Sticholysin I (StI) is a toxin produced by the sea anemone Stichodactyla helianthus and belonging... more Sticholysin I (StI) is a toxin produced by the sea anemone Stichodactyla helianthus and belonging to the actinoporins family. Upon binding to sphingomyelin-containing membranes StI forms oligomeric pores, thereby leading to cell death. According to recent controversial experimental evidences, the pore architecture of actinoporins is a debated topic. Here, we investigated the StI topology in membranes by site-directed spin labeling and electron paramagnetic resonance spectroscopy. The results reveal that StI in membrane exhibits an oligomeric architecture with heterogeneous stoichiometry of predominantly eight or nine protomers, according to the available structural models. The StI topology resembles the conic pore structure reported for the actinoporin fragaceatoxin C. Our data show that StI coexists in two membrane-associated conformations, with the N-terminal segment either attached to the protein core or inserted in the membrane 2 forming the pore. This finding suggests a 'pre-pore' to 'pore' transition determined by a conformational change that detaches the N-terminal segment. Highlights: The topology of membrane bound StI resembles a conical pore model Membrane bound StI adopts 'pre-pore' and 'pore' conformations
<p>The inter-fluorophore distances obtained by determination of the FRET efficiencies and c... more <p>The inter-fluorophore distances obtained by determination of the FRET efficiencies and calculation using equation (2) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#s4" target="_blank">Materials and Methods</a>) are compared to the respective Cα-Cα distances from the crystal structure (pdb: 1GO3).</p
<p>Distance distributions resulting from the DEER experiments are shown in gray. Simulated ... more <p>Distance distributions resulting from the DEER experiments are shown in gray. Simulated distance distributions were obtained from the RLA (red) and from a rotamer selection according to the crystal structures of spin labeled T4 lysozyme (green) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#pone.0039492-Fleissner1" target="_blank">[37]</a>. The inset in the upper left panel shows the two dihedral angles X1 and X2 discussed in the text (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#pone.0039492.s007" target="_blank">Figure S7</a>).</p
<p>deduced from <i>in vacuo</i> MD simulations (A), <i>in aqua</i> ... more <p>deduced from <i>in vacuo</i> MD simulations (A), <i>in aqua</i> MD simulations (B) and MC sampling (C) in Rpo4/7 (as magenta/blue ribbons). Clouds envelope 99.5% (gray) and 50% (red) of the total probability. Rotamers (D, depicted as sticks) calculated from a given rotamer library <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#pone.0039492-Polyhach1" target="_blank">[29]</a> span 99.5% of the population. <i>In aqua</i> MD simulations have not been performed for the fluorophore labels as they are currently computationally to intensive for standard hardware. A pre-calculated FRET-label rotamer library is currently not available.</p
The light-gated dimeric cation channel channelrhodopsin-2 (ChR2) has been established as one of t... more The light-gated dimeric cation channel channelrhodopsin-2 (ChR2) has been established as one of the most important optogenetic tools. During its functional cycle, ChR2 undergoes conformational changes, the most prominent ones include a movement of transmembrane helix B. In the present work, we assign this movement to a trapped photocycle intermediate using DEER spectroscopy combined with sample illumination inside the microwave resonator, allowing trapping and relaxation of defined ChR2 intermediates at different temperatures between 180 and 278 K. Intradimer distances measured between spin-labeled positions 79 located in helix B of ChR2 in the dark state and upon light activation and relaxation at 180 K were similar. In contrast, light activation at 180 K and 30 min relaxation at between 230 and 255 K results in significant changes of the distance distribution. We show that the light-induced movement of helix B is correlated with the presence of the P480 state of ChR2. We hypothesi...
