The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

José G. Gavilanes - One of the best experts on this subject based on the ideXlab platform.

  • a peptide of nine amino acid residues from Alpha Sarcin cytotoxin is a membrane perturbing structure
    Journal of Peptide Research, 2009
    Co-Authors: J.m. Mancheño, Mercedes Oñaderra, Alvaro Martinez Del Pozo, Juan Pablo Albar, José G. Gavilanes
    Abstract:

    A water-soluble synthetic peptide with only nine amino acid residues, comprising the 131-139 sequence region of the cytotoxic protein Alpha-Sarcin (secreted by the mold Aspergillus giganteus), interacts with large unilamellar vesicles composed of acid phospholipids. It promotes lipid mixing between bilayers and leakage of vesicle aqueous contents, and it also abolishes the phospholipid phase transition. Other larger peptides containing such an amino acid sequence also produce these effects. These peptides acquire Alpha-helical conformation in the presence of trifluoroethanol, but display beta-strand conformation in the presence of sodium dodecyl sulfate. The interaction of these peptides with the lipid vesicles also results in beta-structure. The obtained data are discussed in terms of the involvement of the 131-139 stretch of Alpha-Sarcin in its interaction with lipid membranes.

  • tyr 48 a conserved residue in ribotoxins is involved in the rna degrading activity of Alpha Sarcin
    Biological Chemistry, 2006
    Co-Authors: Elisa Alvarezgarcia, Lucia Garciaortega, Marta Bruix, Alvaro Martinez Del Pozo, Yolanda Verdun, José G. Gavilanes
    Abstract:

    Residue Tyr-48 in Alpha-Sarcin is conserved not only within the ribotoxin family, but also within the larger group of extracellular fungal ribonucleases, best represented by RNase T1. A mutant protein in which this Tyr residue was substituted by Phe has been produced and isolated to homogeneity. It was spectroscopically analyzed by means of circular dichroism, fluorescence emission and NMR. Taken together, these results and those from enzyme characterization have revealed the essential role of the -OH group from the Tyr-48 phenolic ring in the cleavage of polymeric RNA substrates, including the ribosome-embedded 28S rRNA, the natural substrate of ribotoxins. Thus, the mutant protein does not degrade its natural ribosomal RNA substrate. However, it has been shown that this Y48F mutant still retains its ability to cleave a phosphodiester bond in a minimal substrate such as the dinucleoside phosphate ApA. The role of different Alpha-Sarcin residues within the enzyme reaction catalyzed by this protein is discussed.

  • conserved asparagine residue 54 of α Sarcin plays a role in protein stability and enzyme activity
    Biological Chemistry, 2004
    Co-Authors: Ansgar Siemer, Mercedes Oñaderra, Manuel Masip, Nelson Carreras, Lucia Garciaortega, Marta Bruix, Alvaro Martinez Del Pozo, José G. Gavilanes
    Abstract:

    Asparagine 54 of Alpha-Sarcin is a conserved residue within the proteins of the ribotoxin family of microbial ribonucleases. It is located in loop 2 of the protein, which lacks repetitive secondary structure elements but exhibits a well-defined conformation. Five mutant variants at this residue have been produced and characterized. The spectroscopic characterization of these proteins indicates that the overall conformation is not changed upon mutation. Activity and denaturation assays show that Asn-54 largely contributes to protein stability, and its presence is a requirement for the highly specific inhibitory activity of these ribotoxins on ribosomes.

  • backbone dynamics of the cytotoxic ribonuclease Alpha Sarcin by 15n nmr relaxation methods
    Journal of Biomolecular NMR, 2002
    Co-Authors: Jose Manuel Perezcanadillas, José G. Gavilanes, Alvaro Martinez Del Pozo, Ramon Camposolivas, Jorge Santoro, Manuel Rico, Marc Guenneugues, Marta Bruix
    Abstract:

    The cytotoxic ribonuclease α-Sarcin is a 150-residue protein that inactivates ribosomes by selectively cleaving a single phosphodiester bond in a strictly conserved rRNA loop. In order to gain insights on the molecular basis of its highly specific activity, we have previously determined its solution structure and studied its electrostatics properties. Here, we complement those studies by analysing the backbone dynamics of α-Sarcin through measurement of longitudinal relaxation rates R1, off resonance rotating frame relaxation rates R1ρ, and the 15N1HNOE of the backbone amide 15N nuclei at two different magnetic field strengths (11.7 and 17.6 T). The two sets of relaxation parameters have been analysed in terms of the reduced spectral density mapping formalism, as well as by the model-free approach. α-Sarcin behaves as an axial symmetric rotor of the prolate type (D∥/D⊥=1.16 ± 0.02) which tumbles with a correlation time τm of 7.54 ± 0.02 ns. The rotational diffusion properties have been also independently evaluated by hydrodynamic calculations and are in good agreement with the experimental results. The analysis of the internal dynamics reveals that α-Sarcin is composed of a rigid hydrophobic core and some exposed segments which undergo fast (ps to ns) internal motions. Slower motions in the μs to ms time scale are less abundant and in some cases can be assigned to specific motional processes. All dynamic data are discussed in relation to the role of some particular residues of α-Sarcin in the process of recognition of its ribosomal target.

  • assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin Alpha Sarcin
    Proteins, 2000
    Co-Authors: C De Antonio, Javier Lacadena, Mercedes Oñaderra, J.m. Mancheño, Marta Bruix, Antonio Martinezruiz, Martinez A Del Pozo, Jose Manuel Perezcanadillas, José G. Gavilanes
    Abstract:

    α-Sarcin, a potent cytotoxic protein from Aspergillus giganteus, contains two tryptophan residues at positions 4 and 51. Two single, W4F and W51F, and the double mutant, W4/51F, have been produced and purified to homogeneity. These two residues are neither required for the highly specific ribonucleolytic activity of the protein on the ribosomes (production of the so called α-fragment) nor for its interaction with lipid membranes (aggregation and fusion of vesicles), although the mutant forms involving Trp-51 show a decreased ribonuclease activity. Proton NMR data reveal that no significant changes in the global structure of the enzyme occur upon replacement of Trp-51 by Phe. Substitution of each Trp residue results in a 4 °C drop in the thermal denaturation midpoint, and the double mutant's midpoint is 9°C lower. Trp-51 is responsible for most of the near-UV circular dichroism of the protein and also contributes to the overall ellipticity of the protein in the peptide bond region. Trp-51 does not show fluorescence emission. The membrane-bound proteins undergo a thermal denaturation at a lower temperature than the corresponding free forms. The interaction of the protein with phospholipid bilayers promotes a large increase of the quantum yield of Trp-51 and its fluorescence emission is quenched by anthracene incorporated into the hydrophobic region of such bilayers. This indicates that the region around this residue is located in the hydrophobic core of the bilayer following protein–vesicle interaction. Proteins 2000;41:350–361. © 2000 Wiley-Liss, Inc.

Javier Lacadena - One of the best experts on this subject based on the ideXlab platform.

  • assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin Alpha Sarcin
    Proteins, 2000
    Co-Authors: C De Antonio, Javier Lacadena, Mercedes Oñaderra, J.m. Mancheño, Marta Bruix, Antonio Martinezruiz, Martinez A Del Pozo, Jose Manuel Perezcanadillas, José G. Gavilanes
    Abstract:

    α-Sarcin, a potent cytotoxic protein from Aspergillus giganteus, contains two tryptophan residues at positions 4 and 51. Two single, W4F and W51F, and the double mutant, W4/51F, have been produced and purified to homogeneity. These two residues are neither required for the highly specific ribonucleolytic activity of the protein on the ribosomes (production of the so called α-fragment) nor for its interaction with lipid membranes (aggregation and fusion of vesicles), although the mutant forms involving Trp-51 show a decreased ribonuclease activity. Proton NMR data reveal that no significant changes in the global structure of the enzyme occur upon replacement of Trp-51 by Phe. Substitution of each Trp residue results in a 4 °C drop in the thermal denaturation midpoint, and the double mutant's midpoint is 9°C lower. Trp-51 is responsible for most of the near-UV circular dichroism of the protein and also contributes to the overall ellipticity of the protein in the peptide bond region. Trp-51 does not show fluorescence emission. The membrane-bound proteins undergo a thermal denaturation at a lower temperature than the corresponding free forms. The interaction of the protein with phospholipid bilayers promotes a large increase of the quantum yield of Trp-51 and its fluorescence emission is quenched by anthracene incorporated into the hydrophobic region of such bilayers. This indicates that the region around this residue is located in the hydrophobic core of the bilayer following protein–vesicle interaction. Proteins 2000;41:350–361. © 2000 Wiley-Liss, Inc.

