in pluripotency, differentiation, and reprogramming

    PubMed Central

    GUALLAR, Diana; WANG, Jianlong

    2014-01-01

    Embryonic stem cell maintenance, differentiation, and somatic cell reprogramming require the interplay of multiple pluripotency factors, epigenetic remodelers, and extracellular signaling pathways. RNA-binding proteins (RBPs) are involved in a wide range of regulatory pathways, from RNA metabolism to epigenetic modifications. In recent years we have witnessed more and more studies on the discovery of new RBPs and the assessment of their functions in a variety of biological systems, including stem cells. We review the current studies on RBPs and focus on those that have functional implications in pluripotency, differentiation, and/or reprogramming in both the human and mouse systems. PMID:25554730

  14. Targeting the inhibitor of Apoptosis Protein BIR3 binding domains.

    PubMed

    Jaquith, James B

    2014-05-01

    The Inhibitor of Apoptosis Proteins (IAPs) play a critical role in the regulation of cellular apoptosis and cytokine signaling. IAP family members include XIAP, cIAP1, cIAP2, NAIP, survivin, Apollon/Bruce, ML-IAP/livin and TIAP. The IAPs have been targeted using both antisense oligonucleotides and small molecule inhibitors. Several research teams have advanced compounds that bind the highly conserved BIR3 domains of the IAPs into clinical trials, as single agents and in combination with standard of care. This patent review highlights the medicinal chemistry strategies that have been applied to the development of clinical compounds. PMID:24998289

  15. Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR-DNA complexes.

    PubMed

    Fisher, Susan H; Wray, Lewis V

    2008-01-22

    The Bacillus subtilis GlnR repressor controls gene expression in response to nitrogen availability. Because all GlnR-regulated genes are expressed constitutively in mutants lacking glutamine synthetase (GS), GS is required for repression by GlnR. Feedback-inhibited GS (FBI-GS) was shown to activate GlnR DNA binding with an in vitro electophoretic mobility shift assay (EMSA). The activation of GlnR DNA binding by GS in these experiments depended on the feedback inhibitor glutamine and did not occur with mutant GS proteins defective in regulating GlnR activity in vivo. Although stable GS-GlnR-DNA ternary complexes were not observed in the EMSA experiments, cross-linking experiments showed that a protein-protein interaction occurs between GlnR and FBI-GS. This interaction was reduced in the absence of the feedback inhibitor glutamine and with mutant GS proteins. Because FBI-GS significantly reduced the dissociation rate of the GlnR-DNA complexes, the stability of these complexes is enhanced by FBI-GS. These results argue that FBI-GS acts as a chaperone that activates GlnR DNA binding through a transient protein-protein interaction that stabilizes GlnR-DNA complexes. GS was shown to control the activity of the B. subtilis nitrogen transcription factor TnrA by forming a stable complex between FBI-GS and TnrA that inhibits TnrA DNA binding. Thus, B. subtilis GS is an enzyme with dual catalytic and regulatory functions that uses distinct mechanisms to control the activity of two different transcription factors.

  16. A vicilin-like seed protein of cycads: similarity to sucrose-binding proteins.

    PubMed

    Braun, H; Czihal, A; Shutov, A D; Bäumlein, H

    1996-04-01

    Seed storage globulins of the 7S and 11S type are synthesized in the seeds of angiosperms and gymnosperms. We have isolated and characterized a vicilin-like gene expressed in the cycad Zamia furfuraceae. Sequence comparisons reveal clear similarities to a sucrose-binding protein isolated from soybean. We suggest the existence of a superfamily of related genes including both vicilin-like and legumin-like seed globulin genes as well as genes coding for spherulins, germins and sucrose-binding-proteins.

  17. Identification and Investigation of Novel Binding Fragments in the Fatty Acid Binding Protein 6 (FABP6).

    PubMed

    Hendrick, Alan G; Müller, Ilka; Willems, Henriëtte; Leonard, Philip M; Irving, Steve; Davenport, Richard; Ito, Takashi; Reeves, Jenny; Wright, Susanne; Allen, Vivienne; Wilkinson, Stephen; Heffron, Helen; Bazin, Richard; Turney, Jennifer; Mitchell, Philip J

    2016-09-01

    Fatty acid binding protein 6 (FABP6) is a potential drug discovery target, which, if inhibited, may have a therapeutic benefit for the treatment of diabetes. Currently, there are no published inhibitors of FABP6, and with the target believed to be amenable to fragment-based drug discovery, a structurally enabled program was initiated. This program successfully identified fragment hits using the surface plasmon resonance (SPR) platform. Several hits were validated with SAR and were found to be displaced by the natural ligand taurocholate. We report the first crystal structure of human FABP6 in the unbound form, in complex with cholate, and with one of the key fragments. PMID:27500412

