The Experts below are selected from a list of 204 Experts worldwide ranked by ideXlab platform
John R. Silvius - One of the best experts on this subject based on the ideXlab platform.
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Mutational and biochemical analysis of plasma membrane targeting mediated by the farnesylated, polybasic Carboxy terminus of K-ras4B.
Biochemistry, 2000Co-Authors: Marie-odile Roy, Rania Leventis, John R. SilviusAbstract:Mutational analysis and in vitro assays of membrane association have been combined to investigate the mechanism of plasma membrane targeting mediated by the farnesylated, polybasic Carboxy-Terminal Sequence of K-ras4B in mammalian cells. Fluorescence-microscopic localization of chimeric proteins linking the enhanced green fluorescent protein (EGFP) to the K-ras4B Carboxy-Terminal Sequence, or to variant forms of this Sequence, reveals that the normal structure of this targeting motif can be greatly altered without compromising plasma membrane-targeting activity so long as an overall strongly polybasic/amphiphilic character is retained. An EGFP/K-ras4B(171-188) chimeric protein was readily abstracted from isolated cell membranes by negatively charged lipid vesicles, and this abstraction was markedly enhanced by the anionic lipid-binding agent neomycin. Our results strongly favor a mechanism in which at the plasma membrane the Carboxy-Terminal Sequence of K-ras4B associates not with a classical specific proteinaceous receptor but rather with nonspecific but highly anionic 'sites' formed at least in part by the membrane lipid bilayer. Our findings also suggest that the recently demonstrated prenylation-dependent trafficking of immature forms of K-ras4B through the endoplasmic reticulum [Choy et al. (1999) Cell 98, 69-80], while required for maturation of the protein, beyond this stage may not be essential to allow the ultimate delivery of the mature protein to the plasma membrane.
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Lipid-binding characteristics of the polybasic Carboxy-Terminal Sequence of K-ras4B.
Biochemistry, 1998Co-Authors: Rania Leventis, John R. SilviusAbstract:We have examined the association with lipid vesicles of fluorescent lipidated peptides based on the farnesylated, polybasic Carboxy-Terminal region of mature K-ras4B, which functions physiologically as an autonomous plasma membrane-targeting motif. While the peptides bind to neutral lipid (phosphatidylcholine/phosphatidylethanolamine) vesicles with relatively low affinity, the vesicle-binding affinity increases exponentially as increasing amounts of anionic lipids are incorporated into the vesicle bilayers. Competitive vesicle-binding experiments reveal that the K-ras4B Carboxy-Terminal Sequence accordingly discriminates strongly between lipid surfaces of differing surface charge, such that two lipid bilayers differing in anionic lipid content by 10 mol % will show a 45-fold preferential accumulation of the lipidated peptide in the more negatively charged surface. At the same time, the Carboxyl-Terminal region of K-ras4B exhibits no preferential binding to particular anionic lipids, including the polyanionic species phosphatidylinositol-4'-phosphate and phosphatidylinositol-4',5'-bisphosphate, beyond that predicted on the basis of surface-charge effects. The K-ras4B Carboxyl-Terminal Sequence dissociates rapidly (with half-times of seconds or less) from lipid bilayers containing up to 40 mol % anionic lipid. These results suggest that the targeting of the mature K-ras4B Carboxy-terminus to the plasma membrane, if it is based on interactions with plasma membrane lipids, is not mediated by a kinetic-trapping mechanism or by specific binding to particular anionic lipids but may rest on the sensitive surface potential-sensing function of this region of the protein.
Hannu Myllykallio - One of the best experts on this subject based on the ideXlab platform.