Background-Hypoxic vasodilation is a physiological response to low oxygen tension that increases ... more Background-Hypoxic vasodilation is a physiological response to low oxygen tension that increases blood supply to match metabolic demands. Although this response has been characterized for Ͼ100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin in the heart can reduce nitrite to nitric oxide (NO ⅐) and thereby contribute to cardiomyocyte NO ⅐ signaling during ischemia. On the basis of recent observations that myoglobin is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular myoglobin to form NO ⅐. Methods and Results-We show in the present study that myoglobin is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO ⅐ from nitrite reduction by deoxygenated myoglobin activates canonical soluble guanylate cyclase/cGMP signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO ⅐ , and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin. Hypoxic vasodilation studies in myoglobin and endothelial and inducible NO synthase knockout models suggest that only myoglobin contributes to systemic hypoxic vasodilatory responses in mice. Conclusions-Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO ⅐ via the heme globin myoglobin enhances blood flow and matches O 2 supply to increased metabolic demands under hypoxic conditions. (Circulation. 2012;126:325-334.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2020
Amphiphilic maleic acid-containing copolymers account for a recent methodical breakthrough in the... more Amphiphilic maleic acid-containing copolymers account for a recent methodical breakthrough in the study of membrane proteins. Their application enables a detergent-free extraction of membrane proteins from lipid bilayers, yielding stable water-soluble, discoidal lipid bilayer particles with incorporated proteins, which are wrapped with copolymers. Although many studies confirm the potential of this approach for membrane protein research, the interactions between the maleic acid-containing copolymers and extracted lipids, as well as possible effects of the copolymers on lipid-embedded proteins deserve further scrutinization. Here, we combine electron paramagnetic resonance spectroscopy and coarse-grain molecular dynamics simulations to compare the distribution and dynamics of lipids in lipid particles of phospholipid bilayers encased either by an aliphatic diisobutylene/maleic acid copolymer (DIBMALPs) or by an aromatic styrene/maleic acid copolymer (SMALPs). Nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels experience restrictions of their reorientational motion depending on the type of encasing copolymer. The dynamics of the lipids was less constrained in DIBMALPs than in SMALPs with the affinity of spin labeled lipids to the polymeric rim being more pronounced in SMALPs.
Vacuolar-type H(+) -ATPases (V-ATPases) have gained recent attention as highly promising anticanc... more Vacuolar-type H(+) -ATPases (V-ATPases) have gained recent attention as highly promising anticancer drug targets, and therefore detailed structural analyses and studies of inhibitor interactions are very important research objectives. Spin labeling of the V-ATPase holoenzyme from the tobacco hornworm Manduca sexta and V-ATPase in isolated yeast (Saccharomyces cerevisiae) vacuoles was accomplished by two novel methods involving the covalent binding of a (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) derivative of N,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-dicyclohexylcarbodiimide (DCC) to the essential glutamate residue in the active site and the noncovalent interaction of a radical analogue of the highly potent inhibitor archazolid, a natural product from myxobacteria. Both complexes were evaluated in detail by electron paramagnetic resonance (EPR) spectroscopic studies and double electron-electron resonance (DEER) measurements, revealing insight into the inhibitor binding mode, dynamics, and stoichiometry as well as into the structure of the central functional subunit c of these medicinally important hetero-multimeric proton-translocating proteins. This study also demonstrates the usefulness of natural product derived spin labels as tools in medicinal chemistry.
The function of a living cell, independent of we are talking about a prokaryotic singlecellular o... more The function of a living cell, independent of we are talking about a prokaryotic singlecellular organism or a cell in the context of an complex organism like a human, depends on intricate and balanced interaction between its components. Proteins are playing a central role in this complex cellular interaction network: Proteins interact with nucleic acids, with membranes of all cellular compartments, and, what will be in the focus of this article, with other proteins. Proteins interact to form functional units, to transmit signals for example perceived at the surface of the cell to cytoplasmic or nuclear components, or to target them to specific locations. Thus, the study of protein-protein interactions on the molecular level provides insights into the basic functional concepts of living cells and emerged as a wide field of intense research, steadily developing with the introduction of new and refined biochemical and biophysical methods.
