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Sample records for transmembrane helix structure

  1. Transmembrane helix structure, dynamics, and interactions: multi-nanosecond molecular dynamics simulations.

    PubMed Central

    Shen, L; Bassolino, D; Stouch, T

    1997-01-01

    To probe the fundamentals of membrane/protein interactions, all-atom multi-nanosecond molecular dynamics simulations were conducted on a single transmembrane poly(32)alanine helix in a fully solvated dimyristoyphosphatidylcholine (DMPC) bilayer. The central 12 residues, which interact only with the lipid hydrocarbon chains, maintained a very stable helical structure. Helical regions extended beyond these central 12 residues, but interactions with the lipid fatty-acyl ester linkages, the lipid headgroups, and water molecules made the helix less stable in this region. The C and N termini, exposed largely to water, existed as random coils. As a whole, the helix tilted substantially, from perpendicular to the bilayer plane (0 degree) to a 30 degrees tilt. The helix experienced a bend at its middle, and the two halves of the helix at times assumed substantially different tilts. Frequent hydrogen bonding, of up to 0.7 ns in duration, occurred between peptide and lipid molecules. This resulted in correlated translational diffusion between the helix and a few lipid molecules. Because of the large variation in lipid conformation, the lipid environment of the peptide was not well defined in terms of "annular" lipids and on average consisted of 18 lipid molecules. When compared with a "neat" bilayer without peptide, no significant difference was seen in the bilayer thickness, lipid conformations or diffusion, or headgroup orientation. However, the lipid hydrocarbon chain order parameters showed a significant decrease in order, especially in those methylene groups closest to the headgroup. Images FIGURE 1 FIGURE 14 PMID:9199766

  2. High-accuracy prediction of transmembrane inter-helix contacts and application to GPCR 3D structure modeling

    PubMed Central

    Yang, Jing; Jang, Richard; Zhang, Yang; Shen, Hong-Bin

    2013-01-01

    Motivation: Residue–residue contacts across the transmembrane helices dictate the three-dimensional topology of alpha-helical membrane proteins. However, contact determination through experiments is difficult because most transmembrane proteins are hard to crystallize. Results: We present a novel method (MemBrain) to derive transmembrane inter-helix contacts from amino acid sequences by combining correlated mutations and multiple machine learning classifiers. Tested on 60 non-redundant polytopic proteins using a strict leave-one-out cross-validation protocol, MemBrain achieves an average accuracy of 62%, which is 12.5% higher than the current best method from the literature. When applied to 13 recently solved G protein-coupled receptors, the MemBrain contact predictions helped increase the TM-score of the I-TASSER models by 37% in the transmembrane region. The number of foldable cases (TM-score >0.5) increased by 100%, where all G protein-coupled receptor templates and homologous templates with sequence identity >30% were excluded. These results demonstrate significant progress in contact prediction and a potential for contact-driven structure modeling of transmembrane proteins. Availability: www.csbio.sjtu.edu.cn/bioinf/MemBrain/ Contact: hbshen@sjtu.edu.cn or zhng@umich.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23946502

  3. Entrapment of Water at the Transmembrane Helix-Helix Interface of Quiescin Sulfhydryl Oxidase 2.

    PubMed

    Ried, Christian L; Scharnagl, Christina; Langosch, Dieter

    2016-03-01

    Little is known about how a membrane can regulate interactions between transmembrane helices. Here, we show that strong self-interaction of the transmembrane helix of human quiescin sulfhydryl oxidase 2 rests on a motif of conserved amino acids comprising one face of the helix. Atomistic molecular dynamics simulations suggest that water molecules enter the helix-helix interface and connect serine residues of both partner helices. In addition, an interfacial tyrosine can interact with noninterfacial water or lipid. Dimerization of this transmembrane helix might therefore be controlled by membrane properties controlling water permeation and/or by the lipid composition of the membrane. PMID:26894260

  4. Assignment of Oriented Sample NMR Resonances from a Three Transmembrane Helix Protein

    PubMed Central

    Murray, D. T.; Hung, I.; Cross, T. A.

    2014-01-01

    Oriented sample solid state NMR techniques have been routinely employed to determine the structures of membrane proteins with one or two transmembrane helices. For larger proteins the technique has been limited by spectral resolution and lack of assignment strategies. Here, a strategy for resonance assignment is devised and applied to a three transmembrane helix protein. Sequence specific assignments for all labeled transmembrane amino acid sites are obtained, which provide a set of orientational restraints and helix orientation in the bilayer. Our experiments expand the utility of solid state NMR in membrane protein structure characterization to three transmembrane helix proteins and represent a straightforward strategy for routinely characterizing multiple transmembrane helix protein structures. PMID:24509383

  5. Transmembrane Helix Assembly by Max-Min Ant System Algorithm.

    PubMed

    Sujaree, Kanon; Kitjaruwankul, Sunan; Boonamnaj, Panisak; Supunyabut, Chirayut; Sompornpisut, Pornthep

    2015-12-01

    Because of the rapid progress in biochemical and structural studies of membrane proteins, considerable attention has been given on developing efficient computational methods for solving low-to-medium resolution structures using sparse structural data. In this study, we demonstrate a novel algorithm, max-min ant system (MMAS), designed to find an assembly of α-helical transmembrane proteins using a rigid helix arrangement guided by distance constraints. The new algorithm generates a large variety with finite number of orientations of transmembrane helix bundle and finds the solution that is matched with the provided distance constraints based on the behavior of ants to search for the shortest possible path between their nest and the food source. To demonstrate the efficiency of the novel search algorithm, MMAS is applied to determine the transmembrane packing of KcsA and MscL ion channels from a limited distance information extracted from the crystal structures, and the packing of KvAP voltage sensor domain using a set of 10 experimentally determined constraints, and the results are compared with those of two popular used stochastic methods, simulated annealing Monte Carlo method and genetic algorithm. PMID:26058409

  6. The homology model of PMP22 suggests mutations resulting in peripheral neuropathy disrupt transmembrane helix packing.

    PubMed

    Mittendorf, Kathleen F; Kroncke, Brett M; Meiler, Jens; Sanders, Charles R

    2014-10-01

    Peripheral myelin protein 22 (PMP22) is a tetraspan membrane protein strongly expressed in myelinating Schwann cells of the peripheral nervous system. Myriad missense mutations in PMP22 result in varying degrees of peripheral neuropathy. We used Rosetta 3.5 to generate a homology model of PMP22 based on the recently published crystal structure of claudin-15. The model suggests that several mutations known to result in neuropathy act by disrupting transmembrane helix packing interactions. Our model also supports suggestions from previous studies that the first transmembrane helix is not tightly associated with the rest of the helical bundle. PMID:25243937

  7. The Homology Model of PMP22 Suggests Mutations Resulting in Peripheral Neuropathy Disrupt Transmembrane Helix Packing

    PubMed Central

    2015-01-01

    Peripheral myelin protein 22 (PMP22) is a tetraspan membrane protein strongly expressed in myelinating Schwann cells of the peripheral nervous system. Myriad missense mutations in PMP22 result in varying degrees of peripheral neuropathy. We used Rosetta 3.5 to generate a homology model of PMP22 based on the recently published crystal structure of claudin-15. The model suggests that several mutations known to result in neuropathy act by disrupting transmembrane helix packing interactions. Our model also supports suggestions from previous studies that the first transmembrane helix is not tightly associated with the rest of the helical bundle. PMID:25243937

  8. SPONTANEOUS TRANSMEMBRANE HELIX INSERTION THERMODYNAMICALLY MIMICS TRANSLOCON-GUIDED INSERTION

    PubMed Central

    Ulmschneider, Martin B.; Ulmschneider, Jakob P.; Schiller, Nina; Wallace, B. A.; von Heijne, Gunnar; White, Stephen H.

    2014-01-01

    The favorable transfer free energy for a transmembrane (TM) α-helix between the aqueous phase and lipid bilayer underlies the stability of membrane proteins. However, the connection between the energetics and process of membrane protein assembly by the Sec61/SecY translocon complex in vivo is not clear. Here, we directly determine the partitioning free energies of a family of designed peptides using three independent approaches: an experimental microsomal Sec61 translocon assay, a biophysical (spectroscopic) characterization of peptide insertion into hydrated planar lipid bilayer arrays, and an unbiased atomic-detail equilibrium folding-partitioning molecular dynamics simulation. Remarkably, the measured free energies of insertion are quantitatively similar for all three approaches. The molecular dynamics simulations show that TM helix insertion involves equilibrium with the membrane interface, suggesting that the interface may play a role in translocon-guided insertion. PMID:25204588

  9. Reversible transition between alpha-helix and beta-sheet conformation of a transmembrane domain.

    PubMed

    Yassine, Wissam; Taib, Nada; Federman, Silvina; Milochau, Alexandra; Castano, Sabine; Sbi, Walid; Manigand, Claude; Laguerre, Michel; Desbat, Bernard; Oda, Reiko; Lang, Jochen

    2009-09-01

    Despite the important functions of protein transmembrane domains, their structure and dynamics are often scarcely known. The SNARE proteins VAMP/synaptobrevin and syntaxin 1 are implicated in membrane fusion. Using different spectroscopic approaches we observed a marked sensitivity of their transmembrane domain structure in regard to the lipid/peptide ratio. In the dilute condition, peptides corresponding to the complete transmembrane domain fold into an alpha-helix inserted at approximately 35 degrees to the normal of the membranes, an observation in line with molecular simulations. Upon an increase in the peptide/lipid ratio, the peptides readily exhibited transition to beta-sheet structure. Moreover, the insertion angle of these beta-sheets increased to 54 degrees and was accompanied by a derangement of lipid acyl chains. For both proteins the transition from alpha-helix to beta-sheet was reversible under certain conditions by increasing the peptide/lipid ratio. This phenomenon was observed in different model systems including multibilayers and small unilamellar vesicles. In addition, differences in peptide structure and transitions were observed when using distinct lipids (DMPC, DPPC or DOPC) thus indicating parameters influencing transmembrane domain structure and conversion from helices to sheets. The putative functional consequences of this unprecedented dynamic behavior of a transmembrane domain are discussed. PMID:19482005

  10. Helix packing and orientation in the transmembrane dimer of gp55-P of the spleen focus forming virus.

    PubMed

    Liu, Wei; Crocker, Evan; Constantinescu, Stefan N; Smith, Steven O

    2005-08-01

    gp55-P is a dimeric membrane protein with a single transmembrane helix that is coded by the env gene of the polycythemic strain of the spleen focus forming virus. gp55-P activates the erythropoietin (Epo) receptor through specific transmembrane helix interactions, leading to Epo-independent growth of erythroid progenitors and eventually promoting erythroleukemia. We describe the use of magic angle spinning deuterium NMR to establish the structure of the transmembrane dimer of gp55-P in model membranes. Comparison of the deuterium lineshapes of leucines in the center (Leu(396-399)) and at the ends (Leu(385), Leu(407)) of the transmembrane sequence shows that gp55-P has a right-handed crossing angle with Leu(399) packed in the dimer interface. We discuss the implications of the structure of the gp55-P transmembrane dimer for activation of the Epo receptor. PMID:15894629

  11. De novo design of transmembrane helix-helix interactions and measurement of stability in a biological membrane.

    PubMed

    Nash, Anthony; Notman, Rebecca; Dixon, Ann M

    2015-05-01

    Membrane proteins regulate a large number of cellular functions, and have great potential as tools for manipulation of biological systems. Developing these tools requires a robust and quantitative understanding of membrane protein folding and interactions within the bilayer. With this in mind, we have designed a series of proteins to probe the net thermodynamic contribution of well-known sequence motifs to transmembrane helix-helix association in a biological membrane. The proteins were designed from first principles (de novo) using current knowledge about membrane insertion and stabilizing interaction motifs. A simple poly-Leu "scaffold" was decorated with individual helix interaction motifs (G-XXX-G, polar residues, heptad repeat) to create transmembrane helix-helix interactions of increasing strength. The GALLEX assay, an in vivo assay for measurement of transmembrane helix self-association, was combined with computational methods to characterize the relative strength and mode of interaction for each sequence. In addition, the apparent free energy contribution (ΔΔGapp) of each motif to transmembrane helix self-association was measured in a biological membrane, results that are the first of their kind for these de novo designed sequences, and suggest that the free energy barrier to overcoming weak association is quite small (<1.4 kcal mol(-1)) in a natural membrane. By quantifying and rationalizing the contribution of key motifs to transmembrane helix association, our work offers a route to direct the design of novel sequences for use in biotechnology or synthetic biology (e.g. molecular switches) and to predict the effects of sequence modification in known transmembrane domains (for control of cellular processes). PMID:25732028

  12. Enzymatic analysis of a rhomboid intramembrane protease implicates transmembrane helix 5 as the lateral substrate gate.

    PubMed

    Baker, Rosanna P; Young, Keith; Feng, Liang; Shi, Yigong; Urban, Sinisa

    2007-05-15

    Intramembrane proteolysis is a core regulatory mechanism of cells that raises a biochemical paradox of how hydrolysis of peptide bonds is accomplished within the normally hydrophobic environment of the membrane. Recent high-resolution crystal structures have revealed that rhomboid proteases contain a catalytic serine recessed into the plane of the membrane, within a hydrophilic cavity that opens to the extracellular face, but protected laterally from membrane lipids by a ring of transmembrane segments. This architecture poses questions about how substrates enter the internal active site laterally from membrane lipid. Because structures are static glimpses of a dynamic enzyme, we have taken a structure-function approach analyzing >40 engineered variants to identify the gating mechanism used by rhomboid proteases. Importantly, our analyses were conducted with a substrate that we show is cleaved at two intramembrane sites within the previously defined Spitz substrate motif. Engineered mutants in the L1 loop and active-site region of the GlpG rhomboid protease suggest an important structural, rather than dynamic, gating function for the L1 loop that was first proposed to be the substrate gate. Conversely, three classes of mutations that promote transmembrane helix 5 displacement away from the protease core dramatically enhanced enzyme activity 4- to 10-fold. Our functional analyses have identified transmembrane helix 5 movement to gate lateral substrate entry as a rate-limiting step in intramembrane proteolysis. Moreover, our mutagenesis also underscores the importance of other residue interactions within the enzyme that warrant further scrutiny. PMID:17463085

  13. Integrin α1 Has a Long Helix, Extending from the Transmembrane Region to the Cytoplasmic Tail in Detergent Micelles

    PubMed Central

    Lai, Chaohua; Liu, Xiaoxi; Tian, Changlin; Wu, Fangming

    2013-01-01

    Integrin proteins are very important adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. They play essential roles in cell signaling and the regulation of cellular shape, motility, and the cell cycle. Here, the transmembrane and cytoplasmic (TMC) domains of integrin α1 and β1 were over-expressed and purified in detergent micelles. The structure and backbone relaxations of α1-TMC in LDAO micelles were determined and analyzed using solution NMR. A long helix, extending from the transmembrane region to the cytoplasmic tail, was observed in α1-TMC. Structural comparisons of α1-TMC with reported αIIb-TMC domains indicated different conformations in the transmembrane regions and cytoplasmic tails. An NMR titration experiment indicated weak interactions between α1-TMC and β1-TMC through several α1-TMC residues located at its N-terminal juxta-transmembrane region and C-terminal extended helix region. PMID:23646163

  14. Combined approaches of EPR and NMR illustrate only one transmembrane helix in the human IFITM3.

    PubMed

    Ling, Shenglong; Zhang, Chengwei; Wang, Wei; Cai, Xiaoying; Yu, Lu; Wu, Fangming; Zhang, Longhua; Tian, Changlin

    2016-01-01

    Interferon-inducible transmembrane protein IFITM3 was known to restrict the entry of a wide spectrum of viruses to the cytosol of the host. The mechanism used by the protein to restrict viral entry is unclear given the unavailability of the membrane topology and structures of the IFITM family proteins. Systematic site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) studies of IFITM3 in detergent micelles identified a single, long transmembrane helix in the C-terminus and an intramembrane segment in the N-terminal hydrophobic region. Solution NMR studies of the same sample verified the secondary structure distribution and demonstrated two rigid regions interacting with the micellar surface. The resulting membrane topology of IFITM3 supports the mechanism of an enhanced restricted membrane hemi-fusion. PMID:27046158

  15. Combined approaches of EPR and NMR illustrate only one transmembrane helix in the human IFITM3

    PubMed Central

    Ling, Shenglong; Zhang, Chengwei; Wang, Wei; Cai, Xiaoying; Yu, Lu; Wu, Fangming; Zhang, Longhua; Tian, Changlin

    2016-01-01

    Interferon-inducible transmembrane protein IFITM3 was known to restrict the entry of a wide spectrum of viruses to the cytosol of the host. The mechanism used by the protein to restrict viral entry is unclear given the unavailability of the membrane topology and structures of the IFITM family proteins. Systematic site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) studies of IFITM3 in detergent micelles identified a single, long transmembrane helix in the C-terminus and an intramembrane segment in the N-terminal hydrophobic region. Solution NMR studies of the same sample verified the secondary structure distribution and demonstrated two rigid regions interacting with the micellar surface. The resulting membrane topology of IFITM3 supports the mechanism of an enhanced restricted membrane hemi-fusion. PMID:27046158

  16. Use of Molecular Dynamics Data in Biochemistry Courses: An Amphipathy Scale to Determine Protein [alpha]-Helix Transmembrane Segments

    ERIC Educational Resources Information Center

    Mazze, Fernanda M.; Fuzo, Carlos A.; Degreve, Leo; Ciancaglini, Pietro

    2008-01-01

    The aim of this manuscript is to explain the application of an amphipathy scale obtained from molecular dynamics simulations and to demonstrate how it can be useful in the protein structure field. It is shown that this scale is easy to be used with the advantage of revealing domains of transmembrane [alpha]-helix of proteins without the need of…

  17. Amide vibrations are delocalized across the hydrophobic interface of a transmembrane helix dimer

    PubMed Central

    Fang, Chong; Senes, Alessandro; Cristian, Lidia; DeGrado, William F.; Hochstrasser, Robin M.

    2006-01-01

    The tertiary interactions between amide-I vibrators on the separate helices of transmembrane helix dimers were probed by ultrafast 2D vibrational photon echo spectroscopy. The 2D IR approach proves to be a useful structural method for the study of membrane-bound structures. The 27-residue human erythrocyte protein Glycophorin A transmembrane peptide sequence: KKITLIIFG79VMAGVIGTILLISWG94IKK was labeled at G79 and G94 with 13C16O or 13C18O. The isotopomers and their 50:50 mixtures formed helical dimers in SDS micelles whose 2D IR spectra showed components from homodimers when both helices had either 13C16O or 13C18O substitution and a heterodimer when one had 13C16O substitution and the other had 13C18O substitution. The cross-peaks in the pure heterodimer 2D IR difference spectrum and the splitting of the homodimer peaks in the linear IR spectrum show that the amide-I mode is delocalized across a pair of helices. The excitation exchange coupling in the range 4.3–6.3 cm−1 arises from through-space interactions between amide units on different helices. The angle between the two Gly79 amide-I transition dipoles, estimated at 103° from linear IR spectroscopy and 110° from 2D IR spectroscopy, combined with the coupling led to a structural picture of the hydrophobic interface that is remarkably consistent with results from NMR on helix dimers. The helix crossing angle in SDS is estimated at 45°. Two-dimensional IR spectroscopy also sets limits on the range of geometrical parameters for the helix dimers from an analysis of the coupling constant distribution. PMID:17075037

  18. On the distribution of amino acid residues in transmembrane alpha-helix bundles.

    PubMed Central

    Samatey, F A; Xu, C; Popot, J L

    1995-01-01

    The periodic distribution of residues in the sequence of 469 putative transmembrane alpha-helices from eukaryotic plasma membrane polytopic proteins has been analyzed with correlation matrices. The method does not involve any a priori assumption about the secondary structure of the segments or about the physicochemical properties of individual amino acid residues. Maximal correlation is observed at 3.6 residues per period, characteristic of alpha-helices. A scale extracted from the data describes the propensity of the various residues to lie on the same or on opposite helix faces. The most polar face of transmembrane helices, presumably that buried in the protein core, shows a strong enrichment in aromatic residues, while residues likely to face the fatty acyl chains of lipids are largely aliphatic. PMID:7753846

  19. Molecular Dynamics Simulation of Membranes and a Transmembrane Helix

    NASA Astrophysics Data System (ADS)

    Duong, Tap Ha; Mehler, Ernest L.; Weinstein, Harel

    1999-05-01

    Three molecular dynamics (MD) simulations of 1.5-ns length were carried out on fully hydrated patches of dimyristoyl phosphatidylcholine (DMPC) bilayers in the liquid-crystalline phase. The simulations were performed using different ensembles and electrostatic conditions: a microcanonical ensemble or constant pressure-temperature ensemble, with or without truncated electrostatic interactions. Calculated properties of the membrane patches from the three different protocols were compared to available data from experiments. These data include the resulting overall geometrical dimensions, the order characteristics of the lipid hydrocarbon chains, as well as various measures of the conformations of the polar head groups. The comparisons indicate that the simulation carried out within the microcanonical ensemble with truncated electrostatic interactions yielded results closest to the experimental data, provided that the initial equilibration phase preceding the production run was sufficiently long. The effects of embedding a non-ideal helical protein domain in the membrane patch were studied with the same MD protocols. This simulation was carried out for 2.5 ns. The protein domain corresponds to the seventh transmembrane segment (TMS7) of the human serotonin 5HT 2Areceptor. The peptide is composed of two ?-helical segments linked by a hinge domain around a perturbing Asn-Pro motif that produces at the end of the simulation a kink angle of nearly 80 between the two helices. Several aspects of the TMS7 structure, such as the bending angle, backbone ? and ? torsion angles, the intramolecular hydrogen bonds, and the overall conformation, were found to be very similar to those determined by NMR for the corresponding transmembrane segment of the tachykinin NK-1 receptor. In general, the simulations were found to yield structural and dynamic characteristics that are in good agreement with experiment. These findings support the application of simulation methods to the study of the complex biomolecular systems at the membrane interface of cells.

  20. The transmembrane region of the nicotinic acetylcholine receptor: is it an all-helix bundle?

    PubMed

    Ortells, M O; Lunt, G G

    1994-01-01

    The nicotinic acetylcholine receptor is the best characterised member of the Ligand-Gated-Ion-Channel family of receptors. In spite of a wealth of data from molecular cloning studies these receptors have so far eluded all attempts at crystallisation; quantitative structural data are few and are at relatively low resolution. The widely accepted current model for the topology of the receptors is that of a pentameric cylindrical bundle that spans the membrane. The disposition of the transmembrane region of the individual subunits is based on hydropathy profiles calculated from sequence data which are interpreted as indicating a common structural motif of four antiparallel alpha-helices, M1 to M4. Until very recently this model has been unquestioned even though there are few direct experimental data to support it. We have constructed models of this key functional region for the nicotinic acetylcholine receptor, building out from the ion-channel. The model of the basic ion-channel comprises a five helical M2 bundle with a left-handed twist. The remainder of the region (M1, M3, M4) was homology modelled, together with M2, as a four helix antiparallel bundle per subunit, using the crystal structure of myohaemerythrin as a template. The models strongly suggest that the four helix bundle model is inappropriate and that recent suggestions of a mixed motif of helix and sheet may better accommodate the existing data. PMID:7521734

  1. Transmembrane helix straightening and buckling underlies activation of mechanosensitive and thermosensitive K(2P) channels.

    PubMed

    Lolicato, Marco; Riegelhaupt, Paul M; Arrigoni, Cristina; Clark, Kimberly A; Minor, Daniel L

    2014-12-17

    Mechanical and thermal activation of ion channels is central to touch, thermosensation, and pain. The TRAAK/TREK K(2P) potassium channel subfamily produces background currents that alter neuronal excitability in response to pressure, temperature, signaling lipids, and anesthetics. How such diverse stimuli control channel function is unclear. Here we report structures of K(2P)4.1 (TRAAK) bearing C-type gate-activating mutations that reveal a tilting and straightening of the M4 inner transmembrane helix and a buckling of the M2 transmembrane helix. These conformational changes move M4 in a direction opposite to that in classical potassium channel activation mechanisms and open a passage lateral to the pore that faces the lipid bilayer inner leaflet. Together, our findings uncover a unique aspect of K(2P) modulation, indicate a means for how the K(2P) C-terminal cytoplasmic domain affects the C-type gate which lies ∼40Å away, and suggest how lipids and bilayer inner leaflet deformations may gate the channel. PMID:25500157

  2. Transmembrane Helix Straightening and Buckling Underlies Activation of Mechanosensitive and Thermosensitive K2P Channels

    PubMed Central

    Lolicato, Marco; Riegelhaupt, Paul M.; Arrigoni, Cristina; Clark, Kimberly A.; Minor, Daniel L.