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Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication
Nucleic Acids Research, 2018Co-Authors: Sonoko Ishino, Stéphane Skouloubris, Hanae Kudo, Caroline L’hermitte-stead, Asmae Es-sadik, Jean-christophe Lambry, Yoshizumi Ishino, Hannu MyllykallioAbstract:The mismatch repair (MMR) system, exemplified by the MutS/MutL proteins, is widespread in Bacteria and Eukarya. However, molecular mechanisms how numerous archaea and bacteria lacking the mutS/mutL genes maintain high replication fidelity and genome stability have remained elusive. EndoMS is a recently discovered hyperthermophilic mismatch-specific endonuclease encoded by nucS in Thermococcales. We deleted the nucS from the actinobacterium Corynebacterium glutamicum and demonstrated a drastic increase of spontaneous transition mutations in the nucS deletion strain. The observed spectra of these mutations were consistent with the enzymatic properties of EndoMS in vitro. The robust mismatch-specific endonuclease activity was detected with the purified C. glutamicum EndoMS protein but only in the presence of the β-clamp (DnaN). Our biochemical and genetic data suggest that the frequently occurring G/T mismatch is efficiently repaired by the bacterial EndoMS-β−clamp complex formed via a Carboxy-Terminal Sequence motif of EndoMS proteins. Our study thus has great implications for understanding how the activity of the novel MMR system is coordinated with the replisome and provides new mechanistic insight into genetic diversity and mutational patterns in industrially and clinically (e.g. Mycobacteria) important archaeal and bacterial phyla previously thought to be devoid of the MMR system.
Sonoko Ishino - One of the best experts on this subject based on the ideXlab platform.
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Activation of the mismatch-specific endonuclease EndoMS/NucS by the replication clamp is required for high fidelity DNA replication
Nucleic Acids Research, 2018Co-Authors: Sonoko Ishino, Stéphane Skouloubris, Hanae Kudo, Caroline L’hermitte-stead, Asmae Es-sadik, Jean-christophe Lambry, Yoshizumi Ishino, Hannu MyllykallioAbstract:The mismatch repair (MMR) system, exemplified by the MutS/MutL proteins, is widespread in Bacteria and Eukarya. However, molecular mechanisms how numerous archaea and bacteria lacking the mutS/mutL genes maintain high replication fidelity and genome stability have remained elusive. EndoMS is a recently discovered hyperthermophilic mismatch-specific endonuclease encoded by nucS in Thermococcales. We deleted the nucS from the actinobacterium Corynebacterium glutamicum and demonstrated a drastic increase of spontaneous transition mutations in the nucS deletion strain. The observed spectra of these mutations were consistent with the enzymatic properties of EndoMS in vitro. The robust mismatch-specific endonuclease activity was detected with the purified C. glutamicum EndoMS protein but only in the presence of the β-clamp (DnaN). Our biochemical and genetic data suggest that the frequently occurring G/T mismatch is efficiently repaired by the bacterial EndoMS-β−clamp complex formed via a Carboxy-Terminal Sequence motif of EndoMS proteins. Our study thus has great implications for understanding how the activity of the novel MMR system is coordinated with the replisome and provides new mechanistic insight into genetic diversity and mutational patterns in industrially and clinically (e.g. Mycobacteria) important archaeal and bacterial phyla previously thought to be devoid of the MMR system.
Rania Leventis - One of the best experts on this subject based on the ideXlab platform.
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Mutational and biochemical analysis of plasma membrane targeting mediated by the farnesylated, polybasic Carboxy terminus of K-ras4B.
Biochemistry, 2000Co-Authors: Marie-odile Roy, Rania Leventis, John R. SilviusAbstract:Mutational analysis and in vitro assays of membrane association have been combined to investigate the mechanism of plasma membrane targeting mediated by the farnesylated, polybasic Carboxy-Terminal Sequence of K-ras4B in mammalian cells. Fluorescence-microscopic localization of chimeric proteins linking the enhanced green fluorescent protein (EGFP) to the K-ras4B Carboxy-Terminal Sequence, or to variant forms of this Sequence, reveals that the normal structure of this targeting motif can be greatly altered without compromising plasma membrane-targeting activity so long as an overall strongly polybasic/amphiphilic character is retained. An EGFP/K-ras4B(171-188) chimeric protein was readily abstracted from isolated cell membranes by negatively charged lipid vesicles, and this abstraction was markedly enhanced by the anionic lipid-binding agent neomycin. Our results strongly favor a mechanism in which at the plasma membrane the Carboxy-Terminal Sequence of K-ras4B associates not with a classical specific proteinaceous receptor but rather with nonspecific but highly anionic 'sites' formed at least in part by the membrane lipid bilayer. Our findings also suggest that the recently demonstrated prenylation-dependent trafficking of immature forms of K-ras4B through the endoplasmic reticulum [Choy et al. (1999) Cell 98, 69-80], while required for maturation of the protein, beyond this stage may not be essential to allow the ultimate delivery of the mature protein to the plasma membrane.