  • the highly refined solution structure of the cytotoxic ribonuclease Alpha Sarcin reveals the structural requirements for substrate recognition and ribonucleolytic activity
    Journal of Molecular Biology, 2000
    Co-Authors: Jose Manuel Perezcanadillas, José G. Gavilanes, Javier Lacadena, Martinez A Del Pozo, Ramon Camposolivas, Jorge Santoro, Manuel Rico, Marta Bruix
    Abstract:

    Abstract α-Sarcin selectively cleaves a single phosphodiester bond in a universally conserved sequence of the major rRNA, that inactivates the ribosome. The elucidation of the three-dimensional solution structure of this 150 residue enzyme is a crucial step towards understanding α-Sarcin’s conformational stability, ribonucleolytic activity, and its exceptionally high level of specificity. Here, the solution structure has been determined on the basis of 2658 conformationally relevant distances restraints (including stereoespecific assignments) and 119 torsional angular restraints, by nuclear magnetic resonance spectroscopy methods. A total of 60 converged structures have been computed using the program DYANA. The 47 best DYANA structures, following restrained energy minimization by GROMOS, represent the solution structure of α-Sarcin. The resulting average pairwise root-mean-square-deviation is 0.86 A for backbone atoms and 1.47 A for all heavy atoms. When the more variable regions are excluded from the analysis, the pairwise root-mean-square deviation drops to 0.50 A and 1.00 A, for backbone and heavy atoms, respectively. The α-Sarcin structure is similar to that reported for restrictocin, although some differences are clearly evident, especially in the loop regions. The average rmsd between the structurally aligned backbones of the 47 final α-Sarcin structures and the crystal structure of restrictocin is 1.46 A. On the basis of a docking model constructed with α-Sarcin solution structure and the crystal structure of a 29-nt RNA containing the Sarcin/ricin domain, the regions in the protein that could interact specifically with the substrate have been identified. The structural elements that account for the specificity of RNA recognition are located in two separate regions of the protein. One is composed by residues 51 to 55 and loop 5, and the other region, located more than 11 A away in the structure, is the positively charged segment formed by residues 110 to 114.

  • characterization of pka values and titration shifts in the cytotoxic ribonuclease Alpha Sarcin by nmr relationship between electrostatic interactions structure and catalytic function
    Biochemistry, 1998
    Co-Authors: Jose Manuel Perezcanadillas, José G. Gavilanes, Javier Lacadena, Martínez Del Pozo, A., Ramon Camposolivas, Jorge Santoro, Manuel Rico, Marta Bruix
    Abstract:

    The electrostatic behavior of titrating groups in α-Sarcin was investigated using 1H NMR spectroscopy. A total of 209 chemical shift titration curves corresponding to different protons in the molecule were determined over the pH range of 3.0−8.5. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson−Hasselbalch equation led to the unambiguous determination of pKa values for all glutamic acid and histidine residues, as well as for the C-terminal carboxylate and most of the aspartic acids in the free enzyme. The ionization constants of catalytically relevant histidines, His50 and His137, and glutamic acid, Glu96, in the α-Sarcin−2‘-GMP complex were also determined. The pKa values of 15 ionizable groups (C-carboxylate, six aspartic acids, four glutamic acids, and four histidines) were found to be close to their normal values. On the other hand, a number of side chain groups, including those in the active center, showed pKa values far from their intrinsic values. Thus, th...

  • oligomerization of the cytotoxin Alpha Sarcin associated with phospholipid membranes
    Molecular Membrane Biology, 1998
    Co-Authors: Mercedes Oñaderra, Javier Lacadena, J.m. Mancheño, Martínez Del Pozo, A., De Los Rios, José G. Gavilanes
    Abstract:

    Alpha-Sarcin is a cytotoxic protein that specifically inactivates ribosomes. The protein translocates across phospholipid membranes. Oligomerization of the protein occurs upon interaction with membranes. Chemically cross-linked protein oligomers have been obtained by treatment of protein-vesicle complexes with the membrane impermeant reagent bis-(sulfosuccinimidyl) suberate. These structures are only obtained in the presence of acidic lipid vesicles composed of either natural or synthetic phospholipids. Such oligomers are not produced in concentrated protein solutions in the absence of vesicles. The formation of the chemically stabilized oligomers is saturated at the same lipid to protein molar ratio as all the perturbations caused by Alpha-Sarcin on lipid vesicles. Results are discussed in terms of the involvement of oligomer formation on protein translocation across membranes.

