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Denisovite is a rare mineral occurring as aggregates of fibres typically 200–500 nm diameter. It was confirmed as a new mineral in 1984, but important facts about its chemical formula, lattice parameters, symmetry and structure have remained incompletely known since then. Recently obtained results from studies using microprobe analysis, X-ray powder diffraction (XRPD), electron crystallography, modelling and Rietveld refinement will be reported. The electron crystallography methods include transmission electron microscopy (TEM), selected-area electron diffraction (SAED), high-angle annular dark-field imaging (HAADF), high-resolution transmission electron microscopy (HRTEM), precession electron diffraction (PED) and electron diffraction tomography (EDT). A structural model of denisovite was developed from HAADF images and later completed on the basis of quasi-kinematic EDT data by ab initio structure solution using direct methods and least-squares refinement. The model was confirmed by Rietveld refinement. The lattice parameters are a = 31.024 (1), b = 19.554 (1) and c = 7.1441 (5) Å, β = 95.99 (3)°, V = 4310.1 (5) Å3 and space group P12/a1. The structure consists of three topologically distinct dreier silicate chains, viz. two xonotlite-like dreier double chains, [Si6O17]10−, and a tubular loop-branched dreier triple chain, [Si12O30]12−. The silicate chains occur between three walls of edge-sharing (Ca,Na) octahedra. The chains of silicate tetrahedra and the octahedra walls extend parallel to the z axis and form a layer parallel to (100). Water molecules and K+ cations are located at the centre of the tubular silicate chain. The latter also occupy positions close to the centres of eight-membered rings in the silicate chains. The silicate chains are geometrically constrained by neighbouring octahedra walls and present an ambiguity with respect to their z position along these walls, with displacements between neighbouring layers being either Δz = c/4 or −c/4. Such behaviour is typical for polytypic sequences and leads to disorder along [100]. In fact, the diffraction pattern does not show any sharp reflections with l odd, but continuous diffuse streaks parallel to a* instead. Only reflections with l even are sharp. The diffuse scattering is caused by (100) nanolamellae separated by stacking faults and twin boundaries. The structure can be described according to the order–disorder (OD) theory as a stacking of layers parallel to (100).
In the title compound, C17H18N2O, the central carbon atom with the OH substituent and one of the (E)-benzylideneamino substituents are disordered over two sets of sites with occupancies of 0.851 (4) and 0.149 (4). The relative positions of the two disorder components is equivalent to a rotation of approximately 60° about the C—N single bond. In the crystal, the molecules are held together by O—H...N hydrogen bonds, forming simple C(5) chains along the b-axis direction. In addition, pairs of the chains are further aggregated by weak C—H...π interactions.
The asymmetric unit of the title compound, C21H28N4O, consists of two unique molecules linked by an O—H⋯N hydrogen bond. The conformation of both C=N bonds is E and the azomethine functional groups lie close to the plane of their associated benzene rings in each of the independent molecules. The dihedral angles between the two benzene rings are 83.14 (4) and 75.45 (4)°. The plane of the one of the N(CH3)2 units is twisted away from the benzene ring by 18.8 (2)°, indicating loss of conjugation between the lone electron pair and the benzene ring. In the crystal structure, O—H⋯N hydrogen bonds together with C—H⋯O hydrogen bonds link neighbouring supramolecular dimers into a three-dimensional network.
The asymmetric unit of the title co-crystalline adduct, 1,3,6,8-tetraazatricyclo[4.4.1.13,8]dodecane (TATD)–4-iodophenol (1/2), C8H16N4·2C6H5IO, comprises a half molecule of the aminal cage polyamine plus a 4-iodophenol molecule. A twofold rotation axis generates the other half of the adduct. The components are linked by two intermolecular O—H⋯N hydrogen bonds. The adducts are further linked into a three-dimensional framework structure by a combination of N⋯I halogen bonds and weak non-conventional C—H⋯O and C—H⋯I hydrogen bonds.
In the title compound, C26H24N2O2, the oxazine moiety is fused to a naphthalene ring system. The asymmetric unit consists of one half of the molecule, which lies about an inversion centre. The C atoms of the ethylene spacer group adopt an antiperiplanar arrangement. The oxazine ring adopts a half-chair conformation. In the crystal, supramolecular chains running along the b axis are formed via short C—H⋯π contacts. The crystal studied was a non-merohedral twin with a fractional contribution of 0.168 (2) of the minor twin component.
