Christoph Von Ballmoos - Academia.edu (original) (raw)
Papers by Christoph Von Ballmoos
Journal of Molecular Biology, 2003
The Na þ -translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum cont... more The Na þ -translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c 11 rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7 Å resolution. Different crystal contacts were induced after treatment of the crystals with dicyclohexylcarbodiimide (DCCD), which is consistent with the binding of the inhibitor to glutamate 65 in the C-terminal helix on the outside of the ring. The c subunits of the isolated c 11 ring of I. tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F 1 F o holoenzyme. The reaction rate increased with decreasing pH but was lower in the presence of Na þ . From the pH profile of the second-order rate constants, the pK of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5 mM NaCl, respectively. These pK values are identical with those determined for the F 1 F o complex. The results indicate that the isolated c-ring retains its native structure, and that the glutamate 65, including binding sites near the middle of the membrane, are accessible to Na þ from the cytoplasm through access channels within the c-ring itself.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 2003
The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventi... more The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventional and inverse polymerase chain reaction (i-PCR) techniques. It contains nine open reading frames that were attributed to eight structural genes of the F 1 F o ATP synthase and the atpI gene, which is not part of the enzyme complex. The initiation codons of all atp genes, except that of atpB coding for the a subunit, were identified by the corresponding N-terminal amino acid sequence. The hydrophobic a subunit was identified by MALDI mass spectrometry. The atp genes of I. tartaricus are arranged in one operon with the sequence atpIBEFHAGDC comprising 6992 base pairs with a GC content of 38.1%. The F 1 F o ATP synthase of I. tartaricus has a calculated molecular mass of 510 kDa and includes 4810 amino acids. The gene sequences and products reveal significant identities to atp genes of other Na + -translocating F 1 F o ATP synthases, especially in the F o subunits a and c which are directly involved in ion translocation. D
EMBO reports, 2006
Cycles have a profound role in cellular life at all levels of organization. Well-known cycles in ... more Cycles have a profound role in cellular life at all levels of organization. Well-known cycles in cell metabolism include the tricarboxylic acid and the urea cycle, in which a specific carrier substrate undergoes a sequence of chemical transformations and is regenerated at the end. Other examples include the interconversions of cofactors, such as NADH or ATP, which are present in the cell in limiting amounts and have to be recycled effectively for metabolism to continue. Every living cell performs a rapid turnover of ATP to ADP to fulfil various energetic demands and effectively regenerates the ATP from ADP in an energy-consuming process. The turnover of the ATP cycle is impressive; a human uses about its body weight in ATP per day. Enzymes perform catalytic reaction cycles in which they undergo several chemical and physical transformations before they are converted back to their original states. The ubiquitous F 1 F o ATP synthase is of particular interest not only because of its biological importance, but also owing to its unique rotational mechanism. Here, we give an overview of the membrane-embedded F o sector, particularly with respect to the recent crystal structure of the c ring from Ilyobacter tartaricus, and summarize current hypotheses for the mechanism by which rotation of the c ring is generated.
Molecular Microbiology, 2009
F1F0 ATP synthases use the electrochemical potential of H + or Na + across biological membranes t... more F1F0 ATP synthases use the electrochemical potential of H + or Na + across biological membranes to synthesize ATP by a rotary mechanism. In bacteria, the enzymes can act in reverse as ATP-driven ion pumps creating the indispensable membrane potential. Here, we demonstrate that the F0 parts of a Na + -and H + -dependent enzyme display major asymmetries with respect to their mode of operation, reflected by the requirement of~100 times higher Na + or H + concentrations for the synthesis compared with the hydrolysis of ATP. A similar asymmetry is observed during ion transport through isolated F0 parts, indicating different affinities for the binding sites in the a/c interface. Together with further data, we propose a model that provides a rationale for a differential usage of membrane potential and ion gradient during ATP synthesis as observed experimentally. The functional asymmetry might also reflect an important property of the ATP synthesis mechanism in vivo. In Escherichia coli, we observed respiratory chain-driven ATP production at pH 7-8, while P-site pH values < 6.5 were required for ATP synthesis in vitro. This discrepancy is discussed with respect to the hypothesis that during respiration lateral proton diffusion could lead to significant acidification at the membrane surface.
