The Experts below are selected from a list of 144 Experts worldwide ranked by ideXlab platform
Andreas Engel - One of the best experts on this subject based on the ideXlab platform.
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atomic force microscopy of native Purple Membrane
Biochimica et Biophysica Acta, 2000Co-Authors: Daniel J Muller, Georg Buldt, Clemens Moller, Bernard J Heymann, Filipp Oesterhelt, Hermann E Gaub, Andreas EngelAbstract:Atomic force microscopy (AFM) allows the observation of surface structures of Purple Membrane (PM) in buffer solution with subnanometer resolution. This offers the possibility to classify the major conformations of the native bacteriorhodopsin (BR) surfaces and to map the variability of individual polypeptide loops connecting transMembrane alpha-helices of BR. The position, the variability and the flexibility of these loops depend on the packing arrangement of BR molecules in the lipid bilayer with significant differences observed between the trigonal and orthorhombic crystal forms. Cleavage of the Schiff base bond leads to a disassembly of the trigonal PM crystal, which is restored by regenerating the bleached PM. The combination of single molecule AFM imaging and single molecule force-spectroscopy provides an unique insight into the interactions between individual BR molecules and the PM, and between secondary structure elements within BR.
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reversible loss of crystallinity on photobleaching Purple Membrane in the presence of hydroxylamine
Journal of Molecular Biology, 2000Co-Authors: Clemens Moller, Andreas Engel, Georg Buldt, Norbert A Dencher, Daniel J MullerAbstract:Structural changes of Purple Membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the Purple Membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact Purple Membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of Purple Membrane. The data provide novel insights into factors triggering Purple Membrane formation and structure.
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charting the surfaces of the Purple Membrane
Journal of Structural Biology, 1999Co-Authors: Bernard J Heymann, Daniel J Muller, Georg Buldt, Ehud M Landau, Jurg P Rosenbusch, Eva Pebaypeyroula, Andreas EngelAbstract:Abstract The preponderance of structural data of the Purple Membrane from X-ray diffraction (XRD), electron crystallography (EC), and atomic force microscopy (AFM) allows us to ask questions about the structure of bacteriorhodopsin itself, as well as about the information derived from the different techniques. The transMembrane helices of bacteriorhodopsin are quite similar in both EC and XRD models. In contrast, the loops at the surfaces of the Purple Membrane show the highest variability between the atomic models, comparable to the height variance measured by AFM. The excellent agreement of the AFM topographs with the atomic models from XRD builds confidence in the results. Small technical difficulties in EC lead to poorer resolution of the loop structures, although the combination of atomic models with AFM surfaces allows clear interpretation of the extent and flexibility of the loop structures. While XRD remains the premier technique to determine very-high-resolution structures, EC offers a method to determine loop structures unhindered by three-dimensional crystal contacts, and AFM provides information about surface structures and their flexibility under physiological conditions.
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immuno atomic force microscopy of Purple Membrane
Biophysical Journal, 1996Co-Authors: Daniel J Muller, Cora Ann Schoenenberger, Georg Buldt, Andreas EngelAbstract:The atomic force microscope is a useful tool for imaging native biological structures at high resolution. In analogy to conventional immunolabeling techniques, we have used antibodies directed against the C-terminus of bacteriorhodopsin to distinguish the cytoplasmic and extracellular surface of Purple Membrane while imaging in buffer solution. At forces > or=0.8 nN the antibodies were removed by the scanning stylus and the molecular topography of the cytoplasmic Purple Membrane surface was revealed. When the stylus was retracted, the scanned Membrane area was relabeled with antibodies within 10min. The extracellular surface of Purple Membrane was imaged at 0.7 nm resolution, exhibiting a major and a minor protrusion per bacteriorhodopsin monomer. As confirmed by immuno-dot blot analysis and sodium dodecyl sulfate-gel electrophoresis, labeling of the Purple Membrane was not observed if the C-terminus of bacteriorhodopsin was cleaved off by papain.
Daniel J Muller - One of the best experts on this subject based on the ideXlab platform.
