text:
Laboratory of biomolecular NMR-spectroscopy
Department of structural biology
Head: Alexander Arseniev, D.Sc, professor
Laboratory investigates the structure of proteins and peptides. For this purpose Laboratory uses one of the most powerful modern techniques – nuclear magnetic resonance spectroscopy (NMR).
The research of the Laboratory is focused on the membrane proteins such as receptor tyrosine kinases, ion channels, toll-like receptors, the beta-amyloid precursor protein, GPCR-s and others. The properties of natural luciferins, the blockers of pain receptors, the viral proteins required for the infection s, as well as the mechanisms of ligand-receptor interactions are also under investigation.
Most of the research is directly related to practical issues such as the search for anti-cancer targets, the causes of Alzheimer's disease, the creation of effective painkillers, specific diagnostic systems and others.
At the disposal of the Laboratory are the most advanced devices of the Bruker company: 600, 700 and 800 MHz machines, equipped with the supersensitive cryogenic probes, and a solid-state MAS probe. In addition, the Laboratory has necessary equipment and techniques for bacterial in-cell and cell-free synthesis ofrecombinant proteins and their physicochemical characterization. Methods for the production of isotropicaly and selectively isotope-labeled proteins were developed on their basis.
All aforesaid allows us to successfully solve the most difficult tasks within and beyond the capabilities of modern structural biology.
Laboratory equipment is a part of the IBCh Shared Research Center, so it is possible to analyze the samples of high complexity by NMR spectroscopy on a commercial basis.
The Laboratory has a rich history. In 1965 it was founded by Vladimir F. Bystrov, the associate member of the USSR Academy of Sciences. He was one of the first in the world who began to engage in structural studies of proteins and peptides in solution by NMR spectroscopy and created the largest in the Soviet Union scientific school. In 1990 the laboratory was headed by his apprentice, Professor Alexander Arseniev.
The Laboratory continues to develop the Bystrov's initiatives till now and is considered as the one of the leading schools in the world of NMR. Each year it produces new highly qualified professionals and PhDs who can solve the most complex problems using NMR-spectroscopy.
Nowadays Laboratory has many friends and partners. Among them are the laboratory of Nobel laureate Kurt Wüthrich, one of the strongest NMR-laboratory in the world of Professor Gerhard Wagner, "Novartis", the second-largest pharmaceutical company in the world and others.
Today, our lab is a very friendly team which has set itself the most ambitious goals and is ready for any interesting collaboration!
Methodological developments for biomolecular NMR spectroscopy
NMR pulse sequences (with parameters) for Bruker AVANCE spectrometers for 13C relaxation measurements of 15N/13C uniformly labeled proteins.
Selected publications
(2017). Synthesis of Panal Terpenoid Core. Synlett 28 (5), 583–588 [+]
Panal is a natural bicyclic cadalane-type sesquiterpenoid with an unusual combination of stereocenters. It was isolated in 1988 as an alleged biosynthetic precursor of luciferin (a light-emitting molecule) in a bioluminescent fungus Panellus stipticus. Herein we present the first approach to the synthesis of the terpenoid skeleton of panal, which includes construction of five stereocenters, one of which is easily epimerizable. The key steps in the synthetic approach presented are high-pressure Diels–Alder reaction disobeying the 'endo rule', Barbier reductive allylation, and cyclization of trans-decalin ring via ring-closing metathesis.ID:1623
(2016). Helix-helix interactions in membrane domains of bitopic proteins: Specificity and role of lipid environment. Biochim. Biophys. Acta , [+]
Interaction between transmembrane helices often determines biological activity of membrane proteins. Bitopic proteins, a broad subclass of membrane proteins, form dimers containing two membrane-spanning helices. Some aspects of their structure-function relationship cannot be fully understood without considering the protein-lipid interaction, which can determine the protein conformational ensemble. Experimental and computer modeling data concerning transmembrane parts of bitopic proteins are reviewed in the present paper. They highlight the importance of lipid-protein interactions and resolve certain paradoxes in the behavior of such proteins. Besides, some properties of membrane organization provided a clue to understanding of allosteric interactions between distant parts of proteins. Interactions of these kinds appear to underlie a signaling mechanism, which could be widely employed in the functioning of many membrane proteins. Treatment of membrane proteins as parts of integrated fine-tuned proteolipid system promises new insights into biological function mechanisms and approaches to drug design.ID:1670
(2016). Structure of purotoxin-2 from wolf spider: modular design and membrane-assisted mode of action in arachnid toxins. Biochem. J. 473 (19), 3113–26 [+]
Traditionally, arachnid venoms are known to contain two particularly important groups of peptide toxins. One is disulfide-rich neurotoxins with a predominance of β-structure that specifically target protein receptors in neurons or muscle cells. The other is linear cationic cytotoxins that form amphiphilic α-helices and exhibit rather non-specific membrane-damaging activity. In the present paper, we describe the first 3D structure of a modular arachnid toxin, purotoxin-2 (PT2) from the wolf spider Alopecosa marikovskyi (Lycosidae), studied by NMR spectroscopy. PT2 is composed of an N-terminal inhibitor cystine knot (ICK, or knottin) β-structural domain and a C-terminal linear cationic domain. In aqueous solution, the C-terminal fragment is hyper-flexible, whereas the knottin domain is very rigid. In membrane-mimicking environment, the C-terminal domain assumes a stable amphipathic α-helix. This helix effectively tethers the toxin to membranes and serves as a membrane-access and membrane-anchoring device. Sequence analysis reveals that the knottin + α-helix architecture is quite widespread among arachnid toxins, and PT2 is therefore the founding member of a large family of polypeptides with similar structure motifs. Toxins from this family target different membrane receptors such as P2X in the case of PT2 and calcium channels, but their mechanism of action through membrane access may be strikingly similar.ID:1568
(2016). Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana. Protein Expr. Purif. 130, 13–20 [+]
Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of (13)C,(15)N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.ID:1599
(2016). Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors. Toxicon 119, 274–9 [+]
'Three-finger' toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with (125)I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs.ID:1598
(2016). Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors. Sci Rep 6, 30698 [+]
Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a 'three-finger' fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.ID:1597
(2016). Characterization of Small Isotropic Bicelles with Various Compositions. Langmuir 32 (26), 6624–37 [+]
Structural studies of membrane proteins are of great importance and interest, with solution and solid state NMR spectroscopy being very promising tools for that task. However, such investigations are hindered by a number of obstacles, and in the first place by the fact that membrane proteins need an adequate environment that models the cell membrane. One of the most widely used and prospective membrane mimetics is isotropic bicelles. While large anisotropic bicelles are well-studied, the field of small bicelles contains a lot of "white spots". The present work reports the radii of particles and concentration of the detergents in the monomeric state in solutions of isotropic bicelles, formed by 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and sodium cholate, as a function of lipid/detergent ratio and temperature. These parameters were measured using (1)H NMR diffusion spectroscopy for the bicelles composed of lipids with saturated fatty chains of different length and lipids, containing unsaturated fatty acid residue. The influence of a model transmembrane protein (membrane domain of rat TrkA) on the properties of bicelles and the effect of the bicelle size and composition on the properties of the transmembrane protein were investigated with heteronuclear NMR and nuclear Overhauser effect spectroscopy. We show that isotropic bicelles that are applicable for solution NMR spectroscopy behave as predicted by the theoretical models and are likely to be bicelles rather than mixed micelles. Using the obtained data, we propose a simple approach to control the size of bicelles at low concentrations. On the basis of our results, we compared different rim-forming agents and selected CHAPS as a detergent of choice for structural studies in bicelles, if the deuteration of the detergent is not required.ID:1569
(2016). Cell-free expression of the APP transmembrane fragments with Alzheimer's disease mutations using algal amino acid mixture for structural NMR studies. Protein Expr. Purif. 123, 105–11 [+]
Structural investigations need ready supply of the isotope labeled proteins with inserted mutations n the quantities sufficient for the heteronuclear NMR. Though cell-free expression system has been widely used in the past years, high startup cost and complex compound composition prevent many researches from the developing this technique, especially for membrane protein production. Here we demonstrate the utility of a robust, cost-optimized cell-free expression technique for production of the physiologically important transmembrane fragment of amyloid precursor protein, APP686-726, containing Alzheimer's disease mutations in the juxtamembrane (E693G, Arctic form) and the transmembrane parts (V717G, London form, or L723P, Australian form). The protein cost was optimized by varying the FM/RM ratio as well as the amino acid concentration. We obtained the wild-type and mutant transmembrane fragments in the pellet mode of continuous exchange cell-free system consuming only commercial algal mixture of the (13)C,(15)N-labeled amino acids. Scaling up analytical tests, we achieved milligram quantity yields of isotope labeled wild-type and mutant APP686-726 for structural studies by high resolution NMR spectroscopy in membrane mimicking environment. The described approach has from 5 to 23-fold cost advantage over the bacterial expression methods described earlier and 1.5 times exceeds our previous result obtained with the longer APP671-726WT fragment.ID:1570