Biochimica Et Biophysica Acta-gene Structure and Expression, 2003
The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventi... more The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventional and inverse polymerase chain reaction (i-PCR) techniques. It contains nine open reading frames that were attributed to eight structural genes of the F1Fo ATP synthase and the atpI gene, which is not part of the enzyme complex. The initiation codons of all atp genes, except that
Biochimica Et Biophysica Acta-bioenergetics, 2008
The membrane-embedded F0 part of ATP synthases is responsible for ion translocation during ATP sy... more The membrane-embedded F0 part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F0 complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F0 part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either
Adenosine triphosphate (ATP) is used as a general energy source by all living cells. The free ene... more Adenosine triphosphate (ATP) is used as a general energy source by all living cells. The free energy released by hydrolyzing its terminal phosphoric acid anhydride bond to yield ADP and phosphate is utilized to drive various energy-consuming reactions. The ubiquitous F1F0 ATP synthase produces the majority of ATP by converting the energy stored in a transmembrane electrochemical gradient of H+ or Na+
Analytical Biochemistry, 2004
European Journal of Biochemistry, 2002
Ó FEBS 2002 Localization of ion binding sites in ATP synthases (Eur. J. Biochem. 269) 5583 Ó FEBS... more Ó FEBS 2002 Localization of ion binding sites in ATP synthases (Eur. J. Biochem. 269) 5583 Ó FEBS 2002 Localization of ion binding sites in ATP synthases (Eur. J. Biochem. 269) 5585 Ó FEBS 2002 Localization of ion binding sites in ATP synthases (Eur. J. Biochem. 269) 5587 Ó FEBS 2002 Localization of ion binding sites in ATP synthases (Eur. J. Biochem. 269) 5589
Annual Review of Biophysics, 2008
F1F0 ATP synthases convert energy stored in an electrochemical gra- dient of H+ or Na+ across the... more F1F0 ATP synthases convert energy stored in an electrochemical gra- dient of H+ or Na+ across the membrane into mechanical rotation, which is subsequently converted into the chemical bond energy of ATP. The majority of cellular ATP is produced by the ATP synthase in organisms throughout the biological kingdom and therefore un- der diverse environmental conditions. The ATP synthase of
Structure, 2003
ATP synthesis by F-type ATP synthases consumes energy stored in a transmembrane electrochemical g... more ATP synthesis by F-type ATP synthases consumes energy stored in a transmembrane electrochemical gradient of protons or sodium ions. The electric component of the ion motive force is crucial for ATP synthesis. Here, we incorporate recent results on structure and function of the F0 domain and present a mechanism for torque generation with the fundamental nature of the membrane potential
Annual Review of Biochemistry, 2009
The majority of cellular energy in the form of adenosine triphosphate (ATP) is synthesized by the... more The majority of cellular energy in the form of adenosine triphosphate (ATP) is synthesized by the ubiquitous F(1)F(0) ATP synthase. Power for ATP synthesis derives from an electrochemical proton (or Na(+)) gradient, which drives rotation of membranous F(0) motor components. Efficient rotation not only requires a significant driving force (DeltamuH(+)), consisting of membrane potential (Deltapsi) and proton concentration gradient (DeltapH), but also a high proton concentration at the source P side. In vivo this is maintained by dynamic proton movements across and along the surface of the membrane. The torque-generating unit consists of the interface of the rotating c ring and the stator a subunit. Ion translocation through this unit involves a sophisticated interplay between the c-ring binding sites, the stator arginine, and the coupling ions on both sides of the membrane. c-ring rotation is transmitted to the eccentric shaft gamma-subunit to elicit conformational changes in the catalytic sites of F(1), leading to ATP synthesis.
Journal of Molecular Biology, 2003
The Na+-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contai... more The Na+-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c11 rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7Å resolution. Different crystal contacts were induced after treatment of the crystals with
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2015
The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin... more The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-β-D-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli.
Nature communications, 2014
Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane sig... more Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE protein-based method for incorporation of multiple membrane proteins into artificial membrane vesicles of well-defined composition, and for delivery of large water-soluble substrates into these vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0 ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks ...
