The Experts below are selected from a list of 618 Experts worldwide ranked by ideXlab platform

Martin Engelhard - One of the best experts on this subject based on the ideXlab platform.

  • Minireview The archaeal sensory rhodopsin II/transducer complex: a model for transmembrane signal transfer
    2020
    Co-Authors: Johann P. Klare, Valentin I. Gordeliy, Georg Bu, Martin Engelhard
    Abstract:

    Archaebacterial photoreceptors mediate phototaxis by regulating cell motility through two-component signalling cascades. Homologs of this sensory pathway occur in all three kingdoms of life, most notably in enteric bacteria in which the chemotaxis has been extensively studied. Recent structural and functional studies on the sensory rhodopsin II/transducer com- plex mediating the photophobic response of Natronomonas pharaonis have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data and the atomic resolution structure of the recep- tor molecule in complex with the transmembrane segment of its cognate transducer provided a model for signal transfer from the receptor to the cytoplasmic side of the transducer. This mecha- nism might also be relevant for eubacterial chemoreceptor sig-

  • Microbial Halorhodopsins: Light-Driven Chloride Pumps
    Chemical Reviews, 2018
    Co-Authors: Christopher Engelhard, Igor Chizhov, Friedrich Siebert, Martin Engelhard
    Abstract:

    Early research on the four microbial rhodopsins discovered in the archaeal Halobacterium salinarum revealed a structural template that served as a scaffold for two different functions: light-driven ion transport and phototaxis. Bacteriorhodopsin and halorhodopsin are proton and chloride pumps, respectively, while sensory rhodopsin I and II are responsible for phototactic behavior of the archaea. Halorhodopsins have been identified in various other species. Besides this group of archaeal halorhodopsins distinct chloride transporting rhodopsins groups have recently been identified in other organism like Flavobacteria or Cyanobacteria. Halorhodopsin from Natronomonas pharaonis is the best-studied homologue because of its facile expression and purification and its advantageous properties, which was the reason to introduce this protein as neural silencer into the new field of optogenetics. Two other major families of genetically encoded silencing proteins, proton pumps and anion channels, extended the repertoi...

  • Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes
    Journal of Physical Chemistry B, 2016
    Co-Authors: Hendrik Mohrmann, Martin Engelhard, Ines Kube, Víctor A. Lórenz-fonfría, Joachim Heberle
    Abstract:

    Sensory rhodopsin II (SRII) is the primary light sensor in the photophobic reaction of the halobacterium Natronomonas pharaonis. Photoactivation of SRII results in a movement of helices F and G of this seven-helical transmembrane protein. This conformational change is conveyed to the transducer protein (HtrII). Global changes in the protein backbone have been monitored by IR difference spectroscopy by recording frequency shifts in the amide bands. Here we investigate local structural changes by judiciously inserting thiocyanides at different locations of SRII. These vibrational Stark probes absorb in a frequency range devoid of any protein vibrations and respond to local changes in the dielectric, electrostatics, and hydrogen bonding. As a proof of principle, we demonstrate the use of Stark probes to test the conformational changes occurring in SRII 12 ms after photoexcitation and later. Thus, a methodology is provided to trace local conformational changes in membrane proteins by a minimal invasive probe ...

  • signaling and adaptation modulate the dynamics of the photosensoric complex of Natronomonas pharaonis
    PLOS Computational Biology, 2015
    Co-Authors: Philipp S Orekhov, Johann P. Klare, Armen Y Mulkidjanian, K V Shaitan, Daniel Klose, Martin Engelhard, Heinzjurgen Steinhoff
    Abstract:

    Motile bacteria and archaea respond to chemical and physical stimuli seeking optimal conditions for survival. To this end transmembrane chemo- and photoreceptors organized in large arrays initiate signaling cascades and ultimately regulate the rotation of flagellar motors. To unravel the molecular mechanism of signaling in an archaeal phototaxis complex we performed coarse-grained molecular dynamics simulations of a trimer of receptor/transducer dimers, namely NpSRII/NpHtrII from Natronomonas pharaonis. Signaling is regulated by a reversible methylation mechanism called adaptation, which also influences the level of basal receptor activation. Mimicking two extreme methylation states in our simulations we found conformational changes for the transmembrane region of NpSRII/NpHtrII which resemble experimentally observed light-induced changes. Further downstream in the cytoplasmic domain of the transducer the signal propagates via distinct changes in the dynamics of HAMP1, HAMP2, the adaptation domain and the binding region for the kinase CheA, where conformational rearrangements were found to be subtle. Overall these observations suggest a signaling mechanism based on dynamic allostery resembling models previously proposed for E. coli chemoreceptors, indicating similar properties of signal transduction for archaeal photoreceptors and bacterial chemoreceptors.