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Lipid-binding characteristics of the polybasic Carboxy-Terminal Sequence of K-ras4B.
Biochemistry, 1998Co-Authors: Rania Leventis, John R. SilviusAbstract:We have examined the association with lipid vesicles of fluorescent lipidated peptides based on the farnesylated, polybasic Carboxy-Terminal region of mature K-ras4B, which functions physiologically as an autonomous plasma membrane-targeting motif. While the peptides bind to neutral lipid (phosphatidylcholine/phosphatidylethanolamine) vesicles with relatively low affinity, the vesicle-binding affinity increases exponentially as increasing amounts of anionic lipids are incorporated into the vesicle bilayers. Competitive vesicle-binding experiments reveal that the K-ras4B Carboxy-Terminal Sequence accordingly discriminates strongly between lipid surfaces of differing surface charge, such that two lipid bilayers differing in anionic lipid content by 10 mol % will show a 45-fold preferential accumulation of the lipidated peptide in the more negatively charged surface. At the same time, the Carboxyl-Terminal region of K-ras4B exhibits no preferential binding to particular anionic lipids, including the polyanionic species phosphatidylinositol-4'-phosphate and phosphatidylinositol-4',5'-bisphosphate, beyond that predicted on the basis of surface-charge effects. The K-ras4B Carboxyl-Terminal Sequence dissociates rapidly (with half-times of seconds or less) from lipid bilayers containing up to 40 mol % anionic lipid. These results suggest that the targeting of the mature K-ras4B Carboxy-terminus to the plasma membrane, if it is based on interactions with plasma membrane lipids, is not mediated by a kinetic-trapping mechanism or by specific binding to particular anionic lipids but may rest on the sensitive surface potential-sensing function of this region of the protein.
John E. Shively - One of the best experts on this subject based on the ideXlab platform.
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Automated Carboxy-Terminal Sequence analysis of polypeptides containing C-Terminal proline.
Analytical biochemistry, 1995Co-Authors: Jerome M. Bailey, G. Issai, John E. ShivelyAbstract:Abstract Proteins and peptides can be Sequenced from the Carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on automation of the thiocyanate chemistry using diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine to derivatize the C-Terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-Terminal amino acid (Bailey, J. M., Nikfarjam, F., Shenoy, N. S., and Shively, J. E. (1992) Protein Sci. 1, 1622-1633). A major limitation of this approach was the inability to derivatize C-Terminal proline. We now describe chemistry based on the DPP-ITC/pyridine reaction which is capable of derivatizing C-Terminal proline to a thiohydantoin. The reaction of DPP-ITC/pyridine with C-Terminal proline rapidly forms an acyl isothiocyanate which is capable of forming a quaternary amine containing thiohydantoin. Unlike formation of peptidylthiohydantoins with the other 19 commonly occurring amino acids in which cyclization to a thiohydantoin is concomitant with loss of a proton from the amide nitrogen, proline has no amide proton and as a result the newly formed proline thiohydantoin contains an unprotonated ring nitrogen. This cyclic structure if left unprotonated will regenerate C-Terminal proline during the cleavage reaction. However, if protonated by the addition of acid, the proline thiohydantoin ring is stabilized and can be readily hydrolyzed to proline thiohydantoin and a shortened peptide by the addition of water vapor or alternatively by sodium or potassium trimethylsilanolate, the reagent normally used for the cleavage reaction. By introducing vapor-phase trifluoroacetic acid (TFA) for the protonation reaction and water vapor for the hydrolysis reaction we have been able to automate the chemistry required for derivatization of C-Terminal proline. Since the TFA/water steps have no effect on peptidylthiohydantoins formed from the other 19 amino acids, the additional steps required for proline were readily integrated into the automated sequencing program, providing for the first time an automated sequencing program which permits the C-Terminal Sequence analysis of all 20 of the commonly occurring amino acids. Automated programs are described for the C-Terminal sequencing of peptides covalently attached to Carboxylic acid-modified polyethylene and larger polypeptides noncovalently applied to Zitex (porous Teflon).
