Refine
Year of publication
Document Type
- Article (135)
- Preprint (23)
- Doctoral Thesis (5)
- Bachelor Thesis (1)
- Part of a Book (1)
- Conference Proceeding (1)
- Review (1)
Has Fulltext
- yes (167)
Is part of the Bibliography
- no (167)
Keywords
- Research article (8)
- Cryoelectron microscopy (6)
- Biophysics and structural biology (4)
- X-ray crystallography (4)
- mitochondria (4)
- Bacterial structural biology (3)
- Biochemistry (3)
- Bioenergetics (3)
- Cellular microbiology (3)
- Enzyme mechanisms (3)
Institute
- MPI für Biophysik (167) (remove)
Membrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the "catalytic core" conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer.
The lysosomal ABC transporter associated with antigen processing-like (TAPL, ABCB9) acts as an ATP-dependent polypeptide transporter with broad length selectivity. To characterize in detail its substrate specificity, a procedure for functional reconstitution of human TAPL was developed. By intensive screening of detergents, ideal solubilization conditions were evolved with respect to efficiency, long term stability, and functionality of TAPL. TAPL was isolated in a two-step procedure with high purity and, subsequently, reconstituted into proteoliposomes. The peptide transport activity of reconstituted TAPL strongly depends on the lipid composition. With the help of combinatorial peptide libraries, the key positions of the peptides were localized to the N- and C-terminal residues with respect to peptide transport. At both ends, TAPL favors positively charged, aromatic, or hydrophobic residues and disfavors negatively charged residues as well as asparagine and methionine. Besides specific interactions of both terminal residues, electrostatic interactions are important, since peptides with positive net charge are more efficiently transported than negatively charged ones.
Cyclophilins, or immunophilins, are proteins found in many organisms including bacteria, plants and humans. Most of them display peptidyl-prolyl cis-trans isomerase activity, and play roles as chaperones or in signal transduction. Here, we show that cyclophilin anaCyp40 from the cyanobacterium Anabaena sp. PCC 7120 is enzymatically active, and seems to be involved in general stress responses and in assembly of photosynthetic complexes. The protein is associated with the thylakoid membrane and interacts with phycobilisome and photosystem components. Knockdown of anacyp40 leads to growth defects under high-salt and high-light conditions, and reduced energy transfer from phycobilisomes to photosystems. Elucidation of the anaCyp40 crystal structure at 1.2-Å resolution reveals an N-terminal helical domain with similarity to PsbQ components of plant photosystem II, and a C-terminal cyclophilin domain with a substrate-binding site. The anaCyp40 structure is distinct from that of other multi-domain cyclophilins (such as Arabidopsis thaliana Cyp38), and presents features that are absent in single-domain cyclophilins.
Hydride transfers play a crucial role in a multitude of biological redox reactions and are mediated by flavin, deazaflavin or nicotinamide adenine dinucleotide cofactors at standard redox potentials ranging from 0 to –340 mV. 2-Naphthoyl-CoA reductase, a key enzyme of oxygen-independent bacterial naphthalene degradation, uses a low-potential one-electron donor for the two-electron dearomatization of its substrate below the redox limit of known biological hydride transfer processes at E°’ = −493 mV. Here we demonstrate by X-ray structural analyses, QM/MM computational studies, and multiple spectroscopy/activity based titrations that highly cooperative electron transfer (n = 3) from a low-potential one-electron (FAD) to a two-electron (FMN) transferring flavin cofactor is the key to overcome the resonance stabilized aromatic system by hydride transfer in a highly hydrophobic pocket. The results evidence how the protein environment inversely functionalizes two flavins to switch from low-potential one-electron to hydride transfer at the thermodynamic limit of flavin redox chemistry.
In weiteren Versuchen wurden Streptokokken der Gruppe A und C dem in der I. Mitteilung beschriebenen Trennungsgang unterworfen. Es gelang dabei, aus diesen Streptokokken ein Nucleoprotein zu isolieren, das in bezug auf seine elektrophoretischen Eigenschaften, Farbreaktionen, UV-Absorptionskurve, Zuckeranalyse mit dem aus menschlichen und tierischen Organen gewonnenen Nucleoprotein identisch ist.
Das in der I. Mitteilung beschriebene Nucleoprotein führt bei Zugabe zu Tropokollagen-Lösungen zur Bildung von kollagenen Fibrillen. Die Fibrillen-bildende Potenz des Nucleoproteins ist sehr groß. Die gebildeten Fibrillen zeigen eine sehr gute Feinstruktur und weisen eine 640 Å Hauptperiodik auf. Es ist zu vermuten, daß dem ubiquitär im Organismus vorkommenden Nucleoprotein eine bedeutende oder gar ausschlaggebende Rolle bei der Bildung von kollagenen Fibrillen aus Tropokollagen im Organismus zukommt.