  • Ground state structure of D75N mutant of sensory rhodopsin II in complex with its cognate transducer.
    Journal of Photochemistry and Photobiology B-biology, 2013
    Co-Authors: Andrii Ishchenko, Johann P. Klare, Martin Engelhard, Ekaterina Round, Valentin Borshchevskiy, Sergei Grudinin, Ivan Gushchin, Taras Balandin, A. Remeeva, Georg Buldt
    Abstract:

    The complex of sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII) mediates negative phototaxis in halobacteria Natronomonas pharaonis. Upon light activation NpSRII triggers, by means of NpHtrII, a signal transduction chain homologous to the two component system in eubacterial chemotaxis. Here we report on the crystal structure of the ground state of the mutant NpSRII-D75N/NpHtrII complex in the space group I212121. Mutations of this aspartic acid in light-driven proton pumps dramatically modify or/and inhibit protein functions. However, in vivo studies show that the similar D75N mutation retains functionality of the NpSRII/NpHtrII complex. The structure provides the molecular basis for the explanation of the unexpected observation that the wild and the mutant complexes display identical physiological response on light excitation.

Heinzjurgen Steinhoff - One of the best experts on this subject based on the ideXlab platform.

  • Conformational Dynamics of Sensory Rhodopsin II in Nanolipoprotein and Styrene-Maleic Acid Lipid Particles.
    Photochemistry and Photobiology, 2019
    Co-Authors: Wageiha Mosslehy, Johann P. Klare, Natalia Voskoboynikova, Armen Y Mulkidjanian, Alexandr Colbasevici, Daniel Klose, Adrian Ricke, Heinzjurgen Steinhoff
    Abstract:

    : Styrene-maleic acid lipid particles (SMALPs) provide stable water-soluble nanocontainers for lipid-encased membrane proteins. Possible effects of the SMA-stabilized lipid environment on the interaction dynamics between functionally coupled membrane proteins remain to be elucidated. The photoreceptor sensory rhodopsin II, NpSRII and its cognate transducer, NpHtrII, of Natronomonas pharaonis form a transmembrane complex, NpSRII2 /NpHtrII2 that plays a key role in negative phototaxis and provides a unique model system to study the light-induced transfer of a conformational signal between two integral membrane proteins. Photon absorption induces transient structural changes in NpSRII comprising an outward movement of helix F that cause further conformational alterations in NpHtrII. We applied site-directed spin labeling and time-resolved optical and EPR spectroscopy to compare the conformational dynamics of NpSRII2 /NpHtrII2 reconstituted in SMALPs with that of nanolipoprotein particle and liposome preparations. NpSRII and NpSRII2 /NpHtrII2 show similar photocycles in liposomes and nanolipoprotein particles. An accelerated decay of the M photointermediate found for SMALPs can be explained by a high local proton concentration provided by the carboxylic groups of the SMA polymer. Light-induced large-scale conformational changes of NpSRII2 /NpHtrII2 observed in liposomes and nanolipoprotein particles are affected in SMALPs, indicating restrictions of the protein's conformational freedom.

  • characterization of lipodisc nanoparticles containing sensory rhodopsin ii and its cognate transducer from Natronomonas pharaonis
    Biophysics, 2016
    Co-Authors: D V Bagrov, Natalia Voskoboynikova, G A Armeev, Wageiha Mosslehy, G S Gluhov, T T Ismagulova, Armen Y Mulkidjanian, M P Kirpichnikov, Heinzjurgen Steinhoff, K V Shaitan
    Abstract:

    We describe the preparation and properties of lipodisc nanoparticles–lipid membrane fragments with a diameter of about 10 nm, stabilized by amphiphilic synthetic polymer molecules. We used the lipodisc nanoparticles made of Escherichia coli polar lipids and compared lipodisc nanoparticles that contained the photosensitive protein complex of the sensory rhodopsin with its cognate transducer from the halobacterium Natronomonas pharaonis with empty lipodisc nanoparticles that contained no protein. The lipodisc nanoparticles were characterized by dynamic light scattering, transmission electron microscopy and atomic force microscopy. We found that the diameter of lipodisc nanoparticles was not affected by incorporation of the protein complexes, which makes them a prospective platform for single-molecule studies of membrane proteins.