(2016). Structural Basis of p75 Transmembrane Domain Dimerization. J. Biol. Chem. 291 (23), 12346–57 [+]
Dimerization of single span transmembrane receptors underlies their mechanism of activation. p75 neurotrophin receptor plays an important role in the nervous system, but the understanding of p75 activation mechanism is still incomplete. The transmembrane (TM) domain of p75 stabilizes the receptor dimers through a disulfide bond, essential for the NGF signaling. Here we solved by NMR the three-dimensional structure of the p75-TM-WT and the functionally inactive p75-TM-C257A dimers. Upon reconstitution in lipid micelles, p75-TM-WT forms the disulfide-linked dimers spontaneously. Under reducing conditions, p75-TM-WT is in a monomer-dimer equilibrium with the Cys(257) residue located on the dimer interface. In contrast, p75-TM-C257A forms dimers through the AXXXG motif on the opposite face of the α-helix. Biochemical and cross-linking experiments indicate that AXXXG motif is not on the dimer interface of p75-TM-WT, suggesting that the conformation of p75-TM-C257A may be not functionally relevant. However, rather than mediating p75 homodimerization, mutagenesis of the AXXXG motif reveals its functional role in the regulated intramembrane proteolysis of p75 catalyzed by the γ-secretase complex. Our structural data provide an insight into the key role of the Cys(257) in stabilization of the weak transmembrane dimer in a conformation required for the NGF signaling.ID:1571
(2016). Alternative packing of EGFR transmembrane domain suggests that protein-lipid interactions underlie signal conduction across membrane. Biochim. Biophys. Acta 1858 (6), 1254–61 [+]
The human epidermal growth factor receptor (EGFR) of HER/ErbB receptor tyrosine kinase family mediates a broad spectrum of cellular responses transducing biochemical signals via lateral dimerization in plasma membrane, while inactive receptors can exist in both monomeric and dimeric forms. Recently, the dimeric conformation of the helical single-span transmembrane domains of HER/ErbB employing the relatively polar N-terminal motifs in a fashion permitting proper kinase activation was experimentally determined. Here we describe the EGFR transmembrane domain dimerization via an alternative weakly polar C-terminal motif A(661)xxxG(665) presumably corresponding to the inactive receptor state. During association, the EGFR transmembrane helices undergo a structural adjustment with adaptation of inter-molecular polar and hydrophobic interactions depending upon the surrounding membrane properties that directly affect the transmembrane helix packing. This might imply that signal transduction through membrane and allosteric regulation are inclusively mediated by coupled protein-protein and protein-lipid interactions, elucidating paradoxically loose linkage between ligand binding and kinase activation.ID:1596
(2016). Cell Free Expression and Purification of the Fragments of the Receptor Tyrosine Kynases of the EGFR Family, Containing the Transmembrane Domain with the Juxtamembrane Region, for Structural Studies. BIOLOGICHESKIE MEMBRANY 33 (2), 124–132 [+]
The EGFR/HER receptor family of an epidermal growth factor represents an important class of the receptor tyrosine kinases playing the key role in the control of cell growth and differentiation in mammalian cells, as well as in the development of a number of pathological processes, including oncogenesis. Binding of a ligand to the extracellular domains initiates switching of the EGFR/HER receptor between the alternative dimeric states that causes the allosteric activation of kinase domains in cell cytoplasm. The transmembrane (TM) domain and adjacent flexible regions alternatively interacting with either membrane surface or kinase domains are directly involved in the complex conformational transition in EGFR/HERs. Here we report on a highly efficient system of the cell free production of the EGFR/HER TM domains with functionally important juxtamembrane (JM) regions for the investigation of the molecular basis of biochemical signal transduction across the cell membrane. To increase the efficiency of synthesis of the EGFR/HER TM-JM fragments of the receptors, we used two N-terminal expression tags, which significantly increased the protein yield. In the case of the TM-JM fragments of EGFR (residues 638–692) and HER2 (residues 644–700), the method allowed us to obtain milligram quantities of the 13C,15N-labeled protein for structural and biophysical investigations in the membrane-mimicking environments using high-resolution heteronuclear NMR spectroscopy.ID:1622
(2016). HER2 Transmembrane Domain Dimerization Coupled with Self-Association of Membrane-Embedded Cytoplasmic Juxtamembrane Regions. J. Mol. Biol. 428 (1), 52–61 [+]
Receptor tyrosine kinases of the human epidermal growth factor receptor (HER or ErbB) family transduce biochemical signals across plasma membrane, playing a significant role in vital cellular processes and in various cancers. Inactive HER/ErbB receptors exist in equilibrium between the monomeric and unspecified pre-dimerized states. After ligand binding, the receptors are involved in strong lateral dimerization with proper assembly of their extracellular ligand-binding, single-span transmembrane, and cytoplasmic kinase domains. The dimeric conformation of the HER2 transmembrane domain that is believed to support the cytoplasmic kinase domain configuration corresponding to the receptor active state was previously described in lipid bicelles. Here we used high-resolution NMR spectroscopy in another membrane-mimicking micellar environment and identified an alternative HER2 transmembrane domain dimerization coupled with self-association of membrane-embedded cytoplasmic juxtamembrane region. Such a dimerization mode appears to be capable of effectively inhibiting the receptor kinase activity. This finding refines the molecular mechanism regarding the signal propagation steps from the extracellular to cytoplasmic domains of HER/ErbB receptors.ID:1369
(2016). A novel lipid transfer protein from the dill Anethum graveolens L.: isolation, structure, heterologous expression, and functional characteristics. J. Pept. Sci. 22 (1), 59–66 [+]
A novel lipid transfer protein, designated as Ag-LTP, was isolated from aerial parts of the dill Anethum graveolens L. Structural, antimicrobial, and lipid binding properties of the protein were studied. Complete amino acid sequence of Ag-LTP was determined. The protein has molecular mass of 9524.4 Da, consists of 93 amino acid residues including eight cysteines forming four disulfide bonds. The recombinant Ag-LTP was overexpressed in Escherichia coli and purified. NMR investigation shows that the Ag-LTP spatial structure contains four α-helices, forming the internal hydrophobic cavity, and a long C-terminal tail. The measured volume of the Ag-LTP hydrophobic cavity is equal to ~800 A(3) , which is much larger than those of other plant LTP1s. Ag-LTP has weak antifungal activity and unpronounced lipid binding specificity but effectively binds plant hormone jasmonic acid. Our results afford further molecular insight into biological functions of LTP in plants. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.ID:1370
(2016). Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor. PLoS ONE 11 (2), e0149733 [+]
SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,-non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.ID:1420
(2015). Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors. J. Biol. Chem. 290 (39), 23616–30 [+]
Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.ID:1394
(2015). NMR Dynamics of Transmembrane and Intracellular Domains of p75NTR in Lipid-Protein Nanodiscs. Biophys. J. 109 (4), 772–82 [+]
P75NTR is a type I integral membrane protein that plays a key role in neurotrophin signaling. However, structural data for the receptor in various functional states are sparse and controversial. In this work, we studied the spatial structure and mobility of the transmembrane and intracellular parts of p75NTR, incorporated into lipid-protein nanodiscs of various sizes and compositions, by solution NMR spectroscopy. Our data reveal a high level of flexibility and disorder in the juxtamembrane chopper domain of p75NTR, which results in the motions of the receptor death domain being uncoupled from the motions of the transmembrane helix. Moreover, none of the intracellular domains of p75NTR demonstrated a propensity to interact with the membrane or to self-associate under the experimental conditions. The obtained data are discussed in the context of the receptor activation mechanism.ID:1312
(2015). Structure of Membrane-active Toxin from Crab Spider Heriaeus melloteei Suggests Parallel Evolution of Sodium Channel Gating Modifiers in Araneomorphae and Mygalomorphae. J. Biol. Chem. 290 (1), 492–504 [+]
We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called "inhibitor cystine knot" or "knottin" fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μm Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and features a "membrane access" mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of inhibitor cystine knot toxins from Araneomorphae and Mygalomorphae suborders.ID:1138
(2014). Cobra cytotoxins: determinants of antibacterial activity. Mendeleev Communications 25 (1), 70–71 [+]