    2014-01-01

    SUMMARY Mechanical and thermal activation of ion channels is central to touch, thermosensation, and pain. The TRAAK/TREK K2P potassium channel subfamily produces background currents that alter neuronal excitability in response to pressure, temperature, signaling lipids, and anesthetics. How such diverse stimuli control channel function is unclear. Here we report structures of K2P4.1 (TRAAK) bearing C-type gate-activating mutations that reveal a tilting and straightening of the M4 inner transmembrane helix and a buckling of the M2 transmembrane helix. These conformational changes move M4 in a direction opposite to that in classical potassium channel activation mechanisms and open a passage lateral to the pore that faces the lipid bilayer inner leaflet. Together, our findings uncover a unique aspect of K2P modulation, indicate a means for how the K2P C-terminal cytoplasmic domain affects the C-type gate which lies ~40 away, and suggest how lipids and bilayer inner leaflet deformations may gate the channel. PMID:25500157

  3. A mixed helix-beta-sheet model of the transmembrane region of the nicotinic acetylcholine receptor.

    PubMed

    Ortells, M O; Lunt, G G

    1996-01-01

    We have modelled the transmembrane region of the alpha 7 nicotinic acetylcholine receptor as a mixed alpha-helical/beta-sheet structure. The model was mainly based on the crystal structure of a pore-forming toxin, heat-labile enterotoxin. This is a pentameric protein having a central pore or channel composed of five alpha-helices, one from each of the 5 B subunits that form this pentamer. The remainder of this structure is beta-sheet, loops and a short alpha-helix, not included in the model. The model uses this channel as a template to build the transmembrane region, from M1 to the middle of M3. The remainder of M3 and M4 were built de novo as alpha-helices. Great consideration was given to labelling data available for the transmembrane region. In general terms, the shape of the model agrees very well with that obtained independently by electron microscopic analysis and the secondary structure predicted by the model is in accord with that estimated independently by Fourier transform infrared spectroscopy. The M2 helical region of the model is only slightly kinked, contrary to what is inferred from electron microscopic analysis, but has the same overall shape and form. On the membrane face of the model, the presence of deep pockets may provide the structural basis for the distinction between annular and non-annular lipid binding sites. Also, the transmembrane region is clearly asymmetric in the direction perpendicular to the membrane, and this may have strong influence on the surrounding lipid composition of each leaflet of the cytoplasmic membrane. PMID:9053903

  4. PSOFuzzySVM-TMH: identification of transmembrane helix segments using ensemble feature space by incorporated fuzzy support vector machine.

    PubMed

    Hayat, Maqsood; Tahir, Muhammad

    2015-08-01

    Membrane protein is a central component of the cell that manages intra and extracellular processes. Membrane proteins execute a diversity of functions that are vital for the survival of organisms. The topology of transmembrane proteins describes the number of transmembrane (TM) helix segments and its orientation. However, owing to the lack of its recognized structures, the identification of TM helix and its topology through experimental methods is laborious with low throughput. In order to identify TM helix segments reliably, accurately, and effectively from topogenic sequences, we propose the PSOFuzzySVM-TMH model. In this model, evolutionary based information position specific scoring matrix and discrete based information 6-letter exchange group are used to formulate transmembrane protein sequences. The noisy and extraneous attributes are eradicated using an optimization selection technique, particle swarm optimization, from both feature spaces. Finally, the selected feature spaces are combined in order to form ensemble feature space. Fuzzy-support vector Machine is utilized as a classification algorithm. Two benchmark datasets, including low and high resolution datasets, are used. At various levels, the performance of the PSOFuzzySVM-TMH model is assessed through 10-fold cross validation test. The empirical results reveal that the proposed framework PSOFuzzySVM-TMH outperforms in terms of classification performance in the examined datasets. It is ascertained that the proposed model might be a useful and high throughput tool for academia and research community for further structure and functional studies on transmembrane proteins. PMID:26054033

  5. Predicting transmembrane helix packing arrangements using residue contacts and a force-directed algorithm.

    PubMed

    Nugent, Timothy; Jones, David T

    2010-03-01

    Alpha-helical transmembrane proteins constitute roughly 30% of a typical genome and are involved in a wide variety of important biological processes including cell signalling, transport of membrane-impermeable molecules and cell recognition. Despite significant efforts to predict transmembrane protein topology, comparatively little attention has been directed toward developing a method to pack the helices together. Here, we present a novel approach to predict lipid exposure, residue contacts, helix-helix interactions and finally the optimal helical packing arrangement of transmembrane proteins. Using molecular dynamics data, we have trained and cross-validated a support vector machine (SVM) classifier to predict per residue lipid exposure with 69% accuracy. This information is combined with additional features to train a second SVM to predict residue contacts which are then used to determine helix-helix interaction with up to 65% accuracy under stringent cross-validation on a non-redundant test set. Our method is also able to discriminate native from decoy helical packing arrangements with up to 70% accuracy. Finally, we employ a force-directed algorithm to construct the optimal helical packing arrangement which demonstrates success for proteins containing up to 13 transmembrane helices. This software is freely available as source code from http://bioinf.cs.ucl.ac.uk/memsat/mempack/. PMID:20333233

  6. Second transmembrane helix (M2) and long range coupling in Ca?-ATPase.

    PubMed

    Daiho, Takashi; Yamasaki, Kazuo; Danko, Stefania; Suzuki, Hiroshi

    2014-11-01

    The actuator (A) domain of sarco(endo)plasmic reticulum Ca(2+)-ATPase not only plays a catalytic role but also undergoes large rotational movements that influence the distant transport sites through connections with transmembrane helices M1 and M2. Here we explore the importance of long helix M2 and its junction with the A domain by disrupting the helix structure and elongating with insertions of five glycine residues. Insertions into the membrane region of M2 and the top junctional segment impair Ca(2+) transport despite reasonable ATPase activity, indicating that they are uncoupled. These mutants fail to occlude Ca(2+). Those at the top segment also exhibited accelerated phosphoenzyme isomerization E1P ? E2P. Insertions into the middle of M2 markedly accelerate E2P hydrolysis and cause strong resistance to inhibition by luminal Ca(2+). Insertions along almost the entire M2 region inhibit the dephosphorylated enzyme transition E2 ? E1. The results pinpoint which parts of M2 control cytoplasm gating and which are critical for luminal gating at each stage in the transport cycle and suggest that proper gate function requires appropriate interactions, tension, and/or rigidity in the M2 region at appropriate times for coupling with A domain movements and catalysis. PMID:25246522

  7. Juxta-terminal Helix Unwinding as a Stabilizing Factor to Modulate the Dynamics of Transmembrane Helices.

    PubMed

    Mortazavi, Armin; Rajagopalan, Venkatesan; Sparks, Kelsey A; Greathouse, Denise V; Koeppe, Roger E

    2016-03-15

    Transmembrane helices of integral membrane proteins often are flanked by interfacial aromatic residues that can serve as anchors to aid the stabilization of a tilted transmembrane orientation. Yet, physical factors that govern the orientation or dynamic averaging of individual transmembrane helices are not well understood and have not been adequately explained. Using solid-state (2) H NMR spectroscopy to examine lipid bilayer-incorporated model peptides of the GWALP23 (acetyl-GGALW(LA)6 LWLAGA-amide) family, we observed substantial unwinding at the terminals of several tilted helices spanning the membranes of DLPC, DMPC, or DOPC lipid bilayers. The fraying of helix ends might be vital for defining the dynamics and orientations of transmembrane helices in lipid bilayer membranes. PMID:26749271

  8. Revisiting Hydrophobic Mismatch with Free Energy Simulation Studies of Transmembrane Helix Tilt and Rotation

    PubMed Central

    Kim, Taehoon; Im, Wonpil

    2010-01-01

    Abstract Protein-lipid interaction and bilayer regulation of membrane protein functions are largely controlled by the hydrophobic match between the transmembrane (TM) domain of membrane proteins and the surrounding lipid bilayer. To systematically characterize responses of a TM helix and lipid adaptations to a hydrophobic mismatch, we have performed a total of 5.8-μs umbrella sampling simulations and calculated the potentials of mean force (PMFs) as a function of TM helix tilt angle under various mismatch conditions. Single-pass TM peptides called WALPn (n = 16, 19, 23, and 27) were used in two lipid bilayers with different hydrophobic thicknesses to consider hydrophobic mismatch caused by either the TM length or the bilayer thickness. In addition, different flanking residues, such as alanine, lysine, and arginine, instead of tryptophan in WALP23 were used to examine their influence. The PMFs, their decomposition, and trajectory analysis demonstrate that 1), tilting of a single-pass TM helix is the major response to a hydrophobic mismatch; 2), TM helix tilting up to ∼10° is inherent due to the intrinsic entropic contribution arising from helix precession around the membrane normal even under a negative mismatch; 3), the favorable helix-lipid interaction provides additional driving forces for TM helix tilting under a positive mismatch; 4), the minimum-PMF tilt angle is generally located where there is the hydrophobic match and little lipid perturbation; 5), TM helix rotation is dependent on the specific helix-lipid interaction; and 6), anchoring residues at the hydrophilic/hydrophobic interface can be an important determinant of TM helix orientation. PMID:20655845

  9. Structural basis of integrin transmembrane activation.

    PubMed

    Wang, Wei; Luo, Bing-Hao

    2010-02-15

    Integrins are cell adhesion receptors that transmit bidirectional signals across plasma membrane and are crucial for many biological functions. Recent structural studies of integrin transmembrane (TM) and cytoplasmic domains have shed light on their conformational changes during integrin activation. A structure of the resting state was solved based on Rosetta computational modeling and experimental data using intact integrins on mammalian cell surface. In this structure, the alpha(IIb) GXXXG motif and their beta(3) counterparts of the TM domains associate with ridge-in-groove packing, and the alpha(IIb) GFFKR motif and the beta(3) Lys-716 in the cytoplasmic segments play a critical role in the alpha/beta association. Comparing this structure with the NMR structures of the monomeric alpha(IIb) and beta(3) (represented as active conformations), the alpha subunit helix remains similar after dissociation whereas beta subunit helix is tilted by embedding additional 5-6 residues into the lipid bilayer. These conformational changes are critical for integrin activation and signaling across the plasma membrane. We thus propose a new model of integrin TM activation in which the recent NMR structure of the alpha(IIb)beta(3) TM/cytoplasmic complex represents an intermediate or transient state, and the electrostatic interaction in the cytoplasmic region is important for priming the initial alpha/beta association, but not absolutely necessary for the resting state. PMID:19950197

  10. Assembly of light-harvesting bacteriochlorophyll in a model transmembrane helix in its natural environment.

    PubMed

    Braun, P; Olsen, J D; Strohmann, B; Hunter, C N; Scheer, H

    2002-05-10

    The transmembrane, bacteriochlorophyll-binding region of a bacterial light-harvesting complex, (LH2-alpha from the photosynthetic bacterium Rhodobacter sphaeroides) was redesigned and overexpressed in a mutant of Rb. sphaeroides lacking LH2. Bacteriochlorophyll served as internal probe for the fitness of this new region for the assembly and energy transfer function of the LH2 complex. The ability to absorb and transfer light energy is practically undisturbed by the exchange of the transmembrane segment, valine -7 to threonine +6, of LH2-alpha with a 14 residue Ala-Leu sequence. This stretch makes up the residues of the transmembrane helix that are in close contact (< or =4.5 A) with the bacteriochlorophyll molecules that are coordinated through His of both the alpha and beta-subunits. In this Ala-Leu stretch, neither alpha-His0, which binds the bacteriochlorophyll, nor the adjacent alpha-Ile-1, were replaced. Novel LH2 complexes composed of LH2-alpha with a model transmembrane sequence and a normal LH2-beta are assembled in vivo into a complex, the biochemical and spectroscopic properties of which closely resemble the native one. In contrast, the additional insertion of four residues just outside the C-terminal end of the model transmembrane helix leads to complete loss of functional antenna complex. The results suggest that light energy can be harvested and transferred efficiently by bacteriochlorophyll molecules attached to only few key residues distributed over the polypeptide, while residues at the bacteriochlorophyll-helix interface seem to be largely dispensable for the functional assembly of this membrane protein complex. This novel antenna with a simplified transmembrane domain and a built-in probe for assembly and function provides a powerful model system for investigation of the factors that contribute to the assembly of chromophores in membrane-embedded proteins. PMID:12054804

  11. Quantification of structural distortions in the transmembrane helices of GPCRs.

    PubMed

    Deupi, Xavier

    2012-01-01

    A substantial part of the structural and much of the functional information about G protein-coupled receptors (GPCRs) comes from studies on rhodopsin. Thus, analysis tools for detailed structure comparison are key to see to what extent this information can be extended to other GPCRs. Among the methods to evaluate protein structures and, in particular, helix distortions, HELANAL has the advantage that it provides data (local bend and twist angles) that can be easily translated to structural effects, as a local opening/tightening of the helix.In this work I show how HELANAL can be used to extract detailed structural information of the transmembrane bundle of GPCRs, and I provide some examples on how these data can be interpreted to study basic principles of protein structure, to compare homologous proteins and to study mechanisms of receptor activation. Also, I show how in combination with the sequence analysis tools provided by the program GMoS, distortions in individual receptors can be put in the context of the whole Class A GPCR family. Specifically, quantification of the strong proline-induced distortions in the transmembrane bundle of rhodopsin shows that they are not standard proline kinks. Moreover, the helix distortions in transmembrane helix (TMH) 5 and TMH 6 of rhodopsin are also present in the rest of GPCR crystal structures obtained so far, and thus, rhodopsin-based homology models have modeled correctly these strongly distorted helices. While in some cases the inherent "rhodopsin bias" of many of the GPCR models to date has not been a disadvantage, the availability of more templates will clearly result in better homology models. This type of analysis can be, of course, applied to any protein, and it may be particularly useful for the structural analysis of other membrane proteins. A detailed knowledge of the local structural changes related to ligand binding and how they are translated into larger-scale movements of transmembrane domains is key to understand receptor activation. PMID:22976031

  12. Differential Regulation of 6- and 7-Transmembrane Helix Variants of μ-Opioid Receptor in Response to Morphine Stimulation

    PubMed Central

    Convertino, Marino; Samoshkin, Alexander; Viet, Chi T.; Gauthier, Josee; Li Fraine, Steven P.; Sharif-Naeini, Reza; Schmidt, Brian L.; Maixner, William; Diatchenko, Luda; Dokholyan, Nikolay V.

    2015-01-01

    The pharmacological effect of opioids originates, at the cellular level, by their interaction with the μ-opioid receptor (mOR) resulting in the regulation of voltage-gated Ca2+ channels and inwardly rectifying K+ channels that ultimately modulate the synaptic transmission. Recently, an alternative six trans-membrane helix isoform of mOR, (6TM-mOR) has been identified, but its function and signaling are still largely unknown. Here, we present the structural and functional mechanisms of 6TM-mOR signaling activity upon binding to morphine. Our data suggest that despite the similarity of binding modes of the alternative 6TM-mOR and the dominant seven trans-membrane helix variant (7TM-mOR), the interaction with morphine generates different dynamic responses in the two receptors, thus, promoting the activation of different mOR-specific signaling pathways. We characterize a series of 6TM-mOR-specific cellular responses, and observed that they are significantly different from those for 7TM-mOR. Morphine stimulation of 6TM-mOR does not promote a cellular cAMP response, while it increases the intracellular Ca2+ concentration and reduces the cellular K+ conductance. Our findings indicate that 6TM-mOR has a unique contribution to the cellular opioid responses. Therefore, it should be considered as a relevant target for the development of novel pharmacological tools and medical protocols involving the use of opioids. PMID:26554831

  13. Observation of helix associations for insertion of a retinal molecule and distortions of helix structures in bacteriorhodopsin

    NASA Astrophysics Data System (ADS)

    Urano, Ryo; Okamoto, Yuko

    2015-12-01

    We applied a newly proposed prediction method for membrane protein structures to bacteriorhodopsin that has distorted transmembrane helices in the native structure. This method uses an implicit membrane model, which restricts sampling space during folding in a membrane region, and includes helix bending. Replica-exchange simulations were performed with seven transmembrane helices only without a retinal molecule. Obtained structures were classified into clusters of similar structures, which correspond to local-minimum free energy states. The two lowest free energy states corresponded to a native-like structure with the correct empty space for retinal and a structure with this empty space filled with a helix. Previous experiments of bacteriorhodopsin suggested that association of transmembrane helices enables them to make a room for insertion of a retinal. Our results are consistent with these results. Moreover, distortions of helices in the native-like structures were successfully reproduced. In the distortions, whereas the locations of kinks for all helices were similar to those of Protein Data Bank's data, the amount of bends was more similar for helices away from the retinal than for those close to the retinal in the native structure. This suggests a hypothesis that the amino-acid sequence specifies the location of kinks in transmembrane helices and that the amount of distortions depends on the interactions with the surrounding molecules such as neighboring helices, lipids, and retinal.

  14. Functional importance of the Gly cluster in transmembrane helix 2 of the Bordetella pertussis CyaA-hemolysin: Implications for toxin oligomerization and pore formation.

    PubMed

    Juntapremjit, Sirikran; Thamwiriyasati, Niramon; Kurehong, Chattip; Prangkio, Panchika; Shank, Lalida; Powthongchin, Busaba; Angsuthanasombat, Chanan

    2015-11-01

    Adenylate cyclase-hemolysin (CyaA) is a major virulence factor of Bordetella pertussis causing whooping cough in humans. We previously showed that two transmembrane helices (α2 and α3) in the hemolysin domain (CyaA-Hly) are crucially involved in hemolytic activity. Here, PCR-based substitutions were employed to investigate a potential involvement in hemolysis of a series of four Gly residues (Gly(530), Gly(533), Gly(537) and Gly(544)) which map onto one face of a helical wheel plot of pore-lining helix 2. All CyaA-Hly mutant toxins were over-expressed in Escherichia coli as 126-kDa soluble proteins at levels comparable to the wild-type toxin. A drastic reduction in hemolytic activity against sheep erythrocytes was observed for three CyaA-Hly mutants, i.e. G530A, G533A and G537A, but not G544A, suggesting a functional importance of the Gly(530)_Gly(533)_Gly(537) cluster. A homology-based structure of the α2-loop-α3 hairpin revealed that this crucial Gly cluster arranged as a GXXGXXXG motif is conceivably involved in helix-helix association. Furthermore, a plausible pore model comprising three α2-loop-α3 hairpins implicated that Gly(530)XXGly(533)XXXGly(537) could function as an important framework for toxin oligomerization. Altogether, our present data signify for the first time that the Gly(530)_Gly(533)_Gly(537) cluster in transmembrane helix 2 serves as a crucial constituent of the CyaA-Hly trimeric pore structure. PMID:26363293

  15. Transmembrane Protein Structure: Spin Labeling of Bacteriorhodopsin Mutants

    NASA Astrophysics Data System (ADS)

    Altenbach, Christian; Marti, Thomas; Gobind Khorana, H.; Hubbell, Wayne L.

    1990-06-01

    Transmembrane proteins serve important biological functions, yet precise information on their secondary and tertiary structure is very limited. The boundaries and structures of membrane-embedded domains in integral membrane proteins can be determined by a method based on a combination of site-specific mutagenesis and nitroxide spin labeling. The application to one polypeptide segment in bacteriorhodopsin, a transmembrane chromoprotein that functions as a light-driven proton pump is described. Single cysteine residues were introduced at 18 consecutive positions (residues 125 to 142). Each mutant was reacted with a specific spin label and reconstituted into vesicles that were shown to be functional. The relative collision frequency of each spin label with freely diffusing oxygen and membrane-impermeant chromium oxalate was estimated with power saturation EPR (electron paramagnetic resonance) spectroscopy. The results indicate that residues 129 to 131 form a short waterexposed loop, while residues 132 to 1a,2 are membraneembedded. The oxygen accessibility for positions 131 to 138 varies with a periodicity of 3.6 residues, thereby providing a striking demonstration of an a helix. The orientation of this helical segment with respect to the remainder of the protein was determined.

  16. Side-Chain to Main-Chain Hydrogen Bonding Controls the Intrinsic Backbone Dynamics of the Amyloid Precursor Protein Transmembrane Helix

    PubMed Central

    Scharnagl, Christina; Pester, Oxana; Hornburg, Philipp; Hornburg, Daniel; Götz, Alexander; Langosch, Dieter

    2014-01-01

    Many transmembrane helices contain serine and/or threonine residues whose side chains form intrahelical H-bonds with upstream carbonyl oxygens. Here, we investigated the impact of threonine side-chain/main-chain backbonding on the backbone dynamics of the amyloid precursor protein transmembrane helix. This helix consists of a N-terminal dimerization region and a C-terminal cleavage region, which is processed by γ-secretase to a series of products. Threonine mutations within this transmembrane helix are known to alter the cleavage pattern, which can lead to early-onset Alzheimer’s disease. Circular dichroism spectroscopy and amide exchange experiments of synthetic transmembrane domain peptides reveal that mutating threonine enhances the flexibility of this helix. Molecular dynamics simulations show that the mutations reduce intrahelical amide H-bonding and H-bond lifetimes. In addition, the removal of side-chain/main-chain backbonding distorts the helix, which alters bending and rotation at a diglycine hinge connecting the dimerization and cleavage regions. We propose that the backbone dynamics of the substrate profoundly affects the way by which the substrate is presented to the catalytic site within the enzyme. Changing this conformational flexibility may thus change the pattern of proteolytic processing. PMID:24655507

  17. The Transmembrane Helix Tilt May Be Determined by the Balance between Precession Entropy and Lipid Perturbation

    PubMed Central

    2012-01-01

    Hydrophobic helical peptides interact with lipid bilayers in various modes, determined by the match between the length of the helix’s hydrophobic core and the thickness of the hydrocarbon region of the bilayer. For example, long helices may tilt with respect to the membrane normal to bury their hydrophobic cores in the membrane, and the lipid bilayer may stretch to match the helix length. Recent molecular dynamics simulations and potential of mean force calculations have shown that some TM helices whose lengths are equal to, or even shorter than, the bilayer thickness may also tilt. The tilt is driven by a gain in the helix precession entropy, which compensates for the free energy penalty resulting from membrane deformation. Using this free energy balance, we derived theoretically an equation of state, describing the dependence of the tilt on the helix length and membrane thickness. To this end, we conducted coarse-grained Monte Carlo simulations of the interaction of helices of various lengths with lipid bilayers of various thicknesses, reproducing and expanding the previous molecular dynamics simulations. Insight from the simulations facilitated the derivation of the theoretical model. The tilt angles calculated using the theoretical model agree well with our simulations and with previous calculations and measurements. PMID:24932138

  18. A hidden Markov model with molecular mechanics energy-scoring function for transmembrane helix prediction.

    PubMed

    Zheng, W Jim; Spassov, Velin Z; Yan, Lisa; Flook, Paul K; Szalma, Sándor

    2004-10-01

    A range of methods has been developed to predict transmembrane helices and their topologies. Although most of these algorithms give good predictions, no single method consistently outperforms the others. However, combining different algorithms is one approach that can potentially improve the accuracy of the prediction. We developed a new method that initially uses a hidden Markov model to predict alternative models for membrane spanning helices in proteins. The algorithm subsequently identifies the best among models by ranking them using a novel scoring function based on the folding energy of transmembrane helical fragments. This folding of helical fragments and the incorporation into membrane is modeled using CHARMm, extended with the Generalized Born surface area solvent model (GBSA/IM) with implicit membrane. The combined method reported here, TMHGB significantly increases the accuracy of the original hidden Markov model-based algorithm. PMID:15548453

  19. Order Parameters of a Transmembrane Helix in a Fluid Bilayer: Case Study of a WALP Peptide

    PubMed Central

    Holt, Andrea; Rougier, La; Rat, Valrie; Jolibois, Franck; Saurel, Olivier; Czaplicki, Jerzy; Killian, J. Antoinette; Milon, Alain

    2010-01-01

    Abstract A new solid-state NMR-based strategy is established for the precise and efficient analysis of orientation and dynamics of transmembrane peptides in fluid bilayers. For this purpose, several dynamically averaged anisotropic constraints, including 13C and 15N chemical shift anisotropies and 13C-15N dipolar couplings, were determined from two different triple-isotope-labeled WALP23 peptides (2H, 13C, and 15N) and combined with previously published quadrupolar splittings of the same peptide. Chemical shift anisotropy tensor orientations were determined with quantum chemistry. The complete set of experimental constraints was analyzed using a generalized, four-parameter dynamic model of the peptide motion, including tilt and rotation angle and two associated order parameters. A tilt angle of 21 was determined for WALP23 in dimyristoylphosphatidylcholine, which is much larger than the tilt angle of 5.5 previously determined from 2H NMR experiments. This approach provided a realistic value for the tilt angle of WALP23 peptide in the presence of hydrophobic mismatch, and can be applied toany transmembrane helical peptide. The influence of the experimental data set on the solution space is discussed, as are potential sources of error. PMID:20441750

  20. Aromatic–aromatic interactions between residues in KCa3.1 pore helix and S5 transmembrane segment control the channel gating process

    PubMed Central

    Garneau, Line; Klein, Hélène; Lavoie, Marie-France; Brochiero, Emmanuelle; Parent, Lucie

    2014-01-01

    The Ca2+-activated potassium channel KCa3.1 is emerging as a therapeutic target for a large variety of health disorders. One distinguishing feature of KCa3.1 is that the channel open probability at saturating Ca2+ concentrations (Pomax) is low, typically 0.1–0.2 for KCa3.1 wild type. This observation argues for the binding of Ca2+ to the calmodulin (CaM)–KCa3.1 complex, promoting the formation of a preopen closed-state configuration leading to channel opening. We have previously shown that the KCa3.1 active gate is most likely located at the level of the selectivity filter. As Ca2+-dependent gating of KCa3.1 originates from the binding of Ca2+ to CaM in the C terminus, the hypothesis of a gate located at the level of the selectivity filter requires that the conformational change initiated in the C terminus be transmitted to the S5 and S6 transmembrane helices, with a resulting effect on the channel pore helix directly connected to the selectivity filter. A study was thus undertaken to determine to what extent the interactions between the channel pore helix with the S5 and S6 transmembrane segments contribute to KCa3.1 gating. Molecular dynamics simulations first revealed that the largest contact area between the pore helix and the S5 plus S6 transmembrane helices involves residue F248 at the C-terminal end of the pore helix. Unitary current recordings next confirmed that modulating aromatic–aromatic interactions between F248 and W216 of the S5 transmembrane helical segment and/or perturbing the interactions between F248 and residues in S6 surrounding the glycine hinge G274 cause important changes in Pomax. This work thus provides the first evidence for a key contribution of the pore helix in setting Pomax by stabilizing the channel closed configuration through aromatic–aromatic interactions involving F248 of the pore helix. We propose that the interface pore helix/S5 constitutes a promising site for designing KCa3.1 potentiators. PMID:24470490

  1. The nop-1 gene of Neurospora crassa encodes a seven transmembrane helix retinal-binding protein homologous to archaeal rhodopsins

    PubMed Central

    Bieszke, Jennifer A.; Braun, Edward L.; Bean, Laura E.; Kang, Seogchan; Natvig, Donald O.; Borkovich, Katherine A.