  • secretion of recombinant pro and mature fungal Alpha Sarcin ribotoxin by the methylotrophic yeast pichia pastoris the lys arg motif is required for maturation
    Protein Expression and Purification, 1998
    Co-Authors: Antonio Martinezruiz, Javier Lacadena, Mercedes Oñaderra, J.m. Mancheño, Alvaro Martinez Del Pozo, Carlos Lopezotin, José G. Gavilanes
    Abstract:

    Abstract α-Sarcin is a ribosome-inactivating protein from the moldAspergillus giganteus.The methylotrophic yeastPichia pastorishas been transformed with two plasmids (pHILD2preαS and pHILS1preαS), which contain the complete α-Sarcin cDNA, including its original fungal leader peptide, under the control of yeast alcohol oxidase promoter. The second one is indeed fused to the signal sequence ofP. pastorisacid phosphatase. The transformed yeasts secreted both mature and pro-α-Sarcin. The presence of this pro-α-Sarcin in the yeast extracellular medium is due to an inefficient recognition of the pro-sequence by a putative Kex2p-like endopeptidase. A third plasmid accounting for a single mutation of the α-Sarcin leader peptide was designed to produce a more efficient Kex2p recognition motif. This approach resulted in the extracellular production of only the mature protein, suggesting the existence of a two-step mechanism for processing its leader peptide. This recombinant α-Sarcin is identical to the original fungal protein, according to activity and spectroscopic criteria. In addition, pro-α-Sarcin, which has been characterized for the first time, also exhibits ribonucleolytic activity as the mature protein does. Therefore, protection of the producing cells against this kind of ribotoxins may depend on an efficient recognition of the signal sequence followed by translocation of the nascent polypeptide to the endoplasmic reticulum.

J.m. Mancheño - One of the best experts on this subject based on the ideXlab platform.

  • a peptide of nine amino acid residues from Alpha Sarcin cytotoxin is a membrane perturbing structure
    Journal of Peptide Research, 2009
    Co-Authors: J.m. Mancheño, Mercedes Oñaderra, Alvaro Martinez Del Pozo, Juan Pablo Albar, José G. Gavilanes
    Abstract:

    A water-soluble synthetic peptide with only nine amino acid residues, comprising the 131-139 sequence region of the cytotoxic protein Alpha-Sarcin (secreted by the mold Aspergillus giganteus), interacts with large unilamellar vesicles composed of acid phospholipids. It promotes lipid mixing between bilayers and leakage of vesicle aqueous contents, and it also abolishes the phospholipid phase transition. Other larger peptides containing such an amino acid sequence also produce these effects. These peptides acquire Alpha-helical conformation in the presence of trifluoroethanol, but display beta-strand conformation in the presence of sodium dodecyl sulfate. The interaction of these peptides with the lipid vesicles also results in beta-structure. The obtained data are discussed in terms of the involvement of the 131-139 stretch of Alpha-Sarcin in its interaction with lipid membranes.

  • assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin Alpha Sarcin
    Proteins, 2000
    Co-Authors: C De Antonio, Javier Lacadena, Mercedes Oñaderra, J.m. Mancheño, Marta Bruix, Antonio Martinezruiz, Martinez A Del Pozo, Jose Manuel Perezcanadillas, José G. Gavilanes
    Abstract:

    α-Sarcin, a potent cytotoxic protein from Aspergillus giganteus, contains two tryptophan residues at positions 4 and 51. Two single, W4F and W51F, and the double mutant, W4/51F, have been produced and purified to homogeneity. These two residues are neither required for the highly specific ribonucleolytic activity of the protein on the ribosomes (production of the so called α-fragment) nor for its interaction with lipid membranes (aggregation and fusion of vesicles), although the mutant forms involving Trp-51 show a decreased ribonuclease activity. Proton NMR data reveal that no significant changes in the global structure of the enzyme occur upon replacement of Trp-51 by Phe. Substitution of each Trp residue results in a 4 °C drop in the thermal denaturation midpoint, and the double mutant's midpoint is 9°C lower. Trp-51 is responsible for most of the near-UV circular dichroism of the protein and also contributes to the overall ellipticity of the protein in the peptide bond region. Trp-51 does not show fluorescence emission. The membrane-bound proteins undergo a thermal denaturation at a lower temperature than the corresponding free forms. The interaction of the protein with phospholipid bilayers promotes a large increase of the quantum yield of Trp-51 and its fluorescence emission is quenched by anthracene incorporated into the hydrophobic region of such bilayers. This indicates that the region around this residue is located in the hydrophobic core of the bilayer following protein–vesicle interaction. Proteins 2000;41:350–361. © 2000 Wiley-Liss, Inc.