Zusammenfassend läßt sich sagen, daß im menschlichen Serum ein Serumprotein vorhanden ist, welches das aus menschlichem Gewebe gewonnene Nucleoprotein spezifisch bindet. Zur Zeit sind Untersuchungen im Gang. den Serumgehalt des Nucleoproteins und des spezifisch bindenden Serumproteins bei verschiedenen Erkrankungen des Bindegewebsapparates quantitativ zu erfassen.
Das aufgefundene Ribonucleoprotein besitzt die Eigenschaft eines Vollantigens. Die durchgeführten Immunisierungsversuche ergaben eine Artspezifität des Antigens. Eine Organspezifität war nicht nachweisbar. Erste Lokalisierungsversuche mit Hilfe der Immunfluoreszenz-Methode ergaben in der Niere eine besondere Anreicherung des Nucleoproteins in den Glomerulokapseln.
As cryo-EM approaches the physical resolution limits imposed by electron optics and radiation damage, it becomes increasingly urgent to address the issues that impede high-resolution structure determination of biological specimens. One of the persistent problems has been beam-induced movement, which occurs when the specimen is irradiated with high-energy electrons. Beam-induced movement results in image blurring and loss of high-resolution information. It is particularly severe for biological samples in unsupported thin films of vitreous water. By controlled devitrification of conventionally plunge-frozen samples, the suspended film of vitrified water was converted into cubic ice, a polycrystalline, mechanically stable solid. It is shown that compared with vitrified samples, devitrification reduces beam-induced movement in the first 5 e Å−2 of an exposure by a factor of ∼4, substantially enhancing the contribution of the initial, minimally damaged frames to a structure. A 3D apoferritin map reconstructed from the first frames of 20 000 particle images of devitrified samples resolved undamaged side chains. Devitrification of frozen-hydrated specimens helps to overcome beam-induced specimen motion in single-particle cryo-EM, as a further step towards realizing the full potential of cryo-EM for high-resolution structure determination.
Movement of the Rieske domain of the iron–sulfur protein is essential for intramolecular electron transfer within complex III2 (CIII2) of the respiratory chain as it bridges a gap in the cofactor chain towards the electron acceptor cytochrome c. We present cryo-EM structures of CIII2 from Yarrowia lipolytica at resolutions up to 2.0 Å under different conditions, with different redox states of the cofactors of the high-potential chain. All possible permutations of three primary positions were observed, indicating that the two halves of the dimeric complex act independently. Addition of the substrate analogue decylubiquinone to CIII2 with a reduced high-potential chain increased the occupancy of the Qo site. The extent of Rieske domain interactions through hydrogen bonds to the cytochrome b and cytochrome c1 subunits varied depending on the redox state and substrate. In the absence of quinols, the reduced Rieske domain interacted more closely with cytochrome b and cytochrome c1 than in the oxidized state. Upon addition of the inhibitor antimycin A, the heterogeneity of the cd1-helix and ef-loop increased, which may be indicative of a long-range effect on the Rieske domain.
Calcification, Collagen Membrane, Ca/P Ratio Dependence Spontaneous calcification of a membrane made of native collagen has been investigated. The method permits independent variation of calcium and phosphate concentrations. With increasing phosphate concentration the precipitation of calcium-phosphate on the collogen occurs at a conspicuously lower calcium concentration as with a number of other membranes.
The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.
Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and 2) are major facilitator superfamily transporters from the solute carrier family 49. Dysregulation of these ubiquitous transporters has been linked to various haematological and neurological disorders. While both FLVCRs were initially proposed to hold a physiological function in heme transport, subsequent studies questioned this notion. Here, we used structural, computational and biochemical methods and conclude that these two FLVCRs function as human choline transporters. We present cryo-electron microscopy structures of FLVCRs in different inward- and outward-facing conformations, captured in the apo state or in complex with choline in their translocation pathways. Our findings provide insights into the molecular framework of choline coordination and transport, largely mediated by conserved cation-π interactions, and further illuminate the conformational dynamics of the transport cycle. Moreover, we identified a heme binding site on the protein surface of the FLVCR2 N-domain, and observed that heme actively drives the conformational transitions of the protein. This auxiliary binding site might indicate a potential regulatory role of heme in the FLVCR2 transport mechanisms. Our work resolves the contested substrate specificity of the FLVCRs, and sheds light on the process of maintaining cellular choline homeostasis at the molecular level.