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atomic force microscopy of native Purple Membrane
Biochimica et Biophysica Acta, 2000Co-Authors: Daniel J Muller, Georg Buldt, Clemens Moller, Bernard J Heymann, Filipp Oesterhelt, Hermann E Gaub, Andreas EngelAbstract:Atomic force microscopy (AFM) allows the observation of surface structures of Purple Membrane (PM) in buffer solution with subnanometer resolution. This offers the possibility to classify the major conformations of the native bacteriorhodopsin (BR) surfaces and to map the variability of individual polypeptide loops connecting transMembrane alpha-helices of BR. The position, the variability and the flexibility of these loops depend on the packing arrangement of BR molecules in the lipid bilayer with significant differences observed between the trigonal and orthorhombic crystal forms. Cleavage of the Schiff base bond leads to a disassembly of the trigonal PM crystal, which is restored by regenerating the bleached PM. The combination of single molecule AFM imaging and single molecule force-spectroscopy provides an unique insight into the interactions between individual BR molecules and the PM, and between secondary structure elements within BR.
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reversible loss of crystallinity on photobleaching Purple Membrane in the presence of hydroxylamine
Journal of Molecular Biology, 2000Co-Authors: Clemens Moller, Andreas Engel, Georg Buldt, Norbert A Dencher, Daniel J MullerAbstract:Structural changes of Purple Membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the Purple Membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact Purple Membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of Purple Membrane. The data provide novel insights into factors triggering Purple Membrane formation and structure.
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charting the surfaces of the Purple Membrane
Journal of Structural Biology, 1999Co-Authors: Bernard J Heymann, Daniel J Muller, Georg Buldt, Ehud M Landau, Jurg P Rosenbusch, Eva Pebaypeyroula, Andreas EngelAbstract:Abstract The preponderance of structural data of the Purple Membrane from X-ray diffraction (XRD), electron crystallography (EC), and atomic force microscopy (AFM) allows us to ask questions about the structure of bacteriorhodopsin itself, as well as about the information derived from the different techniques. The transMembrane helices of bacteriorhodopsin are quite similar in both EC and XRD models. In contrast, the loops at the surfaces of the Purple Membrane show the highest variability between the atomic models, comparable to the height variance measured by AFM. The excellent agreement of the AFM topographs with the atomic models from XRD builds confidence in the results. Small technical difficulties in EC lead to poorer resolution of the loop structures, although the combination of atomic models with AFM surfaces allows clear interpretation of the extent and flexibility of the loop structures. While XRD remains the premier technique to determine very-high-resolution structures, EC offers a method to determine loop structures unhindered by three-dimensional crystal contacts, and AFM provides information about surface structures and their flexibility under physiological conditions.
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immuno atomic force microscopy of Purple Membrane
Biophysical Journal, 1996Co-Authors: Daniel J Muller, Cora Ann Schoenenberger, Georg Buldt, Andreas EngelAbstract:The atomic force microscope is a useful tool for imaging native biological structures at high resolution. In analogy to conventional immunolabeling techniques, we have used antibodies directed against the C-terminus of bacteriorhodopsin to distinguish the cytoplasmic and extracellular surface of Purple Membrane while imaging in buffer solution. At forces > or=0.8 nN the antibodies were removed by the scanning stylus and the molecular topography of the cytoplasmic Purple Membrane surface was revealed. When the stylus was retracted, the scanned Membrane area was relabeled with antibodies within 10min. The extracellular surface of Purple Membrane was imaged at 0.7 nm resolution, exhibiting a major and a minor protrusion per bacteriorhodopsin monomer. As confirmed by immuno-dot blot analysis and sodium dodecyl sulfate-gel electrophoresis, labeling of the Purple Membrane was not observed if the C-terminus of bacteriorhodopsin was cleaved off by papain.
Georg Buldt - One of the best experts on this subject based on the ideXlab platform.
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atomic force microscopy of native Purple Membrane
Biochimica et Biophysica Acta, 2000Co-Authors: Daniel J Muller, Georg Buldt, Clemens Moller, Bernard J Heymann, Filipp Oesterhelt, Hermann E Gaub, Andreas EngelAbstract:Atomic force microscopy (AFM) allows the observation of surface structures of Purple Membrane (PM) in buffer solution with subnanometer resolution. This offers the possibility to classify the major conformations of the native bacteriorhodopsin (BR) surfaces and to map the variability of individual polypeptide loops connecting transMembrane alpha-helices of BR. The position, the variability and the flexibility of these loops depend on the packing arrangement of BR molecules in the lipid bilayer with significant differences observed between the trigonal and orthorhombic crystal forms. Cleavage of the Schiff base bond leads to a disassembly of the trigonal PM crystal, which is restored by regenerating the bleached PM. The combination of single molecule AFM imaging and single molecule force-spectroscopy provides an unique insight into the interactions between individual BR molecules and the PM, and between secondary structure elements within BR.