The investigation of antibacterial activity of three-finger cobra cytotoxins towards Gram-negative and Gram-positive bacteria showed no activity against the former species, whereas M. luteus was found most susceptible to cytotoxins. A correlation was revealed between this activity and hydrophobicity of the toxins (HTL scores), total charge and its distribution over the toxin molecule: the absence of Glu-16 residue and the presence of positively charged residues (Lys30/His31) in the tip of the loop 2.ID:1398
(2014). End-group differentiating ozonolysis of furocoumarins. RSC Advances 4 (106), 61277–61280 [+]
Ozonolysis of furocoumarins followed by reductive work-up yields not only common symmetrical dialdehydes, but also o-formylumbelliferones with moderate-to-high yields. Simultaneous formation of both products accounts for the transformation of carbonyl oxides – products of primary ozonide ring opening.ID:1097
(2014). Toll-like receptor 3 transmembrane domain is able to perform various homotypic interactions: An NMR structural study. FEBS Lett. , [+]
Toll-like receptors (TLRs) take part in both the innate and adaptive immune systems. The role of the transmembrane domain in TLR signaling is still elusive, while its importance for the TLR activation was clearly demonstrated. In the present study the ability of the TLR3 transmembrane domain to form dimers and trimers in detergent micelles was shown by solution NMR spectroscopy. Spatial structures and free energy magnitudes were determined for the TLR3 transmembrane domain in dimeric and trimeric states, and two possible surfaces that may be used for the helix-helix interaction by the full-length TLR3 were revealed.ID:1107
(2014). Structural and functional characterization of alternative transmembrane domain conformations in VEGF receptor 2 activation. Structure 22 (8), 1077–89 [+]
Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization and structural rearrangement of the extracellular domains. RTK activation also depends on the specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutant TMDs carry polar amino acids promoting stable transmembrane helix dimerization, which is essential for kinase activation. We investigated the effect of polar amino acids introduced into the TMD of vascular endothelial growth factor receptor 2, regulating blood vessel homeostasis. Two mutants showed constitutive kinase activity, suggesting that precise TMD orientation is mandatory for kinase activation. Nuclear magnetic resonance spectroscopy revealed that TMD helices in activated constructs were rotated by 180° relative to the interface of the wild-type conformation, confirming that ligand-mediated receptor activation indeed results from transmembrane helix rearrangement. A molecular dynamics simulation confirmed the transmembrane helix arrangement of wild-type and mutant TMDs revealed by nuclear magnetic resonance spectroscopy.ID:1104
(2014). Point mutations in dimerization motifs of the transmembrane domain stabilize active or inactive state of the EphA2 receptor tyrosine kinase. J. Biol. Chem. 289 (21), 14955–64 [+]
The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L(535)X3G(539)X2A(542)X3V(546)X2L(549) rather than through the alternative glycine zipper motif A(536)X3G(540)X3G(544) (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr(588) and/or Tyr(594)) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.ID:1139
(2014). A Novel Type of Luciferin from the Siberian Luminous Earthworm Fridericia heliota: Structure Elucidation by Spectral Studies and Total Synthesis. Angew. Chem. Int. Ed. Engl. 53 (22), 5566–5568 [+]
Press-release on this article: "Novel luciferin from Siberian bioluminescent worm".ID:1016
- (2014). AsLn2, a luciferin-related modified tripeptide from the bioluminescent earthworm Fridericia heliota. Tetrahedron Lett. 55 (2), 463–465 ID:1039
- (2014). CompX, a luciferin-related tyrosine derivative from the bioluminescent earthworm Fridericia heliota. Tetrahedron Lett. 55 (2), 460–462 ID:1040
(2013). NMR-based approach to measure the free energy of transmembrane helix-helix interactions. Biochim. Biophys. Acta 1838 (1PB), 164–172 [+]
Knowledge of the energetic parameters of transmembrane helix-helix interactions is necessary for the establishment of a structure-energy relationship for α-helical membrane domains. A number of techniques have been developed to measure the free energies of dimerization and oligomerization of transmembrane α-helices, and all of these have their advantages and drawbacks. In this study we propose a methodology to determine the magnitudes of the free energy of interactions between transmembrane helices in detergent micelles. The suggested approach employs solution nuclear magnetic resonance (NMR) spectroscopy to determine the population of the oligomeric states of the transmembrane domains and introduces a new formalism to describe the oligomerization equilibrium, which is based on the assumption that both the dimerization of the transmembrane domains and the dissociation of the dimer can occur only upon the collision of detergent micelles. The technique has three major advantages compared with other existing approaches: it may be used to analyze both weak and relatively strong dimerization/oligomerization processes, it works well for the analysis of complex equilibria, e.g. when monomer, dimer and high-order oligomer populations are simultaneously present in the solution, and it can simultaneously yield both structural and energetic characteristics of the helix-helix interaction under study. The proposed methodology was applied to investigate the oligomerization process of transmembrane domains of fibroblast growth factor receptor 3 (FGFR3) and vascular endothelium growth factor receptor 2 (VEGFR2), and allowed the measurement of the free energy of dimerization of both of these objects. In addition the proposed method was able to describe the multi-state oligomerization process of the VEGFR2 transmembrane domain.ID:967
(2013). Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria. Proc. Natl. Acad. Sci. U.S.A. 110 (31), 12631–6 [+]
Light-driven proton pumps are present in many organisms. Here, we present a high-resolution structure of a proteorhodopsin from a permafrost bacterium, Exiguobacterium sibiricum rhodopsin (ESR). Contrary to the proton pumps of known structure, ESR possesses three unique features. First, ESR's proton donor is a lysine side chain that is situated very close to the bulk solvent. Second, the α-helical structure in the middle of the helix F is replaced by 3(10)- and π-helix-like elements that are stabilized by the Trp-154 and Asn-224 side chains. This feature is characteristic for the proteorhodopsin family of proteins. Third, the proton release region is connected to the bulk solvent by a chain of water molecules already in the ground state. Despite these peculiarities, the positions of water molecule and amino acid side chains in the immediate Schiff base vicinity are very well conserved. These features make ESR a very unusual proton pump. The presented structure sheds light on the large family of proteorhodopsins, for which structural information was not available previously.ID:1260
(2013). Role of Dimerization Efficiency of Transmembrane Domains in Activation of Fibroblast Growth Factor Receptor 3. J. Am. Chem. Soc. , [+]
Mutations in transmembrane (TM) domains of receptor tyrosine kinases are shown to cause a number of inherited diseases and cancer development. Here, we use a combined molecular modeling approach to understand molecular mechanism of effect of G380R and A391E mutations on dimerization of TM domains of human fibroblast growth factor receptor 3 (FGFR3). According to results of Monte Carlo conformational search in the implicit membrane and further molecular dynamics simulations, TM dimer of this receptor is able to form a number of various conformations, which differ significantly by the free energy of association in a full-atom model bilayer. The aforementioned mutations affect dimerization efficiency of TM segments and lead to repopulation of conformational ensemble for the dimer. Particularly, both mutations do not change the dimerization free energy of the predominant (putative "non-active") symmetric conformation of TM dimer, while affect dimerization efficiency of its asymmetric ("intermediate") and alternative symmetric (putative "active") models. Results of our simulations provide novel atomistic prospective of the role of G380 and A391E mutations in dimerization of TM domains of FGFR3 and their consecutive contributions to the activation pathway of the receptor.ID:972
(2013). Peptaibol antiamoebin I: spatial structure, backbone dynamics, interaction with bicelles and lipid-protein nanodiscs, and pore formation in context of barrel-stave model. Chem. Biodivers. 10 (5), 838–63 [+]
Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. In comparison with other 16-amino acid-residue peptaibols, e.g., zervamicin IIB (Zrv-IIB), Aam-I possesses relatively weak biological and channel-forming activities. In MeOH solution, Aam-I demonstrates fast cooperative transitions between right-handed and left-handed helical conformation of the N-terminal (1-8) region. We studied Aam-I spatial structure and backbone dynamics in the membrane-mimicking environment (DMPC/DHPC bicelles)(1) ) by heteronuclear (1) H,(13) C,(15) N-NMR spectroscopy. Interaction with the bicelles stabilizes the Aam-I right-handed helical conformation retaining significant intramolecular mobility on the ms-μs time scale. Extensive ms-μs dynamics were also detected in the DPC and DHPC micelles and DOPG nanodiscs. In contrast, Zrv-IIB in the DPC micelles demonstrates appreciably lesser mobility on the μs-ms time scale. Titration with Mn(2+) and 16-doxylstearate paramagnetic probes revealed Aam-I binding to the bicelle surface with the N-terminus slightly immersed into hydrocarbon region. Fluctuations of the Aam-I helix between surface-bound and transmembrane (TM) state were observed in the nanodisc membranes formed from the short-chain (diC12 : 0) DLPC/DLPG lipids. All the obtained experimental data are in agreement with the barrel-stave model of TM pore formation, similarly to the mechanism proposed for Zrv-IIB and other peptaibols. The observed extensive intramolecular dynamics explains the relatively low activity of Aam-I.ID:978