  18. Revisiting Apoplastic Auxin Signaling Mediated by AUXIN BINDING PROTEIN 1

    PubMed Central

    Feng, Mingxiao; Kim, Jae-Yean

    2015-01-01

    It has been suggested that AUXIN BINDING PROTEIN 1 (ABP1) functions as an apoplastic auxin receptor, and is known to be involved in the post-transcriptional process, and largely independent of the already well-known SKP-cullin-F-box-transport inhibitor response (TIR1) /auxin signaling F-box (AFB) (SCFTIR1/AFB) pathway. In the past 10 years, several key components downstream of ABP1 have been reported. After perceiving the auxin signal, ABP1 interacts, directly or indirectly, with plasma membrane (PM)-localized transmembrane proteins, transmembrane kinase (TMK) or SPIKE1 (SPK1), or other unidentified proteins, which transfer the signal into the cell to the Rho of plants (ROP). ROPs interact with their effectors, such as the ROP interactive CRIB motif-containing protein (RIC), to regulate the endocytosis/exocytosis of the auxin efflux carrier PIN-FORMED (PIN) proteins to mediate polar auxin transport across the PM. Additionally, ABP1 is a negative regulator of the traditional SCFTIR1/AFB auxin signaling pathway. However, Gao et al. (2015) very recently reported that ABP1 is not a key component in auxin signaling, and the famous abp1-1 and abp1-5 mutant Arabidopsis lines are being called into question because of possible additional mutantion sites, making it necessary to reevaluate ABP1. In this review, we will provide a brief overview of the history of ABP1 research. PMID:26467289

  19. Revisiting Apoplastic Auxin Signaling Mediated by AUXIN BINDING PROTEIN 1.

    PubMed

    Feng, Mingxiao; Kim, Jae-Yean

    2015-10-01

    It has been suggested that AUXIN BINDING PROTEIN 1 (ABP1) functions as an apoplastic auxin receptor, and is known to be involved in the post-transcriptional process, and largely independent of the already well-known SKP-cullin-F-box-transport inhibitor response (TIR1) /auxin signaling F-box (AFB) (SCF(TIR1/AFB)) pathway. In the past 10 years, several key components downstream of ABP1 have been reported. After perceiving the auxin signal, ABP1 interacts, directly or indirectly, with plasma membrane (PM)-localized transmembrane proteins, transmembrane kinase (TMK) or SPIKE1 (SPK1), or other unidentified proteins, which transfer the signal into the cell to the Rho of plants (ROP). ROPs interact with their effectors, such as the ROP interactive CRIB motif-containing protein (RIC), to regulate the endocytosis/exocytosis of the auxin efflux carrier PIN-FORMED (PIN) proteins to mediate polar auxin transport across the PM. Additionally, ABP1 is a negative regulator of the traditional SCF(TIR1/AFB) auxin signaling pathway. However, Gao et al. (2015) very recently reported that ABP1 is not a key component in auxin signaling, and the famous abp1-1 and abp1-5 mutant Arabidopsis lines are being called into question because of possible additional mutantion sites, making it necessary to reevaluate ABP1. In this review, we will provide a brief overview of the history of ABP1 research.

  20. Computational characterization of TTHA0379: A potential glycerophosphocholine binding protein of Ugp ATP-binding cassette transporter.

    PubMed

    Chandravanshi, Monika; Gogoi, Prerana; Kanaujia, Shankar Prasad

    2016-11-01

    For the de novo biosynthesis of phospholipids, byproducts such as sn-glycerol-3-phosphate (G3P) and glycerophosphocholine (GPC) of glycerophospholipid metabolic pathway are imported inside the cell by an ATP-binding cassette (ABC) transporter known as UgpABCE. Of which, UgpA and UgpE constitutes the transmembrane domains (TMDs), UgpC forms the dimer of ATP-hydrolyzing component and UgpB is the periplasmic substrate binding protein. Structurally, UgpABCE transporter displays similarity to the maltose ABC transporter of Escherichia coli; thus, has been grouped into the CUT1 (Carbohydrate Uptake Transporter-1) family of bacterial ABC transporters. Being a member of CUT1 family, several Ugp (Uptake glycerol phosphate) protein sequences in biological database(s) exhibit sequence and structure similarity to sugar ABC transporters and have been annotated as sugar binding proteins; one of such proteins is TTHA0379 from Thermus thermophilus HB8. Here, in this study, we used computational method(s) to distinguish UgpB and sugar binding proteins based on their primary and tertiary structure features. A comprehensive analysis of these proteins indicates that they are evolutionarily related to each other having common conserved features at their primary and tertiary structure levels. However, they display differences at their active sites owing to the dissimilarity in their ligand preferences. In addition, phylogenetic analysis of TTHA0379 along with UgpB and sugar binding proteins reveals that both the groups of proteins forms two distinct clades and TTHA0379 groups with UgpB proteins. Furthermore, analysis of the ligand binding pocket shows that all the essential features of glycerophosphocholine binding protein i.e. UgpB, are conserved in TTHA0379 as well. Combining these features, here, we designate TTHA0379 to be a GPC binding protein.