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Automated Carboxy-Terminal Sequence analysis of peptides.
Protein science : a publication of the Protein Society, 1992Co-Authors: Jerome M. Bailey, Narmada R. Shenoy, Michael Ronk, John E. ShivelyAbstract:Proteins and peptides can be Sequenced from the Carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-Terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-Terminal amino acid, and the development of methodology to Sequence through the difficult amino acid, aspartate. Automated programs are described for the C-Terminal sequencing of peptides covalently attached to Carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-Terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-Terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to Sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C-Terminal Sequence analysis of peptides.
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Automated Carboxy-Terminal Sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate
Protein science : a publication of the Protein Society, 1992Co-Authors: Jerome M. Bailey, Narmada R. Shenoy, Firoozeh Nikfarjam, John E. ShivelyAbstract:Proteins and peptides can be Sequenced from the Carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on the automation of the thiocyanate chemistry using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) to derivatize the C-Terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-Terminal amino acid (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80). A major limitation of this approach was the need to activate the C-terminus with acetic anhydride. We now describe the use of a new reagent, diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine, which combines the activation and derivatization steps to produce peptidylthiohydantoins. Previous work by Kenner et al. (Kenner, G.W., Khorana, H.G., & Stedman, R.J., 1953, Chem. Soc. J., 673-678) with this reagent demonstrated slow kinetics. Several days were required for complete reaction. We show here that the inclusion of pyridine was found to promote the formation of C-Terminal thiohydantoins by DPP-ITC resulting in complete conversion of the C-Terminal amino acid to a thiohydantoin in less than 1 h. Reagents such as imidazole, triazine, and tetrazole were also found to promote the reaction with DPP-ITC as effectively as pyridine. General base catalysts, such as triethylamine, do not promote the reaction, but are required to convert the C-Terminal Carboxylic acid to a salt prior to the reaction with DPP-ITC and pyridine. By introducing the DPP-ITC reagent and pyridine in separate steps in an automated Sequencer, we observed improved sequencing yields for amino acids normally found difficult to derivatize with acetic anhydride/TMS-ITC. This was particularly true for aspartic acid, which now can be Sequenced in yields comparable to most of the other amino acids. Automated programs are described for the C-Terminal sequencing of peptides covalently attached to Carboxylic acid-modified polyethylene and proteins (200 pmol to 5 nmol) noncovalently applied to Zitex (porous Teflon). The generality of our automated C-Terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids tested were found to Sequence in good yield except for proline, which was found not to be capable of derivatization. In spite of this limitation, the methodology should be a valuable tool for the C-Terminal Sequence analysis of peptides and proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
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Amino acid Sequence of the blue copper protein rusticyanin from Thiobacillus ferrooxidans
Biochemistry, 1991Co-Authors: Michael Ronk, John E. Shively, Elizabeth A. Shute, Robert C. BlakeAbstract:Rusticyanin is a small blue copper protein isolated from Thiobacillus ferrooxidans. The amino acid Sequence of the rusticyanin has been determined by the structural characterization of tryptic and endoproteinase Asp-N peptides with use of amino Terminal microsequencing, fast atom bombardment mass spectrometry, and electrospray triple-quadrupole mass spectrometry techniques. Amino acid analysis, Carboxy-Terminal Sequence analysis, and circular dichroism spectroscopy were also performed on the protein. Amino acid Sequence identity among rusticyanin and six other small blue copper proteins is apparent only in the limited C-Terminal region of each protein bearing three of the four putative copper ligands. A structural model of the rusticyanin is proposed where the protein is principally a beta-barrel comprised of six strands. This model is consistent with the circular dichroism data and computational predictions of the secondary structure of rusticyanin. A feature of the model is the hypothesis that Asp 73 may serve as a fourth copper ligand.