(2012). Recombinant expression and solution structure of antimicrobial peptide aurelin from jellyfish Aurelia aurita. Biochem. Biophys. Res. Commun. 429 (1-2), 63–9 [+]
Aurelin is a 40-residue cationic antimicrobial peptide isolated from the mezoglea of a scyphoid jellyfish Aurelia aurita. Aurelin and its (15)N-labeled analogue were overexpressed in Escherichia coli and purified. Antimicrobial activity of the recombinant peptide was examined, and its spatial structure was studied by NMR spectroscopy. Aurelin represents a compact globule, enclosing one 3(10)-helix and two α-helical regions cross-linked by three disulfide bonds. The peptide binds to anionic lipid (POPC/DOPG, 3:1) vesicles even at physiological salt concentration, it does not interact with zwitterionic (POPC) vesicles and interacts with the DPC micelle surface with moderate affinity via two α-helical regions. Although aurelin shows structural homology to the BgK and ShK toxins of sea anemones, its surface does not possess the "functional dyad" required for the high-affinity interaction with the K(+)-channels. The obtained data permit to correlate the modest antibacterial properties and membrane activity of aurelin.ID:977
(2012). Structure and dynamics of cardiotoxins. Curr. Protein Pept. Sci. 13 (6), 570–84 [+]
Cytotoxins (or cardiotoxins; CTs) are toxins from cobra venom characterized by the three-finger (TF) fold. CTs are on average 60-residue-long peptides, possessing as many as 4 disulfide bonds. The elements of antiparallel β-structure take origin from the hydrophobic core formed by the disulfides. The β-strands adopt the shape of the three loops, giving the name of the fold. While neurotoxins (NTs) - also TF proteins from snake venom - exert their effect through specific interactions with protein receptors, no specific protein target has been found for CTs. Unlike NTs, CTs are amphiphilic and cytotoxic against a variety of cells, including cancer ones. Thus, the hypothesis that the activity of CTs is caused by their interactions with lipid membranes is currently central. To understand molecular basis behind variations in toxicities of CTs highly homologous in their sequences, detailed knowledge of their structure and dynamics is required. The present review summarizes experimental and computational data on the spatial organization of CTs and their dynamics in various environments (aqueous solution, membranous milieus).ID:772
(2011). The role of chain rigidity in lipid self-association: Comparative study of dihexanoyl- and disorbyl-phosphatidylcholines. Chem. Phys. Lipids 165, 382–386 [+]
In the course of structure-function investigations of lipids a phosphatidylcholine molecule with short and rigid tails, di-2,4-hexadienoylphosphatidylcholine (DiSorbPC), was synthesized and studied in comparison with its saturated analog, dihexanoylphosphatidylcholine (DHPC). Conjugated double bonds in the acyl chains in DiSorbPC reduce considerably the number of possible conformers of the lipid within an aggregate. This leads to impaired packing of unsaturated acyl chains and thus, to a surprisingly high (115Å(2)) area per molecule for DiSorbPC at the air-water interface and failure to form micelles of regular size and shape. Details on DiSorbPC aggregation and packing provided by a set of experimental techniques combined with molecular dynamics simulations are presented.ID:669
(2011). Molecular mechanism of action of β-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers. Biochemistry 50 (28), 6255–65 [+]
The membrane-active, cationic, β-hairpin peptide, arenicin, isolated from marine polychaeta Arenicola marina exhibits a broad spectrum of antimicrobial activity. The peptide in aqueous solution adopts the significantly twisted β-hairpin conformation without pronounced amphipathicity. To assess the mechanism of arenicin action, the spatial structure and backbone dynamics of the peptide in membrane-mimicking media and its pore-forming activity in planar lipid bilayers were studied. The spatial structure of the asymmetric arenicin dimer stabilized by parallel association of N-terminal strands of two β-hairpins was determined using triple-resonance nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles. Interaction of arenicin with micelles and its oligomerization significantly decreased the right-handed twist of the β-hairpin, increased its amphipathicity, and led to stabilization of the peptide backbone on a picosecond to nanosecond time scale. Relaxation enhancement induced by water-soluble (Mn(2+)) and lipid-soluble (16-doxylstearate) paramagnetic probes pointed to the dimer transmembrane arrangement. Qualitative NMR and circular dichroism study of arenicin-2 in mixed DPC/1,2-dioleoyl-sn-glycero-3-phosphoglycerol bicelles, sodium dodecyl sulfate micelles, and lipid vesicles confirmed that a similar dimeric assembly of the peptide was retained in membrane-mimicking systems containing negatively charged lipids and detergents. Arenicin-induced conductance was dependent on the lipid composition of the membrane. Arenicin low-conductivity pores were detected in the phosphatidylethanolamine-containing lipid mixture, whereas the high-conductivity pores were observed in an exclusively anionic lipid system. The measured conductivity levels agreed with the model in which arenicin antimicrobial activity was mediated by the formation of toroidal pores assembled of two, three, or four β-structural peptide dimers and lipid molecules. The structural transitions involved in arenicin membrane-disruptive action are discussed.ID:535
(2010). Isolation, structure elucidation, and synergistic antibacterial activity of a novel two-component lantibiotic lichenicidin from Bacillus licheniformis VK21. Biochemistry 49 (30), 6462–72 [+]
A novel synergetic lantibiotic pair, Lchalpha(3249.51 Da) and Lchbeta(3019.36 Da), termed lichenicidin VK21, was isolated from the producer strain Bacillus licheniformis VK21. Chemical and spatial structures of Lchalphaand Lchbeta were determined. Each peptide contains 31 amino acid residues linked by 4 intramolecular thioether bridges and the N-terminal 2-oxobutyryl group. Spatial structures of Lchalpha and Lchbetawere studied by NMR spectroscopy in methanol solution. Lchalpha peptide displays structural homology with mersacidin-like lantibiotics and involves relatively well-structured N- and C-terminal domains connected by a flexible loop stabilized by thioether bridge Ala11-S-Ala21. In contrast, the Lchbetapeptide represents prolonged hydrophobic alpha-helix flanked with more flexible N- and C-terminal domains. A lantibiotic cluster of the Bacillus licheniformis VK21 genome which comprises the structural genes, lchA1 and lchA2, encoding the lantibiotics precursors, as well as the gene of a modifying enzyme lchM1, was amplified and sequenced. The mature peptides, Lchalphaand Lchbetainteract synergistically to possess antibiotic activity against Gram-positive bacteria within a nanomolar concentration range, though the individual peptides were shown to be active at micromolar concentrations. Our results afford molecular insight into mechanism of lichenicidin VK21 action.ID:359
(2010). Spatial Structure of the Transmembrane Domain Heterodimer of ErbB1 and ErbB2 Receptor Tyrosine Kinases. J. Mol. Biol. 400 (2), 231–243 [+]
Growth factor receptor tyrosine kinases of the ErbB family play a significant role in vital cellular processes and various cancers. During signal transduction across plasma membrane, ErbB receptors are involved in lateral homodimerization and heterodimerization with proper assembly of their extracellular single-span transmembrane (TM) and cytoplasmic domains. The ErbB1/ErbB2 heterodimer appears to be the strongest and most potent inducer of cellular transformation and mitogenic signaling compared to other ErbB homodimers and heterodimers. Spatial structure of the heterodimeric complex formed by TM domains of ErbB1 and ErbB2 receptors embedded into lipid bicelles was obtained by solution NMR. The ErbB1 and ErbB2 TM domains associate in a right-handed alpha-helical bundle through their N-terminal double GG4-like motif T(648)G(649)X(2)G(652)A(653) and glycine zipper motif T(652)X(3)S(656)X(3)G(660), respectively. The described heterodimer conformation is believed to support the juxtamembrane and kinase domain configuration corresponding to the receptor active state. The capability for multiple polar interactions, along with hydrogen bonding between TM segments, correlates with the observed highest affinity of the ErbB1/ErbB2 heterodimer, implying an important contribution of the TM helix-helix interaction to signal transduction.ID:346
(2010). Left-handed dimer of EphA2 transmembrane domain: Helix packing diversity among receptor tyrosine kinases. Biophys. J. 98 (5), 881–9 [+]
The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands control a diverse array of cell-cell interactions in the developing and adult organisms. During signal transduction across plasma membrane, Eph receptors, like other receptor tyrosine kinases, are involved in lateral dimerization and subsequent oligomerization presumably with proper assembly of their single-span transmembrane domains. Spatial structure of dimeric transmembrane domain of EphA2 receptor embedded into lipid bicelle was obtained by solution NMR, showing a left-handed parallel packing of the transmembrane helices (535-559)(2). The helices interact through the extended heptad repeat motif L(535)X(3)G(539)X(2)A(542)X(3)V(546)X(2)L(549) assisted by intermolecular stacking interactions of aromatic rings of (FF(557))(2), whereas the characteristic tandem GG4-like motif A(536)X(3)G(540)X(3)G(544) is not used, enabling another mode of helix-helix association. Importantly, a similar motif AX(3)GX(3)G as was found is responsible for right-handed dimerization of transmembrane domain of the EphA1 receptor. These findings serve as an instructive example of the diversity of transmembrane domain formation within the same family of protein kinases and seem to favor the assumption that the so-called rotation-coupled activation mechanism may take place during the Eph receptor signaling. A possible role of membrane lipid rafts in relation to Eph transmembrane domain oligomerization and Eph signal transduction was also discussed.ID:320