  • oligomerization of the cytotoxin Alpha Sarcin associated with phospholipid membranes
    Molecular Membrane Biology, 1998
    Co-Authors: Mercedes Oñaderra, Javier Lacadena, J.m. Mancheño, Martínez Del Pozo, A., De Los Rios, José G. Gavilanes
    Abstract:

    Alpha-Sarcin is a cytotoxic protein that specifically inactivates ribosomes. The protein translocates across phospholipid membranes. Oligomerization of the protein occurs upon interaction with membranes. Chemically cross-linked protein oligomers have been obtained by treatment of protein-vesicle complexes with the membrane impermeant reagent bis-(sulfosuccinimidyl) suberate. These structures are only obtained in the presence of acidic lipid vesicles composed of either natural or synthetic phospholipids. Such oligomers are not produced in concentrated protein solutions in the absence of vesicles. The formation of the chemically stabilized oligomers is saturated at the same lipid to protein molar ratio as all the perturbations caused by Alpha-Sarcin on lipid vesicles. Results are discussed in terms of the involvement of oligomer formation on protein translocation across membranes.

  • secretion of recombinant pro and mature fungal Alpha Sarcin ribotoxin by the methylotrophic yeast pichia pastoris the lys arg motif is required for maturation
    Protein Expression and Purification, 1998
    Co-Authors: Antonio Martinezruiz, Javier Lacadena, Mercedes Oñaderra, J.m. Mancheño, Alvaro Martinez Del Pozo, Carlos Lopezotin, José G. Gavilanes
    Abstract:

    Abstract α-Sarcin is a ribosome-inactivating protein from the moldAspergillus giganteus.The methylotrophic yeastPichia pastorishas been transformed with two plasmids (pHILD2preαS and pHILS1preαS), which contain the complete α-Sarcin cDNA, including its original fungal leader peptide, under the control of yeast alcohol oxidase promoter. The second one is indeed fused to the signal sequence ofP. pastorisacid phosphatase. The transformed yeasts secreted both mature and pro-α-Sarcin. The presence of this pro-α-Sarcin in the yeast extracellular medium is due to an inefficient recognition of the pro-sequence by a putative Kex2p-like endopeptidase. A third plasmid accounting for a single mutation of the α-Sarcin leader peptide was designed to produce a more efficient Kex2p recognition motif. This approach resulted in the extracellular production of only the mature protein, suggesting the existence of a two-step mechanism for processing its leader peptide. This recombinant α-Sarcin is identical to the original fungal protein, according to activity and spectroscopic criteria. In addition, pro-α-Sarcin, which has been characterized for the first time, also exhibits ribonucleolytic activity as the mature protein does. Therefore, protection of the producing cells against this kind of ribotoxins may depend on an efficient recognition of the signal sequence followed by translocation of the nascent polypeptide to the endoplasmic reticulum.

  • substitution of histidine 137 by glutamine abolishes the catalytic activity of the ribosome inactivating protein Alpha Sarcin
    Biochemical Journal, 1995
    Co-Authors: Javier Lacadena, Mercedes Oñaderra, J.m. Mancheño, María Gasset, Antonio Martinezruiz, Martinez A Del Pozo, José G. Gavilanes
    Abstract:

    The Alpha-Sarcin cytotoxin is an extracellular fungal protein that inhibits protein biosynthesis by specifically cleaving one phosphodiester bond of the 28 S rRNA. The His137 residue of Alpha-Sarcin is suggested to be involved in the catalytic activity of this protein, based on the observed sequence similarity with some fungal ribonucleases. Replacement of this residue by Gln (H137Q mutant variant of Alpha-Sarcin) abolishes the ribonuclease activity of the protein. This has been demonstrated for an homogeneous preparation of the H137Q Alpha-Sarcin by measuring its effect against both intact rabbit ribosomes and the homopolymer poly(A). The conformation of H137Q Alpha-Sarcin is highly similar to that of the wild-type protein, which has been analysed by CD and fluorescence spectroscopy. Both H137Q and wild-type Alpha-Sarcin exhibit identical CD spectra in the peptide-bond region, indicating that no changes at the level of the secondary structure are produced upon mutation. Only minor differences are observed in both near-UV CD and fluorescence emission spectra in comparison to those of the wild-type protein. Moreover, H137Q Alpha-Sarcin interacts with phospholipid vesicles, promoting the same effects as the native cytotoxin. Therefore, we propose that His137 is part of the ribonucleolytic active site of the cytotoxin Alpha-Sarcin.

Mercedes Oñaderra - One of the best experts on this subject based on the ideXlab platform.