    1999-01-01

    Opsins are a class of retinal-binding, seven transmembrane helix proteins that function as light-responsive ion pumps or sensory receptors. Previously, genes encoding opsins had been identified in animals and the Archaea but not in fungi or other eukaryotic microorganisms. Here, we report the identification and mutational analysis of an opsin gene, nop-1, from the eukaryotic filamentous fungus Neurospora crassa. The nop-1 amino acid sequence predicts a protein that shares up to 81.8% amino acid identity with archaeal opsins in the 22 retinal binding pocket residues, including the conserved lysine residue that forms a Schiff base linkage with retinal. Evolutionary analysis revealed relatedness not only between NOP-1 and archaeal opsins but also between NOP-1 and several fungal opsin-related proteins that lack the Schiff base lysine residue. The results provide evidence for a eukaryotic opsin family homologous to the archaeal opsins, providing a plausible link between archaeal and visual opsins. Extensive analysis of Δnop-1 strains did not reveal obvious defects in light-regulated processes under normal laboratory conditions. However, results from Northern analysis support light and conidiation-based regulation of nop-1 gene expression, and NOP-1 protein heterologously expressed in Pichia pastoris is labeled by using all-trans [3H]retinal, suggesting that NOP-1 functions as a rhodopsin in N. crassa photobiology. PMID:10393943

  2. Structural Model of the Bilitranslocase Transmembrane Domain Supported by NMR and FRET Data

    PubMed Central

    Choudhury, Amrita Roy; Sikorska, Emilia; van den Boom, Johannes; Bayer, Peter; Popenda, Łukasz; Szutkowski, Kosma; Jurga, Stefan; Bonomi, Massimiliano; Sali, Andrej; Zhukov, Igor; Passamonti, Sabina; Novič, Marjana

    2015-01-01

    We present a 3D model of the four transmembrane (TM) helical regions of bilitranslocase (BTL), a structurally uncharacterized protein that transports organic anions across the cell membrane. The model was computed by considering helix-helix interactions as primary constraints, using Monte Carlo simulations. The interactions between the TM2 and TM3 segments have been confirmed by Förster resonance energy transfer (FRET) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, increasing our confidence in the model. Several insights into the BTL transport mechanism were obtained by analyzing the model. For example, the observed cis-trans Leu-Pro peptide bond isomerization in the TM3 fragment may indicate a key conformational change during anion transport by BTL. Our structural model of BTL may facilitate further studies, including drug discovery. PMID:26291722

  3. De novo design of a transmembrane Zn²⁺-transporting four-helix bundle.

    PubMed

    Joh, Nathan H; Wang, Tuo; Bhate, Manasi P; Acharya, Rudresh; Wu, Yibing; Grabe, Michael; Hong, Mei; Grigoryan, Gevorg; DeGrado, William F

    2014-12-19

    The design of functional membrane proteins from first principles represents a grand challenge in chemistry and structural biology. Here, we report the design of a membrane-spanning, four-helical bundle that transports first-row transition metal ions Zn(2+) and Co(2+), but not Ca(2+), across membranes. The conduction path was designed to contain two di-metal binding sites that bind with negative cooperativity. X-ray crystallography and solid-state and solution nuclear magnetic resonance indicate that the overall helical bundle is formed from two tightly interacting pairs of helices, which form individual domains that interact weakly along a more dynamic interface. Vesicle flux experiments show that as Zn(2+) ions diffuse down their concentration gradients, protons are antiported. These experiments illustrate the feasibility of designing membrane proteins with predefined structural and dynamic properties. PMID:25525248

  4. Structural biology of transmembrane domains: Efficient production and characterization of transmembrane peptides by NMR

    PubMed Central

    Hu, Jian; Qin, Huajun; Li, Conggang; Sharma, Mukesh; Cross, Timothy A.; Gao, Fei Philip

    2007-01-01

    Structural characterization of transmembrane peptides (TMPs) is justified because transmembrane domains of membrane proteins appear to often function independently of the rest of the protein. However, the challenge in obtaining milligrams of isotopically labeled TMPs to study these highly hydrophobic peptides by nuclear magnetic resonance (NMR) is significant. In the present work, a protocol is developed to produce, isotopically label, and purify TMPs in high yield as well as to initially characterize the TMPs with CD and both solution and solid-state NMR. Six TMPs from three integral membrane proteins, CorA, M2, and KdpF, were studied. CorA and KdpF are from Mycobacterium tuberculosis, while M2 is from influenza A virus. Several milligrams of each of these TMPs ranging from 25 to 89 residues were obtained per liter of M9 culture. The initial structural characterization results showed that these peptides were well folded in both detergent micelles and lipid bilayer preparations. The high yield, the simplicity of purification, and the convenient protocol represents a suitable approach for NMR studies and a starting point for characterizing the transmembrane domains of membrane proteins. PMID:17893361

  5. Mutations of charged amino acids at the cytoplasmic end of transmembrane helix 2 affect transport activity of the budding yeast multidrug resistance protein Pdr5p.

    PubMed

    Dou, Weiwang; Zhu, Jianhua; Wang, Tanjun; Wang, Wei; Li, Han; Chen, Xin; Guan, Wenjun

    2016-06-01

    Pdr5p is a major ATP-binding cassette (ABC) transporter in Saccharomyces cerevisiae. It displays a sequence and functional homology to the pathogenic Candida albicans multidrug resistance protein Cdr1p. The transmembrane helices of Pdr5p act in substrate recognition, binding, translocation and eventual removal of toxic substances out of the plasma membrane via the formation of a binding pocket. In this study, we identify two novel Pdr5 mutants (E574K and E580K), which exhibit impaired substrate efflux functions. Both mutants remained hypersensitive to all tested Pdr5p substrates without affecting their protein expression levels, localization or ATPase activities. As E574 and E580 are both located adjacent to the predicted cytoplasmic end of transmembrane helix 2, this implies that such charged residues are functionally essential for Pdr5p. Molecular docking studies suggest the possibility that oppositely charged substitution at residue E574 may disturb the interaction between the substrates and Pdr5p, resulting in impaired transport activity. Our results present new evidence, suggesting that transmembrane helix 2 plays an important role for the efflux function of Pdr5p. PMID:27189366

  6. Structure elucidation of dimeric transmembrane domains of bitopic proteins

    PubMed Central

    Volynsky, Pavel E.; Pavlov, Konstantin V.; Efremov, Roman G.; Arseniev, Alexander S.

    2010-01-01

    The interaction between transmembrane helices is of great interest because it directly determines biological activity of a membrane protein. Either destroying or enhancing such interactions can result in many diseases related to dysfunction of different tissues in human body. One much studied form of membrane proteins known as bitopic protein is a dimer containing two membrane-spanning helices associating laterally. Establishing structure-function relationship as well as rational design of new types of drugs targeting membrane proteins requires precise structural information about this class of objects. At present time, to investigate spatial structure and internal dynamics of such transmembrane helical dimers, several strategies were developed based mainly on a combination of NMR spectroscopy, optical spectroscopy, protein engineering and molecular modeling. These approaches were successfully applied to homo- and heterodimeric transmembrane fragments of several bitopic proteins, which play important roles in normal and in pathological conditions of human organism. PMID:20421711

  7. Structural plasticity of a transmembrane peptide allows self-assembly into biologically active nanoparticles

    PubMed Central

    Tarasov, Sergey G.; Gaponenko, Vadim; Howard, O. M. Zack; Chen, Yuhong; Oppenheim, Joost J.; Dyba, Marzena A.; Subramaniam, Sriram; Lee, Youngshim; Michejda, Christopher; Tarasova, Nadya I.

    2011-01-01

    Significant efforts have been devoted to the development of nanoparticular delivering systems targeting tumors. However, clinical application of nanoparticles is hampered by insufficient size homogeneity, difficulties in reproducible synthesis and manufacturing, frequent high uptake in the liver, systemic toxicity of the carriers (particularly for inorganic nanoparticles), and insufficient selectivity for tumor cells. We have found that properly modified synthetic analogs of transmembrane domains of membrane proteins can self-assemble into remarkably uniform spherical nanoparticles with innate biological activity. Self-assembly is driven by a structural transition of the peptide that adopts predominantly a beta-hairpin conformation in aqueous solutions, but folds into an alpha-helix upon spontaneous fusion of the nanoparticles with cell membrane. A 24-amino acid peptide corresponding to the second transmembrane helix of the CXCR4 forms self-assembled particles that inhibit CXCR4 function in vitro and hamper CXCR4-dependent tumor metastasis in vivo. Furthermore, such nanoparticles can encapsulate hydrophobic drugs, thus providing a delivery system with the potential for dual biological activity. PMID:21628584

  8. Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR

    PubMed Central

    Huang, Rui; Yamamoto, Kazutoshi; Zhang, Meng; Popovych, Nataliya; Hung, Ivan; Im, Sang-Choul; Gan, Zhehong; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2014-01-01

    NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ∼75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ∼13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment. PMID:24853741

  9. Ring Substituents on Substituted Benzamide Ligands Indirectly Mediate Interactions with Position 7.39 of Transmembrane Helix 7 of the D4 Dopamine Receptor

    PubMed Central

    Ericksen, Spencer S.; Cummings, David F.; Teer, Michael E.; Amdani, Shahnawaz

    2012-01-01

    In an effort to delineate how specific molecular interactions of dopamine receptor ligand classes vary between D2-like dopamine receptor subtypes, a conserved threonine in transmembrane (TM) helix 7 (Thr7.39), implicated as a key ligand interaction site with biogenic amine G protein-coupled receptors, was substituted with alanine in D2 and D4 receptors. Interrogation of different ligand chemotypes for sensitivity to this substitution revealed enhanced affinity in the D4, but not the D2 receptor, specifically for substituted benzamides (SBAs) having polar 4- (para) and/or 5- (meta) benzamide ring substituents. D4-T7.39A was fully functional, and the mutation did not alter the sodium-mediated positive and negative allostery observed with SBAs and agonists, respectively. With the exception of the non-SBA ligand (+)-butaclamol, which, in contrast to certain SBAs, had decreased affinity for the D4-T7.39A mutant, the interactions of numerous other ligands were unaffected by this mutation. SBAs were docked into D4 models in the same mode as observed for eticlopride in the D3 crystal structure. In this mode, interactions with TM5 and TM6 residues constrain the SBA ring position that produces distal steric crowding between pyrrolidinyl/diethylamine moieties and D4-Thr7.39. Ligand-residue interaction energy profiles suggest this crowding is mitigated by substitution with a smaller alanine. The profiles indicate sites that contribute to the SBA binding interaction and site-specific energy changes imparted by the D4-T7.39A mutation. Substantial interaction energy changes are observed at only a few positions, some of which are not conserved among the dopamine receptor subtypes and thus seem to account for this D4 subtype-specific structure-activity relationship. PMID:22588261

  10. Recognition and Binding of a Helix-Loop-Helix Peptide to Carbonic Anhydrase Occurs via Partly Folded Intermediate Structures

    PubMed Central

    Lignell, Martin; Becker, Hans-Christian

    2010-01-01

    Abstract We have studied the association of a helix-loop-helix peptide scaffold carrying a benzenesulfonamide ligand to carbonic anhydrase using steady-state and time-resolved fluorescence spectroscopy. The helix-loop-helix peptide, developed for biosensing applications, is labeled with the fluorescent probe dansyl, which serves as a polarity-sensitive reporter of the binding event. Using maximum entropy analysis of the fluorescence lifetime of dansyl at 1:1 stoichiometry reveals three characteristic fluorescence lifetime groups, interpreted as differently interacting peptide/protein structures. We characterize these peptide/protein complexes as mostly bound but unfolded, bound and partly folded, and strongly bound and folded. Furthermore, analysis of the fluorescence anisotropy decay resulted in three different dansyl rotational correlation times, namely 0.18, 1.2, and 23 ns. Using the amplitudes of these times, we can correlate the lifetime groups with the corresponding fluorescence anisotropy component. The 23-ns rotational correlation time, which appears with the same amplitude as a 17-ns fluorescence lifetime, shows that the dansyl fluorophore follows the rotational diffusion of carbonic anhydrase when it is a part of the folded peptide/protein complex. A partly folded and partly hydrated interfacial structure is manifested in an 8-ns dansyl fluorescence lifetime and a 1.2-ns rotational correlation time. This structure, we believe, is similar to a molten-globule-like interfacial structure, which allows segmental movement and has a higher degree of solvent exposure of dansyl. Indirect excitation of dansyl on the helix-loop-helix peptide through Förster energy transfer from one or several tryptophans in the carbonic anhydrase shows that the helix-loop-helix scaffold binds to a tryptophan-rich domain of the carbonic anhydrase. We conclude that binding of the peptide to carbonic anhydrase involves a transition from a disordered to an ordered structure of the helix-loop-helix scaffold. PMID:20141756

  11. Recognition and binding of a helix-loop-helix peptide to carbonic anhydrase occurs via partly folded intermediate structures.

    PubMed

    Lignell, Martin; Becker, Hans-Christian

    2010-02-01

    We have studied the association of a helix-loop-helix peptide scaffold carrying a benzenesulfonamide ligand to carbonic anhydrase using steady-state and time-resolved fluorescence spectroscopy. The helix-loop-helix peptide, developed for biosensing applications, is labeled with the fluorescent probe dansyl, which serves as a polarity-sensitive reporter of the binding event. Using maximum entropy analysis of the fluorescence lifetime of dansyl at 1:1 stoichiometry reveals three characteristic fluorescence lifetime groups, interpreted as differently interacting peptide/protein structures. We characterize these peptide/protein complexes as mostly bound but unfolded, bound and partly folded, and strongly bound and folded. Furthermore, analysis of the fluorescence anisotropy decay resulted in three different dansyl rotational correlation times, namely 0.18, 1.2, and 23 ns. Using the amplitudes of these times, we can correlate the lifetime groups with the corresponding fluorescence anisotropy component. The 23-ns rotational correlation time, which appears with the same amplitude as a 17-ns fluorescence lifetime, shows that the dansyl fluorophore follows the rotational diffusion of carbonic anhydrase when it is a part of the folded peptide/protein complex. A partly folded and partly hydrated interfacial structure is manifested in an 8-ns dansyl fluorescence lifetime and a 1.2-ns rotational correlation time. This structure, we believe, is similar to a molten-globule-like interfacial structure, which allows segmental movement and has a higher degree of solvent exposure of dansyl. Indirect excitation of dansyl on the helix-loop-helix peptide through Förster energy transfer from one or several tryptophans in the carbonic anhydrase shows that the helix-loop-helix scaffold binds to a tryptophan-rich domain of the carbonic anhydrase. We conclude that binding of the peptide to carbonic anhydrase involves a transition from a disordered to an ordered structure of the helix-loop-helix scaffold. PMID:20141756

  12. Geometry and intrinsic tilt of a tryptophan-anchored transmembrane alpha-helix determined by (2)H NMR.

    PubMed Central

    van der Wel, Patrick C A; Strandberg, Erik; Killian, J Antoinette; Koeppe, Roger E

    2002-01-01

    We used solid-state deuterium NMR spectroscopy and an approach involving geometric analysis of labeled alanines (GALA method) to examine the structure and orientation of a designed synthetic hydrophobic, membrane-spanning alpha-helical peptide in phosphatidylcholine (PC) bilayers. The 19-amino-acid peptide consists of an alternating leucine and alanine core, flanked by tryptophans that serve as interfacial anchors: acetyl-GWW(LA)(6)LWWA-ethanolamine (WALP19). A single deuterium-labeled alanine was introduced at different positions within the peptide. Peptides were incorporated in oriented bilayers of dilauroyl- (di-C12:0-), dimyristoyl- (di-C14:0-), or dioleoyl- (di-C18:1(c)-) phosphatidylcholine. The NMR data fit well to a WALP19 orientation characterized by a distinctly nonzero tilt, approximately 4 degrees from the membrane normal, and rapid reorientation about the membrane normal in all three lipids. Although the orientation of WALP19 varies slightly in the different lipids, hydrophobic mismatch does not seem to be the dominant factor causing the tilt. We suggest rather that the peptide itself has an inherently preferred tilted orientation, possibly related to peptide surface characteristics or the disposition of tryptophan indole anchors relative to the lipids, the peptide backbone, and the membrane/water interface. Additionally, the data allow us to define more precisely the local alanine geometry in this membrane-spanning alpha-helix. PMID:12202373

  13. Homology modeling of major intrinsic proteins in rice, maize and Arabidopsis: comparative analysis of transmembrane helix association and aromatic/arginine selectivity filters

    PubMed Central

    Bansal, Anjali; Sankararamakrishnan, Ramasubbu

    2007-01-01

    Background The major intrinsic proteins (MIPs) facilitate the transport of water and neutral solutes across the lipid bilayers. Plant MIPs are believed to be important in cell division and expansion and in water transport properties in response to environmental conditions. More than 30 MIP sequences have been identified in Arabidopsis thaliana, maize and rice. Plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), Nod26-like intrinsic protein (NIPs) and small and basic intrinsic proteins (SIPs) are subfamilies of plant MIPs. Despite sequence diversity, all the experimentally determined structures belonging to the MIP superfamily have the same "hour-glass" fold. Results We have structurally characterized 39 rice and 31 maize MIPs and compared them with that of Arabidopsis. Homology models of 105 MIPs from all three plant species were built. Structure-based sequence alignments were generated and the residues in the helix-helix interfaces were analyzed. Small residues (Gly/Ala/Ser/Thr) are found to be highly conserved as a group in the helix-helix interface of MIP structures. Individual families sometimes prefer one or another of the residues from this group. The narrow aromatic/arginine (ar/R) selectivity filter in MIPs has been shown to provide an important constriction for solute permeability. Ar/R regions were analyzed and compared between the three plant species. Seventeen TIP, NIP and SIP members from rice and maize have ar/R signatures that are not found in Arabidopsis. A subgroup of rice and maize NIPs has small residues in three of the four positions in the ar/R tetrad, resulting in a wider constriction. These MIP members could transport larger solute molecules. Conclusion Small residues are group-conserved in the helix-helix interface of MIP structures and they seem to be important for close helix-helix interactions. Such conservation might help to preserve the hour-glass fold in MIP structures. Analysis and comparison of ar/R selectivity filters suggest that rice and maize MIPs could transport more diverse solutes than Arabidopsis MIPs. Thus the MIP members show conservation in helix-helix interfaces and diversity in aromatic/arginine selectivity filters. The former is related to structural stability and the later can be linked to functional diversity. PMID:17445256

  14. Natural constraints, folding, motion, and structural stability in transmembrane helical proteins

    NASA Astrophysics Data System (ADS)

    Harrington, Susan E.; Ben-Tal, Nir

    Transmembrane (TM) helical proteins are of fundamental importance in many diverse biological processes. To understand these proteins functionally, it is necessary to characterize the forces that stabilize them. What are these forces (both within the protein itself and between the protein and membrane) and how do they give rise to the multiple conformational states and complex activity of TM helical proteins? How do they act in concert to fold TM helical proteins, create their low-energy stable states, and guide their motion? These central questions have led to the description of critical natural constraints and partial answers, which we will review. We will then describe how these constraints can be tracked through homologs and proteins of similar folds in order to better understand how amino acid sequence can specify structure and guide motion. Our emphasis throughout will be on structural features of TM helix bundles themselves, but we will also sketch the membrane-related aspects of these questions.

  15. Cystic fibrosis transmembrane conductance regulator (ABCC7) structure.

    PubMed

    Hunt, John F; Wang, Chi; Ford, Robert C

    2013-02-01

    Structural studies of the cystic fibrosis transmembrane conductance regulator (CFTR) are reviewed. Like many membrane proteins, full-length CFTR has proven to be difficult to express and purify, hence much of the structural data available is for the more tractable, independently expressed soluble domains. Therefore, this chapter covers structural data for individual CFTR domains in addition to the sparser data available for the full-length protein. To set the context for these studies, we will start by reviewing structural information on model proteins from the ATP-binding cassette (ABC) transporter superfamily, to which CFTR belongs. PMID:23378596

  16. Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7) Structure

    PubMed Central

    Hunt, John F.; Wang, Chi; Ford, Robert C.

    2013-01-01

    Structural studies of the cystic fibrosis transmembrane conductance regulator (CFTR) are reviewed. Like many membrane proteins, full-length CFTR has proven to be difficult to express and purify, hence much of the structural data available is for the more tractable, independently expressed soluble domains. Therefore, this chapter covers structural data for individual CFTR domains in addition to the sparser data available for the full-length protein. To set the context for these studies, we will start by reviewing structural information on model proteins from the ATP-binding cassette (ABC) transporter superfamily, to which CFTR belongs. PMID:23378596

  17. Structure of single-wall carbon nanotubes: a graphene helix.

    PubMed

    Lee, Jae-Kap; Lee, Sohyung; Kim, Jin-Gyu; Min, Bong-Ki; Kim, Yong-Il; Lee, Kyung-Il; An, Kay Hyeok; John, Phillip

    2014-08-27

    Evidence is presented in this paper that certain single-wall carbon nanotubes are not seamless tubes, but rather adopt a graphene helix resulting from the spiral growth of a nano-graphene ribbon. The residual traces of the helices are confirmed by high-resolution transmission electron microscopy and atomic force microscopy. The analysis also shows that the tubular graphene material may exhibit a unique armchair structure and the chirality is not a necessary condition for the growth of carbon nanotubes. The description of the structure of the helical carbon nanomaterials is generalized using the plane indices of hexagonal space groups instead of using chiral vectors. It is also proposed that the growth model, via a graphene helix, results in a ubiquitous structure of single-wall carbon nanotubes. PMID:24838196

  18. Structural Basis for High Affinity Volatile Anesthetic Binding in a Natural 4-helix Bundle Protein

    SciTech Connect

    Liu,R.; Loll, P.; Eckenhoff, R.

    2005-01-01

    Physiologic sites for inhaled anesthetics are presumed to be cavities within transmembrane 4-{alpha}-helix bundles of neurotransmitter receptors, but confirmation of binding and structural detail of such sites remains elusive. To provide such detail, we screened soluble proteins containing this structural motif, and found only one that exhibited evidence of strong anesthetic binding. Ferritin is a 24-mer of 4-{alpha}-helix bundles; both halothane and isoflurane bind with K{sub A} values of {approx}10{sup 5} M{sup -1, } higher than any previously reported inhaled anesthetic-protein interaction. The crystal structures of the halothane/apoferritin and isoflurane/apoferritin complexes were determined at 1.75 Angstroms resolution, revealing a common anesthetic binding pocket within an interhelical dimerization interface. The high affinity is explained by several weak polar contacts and an optimal host/guest packing relationship. Neither the acidic protons nor ether oxygen of the anesthetics contribute to the binding interaction. Compared with unliganded apoferritin, the anesthetic produced no detectable alteration of structure or B factors. The remarkably high affinity of the anesthetic/apoferritin complex implies greater selectivity of protein sites than previously thought, and suggests that direct protein actions may underlie effects at lower than surgical levels of anesthetic, including loss of awareness.

  19. The first N-terminal transmembrane helix of each subunit of the antigenic peptide transporter TAP is essential for independent tapasin binding.