<p>(A) Distance distributions for the spin label pairs Rpo4<sup>C36R1</sup>/Rpo... more <p>(A) Distance distributions for the spin label pairs Rpo4<sup>C36R1</sup>/Rpo7<sup>V49R1</sup> (left), Rpo4<sup>C36R1</sup>/Rpo7<sup>S65R1</sup> (center) and Rpo4<sup>C36R1</sup>/Rpo7<sup>K123R1</sup> (right). Distance distributions obtained from the DEER experiments are shown in gray, the results of the <i>in vacuo</i> MD, <i>in aqua</i> MD and MC simulation are shown in dark blue, cyan and red, respectively. Cα-Cα distances obtained from the crystal structure are marked by gray dashed lines. (B) Results of the <i>in vacuo</i> MD simulation for Rpo4<sup>C36R1</sup>/Rpo7<sup>V49R1</sup>. Left panel: Distance trajectory; center and right panel: Volume sampled by the spin labels over simulation time for labels Rpo4<sup>C36R1</sup> and Rpo7<sup>V49R1</sup>, respectively. (C) Corresponding results for the <i>in aqua</i> MD simulation. (D) Results of the corresponding MC samplings. For the <i>in vacuo</i> MD simulations and the MC samplings of the other spin label pairs the distance trajectories and volume plots are given in the Supplementary Information, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#pone.0039492.s005" target="_blank">Figure S5</a>. The corresponding data for the <i>in aqua</i> MD simulation are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#pone.0039492.s006" target="_blank">Figure S6</a>.</p
Site-directed spin labelling (SDSL) combined with electron paramagnetic resonance (EPR) spectrosc... more Site-directed spin labelling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy is an efficient method to study the structure and the conformational dynamics of proteins. In particular, long range distance measurements (up to ∼100 A) between pairs of spin labels by pulse EPR methods enable quantitative analysis of conformational equilibrium dynamics and allow identification and characterization of conformational changes in the course of a proteins function. This review summarizes contributions SDSL EPR made to understanding of how the structure and dynamics of a specific group of GTP-hydrolyzing proteins, G proteins activated by nucleotide-dependent dimerization (GADs), change during their functional cycle.
We have performed a detailed study of the magnetic and electronic properties of highly manganese-... more We have performed a detailed study of the magnetic and electronic properties of highly manganese-doped β-NaGdF4 and β-NaEuF4 nanoparticles with a narrow size distribution. XPS as well as XRF experi...
Transmembrane signaling proteins play a crucial role in the transduction of information across ce... more Transmembrane signaling proteins play a crucial role in the transduction of information across cell membranes. One function of regulated intramembrane proteolysis (RIP) is the release of signaling factors from transmembrane proteins. To study the role of transmembrane domains (TMDs) in modulating structure and activity of released signaling factors, we purified heterologously expressed human transmembrane proteins and their proteolytic processing products from Escherichia coli. Here we show that CD74 and TNFα are heme binding proteins. Heme coordination depends on both a cysteine residue proximal to the membrane and on the oligomerization of the TMD. Furthermore, we show that the various processing products have different modes of heme coordination. We suggest that RIP changes the mode of heme binding of these proteins and generates heme binding peptides with yet unexplored functions. The identification of a RIP modulated cofactor binding of transmembrane signaling proteins sheds ne...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Styrene-maleic acid lipid particles (SMALPs) provide stable water-soluble nanocontainers for lipi... more 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 Accepted Article This article is protected by copyright. All rights reserved. transducer, NpHtrII, of Natronomonas pharaonis form a transmembrane complex, NpSRII 2 /NpHtrII 2 , 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 NpSRII 2 /NpHtrII 2 reconstituted in SMALPs with that of nanolipoprotein particle and liposome preparations. NpSRII and NpSRII 2 /NpHtrII 2 show similar photocyles 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 NpSRII 2 /NpHtrII 2 observed in liposomes and nanolipoprotein particles are affected in SMALPs, indicating restrictions of the protein's conformational freedom.
In linear photosynthetic electron transport, ferredoxin:NADP(H) oxidoreductase (FNR) transfers el... more In linear photosynthetic electron transport, ferredoxin:NADP(H) oxidoreductase (FNR) transfers electrons from ferredoxin (Fd) to NADP +. Both NADPH and reduced Fd (Fd red), are required for reductive assimilation and light/dark activation/deactivation of enzymes. FNR is therefore a hub, connecting photosynthetic electron transport to chloroplast redox metabolism. A correlation between FNR content and tolerance to oxidative stress is well established, although the precise mechanism remains unclear. We investigated the impact of altered FNR content and localization on electron transport and superoxide radical evolution in isolated thylakoids, and probed resulting changes in redox homeostasis, expression of oxidative stress markers and tolerance to high light in planta. Our data indicate that the ratio of Fd red to FNR is critical, with either too much or too little FNR potentially leading to increased superoxide production, and perception of oxidative stress at the level of gene transcription. In FNR overexpressing plants, which show more reduced NADP(H) and glutathione pools, improved tolerance to highlight stress indicates that disturbance of chloroplast redox poise and increased free radical generation may help "prime" the plant and induce protective mechanisms. In fnr1 knockouts , the NADP(H) and glutathione pools are more oxidized relative to the wt, and the photoprotective effect is absent despite perception of oxidative stress at the level of gene transcription.