  • a peptide of nine amino acid residues from Alpha Sarcin cytotoxin is a membrane perturbing structure
    Journal of Peptide Research, 2009
    Co-Authors: J.m. Mancheño, Mercedes Oñaderra, Alvaro Martinez Del Pozo, Juan Pablo Albar, José G. Gavilanes
    Abstract:

    A water-soluble synthetic peptide with only nine amino acid residues, comprising the 131-139 sequence region of the cytotoxic protein Alpha-Sarcin (secreted by the mold Aspergillus giganteus), interacts with large unilamellar vesicles composed of acid phospholipids. It promotes lipid mixing between bilayers and leakage of vesicle aqueous contents, and it also abolishes the phospholipid phase transition. Other larger peptides containing such an amino acid sequence also produce these effects. These peptides acquire Alpha-helical conformation in the presence of trifluoroethanol, but display beta-strand conformation in the presence of sodium dodecyl sulfate. The interaction of these peptides with the lipid vesicles also results in beta-structure. The obtained data are discussed in terms of the involvement of the 131-139 stretch of Alpha-Sarcin in its interaction with lipid membranes.

  • conserved asparagine residue 54 of α Sarcin plays a role in protein stability and enzyme activity
    Biological Chemistry, 2004
    Co-Authors: Ansgar Siemer, Mercedes Oñaderra, Manuel Masip, Nelson Carreras, Lucia Garciaortega, Marta Bruix, Alvaro Martinez Del Pozo, José G. Gavilanes
    Abstract:

    Asparagine 54 of Alpha-Sarcin is a conserved residue within the proteins of the ribotoxin family of microbial ribonucleases. It is located in loop 2 of the protein, which lacks repetitive secondary structure elements but exhibits a well-defined conformation. Five mutant variants at this residue have been produced and characterized. The spectroscopic characterization of these proteins indicates that the overall conformation is not changed upon mutation. Activity and denaturation assays show that Asn-54 largely contributes to protein stability, and its presence is a requirement for the highly specific inhibitory activity of these ribotoxins on ribosomes.

  • assignment of the contribution of the tryptophan residues to the spectroscopic and functional properties of the ribotoxin Alpha Sarcin
    Proteins, 2000
    Co-Authors: C De Antonio, Javier Lacadena, Mercedes Oñaderra, J.m. Mancheño, Marta Bruix, Antonio Martinezruiz, Martinez A Del Pozo, Jose Manuel Perezcanadillas, José G. Gavilanes
    Abstract:

    α-Sarcin, a potent cytotoxic protein from Aspergillus giganteus, contains two tryptophan residues at positions 4 and 51. Two single, W4F and W51F, and the double mutant, W4/51F, have been produced and purified to homogeneity. These two residues are neither required for the highly specific ribonucleolytic activity of the protein on the ribosomes (production of the so called α-fragment) nor for its interaction with lipid membranes (aggregation and fusion of vesicles), although the mutant forms involving Trp-51 show a decreased ribonuclease activity. Proton NMR data reveal that no significant changes in the global structure of the enzyme occur upon replacement of Trp-51 by Phe. Substitution of each Trp residue results in a 4 °C drop in the thermal denaturation midpoint, and the double mutant's midpoint is 9°C lower. Trp-51 is responsible for most of the near-UV circular dichroism of the protein and also contributes to the overall ellipticity of the protein in the peptide bond region. Trp-51 does not show fluorescence emission. The membrane-bound proteins undergo a thermal denaturation at a lower temperature than the corresponding free forms. The interaction of the protein with phospholipid bilayers promotes a large increase of the quantum yield of Trp-51 and its fluorescence emission is quenched by anthracene incorporated into the hydrophobic region of such bilayers. This indicates that the region around this residue is located in the hydrophobic core of the bilayer following protein–vesicle interaction. Proteins 2000;41:350–361. © 2000 Wiley-Liss, Inc.

  • oligomerization of the cytotoxin Alpha Sarcin associated with phospholipid membranes
    Molecular Membrane Biology, 1998
    Co-Authors: Mercedes Oñaderra, Javier Lacadena, J.m. Mancheño, Martínez Del Pozo, A., De Los Rios, José G. Gavilanes
    Abstract:

    Alpha-Sarcin is a cytotoxic protein that specifically inactivates ribosomes. The protein translocates across phospholipid membranes. Oligomerization of the protein occurs upon interaction with membranes. Chemically cross-linked protein oligomers have been obtained by treatment of protein-vesicle complexes with the membrane impermeant reagent bis-(sulfosuccinimidyl) suberate. These structures are only obtained in the presence of acidic lipid vesicles composed of either natural or synthetic phospholipids. Such oligomers are not produced in concentrated protein solutions in the absence of vesicles. The formation of the chemically stabilized oligomers is saturated at the same lipid to protein molar ratio as all the perturbations caused by Alpha-Sarcin on lipid vesicles. Results are discussed in terms of the involvement of oligomer formation on protein translocation across membranes.