    PubMed

    Koch, Joachim; Guntrum, Renate; Tampé, Robert

    2006-07-24

    The heterodimeric ABC transporter TAP translocates proteasomal degradation products from the cytosol into the lumen of the endoplasmic reticulum, where these peptides are loaded onto MHC class I molecules by a macromolecular peptide-loading complex (PLC) and subsequently shuttled to the cell surface for inspection by cytotoxic T lymphocytes. Tapasin recruits, as a central adapter protein, other components of the PLC at the unique N-terminal domains of TAP. We found that the N-terminal domains of human TAP1 and TAP2 can independently bind to tapasin, thus providing two separate loading platforms for PLC assembly. Moreover, tapasin binding is dependent on the first N-terminal transmembrane helix of TAP1 and TAP2, demonstrating that these two helices contribute independently to the recruitment of tapasin and associated factors. PMID:16828748

  20. Transmembrane signaling in the sensor kinase DcuS of Escherichia coli: A long-range piston-type displacement of transmembrane helix 2.

    PubMed

    Monzel, Christian; Unden, Gottfried

    2015-09-01

    The C4-dicarboxylate sensor kinase DcuS is membrane integral because of the transmembrane (TM) helices TM1 and TM2. Fumarate-induced movement of the helices was probed in vivo by Cys accessibility scanning at the membrane-water interfaces after activation of DcuS by fumarate at the periplasmic binding site. TM1 was inserted with amino acid residues 21-41 in the membrane in both the fumarate-activated (ON) and inactive (OFF) states. In contrast, TM2 was inserted with residues 181-201 in the OFF state and residues 185-205 in the ON state. Replacement of Trp 185 by an Arg residue caused displacement of TM2 toward the outside of the membrane and a concomitant induction of the ON state. Results from Cys cross-linking of TM2/TM2' in the DcuS homodimer excluded rotation; thus, data from accessibility changes of TM2 upon activation, either by ligand binding or by mutation of TM2, and cross-linking of TM2 and the connected region in the periplasm suggest a piston-type shift of TM2 by four residues to the periplasm upon activation (or fumarate binding). This mode of function is supported by the suggestion from energetic calculations of two preferred positions for TM2 insertion in the membrane. The shift of TM2 by four residues (or 4-6 Å) toward the periplasm upon activation is complementary to the periplasmic displacement of 3-4 Å of the C-terminal part of the periplasmic ligand-binding domain upon ligand occupancy in the citrate-binding domain in the homologous CitA sensor kinase. PMID:26283365

  1. Structural Dynamics of Insulin Receptor and Transmembrane Signaling.

    PubMed

    Tatulian, Suren A

    2015-09-15

    The insulin receptor (IR) is a (αβ)2-type transmembrane tyrosine kinase that plays a central role in cell metabolism. Each αβ heterodimer consists of an extracellular ligand-binding α-subunit and a membrane-spanning β-subunit that comprises the cytoplasmic tyrosine kinase (TK) domain and the phosphorylation sites. The α- and β-subunits are linked via a single disulfide bridge, and the (αβ)2 tetramer is formed by disulfide bonds between the α-chains. Insulin binding induces conformational changes in IR that reach the intracellular β-subunit followed by a protein phosphorylation and activation cascade. Defects in this signaling process, including IR dysfunction caused by mutations, result in type 2 diabetes. Rational drug design aimed at treatment of diabetes relies on knowledge of the detailed structure of IR and the dynamic structural transformations during transmembrane signaling. Recent X-ray crystallographic studies have provided important clues about the mode of binding of insulin to IR, the resulting structural changes and their transmission to the TK domain, but a complete understanding of the structural basis underlying insulin signaling has not been achieved. This review presents a critical analysis of the current status of the structure-function relationship of IR, with a comparative assessment of the other IR family receptors, and discusses potential advancements that may provide insight into the molecular mechanism of insulin signaling. PMID:26322622

  2. Helix Bundle Quaternary Structure from [alpha]/[beta]-Peptide Foldamers

    SciTech Connect

    Horne, W. Seth; Price, Joshua L.; Keck, James L.; Gellman, Samuel H.

    2008-11-18

    The function of a protein generally depends on adoption of a specific folding pattern, which in turn is determined by the side chain sequence along the polypeptide backbone. Here we show that the sequence-encoded structural information in peptides derived from yeast transcriptional activator GCN4 can be used to prepare hybrid {alpha}/{beta}-peptide foldamers that adopt helix bundle quaternary structures. Crystal structures of two hybrid {alpha}/{beta}-peptides are reported along with detailed structural comparison to {alpha}-peptides of analogous side chain sequence. There is considerable homology between {alpha}- and {alpha}/{beta}-peptides at the level of helical secondary structure, with modest but significant differences in the association geometry of helices in the quaternary structure.

  3. Assembly of transmembrane proteins on oil-water interfaces

    NASA Astrophysics Data System (ADS)

    Yunker, Peter; Landry, Corey; Chong, Shaorong; Weitz, David

    2015-03-01

    Transmembrane proteins are difficult to handle by aqueous solution-based biochemical and biophysical approaches, due to the hydrophobicity of transmembrane helices. Detergents can solubilize transmembrane proteins; however, surfactant coated transmembrane proteins are not always functional, and purifying detergent coated proteins in a micellar solution can be difficult. Motivated by this problem, we study the self-assembly of transmembrane proteins on oil-water interfaces. We found that the large water-oil interface of oil drops prevents nascent transmembrane proteins from forming non-functional aggregates. The oil provides a hydrophobic environment for the transmembrane helix, allowing the ectodomain to fold into its natural structure and orientation. Further, modifying the strength or valency of hydrophobic interactions between transmembrane proteins results in the self-assembly of spatially clustered, active proteins on the oil-water interface. Thus, hydrophobic interactions can facilitate, rather than inhibit, the assembly of transmembrane proteins.

  4. X-ray structure breakthroughs in the GPCR transmembrane region.

    PubMed

    Topiol, Sid; Sabio, Michael

    2009-07-01

    G-protein-coupled receptor (GPCR) proteins [Lundstrom KH, Chiu ML, editors. G protein-coupled receptors in drug discovery. CRC Press; 2006] are the single largest drug target, representing 25-50% of marketed drugs [Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat Rev Drug Discov 2006;5(12):993-6; Parrill AL. Crystal structures of a second G protein-coupled receptor: triumphs and implications. ChemMedChem 2008;3:1021-3]. While there are six subclasses of GPCR proteins, the hallmark of all GPCR proteins is the transmembrane-spanning region. The general architecture of this transmembrane (TM) region has been known for some time to contain seven alpha-helices. From a drug discovery and design perspective, structural information of the GPCRs has been sought as a tool for structure-based drug design. The advances in the past decade of technologies for structure-based design have proven to be useful in a number of areas. Invoking these approaches for GPCR targets has remained challenging. Until recently, the most closely related structures available for GPCR modeling have been those of bovine rhodopsin. While a representative of class A GPCRs, bovine rhodopsin is not a ligand-activated GPCR and is fairly distant in sequence homology to other class A GPCRs. Thus, there is a variable degree of uncertainty in the use of the rhodopsin X-ray structure as a template for homology modeling of other GPCR targets. Recent publications of X-ray structures of class A GPCRs now offer the opportunity to better understand the molecular mechanism of action at the atomic level, to deploy X-ray structures directly for their use in structure-based design, and to provide more promising templates for many other ligand-mediated GPCRs. We summarize herein some of the recent findings in this area and provide an initial perspective of the emerging opportunities, possible limitations, and remaining questions. Other aspects of the recent X-ray structures are described by Weis and Kobilka [Weis WI, Kobilka BK. Structural insights into G-protein-coupled receptor activation. Curr Opin Struct Biol 2008;18:734-40] and Mustafi and Palczewski [Mustafi D, Palczewski K. Topology of class A G protein-coupled receptors: insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors. Mol Pharmacol 2009;75:1-12]. PMID:19447219

  5. Three-Dimensional Structure of the Transmembrane Domain of Vpu from HIV-1 in Aligned Phospholipid Bicelles

    PubMed Central

    Park, Sang Ho; De Angelis, Anna A.; Nevzorov, Alexander A.; Wu, Chin H.; Opella, Stanley J.

    2006-01-01

    The three-dimensional backbone structure of the transmembrane domain of Vpu from HIV-1 was determined by solid-state NMR spectroscopy in two magnetically-aligned phospholipid bilayer environments (bicelles) that differed in their hydrophobic thickness. Isotopically labeled samples of Vpu2–30+, a 36-residue polypeptide containing residues 2–30 from the N-terminus of Vpu, were incorporated into large (q = 3.2 or 3.0) phospholipid bicelles composed of long-chain ether-linked lipids (14-O-PC or 16-O-PC) and short-chain lipids (6-O-PC). The protein-containing bicelles are aligned in the static magnetic field of the NMR spectrometer. Wheel-like patterns of resonances characteristic of tilted transmembrane helices were observed in two-dimensional 1H/15N PISEMA spectra of uniformly 15N-labeled Vpu2–30+ obtained on bicelle samples with their bilayer normals aligned perpendicular or parallel to the direction of the magnetic field. The NMR experiments were performed at a 1H resonance frequency of 900 MHz, and this resulted in improved data compared to lower-resonance frequencies. Analysis of the polarity-index slant-angle wheels and dipolar waves demonstrates the presence of a transmembrane α-helix spanning residues 8–25 in both 14-O-PC and 16-O-PC bicelles, which is consistent with results obtained previously in micelles by solution NMR and mechanically aligned lipid bilayers by solid-state NMR. The three-dimensional backbone structures were obtained by structural fitting to the orientation-dependent 15N chemical shift and 1H-15N dipolar coupling frequencies. Tilt angles of 30° and 21° are observed in 14-O-PC and 16-O-PC bicelles, respectively, which are consistent with the values previously determined for the same polypeptide in mechanically-aligned DMPC and DOPC bilayers. The difference in tilt angle in C14 and C16 bilayer environments is also consistent with previous results indicating that the transmembrane helix of Vpu responds to hydrophobic mismatch by changing its tilt angle. The kink found in the middle of the helix in the longer-chain C18 bilayers aligned on glass plates was not found in either of these shorter-chain (C14 or C16) bilayers. PMID:16861273

  6. The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region*♦

    PubMed Central

    Apellániz, Beatriz; Rujas, Edurne; Serrano, Soraya; Morante, Koldo; Tsumoto, Kouhei; Caaveiro, Jose M. M.; Jiménez, M. Ángeles; Nieva, José L.

    2015-01-01

    The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted α-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct subdomains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments. PMID:25787074

  7. Structure of the transmembrane domain of human nicastrin-a component of γ-secretase

    PubMed Central

    Li, Yan; Liew, Lynette Sin Yee; Li, Qingxin; Kang, CongBao

    2016-01-01

    Nicastrin is the largest component of γ-secretase that is an intramembrane protease important in the development of Alzheimer’s disease. Nicastrin contains a large extracellular domain, a single transmembrane (TM) domain, and a short C-terminus. Its TM domain is important for the γ-secretase complex formation. Here we report nuclear magnetic resonance (NMR) studies of the TM and C-terminal regions of human nicastrin in both sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelles. Structural study and dynamic analysis reveal that the TM domain is largely helical and stable under both SDS and DPC micelles with its N-terminal region undergoing intermediate time scale motion. The TM helix contains a hydrophilic patch that is important for TM-TM interactions. The short C-terminus is not structured in solution and a region formed by residues V697-A702 interacts with the membrane, suggesting that these residues may play a role in the γ-secretase complex formation. Our study provides structural insight into the function of the nicastrin TM domain and the C-terminus in γ-secretase complex. PMID:26776682

  8. Crystal Structure of the Glycophorin A Transmembrane Dimer in Lipidic Cubic Phase.

    PubMed

    Trenker, Raphael; Call, Matthew E; Call, Melissa J

    2015-12-23

    The mechanisms of assembly and function for many important type I/II (single-pass) transmembrane (TM) receptors are proposed to involve the formation and/or alteration of specific interfaces among their membrane-embedded ?-helical TM domains. The application of lipidic cubic phase (LCP) bilayer media for crystallization of single-?-helical TM complexes has the potential to provide valuable structural and mechanistic insights into many such systems. However, the fidelity of the interfaces observed in crowded crystalline arrays has been difficult to establish from the very limited number of such structures determined using X-ray diffraction data. Here we examine this issue using the glycophorin A (GpA) model system, whose homodimeric TM helix interface has been characterized by solution and solid-state NMR and biochemical techniques but never crystallographically. We report that a GpA-TM peptide readily crystallized in a monoolein cubic phase bilayer, yielding a dimeric ?-helical structure that is in excellent agreement with previously reported NMR measurements made in several different types of host media. These results provide compelling support for the wider application of LCP techniques to enable X-ray crystallographic analysis of single-pass TM interactions. PMID:26642914

  9. An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

    DOE PAGESBeta

    AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

    2015-11-19

    Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database inmore » which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.« less

  10. An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

    SciTech Connect

    AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

    2015-11-19

    Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database in which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.

  11. 5-Formylcytosine alters the structure of the DNA double helix

    PubMed Central

    Chirgadze, Dimitri Y.; Beraldi, Dario; Luisi, Ben F.; Balasubramanian, Shankar

    2014-01-01

    The modified base 5-formylcytosine (5fC) was recently identified in mammalian DNA and might be considered as the “seventh” base of the genome. This nucleotide has been implicated in active demethylation mediated by the base excision repair enzyme thymine DNA glycosylase (TDG). Genomics and proteomics studies have suggested a further role for 5fC in transcription regulation through chromatin remodeling. Herein we propose how 5fC might signal these processes through its effect on DNA conformation. Biophysical and structural analysis revealed that 5fC alters the structure of the DNA double helix leading to a conformation unique amongst known DNA structures including those comprising other cytosine modifications. The 1.4 Å resolution X-ray crystal structure of a DNA dodecamer comprising three 5fCpG sites shown how 5fC changes the geometry of the grooves and base pairs associated with the modified base, which lead to helical under-winding. PMID:25504322

  12. patGPCR: A Multitemplate Approach for Improving 3D Structure Prediction of Transmembrane Helices of G-Protein-Coupled Receptors

    PubMed Central

    Wu, Hongjie; Lü, Qiang; Quan, Lijun; Qian, Peide; Xia, Xiaoyan

    2013-01-01

    The structures of the seven transmembrane helices of G-protein-coupled receptors are critically involved in many aspects of these receptors, such as receptor stability, ligand docking, and molecular function. Most of the previous multitemplate approaches have built a “super” template with very little merging of aligned fragments from different templates. Here, we present a parallelized multitemplate approach, patGPCR, to predict the 3D structures of transmembrane helices of G-protein-coupled receptors. patGPCR, which employs a bundle-packing related energy function that extends on the RosettaMem energy, parallelizes eight pipelines for transmembrane helix refinement and exchanges the optimized helix structures from multiple templates. We have investigated the performance of patGPCR on a test set containing eight determined G-protein-coupled receptors. The results indicate that patGPCR improves the TM RMSD of the predicted models by 33.64% on average against a single-template method. Compared with other homology approaches, the best models for five of the eight targets built by patGPCR had a lower TM RMSD than that obtained from SWISS-MODEL; patGPCR also showed lower average TM RMSD than single-template and multiple-template MODELLER. PMID:23554839

  13. Structure of bacteriophage [phi]29 head fibers has a supercoiled triple repeating helix-turn-helix motif

    SciTech Connect

    Xiang, Ye; Rossmann, Michael G.

    2011-12-22

    The tailed bacteriophage {phi}29 capsid is decorated with 55 fibers attached to quasi-3-fold symmetry positions. Each fiber is a homotrimer of gene product 8.5 (gp8.5) and consists of two major structural parts, a pseudohexagonal base and a protruding fibrous portion that is about 110 {angstrom} in length. The crystal structure of the C-terminal fibrous portion (residues 112-280) has been determined to a resolution of 1.6 {angstrom}. The structure is about 150 {angstrom} long and shows three distinct structural domains designated as head, neck, and stem. The stem region is a unique three-stranded helix-turn-helix supercoil that has not previously been described. When fitted into a cryoelectron microscope reconstruction of the virus, the head structure corresponded to a disconnected density at the distal end of the fiber and the neck structure was located in weak density connecting it to the fiber. Thin section studies of Bacillus subtilis cells infected with fibered or fiberless {phi}29 suggest that the fibers might enhance the attachment of the virions onto the host cell wall.

  14. Coordinating the impact of structural genomics on the human α-helical transmembrane proteome

    PubMed Central

    Pieper, Ursula; Schlessinger, Avner; Kloppmann, Edda; Chang, Geoffrey A.; Chou, James J.; Dumont, Mark E.; Fox, Brian G.; Fromme, Petra; Hendrickson, Wayne A.; Malkowski, Michael G.; Rees, Douglas C.; Stokes, David L.; Stowell, Michael H.B.; Wiener, Michael C.; Rost, Burkhard; Stroud, Robert M.; Stevens, Raymond C.; Sali, Andrej

    2013-01-01

    With the recent successes in determining membrane protein structures, we explore the tractability of determining representatives for the entire human membrane proteome. This proteome contains 2,925 unique integral α-helical transmembrane domain sequences that cluster into 1,201 families sharing more than 25% sequence identity. Structures of 100 optimally selected targets would increase the fraction of modelable human α-helical transmembrane domains from 26% to 58%, thus providing structure/function information not otherwise available. PMID:23381628

  15. Synaptobrevin transmembrane domain determines the structure and dynamics of the SNARE motif and the linker region.

    PubMed

    Han, Jing; Pluhackova, Kristyna; Bruns, Dieter; Böckmann, Rainer A

    2016-04-01

    The vesicular protein synaptobrevin II (sybII) constitutes a central component of the SNARE complex, which mediates vesicle fusion in neuronal exocytosis. Previous studies revealed that the transmembrane domain (TMD) of sybII is playing a critical role in the fusion process and is involved in all distinct fusion stages from priming to fusion pore opening. Here, we analyzed sequence-dependent effects of sybII and of mutants of sybII on both structure and flexibility of the protein and the interactions with a phospholipid bilayer by means of microsecond atomistic simulations. The sybII TMD was found to direct the folding of both the juxtamembrane helix and of the connecting linker and thus to influence both the intrinsic helicity and flexibility. Fusion active peptides revealed two helical segments, one for the juxtamembrane region and one for the TMD, connected by a flexible linker. In contrast, a fusion-inactive poly-leucine TMD mutant assumes a structure with a comparably rigid linker that is suggested to hinder the formation of the trans-SNARE complex during fusion. Kinking of the TMD at the central glycine together with anchoring of the TMD via conserved tryptophans and a lysine in position 94 likely yields an enhanced flexibility of sybII for different membrane thickness. All studied peptides were found to deform the outer membrane layer by altering the lipid head group orientation, causing partial membrane dehydration and enhancing lipid protrusions. These effects weaken the integrity of the outer membrane layer and are attributed mainly to the highly charged linker and JM regions of sybII. PMID:26851777

  16. Unraveling the Helix Nebula: Its Structure and Knots

    NASA Astrophysics Data System (ADS)

    O'Dell, C. R.; McCullough, Peter R.; Meixner, Margaret

    2004-11-01

    Through Hubble Space Telescope (HST) imaging of the inner part of the main ring of the Helix Nebula, together with CTIO 4 m images of the fainter outer parts, we have a view of unprecedented quality of the nearest bright planetary nebula. These images have allowed us to determine that the main ring of the nebula is composed of an inner disk of about 499" diameter (0.52 pc) surrounded by an outer ring (in reality a torus) of 742" diameter (0.77 pc) whose plane is highly inclined to the plane of the disk. This outer ring is surrounded by an outermost ring of 1500" (1.76 pc) diameter, which is flattened on the side colliding with the ambient interstellar medium. The inner disk has an extended distribution of low-density gas along its rotational axis of symmetry, and the disk is optically thick to ionizing radiation, as is the outer ring. Published radial velocities of the knots provide support for the two-component structure of the main ring of the nebula and for the idea that the knots found there are expanding along with the nebular material from which they recently originated. These velocities indicate a spatial expansion velocity of the inner disk of 40 and 32 km s-1 for the outer ring, which yields expansion ages of 6560 and 12,100 yr, respectively. The outermost ring may be partially ionized through scattered recombination continuum from the inner parts of the nebula, but shocks certainly are occurring in it. This outermost ring probably represents a third period of mass loss by the central star. There is one compact, outer object that is unexplained, showing shock structures indicating a different orientation of the gas flow from that of the nebula. There is a change in the morphology of the knots as a function of the distance from the local ionization front. This supports a scenario in which the knots are formed in or near the ionization front and are then sculpted by the stellar radiation from the central star as the ionization front advances beyond them. Based in part on observations with the NASA/ESA Hubble Space Telescope, obtained at the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS5-26555. Based in part on observations obtained at the Cerro Tololo Inter-American Observatory, which is operated by the Association of Universities for Research in Astronomy, Inc., under a Cooperative Agreement with the National Science Foundation.

  17. The stability of the three transmembrane and the four transmembrane human vitamin K epoxide reductase models

    NASA Astrophysics Data System (ADS)

    Wu, Sangwook

    2016-04-01

    The three transmembrane and the four transmembrane helix models are suggested for human vitamin K epoxide reductase (VKOR). In this study, we investigate the stability of the human three transmembrane/four transmembrane VKOR models by employing a coarse-grained normal mode analysis and molecular dynamics simulation. Based on the analysis of the mobility of each transmembrane domain, we suggest that the three transmembrane human VKOR model is more stable than the four transmembrane human VKOR model.

  18. Helix-packing motifs in membrane proteins.

    PubMed

    Walters, R F S; DeGrado, W F

    2006-09-12

    The fold of a helical membrane protein is largely determined by interactions between membrane-imbedded helices. To elucidate recurring helix-helix interaction motifs, we dissected the crystallographic structures of membrane proteins into a library of interacting helical pairs. The pairs were clustered according to their three-dimensional similarity (rmsd structural features can be understood in terms of simple principles of helix-helix packing. Thus, the universe of common transmembrane helix-pairing motifs is relatively simple. The largest cluster, which comprises 29% of the library members, consists of an antiparallel motif with left-handed packing angles, and it is frequently stabilized by packing of small side chains occurring every seven residues in the sequence. Right-handed parallel and antiparallel structures show a similar tendency to segregate small residues to the helix-helix interface but spaced at four-residue intervals. Position-specific sequence propensities were derived for the most populated motifs. These structural and sequential motifs should be quite useful for the design and structural prediction of membrane proteins. PMID:16954199

  19. Contact-Induced Structure Transformation in Transmembrane Prion Propagation

    PubMed Central

    Ou, D.-M.; Chen, C.-C.; Chen, C.-M.

    2007-01-01

    Based on recent experimental evidences of the transmission of prion diseases due to a particular transmembrane form (termed CtmPrP), we propose a theoretical model for the molecular mechanism of such conformational diseases, in which a misfolded CtmPrP induces a similar misfolding of another CtmPrP. Computer simulations are performed to investigate the correlation between folding time and the concentration of misfolded PrP in various processes, including dimerization, trimerization, and cooperative dimerization. By comparing with the experimental correlation curve between incubation time and injected dose of scrapie prions, we conclude that cooperative dimerization may play an important role in the pathological mechanism of prion diseases. PMID:17259269

  20. Structural basis of DNA recognition by PCG2 reveals a novel DNA binding mode for winged helix-turn-helix domains

    PubMed Central

    Liu, Junfeng; Huang, Jinguang; Zhao, Yanxiang; Liu, Huaian; Wang, Dawei; Yang, Jun; Zhao, Wensheng; Taylor, Ian A.; Peng, You-Liang

    2015-01-01

    The MBP1 family proteins are the DNA binding subunits of MBF cell-cycle transcription factor complexes and contain an N terminal winged helix-turn-helix (wHTH) DNA binding domain (DBD). Although the DNA binding mechanism of MBP1 from Saccharomyces cerevisiae has been extensively studied, the structural framework and the DNA binding mode of other MBP1 family proteins remains to be disclosed. Here, we determined the crystal structure of the DBD of PCG2, the Magnaporthe oryzae orthologue of MBP1, bound to MCB–DNA. The structure revealed that the wing, the 20-loop, helix A and helix B in PCG2–DBD are important elements for DNA binding. Unlike previously characterized wHTH proteins, PCG2–DBD utilizes the wing and helix-B to bind the minor groove and the major groove of the MCB–DNA whilst the 20-loop and helix A interact non-specifically with DNA. Notably, two glutamines Q89 and Q82 within the wing were found to recognize the MCB core CGCG sequence through making hydrogen bond interactions. Further in vitro assays confirmed essential roles of Q89 and Q82 in the DNA binding. These data together indicate that the MBP1 homologue PCG2 employs an unusual mode of binding to target DNA and demonstrate the versatility of wHTH domains. PMID:25550425

  1. Structural basis of DNA recognition by PCG2 reveals a novel DNA binding mode for winged helix-turn-helix domains.

    PubMed

    Liu, Junfeng; Huang, Jinguang; Zhao, Yanxiang; Liu, Huaian; Wang, Dawei; Yang, Jun; Zhao, Wensheng; Taylor, Ian A; Peng, You-Liang

    2015-01-01

    The MBP1 family proteins are the DNA binding subunits of MBF cell-cycle transcription factor complexes and contain an N terminal winged helix-turn-helix (wHTH) DNA binding domain (DBD). Although the DNA binding mechanism of MBP1 from Saccharomyces cerevisiae has been extensively studied, the structural framework and the DNA binding mode of other MBP1 family proteins remains to be disclosed. Here, we determined the crystal structure of the DBD of PCG2, the Magnaporthe oryzae orthologue of MBP1, bound to MCB-DNA. The structure revealed that the wing, the 20-loop, helix A and helix B in PCG2-DBD are important elements for DNA binding. Unlike previously characterized wHTH proteins, PCG2-DBD utilizes the wing and helix-B to bind the minor groove and the major groove of the MCB-DNA whilst the 20-loop and helix A interact non-specifically with DNA. Notably, two glutamines Q89 and Q82 within the wing were found to recognize the MCB core CGCG sequence through making hydrogen bond interactions. Further in vitro assays confirmed essential roles of Q89 and Q82 in the DNA binding. These data together indicate that the MBP1 homologue PCG2 employs an unusual mode of binding to target DNA and demonstrate the versatility of wHTH domains. PMID:25550425

  2. Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus

    PubMed Central

    Acharya, Rudresh; Carnevale, Vincenzo; Fiorin, Giacomo; Levine, Benjamin G.; Polishchuk, Alexei L.; Balannik, Victoria; Samish, Ilan; Lamb, Robert A.; Pinto, Lawrence H.; DeGrado, William F.; Klein, Michael L.