Sticholysin I (StI) is a toxin produced by the sea anemone Stichodactyla helianthus and belonging... more Sticholysin I (StI) is a toxin produced by the sea anemone Stichodactyla helianthus and belonging to the actinoporins family. Upon binding to sphingomyelin-containing membranes StI forms oligomeric pores, thereby leading to cell death. According to recent controversial experimental evidences, the pore architecture of actinoporins is a debated topic. Here, we investigated the StI topology in membranes by site-directed spin labeling and electron paramagnetic resonance spectroscopy. The results reveal that StI in membrane exhibits an oligomeric architecture with heterogeneous stoichiometry of predominantly eight or nine protomers, according to the available structural models. The StI topology resembles the conic pore structure reported for the actinoporin fragaceatoxin C. Our data show that StI coexists in two membrane-associated conformations, with the N-terminal segment either attached to the protein core or inserted in the membrane 2 forming the pore. This finding suggests a 'pre-pore' to 'pore' transition determined by a conformational change that detaches the N-terminal segment. Highlights: The topology of membrane bound StI resembles a conical pore model Membrane bound StI adopts 'pre-pore' and 'pore' conformations
<p>The inter-fluorophore distances obtained by determination of the FRET efficiencies and c... more <p>The inter-fluorophore distances obtained by determination of the FRET efficiencies and calculation using equation (2) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#s4" target="_blank">Materials and Methods</a>) are compared to the respective Cα-Cα distances from the crystal structure (pdb: 1GO3).</p
<p>Distance distributions resulting from the DEER experiments are shown in gray. Simulated ... more <p>Distance distributions resulting from the DEER experiments are shown in gray. Simulated distance distributions were obtained from the RLA (red) and from a rotamer selection according to the crystal structures of spin labeled T4 lysozyme (green) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#pone.0039492-Fleissner1" target="_blank">[37]</a>. The inset in the upper left panel shows the two dihedral angles X1 and X2 discussed in the text (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#pone.0039492.s007" target="_blank">Figure S7</a>).</p
<p>deduced from <i>in vacuo</i> MD simulations (A), <i>in aqua</i> ... more <p>deduced from <i>in vacuo</i> MD simulations (A), <i>in aqua</i> MD simulations (B) and MC sampling (C) in Rpo4/7 (as magenta/blue ribbons). Clouds envelope 99.5% (gray) and 50% (red) of the total probability. Rotamers (D, depicted as sticks) calculated from a given rotamer library <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#pone.0039492-Polyhach1" target="_blank">[29]</a> span 99.5% of the population. <i>In aqua</i> MD simulations have not been performed for the fluorophore labels as they are currently computationally to intensive for standard hardware. A pre-calculated FRET-label rotamer library is currently not available.</p
The light-gated dimeric cation channel channelrhodopsin-2 (ChR2) has been established as one of t... more The light-gated dimeric cation channel channelrhodopsin-2 (ChR2) has been established as one of the most important optogenetic tools. During its functional cycle, ChR2 undergoes conformational changes, the most prominent ones include a movement of transmembrane helix B. In the present work, we assign this movement to a trapped photocycle intermediate using DEER spectroscopy combined with sample illumination inside the microwave resonator, allowing trapping and relaxation of defined ChR2 intermediates at different temperatures between 180 and 278 K. Intradimer distances measured between spin-labeled positions 79 located in helix B of ChR2 in the dark state and upon light activation and relaxation at 180 K were similar. In contrast, light activation at 180 K and 30 min relaxation at between 230 and 255 K results in significant changes of the distance distribution. We show that the light-induced movement of helix B is correlated with the presence of the P480 state of ChR2. We hypothesi...