  • signaling and adaptation modulate the dynamics of the photosensoric complex of Natronomonas pharaonis
    PLOS Computational Biology, 2015
    Co-Authors: Philipp S Orekhov, Johann P. Klare, Armen Y Mulkidjanian, K V Shaitan, Daniel Klose, Martin Engelhard, Heinzjurgen Steinhoff
    Abstract:

    Motile bacteria and archaea respond to chemical and physical stimuli seeking optimal conditions for survival. To this end transmembrane chemo- and photoreceptors organized in large arrays initiate signaling cascades and ultimately regulate the rotation of flagellar motors. To unravel the molecular mechanism of signaling in an archaeal phototaxis complex we performed coarse-grained molecular dynamics simulations of a trimer of receptor/transducer dimers, namely NpSRII/NpHtrII from Natronomonas pharaonis. Signaling is regulated by a reversible methylation mechanism called adaptation, which also influences the level of basal receptor activation. Mimicking two extreme methylation states in our simulations we found conformational changes for the transmembrane region of NpSRII/NpHtrII which resemble experimentally observed light-induced changes. Further downstream in the cytoplasmic domain of the transducer the signal propagates via distinct changes in the dynamics of HAMP1, HAMP2, the adaptation domain and the binding region for the kinase CheA, where conformational rearrangements were found to be subtle. Overall these observations suggest a signaling mechanism based on dynamic allostery resembling models previously proposed for E. coli chemoreceptors, indicating similar properties of signal transduction for archaeal photoreceptors and bacterial chemoreceptors.

  • Structural Information from Spin-Labelled Membrane-Bound Proteins
    Structural Information from Spin-Labels and Intrinsic Paramagnetic Centres in the Biosciences, 2013
    Co-Authors: Johann P. Klare, Heinzjurgen Steinhoff
    Abstract:

    Site-directed spin labelling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for the investigation of the structure and conformational dynamics of biomolecules including membrane proteins under native-like conditions. EPR spectroscopy of the spin-labelled molecules provides information about the spin label side chain mobility, its solvent accessibility, the polarity of its immediate environment and intra- or intermolecular distances to another paramagnetic centre or spin label. This chapter provides an overview of the basics as well as recent progress in SDSL and related EPR techniques. Continuous wave EPR spectra analyses and pulse EPR techniques are reviewed with special emphasis on applications to the membrane-embedded sensory rhodopsin–transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis, the maltose ABC importer MalFGK2 and the mechanosensitive channel MscS.

  • Transmembrane signal transduction in archaeal phototaxis: the sensory rhodopsin II-transducer complex studied by electron paramagnetic resonance spectroscopy.
    European Journal of Cell Biology, 2011
    Co-Authors: Johann P. Klare, Martin Engelhard, Enrica Bordignon, Heinzjurgen Steinhoff
    Abstract:

    Abstract Archaeal photoreceptors, together with their cognate transducer proteins, mediate phototaxis by regulating cell motility through two-component signal transduction pathways. This sensory pathway is closely related to the bacterial chemotactic system, which has been studied in detail during the past 40 years. Structural and functional studies applying site-directed spin labelling and electron paramagnetic resonance spectroscopy on the sensory rhodopsin II/transducer (NpSRII/NpHtrII) complex of Natronomonas pharaonis have yielded insights into the structure, the mechanisms of signal perception, the signal transduction across the membrane and provided information about the subsequent information transfer within the transducer protein towards the components of the intracellular signalling pathway. Here, we provide an overview about the findings of the last decade, which, combined with the wealth of data from research on the Escherichia coli chemotaxis system, served to understand the basic principles microorganisms use to adapt to their environment. We document the time course of a signal being perceived at the membrane, transferred across the membrane and, for the first time, how this signal modulates the dynamic properties of a HAMP domain, a ubiquitous signal transduction module found in various protein classes.

Nadine Mennes - One of the best experts on this subject based on the ideXlab platform.