(2009). Bacterial synthesis, purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels. Biochemistry Mosc. 74 (12), 1344–9 [+]
An efficient method is described for production of membrane protein KCNE3 and its isotope labeled derivatives ((15)N-, (15)N-/13C-) in amounts sufficient for structural-functional investigations. The purified protein preparation within different detergent micelles was characterized using dynamic light scattering, CD spectroscopy, and NMR spectroscopy. It is shown that within DPC/LDAO micelles the protein is in monomeric form and acquires mainly alpha-helical conformation. The existence of cross-peaks for all glycines of the (15)N-HSQC NMR spectra as well as relatively small line widths (~20 Hz) confirm the high quality of the preparation and the possibility of obtaining structural-dynamic information on KCNE3 by high resolution heteronuclear NMR spectroscopy.ID:273
(2009). Lipid-protein nanodiscs: possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides. Biochemistry Mosc. 74 (7), 756–65 [+]
High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). (15)N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and (31)P-NMR spectroscopy. The 2D 1H-(15)N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased (1)H(N) line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. (15)N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (approximately 40 nsec at 45 degrees C) is indicative of additional peptide motions within the Aam-I/LPN complex.ID:353
(2009). Molecular dynamics simulation of antimicrobial peptide arenicin-2: beta-hairpin stabilization by noncovalent interactions. Biopolymers 92 (3), 143–55 [+]
Arenicin-2 is a 21 residue antimicrobial cyclic peptide, possessing one disulphide bond between residues Cys(3) and Cys(20). NMR and CD studies suggested that the structure of arenicin-2 in water represented a well formed, but highly twisted beta-hairpin. To investigate the spatial arrangement of the peptide side chains and to get a clear view of its possible amphipathic properties we performed molecular dynamics in explicit water. Four independent trajectories, 50 ns in length, were produced, starting from various initial conformations or by applying different simulation conditions. Arenicin-2 retained its beta-hairpin structure during simulations, although the residues close to strand ends were found to escape from the ideal hairpin conformation. The type I' beta-turn connecting the two strands fluctuated between type IV and II' beta-turn. Conversely, the right-handed twist of the beta-hairpin was well conserved with average twist value 203 degrees +/- 19 degrees per eight residues. Several nonbonded interactions, like hydrophobic interactions between aliphatic side chains, cation/pi-aromatic interactions, CH...pi aromatic bond and water bridges, contributed to the hairpin stabilization.ID:410
(2008). Molecular insight into mechanism of antimicrobial action of the beta-hairpin peptide arenicin: specific oligomerization in detergent micelles. Biopolymers 89 (5), 455–64 [+]
Arenicins are 21-residue cationic antimicrobial peptides isolated from marine polychaeta Arenicola marina. The peptides exhibit potent broad-spectrum antimicrobial activity. In water solution arenicin-2 adopts a beta-hairpin conformation, stabilized by one disulfide and nine hydrogen bonds. To determine the propensity for the peptide oligomerization in membrane mimetic systems, the recombinant arenicin-2 was overexpressed as a fused form in Escherichia coli. The arenicin-2 oligomerization and intermolecular packing in membrane mimicking environment were investigated using high-resolution NMR spectroscopy. The present studies show that arenicin-2 preserves a beta-hairpin structure and forms asymmetric dimers upon incorporation into the dodecylphosphocholine micelle. Two monomers of arenicin-2 are aligned parallel to each other by the N-terminal strands of the beta-hairpin (CN upward arrow upward arrowNC type of association). Polyacrylamide gel electrophoresis analysis indicated that in environment of anionic SDS micelles the arenicin-2 might undergo further oligomerization and form tetramers. Our results afford further molecular insight into possible mechanism of antimicrobial action of arenicins.ID:414
(2008). Three-dimensional structure/hydrophobicity of latarcins specifies their mode of membrane activity. Biochemistry 47 (11), 3525–33 [+]
Latarcins, linear peptides from the Lachesana tarabaevi spider venom, exhibit a broad-spectrum antimicrobial activity, likely acting on the bacterial cytoplasmic membrane. We study their spatial structures and interaction with model membranes by a combination of experimental and theoretical methods to reveal the structure-activity relationship. In this work, a 26 amino acid peptide, Ltc1, was investigated. Its spatial structure in detergent micelles was determined by (1)H nuclear magnetic resonance (NMR) and refined by Monte Carlo simulations in an implicit water-octanol slab. The Ltc1 molecule was found to form a straight uninterrupted amphiphilic helix comprising 8-23 residues. A dye-leakage fluorescent assay and (31)P NMR spectroscopy established that the peptide does not induce the release of fluorescent marker nor deteriorate the bilayer structure of the membranes. The voltage-clamp technique showed that Ltc1 induces the current fluctuations through planar membranes when the sign of the applied potential coincides with the one across the bacterial inner membrane. This implies that Ltc1 acts on the membranes via a specific mechanism, which is different from the carpet mode demonstrated by another latarcin, Ltc2a, featuring a helix-hinge-helix structure with a hydrophobicity gradient along the peptide chain. In contrast, the hydrophobic surface of the Ltc1 helix is narrow-shaped and extends with no gradient along the axis. We have also disclosed a number of peptides, structurally homologous to Ltc1 and exhibiting similar membrane activity. This indicates that the hydrophobic pattern of the Ltc1 helix and related antimicrobial peptides specifies their activity mechanism. The latter assumes the formation of variable-sized lesions, which depend upon the potential across the membrane.ID:315
(2008). Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state. J. Biol. Chem. 283 (11), 6950–6 [+]
Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed alpha-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations.ID:314
- (2008). Lipid-protein nanoscale bilayers: a versatile medium for NMR investigations of membrane proteins and membrane-active peptides. J. Am. Chem. Soc. 130 (7), 2140–1 ID:356
(2007). Recombinant expression, synthesis, purification, and solution structure of arenicin. Biochem. Biophys. Res. Commun. 360 (1), 156–62 [+]
Arenicins are 21-residue cationic antimicrobial peptides, isolated from marine polychaeta Arenicola marina. In order to determine a high-resolution three-dimensional structure of arenicin-2, the recombinant peptide was overexpressed as a fused form in Escherichia coli. Both arenicin isoforms were synthesized using the Fmoc-based solid-phase strategy. Recombinant and synthetic arenicins were purified, and their antimicrobial and spectroscopic properties were analyzed. NMR investigation shows that in water solution arenicin-2 displays a prolonged beta-hairpin, formed by two antiparallel beta-strands and stabilized by one disulfide and nine hydrogen bonds. A significant right-handed twist in the beta-sheet is deprived the peptide surface of amphipathicity. CD spectroscopic analysis indicates that arenicin-2 binds to the SDS and DPC micelles, and conformation of the peptide is significantly changed upon binding. Arenicin strongly binds to anionic lipid (POPE/POPG) vesicles in contrast with zwitterionic (POPC) ones. These results suggest that arenicins are membrane active peptides and point to possible mechanism of their selectivity toward bacterial cells.ID:419
(2007). Antiamoebin I in methanol solution: rapid exchange between right-handed and left-handed 3(10)-helical conformations. Chem. Biodivers. 4 (6), 1219–42 [+]
Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. Antiamoebin I has the amino acid sequence: Ac-Phe(1)-Aib-Aib-Aib-Iva-Gly-Leu-Aib(8)-Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phl(16). By using the uniformly (13)C,(15)N-labeled sample of Aam-I, the set of conformationally dependent J couplings and (3h)J(NC) couplings through H-bonds were measured. Analysis of these data along with the data on magnetic nonequivalence of the (13)C(beta) nuclei (Deltadelta((13)C(beta))) in Aib and Iva residues allowed us to draw the univocal conclusion that the N-terminal part (Phe(1)-Gly(6)) of Aam-I in MeOH solution is in fast exchange between the right-handed and left-handed 3(10)-helical conformations, with an approximately equal population of both states. An additional conformational exchange process was found at the Aib(8) residue. The (15)N-NMR-relaxation and CD-spectroscopy measurements confirmed these findings. Molecular modeling and Monte Carlo simulations revealed that both exchange processes are correlated and coupled with significant hinge-bending motions around the Aib(8) residue. Our results explain relatively low activity of Aam-I with respect to other 15-amino acid residue peptaibols (for example, zervamicin) in functional and biological tests. The high dynamic 'propensity' possibly prevents both initial binding of the antiamoebin to the membrane and subsequent formation of stable ionic channels according to the barrel-stave mechanism.ID:418
(2006). Solution of the Spatial Structure of Dimeric Transmembrane Domains of Proteins by Heteronuclear NMR Spectroscopy and Molecular Modeling. Biophysics 51 (S1), S23–S27 [+]
Membrane proteins play an important role in various biological processes. An approach combiningID:347
NMR spectroscopy with molecular modeling was used to study the spatial structure and intramolecular dynamics of protein transmembrane domains consisting of two interacting α-helices. The approach was tested with model transmembrane domains and yielded detailed atomic-level data on the protein–protein and protein–lipid interactions.