  1. Roles of RNA-Binding Proteins in DNA Damage Response.

    PubMed

    Kai, Mihoko

    2016-01-01

    Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR)), and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR) pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, "sensor" proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM)'s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP) with low complexity domains, called intrinsically disordered proteins (IDPs), and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs) in a poly(ADP-ribose) (PAR)-dependent manner (unpublished data). DNA-dependent PARP1 (poly-(ADP) ribose polymerase 1) makes key contributions in the DNA damage response (DDR) network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as "liquid-demixing"). Among the

  2. Accounting for conformational entropy in predicting binding free energies of protein-protein interactions.

    PubMed

    Kamisetty, Hetunandan; Ramanathan, Arvind; Bailey-Kellogg, Chris; Langmead, Christopher James

    2011-02-01

    Protein-protein interactions are governed by the change in free energy upon binding, ΔG = ΔH - TΔS. These interactions are often marginally stable, so one must examine the balance between the change in enthalpy, ΔH, and the change in entropy, ΔS, when investigating known complexes, characterizing the effects of mutations, or designing optimized variants. To perform a large-scale study into the contribution of conformational entropy to binding free energy, we developed a technique called GOBLIN (Graphical mOdel for BiomoLecular INteractions) that performs physics-based free energy calculations for protein-protein complexes under both side-chain and backbone flexibility. Goblin uses a probabilistic graphical model that exploits conditional independencies in the Boltzmann distribution and employs variational inference techniques that approximate the free energy of binding in only a few minutes. We examined the role of conformational entropy on a benchmark set of more than 700 mutants in eight large, well-studied complexes. Our findings suggest that conformational entropy is important in protein-protein interactions--the root mean square error (RMSE) between calculated and experimentally measured ΔΔGs decreases by 12% when explicit entropic contributions were incorporated. GOBLIN models all atoms of the protein complex and detects changes to the binding entropy along the interface as well as positions distal to the binding interface. Our results also suggest that a variational approach to entropy calculations may be quantitatively more accurate than the knowledge-based approaches used by the well-known programs FOLDX and Rosetta--GOBLIN's RMSEs are 10 and 36% lower than these programs, respectively. PMID:21120864

  3. The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

    SciTech Connect

    Bernard, S.M.; Habash, D.Z.

    2009-07-02

    Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

  4. Macrocycles that inhibit the binding between heat shock protein 90 and TPR-containing proteins.

    PubMed

    Ardi, Veronica C; Alexander, Leslie D; Johnson, Victoria A; McAlpine, Shelli R

    2011-12-16

    Heat shock protein 90 (Hsp90) accounts for 1-2% of the total proteins in normal cells and functions as a molecular chaperone that folds, assembles, and stabilizes client proteins. Hsp90 is overexpressed (3- to 6-fold increase) in stressed cells, including cancer cells, and regulates over 200 client and co-chaperone proteins. Hsp90 client proteins are involved in a plethora of cellular signaling events including numerous growth and apoptotic pathways. Since pathway-specific inhibitors can be problematic in drug-resistant cancers, shutting down multiple pathways at once is a promising approach when developing new therapeutics. Hsp90's ability to modulate many growth and signaling pathways simultaneously makes this protein an attractive target in the field of cancer therapeutics. Herein we present evidence that a small molecule modulates Hsp90 via binding between the N and middle domain and allosterically inhibiting the binding interaction between Hsp90 and four C-terminal binding client proteins: IP6K2, FKBP38, FKBP52, and HOP. These last three clients contain a tetratricopeptide-repeat (TPR) region, which is known to interact with the MEEVD sequence on the C-terminus of Hsp90. Thus, this small molecule modulates the activity between co-chaperones that contain TPR motifs and Hsp90's MEEVD region. This mechanism of action is unique from that of all Hsp90 inhibitors currently in clinical trials where these molecules have no effect on proteins that bind to the C-terminus of Hsp90. Further, our small molecule induces a Caspase-3 dependent apoptotic event. Thus, we describe the mechanism of a novel scaffold that is a useful tool for studying cell-signaling events that result when blocking the MEEVD-TPR interaction between Hsp90 and co-chaperone proteins.