The Kinase Chemogenomic Set (KCGS): An open science resource for kinase vulnerability identification
(2019)
We describe the assembly and annotation of a chemogenomic set of protein kinase inhibitors as an open science resource for studying kinase biology. The set only includes inhibitors that show potent kinase inhibition and a narrow spectrum of activity when screened across a large panel of kinase biochemical assays. Currently, the set contains 187 inhibitors that cover 215 human kinases. The kinase chemogenomic set (KCGS) is the most highly annotated set of selective kinase inhibitors available to researchers for use in cell-based screens.
Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus. We show that the dynamics of Q is redox-state-dependent, and that quinol, QH2, moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica. We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.
Lunapark (Lnp) is a conserved membrane protein that localizes to and stabilizes three-way junctions of the tubular ER network. In higher eukaryotes, phosphorylation of Lnp may contribute to the conversion of the ER from tubules to sheets during mitosis. Here, we report on the reconstitution of purified Lnp with phospholipids. Surprisingly, Lnp induces the formation of stacked membrane discs. Each disc is a bicelle, with Lnp sitting in the bilayer facing both directions. The interaction between bicelles is mediated by the cytosolic domains of Lnp, resulting in a constant distance between the discs. A phosphomimetic Lnp mutant shows reduced bicelle stacking. Based on these results, we propose that Lnp tethers ER membranes in vivo in a cell cycle–dependent manner. Lnp appears to be the first membrane protein that induces the formation of stacked bicelles.
Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1
(2021)
The ER is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine cross-linking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1.
The carnitine transporter CaiT from Escherichia coli belongs to the betaine, choline, and carnitine transporter family of secondary transporters. It acts as an L-carnitine/gamma-butyrobetaine exchanger and is predicted to span the membrane 12 times. Unlike the other members of this transporter family, it does not require an ion gradient and does not respond to osmotic stress (Jung, H., Buchholz, M., Clausen, J., Nietschke, M., Revermann, A., Schmid, R., and Jung, K. (2002) J. Biol. Chem. 277, 39251-39258). The structure and oligomeric state of the protein was examined in detergent and in lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. This result was further supported by gel filtration and cross-linking studies. Electron microscopy and single particle analysis of the protein showed a triangular structure of three masses or two parallel elongated densities. Reconstitution of CaiT into lipid bilayers yielded two-dimensional crystals that indicated that CaiT was a trimer in the membrane, similar to its homologue BetP. The implications of the trimeric structure on the function of CaiT are discussed.
IHMCIF: an extension of the PDBx/mmCIF data standard for integrative structure determination methods
(2024)
IHMCIF (github.com/ihmwg/IHMCIF) is a data information framework that supports archiving and disseminating macromolecular structures determined by integrative or hybrid modeling (IHM), and making them Findable, Accessible, Interoperable, and Reusable (FAIR). IHMCIF is an extension of the Protein Data Bank Exchange/macromolecular Crystallographic Information Framework (PDBx/mmCIF) that serves as the framework for the Protein Data Bank (PDB) to archive experimentally determined atomic structures of biological macromolecules and their complexes with one another and small molecule ligands (e.g., enzyme cofactors and drugs). IHMCIF serves as the foundational data standard for the PDB-Dev prototype system, developed for archiving and disseminating integrative structures. It utilizes a flexible data representation to describe integrative structures that span multiple spatiotemporal scales and structural states with definitions for restraints from a variety of experimental methods contributing to integrative structural biology. The IHMCIF extension was created with the benefit of considerable community input and recommendations gathered by the Worldwide Protein Data Bank (wwPDB) Task Force for Integrative or Hybrid Methods (wwpdb.org/task/hybrid). Herein, we describe the development of IHMCIF to support evolving methodologies and ongoing advancements in integrative structural biology. Ultimately, IHMCIF will facilitate the unification of PDB-Dev data and tools with the PDB archive so that integrative structures can be archived and disseminated through PDB.
Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na+ across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430 kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.
Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness is a key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned by cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen thickness for cryo-ET on lamellae has not been systematically investigated. Furthermore, the ions used to ablate material can cause damage in the lamellae, thereby reducing STA resolution. Here, we systematically benchmark the resolution depending on lamella thickness and the depth of the particles within the sample. Up to ca. 180 nm, lamella thickness does not negatively impact resolution. This shows that there is no need to generate very thin lamellae and thickness can be chosen such that it captures major cellular features. Furthermore, we show that gallium-ion-induced damage extends to depths of up to 30 nm from either lamella surface.