    2010-01-01

    The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2+ and 3+ with a pKa near 6. A 1.65 Å resolution X-ray structure of the transmembrane protein (residues 25–46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models. PMID:20689043

  3. Structure and Mechanism of Proton Transport Through the Transmembrane Tetrameric M2 Protein Bundle of the Influenza A Virus

    SciTech Connect

    R Acharya; V Carnevale; G Fiorin; B Levine; A Polishchuk; V Balannick; I Samish; R Lamb; L Pinto; et al.

    2011-12-31

    The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2{sup +} and 3{sup +} with a pK{sub a} near 6. A 1.65 {angstrom} resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.

  4. Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

    PubMed Central

    del Val, Coral; White, Stephen H.

    2014-01-01

    We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide. PMID:22836667

  5. Ser/Thr motifs in transmembrane proteins: conservation patterns and effects on local protein structure and dynamics.

    PubMed

    Del Val, Coral; White, Stephen H; Bondar, Ana-Nicoleta

    2012-11-01

    We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide. PMID:22836667

  6. Enhanced Sampling of Coarse-Grained Transmembrane-Peptide Structure Formation from Hydrogen-Bond Replica Exchange.

    PubMed

    Bereau, Tristan; Deserno, Markus

    2015-06-01

    Protein structure formation in the membrane highlights a grand challenge of sampling in computer simulations, because kinetic traps and slow dynamics make it difficult to find the native state. Exploiting increased fluctuations at higher temperatures can help overcome free-energy barriers, provided the membrane's structure remains stable. In this work, we apply Hamiltonian replica-exchange molecular dynamics, where we only tune the backbone hydrogen-bond strength to help reduce the propensity of long-lived misfolded states. Using a recently developed coarse-grained model, we illustrate the robustness of the method by folding different WALP transmembrane helical peptides starting from stretched, unstructured conformations. We show the efficiency of the method by comparing to simulations without enhanced sampling, achieving folding in one example after significantly longer simulation times. Analysis of the bilayer structure during folding provides insight into the local membrane deformation during helix formation as a function of chain length (from 16 to 23 residues). Finally, we apply our method to fold the 50-residue-long major pVIII coat protein (fd coat) of the filamentous fd bacteriophage. Our results agree well with experimental structures and atomistic simulations based on implicit membrane models, suggesting that our explicit CG folding protocol can serve as a starting point for better-refined atomistic simulations in a multiscale framework. PMID:25311530

  7. Computer simulations and modeling-assisted ToxR screening in deciphering 3D structures of transmembrane ?-helical dimers: ephrin receptor A1

    NASA Astrophysics Data System (ADS)

    Volynsky, P. E.; Mineeva, E. A.; Goncharuk, M. V.; Ermolyuk, Ya S.; Arseniev, A. S.; Efremov, R. G.

    2010-03-01

    Membrane-spanning segments of numerous proteins (e.g. receptor tyrosine kinases) represent a novel class of pharmacologically important targets, whose activity can be modulated by specially designed artificial peptides, the so-called interceptors. Rational construction of such peptides requires understanding of the main factors driving peptide-peptide association in lipid membranes. Here we present a new method for rapid prediction of the spatial structure of transmembrane (TM) helix-helix complexes. It is based on computer simulations in membrane-like media and subsequent refinement/validation of the results using experimental studies of TM helix dimerization in a bacterial membrane by means of the ToxR system. The approach was applied to TM fragments of the ephrin receptor A1 (EphA1). A set of spatial structures of the dimer was proposed based on Monte Carlo simulations in an implicit membrane followed by molecular dynamics relaxation in an explicit lipid bilayer. The resulting models were employed for rational design of wild-type and mutant genetic constructions for ToxR assays. The computational and the experimental data are self-consistent and provide an unambiguous spatial model of the TM dimer of EphA1. The results of this work can be further used to develop new biologically active 'peptide interceptors' specifically targeting membrane domains of proteins.

  8. Structural basis for amino-acid recognition and transmembrane signalling by tandem Per-Arnt-Sim (tandem PAS) chemoreceptor sensory domains.

    PubMed

    Liu, Yu C; Machuca, Mayra A; Beckham, Simone A; Gunzburg, Menachem J; Roujeinikova, Anna

    2015-10-01

    Chemotaxis, mediated by methyl-accepting chemotaxis protein (MCP) receptors, plays an important role in the ecology of bacterial populations. This paper presents the first crystallographic analysis of the structure and ligand-induced conformational changes of the periplasmic tandem Per-Arnt-Sim (PAS) sensing domain (PTPSD) of a characterized MCP chemoreceptor. Analysis of the complex of the Campylobacter jejuni Tlp3 PTPSD with isoleucine (a chemoattractant) revealed that the PTPSD is a dimer in the crystal. The two ligand-binding sites are located in the membrane-distal PAS domains on the faces opposite to the dimer interface. Mutagenesis experiments show that the five strongly conserved residues that stabilize the main-chain moiety of isoleucine are essential for binding, suggesting that the mechanism by which this family of chemoreceptors recognizes amino acids is highly conserved. Although the fold and mode of ligand binding of the PTPSD are different from the aspartic acid receptor Tar, the structural analysis suggests that the PTPSDs of amino-acid chemoreceptors are also likely to signal by a piston displacement mechanism. The PTPSD fluctuates between piston (C-terminal helix) `up' and piston `down' states. Binding of an attractant to the distal PAS domain locks it in the closed form, weakening its association with the proximal domain and resulting in the transition of the latter into an open form, concomitant with a downward (towards the membrane) 4 Å piston displacement of the C-terminal helix. In vivo, this movement would generate a transmembrane signal by driving a downward displacement of the transmembrane helix 2 towards the cytoplasm. PMID:26457436

  9. Helix compactness and stability: Electron structure calculations of conformer dependent thermodynamic functions

    NASA Astrophysics Data System (ADS)

    Jákli, Imre; Csizmadia, Imre G.; Fejer, Szilard N.; Farkas, Ödön; Viskolcz, Bela; Knak Jensen, Svend J.; Perczel, Andras

    2013-03-01

    Structure, stability, cooperativity and molecular packing of two major backbone forms: 310-helix and β-strand are investigated. Long models HCO-(Xxx)n-NH2 Xxx = Gly and (L-)Ala, n ⩽ 34, are studied at two levels of theory including the effect of dispersion forces. Structure and folding preferences are established, the length modulated cooperativity and side-chain determined fold compactness is quantified. By monitoring ΔG°β→α rather than the electronic energy, ΔEβ→α, it appears that Ala is a much better helix forming residue than Gly. The achiral Gly forms a more compact 310-helix than any chiral amino acid residue probed here for L-Ala.

  10. Degeneracy and inversion of band structure for Wigner crystals on a closed helix

    NASA Astrophysics Data System (ADS)

    Zampetaki, A. V.; Stockhofe, J.; Schmelcher, P.

    2015-02-01

    Constraining long-range interacting particles to move on a curved manifold can drastically alter their effective interactions. As a prototype we explore the structure and vibrational dynamics of crystalline configurations formed on a closed helix. We show that the ground state undergoes a pitchfork bifurcation from a symmetric polygonic to a zigzag-like configuration with increasing radius of the helix. Remarkably, we find that, for a specific value of the helix radius, below the bifurcation point, the vibrational frequency spectrum collapses to a single frequency. This allows for an essentially independent small-amplitude motion of the individual particles and, consequently, localized excitations can propagate in time without significant spreading. Upon increasing the radius beyond the degeneracy point, the band structure is inverted, with the out-of-phase oscillation mode becoming lower in frequency than the mode corresponding to the center-of-mass motion.

  11. DEAD-Box Helicase Proteins Disrupt RNA Tertiary Structure Through Helix Capture

    PubMed Central

    Pan, Cynthia; Potratz, Jeffrey P.; Cannon, Brian; Simpson, Zachary B.; Ziehr, Jessica L.; Tijerina, Pilar; Russell, Rick

    2014-01-01

    DEAD-box helicase proteins accelerate folding and rearrangements of highly structured RNAs and RNA–protein complexes (RNPs) in many essential cellular processes. Although DEAD-box proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt RNA tertiary structure. Here, we use single molecule fluorescence to show that the DEAD-box protein CYT-19 disrupts tertiary structure in a group I intron using a helix capture mechanism. CYT-19 binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then CYT-19 uses ATP to unwind the helix, liberating the product strands. Ded1, a multifunctional yeast DEAD-box protein, gives analogous results with small but reproducible differences that may reflect its in vivo roles. The requirement for spontaneous dynamics likely targets DEAD-box proteins toward less stable RNA structures, which are likely to experience greater dynamic fluctuations, and provides a satisfying explanation for previous correlations between RNA stability and CYT-19 unfolding efficiency. Biologically, the ability to sense RNA stability probably biases DEAD-box proteins to act preferentially on less stable misfolded structures and thereby to promote native folding while minimizing spurious interactions with stable, natively folded RNAs. In addition, this straightforward mechanism for RNA remodeling does not require any specific structural environment of the helicase core and is likely to be relevant for DEAD-box proteins that promote RNA rearrangements of RNP complexes including the spliceosome and ribosome. PMID:25350280

  12. Structural Analysis of a Peptide Fragment of Transmembrane Transporter Protein Bilitranslocase

    PubMed Central

    Župerl, Špela; Sikorska, Emilia; Zhukov, Igor; Solmajer, Tom; Novič, Marjana

    2012-01-01

    Using a combination of genomic and post-genomic approaches is rapidly altering the number of identified human influx carriers. A transmembrane protein bilitranslocase (TCDB 2.A.65) has long attracted attention because of its function as an organic anion carrier. It has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its structure. However, at present, only the primary structure of bilitranslocase is known. In our work, transmembrane subunits of bilitranslocase were predicted by a previously developed chemometrics model and the stability of these polypeptide chains were studied by molecular dynamics (MD) simulation. Furthermore, sodium dodecyl sulfate (SDS) micelles were used as a model of cell membrane and herein we present a high-resolution 3D structure of an 18 amino acid residues long peptide corresponding to the third transmembrane part of bilitranslocase obtained by use of multidimensional NMR spectroscopy. It has been experimentally confirmed that one of the transmembrane segments of bilitranslocase has alpha helical structure with hydrophilic amino acid residues oriented towards one side, thus capable of forming a channel in the membrane. PMID:22745694

  13. Analysis of negative material supported helix slow wave structure for traveling-wave tubes

    NASA Astrophysics Data System (ADS)

    Purushothaman, N.; Srivastava, V.; Ghosh, S. K.

    2013-06-01

    We investigate the effect of negative materials used as support structures for helix travelling wave tubes (TWTs). Analysis is carried out with materials having negative permittivity or negative permeability and compared with the positive dielectric support materials. The work attempts to focus on the dispersion relation and interaction impedance as a measure to check for the feasibility of using negative materials in TWT.

  14. Effects of Transmembrane α-Helix Length and Concentration on Phase Behavior in Four-Component Lipid Mixtures: A Molecular Dynamics Study.

    PubMed

    Ackerman, David G; Feigenson, Gerald W

    2016-05-01

    We used coarse-grained molecular dynamics simulations to examine the effects of transmembrane α-helical WALP peptides on the behavior of four-component lipid mixtures. These mixtures contain a high-melting temperature (high-Tm) lipid, a nanodomain-inducing low-Tm lipid, a macrodomain-inducing low-Tm lipid and cholesterol to model the outer leaflet of cell plasma membranes. In a series of simulations, we incrementally replace the nanodomain-inducing low-Tm lipid by the macrodomain-inducing low-Tm lipid and measure how lipid and phase properties are altered by the addition of WALPs of different length. Regardless of the ratio of the two low-Tm lipids, shorter WALPs increase domain size and all WALPs increase domain alignment between the two leaflets. These effects are smallest for the longest WALP tested, and increase with increasing WALP concentration. Thus, our simulations explain the experimental observation that WALPs induce macroscopic domains in otherwise nanodomain-forming lipid-only mixtures (unpublished). Since the cell plasma membrane contains a large fraction of transmembrane proteins, these findings link the behavior of lipid-only model membranes in vitro to phase behavior in vivo. PMID:27081858

  15. Biological insertion of computationally designed short transmembrane segments

    PubMed Central

    Baeza-Delgado, Carlos; von Heijne, Gunnar; Marti-Renom, Marc A.; Mingarro, Ismael

    2016-01-01

    The great majority of helical membrane proteins are inserted co-translationally into the ER membrane through a continuous ribosome-translocon channel. The efficiency of membrane insertion depends on transmembrane (TM) helix amino acid composition, the helix length and the position of the amino acids within the helix. In this work, we conducted a computational analysis of the composition and location of amino acids in transmembrane helices found in membrane proteins of known structure to obtain an extensive set of designed polypeptide segments with naturally occurring amino acid distributions. Then, using an in vitro translation system in the presence of biological membranes, we experimentally validated our predictions by analyzing its membrane integration capacity. Coupled with known strategies to control membrane protein topology, these findings may pave the way to de novo membrane protein design. PMID:26987712

  16. End-to-end and end-to-middle interhelical interactions: new classes of interacting helix pairs in protein structures

    SciTech Connect

    Ghosh, Tarini Shankar; Chaitanya, S. Krishna; Sankararamakrishnan, Ramasubbu

    2009-10-01

    New classes of helix–helix interactions in protein structures are reported in which interactions only occur at the terminal regions or between the terminal region of one helix and the middle region of another helix. Helix–helix interactions are important for the structure, stability and function of α-helical proteins. Helices that either cross in the middle or show extensive contacts between each other, such as coiled coils, have been investigated in previous studies. Interactions between two helices can also occur only at the terminal regions or between the terminal region of one helix and the middle region of another helix. Examples of such helix pairs are found in aquaporin, H{sup +}/Cl{sup −} transporter and Bcl-2 proteins. The frequency of the occurrence of such ‘end-to-end’ (EE) and ‘end-to-middle’ (EM) helix pairs in protein structures is not known. Questions regarding the residue preferences in the interface and the mode of interhelical interactions in such helix pairs also remain unanswered. In this study, high-resolution structures of all-α proteins from the PDB have been systematically analyzed and the helix pairs that interact only in EE or EM fashion have been extracted. EE and EM helix pairs have been categorized into five classes (N–N, N–C, C–C, N–MID and C–MID) depending on the region of interaction. Nearly 13% of 5725 helix pairs belonged to one of the five classes. Analysis of single-residue propensities indicated that hydrophobic and polar residues prefer to occur in the C-terminal and N-terminal regions, respectively. Hydrophobic C-terminal interacting residues and polar N-terminal interacting residues are also highly conserved. A strong correlation exists between some of the residue properties (surface area/volume and length of side chains) and their preferences for occurring in the interface of EE and EM helix pairs. In contrast to interacting non-EE/EM helix pairs, helices in EE and EM pairs are farther apart. In these helix pairs, residues with large surface area/volume and longer side chains are preferred in the interfacial region.

  17. Binding of MgtR, a Salmonella Transmembrane Regulatory Peptide, to MgtC, a Mycobacterium tuberculosis Virulence Factor: a Structural Study

    PubMed Central

    Jean-Francois, Frantz L.; Dai, Jian; Yu, Lu; Myrick, Alissa; Rubin, Eric; Fajer, Piotr G.; Song, Likai; Zhou, Huan-Xiang; Cross, Timothy A.

    2013-01-01

    SUMMARY MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium,inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologuous to its S. Typhimurium analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhimurium MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC. PMID:24140750

  18. Shielded helix traveling wave cathode ray tube deflection structure

    DOEpatents

    Norris, Neil J.; Hudson, Charles L.

    1992-01-01

    Various embodiments of a helical coil deflection structure of a CRT are described and illustrated which provide shielding between adjacent turns of the coil on either three or four sides of each turn in the coil. Threaded members formed with either male or female threads and having the same pitch as the deflection coil are utilized for shielding the deflection coil with each turn of the helical coil placed between adjacent threads which act to shield each coil turn from adjacent turns and to confine the field generated by the coil to prevent or inhibit cross-coupling between adjacent turns of the coil to thereby prevent generation of fast fields which might otherwise deflect the beam out of time synchronization with the electron beam pulse.

  19. Shielded helix traveling wave cathode ray tube deflection structure

    DOEpatents

    Norris, N.J.; Hudson, C.L.

    1992-12-15

    Various embodiments of a helical coil deflection structure of a CRT are described and illustrated which provide shielding between adjacent turns of the coil on either three or four sides of each turn in the coil. Threaded members formed with either male or female threads and having the same pitch as the deflection coil are utilized for shielding the deflection coil with each turn of the helical coil placed between adjacent threads which act to shield each coil turn from adjacent turns and to confine the field generated by the coil to prevent or inhibit cross-coupling between adjacent turns of the coil to thereby prevent generation of fast fields which might otherwise deflect the beam out of time synchronization with the electron beam pulse. 13 figs.

  20. Mutations in the transmembrane helix S6 of domain IV confer cockroach sodium channel resistance to sodium channel blocker insecticides and local anesthetics.

    PubMed

    Jiang, Dingxin; Du, Yuzhe; Nomura, Yoshiko; Wang, Xingliang; Wu, Yidong; Zhorov, Boris S; Dong, Ke

    2015-11-01

    Indoxacarb and metaflumizone are two sodium channel blocker insecticides (SCBIs). They preferably bind to and trap sodium channels in the slow-inactivated non-conducting state, a mode of action similar to that of local anesthetics (LAs). Recently, two sodium channel mutations, F1845Y (F(4i15)Y) and V1848I (V(4i18)I), in the transmembrane segment 6 of domain IV (IVS6), were identified to be associated with indoxacarb resistance in Plutella xylostella. F(4i15) is known to be critical for the action of LAs on mammalian sodium channels. Previously, mutation F(4i15)A in a cockroach sodium channel, BgNav1-1a, has been shown to reduce the action of lidocaine, a LA, but not the action of SCBIs. In this study, we introduced mutations F(4i15)Y and V(4i18)A/I individually into the cockroach sodium channel, BgNav1-1a, and conducted functional analysis of the three mutants in Xenopus oocytes. We found that both the F(4i15)Y and V(4i18)I mutations reduced the inhibition of sodium current by indoxacarb, DCJW (an active metabolite of indoxacarb) and metaflumizone. F(4i15)Y and V(4i18)I mutations also reduced the use-dependent block of sodium current by lidocaine. In contrast, substitution V(4i18)A enhanced the action metaflumizone and lidocaine. These results show that both F(4i15)Y and V(4i18)I mutations may contribute to target-site resistance to SCBIs, and provide the first molecular evidence for common amino acid determinants on insect sodium channels involved in action of SCBIs and LA. PMID:26407935

  1. Influence of assignment on the prediction of transmembrane helices in protein structures.

    PubMed

    Pylouster, Jean; Bornot, Aurélie; Etchebest, Catherine; de Brevern, Alexandre G

    2010-11-01

    α-Helical transmembrane proteins (TMPα) are composed of a series of helices embedded in the lipid bilayer. Due to technical difficulties, few 3D structures are available. Therefore, the design of structural models of TMPα is of major interest. We study the secondary structures of TMPα by analyzing the influence of secondary structures assignment methods (SSAMs). For this purpose, a published and updated benchmark databank of TMPα is used and several SSAMs (9) are evaluated. The analysis of the results points to significant differences in SSA depending on the methods used. Pairwise comparisons between SSAMs led to more than 10% of disagreement. Helical regions corresponding to transmembrane zones are often correctly characterized. The study of the sequence-structure relationship shows very limited differences with regard to the structural disagreement. Secondary structure prediction based on Bayes' rule and using only a single sequence give correct prediction rates ranging from 78 to 81%. A structural alphabet approach gives a slightly better prediction, i.e., only 2% less than the best equivalent approach, whereas the prediction rate with a very different assignment bypasses 86%. This last result highlights the importance of the correct assignment choice to evaluate the prediction assessment. PMID:20349322

  2. Structural studies of E73 from a hyperthermophilic archaeal virus identify the RH3 domain, an elaborated ribbon-helix-helix motif involved in DNA recognition

    PubMed Central

    Schlenker, Casey; Goel, Anupam; Tripet, Brian P.; Menon, Smita; Willi, Taylor; Dlaki?, Mensur; Young, Mark J.; Lawrence, C Martin; Copi, Valrie

    2012-01-01

    Hyperthermophilic archaeal viruses including Sulfolobus spindle-shaped viruses (SSVs) such as SSV-1 and SSV-Ragged Hills exhibit remarkable morphology and genetic diversity. However, they remain poorly understood, in part because their genomes exhibit limited or unrecognizable sequence similarity to genes with known function. Here we report structural and functional studies of E73, a 73-residue homodimeric protein encoded within the SSV-Ragged Hills genome. Despite lacking significant sequence similarity, the NMR structure reveals clear similarity to ribbon-helix-helix (RHH) domains present in numerous proteins involved in transcriptional regulation. In vitro dsDNA binding experiments confirm the ability of E73 to bind dsDNA in a non-specific manner with micromolar affinity, and characterization of the K11E variant confirms the location of the predicted DNA binding surface. E73 is distinct, however, from known RHHs. The RHH motif is elaborated upon by the insertion of a third helix that is tightly integrated into the structural domain, giving rise to the RH3 fold. Within the homodimer, this helix results in the formation of a conserved, symmetric cleft distal to the DNA binding surface, where it may mediate protein-protein interactions, or contribute to the high thermal stability of E73. Analysis of backbone amide dynamics by NMR provides evidence for a rigid core, and fast ps-ns timescale NH bond vector motions for residues located within the antiparallel ?-sheet region of the proposed DNA-binding surface, and slower ?s to ms timescale motions for residues in the ?1-?2 loop. The role of E73 and its SSV homologs in the viral life cycle are discussed. PMID:22409376

  3. Structural Basis for Elastic Mechanical Properties of the DNA Double Helix.

    PubMed

    Kim, Young-Joo; Kim, Do-Nyun

    2016-01-01

    In this article, we investigate the principal structural features of the DNA double helix and their effects on its elastic mechanical properties. We develop, in the pursuit of this purpose, a helical continuum model consisting of a soft helical core and two stiff ribbons wrapping around it. The proposed model can reproduce the negative twist-stretch coupling of the helix successfully as well as its global stretching, bending, and torsional rigidities measured experimentally. Our parametric study of the model using the finite element method further reveals that the stiffness of phosphate backbones is a crucial factor for the counterintuitive overwinding behavior of the duplex and its extraordinarily high torsional rigidity, the major-minor grooves augment the twist-stretch coupling, and the change of the helicity might be responsible for the transition from a negative to a positive twist-stretching coupling when a tensile force is applied to the duplex. PMID:27055239

  4. Structural Basis for Elastic Mechanical Properties of the DNA Double Helix

    PubMed Central

    Kim, Young-Joo; Kim, Do-Nyun

    2016-01-01

    In this article, we investigate the principal structural features of the DNA double helix and their effects on its elastic mechanical properties. We develop, in the pursuit of this purpose, a helical continuum model consisting of a soft helical core and two stiff ribbons wrapping around it. The proposed model can reproduce the negative twist-stretch coupling of the helix successfully as well as its global stretching, bending, and torsional rigidities measured experimentally. Our parametric study of the model using the finite element method further reveals that the stiffness of phosphate backbones is a crucial factor for the counterintuitive overwinding behavior of the duplex and its extraordinarily high torsional rigidity, the major-minor grooves augment the twist-stretch coupling, and the change of the helicity might be responsible for the transition from a negative to a positive twist-stretching coupling when a tensile force is applied to the duplex. PMID:27055239