  5. Binding of Y-box proteins to RNA: involvement of different protein domains.

    PubMed Central

    Ladomery, M; Sommerville, J

    1994-01-01

    Eukaryotic Y-box proteins are reported to interact with a wide variety of nucleic acid structures to act as transcription factors and mRNA masking proteins. The modular structure of Y-box proteins includes a highly conserved N-terminal cold-shock domain (CSD, equivalent to the bacterial cold-shock proteins) plus four basic C-terminal domains containing arginine clusters and aromatic residues. In addition, the basic domains are separated by acidic regions which contain several potential sites for serine/threonine phosphorylation. The interaction of Y-box proteins, isolated from Xenopus oocytes (FRGY2 type), with RNA molecules has been studied by UV crosslinking and protein fragmentation. We have identified two distinct binding activities. The CSD interacts preferentially with the polypurines poly(A,G) and poly(G) but not poly(A), this activity being sensitive to 5 mM MgCl2 but not to 5 mM spermidine. In the presence of 1 mM MgCl2 or 1 mM spermidine, the basic domains interact preferentially with poly(C,U), this activity being sensitive to 0.5 M NaCl. Binding of the basic domains is also sensitive to low concentrations of heparin. The basic domains can be crosslinked individually to labelled RNA. These results are discussed with reference to the various specificities noted in the binding of Y-box proteins to RNA and DNA. Images PMID:7530842

  6. Characterization of Transport Proteins for Aromatic Compounds Derived from Lignin: Benzoate Derivative Binding Proteins

    PubMed Central

    Michalska, Karolina; Chang, Changsoo; Mack, Jamey C.; Zerbs, Sarah; Joachimiak, Andrzej; Collart, Frank R.

    2013-01-01

    In vitro growth experiments have demonstrated that aromatic compounds derived from lignin can be metabolized and represent a major carbon resource for many soil bacteria. However, the proteins that mediate the movement of these metabolites across the cell membrane have not been thoroughly characterized. To address this deficiency, we used a library representative of lignin degradation products and a thermal stability screen to determine ligand specificity for a set of solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters. The ligand mapping process identified a set of proteins from Alphaproteobacteria that recognize various benzoate derivatives. Seven high-resolution crystal structures of these proteins in complex with four different aromatic compounds were obtained. The protein–ligand complexes provide details of molecular recognition that can be used to infer binding specificity. This structure–function characterization provides new insight for the biological roles of these ABC transporters and their SBPs, which had been previously annotated as branched-chain amino-acid-binding proteins. The knowledge derived from the crystal structures provides a foundation for development of sequencebased methods to predict the ligand specificity of other uncharacterized transporters. These results also demonstrate that Alphaproteobacteria possess a diverse set of transport capabilities for lignin-derived compounds. Characterization of this new class of transporters improves genomic annotation projects and provides insight into the metabolic potential of soil bacteria. PMID:22925578

  7. Protein-protein binding before and after photo-modification of albumin

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.; Glickman, Randolph D.; Thomas, Robert J.; Brancaleon, Lorenzo

    2016-03-01

    Bioeffects of directed-optical-energy encompass a wide range of applications. One aspect of photochemical interactions involves irradiating a photosensitizer with visible light in order to induce protein unfolding and consequent changes in function. In the past, irradiation of several dye-protein combinations has revealed effects on protein structure. Beta lactoglobulin, human serum albumin (HSA) and tubulin have all been photo-modified with meso-tetrakis(4- sulfonatophenyl)porphyrin (TSPP) bound, but only in the case of tubulin has binding caused a verified loss of biological function (loss of ability to form microtubules) as a result of this light-induced structural change. The current work questions if the photo-induced structural changes that occur to HSA, are sufficient to disable its biological function of binding to osteonectin. The albumin-binding protein, osteonectin, is about half the molecular weight of HSA, so the two proteins and their bound product can be separated and quantified by size exclusion high performance liquid chromatography. TSPP was first bound to HSA and irradiated, photo-modifying the structure of HSA. Then native HSA or photo-modified HSA (both with TSPP bound) were compared, to assess loss in HSA's innate binding ability as a result of light-induced structure modification.