Background-Hypoxic vasodilation is a physiological response to low oxygen tension that increases ... more Background-Hypoxic vasodilation is a physiological response to low oxygen tension that increases blood supply to match metabolic demands. Although this response has been characterized for Ͼ100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin in the heart can reduce nitrite to nitric oxide (NO ⅐) and thereby contribute to cardiomyocyte NO ⅐ signaling during ischemia. On the basis of recent observations that myoglobin is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular myoglobin to form NO ⅐. Methods and Results-We show in the present study that myoglobin is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO ⅐ from nitrite reduction by deoxygenated myoglobin activates canonical soluble guanylate cyclase/cGMP signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO ⅐ , and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin. Hypoxic vasodilation studies in myoglobin and endothelial and inducible NO synthase knockout models suggest that only myoglobin contributes to systemic hypoxic vasodilatory responses in mice. Conclusions-Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO ⅐ via the heme globin myoglobin enhances blood flow and matches O 2 supply to increased metabolic demands under hypoxic conditions. (Circulation. 2012;126:325-334.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2020
Amphiphilic maleic acid-containing copolymers account for a recent methodical breakthrough in the... more Amphiphilic maleic acid-containing copolymers account for a recent methodical breakthrough in the study of membrane proteins. Their application enables a detergent-free extraction of membrane proteins from lipid bilayers, yielding stable water-soluble, discoidal lipid bilayer particles with incorporated proteins, which are wrapped with copolymers. Although many studies confirm the potential of this approach for membrane protein research, the interactions between the maleic acid-containing copolymers and extracted lipids, as well as possible effects of the copolymers on lipid-embedded proteins deserve further scrutinization. Here, we combine electron paramagnetic resonance spectroscopy and coarse-grain molecular dynamics simulations to compare the distribution and dynamics of lipids in lipid particles of phospholipid bilayers encased either by an aliphatic diisobutylene/maleic acid copolymer (DIBMALPs) or by an aromatic styrene/maleic acid copolymer (SMALPs). Nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels experience restrictions of their reorientational motion depending on the type of encasing copolymer. The dynamics of the lipids was less constrained in DIBMALPs than in SMALPs with the affinity of spin labeled lipids to the polymeric rim being more pronounced in SMALPs.
Vacuolar-type H(+) -ATPases (V-ATPases) have gained recent attention as highly promising anticanc... more Vacuolar-type H(+) -ATPases (V-ATPases) have gained recent attention as highly promising anticancer drug targets, and therefore detailed structural analyses and studies of inhibitor interactions are very important research objectives. Spin labeling of the V-ATPase holoenzyme from the tobacco hornworm Manduca sexta and V-ATPase in isolated yeast (Saccharomyces cerevisiae) vacuoles was accomplished by two novel methods involving the covalent binding of a (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) derivative of N,N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-dicyclohexylcarbodiimide (DCC) to the essential glutamate residue in the active site and the noncovalent interaction of a radical analogue of the highly potent inhibitor archazolid, a natural product from myxobacteria. Both complexes were evaluated in detail by electron paramagnetic resonance (EPR) spectroscopic studies and double electron-electron resonance (DEER) measurements, revealing insight into the inhibitor binding mode, dynamics, and stoichiometry as well as into the structure of the central functional subunit c of these medicinally important hetero-multimeric proton-translocating proteins. This study also demonstrates the usefulness of natural product derived spin labels as tools in medicinal chemistry.
The function of a living cell, independent of we are talking about a prokaryotic singlecellular o... more The function of a living cell, independent of we are talking about a prokaryotic singlecellular organism or a cell in the context of an complex organism like a human, depends on intricate and balanced interaction between its components. Proteins are playing a central role in this complex cellular interaction network: Proteins interact with nucleic acids, with membranes of all cellular compartments, and, what will be in the focus of this article, with other proteins. Proteins interact to form functional units, to transmit signals for example perceived at the surface of the cell to cytoplasmic or nuclear components, or to target them to specific locations. Thus, the study of protein-protein interactions on the molecular level provides insights into the basic functional concepts of living cells and emerged as a wide field of intense research, steadily developing with the introduction of new and refined biochemical and biophysical methods.