The Biochemical journal, Jan 15, 2004
There is much interest in chemokine receptors as therapeutic targets in diseases such as AIDS, au... more There is much interest in chemokine receptors as therapeutic targets in diseases such as AIDS, autoimmune and inflammatory disorders, and cancer. Hampering such studies is the lack of accurate three-dimensional structural models of these molecules. The CC-chemokine receptor D6 is expressed at exceptionally high levels in heterologous transfectants. Here we report the purification and biochemical characterization of milligram quantities of D6 protein from relatively small cultures of transfected mammalian cells. Importantly, purified D6 retains full functional activity, shown by displaceable binding of 125I-labelled MIP-1beta (macrophage inflammatory protein-1beta) and by complete binding of the receptor to a MIP-1alpha affinity column. In addition, we show that D6 is decorated on the N-terminus by N-linked glycosylation. Mutational analysis reveals that this glycosylation is dispensable for ligand binding and high expression in transfected cells. Metabolic labelling has revealed the...
A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with t... more A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na ؉translocating F 1 F 0 ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na ؉ ions but not in its presence, indicating the specific reaction with the Na ؉ binding c-Glu 65 residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu 65 was therefore in close contact with phospholipid but does not contact subunit a. Na ؉ in / 22 Na ؉ out exchange activity of the ATP synthase was not affected by modifying the c-Glu 65 sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na ؉ ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospolipases C and A 2 and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu 65 is located within the core of the membrane.
ATP is the universal energy currency of living cells, and the majority of it is synthesized by th... more ATP is the universal energy currency of living cells, and the majority of it is synthesized by the F 1 F 0 ATP synthase. Inhibitors of this enzyme are therefore potentially detrimental for all life forms. Tributyltin chloride (TBT-Cl) inhibits ATP hydrolysis by the Na ؉translocating ATP synthase of Ilyobacter tartaricus or the H ؉translocating counterpart of Escherichia coli with apparent K i of 200 nM. To target the site of this inhibition, we synthesized a tritium-labeled derivative of TBT-Cl in which one of the butyl groups was replaced by a photoactivatable aryldiazirine residue. Upon illumination, subunit a of the ATP synthase becomes specifically modified, and this labeling is suppressed in the presence of the original inhibitor. In case of the Na ؉ ATP synthase, labeling is also suppressed in the presence of Na ؉ ions, suggesting an interference in Na ؉ or TBT-Cl binding to subunit a. This interference is corroborated by the protection of ATP hydrolysis from TBT-Cl inhibition by 105 mM Na ؉ . TBT-Cl strongly inhibits Na ؉ exchange by the reconstituted I. tartaricus ATP synthase. Taken together these results indicate that the subunit a ion channel is the target site for ATPase inhibition by toxic organotin compounds. An inhibitor interacting specifically with this site has not been reported previously.
The F1F0 ATP synthase utilizes energy stored in an electrochemical gradient of protons (or Na+ io... more The F1F0 ATP synthase utilizes energy stored in an electrochemical gradient of protons (or Na+ ions) across the membrane to synthesize ATP from ADP and phosphate. Current models predict that the protonation/deprotonation of specific acidic c ring residues is at the core of the proton translocation mechanism by this enzyme. To probe the mode of proton binding, we measured the covalent modification of the acidic c ring residues with the inhibitor dicyclohexylcarbodiimide (DCCD) over the pH range from 5 to 11. With the H+-translocating ATP synthase from the archaeum Halobacterium salinarium or the Na+-translocating ATP synthase from Ilyobacter tartaricus, the pH profile of DCCD labeling followed a titration curve with a pKa around neutral, reflecting protonation of the acidic c ring residues. However, with the ATP synthases from Escherichia coli, mitochondria, or chloroplasts, a clearly different, bell-shaped pH profile for DCCD labeling was observed which is not compatible with carboxylate protonation but might be explained by the coordination of a hydronium ion as proposed earlier [Boyer, P. D. (1988) Trends Biochem. Sci. 13, 5-7]. Upon site-directed mutagenesis of single binding site residues of the structurally resolved c ring, the sigmoidal pH profile for DCCD labeling could be converted to a more bell-shaped one, demonstrating that the different ion binding modes are based on subtle changes in the amino acid sequence of the protein. The concept of two different binding sites in the ATP synthase family is supported by the ATP hydrolysis pH profiles of the investigated enzymes.