  5. Structure of the Membrane Anchor of Pestivirus Glycoprotein Erns, a Long Tilted Amphipathic Helix

    PubMed Central

    Aberle, Daniel; Muhle-Goll, Claudia; Bürck, Jochen; Wolf, Moritz; Reißer, Sabine; Luy, Burkhard; Wenzel, Wolfgang; Ulrich, Anne S.; Meyers, Gregor

    2014-01-01

    Erns is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the Erns membrane contact, processing and secretion. PMID:24586172

  6. Structure of the membrane anchor of pestivirus glycoprotein E(rns), a long tilted amphipathic helix.

    PubMed

    Aberle, Daniel; Muhle-Goll, Claudia; Bürck, Jochen; Wolf, Moritz; Reißer, Sabine; Luy, Burkhard; Wenzel, Wolfgang; Ulrich, Anne S; Meyers, Gregor

    2014-02-01

    E(rns) is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the E(rns) membrane contact, processing and secretion. PMID:24586172

  7. Structural investigation of the transmembrane domain of KCNE1 in proteoliposomes.

    PubMed

    Sahu, Indra D; Kroncke, Brett M; Zhang, Rongfu; Dunagan, Megan M; Smith, Hubbell J; Craig, Andrew; McCarrick, Robert M; Sanders, Charles R; Lorigan, Gary A

    2014-10-14

    KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodisq nanoparticles to directly compare the structure of the TMD in a variety of different membrane environments. Experimentally derived DEER distances coupled with simulated annealing molecular dynamic simulations were used to probe the bilayer structure of the TMD of KCNE1. The results indicate that the structure is helical in proteoliposomes and is slightly curved, which is consistent with the previously determined solution NMR structure in micelles. The evident resilience of the curvature in the KCNE1 TMD leads us to hypothesize that the curvature is likely to be maintained upon binding of the protein to the KCNQ1 channel. PMID:25234231

  8. Structural Investigation of the Transmembrane Domain of KCNE1 in Proteoliposomes

    PubMed Central

    2015-01-01

    KCNE1 is a single-transmembrane protein of the KCNE family that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in KCNE1 have been linked to diseases such as long QT syndrome (LQTS), atrial fibrillation, sudden infant death syndrome, and deafness. The transmembrane domain (TMD) of KCNE1 plays a key role in mediating the physical association with KCNQ1 and in subsequent modulation of channel gating kinetics and conductance. However, the mechanisms associated with these roles for the TMD remain poorly understood, highlighting a need for experimental structural studies. A previous solution NMR study of KCNE1 in LMPG micelles revealed a curved transmembrane domain, a structural feature proposed to be critical to KCNE1 function. However, this curvature potentially reflects an artifact of working in detergent micelles. Double electron electron resonance (DEER) measurements were conducted on KCNE1 in LMPG micelles, POPC/POPG proteoliposomes, and POPC/POPG lipodisq nanoparticles to directly compare the structure of the TMD in a variety of different membrane environments. Experimentally derived DEER distances coupled with simulated annealing molecular dynamic simulations were used to probe the bilayer structure of the TMD of KCNE1. The results indicate that the structure is helical in proteoliposomes and is slightly curved, which is consistent with the previously determined solution NMR structure in micelles. The evident resilience of the curvature in the KCNE1 TMD leads us to hypothesize that the curvature is likely to be maintained upon binding of the protein to the KCNQ1 channel. PMID:25234231

  9. Stoichiometry of lipid interactions with transmembrane proteins—Deduced from the 3D structures

    PubMed Central

    Páli, Tibor; Bashtovyy, Denys; Marsh, Derek

    2006-01-01

    The stoichiometry of the first shell of lipids interacting with a transmembrane protein is defined operationally by the population of spin-labeled lipid chains whose motion is restricted directly by the protein. Interaction stoichiometries have been determined experimentally for a wide range of α-helical integral membrane proteins by using spin-label ESR spectroscopy. Here, we determine the spatially defined number of first-shell lipids at the hydrophobic perimeter of integral membrane proteins whose 3D structure has been determined by X-ray crystallography and lipid–protein interactions characterized by spin-labeling. Molecular modeling is used to build a single shell of lipids surrounding transmembrane structures derived from the PDB. Constrained energy optimization of the protein–lipid assemblies is performed by molecular mechanics. For relatively small proteins (up to 7–12 transmembrane helices), the geometrical first shell corresponds to that defined experimentally by perturbation of the lipid-chain dynamics. For larger, multi-subunit α-helical proteins, the lipids perturbed directly by the protein may either exceed or be less in number than those that can be accommodated at the intramembranous perimeter. In these latter cases, the motionally restricted spin-labeled lipids can be augmented by intercalation, or can correspond to a specific subpopulation at the protein interface, respectively. For monomeric β-barrel proteins, the geometrical lipid stoichiometry corresponds to that determined from lipid mobility for a 22-stranded barrel, but fewer lipids are motionally restricted than can be accommodated around an eight-stranded barrel. Deviations from the geometrical first shell, in the β-barrel case, are for the smaller protein with a highly curved barrel. PMID:16641489

  10. Stoichiometry of lipid interactions with transmembrane proteins--Deduced from the 3D structures.

    PubMed

    Páli, Tibor; Bashtovyy, Denys; Marsh, Derek

    2006-05-01

    The stoichiometry of the first shell of lipids interacting with a transmembrane protein is defined operationally by the population of spin-labeled lipid chains whose motion is restricted directly by the protein. Interaction stoichiometries have been determined experimentally for a wide range of alpha-helical integral membrane proteins by using spin-label ESR spectroscopy. Here, we determine the spatially defined number of first-shell lipids at the hydrophobic perimeter of integral membrane proteins whose 3D structure has been determined by X-ray crystallography and lipid-protein interactions characterized by spin-labeling. Molecular modeling is used to build a single shell of lipids surrounding transmembrane structures derived from the PDB. Constrained energy optimization of the protein-lipid assemblies is performed by molecular mechanics. For relatively small proteins (up to 7-12 transmembrane helices), the geometrical first shell corresponds to that defined experimentally by perturbation of the lipid-chain dynamics. For larger, multi-subunit alpha-helical proteins, the lipids perturbed directly by the protein may either exceed or be less in number than those that can be accommodated at the intramembranous perimeter. In these latter cases, the motionally restricted spin-labeled lipids can be augmented by intercalation, or can correspond to a specific subpopulation at the protein interface, respectively. For monomeric beta-barrel proteins, the geometrical lipid stoichiometry corresponds to that determined from lipid mobility for a 22-stranded barrel, but fewer lipids are motionally restricted than can be accommodated around an eight-stranded barrel. Deviations from the geometrical first shell, in the beta-barrel case, are for the smaller protein with a highly curved barrel. PMID:16641489

  11. Symmetry of helicoidal biopolymers in the frameworks of algebraic geometry: α-helix and DNA structures.

    PubMed

    Samoylovich, Mikhail; Talis, Alexander

    2014-03-01

    The chain of algebraic geometry and topology constructions is mapped on a structural level that allows one to single out a special class of discrete helicoidal structures. A structure that belongs to this class is locally periodic, topologically stable in three-dimensional Euclidean space and corresponds to the bifurcation domain. Singular points of its bounding minimal surface are related by transformations determined by symmetries of the second coordination sphere of the eight-dimensional crystallographic lattice E8. These points represent cluster vertices, whose helicoid joining determines the topology and structural parameters of linear biopolymers. In particular, structural parameters of the α-helix are determined by the seven-vertex face-to-face joining of tetrahedra with the E8 non-integer helical axis 40/11 having a rotation angle of 99°, and the development of its surface coincides with the cylindrical development of the α-helix. Also, packing models have been created which determine the topology of the A, B and Z forms of DNA. PMID:24572320

  12. Sequence-structure relationship study in all-α transmembrane proteins using an unsupervised learning approach.

    PubMed

    Esque, Jérémy; Urbain, Aurélie; Etchebest, Catherine; de Brevern, Alexandre G

    2015-11-01

    Transmembrane proteins (TMPs) are major drug targets, but the knowledge of their precise topology structure remains highly limited compared with globular proteins. In spite of the difficulties in obtaining their structures, an important effort has been made these last years to increase their number from an experimental and computational point of view. In view of this emerging challenge, the development of computational methods to extract knowledge from these data is crucial for the better understanding of their functions and in improving the quality of structural models. Here, we revisit an efficient unsupervised learning procedure, called Hybrid Protein Model (HPM), which is applied to the analysis of transmembrane proteins belonging to the all-α structural class. HPM method is an original classification procedure that efficiently combines sequence and structure learning. The procedure was initially applied to the analysis of globular proteins. In the present case, HPM classifies a set of overlapping protein fragments, extracted from a non-redundant databank of TMP 3D structure. After fine-tuning of the learning parameters, the optimal classification results in 65 clusters. They represent at best similar relationships between sequence and local structure properties of TMPs. Interestingly, HPM distinguishes among the resulting clusters two helical regions with distinct hydrophobic patterns. This underlines the complexity of the topology of these proteins. The HPM classification enlightens unusual relationship between amino acids in TMP fragments, which can be useful to elaborate new amino acids substitution matrices. Finally, two challenging applications are described: the first one aims at annotating protein functions (channel or not), the second one intends to assess the quality of the structures (X-ray or models) via a new scoring function deduced from the HPM classification. PMID:26043903

  13. Investigation and direct mapping of the persistent spin helix in confined structures

    NASA Astrophysics Data System (ADS)

    Schwemmer, Markus; Weingartner, Matthias; Völkl, Roland; Oltscher, Martin; Schuh, Dieter; Bougeard, Dominique; Korn, Tobias; Schüller, Christian

    The spin-orbit field in GaAs-based quantum well (QW) structures typically consists of two different contributions: Dresselhaus and Rashba field. The geometry of the Dresselhaus field, which arises due to the bulk inversion asymmetry, is mostly determined by the growth direction of the quantum well. The Rashba field instead is caused by a structure inversion asymmetry, which can be controlled, e.g. by the modulation doping. For the specific case of a (001)-grown GaAs quantum well with equal strength of Dresselhaus and Rashba fields, the effective spin-orbit field is oriented along the in-plane [110] direction for all k values and the spin splitting for this direction vanishes. For optically injected spins, which are initially oriented perpendicular to the QW plane, a persistent spin helix (PSH) state forms. We use a femtosecond pulsed TiSa-Laser system combined with a magneto-optical Kerr effect microscope for time- and space-resolved mapping of the PSH. With this technique, we investigate the PSH behavior in confined structures, e.g., thin channels along the helix direction. Hence we find that lateral confinement increases the effective PSH lifetime drastically. In more complex structures, we observe that PSH formation is even stable under a forced direction change. Financial support by the DFG via SFB 689 and SPP 1285 is gratefully acknowledged.

  14. Structural Organization of a Full-Length Gp130/LIF-R Cytokine Receptor Transmembrane Complex

    SciTech Connect

    Skiniotis, G.; Lupardus, P.J.; Martick, M.; Walz, T.; Garcia, K.C.

    2009-05-26

    gp130 is a shared receptor for at least nine cytokines, and can signal either as a homodimer, or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-R{alpha}). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6R{alpha} hexameric complex, CNTF/CNTF-R{alpha} heterodimerizes gp130 and LIF-R via non-cooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic (EM) analysis of the full-length gp130/LIF-R/CNTF-R{alpha}/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the 'tall' class of gp130-family cytokine receptor complexes including LIF, IL-27, IL-12, and others.

  15. All-atom 3D structure prediction of transmembrane β-barrel proteins from sequences.

    PubMed

    Hayat, Sikander; Sander, Chris; Marks, Debora S; Elofsson, Arne

    2015-04-28

    Transmembrane β-barrels (TMBs) carry out major functions in substrate transport and protein biogenesis but experimental determination of their 3D structure is challenging. Encouraged by successful de novo 3D structure prediction of globular and α-helical membrane proteins from sequence alignments alone, we developed an approach to predict the 3D structure of TMBs. The approach combines the maximum-entropy evolutionary coupling method for predicting residue contacts (EVfold) with a machine-learning approach (boctopus2) for predicting β-strands in the barrel. In a blinded test for 19 TMB proteins of known structure that have a sufficient number of diverse homologous sequences available, this combined method (EVfold_bb) predicts hydrogen-bonded residue pairs between adjacent β-strands at an accuracy of ∼70%. This accuracy is sufficient for the generation of all-atom 3D models. In the transmembrane barrel region, the average 3D structure accuracy [template-modeling (TM) score] of top-ranked models is 0.54 (ranging from 0.36 to 0.85), with a higher (44%) number of residue pairs in correct strand-strand registration than in earlier methods (18%). Although the nonbarrel regions are predicted less accurately overall, the evolutionary couplings identify some highly constrained loop residues and, for FecA protein, the barrel including the structure of a plug domain can be accurately modeled (TM score = 0.68). Lower prediction accuracy tends to be associated with insufficient sequence information and we therefore expect increasing numbers of β-barrel families to become accessible to accurate 3D structure prediction as the number of available sequences increases. PMID:25858953

  16. All-atom 3D structure prediction of transmembrane β-barrel proteins from sequences

    PubMed Central

    Hayat, Sikander; Sander, Chris; Marks, Debora S.

    2015-01-01

    Transmembrane β-barrels (TMBs) carry out major functions in substrate transport and protein biogenesis but experimental determination of their 3D structure is challenging. Encouraged by successful de novo 3D structure prediction of globular and α-helical membrane proteins from sequence alignments alone, we developed an approach to predict the 3D structure of TMBs. The approach combines the maximum-entropy evolutionary coupling method for predicting residue contacts (EVfold) with a machine-learning approach (boctopus2) for predicting β-strands in the barrel. In a blinded test for 19 TMB proteins of known structure that have a sufficient number of diverse homologous sequences available, this combined method (EVfold_bb) predicts hydrogen-bonded residue pairs between adjacent β-strands at an accuracy of ∼70%. This accuracy is sufficient for the generation of all-atom 3D models. In the transmembrane barrel region, the average 3D structure accuracy [template-modeling (TM) score] of top-ranked models is 0.54 (ranging from 0.36 to 0.85), with a higher (44%) number of residue pairs in correct strand–strand registration than in earlier methods (18%). Although the nonbarrel regions are predicted less accurately overall, the evolutionary couplings identify some highly constrained loop residues and, for FecA protein, the barrel including the structure of a plug domain can be accurately modeled (TM score = 0.68). Lower prediction accuracy tends to be associated with insufficient sequence information and we therefore expect increasing numbers of β-barrel families to become accessible to accurate 3D structure prediction as the number of available sequences increases. PMID:25858953

  17. Helix 3 acts as a conformational hinge in Class A GPCR activation: An analysis of interhelical interaction energies in crystal structures.

    PubMed

    Lans, Isaias; Dalton, James A R; Giraldo, Jesús

    2015-12-01

    A collection of crystal structures of rhodopsin, β2-adrenergic and adenosine A2A receptors in active, intermediate and inactive states were selected for structural and energetic analyses to identify the changes involved in the activation/deactivation of Class A GPCRs. A set of helix interactions exclusive to either inactive or active/intermediate states were identified. The analysis of these interactions distinguished some local conformational changes involved in receptor activation, in particular, a packing between the intracellular domains of transmembrane helices H3 and H7 and a separation between those of H2 and H6. Also, differential movements of the extracellular and intracellular domains of these helices are apparent. Moreover, a segment of residues in helix H3, including residues L/I3.40 to L3.43, is identified as a key component of the activation mechanism, acting as a conformational hinge between extracellular and intracellular regions. Remarkably, the influence on the activation process of some glutamic and aspartic acidic residues and, as a consequence, the influence of variations on local pH is highlighted. Structural hypotheses that arose from the analysis of rhodopsin, β2-adrenergic and adenosine A2A receptors were tested on the active and inactive M2 muscarinic acetylcholine receptor structures and further discussed in the context of the new mechanistic insights provided by the recently determined active and inactive crystal structures of the μ-opioid receptor. Overall, the structural and energetic analyses of the interhelical interactions present in this collection of Class A GPCRs suggests the existence of a common general activation mechanism featuring a chemical space useful for drug discovery exploration. PMID:26522273

  18. A benchmark server using high resolution protein structure data, and benchmark results for membrane helix predictions

    PubMed Central

    2013-01-01

    Background Helical membrane proteins are vital for the interaction of cells with their environment. Predicting the location of membrane helices in protein amino acid sequences provides substantial understanding of their structure and function and identifies membrane proteins in sequenced genomes. Currently there is no comprehensive benchmark tool for evaluating prediction methods, and there is no publication comparing all available prediction tools. Current benchmark literature is outdated, as recently determined membrane protein structures are not included. Current literature is also limited to global assessments, as specialised benchmarks for predicting specific classes of membrane proteins were not previously carried out. Description We present a benchmark server at http://sydney.edu.au/pharmacy/sbio/software/TMH_benchmark.shtml that uses recent high resolution protein structural data to provide a comprehensive assessment of the accuracy of existing membrane helix prediction methods. The server further allows a user to compare uploaded predictions generated by novel methods, permitting the comparison of these novel methods against all existing methods compared by the server. Benchmark metrics include sensitivity and specificity of predictions for membrane helix location and orientation, and many others. The server allows for customised evaluations such as assessing prediction method performances for specific helical membrane protein subtypes. We report results for custom benchmarks which illustrate how the server may be used for specialised benchmarks. Which prediction method is the best performing method depends on which measure is being benchmarked. The OCTOPUS membrane helix prediction method is consistently one of the highest performing methods across all measures in the benchmarks that we performed. Conclusions The benchmark server allows general and specialised assessment of existing and novel membrane helix prediction methods. Users can employ this benchmark server to determine the most suitable method for the type of prediction the user needs to perform, be it general whole-genome annotation or the prediction of specific types of helical membrane protein. Creators of novel prediction methods can use this benchmark server to evaluate the performance of their new methods. The benchmark server will be a valuable tool for researchers seeking to extract more sophisticated information from the large and growing protein sequence databases. PMID:23530628

  19. Helix coupling

    DOEpatents

    Ginell, W.S.

    1982-03-17

    A coupling for connecting helix members in series, which consists of a pair of U-shaped elements, one of which is attached to each helix end with the U sections of the elements interlocked. The coupling is particularly beneficial for interconnecting helical Nitinol elements utilized in thermal actuators or engines. Each coupling half is attached to the associated helix at two points, thereby providing axial load while being easily removed from the helix, and reusable.

  20. Helix coupling

    DOEpatents

    Ginell, William S.

    1989-04-25

    A coupling for connecting helix members in series, which consists of a pair of U-shaped elements, one of which is attached to each helix end with the "U" sections of the elements interlocked. The coupling is particularly beneficial for interconnecting helical Nitinol elements utilized in thermal actuators or engines. Each coupling half is attached to the associated helix at two points, thereby providing axial load while being easily removed from the helix, and reusable.

  1. PolyprOnline: polyproline helix II and secondary structure assignment database

    PubMed Central

    Chebrek, Romain; Leonard, Sylvain; de Brevern, Alexandre G.; Gelly, Jean-Christophe

    2014-01-01

    The polyproline helix type II (PPII) is a regular protein secondary structure with remarkable features. Many studies have highlighted different crucial biological roles supported by this local conformation, e.g. in the interactions between biological macromolecules. Although PPII is less frequently present than regular secondary structures such as canonical alpha helices and beta strands, it corresponds to 3–10% of residues. Up to now, PPII is not assigned by most popular assignment tools, and therefore, remains insufficiently studied. PolyprOnline database is, therefore, dedicated to PPII structure assignment and analysis to facilitate the study of PPII structure and functional roles. This database is freely accessible from www.dsimb.inserm.fr/dsimb_tools/polyproline. PMID:25380779

  2. The relationship between insulin bioactivity and structure in the NH2-terminal A-chain helix.

    PubMed

    Olsen, H B; Ludvigsen, S; Kaarsholm, N C

    1998-11-27

    Studies of naturally occuring and chemically modified insulins have established that the NH2-terminal helix of the A-chain is important in conferring affinity in insulin-receptor interactions. Nevertheless, the three-dimensional structural basis for these observations has not previously been studied in detail. To correlate structure and function in this region of the molecule, we have used the solution structure of an engineered monomer (GluB1, GluB10, GluB16, GluB27, desB30)-insulin (4E insulin) as a template for design of A-chain mutants associated with enhanced or greatly diminished affinity for the insulin receptor. In the context of 4E insulin, the employed mutants, i.e. ThrA8-->His and ValA3-->Gly, result in species with 143% and 0.1% biological activity, respectively, relative to human insulin. The high-resolution NMR studies reveal two well-defined structures each resembling the template. However, significant structural differences are evident notably in residues A2-A8 and their immediate environment. In comparison with the template structure, the A8His mutation enhances the helical character of residues A2-A8. This structural change leads to additional exposure of a hydrophobic patch mainly consisting of species invariant residues. In contrast, the A3Gly mutation leads to stretching and disruption of the A2-A8 helix and changes both the dimensions and the access to the hydrophobic patch exposed in the more active insulins. We conclude that the mutations induce small, yet decisive structural changes that either mediate or inhibit the subtle conformational adjustments involved in the presentation of this part of the insulin pharmacophore to the receptor. PMID:9813131

  3. Characterizing diverse orthologues of the cystic fibrosis transmembrane conductance regulator protein for structural studies.

    PubMed

    Pollock, Naomi L; Rimington, Tracy L; Ford, Robert C

    2015-10-01

    As an ion channel, the cystic fibrosis transmembrane conductance regulator (CFTR) protein occupies a unique niche within the ABC family. Orthologues of CFTR are extant throughout the animal kingdom from sharks to platypods to sheep, where the osmoregulatory function of the protein has been applied to differing lifestyles and diverse organ systems. In humans, loss-of-function mutations to CFTR cause the disease cystic fibrosis, which is a significant health burden in populations of white European descent. Orthologue screening has proved fruitful in the pursuit of high-resolution structural data for several membrane proteins, and we have applied some of the princples developed in previous studies to the expression and purification of CFTR. We have overexpressed this protein, along with evolutionarily diverse orthologues, in Saccharomyces cerevisiae and developed a purification to isolate it in quantities sufficient for structural and functional studies. PMID:26517900

  4. Structure and Biophysical Properties of a Triple-Stranded Beta-Helix Comprising the Central Spike of Bacteriophage T4.

    PubMed

    Buth, Sergey A; Menin, Laure; Shneider, Mikhail M; Engel, Jürgen; Boudko, Sergei P; Leiman, Petr G

    2015-08-01

    Gene product 5 (gp5) of bacteriophage T4 is a spike-shaped protein that functions to disrupt the membrane of the target cell during phage infection. Its C-terminal domain is a long and slender β-helix that is formed by three polypeptide chains wrapped around a common symmetry axis akin to three interdigitated corkscrews. The folding and biophysical properties of such triple-stranded β-helices, which are topologically related to amyloid fibers, represent an unsolved biophysical problem. Here, we report structural and biophysical characterization of T4 gp5 β-helix and its truncated mutants of different lengths. A soluble fragment that forms a dimer of trimers and that could comprise a minimal self-folding unit has been identified. Surprisingly, the hydrophobic core of the β-helix is small. It is located near the C-terminal end of the β-helix and contains a centrally positioned and hydrated magnesium ion. A large part of the β-helix interior comprises a large elongated cavity that binds palmitic, stearic, and oleic acids in an extended conformation suggesting that these molecules might participate in the folding of the complete β-helix. PMID:26295253

  5. Structure and Biophysical Properties of a Triple-Stranded Beta-Helix Comprising the Central Spike of Bacteriophage T4

    PubMed Central

    Buth, Sergey A.; Menin, Laure; Shneider, Mikhail M.; Engel, Jürgen; Boudko, Sergei P.; Leiman, Petr G.

    2015-01-01

    Gene product 5 (gp5) of bacteriophage T4 is a spike-shaped protein that functions to disrupt the membrane of the target cell during phage infection. Its C-terminal domain is a long and slender β-helix that is formed by three polypeptide chains wrapped around a common symmetry axis akin to three interdigitated corkscrews. The folding and biophysical properties of such triple-stranded β-helices, which are topologically related to amyloid fibers, represent an unsolved biophysical problem. Here, we report structural and biophysical characterization of T4 gp5 β-helix and its truncated mutants of different lengths. A soluble fragment that forms a dimer of trimers and that could comprise a minimal self-folding unit has been identified. Surprisingly, the hydrophobic core of the β-helix is small. It is located near the C-terminal end of the β-helix and contains a centrally positioned and hydrated magnesium ion. A large part of the β-helix interior comprises a large elongated cavity that binds palmitic, stearic, and oleic acids in an extended conformation suggesting that these molecules might participate in the folding of the complete β-helix. PMID:26295253

  6. High-Resolution Modeling of Transmembrane Helical Protein Structures from Distant Homologues

    PubMed Central

    Chen, Kuang-Yui M.; Sun, Jiaming; Salvo, Jason S.; Baker, David; Barth, Patrick

    2014-01-01

    Eukaryotic transmembrane helical (TMH) proteins perform a wide diversity of critical cellular functions, but remain structurally largely uncharacterized and their high-resolution structure prediction is currently hindered by the lack of close structural homologues. To address this problem, we present a novel and generic method for accurately modeling large TMH protein structures from distant homologues exhibiting distinct loop and TMH conformations. Models of the adenosine A2AR and chemokine CXCR4 receptors were first ranked in GPCR-DOCK blind prediction contests in the receptor structure accuracy category. In a benchmark of 50 TMH protein homolog pairs of diverse topology (from 5 to 12 TMHs), size (from 183 to 420 residues) and sequence identity (from 15% to 70%), the method improves most starting templates, and achieves near-atomic accuracy prediction of membrane-embedded regions. Unlike starting templates, the models are of suitable quality for computer-based protein engineering: redesigned models and redesigned X-ray structures exhibit very similar native interactions. The method should prove useful for the atom-level modeling and design of a large fraction of structurally uncharacterized TMH proteins from a wide range of structural homologues. PMID:24854015

  7. Predicting RNA 3D structure using a coarse-grain helix-centered model

    PubMed Central

    Kerpedjiev, Peter; Höner zu Siederdissen, Christian; Hofacker, Ivo L.