Potassium homeostasis is vital for all organisms, but is challenging in single-celled organisms like bacteria and yeast and immobile organisms like plants that constantly need to adapt to changing external conditions. KUP transporters facilitate potassium uptake by the co-transport of protons. Here, we uncover the molecular basis for transport in this widely distributed family. We identify the potassium importer KimA from Bacillus subtilis as a member of the KUP family, demonstrate that it functions as a K+/H+ symporter and report a 3.7 Å cryo-EM structure of the KimA homodimer in an inward-occluded, trans-inhibited conformation. By introducing point mutations, we identify key residues for potassium and proton binding, which are conserved among other KUP proteins.
Nuclear pore complexes (NPCs) constitute giant channels within the nuclear envelope that mediate nucleocytoplasmic exchange. NPC diameter is thought to be regulated by nuclear envelope tension, but how such diameter changes are physiologically linked to cell differentiation, where mechanical properties of nuclei are remodeled and nuclear mechanosensing occurs, remains unstudied. Here we used cryo-electron tomography to show that NPCs dilate during differentiation of mouse embryonic stem cells into neural progenitors. In Nup133-deficient cells, which are known to display impaired neural differentiation, NPCs however fail to dilate. By analyzing the architectures of individual NPCs with template matching, we revealed that the Nup133-deficient NPCs are structurally heterogeneous and frequently disintegrate, resulting in the formation of large nuclear envelope openings. We propose that the elasticity of the NPC scaffold mechanically safeguards the nuclear envelope. Our studies provide a molecular explanation for how genetic perturbation of scaffolding components of macromolecular complexes causes tissue-specific phenotypes.
Cryo electron tomography (cryo-ET) combined with subtomogram averaging (StA) enables structural determination of macromolecules in their native context. A few structures were reported by StA at resolution higher than 4.5 Å, however all of these are from viral structural proteins or vesicle coats. Reaching high resolution for a broader range of samples is uncommon due to beam-induced sample drift, poor signal-to-noise ratio (SNR) of images, challenges in CTF correction, limited number of particles. Here we propose a strategy to address these issues, which consists of a tomographic data collection scheme and a processing workflow. Tilt series are collected with higher electron dose at zero-degree tilt in order to increase SNR. Next, after performing StA conventionally, we extract 2D projections of the particles of interest from the higher SNR images and use the single particle analysis tools to refine the particle alignment and generate a reconstruction. We benchmarked our proposed hybrid StA (hStA) workflow and improved the resolution for tobacco mosaic virus from 7.2 to 5.2 Å and the resolution for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. We demonstrate that hStA can improve the resolution obtained by conventional StA and promises to be a useful tool for StA projects aiming at subnanometer resolution or higher.
Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.
Under natural conditions, the visual system often sees a given input repeatedly. This provides an opportunity to optimize processing of the repeated stimuli. Stimulus repetition has been shown to strongly modulate neuronal-gamma band synchronization, yet crucial questions remained open. Here we used magnetoencephalography in 30 human subjects and find that gamma decreases across ~10 repetitions and then increases across further repetitions, revealing plastic changes of the activated neuronal circuits. Crucially, changes induced by one stimulus did not affect responses to other stimuli, demonstrating stimulus specificity. Changes partially persisted when the inducing stimulus was repeated after 25 minutes of intervening stimuli. They were strongest in early visual cortex and increased interareal feedforward influences. Our results suggest that early visual cortex gamma synchronization enables adaptive neuronal processing of recurring stimuli. These and previously reported changes might be due to an interaction of oscillatory dynamics with established synaptic plasticity mechanisms.
The major light-harvesting complex (LHC-II) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Native LHC-II isolated from plant tissue consists of three isoforms, Lhcb1, Lhcb2, and Lhcb3, which form homo- and heterotrimers. All three isoforms are highly conserved among different species, suggesting distinct functional roles. We produced the three LHC-II isoforms by heterologous expression of the polypeptide in Escherichia coli and in vitro refolding with purified pigments. Although Lhcb1 and Lhcb2 are very similar in polypeptide sequence and pigment content, Lhcb3 is clearly different because it lacks an N-terminal phosphorylation site and has a higher chlorophyll a/b ratio, suggesting the absence of one chlorophyll b. Low temperature absorption and fluorescence emission spectra of the pure isoforms revealed small but significant differences in pigment organization. The oligomeric state of the pure isoforms and of their permutations was investigated by native gel electrophoresis, sucrose density gradient centrifugation, and SDS-PAGE. Lhcb1 and Lhcb2 formed trimeric complexes by themselves and with one another, but Lhcb3 was able to do so only in combination with one or both of the other isoforms. We conclude that the main role of Lhcb1 and Lhcb2 is in the adaptation of photosynthesis to different light regimes. The most likely role of Lhcb3 is as an intermediary in light energy transfer from the main Lhcb1/Lhcb2 antenna to the photosystem II core.
