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Franc Pattus - One of the best experts on this subject based on the ideXlab platform.
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The interaction between pyoverdin and its outer Membrane receptor in Pseudomonas aeruginosa leads to different conformers: a time-resolved fluorescence study.
Biochemistry, 2002Co-Authors: Nicolas Folschweiller, Mohamed A Abdallah, Franc Pattus, Jacques Gallay, Michel Vincent, Isabelle J SchalkAbstract:In iron limitation conditions, Pseudomonas aeruginosa secretes a major fluorescent siderophore named pyoverdin (PaA). PaA has an extremely high affinity for Fe(3+) but also chelates other ions such as Al(3+) and Ga(3+) with a lower affinity. The transfer of PaA-Fe(3+) across the outer Membrane of the bacteria is mediated by the receptor FpvA, a TonB-dependent outer Membrane Transport Protein. FpvA binds the iron-free and iron-loaded forms of pyoverdin with similar affinities, but only PaA-Fe(3+) is taken up by the cell, suggesting that FpvA adopts different conformations depending on its loading status. We used time-resolved fluorescence spectroscopy to characterize the different forms of FpvA-PaA in vitro. We showed that the FpvA-PaA complex adopts two different conformations depending on how it was prepared (formed in vitro or in vivo prior to purification). The dihydroquinoline moiety of both conformers is fully protonated, or coordinated by Protein charged groups, but the polarity of its environment, its solvent accessibility, and its rotational dynamics are much slower when the FpvA-PaA complex is formed in vivo than in vitro. In the presence of Ga(3+) or Al(3+) ions, the solvent accessibility and mobility of the dihydroquinoline moiety in the two FpvA-PaA complexes are intermediate between those observed for the metal-free ones. In addition, the F?er resonance energy transfer kinetics from FpvA tryptophan residues to the PaA chromophore differs from one complex to the other, revealing differences in one or more of the donor-acceptor topologies.
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Copurification of the FpvA ferric pyoverdin receptor of Pseudomonas aeruginosa with its iron-free ligand: implications for siderophore-mediated iron Transport.
Biochemistry, 1999Co-Authors: Isabelle J Schalk, P Kyslik, D Prome, A Van Dorsselaer, K Poole, Mohamed A Abdallah, Franc PattusAbstract:The Pseudomonas aeruginosa FpvA receptor is a TonB-dependent outer Membrane Transport Protein that catalyzes uptake of ferric pyoverdin across the outer Membrane. Surprisingly, FpvA expressed in P. aeruginosa grown in an iron-deficient medium copurifies with a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron-free pyoverdin (apo-PaA). PaA was absent from FpvA purified from a PaA-deficient P. aeruginosa strain. The properties of ligand binding in vitro revealed very similar affinities of apo-PaA and ferric-PaA to FpvA. Fluorescence resonance energy transfer was used to study in vitro the formation of the FpvA-PaA-Fe complex in the presence of PaA-Fe or citrate-Fe. The circular dichroism spectrum of FpvA indicated a 57% beta-structure content typical of porins and in agreement with the 3D structures of the siderophore receptors FhuA and FepA. In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus was purified. This truncated form still bound PaA, and its beta-sheet content was conserved. This N-terminal region displays significant homology to the N-terminal periplasmic extensions of FecA from Escherichia coli and PupB from Pseudomonas putida, which were previously shown to be involved in signal transduction. This suggests a similar function for FpvA. The mechanism of iron Transport in P. aeruginosa via the pyoverdin pathway is discussed in the light of all these new findings.
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copurification of the fpva ferric pyoverdin receptor of pseudomonas aeruginosa with its iron free ligand implications for siderophore mediated iron Transport
Biochemistry, 1999Co-Authors: Isabelle J Schalk, P Kyslik, D Prome, A Van Dorsselaer, K Poole, Mohamed A Abdallah, Franc PattusAbstract:The Pseudomonas aeruginosa FpvA receptor is a TonB-dependent outer Membrane Transport Protein that catalyzes uptake of ferric pyoverdin across the outer Membrane. Surprisingly, FpvA expressed in P. aeruginosa grown in an iron-deficient medium copurifies with a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron-free pyoverdin (apo-PaA). PaA was absent from FpvA purified from a PaA−deficient P. aeruginosa strain. The properties of ligand binding in vitro revealed very similar affinities of apo-PaA and ferric−PaA to FpvA. Fluorescence resonance energy transfer was used to study in vitro the formation of the FpvA−PaA−Fe complex in the presence of PaA−Fe or citrate−Fe. The circular dichroism spectrum of FpvA indicated a 57% β-structure content typical of porins and in agreement with the 3D structures of the siderophore receptors FhuA and FepA. In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus was purified. Th...