  8. Interaction of ice binding proteins with ice, water and ions.

    PubMed

    Oude Vrielink, Anneloes S; Aloi, Antonio; Olijve, Luuk L C; Voets, Ilja K

    2016-03-01

    Ice binding proteins (IBPs) are produced by various cold-adapted organisms to protect their body tissues against freeze damage. First discovered in Antarctic fish living in shallow waters, IBPs were later found in insects, microorganisms, and plants. Despite great structural diversity, all IBPs adhere to growing ice crystals, which is essential for their extensive repertoire of biological functions. Some IBPs maintain liquid inclusions within ice or inhibit recrystallization of ice, while other types suppress freezing by blocking further ice growth. In contrast, ice nucleating proteins stimulate ice nucleation just below 0 °C. Despite huge commercial interest and major scientific breakthroughs, the precise working mechanism of IBPs has not yet been unraveled. In this review, the authors outline the state-of-the-art in experimental and theoretical IBP research and discuss future scientific challenges. The interaction of IBPs with ice, water and ions is examined, focusing in particular on ice growth inhibition mechanisms. PMID:26787386

  9. Light-activated DNA binding in a designed allosteric protein

    SciTech Connect

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R.

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  10. Interaction of ice binding proteins with ice, water and ions.

    PubMed

    Oude Vrielink, Anneloes S; Aloi, Antonio; Olijve, Luuk L C; Voets, Ilja K

    2016-03-19

    Ice binding proteins (IBPs) are produced by various cold-adapted organisms to protect their body tissues against freeze damage. First discovered in Antarctic fish living in shallow waters, IBPs were later found in insects, microorganisms, and plants. Despite great structural diversity, all IBPs adhere to growing ice crystals, which is essential for their extensive repertoire of biological functions. Some IBPs maintain liquid inclusions within ice or inhibit recrystallization of ice, while other types suppress freezing by blocking further ice growth. In contrast, ice nucleating proteins stimulate ice nucleation just below 0 °C. Despite huge commercial interest and major scientific breakthroughs, the precise working mechanism of IBPs has not yet been unraveled. In this review, the authors outline the state-of-the-art in experimental and theoretical IBP research and discuss future scientific challenges. The interaction of IBPs with ice, water and ions is examined, focusing in particular on ice growth inhibition mechanisms.

  11. Identification of Actin-Binding Proteins from Maize Pollen

    SciTech Connect

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  12. The Use of Multimeric Protein Scaffolds for Identifying Multi-SUMO Binding Proteins.

    PubMed

    Aguilar-Martínez, Elisa; Sharrocks, Andrew D

    2016-01-01

    The use of in vitro assays, such as glutathione S-transferase (GST) pull-downs, enables the study of complex cellular processes in a simplified form. Pull-down assays facilitate the discovery and detailed study of protein-protein interactions, which can then be extrapolated to the cellular environment. Here, we describe the expression, purification and use of a multi-SUMO platform to identify SUMO-interacting proteins. This SUMO-platform can be easily expressed and purified from bacterial cells for use as baits in pull-down assays. This methodology facilitates the discovery of novel SUMO-binding proteins or further characterization of SUMO with known binding partners. PMID:27631807

  13. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  14. Synthesis, characterization, DNA binding, DNA cleavage, protein binding and cytotoxic activities of Ru(II) complexes.

    PubMed

    Thota, Sreekanth; Vallala, Srujana; Yerra, Rajeshwar; Rodrigues, Daniel Alencar; Raghavendra, Nulgumnalli Manjunathaiah; Barreiro, Eliezer J

    2016-01-01

    We report on the synthesis of novel Ru(II) compounds (Ru-1 to Ru-8) bearing R-pdc, 4-Cl-pbinh ligands (where R=4-CF3, 4-F, 4-OH pdc=3-phenyl-5-(1H-pyrrol-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide, pbinh=phenoxybenzylidene isonicotinyl hydrazides) and their in vitro antitumor activity toward the cell lines murine leukemia L1210, human lymphocyte CEM, human epithelial cervical carcinoma HeLa, BEL-7402 and Molt4/C8. Some of the complexes exhibited more potent antiproliferative activity against cell lines than the standard drug cisplatin. Ruthenium complex Ru-2 displayed potent cytotoxicity with than that of cisplatin. DNA-binding, DNA cleavage and protein binding properties of ruthenium complexes with these ligands are reported. Interactions of these ruthenium complexes with DNA revealed an intercalative mode of binding between them. Synchronous fluorescence spectra proved that the interaction of ruthenium complexes with bovine serum albumin (BSA) resulted in a conformational change of the latter.

  15. Periplasmic Binding Proteins in Thermophiles: Characterization and Potential Application of an Arginine-Binding Protein from Thermotoga maritima: A Brief Thermo-Story.