    2015-01-01

    A 3D model of RNA structure can provide information about its function and regulation that is not possible with just the sequence or secondary structure. Current models suffer from low accuracy and long running times and either neglect or presume knowledge of the long-range interactions which stabilize the tertiary structure. Our coarse-grained, helix-based, tertiary structure model operates with only a few degrees of freedom compared with all-atom models while preserving the ability to sample tertiary structures given a secondary structure. It strikes a balance between the precision of an all-atom tertiary structure model and the simplicity and effectiveness of a secondary structure representation. It provides a simplified tool for exploring global arrangements of helices and loops within RNA structures. We provide an example of a novel energy function relying only on the positions of stems and loops. We show that coupling our model to this energy function produces predictions as good as or better than the current state of the art tools. We propose that given the wide range of conformational space that needs to be explored, a coarse-grain approach can explore more conformations in less iterations than an all-atom model coupled to a fine-grain energy function. Finally, we emphasize the overarching theme of providing an ensemble of predicted structures, something which our tool excels at, rather than providing a handful of the lowest energy structures. PMID:25904133

  8. Genomic structure of SAS, a member of the transmembrane 4 superfamily amplified in human sarcomas

    SciTech Connect

    Jankowski, S.A.; De Jong, P.; Meltzer, P.S.

    1995-01-20

    SAS is a recently identified member of the transmembrane 4 superfamily (TM4SF) that is frequently amplified in human sarcomas. To further its characterization and to confirm its classification, the genomic structure of the SAS gene was determined. The SAS gene covers approximately 3.2 kb of DNA. It contains six exons within its translated region, three of which are highly conserved in the TM4SF. 5{prime} to the translation start site are two putative transcription start sites, two CCAAT consensus sequences, and potential binding sites for both Sp1 and ATF transcription factors. Comparison of SAS organization to human ME491, CD9, and CD53 and murine CD53 and TAPA-1 confirms that SAS is a member of this family of genes and is consistent with the theory that these genes arose through duplication and divergent evolution. 44 refs., 4 figs., 2 tabs.

  9. Crystal Structure of the Signaling Helix Coiled-coil Domain of the b1 Subunit of the Soluble guanylyl Cyclase

    SciTech Connect

    Ma, X.; Beuve, A; van den Akker, F

    2010-01-01

    The soluble guanylyl cyclase (sGC) is a heterodimeric enzyme that, upon activation by nitric oxide, stimulates the production of the second messenger cGMP. Each sGC subunit harbor four domains three of which are used for heterodimerization: H-NOXA/H-NOBA domain, coiled-coil domain (CC), and catalytic guanylyl cyclase domain. The CC domain has previously been postulated to be part of a larger CC family termed the signaling helix (S-helix) family. Homodimers of sGC have also been observed but are not functionally active yet are likely transient awaiting their intended heterodimeric partner. To investigate the structure of the CC S-helix region, we crystallized and determined the structure of the CC domain of the sGC{beta}1 subunit comprising residues 348-409. The crystal structure was refined to 2.15 {angstrom} resolution. The CC structure of sGC{beta}1 revealed a tetrameric arrangement comprised of a dimer of CC dimers. Each monomer is comprised of a long a-helix, a turn near residue P399, and a short second a-helix. The CC structure also offers insights as to how sGC homodimers are not as stable as (functionally) active heterodimers via a possible role for inter-helix salt-bridge formation. The structure also yielded insights into the residues involved in dimerization. In addition, the CC region is also known to harbor a number of congenital and man-made mutations in both membrane and soluble guanylyl cyclases and those function-affecting mutations have been mapped onto the CC structure. This mutant analysis indicated an importance for not only certain dimerization residue positions, but also an important role for other faces of the CC dimer which might perhaps interact with adjacent domains. Our results also extend beyond guanylyl cyclases as the CC structure is, to our knowledge, the first S-helix structure and serves as a model for all S-helix containing family members.

  10. An empirical energy function for structural assessment of protein transmembrane domains.

    PubMed

    Postic, Guillaume; Ghouzam, Yassine; Gelly, Jean-Christophe

    2015-08-01

    Knowing the structure of a protein is essential to characterize its function and mechanism at the molecular level. Despite major advances in solving structures experimentally, most membrane protein native conformations remain unknown. This lack of available structures, along with the physical constraints imposed by the lipid bilayer environment, constitutes a difficulty for the modeling of membrane protein structures. Assessing the quality of membrane protein models is therefore critical. Using a non-redundant set of 66 membrane protein structures (41 alpha and 25 beta), we have developed an empirical energy function for the structural assessment of alpha-helical and beta-sheet transmembrane domains. This statistical potential quantifies the interatomic distance between residues located in the lipid bilayer. To minimize the problem of insufficient sampling, we have used kernel density estimations of the distance distributions. Following a leave-one-out cross-validation procedure, we show that our method outperforms current statistical potentials in discriminating correct from incorrect membrane protein models. Furthermore, the comparison of our distance-dependent statistical potential with one optimized on globular proteins provides insights into the rules by which residues interact within the lipid bilayer. PMID:26044650

  11. The Interaction between Influenza HA Fusion Peptide and Transmembrane Domain Affects Membrane Structure.

    PubMed

    Lai, Alex L; Freed, Jack H

    2015-12-15

    Viral glycoproteins, such as influenza hemagglutinin (HA) and human immunodeficiency virus gp41, are anchored by a single helical segment transmembrane domain (TMD) on the viral envelope membrane. The fusion peptides (FP) of the glycoproteins insert into the host membrane and initiate membrane fusion. Our previous study showed that the FP or TMD alone perturbs membrane structure. Interaction between the influenza HA FP and TMD has previously been shown, but its role is unclear. We used PC spin labels dipalmitoylphospatidyl-tempo-choline (on the headgroup), 5PC and 14PC (5-C and 14-C positions on the acyl chain) to detect the combined effect of FP-TMD interaction by titrating HA FP to TMD-reconstituted 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol)/cholesterol lipid bilayers using electron spin resonance. We found that the FP-TMD increases the lipid order at all positions, which has a greater lipid ordering effect than the sum of the FP or TMD alone, and this effect reaches deeper into the membranes. Although HA-mediated membrane fusion is pH dependent, this combined effect is observed at both pH 5 and pH 7. In addition to increasing lipid order, multiple components are found for 5PC at increased concentration of FP-TMD, indicating that distinct domains are induced. However, the mutation of Gly1 in the FP and L187 in the TMD eliminates the perturbations, consistent with their fusogenic phenotypes. Electron spin resonance on spin-labeled peptides confirms these observations. We suggest that this interaction may provide a driving force in different stages of membrane fusion: initialization, transition from hemifusion stalk to transmembrane contact, and fusion pore formation. PMID:26682811

  12. Structures of Two Arabidopsis thaliana Major Latex Proteins Represent Novel Helix-Grip Folds

    PubMed Central

    Lytle, Betsy L.; Song, Jikui; de la Cruz, Norberto B.; Peterson, Francis C.; Johnson, Kenneth A.; Bingman, Craig A.; Phillips, George N.; Volkman, Brian F.

    2010-01-01

    The major latex proteins (MLP) are a protein family first identified in the latex of opium poppy. They are found only in plants and have 24 identified members in Arabidopsis alone as well as in other plants such as peach, strawberry, melon, cucumber, and soybean. While the function of the MLPs is unknown, they have been associated with fruit and flower development and in pathogen defense responses. Based on modest sequence similarity, they have been characterized as members of the Bet v 1 protein superfamily; however, no structures have yet been reported. As part of an ongoing structural genomics effort, we determined the structures of two Arabidopsis thaliana MLPs: the solution structure of MLP28 (gene product of At1g70830.1) and the crystal structure of At1g24000.1. The structures revealed distinct differences when compared to one another and to the typical Bet v 1 fold. Nevertheless, NMR titration experiments demonstrated that the characteristic Bet v 1 hydrophobic binding pocket of At1g24000.1 is able to bind a ligand, suggesting that it plays a role in the function of the MLPs. A structure-based sequence analysis identified conserved hydrophobic residues in the long alpha helix that contribute to the binding cavity and may specify preferred ligands for the MLP family. PMID:19326460

  13. EspR, a key regulator of Mycobacterium tuberculosis virulence, adopts a unique dimeric structure among helix-turn-helix proteins

    PubMed Central

    Rosenberg, Oren S.; Dovey, Cole; Tempesta, Michael; Robbins, Rebecca A.; Finer-Moore, Janet S.; Stroud, Robert M.; Cox, Jeffery S.

    2011-01-01

    EspR is a transcriptional regulator that activates the ESX-1 secretion system during Mycobacterium tuberculosis infection and is critical for pathogenesis. It is unique among DNA-binding proteins as it is secreted as part of a feedback regulatory loop that serves to mitigate transcriptional activity. Here we report the crystal structure of a functional EspR dimer at 2.5-Å resolution. The amino-terminal half of EspR is a helix-turn-helix (HTH) DNA-binding domain and the carboxy terminus consists of a dimerization domain with similarity to the SinR:SinI sporulation regulator of Bacillus subtilis. Surprisingly, the HTH domains of EspR are arranged in an unusual conformation in which they are splayed at an oblique angle to each other, suggesting that EspR binds DNA in a profoundly different way than most other known HTH regulators. By mapping the EspR binding sites in the espACD promoter, using both in vivo and in vitro binding assays, we show that the EspR operators are located unusually far from the promoter. The EspR dimer binds to these sites cooperatively, but the two “half-sites” contacted by each DNA recognition motif are separated by 177 base pairs. The distinctive structure of EspR and the exceptional arrangement of its operator contacts suggest that it could promote DNA looping in its target promoter. We hypothesize that direct DNA looping mediated by single-site binding of each EspR monomer may facilitate transcriptional control of this important virulence system. PMID:21795602

  14. Crystal structures of CusC review conformational changes accompanying folding and transmembrane channel formation

    PubMed Central

    Lei, Hsiang-Ting; Bolla, Jani Reddy; Su, Chih-Chia; Yu, Edward W.

    2016-01-01

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes in the resistance-nodulation-division (RND) family to expel diverse toxic compounds from the cell. These complexes span both the inner and outer membranes of the bacterium via an α-helical, inner membrane transporter; a periplasmic membrane fusion protein; and a β-barrel, outer membrane channel. One such efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. To remove these toxic ions, the CusC outer membrane channel must form a β-barrel structural domain, which creates a pore and spans the entire outer membrane. We here report the crystal structures of wild-type CusC as well as two CusC mutants, suggesting that the first N-terminal cysteine residue plays an important role in protein-membrane interactions and is critical for the insertion of this channel protein into the outer membrane. These structures provide insight into the mechanisms on CusC folding and transmembrane channel formation. It is also found that the interactions between CusC and membrane may be crucial for controlling the opening and closing of this β-barrel, outer membrane channel. PMID:24099674

  15. Transmembrane TNF-α: structure, function and interaction with anti-TNF agents

    PubMed Central

    Mitoma, Hiroki; Harashima, Shin-ichi; Tsukamoto, Hiroshi; Shimoda, Terufumi

    2010-01-01

    Transmembrane TNF-α, a precursor of the soluble form of TNF-α, is expressed on activated macrophages and lymphocytes as well as other cell types. After processing by TNF-α-converting enzyme (TACE), the soluble form of TNF-α is cleaved from transmembrane TNF-α and mediates its biological activities through binding to Types 1 and 2 TNF receptors (TNF-R1 and -R2) of remote tissues. Accumulating evidence suggests that not only soluble TNF-α, but also transmembrane TNF-α is involved in the inflammatory response. Transmembrane TNF-α acts as a bipolar molecule that transmits signals both as a ligand and as a receptor in a cell-to-cell contact fashion. Transmembrane TNF-α on TNF-α-producing cells binds to TNF-R1 and -R2, and transmits signals to the target cells as a ligand, whereas transmembrane TNF-α also acts as a receptor that transmits outside-to-inside (reverse) signals back to the cells after binding to its native receptors. Anti-TNF agents infliximab, adalimumab and etanercept bind to and neutralize soluble TNF-α, but exert different effects on transmembrane TNF-α-expressing cells (TNF-α-producing cells). In the clinical settings, these three anti-TNF agents are equally effective for RA, but etanercept is not effective for granulomatous diseases. Moreover, infliximab induces granulomatous infections more frequently than etanercept. Considering the important role of transmembrane TNF-α in granulomatous inflammation, reviewing the biology of transmembrane TNF-α and its interaction with anti-TNF agents will contribute to understanding the bases of differential clinical efficacy of these promising treatment modalities. PMID:20194223

  16. Delineation of chicken cathepsin L secondary structure; relationship between pH dependence activity and helix content.

    PubMed

    Dufour, E; Dive, V; Toma, F

    1988-06-29

    The secondary structure of the recently sequenced chicken liver cathepsin L (EC 3.4.22.15) has been studied both by circular dichroism and a predictive method. The structural data provided by these approaches allow us to underline the extent of the structural similarities between cathepsin L and papain, one of the best known proteins in the cysteine proteinase family. The predictive method of Garnier et al. (J. Mol. Biol. 120 (1978) 97-120) is used to locate alpha-helix and beta-sheet segments in the cathepsin L sequence. An optimization of decision constants has been performed, using circular dichroism data, to improve good predictions. The combination of these approaches lead us to suggest that the location of ordered structures observed in papain is maintained in cathepsin L, but with an additional alpha-helix in the middle region (residues 85-108) of cathepsin L. Furthermore, we show that cathepsin L inactivation at neutral pH is correlated to the lost of alpha-helix content (40% at pH 5.8 and 17% at pH 7.0) in this protein. It appears that such an effect can be related to the change in the ionization state of histidine side-chains which are shown to be mainly located in the predicted alpha-helix regions. PMID:3382672

  17. Internal structure and visualization of transmembrane domains of the RyR1 calcium release channel by cryo‐EM

    PubMed Central

    Samsó, Montserrat; Wagenknecht, Terence; Allen, PD

    2007-01-01

    RyR1 is an intracellular calcium channel with a central role in muscle contraction. We obtained a three‐dimensional reconstruction of the RyR1 in the closed state at a nominal resolution of ∼10 Å using cryo‐EM. The cytoplasmic assembly consists of a series of interconnected tubular structures that merge into four columns that extend into the transmembrane assembly. The transmembrane assembly, which has at least six transmembrane α‐helices per monomer, has four tilted rods that can be fitted with the inner helices of a closed K+ channel atomic structure. The rods splay out at the lumenal side and converge into a dense ring at the cytoplasmic side. Another set of four rods emerges from this ring and shapes the inner part of the four columns. The resulting constricted axial structure provides direct continuity between cytoplasmic and transmembrane assemblies, and a possible mechanism for control of channel gating through conformational changes in the cytoplasmic assembly. PMID:15908964

  18. Interface connections of a transmembrane voltage sensor

    PubMed Central

    Freites, J. Alfredo; Tobias, Douglas J.; von Heijne, Gunnar; White, Stephen H.

    2005-01-01

    Voltage-sensitive ion channels open and close in response to changes in transmembrane (TM) potential caused by the motion of the S4 voltage sensors. These sensors are α–helices that include four or more positively charged amino acids, most commonly arginine. The so-called paddle model, based on the high-resolution structure of the KvAP K+ channel [Jiang, et al. (2003) Nature 423, 33–41], posits that the S4 sensors move within the membrane bilayer in response to TM voltage changes. Direct exposure of S4 sensors to lipid is contrary to the classical expectation that the dielectric contrast between the membrane hydrocarbon core and water presents an insurmountable energetic penalty to burial of electric charges. Nevertheless, recent experiments have shown that a helix with the sequence of KvAP S4 can be inserted across the endoplasmic reticulum membrane. To reconcile this result with the classical energetics argument, we have carried out a molecular dynamics simulation of an isolated TM S4 helix in a lipid bilayer. The simulation reveals a stabilizing hydrogen-bonded network of water and lipid phosphates around the arginines that reduces the effective thickness of the bilayer hydrocarbon core to ≈10Åinthe vicinity of the helix. It suggests that bilayer phospholipids can adapt locally to strongly perturbing protein elements, causing the phospholipids to become a structural extension of the protein. PMID:16217012

  19. Probabilistic grammatical model for helix‐helix contact site classification

    PubMed Central

    2013-01-01

    Background Hidden Markov Models power many state‐of‐the‐art tools in the field of protein bioinformatics. While excelling in their tasks, these methods of protein analysis do not convey directly information on medium‐ and long‐range residue‐residue interactions. This requires an expressive power of at least context‐free grammars. However, application of more powerful grammar formalisms to protein analysis has been surprisingly limited. Results In this work, we present a probabilistic grammatical framework for problem‐specific protein languages and apply it to classification of transmembrane helix‐helix pairs configurations. The core of the model consists of a probabilistic context‐free grammar, automatically inferred by a genetic algorithm from only a generic set of expert‐based rules and positive training samples. The model was applied to produce sequence based descriptors of four classes of transmembrane helix‐helix contact site configurations. The highest performance of the classifiers reached AUCROC of 0.70. The analysis of grammar parse trees revealed the ability of representing structural features of helix‐helix contact sites. Conclusions We demonstrated that our probabilistic context‐free framework for analysis of protein sequences outperforms the state of the art in the task of helix‐helix contact site classification. However, this is achieved without necessarily requiring modeling long range dependencies between interacting residues. A significant feature of our approach is that grammar rules and parse trees are human‐readable. Thus they could provide biologically meaningful information for molecular biologists. PMID:24350601

  20. Transmembrane and Juxtamembrane Structure of αL Integrin in Bicelles

    PubMed Central

    Millet, Oscar; Diercks, Tammo; Torres, Jaume

    2013-01-01

    The accepted model for the interaction of α and β integrins in the transmembrane (TM) domain is based on the pair αIIbβ3. This involves the so-called outer and inner membrane association clasps (OMC and IMC, respectively). In the α chain, the OMC involves a GxxxG-like motif, whereas in the IMC a conserved juxtamembrane GFFKR motif experiences a backbone reversal that partially fills the void generated by TM separation towards the cytoplasmic half. However, the GFFKR motif of several α integrin cytoplasmic tails in non-bicelle environments has been shown to adopt an α-helical structure that is not membrane-embedded and which was shown to bind a variety of cytoplasmic proteins. Thus it is not known if a membrane-embedded backbone reversal is a conserved structural feature in α integrins. We have studied the system αLβ2 because of its importance in leukocytes, where integrin deactivation is particularly important. Herein we show that the backbone reversal feature is not only present in αIIb but also in αL-TM when reconstituted in bicelles. Additionally, titration with β2 TM showed eight residues clustering along one side of αL-TM, forming a plausible interacting face with β2. The latter orientation is consistent with a previously predicted reported polar interaction between αL Ser-1071 and β2 Thr-686. PMID:24069290

  1. Structure modulation of helix 69 from Escherichia coli 23S ribosomal RNA by pseudouridylations

    PubMed Central

    Jiang, Jun; Aduri, Raviprasad; Chow, Christine S.; SantaLucia, John

    2014-01-01

    Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (?) modifications clustered in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of ? on the conformations of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal the structural differences between H69 with (???) and without (UUU) ? modifications. Comparison of the two structures shows that H69 ??? has the following unique features: (i) the loop region is closed by a WatsonCrick base pair between ?1911 and A1919, which is potentially reinforced by interactions involving ?1911N1H and (ii) ? modifications at loop residues 1915 and 1917 promote base stacking from ?1915 to A1918. In contrast, the H69 UUU loop region, which lacks ? modifications, is less organized. Structure modulation by ? leads to alteration in conformational behavior of the 5' half of the H69 loop region, observed as broadening of C1914 non-exchangeable base proton resonances in the H69 ??? nuclear magnetic resonance spectra, and plays an important biological role in establishing the ribosomal intersubunit bridge B2a and mediating translational fidelity. PMID:24371282

  2. Mimicry of ice structure by surface hydroxyls and water of a beta-helix antifreeze protein.

    PubMed

    Liou, Y C; Tocilj, A; Davies, P L; Jia, Z

    2000-07-20

    Insect antifreeze proteins (AFP) are much more effective than fish AFPs at depressing solution freezing points by ice-growth inhibition. AFP from the beetle Tenebrio molitor is a small protein (8.4 kDa) composed of tandem 12-residue repeats (TCTxSxxCxxAx). Here we report its 1.4-A resolution crystal structure, showing that this repetitive sequence translates into an exceptionally regular beta-helix. Not only are the 12-amino-acid loops almost identical in the backbone, but also the conserved side chains are positioned in essentially identical orientations, making this AFP perhaps the most regular protein structure yet observed. The protein has almost no hydrophobic core but is stabilized by numerous disulphide and hydrogen bonds. On the conserved side of the protein, threonine-cysteine-threonine motifs are arrayed to form a flat beta-sheet, the putative ice-binding surface. The threonine side chains have exactly the same rotameric conformation and the spacing between OH groups is a near-perfect match to the ice lattice. Together with tightly bound co-planar external water, three ranks of oxygen atoms form a two-dimensional array, mimicking an ice section. PMID:10917536

  3. The simulation study of protein-protein interfaces based on the 4-helix bundle structure

    NASA Astrophysics Data System (ADS)

    Fukuda, Masaki; Komatsu, Yu; Morikawa, Ryota; Miyakawa, Takeshi; Takasu, Masako; Akanuma, Satoshi; Yamagishi, Akihiko

    2013-02-01

    Docking of two protein molecules is induced by intermolecular interactions. Our purposes in this study are: designing binding interfaces on the two proteins, which specifically interact to each other; and inducing intermolecular interactions between the two proteins by mixing them. A 4-helix bundle structure was chosen as a scaffold on which binding interfaces were created. Based on this scaffold, we designed binding interfaces involving charged and nonpolar amino acid residues. We performed molecular dynamics (MD) simulation to identify suitable amino acid residues for the interfaces. We chose YciF protein as the scaffold for the protein-protein docking simulation. We observed the structure of two YciF protein molecules (I and II), and we calculated the distance between centroids (center of gravity) of the interfaces' surface planes of the molecules I and II. We found that the docking of the two protein molecules can be controlled by the number of hydrophobic and charged amino acid residues involved in the interfaces. Existence of six hydrophobic and five charged amino acid residues within an interface were most suitable for the protein-protein docking.

  4. Structural and functional analysis of transmembrane segment IV of the salt tolerance protein Sod2.

    PubMed

    Ullah, Asad; Kemp, Grant; Lee, Brian; Alves, Claudia; Young, Howard; Sykes, Brian D; Fliegel, Larry

    2013-08-23

    Sod2 is the plasma membrane Na(+)/H(+) exchanger of the fission yeast Schizosaccharomyces pombe. It provides salt tolerance by removing excess intracellular sodium (or lithium) in exchange for protons. We examined the role of amino acid residues of transmembrane segment IV (TM IV) ((126)FPQINFLGSLLIAGCITSTDPVLSALI(152)) in activity by using alanine scanning mutagenesis and examining salt tolerance in sod2-deficient S. pombe. Two amino acids were critical for function. Mutations T144A and V147A resulted in defective proteins that did not confer salt tolerance when reintroduced into S. pombe. Sod2 protein with other alanine mutations in TM IV had little or no effect. T144D and T144K mutant proteins were inactive; however, a T144S protein was functional and provided lithium, but not sodium, tolerance and transport. Analysis of sensitivity to trypsin indicated that the mutations caused a conformational change in the Sod2 protein. We expressed and purified TM IV (amino acids 125-154). NMR analysis yielded a model with two helical regions (amino acids 128-142 and 147-154) separated by an unwound region (amino acids 143-146). Molecular modeling of the entire Sod2 protein suggested that TM IV has a structure similar to that deduced by NMR analysis and an overall structure similar to that of Escherichia coli NhaA. TM IV of Sod2 has similarities to TM V of the Zygosaccharomyces rouxii Na(+)/H(+) exchanger and TM VI of isoform 1 of mammalian Na(+)/H(+) exchanger. TM IV of Sod2 is critical to transport and may be involved in cation binding or conformational changes of the protein. PMID:23836910

  5. Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure.