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reversible loss of crystallinity on photobleaching Purple Membrane in the presence of hydroxylamine
Journal of Molecular Biology, 2000Co-Authors: Clemens Moller, Andreas Engel, Georg Buldt, Norbert A Dencher, Daniel J MullerAbstract:Structural changes of Purple Membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the Purple Membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact Purple Membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of Purple Membrane. The data provide novel insights into factors triggering Purple Membrane formation and structure.
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charting the surfaces of the Purple Membrane
Journal of Structural Biology, 1999Co-Authors: Bernard J Heymann, Daniel J Muller, Georg Buldt, Ehud M Landau, Jurg P Rosenbusch, Eva Pebaypeyroula, Andreas EngelAbstract:Abstract The preponderance of structural data of the Purple Membrane from X-ray diffraction (XRD), electron crystallography (EC), and atomic force microscopy (AFM) allows us to ask questions about the structure of bacteriorhodopsin itself, as well as about the information derived from the different techniques. The transMembrane helices of bacteriorhodopsin are quite similar in both EC and XRD models. In contrast, the loops at the surfaces of the Purple Membrane show the highest variability between the atomic models, comparable to the height variance measured by AFM. The excellent agreement of the AFM topographs with the atomic models from XRD builds confidence in the results. Small technical difficulties in EC lead to poorer resolution of the loop structures, although the combination of atomic models with AFM surfaces allows clear interpretation of the extent and flexibility of the loop structures. While XRD remains the premier technique to determine very-high-resolution structures, EC offers a method to determine loop structures unhindered by three-dimensional crystal contacts, and AFM provides information about surface structures and their flexibility under physiological conditions.
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x ray diffraction from a single layer of Purple Membrane at the air water interface
Journal of Molecular Biology, 1999Co-Authors: Georg Buldt, Norbert A Dencher, Stephan A W Verclas, P B Howes, Kristian Kjaer, Angelika Wurlitzer, Markus Weygand, Mathias LoscheAbstract:Abstract X-ray diffraction patterns have been recorded from a single layer of Purple Membrane (∼50 A thickness) at the air/water interface in a Langmuir trough. Grazing-incidence X-ray diffraction is demonstrated to be a promising method for obtaining structural information on Membrane proteins under physiological conditions. The method is so sensitive that diffraction can be measured from samples with only 10 13 protein molecules in the beam. Diffraction from hexagonal crystals of Purple Membrane with a lattice constant of 61.3 A was observed up to the order { h,k }={4,3}, corresponding to a resolution of ∼9 A. The work reported here is a first step towards a new way of protein crystallography using grazing-incidence X-ray diffraction at the air/water interface.
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immuno atomic force microscopy of Purple Membrane
Biophysical Journal, 1996Co-Authors: Daniel J Muller, Cora Ann Schoenenberger, Georg Buldt, Andreas EngelAbstract:The atomic force microscope is a useful tool for imaging native biological structures at high resolution. In analogy to conventional immunolabeling techniques, we have used antibodies directed against the C-terminus of bacteriorhodopsin to distinguish the cytoplasmic and extracellular surface of Purple Membrane while imaging in buffer solution. At forces > or=0.8 nN the antibodies were removed by the scanning stylus and the molecular topography of the cytoplasmic Purple Membrane surface was revealed. When the stylus was retracted, the scanned Membrane area was relabeled with antibodies within 10min. The extracellular surface of Purple Membrane was imaged at 0.7 nm resolution, exhibiting a major and a minor protrusion per bacteriorhodopsin monomer. As confirmed by immuno-dot blot analysis and sodium dodecyl sulfate-gel electrophoresis, labeling of the Purple Membrane was not observed if the C-terminus of bacteriorhodopsin was cleaved off by papain.
Norbert A Dencher - One of the best experts on this subject based on the ideXlab platform.