Isabelle J Schalk - One of the best experts on this subject based on the ideXlab platform.
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The interaction between pyoverdin and its outer Membrane receptor in Pseudomonas aeruginosa leads to different conformers: a time-resolved fluorescence study.
Biochemistry, 2002Co-Authors: Nicolas Folschweiller, Mohamed A Abdallah, Franc Pattus, Jacques Gallay, Michel Vincent, Isabelle J SchalkAbstract:In iron limitation conditions, Pseudomonas aeruginosa secretes a major fluorescent siderophore named pyoverdin (PaA). PaA has an extremely high affinity for Fe(3+) but also chelates other ions such as Al(3+) and Ga(3+) with a lower affinity. The transfer of PaA-Fe(3+) across the outer Membrane of the bacteria is mediated by the receptor FpvA, a TonB-dependent outer Membrane Transport Protein. FpvA binds the iron-free and iron-loaded forms of pyoverdin with similar affinities, but only PaA-Fe(3+) is taken up by the cell, suggesting that FpvA adopts different conformations depending on its loading status. We used time-resolved fluorescence spectroscopy to characterize the different forms of FpvA-PaA in vitro. We showed that the FpvA-PaA complex adopts two different conformations depending on how it was prepared (formed in vitro or in vivo prior to purification). The dihydroquinoline moiety of both conformers is fully protonated, or coordinated by Protein charged groups, but the polarity of its environment, its solvent accessibility, and its rotational dynamics are much slower when the FpvA-PaA complex is formed in vivo than in vitro. In the presence of Ga(3+) or Al(3+) ions, the solvent accessibility and mobility of the dihydroquinoline moiety in the two FpvA-PaA complexes are intermediate between those observed for the metal-free ones. In addition, the F?er resonance energy transfer kinetics from FpvA tryptophan residues to the PaA chromophore differs from one complex to the other, revealing differences in one or more of the donor-acceptor topologies.
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Copurification of the FpvA ferric pyoverdin receptor of Pseudomonas aeruginosa with its iron-free ligand: implications for siderophore-mediated iron Transport.
Biochemistry, 1999Co-Authors: Isabelle J Schalk, P Kyslik, D Prome, A Van Dorsselaer, K Poole, Mohamed A Abdallah, Franc PattusAbstract:The Pseudomonas aeruginosa FpvA receptor is a TonB-dependent outer Membrane Transport Protein that catalyzes uptake of ferric pyoverdin across the outer Membrane. Surprisingly, FpvA expressed in P. aeruginosa grown in an iron-deficient medium copurifies with a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron-free pyoverdin (apo-PaA). PaA was absent from FpvA purified from a PaA-deficient P. aeruginosa strain. The properties of ligand binding in vitro revealed very similar affinities of apo-PaA and ferric-PaA to FpvA. Fluorescence resonance energy transfer was used to study in vitro the formation of the FpvA-PaA-Fe complex in the presence of PaA-Fe or citrate-Fe. The circular dichroism spectrum of FpvA indicated a 57% beta-structure content typical of porins and in agreement with the 3D structures of the siderophore receptors FhuA and FepA. In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus was purified. This truncated form still bound PaA, and its beta-sheet content was conserved. This N-terminal region displays significant homology to the N-terminal periplasmic extensions of FecA from Escherichia coli and PupB from Pseudomonas putida, which were previously shown to be involved in signal transduction. This suggests a similar function for FpvA. The mechanism of iron Transport in P. aeruginosa via the pyoverdin pathway is discussed in the light of all these new findings.
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copurification of the fpva ferric pyoverdin receptor of pseudomonas aeruginosa with its iron free ligand implications for siderophore mediated iron Transport
Biochemistry, 1999Co-Authors: Isabelle J Schalk, P Kyslik, D Prome, A Van Dorsselaer, K Poole, Mohamed A Abdallah, Franc PattusAbstract:The Pseudomonas aeruginosa FpvA receptor is a TonB-dependent outer Membrane Transport Protein that catalyzes uptake of ferric pyoverdin across the outer Membrane. Surprisingly, FpvA expressed in P. aeruginosa grown in an iron-deficient medium copurifies with a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron-free pyoverdin (apo-PaA). PaA was absent from FpvA purified from a PaA−deficient P. aeruginosa strain. The properties of ligand binding in vitro revealed very similar affinities of apo-PaA and ferric−PaA to FpvA. Fluorescence resonance energy transfer was used to study in vitro the formation of the FpvA−PaA−Fe complex in the presence of PaA−Fe or citrate−Fe. The circular dichroism spectrum of FpvA indicated a 57% β-structure content typical of porins and in agreement with the 3D structures of the siderophore receptors FhuA and FepA. In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus was purified. Th...