The cell division cycle protein 37 (Cdc37) and the 90-kDa heat shock protein (Hsp90) are molecular chaperones, which are crucial elements in the protein signaling pathway. The largest class of client proteins for Cdc37 and Hsp90 are protein kinases. The catalytic domains of these kinases are stabilized by Cdc37, and their proper folding and functioning is dependent on Hsp90. Here, we present the x-ray crystal structure of the 16-kDa middle domain of human Cdc37 at 1.88 angstroms resolution and the structure of this domain in complex with the 23-kDa N-terminal domain of human Hsp90 based on heteronuclear solution state NMR data and docking. Our results demonstrate that the middle domain of Cdc37 exists as a monomer. NMR and mutagenesis experiments reveal Leu-205 in Cdc37 as a key residue enabling complex formation. These findings can be very useful in the development of small molecule inhibitors against cancer.
Respirasomes are macromolecular assemblies of the respiratory chain complexes I, III and IV in the inner mitochondrial membrane. We determined the structure of supercomplex I1III2IV1 from bovine heart mitochondria by cryo-EM at 9 Å resolution. Most protein-protein contacts between complex I, III and IV in the membrane are mediated by supernumerary subunits. Of the two Rieske iron-sulfur cluster domains in the complex III dimer, one is resolved, indicating that this domain is immobile and unable to transfer electrons. The central position of the active complex III monomer between complex I and IV in the respirasome is optimal for accepting reduced quinone from complex I over a short diffusion distance of 11 nm, and delivering reduced cytochrome c to complex IV. The functional asymmetry of complex III provides strong evidence for directed electron flow from complex I to complex IV through the active complex III monomer in the mammalian supercomplex.
Electron transfer in respiratory chains generates the electrochemical potential that serves as energy source for the cell. Prokaryotes can use a wide range of electron donors and acceptors and may have alternative complexes performing the same catalytic reactions as the mitochondrial complexes. This is the case for the alternative complex III (ACIII), a quinol:cytochrome c/HiPIP oxidoreductase. In order to understand the catalytic mechanism of this respiratory enzyme, we determined the structure of ACIII from Rhodothermus marinus at 3.9 Å resolution by single-particle cryo-electron microscopy. ACIII presents a so-far unique structure, for which we establish the arrangement of the cofactors (four iron–sulfur clusters and six c-type hemes) and propose the location of the quinol-binding site and the presence of two putative proton pathways in the membrane. Altogether, this structure provides insights into a mechanism for energy transduction and introduces ACIII as a redox-driven proton pump.
Poster presentation at 1st International Workshop on Odor Spaces.
Mice are exceptional in their ability to capture their chemical environment, mapping the olfactory world into a basic sensory representation with over one thousand different types of chemical sensors, that is, olfactory sensory neurons (OSNs). OSNs of each type converge in the olfactory bulb onto exclusive distinct physiological areas called glomeruli. The glomeruli constitute the first relay station of olfactory stimulus representation in the mouse brain. Thus, the stimulus induced glomerular input pattern spatially embodies an important part of the sensory representation in the olfactory bulb. Still, topographic organization principles (chemotopy, tunotopy) are under debate. One reason might be that investigation are, due to experimental limitations, only performed on stimuli sets in the size of one hundred odors. But this represents only a tiny snapshot of the vast amount of molecules in the olfactory world and topographic relationships might be disguised in the incomplete representation of molecular receptive ranges (MRR). Therefore we investigated the problem with the MOR18-2 glomerulus as point of reference: First we determined it's MRR. Then, based on a measurement set covering this MRR, we elucidated the topographic embedding. It shows that MOR18-2 is embedded in a hierarchy of patchy tunotopic domains.
Es wurde der Einfluß von Röntgenbestrahlungen auf die Lebensfähigkeit und das Formbildungs-Vermögen kernloser Zellteile von Acetabularia mediterranea in Abhängigkeit von der Strahlendosis untersucht. Dabei erwies sich die Hutbildung stets als der am strahlenempfindlichste Prozeß. Die gefundenen Ergebnisse werden diskutiert und mit den entsprechenden Befunden nach UV-Bestrahlung verglichen.