Journal of Molecular Biology, 2003
The Na þ -translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum cont... more The Na þ -translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c 11 rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7 Å resolution. Different crystal contacts were induced after treatment of the crystals with dicyclohexylcarbodiimide (DCCD), which is consistent with the binding of the inhibitor to glutamate 65 in the C-terminal helix on the outside of the ring. The c subunits of the isolated c 11 ring of I. tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F 1 F o holoenzyme. The reaction rate increased with decreasing pH but was lower in the presence of Na þ . From the pH profile of the second-order rate constants, the pK of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5 mM NaCl, respectively. These pK values are identical with those determined for the F 1 F o complex. The results indicate that the isolated c-ring retains its native structure, and that the glutamate 65, including binding sites near the middle of the membrane, are accessible to Na þ from the cytoplasm through access channels within the c-ring itself.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 2003
The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventi... more The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventional and inverse polymerase chain reaction (i-PCR) techniques. It contains nine open reading frames that were attributed to eight structural genes of the F 1 F o ATP synthase and the atpI gene, which is not part of the enzyme complex. The initiation codons of all atp genes, except that of atpB coding for the a subunit, were identified by the corresponding N-terminal amino acid sequence. The hydrophobic a subunit was identified by MALDI mass spectrometry. The atp genes of I. tartaricus are arranged in one operon with the sequence atpIBEFHAGDC comprising 6992 base pairs with a GC content of 38.1%. The F 1 F o ATP synthase of I. tartaricus has a calculated molecular mass of 510 kDa and includes 4810 amino acids. The gene sequences and products reveal significant identities to atp genes of other Na + -translocating F 1 F o ATP synthases, especially in the F o subunits a and c which are directly involved in ion translocation. D
EMBO reports, 2006
Cycles have a profound role in cellular life at all levels of organization. Well-known cycles in ... more Cycles have a profound role in cellular life at all levels of organization. Well-known cycles in cell metabolism include the tricarboxylic acid and the urea cycle, in which a specific carrier substrate undergoes a sequence of chemical transformations and is regenerated at the end. Other examples include the interconversions of cofactors, such as NADH or ATP, which are present in the cell in limiting amounts and have to be recycled effectively for metabolism to continue. Every living cell performs a rapid turnover of ATP to ADP to fulfil various energetic demands and effectively regenerates the ATP from ADP in an energy-consuming process. The turnover of the ATP cycle is impressive; a human uses about its body weight in ATP per day. Enzymes perform catalytic reaction cycles in which they undergo several chemical and physical transformations before they are converted back to their original states. The ubiquitous F 1 F o ATP synthase is of particular interest not only because of its biological importance, but also owing to its unique rotational mechanism. Here, we give an overview of the membrane-embedded F o sector, particularly with respect to the recent crystal structure of the c ring from Ilyobacter tartaricus, and summarize current hypotheses for the mechanism by which rotation of the c ring is generated.
Molecular Microbiology, 2009
F1F0 ATP synthases use the electrochemical potential of H + or Na + across biological membranes t... more F1F0 ATP synthases use the electrochemical potential of H + or Na + across biological membranes to synthesize ATP by a rotary mechanism. In bacteria, the enzymes can act in reverse as ATP-driven ion pumps creating the indispensable membrane potential. Here, we demonstrate that the F0 parts of a Na + -and H + -dependent enzyme display major asymmetries with respect to their mode of operation, reflected by the requirement of~100 times higher Na + or H + concentrations for the synthesis compared with the hydrolysis of ATP. A similar asymmetry is observed during ion transport through isolated F0 parts, indicating different affinities for the binding sites in the a/c interface. Together with further data, we propose a model that provides a rationale for a differential usage of membrane potential and ion gradient during ATP synthesis as observed experimentally. The functional asymmetry might also reflect an important property of the ATP synthesis mechanism in vivo. In Escherichia coli, we observed respiratory chain-driven ATP production at pH 7-8, while P-site pH values < 6.5 were required for ATP synthesis in vitro. This discrepancy is discussed with respect to the hypothesis that during respiration lateral proton diffusion could lead to significant acidification at the membrane surface.