The asymmetric unit of the title compound, C18H18I2N2O2, consists of one half-molecule, completed by the application of inversion symmetry. The molecule adopts the typical structure for this class of bis-benxozazines, characterized by an anti orientation of the two benzoxazine rings around the central C—C bond. The oxazinic ring adopts a half-chair conformation. In the crystal, molecules are linked by C—I⋯N short contacts [I⋯N = 3.378 (2) Å], generating layers lying parallel to the bc plane.
Infections with multidrug resistant bacterial strains like Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa or Acinetobacter baumanii that can accumulate resistance mechanisms against different groups of drugs cause increasing problems for the health care system. Multidrug efflux pumps are able to transport different classes of substances, providing a basic resistance to different antibiotics. Especially when they are overexpressed they can keep bacterial cells alive under antibiotic pressure unless other high level resistance mechanisms like expression of β-lactamases are established. One example for a clinically relevant multidrug efflux pump is the AcrAB/TolC tripartite system of E. coli, that transports a variety of different substrates, including besides antibiotics dyes, detergents, bile salts and organic compounds from the periplasm or the inner membrane out of the cell. AcrB is the inner membrane component of the protein complex that determines not only the substrate specificity of the tripartite system but energises the transport through the whole system process via proton transduction as well. TolC is the outer membrane spanning protein that forms a pore in the outer membrane enabling the system to transport drugs over the latter out of the cell. The periplasmic membrane fusion protein AcrA connects AcrB and TolC in the periplasm completing the channel from the periplasm, respective the inner membrane to the extracellular space. AcrB assembles as trimers, in asymmetric crystal structures each of the protomers adapts a different conformation designated L(oose), T(ight) and O(pen). In the protomers tunnels open up and collaps in different conformations. In the L protomer a periplasmic cleft opens up that can initially bind substrates to the periplasmic part of AcrB. In the T conformation the deep binding pocket opens that is assumed to bind substrates tightly that were bound to the access pocket before. As well in the T conformation a second pathway leading to the deep binding pocket opens that can guide substrates from a groove between transmembrane helices TM7, TM8 and TM9, the TM8 groove, that is connected with socalled tunnel 1 that ends in the deep binding pocket. In the O conformation a new tunnel opens that connects the collapsing deep binding pocket with the periplasmic space, respective the channel through the periplasmic space formed from AcrA and TolC. Substrates were cocrystallised in access and deep binding pocket verifying their role in substrate transport. In the TM8 groove in high resolution crystal structures DDM molecules were cocrystallised in L and T conformation, indicating that the AcrB substrate DDM may utilise this entrance to the deep binding pocket. The asymmetry observed in the AcrB trimers trongly suggests a peristaltic pump mechanism. The functional rotation cycle demands communication between the subunits and tight control of substrate load of protomers during the transport to optimise the ration between protons that are transduced and substrates transported. Indeed it was shown that AcrB transport mechanism is positively cooperative for some β-lactam substrates. For the communication between the subunits it was assumed that ionic interaction between ion pairs established between charged amino acids at the interfaces of protomers in different conformations are of special importance. Thus the amino acids engaged in ionic interactions, respective ion pairs D73-K131, E130-K110, D174-K110, R168, R259-E734 were substituted with non-charged amino acids pairwise and phenotypes were determined in plate dilution assays and MIC experiments. No evidence for a general, substrate independent, reduction of AcrB activity, that would be expected when the ionic residues are of special importance for AcrB function, could be found with the methods applied. Substitutions were not only combined pairwise according to the putative ion pairs but as well in combinations of R168A with D174N, E130Q and K131M. AcrB activity is reduced for the variant R168A_D174N significantly, activity decreases further for quadruple variant E130Q_K131M_ R168A_D174N. Because the reduced activity is only observed in this combination of substitutions the phenotype must result from accumulation of small effects of the single substitutions. R168A may destabilise the protomer interfaces, as its side chain is oriented in direction to the neighbouring protomer at all interfaces, enhancing substratespecific effects of substitutions E130Q, K131M, D174N that are not in all conformations oriented towards the neighbouring protomer but as well along the substrate transport pathway. Further investigations to figure out the details of the effects observed were not conducted because fluctuating expression of the variants hindered experimental procedures.