Es wird der Einfluß von Röntgenstrahlen und ultravioletten Strahlen verschiedener Wellenlängen auf kernhaltige Zellteile von Acetabularia mediterranea untersucht. Die Röntgenbestrahlung führt zu einer Verminderung des Regenerationsvermögens der Zellteile, zu einer Verringerung der Cystenbildung der Regenerate und zu einer Herabsetzung der Lebensfähigkeit der Cysten. Erst nach einer Dosis von 400 kr ist das Regenerationsvermögen fast völlig zerstört. Die Fähigkeit zur Bildung fortpflanzungsfähiger Gameten geht bereits nach 40 kr verloren. Die Wirkung von UV-Bestrahlungen ist demgegenüber sehr gering. Es wird geschlossen, daß die beobachteten Leistungen der kernhaltigen Zellteile wesentlich durch den Zellkern bestimmt sind, der im Rhizoid gegen die UV-Strahlung weitgehend abgeschirmt ist. Erfolgt eine vollständige Regeneration bis zur Hutbildung, so scheinen Größe und Gestalt der ausgewachsenen Regenerate nicht wesentlich von der Röntgenbestrahlung beeinflußt zu sein. In den meisten Fällen bilden Hutregenerate auch Cysten.
Es wird versucht, die Treffertheorie auf die indirekte Strahlenwirkung auszudehnen. Dazu wird angenommen, daß durch die Strahlung „Energieträger“ erzeugt werden, die durch Diffusion zu den „empfindlichen Bereichen“ gelangen und diese verändern können.
Der Berechnung des Wirkvolumens für derartige „indirekte Treffer“ folgt eine reaktionskinetische Betrachtung der indirekten Wirkung.
Durch die Einschaltung physikalisch-chemischer Prozesse zwischen Strahlenabsorption und „Treffer“ erscheint eine Berücksichtigung der physikalischen und chemischen Gegebenheiten im bestrahlten Objekt viel eher möglich als in der „klassischen“ Theorie der „direkten Trefferwirkung“.
Es wurde die Einwirkung monochromatischer ultravioletter Strahlen der Wellenlänge 254 mμ, 281 mμ und 297 mμ auf kernlose Zellteile von Acetabularia untersucht. Dazu wurden in Abhängigkeit von der Strahlendosis die mittlere Lebensdauer und das Formbildungs-Vermögen der kernlosen Teile bestimmt. Der 254-mμ-Strahlung kam dabei stets die größte biologische Wirksamkeit zu, während die 297-mμ-Strahlung im untersuchten Dosisbereich fast ohne Wirkung blieb. Die gefundene Wellenlängen-Abhängigkeit weist darauf hin, daß der UV-Absorption durch die Purine und Pyrimidine für den Wirkungsmechanismus der beobachteten UV-Schädigungen kernloser Zellteile besondere Bedeutung zukommen muß.
A first model of the three-dimensional structure of the photosynthetic reaction center of the mutant T1 (SerL 223 → Ala, ArgL 217 → His) from Rhodopseudomonas viridis, resistant toward the triazine herbicide terbutryn (2-methylthio-4-ethylamino-6-f-butylamino-5-triazine), has been developed from X-ray data measured to a resolution of 2.5 Å. The secondary quinone, QB, which in T1 binds better than in the wild type, is present in the crystals. Both substituted residues are clearly visible in the difference fourier map. The replacement of these two residues in the QB site causes only minor changes in the overall structure of the protein.
Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.
Bei der UV-Bestrahlung (2537 Å) des Zn-Insulins beobachtet man für kleinere Dosen (bis 10 Einstein/Mol) eine direkte Korrelation zwischen der Inaktivierung und der Photoreduktion einer der drei Disulfidbrücken. Mit steigender Dosis wird die Quantenausbeute für die Reduktion der Disulfidbrücken (Bildung von SH-Gruppen) sehr klein, dagegen führen dann andere Prozesse zunehmend zur photochemischen Zerstörung der Disulfidbrücken. Für größere Strahlendosen (über 100 Einstein/Mol) ergibt die Extrapolation, daß für die völlige Inaktivierung des Insulins sämtliche drei Cystinreste zerstört werden müssen. Von den übrigen Aminosäuren wird durch Dosen um 100 Einstein/Mol nur der Tyrosin-Anteil signifikant vermindert. Mit steigender Strahlendosis ändert sich — wahrscheinlich infolge von Konformationsänderungen der Polypeptidketten — die Photosensibilität der Aminosäuren.