  • salt driven equilibrium between two conformations in the hamp domain from Natronomonas pharaonis the language of signal transfer
    Journal of Biological Chemistry, 2008
    Co-Authors: Meike Doebber, Johann P. Klare, Martin Engelhard, Nadine Mennes, Enrica Bordignon, Julia Holterhues, Swetlana Martell, Lin Li, Heinzjuergen Steinhoff
    Abstract:

    Abstract HAMP domains (conserved in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) perform their putative function as signal transducing units in diversified environments in a variety of protein families. Here the conformational changes induced by environmental agents, namely salt and temperature, on the structure and function of a HAMP domain of the phototransducer from Natronomonas pharaonis (NpHtrII) in complex with sensory rhodopsin II (NpSRII) were investigated by site-directed spin labeling electron paramagnetic resonance. A series of spin labeled mutants were engineered in NpHtrII157, a truncated analog containing only the first HAMP domain following the transmembrane helix 2. This truncated transducer is shown to be a valid model system for a signal transduction domain anchored to the transmembrane light sensor NpSRII. The HAMP domain is found to be engaged in a “two-state” equilibrium between a highly dynamic (dHAMP) and a more compact (cHAMP) conformation. The structural properties of the cHAMP as proven by mobility, accessibility, and intra-transducer-dimer distance data are in agreement with the four helical bundle NMR model of the HAMP domain from Archaeoglobus fulgidus.

  • expression of the halobacterial transducer protein htrii from Natronomonas pharaonis in escherichia coli
    FEBS Letters, 2007
    Co-Authors: Nadine Mennes, Johann P. Klare, Ramona Schlesinger, Igor Chizhov, Ralf Seidel, Martin Engelhard
    Abstract:

    Abstract Archaeal phototaxis is mediated by sensory rhodopsins which form complexes with their cognate transducers. Whereas the receptors sensory rhodopsin I and sensory rhodopsin II (SRII) have been expressed in Escherichia coli (E. coli) only shortened fragments of HtrII from Natronomonas pharaonis (NpHtrII) are available. Here we describe the heterologous expression of full length NpHtrII which was achieved in yields of up to 0.9 mg per litre cell culture. Gel filtration analysis reveals the tendency of the transducer to form dimers and higher-order oligomers which was also observed when complexed to NpSRII. A circular dichroism (CD) spectrum of NpHtrII is comparable to those obtained for the E. coli chemoreceptors indicating a similar folding with predominantly α-helical structure. NpHtrII dissociates from the NpSRII/HtrII complex with an apparent KD of about 0.6 μM. Photocycle kinetics of the complex is comparable to that obtained for NpSRII in complex with a truncated transducer with slight differences in the M-decay. The data indicate that the heterologously expressed NpHtrII adopt a native like structure, providing the means for elucidating transmembrane signal transduction and activation of microbial signalling cascades.

  • strukturelle und funktionelle untersuchungen des transducers htrii aus Natronomonas pharaonis und von htrii tar chimaren
    2007
    Co-Authors: Nadine Mennes, Martin Engelhard
    Abstract:

    Das halophile Archaebakterium Natronomonas pharaonis besitzt die Fahigkeit zur Phototaxis. Das Lichtsignal wird von dem sensorischen Rhodopsin NpSRII wahrgenommen und auf den Photo-Transducer NpHtrII ubertragen, wodurch es zu einer photophoben Antwort auf blaues Licht kommt. Der NpHtrII wurde erfolgreich heterolog im Bakterium E. coli mit einer Ausbeute von ca. 0,6 – 0,9 mg/L Zellkultur exprimiert und nachfolgend mit biochemischen und biophysikalischen Methoden charakterisiert. Durch ITC- und Photozyklus Messungen konnte eine Bindung des sensorischen Rezeptors NpSRII an NpHtrII nachgewiesen werden. CD- und FTIR Messungen zeigen dabei einen hohen alpha-helikalen Sekundarstruktur-Anteil. Untersuchungen durch Grosenausschluss-Chromatographie lieferten Hinweise auf eine Oligomerisierung der Transducer-Molekule. Mittels ortsspezifischer Spinmarkierung und ESR-Spektroskopie konnte eine coiled-coil bzw. eine 4-Helix-Bundel Struktur fur den Interaktionsbereich des Transducers mit der nachgeschalteten Histidin-Kinase CheA bestatigt werden. Zudem liesen sich erste Hinweise auf reizinduzierte Anderungen der Protein-Dynamik in der Signaldomane beobachten, wodurch erste Hinweise auf den Mechanismus der intra- und intermolekularen Signalweiterleitung durch Konformationsanderungen erhalten wurden. Auf Grundlage der grosen Homologie zwischen der archaebakteriellen Phototaxis und der eubakteriellen Chemotaxis wurden im Weiteren Chimaren entwickelt, die aus dem N-terminalen Bereich des NpHtrII fusioniert mit dem C-terminalen Bereich des Tar-Rezeptors aus E. coli bestehen. Eine Charakterisierung dieser Fusionsproteine wurde ebenfalls mit Hilfe von ESR-Messungen und Funktionalitatsuntersuchungen durchgefuhrt. Zu letzteren gehorten ein in vitro Phosphorylierungs-Assay und ein in vivo Schwimmversuch zur Etablierung eines Modellsystems der Phototaxis im Bakterium E. coli.