    PubMed

    Ausili, Alessio; Staiano, Maria; Dattelbaum, Jonathan; Varriale, Antonio; Capo, Alessandro; D'Auria, Sabato

    2013-01-01

    Arginine-binding protein from the extremophile Thermotoga maritima is a 27.7 kDa protein possessing the typical two-domain structure of the periplasmic binding proteins family. The protein is characterized by a very high specificity and affinity to bind to arginine, also at high temperatures. Due to its features, this protein could be taken into account as a potential candidate for the design of a biosensor for arginine. It is important to investigate the stability of proteins when they are used for biotechnological applications. In this article, we review the structural and functional features of an arginine-binding protein from the extremophile Thermotoga maritima with a particular eye on its potential biotechnological applications. PMID:25371336

  16. Direct protein-protein interactions and substrate channeling between cellular retinoic acid binding proteins and CYP26B1.

    PubMed

    Nelson, Cara H; Peng, Chi-Chi; Lutz, Justin D; Yeung, Catherine K; Zelter, Alex; Isoherranen, Nina

    2016-08-01

    Cellular retinoic acid binding proteins (CRABPs) bind all-trans-retinoic acid (atRA) tightly. This study aimed to determine whether atRA is channeled directly to cytochrome P450 (CYP) CYP26B1 by CRABPs, and whether CRABPs interact directly with CYP26B1. atRA bound to CRABPs (holo-CRABP) was efficiently metabolized by CYP26B1. Isotope dilution experiments showed that delivery of atRA to CYP26B1 in solution was similar with or without CRABP. Holo-CRABPs had higher affinity for CYP26B1 than free atRA, but both apo-CRABPs inhibited the formation of 4-OH-RA by CYP26B1. Similar protein-protein interactions between soluble binding proteins and CYPs may be important for other lipophilic CYP substrates.

  17. Riboflavin-binding protein exhibits selective sweet suppression toward protein sweeteners.

    PubMed

    Maehashi, Kenji; Matano, Mami; Kondo, Azusa; Yamamoto, Yasushi; Udaka, Shigezo

    2007-02-01

    Riboflavin-binding protein (RBP) is well known as a riboflavin carrier protein in chicken egg and serum. A novel function of RBP was found as a sweet-suppressing protein. RBP, purified from hen egg white, suppressed the sweetness of protein sweeteners such as thaumatin, monellin, and lysozyme, whereas it did not suppress the sweetness of low molecular weight sweeteners such as sucrose, glycine, D-phenylalanine, saccharin, cyclamate, aspartame, and stevioside. Therefore, the sweet-suppressing activity of RBP was apparently selective to protein sweeteners. The sweet suppression by RBP was independent of binding of riboflavin with its molecule. Yolk RBP, with minor structural differences compared with egg white RBP, also elicited a weaker sweet suppression. However, other commercially available proteins including ovalbumin, ovomucoid, beta-lactogloblin, myoglobin, and albumin did not substantially alter the sweetness of protein sweeteners. Because a prerinse with RBP reduced the subsequent sweetness of protein sweeteners, whereas the enzymatic activity of lysozyme and the elution profile of lysozyme on gel permeation chromatography were not affected by RBP, it is suggested that the sweet suppression is caused by an interaction of RBP with a sweet taste receptor rather than with the protein sweeteners themselves. The selectivity in the sweet suppression by RBP is consistent with the existence of multiple interaction sites within a single sweet taste receptor.

  18. Glutamine and leucine nitrogen kinetics and their relation to urea nitrogen in newborn infants.

    PubMed

    Parimi, Prabhu S; Devapatla, Srisatish; Gruca, Lourdes; O'Brien, Alicia M; Hanson, Richard W; Kalhan, Satish C

    2002-03-01

    Glutamine kinetics and its relation to transamination of leucine and urea synthesis were quantified in 16 appropriate-for-gestational-age infants, four small-for-gestational-age infants, and seven infants of diabetic mothers. Kinetics were measured between 4 and 5 h after the last feed (fasting) and in response to formula feeding using [5-(15)N]glutamine, [1-(13)C,(15)N]leucine, [(2)H(5)]phenylalanine, and [(15)N(2)]urea tracers. Leucine nitrogen and glutamine kinetics during fasting were significantly higher than those reported in adults. De novo synthesis accounted for approximately 85% of glutamine turnover. In response to formula feeding, a significant increase (P = 0.04) in leucine nitrogen turnover was observed, whereas a significant decrease (P = 0.002) in glutamine and urea rate of appearance was seen. The rate of appearance of leucine nitrogen was positively correlated (r(2) = 0.59, P = 0.001) with glutamine turnover. Glutamine flux was negatively correlated (r(2) = 0.39, P = 0.02) with the rate of urea synthesis. These data suggest that, in the human newborn, glutamine turnover is related to a high anaplerotic flux into the tricarboxylic acid cycle as a consequence of a high rate of protein turnover. The negative relationship between glutamine turnover and the irreversible oxidation of protein (urea synthesis) suggests an important role of glutamine as a nitrogen source for other synthetic processes and accretion of body proteins.