Der Einfluß von Elektrodenpolarisation auf die Bestimmung der elektrischen Konstanten leitfähiger Substanzen wird untersucht; es wird gezeigt, daß der durch Polarisation bedingte Effekt auf die Kapazität um mehrere Zehnerpotenzen größer ist als der auf die Wirkwiderstandskomponente. Die Bestimmung der Dielektrizitätskonstante stark leitfähiger Materialien bei Niederfrequenz wird dadurch sehr erschwert. Die verschiedenen Möglichkeiten, Polarisationseinflüsse herabzusetzen, werden diskutiert und es wird gezeigt, daß einzig eine Messung mit verschiedenem Elektrodenabstand einwandfreie Ergebnisse gewährleistet, wenn Polarisation merklich auftritt. Eine bei biologischen Arbeiten öfter angewandte Methodik geht von der Voraussetzung aus, daß beim Austausch biologischen Materials gegen eine Salzlösung gleicher Beschaffenheit, wie sie intrazellular im biologischen Material vorliegt und in Kontakt mit den Elektroden steht, die Polarisationsimpedanz keiner Änderung unterliegt. Die Analyse eigener experimenteller Untersuchungen, über die berichtet wird, zeigt, daß diese Annahme nur berechtigt ist, wenn das biologische Material in so hinreichend großem Abstand von den Elektroden angeordnet wird, daß keine Schattenwirkung auftreten kann. In allen anderen Fällen ist sie falsch, und Arbeiten, die dem nicht Rechnung tragen, sind kritisch zu bewerten.
Es wird eine einfache und billig herzustellende Resonanzanordnung beschrieben, mit der genaue Bestimmungen elektrischer Impedanzwerte und Materialkonstanten im Dezimeterwellenbereich durchführbar sind. Prinzip der Methode, Meßbereich, Genauigkeit und Einzelheiten des Aufbaues werden angegeben und an einigen Beispielen erläutert.
Eine einfache Methode wird erklärt, die es gestattet, genaue Aussagen über das Verteilungsgesetz elektrischer Relaxationszeiten in frequenzabhängigen Dielektrika zu machen. Die Methode setzt die Gültigkeit einer verallgemeinerten Form des von Cole und Cole formulierten Verteilungsgesetzes elektrischer Relaxationszeiten voraus. Sie basiert auf der Tatsache, daß dielektrische Verluste. die bei wesentlich kleineren Frequenzen als der mittleren charakteristischen Frequenz bestimmt werden, außerordentlich empfindlich sind gegen geringe Änderungen im Verteilungsgesetz. Die Methode wird am Beispiel dielektrischer Messungen an Wasser demonstriert. Die Auswertung eigener Messungen ergibt, daß sich im Rahmen der erzielbaren hohen Genauigkeit das dielektrische Verhalten von Wasser durch eine einzige Relaxationszeit charakterisieren läßt.
A new type of Na+-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif
(2013)
Abstract: The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na+. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F1Fo-ATP synthase with a novel Na+ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na+ specificity in physiological settings. Consistently, activity measurements showed Na+ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na+ ionophore monensin. Furthermore, Na+ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na+ coupling is provided by two identical crystal structures of the c11 ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na+ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na+ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.
Author Summary: Essential cellular processes such as biosynthesis, transport, and motility are sustained by the energy released in the hydrolysis of ATP, the universal energy carrier in living cells. Most ATP in the cell is produced by a membrane-bound enzyme, the ATP synthase, through a rotary mechanism that is coupled to the translocation of ions across the membrane. The majority of ATP synthases are energized by transmembrane electrochemical gradients of protons (proton-motive force), but a number of organisms, including some important human pathogens, use gradients of sodium ions instead (sodium-motive force). The ion specificity of ATP synthases is determined by a membrane-embedded sub-complex, the c-ring, which is the smallest known biological rotor. The functional mechanism of the rotor ring and its variations among different organisms are of wide interest, because of this enzyme's impact on metabolism and disease, and because of its potential for nanotechnology applications. Here, we characterize a previously unrecognized type of Na+-driven ATP synthase from the opportunistic human pathogen Fusobacterium nucleatum, which is implicated in periodontal diseases. We analyzed this ATP synthase and its rotor ring through a multi-disciplinary approach, combining cell-growth and biochemical assays, X-ray crystallography and computer-simulation methods. Two crystal structures of the membrane rotor were solved, at low and high pH, revealing an atypical ion-recognition motif mediated by two carboxylate side-chains. This motif is shared by other human pathogens, such as Mycobacterium tuberculosis or Streptococcus pneumonia, whose ATP synthases are targets of novel antibiotic drugs. The implications of this ion-recognition mode on the mechanism of the ATP synthase and the cellular bioenergetics of F. nucleatum were thus examined. Our results provide the basis for future pharmacological efforts against this important pathogen.