Thomas J Silhavy - One of the best experts on this subject based on the ideXlab platform.
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contact dependent growth inhibition requires the essential outer Membrane Protein bama yaet as the receptor and the inner Membrane Transport Protein acrb
Molecular Microbiology, 2008Co-Authors: Stephanie K Aoki, Juliana C Malinverni, Kyle Jacoby, Benjamin Thomas, Rupinderjit Pamma, Brooke Trinh, Susan Remers, Julia S Webb, B A Braaten, Thomas J SilhavyAbstract:Summary Contact-dependent growth inhibition (CDI) is a phe- nomenon by which bacterial cell growth is regulated by direct cell-to-cell contact via the CdiA/CdiB two-partner secretion system. Characterization of mutants resistant to CDI allowed us to identify BamA (YaeT) as the outer Membrane receptor for CDI and AcrB as a potential downstream target. Notably, both BamA and AcrB are part of distinct multi-component machines. The Bam machine assembles outer mem- brane b-barrel Proteins into the outer Membrane and the Acr machine exports small molecules into the extracellular milieu. We discovered that a mutation that reduces expression of BamA decreased binding of CDI + inhibitor cells, measured by flow cytometry with fluorescently labelled bacteria. In addition, a-BamA antibodies, which recognized extracellular epitopes of BamA based on immunofluorescence, specifically blocked inhibitor-target cells binding and CDI. A second class of CDI-resistant mutants identi- fied carried null mutations in the acrB gene. AcrB is an inner Membrane component of a multidrug efflux pump that normally forms a cell envelope-spanning complex with the Membrane fusion Protein AcrA and the outer Membrane Protein TolC. Strikingly, the requirement for the BamA and AcrB Proteins in CDI is independent of their multi-component machines, and thus their role in the CDI pathway may reflect novel, import-related functions.
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contact dependent growth inhibition requires the essential outer Membrane Protein bama yaet as the receptor and the inner Membrane Transport Protein acrb
Molecular Microbiology, 2008Co-Authors: Stephanie K Aoki, Juliana C Malinverni, Kyle Jacoby, Benjamin Thomas, Rupinderjit Pamma, Brooke Trinh, Susan Remers, Julia S Webb, B A Braaten, Thomas J SilhavyAbstract:Contact-dependent growth inhibition (CDI) is a phenomenon by which bacterial cell growth is regulated by direct cell-to-cell contact via the CdiA/CdiB two-partner secretion system. Characterization of mutants resistant to CDI allowed us to identify BamA (YaeT) as the outer Membrane receptor for CDI and AcrB as a potential downstream target. Notably, both BamA and AcrB are part of distinct multi-component machines. The Bam machine assembles outer Membrane beta-barrel Proteins into the outer Membrane and the Acr machine exports small molecules into the extracellular milieu. We discovered that a mutation that reduces expression of BamA decreased binding of CDI+ inhibitor cells, measured by flow cytometry with fluorescently labelled bacteria. In addition, alpha-BamA antibodies, which recognized extracellular epitopes of BamA based on immunofluorescence, specifically blocked inhibitor-target cells binding and CDI. A second class of CDI-resistant mutants identified carried null mutations in the acrB gene. AcrB is an inner Membrane component of a multidrug efflux pump that normally forms a cell envelope-spanning complex with the Membrane fusion Protein AcrA and the outer Membrane Protein TolC. Strikingly, the requirement for the BamA and AcrB Proteins in CDI is independent of their multi-component machines, and thus their role in the CDI pathway may reflect novel, import-related functions.