  19. Variant-specific surface proteins of Giardia lamblia are zinc-binding proteins.

    PubMed Central

    Nash, T E; Mowatt, M R

    1993-01-01

    Giardia lamblia undergoes surface antigenic variation. The variant-specific surface proteins (VSPs) are a distinct family of cysteine-rich proteins. Characteristically, cysteine residues occur mostly as CXXC tetrapeptides. Four of the reported five VSPs contain a putative metal-binding domain that resembles other metal-binding motifs; the fifth is closely related but lacks an essential histidine. Three different native VSPs bound Zn2+. Co2+, Cu2+, and Cd2+ inhibited Zn2+ binding. Analysis of recombinant VSP fusion proteins showed that the putative binding motif bound Zn2+. Surprisingly, peptide fragments from other regions of the VSP contain numerous CXXCXnCXXC motifs that also bound Zn2+. Analysis of deduced amino acid sequences showed well-conserved CXXC spacing in three out of five VSPs, suggesting conservation of structure despite amino acid sequence divergence. The function of VSPs is unknown, but by binding Zn2+ or other metals in the intestine, VSPs may contribute to Zn2+ malnutrition or inhibition of metal-dependent intestinal enzymes, which would lead to malabsorption, a well-known consequence of giardiasis. Images Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8516291

  20. A Comprehensive Analysis of Plasmodium Circumsporozoite Protein Binding to Hepatocytes.

    PubMed

    Zhao, Jinghua; Bhanot, Purnima; Hu, Junjie; Wang, Qian

    2016-01-01

    Circumsporozoite protein (CSP) is the dominant protein on the surface of Plasmodium sporozoites and plays a critical role in the invasion by sporozoites of hepatocytes. Contacts between CSP and heparin sulfate proteoglycans (HSPGs) lead to the attachment of sporozoites to hepatocytes and trigger signaling events in the parasite that promote invasion of hepatocytes. The precise sequence elements in CSP that bind HSPGs have not been identified. We performed a systematic in vitro analysis to dissect the association between Plasmodium falciparum CSP (PfCSP) and hepatocytes. We demonstrate that interactions between PfCSP and heparin or a cultured hepatoma cell line, HepG2, are mediated primarily by a lysine-rich site in the amino terminus of PfCSP. Importantly, the carboxyl terminus of PfCSP facilitates heparin-binding by the amino-terminus but does not interact directly with heparin. These findings provide insights into how CSP recognizes hepatocytes and useful information for further functional studies of CSP. PMID:27560376

  1. STEROIDOGENIC FACTOR-1 IS A SPHINGOLIPID BINDING PROTEIN

    PubMed Central

    Urs, Aarti N.; Dammer, Eric; Kelly, Samuel; Wang, Elaine; Merrill, Alfred H.; Sewer, Marion B.

    2007-01-01

    Steroidogenic factor (SF1, NR5A1, Ad4BP) is an orphan nuclear receptor that is essential for steroid hormone-biosynthesis and endocrine development. Studies have found that the ability of this receptor to increase target gene expression can be regulated by post-translational modification, subnuclear localization, and protein-protein interactions. Recent crystallographic studies and our mass spectrometric analyses of the endogenous receptor have demonstrated an integral role for ligand-binding in the control of SF1 transactivation activity. Herein, we discuss our findings that sphingosine is an endogenous ligand for SF1. These studies and the structural findings of others have demonstrated that the receptor can bind both sphingolipids and phospholipids. Thus, it is likely that multiple bioactive lipids are ligands for SF1 and that these lipids will differentially act to control SF1 activity in a context-dependent manner. Finally, these findings highlight a central role for bioactive lipids as mediators of trophic-hormone stimulated steroid hormone biosynthesis. PMID:17196738

  2. A Comprehensive Analysis of Plasmodium Circumsporozoite Protein Binding to Hepatocytes

    PubMed Central

    Zhao, Jinghua; Bhanot, Purnima; Hu, Junjie; Wang, Qian

    2016-01-01

    Circumsporozoite protein (CSP) is the dominant protein on the surface of Plasmodium sporozoites and plays a critical role in the invasion by sporozoites of hepatocytes. Contacts between CSP and heparin sulfate proteoglycans (HSPGs) lead to the attachment of sporozoites to hepatocytes and trigger signaling events in the parasite that promote invasion of hepatocytes. The precise sequence elements in CSP that bind HSPGs have not been identified. We performed a systematic in vitro analysis to dissect the association between Plasmodium falciparum CSP (PfCSP) and hepatocytes. We demonstrate that interactions between PfCSP and heparin or a cultured hepatoma cell line, HepG2, are mediated primarily by a lysine-rich site in the amino terminus of PfCSP. Importantly, the carboxyl terminus of PfCSP facilitates heparin-binding by the amino-terminus but does not interact directly with heparin. These findings provide insights into how CSP recognizes hepatocytes and useful information for further functional studies of CSP. PMID:27560376