  • Strukturelle und funktionelle Untersuchungen des Transducers HtrII aus Natronomonas pharaonis und von HtrII/Tar-Chimären
    2007
    Co-Authors: Nadine Mennes, Martin Engelhard
    Abstract:

    Das halophile Archaebakterium Natronomonas pharaonis besitzt die Fahigkeit zur Phototaxis. Das Lichtsignal wird von dem sensorischen Rhodopsin NpSRII wahrgenommen und auf den Photo-Transducer NpHtrII ubertragen, wodurch es zu einer photophoben Antwort auf blaues Licht kommt. Der NpHtrII wurde erfolgreich heterolog im Bakterium E. coli mit einer Ausbeute von ca. 0,6 – 0,9 mg/L Zellkultur exprimiert und nachfolgend mit biochemischen und biophysikalischen Methoden charakterisiert. Durch ITC- und Photozyklus Messungen konnte eine Bindung des sensorischen Rezeptors NpSRII an NpHtrII nachgewiesen werden. CD- und FTIR Messungen zeigen dabei einen hohen alpha-helikalen Sekundarstruktur-Anteil. Untersuchungen durch Grosenausschluss-Chromatographie lieferten Hinweise auf eine Oligomerisierung der Transducer-Molekule. Mittels ortsspezifischer Spinmarkierung und ESR-Spektroskopie konnte eine coiled-coil bzw. eine 4-Helix-Bundel Struktur fur den Interaktionsbereich des Transducers mit der nachgeschalteten Histidin-Kinase CheA bestatigt werden. Zudem liesen sich erste Hinweise auf reizinduzierte Anderungen der Protein-Dynamik in der Signaldomane beobachten, wodurch erste Hinweise auf den Mechanismus der intra- und intermolekularen Signalweiterleitung durch Konformationsanderungen erhalten wurden. Auf Grundlage der grosen Homologie zwischen der archaebakteriellen Phototaxis und der eubakteriellen Chemotaxis wurden im Weiteren Chimaren entwickelt, die aus dem N-terminalen Bereich des NpHtrII fusioniert mit dem C-terminalen Bereich des Tar-Rezeptors aus E. coli bestehen. Eine Charakterisierung dieser Fusionsproteine wurde ebenfalls mit Hilfe von ESR-Messungen und Funktionalitatsuntersuchungen durchgefuhrt. Zu letzteren gehorten ein in vitro Phosphorylierungs-Assay und ein in vivo Schwimmversuch zur Etablierung eines Modellsystems der Phototaxis im Bakterium E. coli.

Johann P. Klare - One of the best experts on this subject based on the ideXlab platform.

  • Minireview The archaeal sensory rhodopsin II/transducer complex: a model for transmembrane signal transfer
    2020
    Co-Authors: Johann P. Klare, Valentin I. Gordeliy, Georg Bu, Martin Engelhard
    Abstract:

    Archaebacterial photoreceptors mediate phototaxis by regulating cell motility through two-component signalling cascades. Homologs of this sensory pathway occur in all three kingdoms of life, most notably in enteric bacteria in which the chemotaxis has been extensively studied. Recent structural and functional studies on the sensory rhodopsin II/transducer com- plex mediating the photophobic response of Natronomonas pharaonis have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data and the atomic resolution structure of the recep- tor molecule in complex with the transmembrane segment of its cognate transducer provided a model for signal transfer from the receptor to the cytoplasmic side of the transducer. This mecha- nism might also be relevant for eubacterial chemoreceptor sig-

  • Conformational Dynamics of Sensory Rhodopsin II in Nanolipoprotein and Styrene-Maleic Acid Lipid Particles.
    Photochemistry and Photobiology, 2019
    Co-Authors: Wageiha Mosslehy, Johann P. Klare, Natalia Voskoboynikova, Armen Y Mulkidjanian, Alexandr Colbasevici, Daniel Klose, Adrian Ricke, Heinzjurgen Steinhoff
    Abstract:

    : Styrene-maleic acid lipid particles (SMALPs) provide stable water-soluble nanocontainers for lipid-encased membrane proteins. Possible effects of the SMA-stabilized lipid environment on the interaction dynamics between functionally coupled membrane proteins remain to be elucidated. The photoreceptor sensory rhodopsin II, NpSRII and its cognate transducer, NpHtrII, of Natronomonas pharaonis form a transmembrane complex, NpSRII2 /NpHtrII2 that plays a key role in negative phototaxis and provides a unique model system to study the light-induced transfer of a conformational signal between two integral membrane proteins. Photon absorption induces transient structural changes in NpSRII comprising an outward movement of helix F that cause further conformational alterations in NpHtrII. We applied site-directed spin labeling and time-resolved optical and EPR spectroscopy to compare the conformational dynamics of NpSRII2 /NpHtrII2 reconstituted in SMALPs with that of nanolipoprotein particle and liposome preparations. NpSRII and NpSRII2 /NpHtrII2 show similar photocycles in liposomes and nanolipoprotein particles. An accelerated decay of the M photointermediate found for SMALPs can be explained by a high local proton concentration provided by the carboxylic groups of the SMA polymer. Light-induced large-scale conformational changes of NpSRII2 /NpHtrII2 observed in liposomes and nanolipoprotein particles are affected in SMALPs, indicating restrictions of the protein's conformational freedom.

  • signaling and adaptation modulate the dynamics of the photosensoric complex of Natronomonas pharaonis
    PLOS Computational Biology, 2015
    Co-Authors: Philipp S Orekhov, Johann P. Klare, Armen Y Mulkidjanian, K V Shaitan, Daniel Klose, Martin Engelhard, Heinzjurgen Steinhoff
    Abstract:

    Motile bacteria and archaea respond to chemical and physical stimuli seeking optimal conditions for survival. To this end transmembrane chemo- and photoreceptors organized in large arrays initiate signaling cascades and ultimately regulate the rotation of flagellar motors. To unravel the molecular mechanism of signaling in an archaeal phototaxis complex we performed coarse-grained molecular dynamics simulations of a trimer of receptor/transducer dimers, namely NpSRII/NpHtrII from Natronomonas pharaonis. Signaling is regulated by a reversible methylation mechanism called adaptation, which also influences the level of basal receptor activation. Mimicking two extreme methylation states in our simulations we found conformational changes for the transmembrane region of NpSRII/NpHtrII which resemble experimentally observed light-induced changes. Further downstream in the cytoplasmic domain of the transducer the signal propagates via distinct changes in the dynamics of HAMP1, HAMP2, the adaptation domain and the binding region for the kinase CheA, where conformational rearrangements were found to be subtle. Overall these observations suggest a signaling mechanism based on dynamic allostery resembling models previously proposed for E. coli chemoreceptors, indicating similar properties of signal transduction for archaeal photoreceptors and bacterial chemoreceptors.

  • Site-directed spin labeling EPR spectroscopy in protein research.
    Biological Chemistry, 2013
    Co-Authors: Johann P. Klare
    Abstract:

    Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has emerged as an efficient tool to elucidate the structure and the conformational dynamics of proteins under conditions close to the native state. This review article summarizes the basics as well as the recent progress in SDSL and EPR methods, especially for investigations on protein structure, protein function, and interaction of proteins with other proteins or nucleic acids. Labeling techniques as well as EPR methods are introduced and exemplified with applications to systems that have been studied in the author's laboratory in the past 15 years, headmost the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis. Further examples underline the application of SDSL EPR spectroscopy to answer specific questions about the system under investigation, such as the nature and influence of interactions of proteins with other proteins or nucleic acids. Finally, it is discussed how SDSL EPR can be combined with other biophysical techniques to combine the strengths of the different methodologies.