  • secretion of recombinant pro and mature fungal Alpha Sarcin ribotoxin by the methylotrophic yeast pichia pastoris the lys arg motif is required for maturation
    Protein Expression and Purification, 1998
    Co-Authors: Antonio Martinezruiz, Javier Lacadena, Mercedes Oñaderra, J.m. Mancheño, Alvaro Martinez Del Pozo, Carlos Lopezotin, José G. Gavilanes
    Abstract:

    Abstract α-Sarcin is a ribosome-inactivating protein from the moldAspergillus giganteus.The methylotrophic yeastPichia pastorishas been transformed with two plasmids (pHILD2preαS and pHILS1preαS), which contain the complete α-Sarcin cDNA, including its original fungal leader peptide, under the control of yeast alcohol oxidase promoter. The second one is indeed fused to the signal sequence ofP. pastorisacid phosphatase. The transformed yeasts secreted both mature and pro-α-Sarcin. The presence of this pro-α-Sarcin in the yeast extracellular medium is due to an inefficient recognition of the pro-sequence by a putative Kex2p-like endopeptidase. A third plasmid accounting for a single mutation of the α-Sarcin leader peptide was designed to produce a more efficient Kex2p recognition motif. This approach resulted in the extracellular production of only the mature protein, suggesting the existence of a two-step mechanism for processing its leader peptide. This recombinant α-Sarcin is identical to the original fungal protein, according to activity and spectroscopic criteria. In addition, pro-α-Sarcin, which has been characterized for the first time, also exhibits ribonucleolytic activity as the mature protein does. Therefore, protection of the producing cells against this kind of ribotoxins may depend on an efficient recognition of the signal sequence followed by translocation of the nascent polypeptide to the endoplasmic reticulum.

Ira G Wool - One of the best experts on this subject based on the ideXlab platform.

  • determination of the 28 s ribosomal rna identity element g4319 for Alpha Sarcin and the relationship of recognition to the selection of the catalytic site
    Journal of Molecular Biology, 1996
    Co-Authors: Anton Gluck, Ira G Wool
    Abstract:

    Ricin A-chin and Alpha-Sarcin are ribotoxins that inactivate eukaryotic ribosomes by modifying 28 S rRNA; ricin A-chain is an RNA N-glycosidase that depurinates the adenosine at position 4324 and Alpha-Sarcin is a ribonuclease that cleaves the phosphodiester bond on the 3' side of the adjacent guanosine (at position 4325). In cartoons of the secondary structure these two residues are seen to be embedded in a 17 base single-stranded loop over a seven base-pair helix. However, NMR spectroscopy of an oligoribonucleotide, a 29-mer that mimics the Sarcin/ricin domain, indicates that the RNA has a compact conformation in which the guanosine at the position analogous to 4319 in 28 S rRNA is bulged out of what otherwise is an extended A-form helix. Since similar structural irregularities are used by proteins to bind to RNA, we have tested the effect of mutations of the bulged guanosine on recognition and covalent modification of the RNA by ricin A-chain and by Alpha-sacrin. For the test a synthetic oligoribonucletide, a 35-mer, was used; the mutations were the deletion, the transition to adenosine, and the transversion to cytidine and uridine of the guanosine that is the analog of G4319. Each of the four mutations abolished cleavage og the RNA by Alpha-sacrin, where depurination by ricin A-chain was little affected. Thus G4319 is an identity element for Alpha-sacrin recognition. Analysis of the effect of Alpha-sacrin on variant oligoribonucleotides in which additional bases were inserted between the identity element guanosine and the site of catalysis suggest that on binding to the RNA the toxin uses the guanosine for orientation and then cleaves at a fixed distance and at a fixed position in space.

  • the ribosomal rna identity elements for ricin and for α Sarcin mutations in the putative cg pair that closes a gaga tetraloop
    Nucleic Acids Research, 1994
    Co-Authors: Anton Gluck, Yaeta Endo, Ira G Wool
    Abstract:

    Alpha-Sarcin is a ribonuclease that cleaves the phosphodiester bond on the 3' side of G4325 in 28S rRNA; ricin A-chain is a RNA N-glycosidase that depurinates the 5' adjacent A4324. These single covalent modifications inactivate the ribosome. An oligoribonucleotide that reproduces the structure of the Sarcin/ricin domain in 28S rRNA was synthesized and mutations were constructed in the 5' C and the 3' G that surround a GAGA tetrad that has the sites of toxin action. Covalent modification of the RNA by ricin, but not by Alpha-Sarcin, requires a Watson-Crick pair to shut off a putative GAGA tetraloop. Either the recognition elements for the two toxins are different despite their catalyzing covalent modification of adjacent nucleotides in 28S rRNA or there are transitions in the conformation of the Alpha-Sarcin/ricin domain in 28S rRNA and one conformer is recognized by Alpha-Sarcin and the other by ricin A-chain.