H. Ronald Kaback - One of the best experts on this subject based on the ideXlab platform.
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Correction for Guan et al., Manipulating phospholipids for crystallization of a Membrane Transport Protein
Proceedings of the National Academy of Sciences, 2006Co-Authors: Lan Guan, Irina N. Smirnova, Gill Verner, Shushi Nagamori, H. Ronald KabackAbstract:biochemistry. For the article “Manipulating phospholipids for crystallization of a Membrane Transport Protein,” by Lan Guan, Irina N. Smirnova, Gill Verner, Shushi Nagamoni, and H. Ronald Kaback, which appeared in issue 6, February 7, 2006, of Proc. Natl. Acad. Sci. USA (103, 1723#x2013;1726; first published January 30, 2006; 10.1073/pnas.0510922103), the author name Shushi Nagamoni should have appeared as Shushi Nagamori. The corrected author line appears below. The online version has been corrected.
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Manipulating phospholipids for crystallization of a Membrane Transport Protein
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Lan Guan, Irina N. Smirnova, Gill Verner, Shushi Nagamori, H. Ronald KabackAbstract:Crystallization is a major bottleneck to obtaining x-ray structures of Membrane Proteins. By applying an established crystallization protocol for the lactose permease (LacY) of Escherichia coli, a systematic study of the effect of phospholipids (PL) on crystallization of LacY was undertaken. We observe three different crystal forms that diffract to increasingly better resolution in a manner that correlates with the concentration of copurified PL. Consistently, progressive addition of E. coli PL to delipidated LacY leads to different crystal forms. Tetragonal crystals are obtained with improved diffraction quality for a stable mutant by carefully adjusting PL content. Furthermore, crystals of good quality from wild-type LacY, a particularly difficult Protein, were also obtained by using same approach. Thus, it is likely that manipulation of PL is a good strategy for predominantly hydrophobic Membrane Proteins like LacY.
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Elucidation of substrate binding interactions in a Membrane Transport Protein by mass spectrometry
The EMBO journal, 2003Co-Authors: Adam B. Weinglass, Julian P. Whitelegge, G. Verner, Kym F. Faull, H. Ronald KabackAbstract:Integration of biochemical and biophysical data on the lactose permease of Escherichia coli has culminated in a molecular model that predicts substrate-Protein proximities which include interaction of a hydroxyl group in the galactopyranosyl ring with Glu269. In order to test this hypothesis, we studied covalent modification of carboxyl groups with carbodiimides using electrospray ionization mass spectrometry (ESI-MS) and demonstrate that substrate protects the permease against carbodiimide reactivity. Further more, a significant proportion of the decrease in carbodiimide reactivity occurs specifically in a nanopeptide containing Glu269. In contrast, carbodiimide reactivity of mutant Glu269-->Asp that exhibits lower affinity is unaffected by substrate. By monitoring the ability of different substrate analogs to protect against carbodiimide modification of Glu269, it is suggested that the C-3 OH group of the galactopyranosyl ring may play an important role in specificity, possibly by H-bonding with Glu269. The approach demonstrates that mass spectrometry can provide a powerful means of analyzing ligand interactions with integral Membrane Proteins.
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Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators
Journal of Biological Chemistry, 2002Co-Authors: Lan Guan, Miklós Sahin-tóth, Tamás Kálai, Kálmán Hideg, H. Ronald KabackAbstract:Abstract Affinity inactivators are useful for probing catalytic mechanisms. Here we describe the synthesis and properties of methanethiosulfonyl (MTS) galactose or glucose derivatives with respect to a well studied Membrane Transport Protein, the lactose permease ofEscherichia coli. The MTS-galactose derivatives behave as affinity inactivators of a functional mutant with Ala122→Cys in a background otherwise devoid of Cys residues. A proton electrochemical gradient (ΔμH+) markedly increases the rate of reaction between Cys122 and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected. When the Ala122→Cys mutation is combined with a mutation (Cys154→Gly) that blocks Transport but increases binding affinity, discrimination between the MTS-galactose and -glucose derivatives is abolished, and ΔμH+ has no effect. The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala122 in helix IV. In addition, the findings demonstrate that the MTS-galactose derivatives do not react with the Cys residue at position 122 upon binding per sebut at a subsequent step in the overall Transport mechanism. Thus, these inactivators behave as unique suicide substrates.