  • Structural Information from Spin-Labelled Membrane-Bound Proteins
    Structural Information from Spin-Labels and Intrinsic Paramagnetic Centres in the Biosciences, 2013
    Co-Authors: Johann P. Klare, Heinzjurgen Steinhoff
    Abstract:

    Site-directed spin labelling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for the investigation of the structure and conformational dynamics of biomolecules including membrane proteins under native-like conditions. EPR spectroscopy of the spin-labelled molecules provides information about the spin label side chain mobility, its solvent accessibility, the polarity of its immediate environment and intra- or intermolecular distances to another paramagnetic centre or spin label. This chapter provides an overview of the basics as well as recent progress in SDSL and related EPR techniques. Continuous wave EPR spectra analyses and pulse EPR techniques are reviewed with special emphasis on applications to the membrane-embedded sensory rhodopsin–transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis, the maltose ABC importer MalFGK2 and the mechanosensitive channel MscS.

Naoki Kamo - One of the best experts on this subject based on the ideXlab platform.

  • Probing the Cl--pumping photocycle of pharaonis halorhodopsin: Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle.
    Biochimica et Biophysica Acta, 2015
    Co-Authors: Takashi Kikukawa, Kunio Ihara, Naoki Kamo, Masakatsu Kamiya, Tomoyasu Aizawa, Chikara Kusakabe, Asami Kokubo, Takashi Tsukamoto, Makoto Demura
    Abstract:

    Abstract Halorhodopsin (HR) functions as a light-driven inward Cl − pump. The Cl − transfer process of HR from Natronomonas pharaonis (NpHR) was examined utilizing a mutant strain, KM-1, which expresses large amount of NpHR in a complex with the carotenoid bacterioruberin (Brub). When Cl − was added to unphotolyzed Cl − -free NpHR–Brub complex, Brub caused the absorption spectral change in response to the Cl − binding to NpHR through the altered electrostatic environment and/or distortion of its own configuration. During the Cl − -puming photocycle, on the other hand, oppositely directed spectral change of Brub appeared during the O intermediate formation and remained until the decay of the last intermediate NpHR′. These results indicate that Cl − is released into the cytoplasmic medium during the N to O transition, and that the subsequent NpHR′ still maintains an altered protein conformation while another Cl − already binds in the vicinity of the Schiff base. Using the cell envelope vesicles, the effect of the interior negative membrane potential on the photocycle was examined. The prominent effect appeared in the shift of the N–O quasi-equilibrium toward N, supporting Cl − release during the N to O transition. The membrane potential had a much larger effect on the Cl − transfer in the cytoplasmic half channel compared to that in the extracellular half channel. This result may reflect the differences in dielectric constants and/or lengths of the pathways for Cl − transfers during N to O and O to NpHR′ transitions.

  • the effects of chloride ion binding on the photochemical properties of sensory rhodopsin ii from Natronomonas pharaonis
    Journal of Photochemistry and Photobiology B-biology, 2014
    Co-Authors: Makoto Demura, Naoki Kamo, Takashi Kikukawa, Jun Tamogami, Toshifumi Nara, Katsunori Iwano, Atsushi Matsuyama
    Abstract:

    Abstract Whether Cl− binds to the sensory rhodopsin II from Natronomonas pharaonis (NpSRII) that acts as a negative phototaxis receptor remains controversial. Two previous photoelectrochemical studies using SnO2 transparent electrodes and ATR–FTIR demonstrated that Cl− binding affects the photoinduced proton release from Asp193 in phospholipid (PC)-reconstituted NpSRII (Iwamoto et al., 2004; Kitade et al., 2009). In this study, we investigated the effects of Cl− on the photochemistry of NpSRII solubilized by detergent (DDM). Even under these conditions, Cl− could bind to NpSRII with a Kd of approximately 250 mM; this value is ∼10-fold larger than that in the PC membrane. The binding of Cl− to NpSRII depended on the pH of the medium. In addition, Cl− binding induced the following effects: (1) a small red shift in the absorbance spectrum originating from the partial protonation of Asp75, (2) the formation of an interaction through a hydrogen-bonding network between Asp75 and Asp193, which is a proton-releasing residue, (3) several changes of the kinetic behavior of the photocycle, and (4) a photoinduced initial proton release from Asp193. The pKa values of Asp193 at various Cl− concentrations were also estimated. Based on the difference between the pKa values of Asp193 in Cl− bound and unbound NpSRII, the distance between the bound Cl− and Asp193 was determined to be approximately 6.1 A, which agrees with the value estimated from the crystal structure presented by Royant et al. (2001). Therefore, the Cl− binding site affecting the photochemical properties of NpSRII is identical to the site proposed by Royant et al. (2001). This assignment was also supported by an experiment that introduced a mutation at Arg72.