Biochimica Et Biophysica Acta-gene Structure and Expression, 2003
The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventi... more The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventional and inverse polymerase chain reaction (i-PCR) techniques. It contains nine open reading frames that were attributed to eight structural genes of the F1Fo ATP synthase and the atpI gene, which is not part of the enzyme complex. The initiation codons of all atp genes, except that
Biochimica Et Biophysica Acta-bioenergetics, 2008
The membrane-embedded F0 part of ATP synthases is responsible for ion translocation during ATP sy... more The membrane-embedded F0 part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F0 complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F0 part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either
Adenosine triphosphate (ATP) is used as a general energy source by all living cells. The free ene... more Adenosine triphosphate (ATP) is used as a general energy source by all living cells. The free energy released by hydrolyzing its terminal phosphoric acid anhydride bond to yield ADP and phosphate is utilized to drive various energy-consuming reactions. The ubiquitous F1F0 ATP synthase produces the majority of ATP by converting the energy stored in a transmembrane electrochemical gradient of H+ or Na+
Analytical Biochemistry, 2004
European Journal of Biochemistry, 2002
Ó FEBS 2002 Localization of ion binding sites in ATP synthases (Eur. J. Biochem. 269) 5583 Ó FEBS... more Ó FEBS 2002 Localization of ion binding sites in ATP synthases (Eur. J. Biochem. 269) 5583 Ó FEBS 2002 Localization of ion binding sites in ATP synthases (Eur. J. Biochem. 269) 5585 Ó FEBS 2002 Localization of ion binding sites in ATP synthases (Eur. J. Biochem. 269) 5587 Ó FEBS 2002 Localization of ion binding sites in ATP synthases (Eur. J. Biochem. 269) 5589
Annual Review of Biophysics, 2008
F1F0 ATP synthases convert energy stored in an electrochemical gra- dient of H+ or Na+ across the... more F1F0 ATP synthases convert energy stored in an electrochemical gra- dient of H+ or Na+ across the membrane into mechanical rotation, which is subsequently converted into the chemical bond energy of ATP. The majority of cellular ATP is produced by the ATP synthase in organisms throughout the biological kingdom and therefore un- der diverse environmental conditions. The ATP synthase of
Structure, 2003
ATP synthesis by F-type ATP synthases consumes energy stored in a transmembrane electrochemical g... more ATP synthesis by F-type ATP synthases consumes energy stored in a transmembrane electrochemical gradient of protons or sodium ions. The electric component of the ion motive force is crucial for ATP synthesis. Here, we incorporate recent results on structure and function of the F0 domain and present a mechanism for torque generation with the fundamental nature of the membrane potential
Annual Review of Biochemistry, 2009
The majority of cellular energy in the form of adenosine triphosphate (ATP) is synthesized by the... more The majority of cellular energy in the form of adenosine triphosphate (ATP) is synthesized by the ubiquitous F(1)F(0) ATP synthase. Power for ATP synthesis derives from an electrochemical proton (or Na(+)) gradient, which drives rotation of membranous F(0) motor components. Efficient rotation not only requires a significant driving force (DeltamuH(+)), consisting of membrane potential (Deltapsi) and proton concentration gradient (DeltapH), but also a high proton concentration at the source P side. In vivo this is maintained by dynamic proton movements across and along the surface of the membrane. The torque-generating unit consists of the interface of the rotating c ring and the stator a subunit. Ion translocation through this unit involves a sophisticated interplay between the c-ring binding sites, the stator arginine, and the coupling ions on both sides of the membrane. c-ring rotation is transmitted to the eccentric shaft gamma-subunit to elicit conformational changes in the catalytic sites of F(1), leading to ATP synthesis.
Journal of Molecular Biology, 2003
The Na+-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contai... more The Na+-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c11 rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7Å resolution. Different crystal contacts were induced after treatment of the crystals with
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2015
The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin... more The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-β-D-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli.
Nature communications, 2014
Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane sig... more Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE protein-based method for incorporation of multiple membrane proteins into artificial membrane vesicles of well-defined composition, and for delivery of large water-soluble substrates into these vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0 ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks ...