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The kamikaze approach to Membrane Transport
Nature Reviews Molecular Cell Biology, 2001Co-Authors: H. Ronald Kaback, Miklós Sahin-tóth, Adam B. WeinglassAbstract:Membrane Transport Proteins catalyse the movement of molecules into and out of cells and organelles, but their hydrophobic and metastable nature often makes them difficult to study by traditional means. Novel approaches that have been developed and applied to one Membrane Transport Protein, the lactose permease from Escherichia coli , are now being used to study various other Membrane Proteins. Membrane Transport Proteins mediate the movement of molecules into, or out of, cells, intracellular organelles and across epithelia. Despite their abundance in the genome and evident importance for the cell, we know little about their structure and function, largely because their hydrophobic and metastable nature makes them difficult to study. Recent developments have allowed initial insight into the structure and mechanism of Membrane Transport Proteins. One example is the lactose permease from Escherichia coli , a member of the major facilitator superfamily. This Membrane Protein uses free energy released from the energetically downhill translocation of H^+ in response to an electrochemical H^+ gradient to drive the accumulation of specific sugars against a concentration gradient. Extensive use of site-directed mutagenesis demonstrates that only six amino acid residues are irreplaceable with respect to active lactose Transport. Furthermore, mutants engineered for various biochemical and biophysical approaches provide structural information about how the helices are packed and how the irreplaceable residues interact to catalyse Transport. Through this work, charge pairs have been identified that mediate substrate binding and H^+ translocation. The residues that are irreplaceable for activity are conserved in other members of the oligosaccharide/H^+ symport subfamily, but are not found in other members of the major facilitator superfamily. Despite this, it is thought that relatively few residues will be critical for Transport in these other families of Membrane Transport Proteins and that the conformational changes involved will be largely rigid body movements of the transMembrane helices.
Mathias Grote - One of the best experts on this subject based on the ideXlab platform.
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Purple Matter, Membranes and ‘Molecular Pumps’ in Rhodopsin Research (1960s–1980s)
Journal of the History of Biology, 2013Co-Authors: Mathias GroteAbstract:In the context of 1960s research on biological Membranes, scientists stumbled upon a curiously coloured material substance, which became called the “purple Membrane.” Interactions with the material as well as chemical analyses led to the conclusion that the microbial Membrane contained a photoactive molecule similar to rhodopsin, the light receptor of animals’ retinae. Until 1975, the find led to the formation of novel objects in science, and subsequently to the development of a field in the molecular life sciences that comprised biophysics, bioenergetics as well as Membrane and structural biology. Furthermore, the purple Membrane and bacteriorhodopsin, as the photoactive Membrane Transport Protein was baptized, inspired attempts at hybrid bio-optical engineering throughout the 1980s. A central motif of the research field was the identification of a functional biological structure, such as a Membrane, with a reactive material substance that could be easily prepared and manipulated. Building on this premise, early purple Membrane research will be taken as a case in point to understand the appearance and transformation of objects in science through work with material substances. Here, the role played by a perceptible material and its spontaneous change of colour, or reactivity, casts a different light on objects and experimental practices in the late twentieth century molecular life sciences. With respect to the impact of chemical working and thinking, the purple Membrane and rhodopsins represent an influential domain straddling the life and chemical sciences as well as bio- and material technologies, which has received only little historical and philosophical attention. Re-drawing the boundary between the living and the non-enlivened, these researches explain and model organismic activity through the reactivity of macromolecular structures, and thus palpable material substances.
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Purple matter, Membranes and 'molecular pumps' in rhodopsin research (1960s-1980s).
Journal of the History of Biology, 2012Co-Authors: Mathias GroteAbstract:In the context of 1960s research on biological Membranes, scientists stumbled upon a curiously coloured material substance, which became called the “purple Membrane.” Interactions with the material as well as chemical analyses led to the conclusion that the microbial Membrane contained a photoactive molecule similar to rhodopsin, the light receptor of animals’ retinae. Until 1975, the find led to the formation of novel objects in science, and subsequently to the development of a field in the molecular life sciences that comprised biophysics, bioenergetics as well as Membrane and structural biology. Furthermore, the purple Membrane and bacteriorhodopsin, as the photoactive Membrane Transport Protein was baptized, inspired attempts at hybrid bio-optical engineering throughout the 1980s. A central motif of the research field was the identification of a functional biological structure, such as a Membrane, with a reactive material substance that could be easily prepared and manipulated. Building on this premise, early purple Membrane research will be taken as a case in point to understand the appearance and transformation of objects in science through work with material substances. Here, the role played by a perceptible material and its spontaneous change of colour, or reactivity, casts a different light on objects and experimental practices in the late twentieth century molecular life sciences. With respect to the impact of chemical working and thinking, the purple Membrane and rhodopsins represent an influential domain straddling the life and chemical sciences as well as bio- and material technologies, which has received only little historical and philosophical attention. Re-drawing the boundary between the living and the non-enlivened, these researches explain and model organismic activity through the reactivity of macromolecular structures, and thus palpable material substances.