<p>(A) Distance distributions for the spin label pairs Rpo4<sup>C36R1</sup>/Rpo... more <p>(A) Distance distributions for the spin label pairs Rpo4<sup>C36R1</sup>/Rpo7<sup>V49R1</sup> (left), Rpo4<sup>C36R1</sup>/Rpo7<sup>S65R1</sup> (center) and Rpo4<sup>C36R1</sup>/Rpo7<sup>K123R1</sup> (right). Distance distributions obtained from the DEER experiments are shown in gray, the results of the <i>in vacuo</i> MD, <i>in aqua</i> MD and MC simulation are shown in dark blue, cyan and red, respectively. Cα-Cα distances obtained from the crystal structure are marked by gray dashed lines. (B) Results of the <i>in vacuo</i> MD simulation for Rpo4<sup>C36R1</sup>/Rpo7<sup>V49R1</sup>. Left panel: Distance trajectory; center and right panel: Volume sampled by the spin labels over simulation time for labels Rpo4<sup>C36R1</sup> and Rpo7<sup>V49R1</sup>, respectively. (C) Corresponding results for the <i>in aqua</i> MD simulation. (D) Results of the corresponding MC samplings. For the <i>in vacuo</i> MD simulations and the MC samplings of the other spin label pairs the distance trajectories and volume plots are given in the Supplementary Information, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#pone.0039492.s005" target="_blank">Figure S5</a>. The corresponding data for the <i>in aqua</i> MD simulation are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039492#pone.0039492.s006" target="_blank">Figure S6</a>.</p
Site-directed spin labelling (SDSL) combined with electron paramagnetic resonance (EPR) spectrosc... more Site-directed spin labelling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy is an efficient method to study the structure and the conformational dynamics of proteins. In particular, long range distance measurements (up to ∼100 A) between pairs of spin labels by pulse EPR methods enable quantitative analysis of conformational equilibrium dynamics and allow identification and characterization of conformational changes in the course of a proteins function. This review summarizes contributions SDSL EPR made to understanding of how the structure and dynamics of a specific group of GTP-hydrolyzing proteins, G proteins activated by nucleotide-dependent dimerization (GADs), change during their functional cycle.
We have performed a detailed study of the magnetic and electronic properties of highly manganese-... more We have performed a detailed study of the magnetic and electronic properties of highly manganese-doped β-NaGdF4 and β-NaEuF4 nanoparticles with a narrow size distribution. XPS as well as XRF experi...
Transmembrane signaling proteins play a crucial role in the transduction of information across ce... more Transmembrane signaling proteins play a crucial role in the transduction of information across cell membranes. One function of regulated intramembrane proteolysis (RIP) is the release of signaling factors from transmembrane proteins. To study the role of transmembrane domains (TMDs) in modulating structure and activity of released signaling factors, we purified heterologously expressed human transmembrane proteins and their proteolytic processing products from Escherichia coli. Here we show that CD74 and TNFα are heme binding proteins. Heme coordination depends on both a cysteine residue proximal to the membrane and on the oligomerization of the TMD. Furthermore, we show that the various processing products have different modes of heme coordination. We suggest that RIP changes the mode of heme binding of these proteins and generates heme binding peptides with yet unexplored functions. The identification of a RIP modulated cofactor binding of transmembrane signaling proteins sheds ne...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Styrene-maleic acid lipid particles (SMALPs) provide stable water-soluble nanocontainers for lipi... more 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 Accepted Article This article is protected by copyright. All rights reserved. transducer, NpHtrII, of Natronomonas pharaonis form a transmembrane complex, NpSRII 2 /NpHtrII 2 , 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 NpSRII 2 /NpHtrII 2 reconstituted in SMALPs with that of nanolipoprotein particle and liposome preparations. NpSRII and NpSRII 2 /NpHtrII 2 show similar photocyles 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 NpSRII 2 /NpHtrII 2 observed in liposomes and nanolipoprotein particles are affected in SMALPs, indicating restrictions of the protein's conformational freedom.
In linear photosynthetic electron transport, ferredoxin:NADP(H) oxidoreductase (FNR) transfers el... more In linear photosynthetic electron transport, ferredoxin:NADP(H) oxidoreductase (FNR) transfers electrons from ferredoxin (Fd) to NADP +. Both NADPH and reduced Fd (Fd red), are required for reductive assimilation and light/dark activation/deactivation of enzymes. FNR is therefore a hub, connecting photosynthetic electron transport to chloroplast redox metabolism. A correlation between FNR content and tolerance to oxidative stress is well established, although the precise mechanism remains unclear. We investigated the impact of altered FNR content and localization on electron transport and superoxide radical evolution in isolated thylakoids, and probed resulting changes in redox homeostasis, expression of oxidative stress markers and tolerance to high light in planta. Our data indicate that the ratio of Fd red to FNR is critical, with either too much or too little FNR potentially leading to increased superoxide production, and perception of oxidative stress at the level of gene transcription. In FNR overexpressing plants, which show more reduced NADP(H) and glutathione pools, improved tolerance to highlight stress indicates that disturbance of chloroplast redox poise and increased free radical generation may help "prime" the plant and induce protective mechanisms. In fnr1 knockouts , the NADP(H) and glutathione pools are more oxidized relative to the wt, and the photoprotective effect is absent despite perception of oxidative stress at the level of gene transcription.
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