The Biochemical journal, Jan 15, 2004
There is much interest in chemokine receptors as therapeutic targets in diseases such as AIDS, au... more There is much interest in chemokine receptors as therapeutic targets in diseases such as AIDS, autoimmune and inflammatory disorders, and cancer. Hampering such studies is the lack of accurate three-dimensional structural models of these molecules. The CC-chemokine receptor D6 is expressed at exceptionally high levels in heterologous transfectants. Here we report the purification and biochemical characterization of milligram quantities of D6 protein from relatively small cultures of transfected mammalian cells. Importantly, purified D6 retains full functional activity, shown by displaceable binding of 125I-labelled MIP-1beta (macrophage inflammatory protein-1beta) and by complete binding of the receptor to a MIP-1alpha affinity column. In addition, we show that D6 is decorated on the N-terminus by N-linked glycosylation. Mutational analysis reveals that this glycosylation is dispensable for ligand binding and high expression in transfected cells. Metabolic labelling has revealed the...
A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with t... more A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na ؉translocating F 1 F 0 ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na ؉ ions but not in its presence, indicating the specific reaction with the Na ؉ binding c-Glu 65 residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu 65 was therefore in close contact with phospholipid but does not contact subunit a. Na ؉ in / 22 Na ؉ out exchange activity of the ATP synthase was not affected by modifying the c-Glu 65 sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na ؉ ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospolipases C and A 2 and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu 65 is located within the core of the membrane.
ATP is the universal energy currency of living cells, and the majority of it is synthesized by th... more ATP is the universal energy currency of living cells, and the majority of it is synthesized by the F 1 F 0 ATP synthase. Inhibitors of this enzyme are therefore potentially detrimental for all life forms. Tributyltin chloride (TBT-Cl) inhibits ATP hydrolysis by the Na ؉translocating ATP synthase of Ilyobacter tartaricus or the H ؉translocating counterpart of Escherichia coli with apparent K i of 200 nM. To target the site of this inhibition, we synthesized a tritium-labeled derivative of TBT-Cl in which one of the butyl groups was replaced by a photoactivatable aryldiazirine residue. Upon illumination, subunit a of the ATP synthase becomes specifically modified, and this labeling is suppressed in the presence of the original inhibitor. In case of the Na ؉ ATP synthase, labeling is also suppressed in the presence of Na ؉ ions, suggesting an interference in Na ؉ or TBT-Cl binding to subunit a. This interference is corroborated by the protection of ATP hydrolysis from TBT-Cl inhibition by 105 mM Na ؉ . TBT-Cl strongly inhibits Na ؉ exchange by the reconstituted I. tartaricus ATP synthase. Taken together these results indicate that the subunit a ion channel is the target site for ATPase inhibition by toxic organotin compounds. An inhibitor interacting specifically with this site has not been reported previously.
The F1F0 ATP synthase utilizes energy stored in an electrochemical gradient of protons (or Na+ io... more The F1F0 ATP synthase utilizes energy stored in an electrochemical gradient of protons (or Na+ ions) across the membrane to synthesize ATP from ADP and phosphate. Current models predict that the protonation/deprotonation of specific acidic c ring residues is at the core of the proton translocation mechanism by this enzyme. To probe the mode of proton binding, we measured the covalent modification of the acidic c ring residues with the inhibitor dicyclohexylcarbodiimide (DCCD) over the pH range from 5 to 11. With the H+-translocating ATP synthase from the archaeum Halobacterium salinarium or the Na+-translocating ATP synthase from Ilyobacter tartaricus, the pH profile of DCCD labeling followed a titration curve with a pKa around neutral, reflecting protonation of the acidic c ring residues. However, with the ATP synthases from Escherichia coli, mitochondria, or chloroplasts, a clearly different, bell-shaped pH profile for DCCD labeling was observed which is not compatible with carboxylate protonation but might be explained by the coordination of a hydronium ion as proposed earlier [Boyer, P. D. (1988) Trends Biochem. Sci. 13, 5-7]. Upon site-directed mutagenesis of single binding site residues of the structurally resolved c ring, the sigmoidal pH profile for DCCD labeling could be converted to a more bell-shaped one, demonstrating that the different ion binding modes are based on subtle changes in the amino acid sequence of the protein. The concept of two different binding sites in the ATP synthase family is supported by the ATP hydrolysis pH profiles of the investigated enzymes.