  • ribosomal rna identity elements for ricin a chain recognition and catalysis analysis with tetraloop mutants
    Journal of Molecular Biology, 1992
    Co-Authors: Anton Gluck, Yaeta Endo, Ira G Wool
    Abstract:

    Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose of the adenosine at position 4324 in eukaryotic 28 S rRNA. Ricin A-chain will also catalyze depurination in naked prokaryotic 16 S rRNA; the adenosine is at position 1014 in a GAGA tetraloop. The rRNA identity elements for recognition by ricin A-chain and for the catalysis of cleavage were examined using synthetic GAGA tetraloop oligoribonucleotides. The RNA designated wild-type, an oligoribonucleotide (19-mer) that approximates the structure of the ricin-sensitive site in 16 S rRNA, and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. With the wild-type tetraloop oligoribonucleotide the ricin A-chain-catalyzed reaction has a Km of 5.7 microM and a Kcat of 0.01 min-1. The toxin Alpha-Sarcin, which cleaves the phosphodiester bond on the 3' side of G4325 in 28 S rRNA, does not recognize the tetraloop RNA, although Alpha-Sarcin does affect a larger synthetic oligoribonucleotide that has a 17-nucleotide loop with a GAGA sequence; thus, there is a clear divergence in the identity elements for the two toxins. Mutants were constructed with all of the possible transitions and transversions of each nucleotide in the GAGA tetraloop; none was recognized by ricin A-chain. Thus, there is an absolute requirement for the integrity of the GAGA sequence in the tetraloop. The helical stem of the tetraloop oligoribonucleotide can be reduced to three base-pairs, indeed, to two base-pairs if the temperature is decreased, without affecting recognition; the nature of these base-pairs does not influence recognition or catalysis by ricin A-chain. If the tetraloop is opened so as to form a GAGA-containing hexaloop, recognition by ricin A-chain is lost. This suggests that during the elongation cycle, a GAGA tetraloop either exists or is formed in the putative 17-member single-stranded region of the ricin domain in 28 S rRNA and this bears on the mechanism of protein synthesis.

  • ribosomal rna identity elements for ricin a chain recognition and catalysis
    Journal of Molecular Biology, 1991
    Co-Authors: Y Endo, Anton Gluck, Ira G Wool
    Abstract:

    Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose at position A4324 in eukaryotic 28 S rRNA. The requirements for the recognition by ricin A-chain of this nucleotide and for the catalysis of cleavage were examined using a synthetic oligoribonucleotide that reproduces the sequence and the secondary structure of the RNA domain (a helical stem, a bulged nucleotide, and a 17-member single-stranded loop). The wild-type RNA (35mer) and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase. With the wild-type oligoribonucleotide the ricin A-chain catalyzed reaction has a Km of 13.55 microM and a Kcat of 0.023 min-1. Recognition and catalysis by ricin A-chain has an absolute requirement for A at the position that corresponds to 4324. The helical stem is also essential; however, the number of base-pairs can be reduced from the seven found in 28 S rRNA to three without loss of identity. The nature of these base-pairs can affect catalysis. A change of the second set from one canonical (G.C) to another (U.A) reduces sensitivity to ricin A-chain; whereas, a change of the third pair (U.A----G.C) produces supersensitivity. The bulged nucleotide does not contribute to identification. Hydrolysis is affected by altering the nucleotides in the universal sequence surrounding A4324 or by changing the position in the loop of the tetranucleotide GA(ricin)GA: all of these mutants have a null phenotype. If ribosomes are treated first with Alpha-Sarcin to cleave the phosphodiester bond at G4325 ricin can still catalyze depurination at A4324. This implies that cleavage by Alpha-Sarcin at the center of what has been presumed to be a 17 nucleotide single-stranded loop in 28 S rRNA produces ends that are constrained in some way. On the other hand, hydrolysis by Alpha-Sarcin of the corresponding position in the synthetic oligoribonucleotide prevents recognition by ricin A-chain. The results suggest that the loop has a complex structure, affected by ribosomal proteins, and this bears on the function in protein synthesis of the Alpha-Sarcin/ricin rRNA domain.