(1995). Processing of heteronuclear NMR relaxation data with the new software DASHA. Appl. Mag. Reson. 9 (4), 581–588 [+]
The new program DASHA is an efficient implementation of common data processing steps for the protein internal dynamic analysis. The "model-free" parameters and their uncertainties (Lipari G., Szabo A.: J. Am. Chem. Soc.104, 4546–4559 (1982) can be calculated from an arbitrary combination of experimental data sets (i.e. heteronuclear1H−15N or1H−13C relaxation times and NOE values at different spectrometer frequencies). Anisotropy of the molecular rotational diffusion could be also taken into account without introduction of the new adjustable parameters into the spectral density functionJ(ω), provided the structure of the molecule is known. Parameters of chemical (conformational) exchange can be estimated from the CPMG spin-lock frequency dependences (Bloomet al.: J. Chem. Phys.42, 1615–1624 (1965); Orekhovet al.: Eur. J. Biochem.219, 887–896 (1994). The program can be used both in the interactive and batch modes. It has sophisticated PostScript plotting facilities.ID:1046
(1985). 1H-NMR study of gramicidin A transmembrane ion channel. Head-to-head right-handed, single-stranded helices. FEBS Lett. 186 (2), 168–174 [+]
The structure of [Val1] gramicidin A incorporated into sodium dodecyl-d25 sulphate micelles has been studied by two-dimensional proton NMR spectroscopy. Analysis of nuclear Overhauser effects, spin-spin couplings and solvent accessibility of NH groups show that the conformation of the Na+ complex of gramicidin A in detergent micelles, which in many ways mimic the phospholipid bilayer of biomembranes, is an N-terminal to N-terminal (head-to-head) dimer formed by two right-handed, single-stranded β6.3 helices with 6.3 residues per turn, differing from Urry's structure by handedness of the helices.ID:144
(1973). Refinement of the angular dependence of the peptide vicinal NH-CaH coupling constant. Tetrahedron 29 (6), 873–877 [+]
The refined dependence of the peptide NHCαH vicinal coupling constant on the dihedral angle θ have been derived on the basis of the accumulated experimental data. The mean permissible values (in Hz) are approximated by 3JNHCH = 9·4 cos2 θ - 1·1 cos θ + 0·4 An analogous relationship for the sum of two vicinal NH-CαH2 coupling constants in the glycyl residue have been calculated from the above dependence. Measurements on N-methylacetamide in various solvents and in the presence of an alkali salt showed the vicinal constant NH-CH to vary by not more than ± 3%. Some of the other proposed 3JNHCH(θ) dependencies give too low values for the cis-oriented NH and CαH bonds. This may be due to the fact that in these correlations the data for compounds with cis-amide bonds have been used for 0° - θ - 90° region of the dependence.ID:1044
(1971). NMR differentiation of the internal and external phospholipid membrane surfaces using paramagnetic Mn2+ and Eu3+ ions. Chem. Phys. Lipids 6 (4), 343–350 [+]
The effect of paramagnetic Mn2+ and Eu3+ ions on the NMR spectra of sonicated lecithin dispersions in water and benzene has been investigated. In aqueous dispersions containing paramagnetic ions the N+(CH3)3 signal of the lecithin molecules in the lipid bilayer consists of two components, one broadened by Mn2+ and shifted to high field by Eu3+ must correspond to molecules in the external surface in contact with the bulk salt solution, and the other corresponding to internal lecithin molecules in contact with the ion free internal aqueous phase. Thus the external and internal surfaces of a lecithin containing membrane can be discriminated. The rates of exchange of the paramagnetic ions, of the lecithin and the water molecules between micelles and/or between micelles and external medium have been evaluated.ID:1045
Alexander Arseniev
- Russia, Moscow, Ul. Miklukho-Maklaya 16/10 On the map
- IBCh RAS, build. 33, office. 129
- Phone:
- E-mail: email@example.com
Alternative dimerization of the EGFR transmembrane domain and protein-lipid mediated signal transduction mechanism of RTK activation (2016-11-18)
The epidermal growth factor receptor EGFR is a representative of HER/ErbB receptor tyrosine kinases family (RTK) and plays important role in cell proliferation and differentiation, both in normal and pathological conditions of the human organism. With the help of high-resolution NMR spectroscopy, we showed that a change of membrane mimicking environment leads to alternative dimerization of the EGFR TM domain. Comparing the investigation results with the published data for the ligand-binding, juxtamembrane and kinase domains, we proposed a novel mechanism of RTK signaling through the cell membrane by means of protein-lipid interactions, explaining a number of paradoxes observed at RTK activation.
- (2016). Alternative packing of EGFR transmembrane domain suggests that protein-lipid interactions underlie signal conduction across membrane. Biochim. Biophys. Acta 1858 (6), 1254–61 [+]
The human epidermal growth factor receptor (EGFR) of HER/ErbB receptor tyrosine kinase family mediates a broad spectrum of cellular responses transducing biochemical signals via lateral dimerization in plasma membrane, while inactive receptors can exist in both monomeric and dimeric forms. Recently, the dimeric conformation of the helical single-span transmembrane domains of HER/ErbB employing the relatively polar N-terminal motifs in a fashion permitting proper kinase activation was experimentally determined. Here we describe the EGFR transmembrane domain dimerization via an alternative weakly polar C-terminal motif A(661)xxxG(665) presumably corresponding to the inactive receptor state. During association, the EGFR transmembrane helices undergo a structural adjustment with adaptation of inter-molecular polar and hydrophobic interactions depending upon the surrounding membrane properties that directly affect the transmembrane helix packing. This might imply that signal transduction through membrane and allosteric regulation are inclusively mediated by coupled protein-protein and protein-lipid interactions, elucidating paradoxically loose linkage between ligand binding and kinase activation.ID:1596
- (2016). Cell Free Expression and Purification of the Fragments of the Receptor Tyrosine Kynases of the EGFR Family, Containing the Transmembrane Domain with the Juxtamembrane Region, for Structural Studies. BIOLOGICHESKIE MEMBRANY 33 (2), 124–132 [+]
The EGFR/HER receptor family of an epidermal growth factor represents an important class of the receptor tyrosine kinases playing the key role in the control of cell growth and differentiation in mammalian cells, as well as in the development of a number of pathological processes, including oncogenesis. Binding of a ligand to the extracellular domains initiates switching of the EGFR/HER receptor between the alternative dimeric states that causes the allosteric activation of kinase domains in cell cytoplasm. The transmembrane (TM) domain and adjacent flexible regions alternatively interacting with either membrane surface or kinase domains are directly involved in the complex conformational transition in EGFR/HERs. Here we report on a highly efficient system of the cell free production of the EGFR/HER TM domains with functionally important juxtamembrane (JM) regions for the investigation of the molecular basis of biochemical signal transduction across the cell membrane. To increase the efficiency of synthesis of the EGFR/HER TM-JM fragments of the receptors, we used two N-terminal expression tags, which significantly increased the protein yield. In the case of the TM-JM fragments of EGFR (residues 638–692) and HER2 (residues 644–700), the method allowed us to obtain milligram quantities of the 13C,15N-labeled protein for structural and biophysical investigations in the membrane-mimicking environments using high-resolution heteronuclear NMR spectroscopy.ID:1622


text:
David Islam:
So proud. #CumLaude #Olympians #newchapter
David Islam:
So proud. #CumLaude #Olympians #newchapter
Luis Fenero's Instagram Story 5/6/2017–Alex and Mitch hanging out with Gabriella Papadais and Luis Fenero :)
Alexandra Paul's Instagram Story
Sochi 2014 ❆ Kaitlyn Weaver/Andrew Poje, Alexandra Paul/Mitchell Islam (Canada) [#4]
(x, November 12 Story)
At Cup of China 2016!
Canadian Skaters @ HPC Karaoke to Journey's "Don't Stop Believing"
Team Canada after Meagan Duhamel and Eric Radford's short program in the team competition.
Can we just take a moment to appreciate the lovely "ghost photographers" who always take the cutest pics of their girls?
Good job Mitch and Scott! 👌
Our mistakes they were bound to be made
But I promise you, I'll keep you safe. (x)
Team Canada and Team USA at the closing banquet at 4CC 2015
Parade of Champions | Canada's Figure Skating Olympic Team
Season 2014/15 + a splash of color

subset: commoncrawl


text:
- Telephone:
- 07441 913394 | 01484 973869
Key factsPrint summary
Nice clean family car..very good spec.... drives really nice...great colour Part exchange to clear...no warning lights on... just a genuine nice clean car...ready to drive away... Don't miss out on this great Family Bargain ! 4 owners, Next MOT due 18/03/2018, Part service history, Good bodywork, Black Cloth interior - Good Condition, Tyre condition Good, Grey, Mot Until March 2018, Please Ring Before Setting Off To View ! FINANCE on used cars !!!! Prices Discounted for Internet, £1,500
This car comes with
Alloy Wheels Upg. 16in Santiaguito to 17in RINJANI, Alloy Wheels Upg. 16in Santiaguito to 17in RINJANI, Telephone Equipment (Bluetooth Interface), Alloy Wheels Upg. 16in Santiaguito to 17in RINJANI, Rain Sensor, Alloy Wheels Upg. 16in Santiaguito to 17in RINJANI, Side Protection Mouldings, Tyre Repair Kit, Tachometer, External Temperature Display, Headlight Elevation, Rear Wash/Wipe, ESP, Grab Handles, Traction Control System, ESP, Seats Split Rear, Air-Conditioning, Upholstery Cloth, Cup Holder, Electronic Brake Force Distribution, Immobiliser, Alloy Wheels (17in), Speakers, Third Brake Light, Seat Belt Pre-Tensioners, Rev Counter, Seat Height Adjustment, Clock, Climate Control, Steering Wheel Mounted Controls, Air Bag Passenger, Spare Wheel, Front Fog Lights, Armrest, Child Locks, Body Coloured Bumpers, Seat - ISOFIX Anchorage Point, Steering Wheel Leather, Power Socket, Trip Computer, Central Door Locking, Power-Assisted Steering, Head Restraints, Air Bag Side, Electric Windows (Front/Rear), Electronic Stability Programme, ESP, Mirrors External, Ashtray/Lighter, Centre Rear Seat Belt, Tinted Glass, Windscreen Wiper, Deadlocks, Adjustable Steering Column/Wheel, Cruise Control, Anti-Lock Brakes, Air Bag Driver, In Car Entertainment (Radio/CD/MP3)
- 201711131221929
- 2007
- 57
- £1,500
- 150
- 106,000
- Cars
- 308
- 9vjzr7h1YIs=
Vehicle summary
|Body style
|Engine size
|1598 cc
|Fuel type
|Number of doors
|Number of seats
|Gearbox type
|CO2 emissions
|159 g/km
|Insurance group
|Standard manufacturer's warranty (miles)
|60000 miles
|Standard manufacturer's warranty (years)
|3 years
|Standard paintwork guarantee
|3 years
Performance & economy
|Fuel consumption (urban)
|30.4 mpg
|Fuel consumption (extra urban)
|54.3 mpg
|Fuel consumption (combined)
|42.2 mpg
|0 - 60 mph
|10.8 seconds
|Top speed
|122 mph
|Engine power
|120 bhp
|Engine torque
|118.02 lbs/ft
|1519 mm
|Height inclusive of roof rails
|No details available
|4276 mm
|2608 mm
|1821 mm
|Width including mirrors
|No details available
|Fuel tank capacity
|60 litres
|Minimum kerb weight
|1362 kg
|Interior features
|Exterior features
|Alloy Wheels (17in)
|Rev Counter
|Rear Wash/Wipe
|Power Socket
|Spare Wheel
|External Temperature Display
|Windscreen Wiper
|Climate Control
|Alloy Wheels Upg. 16in Santiaguito to 17in RINJANI
|Side Protection Mouldings
|Electric Windows (Front/Rear)
|Body Coloured Bumpers
|Cruise Control
|Mirrors External
|Adjustable Steering Column/Wheel
|Steering Wheel Leather
|Trip Computer
|Seats Split Rear
|Rain Sensor
|Tinted Glass
|In Car Entertainment (Radio/CD/MP3)
|Seat Height Adjustment
|Cup Holder
|Upholstery Cloth
|Steering Wheel Mounted Controls
|Grab Handles
|Telephone Equipment (Bluetooth Interface)
|Seat - ISOFIX Anchorage Point
|Head Restraints
|Air Bag Passenger
|Third Brake Light
|Air Bag Side
|Anti-Lock Brakes
|Headlight Elevation
|Air Bag Driver
|Tyre Repair Kit
|Child Locks
|Power-Assisted Steering
|Central Door Locking
|Seat Belt Pre-Tensioners
|Centre Rear Seat Belt
|Electronic Stability Programme
|Front Fog Lights
|Traction Control System
|Electronic Brake Force Distribution
Please note
This includes MPG figures which are calculated under European Standards, in test conditions, and will vary under normal driving conditions. The information displayed above describes the typical specification of the most recent model of this vehicle. It is not the exact specification for the actual vehicle being offered for sale which may vary. Specifications for older models may also vary. Please contact us prior to purchase to confirm the exact specification of this vehicle.