In another approach TM8 was in focus of the interest. As mentioned above it is a possible substrate entrance in the inner membrane. The linker between TM8 and the periplasmic PC2 subdomain undergoes a coil-to-helix transition when AcrB cycles through L, T and O conformations. Linking the transmembrane part of AcrB that provides the energy for the transport process via proton transduction with the periplasmic part harbouring the major part of the substrate pathway assignes TM8 and the periplasmic linker (859-876) an important role in the function of AcrB. Thus it was investigated with an alanine-scan of residues 859 to 884 and G/P respective P/G exchange followed by phenotype characterisation in growth curve and plate dilution assays of selected variants. In the phenotype determinations none of the variants, except G861P that seems to cause massive sterical restriction in an α-helical region, displayed a general, substrate independent decrease of AcrB activity. Thus it is concluded that the individual properties of amino acids in TM8 and the periplasmic linker are not of general importance for the mechanism of AcrB. The substitution of individual amino acids had impact on uptake of different substrates in plate dilution assays in a substrate dependent manner. The uptake of some substrates, like erythromycin or chloramphenicol is more affected than that of others with rhodamine 6G resistance being only reduced for the G861P variant. A relation between the PSA of substrates and reduced activity of AcrB was observed. in Substrates with higher PSA values are more affected by substitutions in TM8 or periplasmic linker, resulting in the conclusion that substrates with higher PSA are more likely to be taken up via the TM8 groove/tunnel 1 pathway than those with lower PSA values.
Ion channel gating is essential for cellular homeostasis and is tightly controlled. In some eukaryotic and most bacterial ligand-gated K+ channels, RCK domains regulate ion fluxes. Until now, a single regulatory mechanism has been proposed for all RCK-regulated channels, involving signal transduction from the RCK domain to the gating area. Here, we present an inactive ADP-bound structure of KtrAB from Vibrio alginolyticus, determined by cryo-electron microscopy, which, combined with EPR spectroscopy and molecular dynamics simulations, uncovers a novel regulatory mechanism for ligand-induced action at a distance. Exchange of activating ATP to inactivating ADP triggers short helical segments in the K+-translocating KtrB dimer to organize into two long helices that penetrate deeply into the regulatory RCK domains, thus connecting nucleotide-binding sites and ion gates. As KtrAB and its homolog TrkAH have been implicated as bacterial pathogenicity factors, the discovery of this functionally relevant inactive conformation may advance structure-guided drug development.
β-barrel proteins mediate nutrient uptake in bacteria and serve vital functions in cell signaling and adhesion. For the 14-strand outer membrane protein G of Escherichia coli, opening and closing is pH-dependent. Different roles of the extracellular loops in this process were proposed, and X-ray and solution NMR studies were divergent. Here, we report the structure of outer membrane protein G investigated in bilayers of E. coli lipid extracts by magic-angle-spinning NMR. In total, 1847 inter-residue 1H–1H and 13C–13C distance restraints, 256 torsion angles, but no hydrogen bond restraints are used to calculate the structure. The length of β-strands is found to vary beyond the membrane boundary, with strands 6–8 being the longest and the extracellular loops 3 and 4 well ordered. The site of barrel closure at strands 1 and 14 is more disordered than most remaining strands, with the flexibility decreasing toward loops 3 and 4. Loop 4 presents a well-defined helix.
The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto‐oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single‐particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B‐bound MET. We show that internalin B‐bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B‐bound receptors than for resting MET. Results of single‐particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect.
In bacteria, the regulation of gene expression by cis-acting transcriptional riboswitches located in the 5'-untranslated regions of messenger RNA requires the temporal synchronization of RNA synthesis and ligand binding-dependent conformational refolding. Ligand binding to the aptamer domain of the riboswitch induces premature termination of the mRNA synthesis of ligand-associated genes due to the coupled formation of 3'-structural elements acting as terminators. To date, there has been no high resolution structural description of the concerted process of synthesis and ligand-induced restructuring of the regulatory RNA element. Here, we show that for the guanine-sensing xpt-pbuX riboswitch from Bacillus subtilis, the conformation of the full-length transcripts is static: it exclusively populates the functional off-state but cannot switch to the on-state, regardless of the presence or absence of ligand. We show that only the combined matching of transcription rates and ligand binding enables transcription intermediates to undergo ligand-dependent conformational refolding.
The synergetic effects of combining structural biology and epr spectroscopy on membrane proteins
(2017)
Protein structures as provided by structural biology such as X-ray crystallography, cryo-electron microscopy and NMR spectroscopy are key elements to understand the function of a protein on the molecular level. Nonetheless, they might be error-prone due to crystallization artifacts or, in particular in case of membrane-imbedded proteins, a mostly artificial environment. In this review, we will introduce different EPR spectroscopy methods as powerful tools to complement and validate structural data gaining insights in the dynamics of proteins and protein complexes such that functional cycles can be derived. We will highlight the use of EPR spectroscopy on membrane-embedded proteins and protein complexes ranging from receptors to secondary active transporters as structural information is still limited in this field and the lipid environment is a particular challenge.