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glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in Purple Membrane preparations
Biochimica et Biophysica Acta, 2007Co-Authors: Angela Corcelli, Simona Lobasso, Matilde Sublimi Saponetti, Andreas Leopold, Norbert A DencherAbstract:Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the Purple Membrane of Halobacterium salinarum. Here Purple Membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that Purple Membranes isolated from envelopes have a lower content of glycocardiolipin than standard Purple Membranes isolated from cells. The properties of bacteriorhodopsin in the two different Purple Membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native Membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the Membrane surface and its equilibration with the aqueous bulk phase are altered.
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reversible loss of crystallinity on photobleaching Purple Membrane in the presence of hydroxylamine
Journal of Molecular Biology, 2000Co-Authors: Clemens Moller, Andreas Engel, Georg Buldt, Norbert A Dencher, Daniel J MullerAbstract:Structural changes of Purple Membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the Purple Membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact Purple Membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of Purple Membrane. The data provide novel insights into factors triggering Purple Membrane formation and structure.
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x ray diffraction from a single layer of Purple Membrane at the air water interface
Journal of Molecular Biology, 1999Co-Authors: Georg Buldt, Norbert A Dencher, Stephan A W Verclas, P B Howes, Kristian Kjaer, Angelika Wurlitzer, Markus Weygand, Mathias LoscheAbstract:Abstract X-ray diffraction patterns have been recorded from a single layer of Purple Membrane (∼50 A thickness) at the air/water interface in a Langmuir trough. Grazing-incidence X-ray diffraction is demonstrated to be a promising method for obtaining structural information on Membrane proteins under physiological conditions. The method is so sensitive that diffraction can be measured from samples with only 10 13 protein molecules in the beam. Diffraction from hexagonal crystals of Purple Membrane with a lattice constant of 61.3 A was observed up to the order { h,k }={4,3}, corresponding to a resolution of ∼9 A. The work reported here is a first step towards a new way of protein crystallography using grazing-incidence X-ray diffraction at the air/water interface.
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protonation dynamics of the extracellular and cytoplasmic surface of bacteriorhodopsin in the Purple Membrane
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Esther Nachliel, Menachem Gutman, S Kiryati, Norbert A DencherAbstract:Abstract The dynamics of proton binding to the extracellular and the cytoplasmic surfaces of the Purple Membrane were measured by laser-induced proton pulses. Purple Membranes, selectively labeled by fluorescein at Lys-129 of bacteriorhodopsin, were pulsed by protons released in the aqueous bulk from excited pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) and the reaction of protons with the indicators was measured. Kinetic analysis of the data imply that the two faces of the Membrane differ in their buffer capacities and in their rates of interaction with bulk protons. The extracellular surface of the Purple Membrane contains one anionic proton binding site per protein molecule with pK = 5.1. This site is within a Coulomb cage radius (approximately 15 A) from Lys-129. The cytoplasmic surface of the Purple Membrane bears 4-5 protonable moieties (pK = 5.1) that, due to close proximity, function as a common proton binding site. The reaction of the proton with this cluster is at a very fast rate (3.10(10) M-1.s-1). The proximity between the elements is sufficiently high that even in 100 mM NaCl they still function as a cluster. Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates of the cytoplasmic surface, and two to three of them assume positions that almost bar their reaction with bulk protons. The protonation dynamics determined at the surface of the Purple Membrane is of relevance both for the vectorial proton transport mechanism of bacteriorhodopsin and for energy coupling, not only in halobacteria, but also in complex chemiosmotic systems such as mitochondrial and thylakoid Membranes.
D Strauch - One of the best experts on this subject based on the ideXlab platform.
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protein protein interaction in Purple Membrane
Physical Review Letters, 2009Co-Authors: Maikel C Rheinstadter, K Schmalzl, Kathleen Wood, D StrauchAbstract:We present experimental evidence for a long-range protein-protein interaction in Purple Membrane (PM). The interprotein dynamics were quantified by measuring the spectrum of the acoustic phonons in the 2D bacteriorhodopsin (BR) protein lattice using inelastic neutron scattering. Phonon energies of about 1 meV were determined. The data are compared to an analytical model, and the effective spring constant for the interaction between neighboring protein trimers are determined to be k ¼ 53 N=m. Additional, optical-like excitations at 0.45 meV were found and assigned to intraprotein dynamics between neighboring BR monomers.