Huddersfield Car Sales Limited
Based on 46 customer reviews
Powered by
Thank you for contacting us!
We will aim to get back to you as soon as possible.
There was a problem sending your email.
There may be a technical issue.
Please try again later.


text:
Aron Ralston: Pinned in a Canyon
by Roberta Baxter
Through narrative nonfiction text, readers learn the shocking story of Aron Ralston, a man who became trapped under a rock and had to cut off his own arm to survive. Additional features to aid comprehension include a table of contents, fact-filled captions, callouts, and sidebars, a glossary, sources for further research, a listing of source notes, and an introduction to the author.
Table of Contents
- Trapped!
- How Did He Get Here?
- Counting the Hours
- I Have to Do This
- Still the Outdoorsman
- Glossary
- Source Notes
- To Learn More
- Index
- Dewey: 92
- Graphics: Full-color photographs, Historical photographs
- Reinforced book (9781634074698): 6.5 x 9, 24 pages, © 2016
- Hosted ebook (9781503807181): 6.5 x 9, 24 pages, © 2016
- Subject: Narrative Nonfiction
- Imprint: MOMENTUM
- Series: True Stories, Real People
- Suggested Interest Level: Grade 3 - Grade 6
- Suggested Reading Level: Grade 4
- Lexile® Measure: 800
- Guided Reading Level: S
- ATOS Reading Level: 5.0
- ATOS Interest Level: MG
- Accelerated Reader® Quiz: 180025
- Accelerated Reader® Points: 0.5
BISAC Subjects
- JUVENILE NONFICTION / Biography & Autobiography / General (JNF007000)
- JUVENILE NONFICTION / Biography & Autobiography / Social Activists (JNF007110)
A Review of "True Stories, Real People" in Horn Book Guide
Reviewed on 1 October 2016
True Stories, Real People series. Each short, inspiring biography is divided into four or five chapters written in an objective, unpretentious manner. Quotation sources are fastidiously footnoted and listed in the back matter. Navigation is easy thanks to clean layouts and a simple color scheme (orange photo captions, navy call-out boxes). Great for school assignments and engaging enough for pleasure reading. Reading list. Glos., ind.
Author: Roberta Baxter
Cart Summary
Your cart is empty
Common Core
- Author/Illustrator biography
- Glossary of key words
- Index
- Informative sidebars
- Primary-source text
- Sources for further research
- Suggested websites
- Table of contents
- Reviewed


text:
China on Thursday criticised Europe\'s refusal to ease two longtime trade restrictions as \"discrimination\", following a high-level meeting between the two sides this week. China has repeatedly sought to end an embargo on arms sales imposed after the deadly 1989 Tiananmen crackdown on democracy protesters. It has similarly pushed to be granted full \"market economy status\", a designation that would lift bans on certain Chinese exports and investments. \"We believe that the two issues are actually an embodiment of the discrimination against China by the European side,\" Hua Chunying, a foreign ministry councillor handling European affairs, told reporters. \"The two issues reflect a lack of trust by the European side,\" she said. \"If the European side can take a small step forward in solving these two issues, then we can make a major step forward in enhancing mutual trust.\" China and Europe have have locked horns over a series of trade issues in recent years, even as Europe has sought Chinese support in overcoming its debt crisis. Some of these issues were raised during the third annual EU-China Strategic Dialogue held in Beijing this week, although both sides emphasised at the end of the talks that they were held in good spirit and helped build ties. China is due to be granted market economy status in 2016 regardless, having agreed on a 15-year delay when it joined the World Trade Organization. Chinese Premier Wen Jiabao last year called on European nations to show their \"friendship\" by granting the status sooner. But some European leaders say that China does not yet qualify, since most of its biggest companies are state-owned and their leaders are selected by the government.
GMT 23:11 2017 Friday ,17 NovemberIMF sees stronger growth in Myanmar, but Rohingya crisis may hurt investment
GMT 22:59 2017 Friday ,17 NovemberBitcoin at all-time high near $8,000
GMT 22:38 2017 Friday ,17 NovemberBitcoin bubble warning, but blockchain takes off
GMT 00:49 2017 Friday ,17 NovemberPhilippine economy grows 6.9 percent in the third quarter, beats forecasts
GMT 07:42 2017 Thursday ,16 NovemberBubble risks in China's financial sector, government official warns
GMT 05:14 2017 Thursday ,16 NovemberFines by top Wall Street cop down in Trump's 1st year
GMT 11:46 2017 Wednesday ,15 NovemberFrankfurt, Paris to get most Goldman Brexit jobs
GMT 11:29 2017 Wednesday ,15 NovemberGlobal wealth up 27% since financial crisis: study


text:
Buy Residential Properties, Apartments and 2, 3 BH...
Property for sale
23/09/2014 Property for sale
Raheja Revanta Sector 78 Gurgaon Spread over 18.39 acres in the heart of Gurgaon at Sector 78, Raheja Revanta features a perfect blend of elegant style & design intelligence, with various options of 1, 2, 3, 4, 5, 6BHK Living Apartments.
{{Raheja Revanta Specification}}
Apartments overall Size 1198 Sq. Ft to 4961 Sq. Ft
Price/SQFT = 7875
Booking and more information Call us + 91 9891856789
For more information: http://www.raheja-revanta.co.in
CONTACT US 607 Time Tower M.G Road Gurgaon - 122002 Haryana
Are you looking for residential property or apartments in Gurgaon? Ashiana Housing is India's biggest real estate compan...
Category : Property for sale Posted : 21/11/2017 Price : Check with seller Location : Gurgaon(Haryana)
Property for sale
Yashada Realty Group (a group company of Vivanta Realty), brings in its new residential launch named Vivanta Life Veroni...
Category : Property for sale Posted : 16/11/2017 Price : Check with seller Location : Pune(Maharashtra)
Property for sale
With the support of the experience engineer, this group g for major site plan, location plan and master plan. Hence it d...
Category : Property for sale Posted : 20/11/2017 Price : 5500000.00 ₹ Location : Noida(Uttar Pradesh)
Property for sale
M3M Heights Gurgaon launch new affordable residential properties for sale in Golf Course Road Sector 65 Gurgaon.You look...
Category : Property for sale Posted : 20/11/2017 Price : 500000.00 ₹ Location : Gurgaon(Haryana)
Property for sale


text:
Missing teen Cayleb Hough was murdered, police say, after bones found in an abandoned mine shaft outside Melbourne were confirmed to be his
- Remains found in abandoned mineshaft identified as missing teenager's
- Cayleb Hough, 17, went missing almost four months ago in Melbourne
- His bones were found in shire of Bacchus Marsh, west of Melbourne
- Police are treating his death as suspicious as they appeal for information
- A detective said he could say 'quite categorically' teenager was murdered
Human remains found at the bottom of an abandoned mine shaft have been identified as those of a teenager who went missing almost four months ago.
Cayleb Hough, 17, was last seen in the area of The Crescent in Highett, in Melbourne's south-east, in the early hours of December 20, 2015.
The remains were found in a shallow mineshaft near Coimadai, in the shire of Bacchus Marsh, on Seerey Track on March 10 as police launch a murder investigation into Cayleb's death.
Scroll down for video
The human remains found at the bottom of an abandoned mineshaft have been identified as those of teenager Cayleb Hough who went missing almost four months ago
Missing Persons Squad detectives and State Emergency Services were searching the area around the mineshaft
Detective Senior Sergeant Craig Rhodes told reporters on Tuesday: 'I can say quite categorically that Cayleb Hough was murdered'
Police have confirmed they are treating the teenager's death as suspicious.
'I can say quite categorically that Cayleb Hough was murdered,' Detective Senior Sergeant Craig Rhodes told reporters on Tuesday.