Es wurde das Leitfähigkeitsverhalten von reinem, lufthaltigem Wasser bei kontinuierlicher und impulsgetasteter Röntgenbestrahlung (60 kV8) untersucht. Hierbei ergaben sich zwei einander überlagerte Effekte: 1. Ein der Röntgen-Dosisleistung proportionaler irreversibler Leitfähigkeitsanstieg, der vermutlich auf eine Strahlenreaktion des gelösten CO2 zurückzuführen ist, 2. eine reversible Leitfähigkeitserhöhung während der Bestrahlung, die sich mit der Entstehung einer Ionenart mit einer mittleren Lebensdauer von ca. 0,15 sec erklären läßt. Es wird angenommen, daß es sich dabei um Radikalionen O2⊖ handelt, welche durch die Reaktion der als Strahlungsprodukt entstehenden Η-Radikale mit dem gelösten Sauerstoff gebildet werden. Ein möglicher chemischer Reaktionsmechanismus wird angegeben, der zu befriedigender quantitativer Übereinstimmung der Versuchsergebnisse mit Ausbeutewerten und Reaktionskonstanten aus der Literatur führt.
The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions.
In recent decades, mass spectrometry has moved more than ever before into the front line of protein-centered research. After being established at the qualitative level, the more challenging question of quantification of proteins and peptides using mass spectrometry has become a focus for further development. In this chapter, we discuss and review actual strategies and problems of the methods for the quantitative analysis of peptides, proteins, and finally proteomes by mass spectrometry. The common themes, the differences, and the potential pitfalls of the main approaches are presented in order to provide a survey of the emerging field of quantitative, mass spectrometry-based proteomics.
Um die von RAJEWSKY und WOLF aufgeworfene Frage nach dem Einfluß der DNS-Struktur auf die radiationschemische Veränderung der Basen zu untersuchen, wurde die DNS-Spirale bei einem Teil der Untersuchungen in dest. Wasser aufgelöst und mit Röntgenstrahlen bestrahlt. Es ergab sich eine Erhöhung der Strahlenempfindlichkeit der Basen, vor allem zu Beginn der Bestrahlung auf den Wert, den man bei der Bestrahlung der Monomerlösungen beobachtet. Bei Bestrahlung in 0,1 und 1-n. NaCl gelöster DNS sind dagegen die Basen gegen die Einwirkung der im Wasser gebildeten Radikale geschützt, solange sie innerhalb der intakten Spirale gebunden sind. Dieser strukturbedingte Schutzeffekt besteht nicht gegenüber der direkten Strahlenwirkung von UV-Licht. Dieses Ergebnis ist von strahlenbiologischem Interesse, da das Optimum der Strahlenwirkung auf den Mitoseablauf nach Arbeiten von CARLSON und GRAY in der frühen Prophase liegt17, also ebenfalls in einem Stadium, in dem die DNS-Spirale (vor der Verdoppelung) völlig aufgelöst ist. (Vgl. auch BACQ-ALEXANDER und FRITZ-NIGGLI.
Funktionelle und strukturelle Charakterisierung von SLC-Transportern in eukaryotischen Systemen
(2018)
Die evolutionäre Voraussetzung für die Entwicklung komplexer, differenzierter Organismen bildet die Separierung der Zelle in Reaktionsräume, die so genannte Kompartimentierung. Das Prinzip der Kompartimentierung ermöglicht zahlreiche lebensnotwendige, biochemische Prozesse, wie die Konservierung von Energie durch Protonengradienten in der Atmungskette oder parallele, gegenläufige Stoffwechselwege. Zelluläre Kompartimente werden häufig durch Biomembranen gebildet, welche aus einer zweilagigen Lipidschicht bestehen. Lipidmoleküle in einer Zelle sind meistens amphipathisch, das bedeutet, sie bestehen aus einer polaren, hydrophilen Kopfgruppe und einem unpolaren, hydrophopen Ende (Abbildung 1). Die Lipidzusammensetzung in einer Biomembran ist sehr divers und unterscheidet sich in verschiedenen Organismen und Organellen. Phosphoglyceride bilden den Hauptbestandteil der Lipidschicht. Phosphoglyceride besteht aus einem Glycerin Rückgrat, welches an dem C1- und C2-Atom mit zwei Fettsäuren verestert und an dem C3-Atom mit einem Phosphorsäurediester verbunden ist. ...
Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2,3,4,5,6,7. Earlier studies concluded that FLVCR1 may function as a haem exporter8,9,10,11,12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14,15,16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation–π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.
Human feline leukemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and 2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN, and Fowler syndrome2–7. Earlier studies concluded that FLVCR1 may function as a putative heme exporter8–12, while FLVCR2 was suggested to act as a heme importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14–17. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across human plasma membranes, utilizing a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unraveled the coordination chemistry underlying their substrate interactions. Within the binding pocket of both transporters, we identify fully conserved tryptophan and tyrosine residues holding a central role in the formation of cation-π interactions, essential for choline and ethanolamine selectivity. Our findings not only clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhancing our comprehension of disease-associated mutations that interfere with these vital processes, but also shed light on the conformational dynamics of these MFS-type proteins during the transport cycle.