  • role of thr218 in the light driven anion pump halorhodopsin from Natronomonas pharaonis
    Biochemistry, 2013
    Co-Authors: Kousuke Shibasaki, Naoki Kamo, Hiroaki Shigemura, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Makoto Demura
    Abstract:

    Halorhodopsin (HR) is an inward-directed light-driven halogen ion pump, and NpHR is a HR from Natronomonas pharaonis. Unphotolyzed NpHR binds halogen ion in the vicinity of the Schiff base, which links retinal to Lys256. This halogen ion is transported during the photocycle. We made various mutants of Thr218, which is located one half-turn up from the Schiff base to the cytoplasm (CP) channel, and analyzed the photocycle using a sequential irreversible model. Four photochemically defined intermediates (Pi, i = 1–4) were adequate to describe the photocycle. The third component, P3, was a quasi-equilibrium complex between the N and O intermediates, where a N ↔ O + Cl– equilibrium was attained. The Kd,N↔O values of this equilibrium for various mutants were determined, and the value of Thr (wild type) was the highest. The partial molar volume differences between N and O, ΔVN→O, were estimated from the pressure dependence of Kd,N↔O. A comparison between Kd,N↔O and ΔVN→O led to the conclusion that water entry b...

  • thermodynamic parameters of anion binding to halorhodopsin from Natronomonas pharaonis by isothermal titration calorimetry
    Biophysical Chemistry, 2013
    Co-Authors: Saori Hayashi, Kazumi Shimono, Makoto Demura, Naoki Kamo, Takashi Kikukawa, Jun Tamogami, Haruka Okamoto, Seiji Miyauchi, Toshifumi Nara
    Abstract:

    Abstract Halorhodopsin (HR), an inwardly directed, light-driven anion pump, is a membrane protein in halobacterial cells that contains the chromophore retinal, which binds to a specific lysine residue forming the Schiff base. An anion binds to the extracellular binding site near the Schiff base, and illumination makes this anion go to the intracellular channel, followed by its release from the protein and re-uptake from the opposite side. The thermodynamic properties of the anion binding in the dark, which have not been previously estimated, are determined using isothermal titration calorimetry (ITC). For Cl− as a typical substrate of HR from Natronomonas pharaonis, ΔG = −RT ln(1/Kd) = − 15.9 kJ/mol, ΔH = − 21.3 kJ/mol and TΔS = − 5.4 kJ/mol at 35 °C, where Kd represents the dissociation constant. In the dark, Kd values have been determined by the usual spectroscopic methods and are in agreement with the values estimated by ITC here. Opsin showed no Cl− binding ability, and the deprotonated Schiff base showed weak binding affinity, suggesting the importance of the positively charged protonated Schiff base for the anion binding.

  • Photo-induced bleaching of sensory rhodopsin II (phoborhodopsin) from Halobacterium salinarum by hydroxylamine: Identification of the responsible intermediates
    Journal of Photochemistry and Photobiology B-biology, 2011
    Co-Authors: Jun Tamogami, Makoto Demura, Takashi Kikukawa, Toshifumi Nara, Yoichi Ikeda, Naoki Kamo
    Abstract:

    Abstract Sensory rhodopsin II from Halobacterium salinarum (HsSRII) is a retinal protein in which retinal binds to a specific lysine residue through a Schiff base. Here, we investigated the photobleaching of HsSRII in the presence of hydroxylamine. For identification of intermediate(s) attacked by hydroxylamine, we employed the flash-induced bleaching method. In order to change the concentration of intermediates, such as M- and O-intermediates, experiments were performed under varying flashlight intensities and concentrations of azide that accelerated only the M-decay. We found the proportional relationship between the bleaching rate and area under the concentration–time curve of M, indicating a preferential attack of hydroxylamine on M. Since hydroxylamine is a water-soluble reagent, we hypothesize that for M, hydrophilicity or water-accessibility increases specifically in the moiety of Schiff base. Thus, hydroxylamine bleaching rates may be an indication of conformational changes near the Schiff base. We also considered the possibility that azide may induce a small conformational change around the Schiff base. We compared the hydroxylamine susceptibility between HsSRII and NpSRII (SRII from Natronomonas pharaonis ) and found that the M of HsSRII is about three times more susceptible than that of the stable NpSRII. In addition, long illumination to HsSRII easily produced M-like photoproduct, P370. We thus infer that the instability of HsSRII under illumination may be related to this increase of hydrophilicity at M and P370.