A post-mortem examination has been conducted but police have declined to comment on the cause of death or if a weapon was used.
'The body took some time to identity because of the way the body was and the DNA process,' Det Sen Sgt Rhodes said.
A police investigator holds a piece a fabric alongside SES workers during the search for clues
Det Sen Sgt Rhodes appealed to the public for any information regarding the case as the search for clues continued
Police are appealing to the public to come forward if they saw anything strange in and around the state park, where the mine is, between December and March.
'It is an out-of-the-way area,' the detective said.
'If someone up here may have seen anything that was unusual... anything that was out of the norm, that little bit of evidence might support us in our investigation.'
Police do not believe the teen was been mixed up with drugs or the wrong crowd.
'We have no idea why that would have occurred,' Det Snr Sgt Rhodes said.
'He was just a young 17-year-old kid who had just left home and was looking for work. There was nothing untoward at all.'
The teen was known to police before he disappeared.
Missing Persons Squad detectives and State Emergency Services were searching the area around the mineshaft.
Hough was reportedly seen travelling in a blue 2003 Ford Falcon XR6 sedan with registration SOG 812 shortly before his disappearance.
Cayleb, 17, was last seen in the area of The Crescent in Highett, in Melbourne's south-east, in the early hours of December 20, 2015
SES members form a search line to uncover clues to Cayleb's disappearance on Tuesday
Police are appealing to the public to come forward if they saw anything strange in and around the state park, where the mine is located, between December and March
Police released photos of the jacket (left) and shorts (right) Cayleb was wearing at the time of his disappearance
The shoes Cayleb had on at the time. Police hope to find answers about Cayleb's death
Police found the sedan in the car park at a Southbank entertainment complex about 1pm on January 16.
The vehicle remains in police possession as the investigation continues.
Det Sen Sgt Rhodes said police were 'particularly keen to speak to anyone who may have seen any suspicious activity in the Lerderderg State Park since 20 December last year'.
'Someone out there knows who is responsible for Cayleb's death and we are appealing for them to come forward and contact police,' he said.
'Cayleb's family deserve some answers in relation to this horrific tragedy.'
Police have released images of clothing similar to what Hough was wearing at the time of his disappearance.
Police found the sedan in the car park at a Southbank entertainment complex about 1pm on January 16
A mock-up of the number plate that was on the vehicle is pictured above
The distinct socks Cayleb had on when he was last seen almost four months ago
Most watched News videos
- Moment off duty cop holds baby while shooting suspected thieves
- Huntswoman appears to batter saboteur with riding crop
- Serial killer and cult leader Charles Manson dies at 83
- Melania and Barron welcome Christmas tree to The White House
- Man living in iron lung for over 60 years talks about experience
- Paul Hollywood and wife Alexandra bicker on the red carpet
- Devastated sister of Gaia Pope gives an emotional tribute
- Woman helps to free deer tangled in her backyard hammock
- Moment woman films a man who sexually harasses her on the street
- CBS News addresses Charlie Rose sex harassment allegations
- Fish and seafood eaten whilst still ALIVE in Chinese tradition
- Lena Dunham and Jenni Konner at Power of Women gala in April
- Taller than mom! Barron Trump towers over Melania - aged...
- Great British Bake Off Star Paul Hollywood and his wife...
- Malia Obama caught kissing a mystery man and smoking a...
- Paul Hollywood's marriage 'crumbled over his...
- Hostages speak out about being gang-raped in captivity,...
- Barbra Streisand says she'll never do anther show after...
- 'I finished her off, I strangled her. I got up and wiped...
- Crew of missing Argentine submarine 'have just TWO DAYS...
- PIERS MORGAN: Is ludicrous Lena (with her massively...
- America's last iron lungs: Incredible video shows Polio...
- Charlie Rose canned: CBS suspends host and his eponymous...
- Mother posts warning online after hibernating hamster...
- Kim bans fun: North Korean dictator outlaws drinking and...
- EXCLUSIVE: 'Aled Jones is as capable of sexual harassment...
- 'Hi, I'm Cait and these are my two boyfriends':...
- 'ISIS is over': Iranian president declares terror group...
- EXCLUSIVE: Wearing a hoodie and shades, Kevin Spacey...
- Mom of Silicon Valley CEO Anne Wojcicki says she knew her...


text:
So Ashton's going on a road trip with his friend, Calum, and he invited me to tag along. Being the adventurous shit I am, I said yes, but I wouldn't have if I knew that Luke Hemmings was going as well. God, I hate that guy, and the feeling's mutual. Why does that annoying brat have to come along? And Ashton and Calum expect us to be friends? Yeah, right. There's no chonce that Luke Hemmings and I, Michael Clifford, are ever going to get along.
Cover: _Jikko
Other pairing(s): Cashton (minor)
© 2014 iCheeseYou
- 5secondsofsummer
- 5secondsofsummerau
- 5sos
- 5sosau
- ashtonirwin
- boyxboy
- calumhood
- highschoolau
- lukehemmings
- michaelclifford
- muke
- muke5sos
- mukeclemmings
- mukefanfiction
- mukefluff
- mukesmut
multiplefd Oct 03
javdjagjdvsizkshwjvrksiydjs, have sex and film it and leak it, thnx
Larry_Ashden Oct 22
I get 3 likes from my mom,my sister and my best friend...that's it
multiplefd Oct 03
i killed a spider with my brother's show and made my mom make sure it was dead. she was like 'why tf are you scared of this tiny ass spider?', and i was just screaming the whole time.
shipsnshit Sep 22
English this year is dull but the teacher always looks annoyed with everyone and he seems cool so I relate
prettythoughtsx_ Nov 08
I thought it said hair dryer and hand dryer so I was confused and was like, "bítch where the fück did you get a hair dryer?" Then I reread it 3 more times and realized it said hand 😂😂
kylalamara 15 hours ago
"really good foreshadowing is called 5-shadowing" -John Greene


text:
- sort by:
53 properties in LincolnshireDid you mean?
- Check you have entered the location correctly.
- Alternatively enter another alternative location or adjust your search criteria.
- If 'no results' continues to show, we may not have any properties for sale in your desired location.
4 Bedroom Detached Bungalow For Sale
Fen Lane, East Keal, Spilsby, PE23
This is a rare opportunity to purchase a superbly presented cottage style detached bungalow with full range of equestrian facilities. This is a spacious Four bedroom property set with 2 acres with a stable block, paddock, Menage, established gardens, pond and a large...
3 Bedroom Detached Bungalow For Sale
Main Street, Horsington, Woodhall Spa, LN10
This superbly presented detached Bungalow is situated within the popular village of Horsington. The accommodation benefits from three bedrooms with an en-suit to the master bedroom, large lounge, Kitchen, Utility Room, Workshop and Garage. The property also offers a...
3 Bedroom Detached Bungalow For Sale
Cole Lane, Stickford, Boston, PE22
Bairstow Eves are pleased to offer this executive bungalow that offers spacious accommodation. Having three double bedrooms with en-suite to the master bedroom this property also has a large family bathroom, lounge, kitchen open plan with separate dining room and a...
3 Bedroom Detached Bungalow For Sale
Lincoln Road, Horncastle, LN9
The accommodation benefits from an updated farmhouse style kitchen with conveniences including dishwasher and double oven, UPVC conservatory, utility and three double bedrooms. The master bedroom benefits from an en-suite and a dressing room. Outside, the mature garden
4 Bedroom Detached Bungalow For Sale
Lady Meers Road, Cherry Willingham, Lincoln, LN3
Fabulous edge of village position this bungalow has it all!One of the best positioned bungalows on the development having a private road access and fields to the side. The spacious accommodation includes fitted kitchen and utility, lounge and dining room, four bedrooms
3 Bedroom Detached Bungalow For Sale
Woodland Drive, Woodhall Spa, LN10
Fantastic opportunity to purchase a spacious detached three bedroom bungalow in the highly desirable village and Spa resort of Woodhall Spa. The bungalow would benefit from some modernisation internally but the price does reflect this. The spacious rooms which extend...
3 Bedroom Detached Bungalow For Sale
North Road, Tattershall Thorpe, Lincoln, LN4
No ChainHaving been fully renovated to a high standard, this three bedroom detached bungalow occupies a well-maintained plot and is deceptively spacious - driving by will not do it justice. With three reception rooms to include a lounge and a dining room that is open...
4 Bedroom Detached Bungalow For Sale
Lyndon Crescent, Louth, LN11
Four bedroom detached bungalow with extensive living accommodation throughout, good size gardens, single garage, and gas central heating. The accommodation comprise of: Lounge, dining room, kitchen with fitted appliances, conservatory, master bedroom with en-suite...
3 Bedroom Detached Bungalow For Sale
Church Drive, Norton Disney, Lincoln, LN6
Immaculately presented detached bungalow in a pleasant non estate position. Features includes long driveway with ample parking and garage, super landscaped gardens extending to approximately 0.23 acres, and pleasant views over pastureland to the rear. Internally there...
3 Bedroom Detached Bungalow For Sale
New York Road, Dogdyke, Lincoln, LN4
Recently reduced by a motivated vendor This charming country cottage style bungalow has been fully refurbished by the current owner and simply has to be viewed to fully appreciate it. Having a breakfast kitchen with double doors leading to a large lounge, the property...
- sort by:
- Single Property
- Multiple Properties