  3. The use of biotin-binding proteins for insect control.

    PubMed

    Christeller, John T; Markwick, Ngaire P; Burgess, Elisabeth P J; Malone, Louise A

    2010-04-01

    Biotin-binding proteins (BBPs), expressed in transgenic plants, are insecticidal to a very wide range of insects. The expression levels required are generally low (approximately 100 ppm), and although higher than required for Bacillus thuringiensis (Bt) delta-endotoxins, BBPs are effective across a broader range of insect orders and other invertebrates than the Bt Cry proteins. Avidin and streptavidin, in particular, have been reported as causing death or severe growth reduction in at least 40 species of insects across five insect orders (Lepidoptera, Coleoptera, Orthoptera, Diptera, and leaf-eating Hymenoptera) and mites. In addition, due largely to its rapid dilution in ecosystems, no adverse impacts on nontarget microorganisms or invertebrates have been recorded. Because the target, biotin, cannot itself be modified to prevent it binding to BBPs and remain effective as a vitamin, the major avenue open to insects to develop resistance is unavailable. Two properties of the biotin-avidin complex make it highly suitable for use in transgenic plant crop protection strategies against a large range of insects; its extreme stability and its resistance to proteolysis. However, because the nutritional value of the plant could potentially be compromised in the absence of biotin supplementation, its use in nonfood crops such as fiber, forestry, and biofuel crops is seen as the most suitable initial focus for this technology. PMID:20429467

  4. Plasma binding proteins as mediators of corticosteroid action in vertebrates.

    PubMed

    Breuner, C W; Orchinik, M

    2002-10-01

    Stressors elicit a complex but variable suite of endocrine events. Comparative studies of the stress response have focused primarily on the adrenocortical response to stress, in particular the measurement of plasma levels of glucocorticoids. However, a number of other factors contribute to and modify cellular and organismal responses to glucocorticoids. Notably, plasma corticosteroid binding globulins (CBGs) can regulate the general availability of steroid to tissues, and/or direct the delivery of hormones to specific sites. In this paper, we discuss possible functions of CBG and mechanisms of CBG action, review CBG characteristics among vertebrates, and discuss our recent studies indicating that CBG may indeed modulate responses to stressors. For example, in house sparrows, we found that basal and stress-induced concentrations of total corticosteroid (cortisol or corticosterone) (CORT) vary seasonally, but CBG concentrations change proportionally, so that free CORT concentrations appear static year-round. In contrast, in white-crowned sparrows and tree lizards, CBG concentrations change under conditions when total CORT levels do not, resulting in significant changes in circulating free CORT. These differences in free CORT are masked if CBG is not accounted for. We have also found that the binding properties of CBG vary considerably between species and need to be determined empirically. Such studies led to the observation that CBG in several species may also serve as a functional androgen binding protein; this is especially important for birds, because previous studies had concluded that birds lack androgen binding globulins. We propose that consideration of CBG is paramount to understanding the role of glucocorticoids in mediating behavioral and physiological responses to stress.

  5. Localization of Cellular Retinol-Binding Protein and Retinol-Binding Protein in Cells Comprising the Blood-Brain Barrier of Rat and Human

    NASA Astrophysics Data System (ADS)

    MacDonald, Paul N.; Bok, Dean; Ong, David E.

    1990-06-01

    Brain is not generally recognized as an organ that requiries vitamin A, perhaps because no obvious histologic lesions have been observed in severely vitamin A-deficient animals. However, brain tissue does contain cellular vitamin A-binding proteins and a nuclear receptor protein for retinoic acid. In the present study, immunohistochemical techniques were used to determine the cell-specific location of cellular retinol-binding protein in human and rat brain tissue. Cellular retinol-binding protein was localized specifically within the endothelial cells of the brain microvasculature and within the cuboidal epithelial cells of the choroid plexus, two primary sites of the mammalian blood-brain barrier. In addition, autoradiographic procedures demonstrated binding sites for serum retinol-binding protein in the choroidal epithelium. These observations suggest that a significant movement of retinol across the blood-brain barrier may occur.

  6. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human

    SciTech Connect

    MacDonald, P.N.; Ong, D.E. ); Bok, D. )

    1990-06-01

    Brain is not generally recognized as an organ that requi