    PubMed

    Banerjee, Saikat; Shi, Heliang; Habte, Habtom H; Qin, Yali; Cho, Michael W

    2016-03-01

    The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; (671)NWFDITNWLWYIK(683)) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-∆10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies. PMID:26803471

  6. Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation.

    PubMed

    Ghosh, Dhiman; Singh, Pradeep K; Sahay, Shruti; Jha, Narendra Nath; Jacob, Reeba S; Sen, Shamik; Kumar, Ashutosh; Riek, Roland; Maji, Samir K

    2015-01-01

    Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases. PMID:25784353

  7. Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation

    PubMed Central

    Ghosh, Dhiman; Singh, Pradeep K.; Sahay, Shruti; Jha, Narendra Nath; Jacob, Reeba S.; Sen, Shamik; Kumar, Ashutosh; Riek, Roland; Maji, Samir K.

    2015-01-01

    Mechanistic understanding of nucleation dependent polymerization by α-synuclein (α-Syn) into toxic oligomers and amyloids is important for the drug development against Parkinson's disease. However the structural and morphological characterization during nucleation and subsequent fibrillation process of α-Syn is not clearly understood. Using a variety of complementary biophysical techniques monitoring entire pathway of nine different synucleins, we found that transition of unstructured conformation into β-sheet rich fibril formation involves helix-rich intermediates. These intermediates are common for all aggregating synucleins, contain high solvent-exposed hydrophobic surfaces, are cytotoxic to SHSY-5Y cells and accelerate α-Syn aggregation efficiently. A multidimensional NMR study characterizing the intermediate accompanied with site-specific fluorescence study suggests that the N-terminal and central portions mainly participate in the helix-rich intermediate formation while the C-terminus remained in an extended conformation. However, significant conformational transitions occur at the middle and at the C-terminus during helix to β-sheet transition as evident from Trp fluorescence study. Since partial helix-rich intermediates were also observed for other amyloidogenic proteins such as Aβ and IAPP, we hypothesize that this class of intermediates may be one of the important intermediates for amyloid formation pathway by many natively unstructured protein/peptides and represent a potential target for drug development against amyloid diseases. PMID:25784353

  8. Occurrence, solution structure and stability of DNA hairpins stabilized by a GA/CG helix unit.

    PubMed Central

    Sandusky, P; Wooten, E W; Kurochkin, A V; Kavanaugh, T; Mandecki, W; Zuiderweg, E R

    1995-01-01

    The occurrence and NMR solution structure of a class of biloop hairpins containing the sequence 5'-CGXYAG are presented. These hairpins, which are variations on a sequence found in the reverse transcript of the human T-cell leukemia virus 2 (HLV2), show elevated melting points and high chemical stability toward denaturation by urea. Hairpins with the 5'-CGXYAG configuration have melting points 18-20 degrees higher than hairpins with 5'-CAXYGG or 5'-GGXYAC configurations. The identities of the looping bases, X and Y above, play a negligible role in determining the stability of this DNA hairpin stability. This is very different from G-A based loops in RNA, where the third base must be a purine for high stability [the GNRA loops; V.P. Antao, S.Y. Lai and I. Tinoco, Jr (1991) Nucleic Acids Res., 19, 5901-5905]. We show that these properties are associated with a four base helix unit that contains both a sheared GA base pair and a Watson-Crick CG base pair upon which it is stacked. As an understanding of the significance of AG base pairs has become increasingly important in the structural biology of nucleic acids, we compute an 0.7-0.9 A precision ensemble of NMR solution structures using iterative relaxation matrix methods. Calculations performed on NMR-derived structures indicate that neither base-base electrostatic interactions, nor base-solvent dispersive interactions, are significant factors in determining the observed differences in hairpin stability. Thus the stability of the 5'-CGXYAG configuration would appear to derive from favorable base-base London/van der Waals interactions. Images PMID:8524666

  9. Crystal Structures of the Response Regulator DosR From Mycobacterium Tuberculosis Suggest a Helix Rearrangement Mechanism for Phosphorylation Activation

    SciTech Connect

    Wisedchaisri, G.; Wu, M.; Sherman, D.R.; Hol, W.G.J.

    2009-05-26

    The response regulator DosR is essential for promoting long-term survival of Mycobacterium tuberculosis under low oxygen conditions in a dormant state and may be responsible for latent tuberculosis in one-third of the world's population. Here, we report crystal structures of full-length unphosphorylated DosR at 2.2 {angstrom} resolution and its C-terminal DNA-binding domain at 1.7 {angstrom} resolution. The full-length DosR structure reveals several features never seen before in other response regulators. The N-terminal domain of the full-length DosR structure has an unexpected ({beta}{alpha}){sub 4} topology instead of the canonical ({beta}{alpha}){sub 5} fold observed in other response regulators. The linker region adopts a unique conformation that contains two helices forming a four-helix bundle with two helices from another subunit, resulting in dimer formation. The C-terminal domain in the full-length DosR structure displays a novel location of helix {alpha}10, which allows Gln199 to interact with the catalytic Asp54 residue of the N-terminal domain. In contrast, the structure of the DosR C-terminal domain alone displays a remarkable unstructured conformation for helix {alpha}10 residues, different from the well-defined helical conformations in all other known structures, indicating considerable flexibility within the C-terminal domain. Our structures suggest a mode of DosR activation by phosphorylation via a helix rearrangement mechanism.

  10. Myriad Triple-Helix-Forming Structures in the Transposable Element RNAs of Plants and Fungi.

    PubMed

    Tycowski, Kazimierz T; Shu, Mei-Di; Steitz, Joan A

    2016-05-10

    The ENE (element for nuclear expression) is a cis-acting RNA structure that protects viral or cellular noncoding RNAs (ncRNAs) from nuclear decay through triple-helix formation with the poly(A) tail or 3'-terminal A-rich tract. We expanded the roster of nine known ENEs by bioinformatic identification of ∼200 distinct ENEs that reside in transposable elements (TEs) of numerous non-metazoan and one fish species and in four Dicistrovirus genomes. Despite variation within the ENE core, none of the predicted triple-helical stacks exceeds five base triples. Increased accumulation of reporter transcripts in human cells demonstrated functionality for representative ENEs. Location close to the poly(A) tail argues that ENEs are active in TE transcripts. Their presence in intronless, but not intron-containing, hAT transposase genes supports the idea that TEs acquired ENEs to counteract the RNA-destabilizing effects of intron loss, a potential evolutionary consequence of TE horizontal transfer in organisms that couple RNA silencing to splicing deficits. PMID:27134163

  11. Myriad Triple-Helix-Forming Structures in the Transposable Element RNAs of Plants and Fungi

    PubMed Central

    Tycowski, Kazimierz T.; Shu, Mei-Di; Steitz, Joan A.

    2016-01-01

    SUMMARY The ENE (element for nuclear expression) is a cis-acting RNA structure that protects viral or cellular noncoding (nc)RNAs from nuclear decay through triple-helix formation with the poly(A) tail or 3′-terminal A-rich tract. We expanded the roster of 9 known ENEs by bioinformatic identification of ~200 distinct ENEs that reside in transposable elements (TEs) of numerous non-metazoan and one fish species, and in four Dicistrovirus genomes. Despite variation within the ENE core, none of the predicted triple-helical stacks exceeds five base triples. Increased accumulation of reporter transcripts in human cells demonstrated functionality for representative ENEs. Location close to the poly(A) tail argues that ENEs are active in TE transcripts. Their presence in intronless but not intron-containing hAT transposase genes supports the idea that TEs acquired ENEs to counteract the RNA-destabilizing effects of intron loss, a potential evolutionary consequence of TE horizontal transfer in organisms that couple RNA silencing to splicing deficits. PMID:27134163

  12. Flanking Polyproline Sequences Inhibit [beta]-Sheet Structure in Polyglutamine Segments by Inducing PPII-like Helix Structure

    SciTech Connect

    Darnell, Gregory; Orgel, Joseph P.R.O.; Pahl, Reinhard; Meredith, Stephen C.

    2008-06-24

    Polyglutamine (poly(Q)) expansion is associated with protein aggregation into {beta}-sheet amyloid fibrils and neuronal cytotoxicity. In the mutant poly(Q) protein huntingtin, associated with Huntington's disease, both aggregation and cytotoxicity may be abrogated by a polyproline (poly(P)) domain flanking the C terminus of the poly(Q) region. To understand structural changes that may occur with the addition of the poly(P) sequence, we synthesized poly(Q) peptides with 3-15 glutamine residues and a corresponding set of poly(Q) peptides flanked on the C terminus by 11 proline residues (poly(Q)-poly(P)), as occurs in the huntingtin sequence. The shorter soluble poly(Q) peptides (three or six glutamine residues) showed polyproline type II-like (PPII)-like helix conformation when examined by circular dichroism spectroscopy and were monomers as judged by size-exclusion chromatography (SEC), while the longer poly(Q) peptides (nine or 15 glutamine residues) showed a {beta}-sheet conformation by CD and defined oligomers by SEC. Soluble poly(Q)-poly(P) peptides showed PPII-like content but SEC showed poorly defined, overlapping oligomeric peaks, and as judged by CD these peptides retained significant PPII-like structure with increasing poly(Q) length. More importantly, addition of the poly(P) domain increased the threshold for fibril formation to {approx} 15 glutamine residues. X-ray diffraction, electron microscopy, and film CD showed that, while poly(Q) peptides with {ge} 6 glutamine residues formed {beta}-sheet-rich fibrils, only the longest poly(Q)-poly(P) peptide (15 glutamine residues) did so. From these and other observations, we propose that poly(Q) domains exist in a 'tug-of-war' between two conformations, a PPII-like helix and a {beta}-sheet, while the poly(P) domain is conformationally constrained into a proline type II helix (PPII). Addition of poly(P) to the C terminus of a poly(Q) domain induces a PPII-like structure, which opposes the aggregation-prone {beta}-sheet. These structural observations may shed light on the threshold phenomenon of poly(Q) aggregation, and support the hypothesized evolution of 'protective' poly(P) tracts adjacent to poly(Q) aggregation domains.

  13. The Membrane- and Soluble-Protein Helix-Helix Interactome: Similar Geometry via Different Interactions

    PubMed Central

    Zhang, Shao-Qing; Kulp, Daniel W.; Schramm, Chaim A.; Mravic, Marco; Samish, Ilan; DeGrado, William F.

    2015-01-01

    Summary Alpha-helices are a basic unit of protein secondary structure and therefore the interaction between helices is crucial to understanding tertiary and higher-order folds. Comparing subtle variations in the structural and sequence motifs between membrane and soluble proteins sheds light on the different constraints faced by each environment and elucidates the complex puzzle of membrane protein folding. Here, we demonstrate that membrane and water-soluble helix pairs share a small number of similar folds with various interhelical distances. The composition of the residues that pack at the interface between corresponding motifs shows that hydrophobic residues tend to be more enriched in the water-soluble class of structures and small residues in the transmembrane class. The latter group facilitates packing via sidechain- and backbone-mediated hydrogen bonds within the low-dielectric membrane milieu. The helix-helix interactome space, with its associated sequence preferences and accompanied hydrogen-bonding patterns, should be useful for protein structure engineering, prediction and design. PMID:25703378

  14. Structures of OppA and PstS from Yersinia pestis indicate variability of interactions with transmembrane domains.

    PubMed

    Tanabe, Mikio; Mirza, Osman; Bertrand, Thomas; Atkins, Helen S; Titball, Richard W; Iwata, So; Brown, Katherine A; Byrne, Bernadette

    2007-11-01

    Bacterial ATP-binding cassette (ABC) transport systems couple ATP hydrolysis with the uptake and efflux of a wide range of substances across bacterial membranes. These systems are comprised of transmembrane domains, nucleotide binding domains and, in the case of uptake systems, periplasmic binding proteins responsible for binding and presentation of substrate to the transmembrane domains. In pathogenic bacteria, ABC systems are known to play roles in virulence and pathogenicity and the surface localization of some components has made them attractive targets for both vaccine and anti-infective development. Here, the crystallization of five proteins (OppA, PstS, PiuA, YrbD and CysP) from Yersinia pestis, the causative agent of plague, are reported that diffracted to resolution limits ranging from 1.6 to 5 A. The first crystal structures of ABC system components from Y. pestis, OppA and PstS, are also reported here as complexes with their substrates. Comparisons of these two structures with known structures of related proteins suggest that these proteins possess versatility in substrate recognition and variations in protein-protein interactions with their cognate transmembrane domains. PMID:18007034

  15. Structure-Based Sequence Alignment of the Transmembrane Domains of All Human GPCRs: Phylogenetic, Structural and Functional Implications

    PubMed Central

    Cvicek, Vaclav; Goddard, William A.; Abrol, Ravinder

    2016-01-01

    The understanding of G-protein coupled receptors (GPCRs) is undergoing a revolution due to increased information about their signaling and the experimental determination of structures for more than 25 receptors. The availability of at least one receptor structure for each of the GPCR classes, well separated in sequence space, enables an integrated superfamily-wide analysis to identify signatures involving the role of conserved residues, conserved contacts, and downstream signaling in the context of receptor structures. In this study, we align the transmembrane (TM) domains of all experimental GPCR structures to maximize the conserved inter-helical contacts. The resulting superfamily-wide GpcR Sequence-Structure (GRoSS) alignment of the TM domains for all human GPCR sequences is sufficient to generate a phylogenetic tree that correctly distinguishes all different GPCR classes, suggesting that the class-level differences in the GPCR superfamily are encoded at least partly in the TM domains. The inter-helical contacts conserved across all GPCR classes describe the evolutionarily conserved GPCR structural fold. The corresponding structural alignment of the inactive and active conformations, available for a few GPCRs, identifies activation hot-spot residues in the TM domains that get rewired upon activation. Many GPCR mutations, known to alter receptor signaling and cause disease, are located at these conserved contact and activation hot-spot residue positions. The GRoSS alignment places the chemosensory receptor subfamilies for bitter taste (TAS2R) and pheromones (Vomeronasal, VN1R) in the rhodopsin family, known to contain the chemosensory olfactory receptor subfamily. The GRoSS alignment also enables the quantification of the structural variability in the TM regions of experimental structures, useful for homology modeling and structure prediction of receptors. Furthermore, this alignment identifies structurally and functionally important residues in all human GPCRs. These residues can be used to make testable hypotheses about the structural basis of receptor function and about the molecular basis of disease-associated single nucleotide polymorphisms. PMID:27028541

  16. Structure-Based Sequence Alignment of the Transmembrane Domains of All Human GPCRs: Phylogenetic, Structural and Functional Implications.

    PubMed

    Cvicek, Vaclav; Goddard, William A; Abrol, Ravinder

    2016-03-01

    The understanding of G-protein coupled receptors (GPCRs) is undergoing a revolution due to increased information about their signaling and the experimental determination of structures for more than 25 receptors. The availability of at least one receptor structure for each of the GPCR classes, well separated in sequence space, enables an integrated superfamily-wide analysis to identify signatures involving the role of conserved residues, conserved contacts, and downstream signaling in the context of receptor structures. In this study, we align the transmembrane (TM) domains of all experimental GPCR structures to maximize the conserved inter-helical contacts. The resulting superfamily-wide GpcR Sequence-Structure (GRoSS) alignment of the TM domains for all human GPCR sequences is sufficient to generate a phylogenetic tree that correctly distinguishes all different GPCR classes, suggesting that the class-level differences in the GPCR superfamily are encoded at least partly in the TM domains. The inter-helical contacts conserved across all GPCR classes describe the evolutionarily conserved GPCR structural fold. The corresponding structural alignment of the inactive and active conformations, available for a few GPCRs, identifies activation hot-spot residues in the TM domains that get rewired upon activation. Many GPCR mutations, known to alter receptor signaling and cause disease, are located at these conserved contact and activation hot-spot residue positions. The GRoSS alignment places the chemosensory receptor subfamilies for bitter taste (TAS2R) and pheromones (Vomeronasal, VN1R) in the rhodopsin family, known to contain the chemosensory olfactory receptor subfamily. The GRoSS alignment also enables the quantification of the structural variability in the TM regions of experimental structures, useful for homology modeling and structure prediction of receptors. Furthermore, this alignment identifies structurally and functionally important residues in all human GPCRs. These residues can be used to make testable hypotheses about the structural basis of receptor function and about the molecular basis of disease-associated single nucleotide polymorphisms. PMID:27028541

  17. Structural regularities of helicoidally-like biopolymers in the framework of algebraic topology: II. {alpha}-Helix and DNA structures

    SciTech Connect

    Samoylovich, M. I.; Talis, A. L.

    2013-09-15

    The developed apparatus of the 'structural application' of algebraic geometry and topology makes it possible to determine topologically stable helicoidally-like packings of polyhedra (clusters). A packing found is limited by a minimal surface with zero instability index; this surface is set by the Weierstrass representation and corresponds to the bifurcation point. The symmetries of the packings under consideration are determined by four-dimensional polyhedra (polytopes) from a closed sequence, which begins with diamondlike polytope (240). One example of these packings is a packing of tetrahedra, which arises as a result of the multiplication of a peculiar starting aggregation of tetrahedra by a fractional 40/11 axis with an angle of helical rotation of 99 Degree-Sign . The arrangement of atoms in particular positions of this starting aggregation allows one to obtain a model of the {alpha}-helix. This apparatus makes it possible to determine a priori the symmetry parameters of DNA double helices.

  18. Energy and Structure of the M2 Helix in Acetylcholine Receptor-Channel Gating

    PubMed Central

    Jha, Archana; Purohit, Prasad; Auerbach, Anthony

    2009-01-01

    We studied single-channel currents from neuromuscular acetylcholine receptor-channels with mutations in the pore-lining, M2 helix of the ɛ-subunit. Three parameters were quantified: 1), the diliganded gating equilibrium constant (E2), which reflects the energy difference between C(losed) and O(pen) conformations; 2), the correlation between the opening rate constant and E2 on a log-log scale (Φ), which illuminates the energy character of the residue (C- versus O-like) within the C↔O isomerization process; and 3), the open-channel current amplitude (i0), which reports whether a mutation alters the energetics of ion permeation. The largest E2 changes were observed in the cytoplasmic half of ɛM2 (5′, 9′, 12′, 13′, and 16′), with smaller changes apparent for residues ≥17′. Φ was ∼0.54 for most ɛM2 residues, but was ∼0.32 at the positions that had largest E2 changes. An arginine substitution reduced i0 significantly at six positions, with the magnitude of the reduction increasing, 16′→2′. The measurements suggest that the 9′, 12′, and 13′ residues experience large and late free-energy changes in the channel-opening process. We speculate that in the gating isomerization the pore-facing residues >6′ and <16′ experience multiple energy perturbations associated with changes in protein structure and, perhaps, hydration. PMID:19450479

  19. Molecular structure of r/GCG/d/TATACGC/ - A DNA-RNA hybrid helix joined to double helical DNA

    NASA Technical Reports Server (NTRS)

    Wang, A. H.-J.; Fujii, S.; Rich, A.; Van Boom, J. H.; Van Der Marel, G. A.; Van Boeckel, S. A. A.

    1982-01-01

    The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA-RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.

  20. Loop-to-helix transition in the structure of multidrug regulator AcrR at the entrance of the drug-binding cavity.

    PubMed

    Manjasetty, Babu A; Halavaty, Andrei S; Luan, Chi-Hao; Osipiuk, Jerzy; Mulligan, Rory; Kwon, Keehwan; Anderson, Wayne F; Joachimiak, Andrzej

    2016-04-01

    Multidrug transcription regulator AcrR from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 belongs to the tetracycline repressor family, one of the largest groups of bacterial transcription factors. The crystal structure of dimeric AcrR was determined and refined to 1.56Å resolution. The tertiary and quaternary structures of AcrR are similar to those of its homologs. The multidrug binding site was identified based on structural alignment with homologous proteins and has a di(hydroxyethyl)ether molecule bound. Residues from helices α4 and α7 shape the entry into this binding site. The structure of AcrR reveals that the extended helical conformation of helix α4 is stabilized by the hydrogen bond between Glu67 (helix α4) and Gln130 (helix α7). Based on the structural comparison with the closest homolog structure, the Escherichia coli AcrR, we propose that this hydrogen bond is responsible for control of the loop-to-helix transition within helix α4. This local conformational switch of helix α4 may be a key step in accessing the multidrug binding site and securing ligands at the binding site. Solution small-molecule binding studies suggest that AcrR binds ligands with their core chemical structure resembling the tetracyclic ring of cholesterol. PMID:26796657

  1. Membrane Protein Crystallization in Lipidic Mesophases. Hosting Lipid Effects on the Crystallization and Structure of a Transmembrane Peptide

    SciTech Connect

    Hfer, Nicole; Aragao, David; Lyons, Joseph A.; Caffrey, Martin

    2011-09-28

    Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating d- and l-amino acids. It functions, in part, by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations. The peptide should be able to traverse the host membrane either as a double-stranded, intertwined double helix (DSDH) or as a head-to-head single-stranded helix (HHSH). Current structure models are based on macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR). However, the HHSH form has only been observed by NMR. The shape and size of the different gramicidin conformations differ. We speculated therefore that reconstituting it into a lipidic mesophase with bilayers of different microstructures would preferentially stabilize one form over the other. By using such mesophases for in meso crystallogenesis, the expectation was that at least one would generate crystals of gramicidin in the HHSH form for structure determination by MX. This was tested using commercial and in-house synthesized lipids that support in meso crystallogenesis. Lipid acyl chain lengths were varied from 14 to 18 carbons to provide mesophases with a range of bilayer thicknesses. Unexpectedly, all lipids produced high-quality, structure-grade crystals with gramicidin only in the DSDH conformation.

  2. Structural studies of the natriuretic peptide receptor: a novel hormone-induced rotation mechanism for transmembrane signal transduction.

    PubMed

    Misono, Kunio S; Ogawa, Haruo; Qiu, Yue; Ogata, Craig M

    2005-06-01

    The atrial natriuretic peptide (ANP) receptor is a single-span transmembrane receptor that is coupled to its intrinsic intracellular guanylate cyclase (GCase) catalytic activity. To investigate the mechanisms of hormone binding and signal transduction, we have expressed the extracellular hormone-binding domain of the ANP receptor (ANPR) and characterized its structure and function. The disulfide-bond structure, state of glycosylation, binding-site residues, chloride-dependence of ANP binding, dimerization, and binding stoichiometry have been determined. More recently, the crystal structures of both the apoANPR dimer and ANP-bound complex have been determined. The structural comparison between the two has shown that, upon ANP binding, two ANPR molecules in the dimer undergo an inter-molecular twist with little intra-molecular conformational change. This motion produces a Ferris wheel-like translocation of two juxtamembrane domains with essentially no change in the inter-domain distance. This movement alters the relative orientation of the two domains equivalent to counter-clockwise rotation of each by 24 degrees . These results suggest that transmembrane signaling by the ANP receptor is mediated by a novel hormone-induced rotation mechanism. PMID:15911065

  3. Temperature dependence of the DNA double helix at the nanoscale: structure, elasticity, and fluctuations.

    PubMed

    Meyer, Sam; Jost, Daniel; Theodorakopoulos, Nikos; Peyrard, Michel; Lavery, Richard; Everaers, Ralf

    2013-10-15

    Biological organisms exist over a broad temperature range of -15C to +120C, where many molecular processes involving DNA depend on the nanoscale properties of the double helix. Here, we present results of extensive molecular dynamics simulations of DNA oligomers at different temperatures. We show that internal basepair conformations are strongly temperature-dependent, particularly in the stretch and opening degrees of freedom whose harmonic fluctuations can be considered the initial steps of the DNA melting pathway. The basepair step elasticity contains a weaker, but detectable, entropic contribution in the roll, tilt, and rise degrees of freedom. To extend the validity of our results to the temperature interval beyond the standard melting transition relevant to extremophiles, we estimate the effects of superhelical stress on the stability of the basepair steps, as computed from the Benham model. We predict that although the average twist decreases with temperature in vitro, the stabilizing external torque in vivo results in an increase of ?1/bp (or a superhelical density of ?? ? +0.03) in the interval 0-100C. In the final step, we show that the experimentally observed apparent bending persistence length of torsionally unconstrained DNA can be calculated from a hybrid model that accounts for the softening of the double helix and the presence of transient denaturation bubbles. Although the latter dominate the behavior close to the melting transition, the inclusion of helix softening is important around standard physiological temperatures. PMID:24138866

  4. Temperature Dependence of the DNA Double Helix at the Nanoscale: Structure, Elasticity, and Fluctuations

    PubMed Central

    Meyer, Sam; Jost, Daniel; Theodorakopoulos, Nikos; Peyrard, Michel; Lavery, Richard; Everaers, Ralf

    2013-01-01

    Biological organisms exist over a broad temperature range of ?15C to+120C, where many molecular processes involving DNA depend on the nanoscale properties of the double helix. Here, we present results of extensive molecular dynamics simulations of DNA oligomers at different temperatures. We show that internal basepair conformations are strongly temperature-dependent, particularly in the stretch and opening degrees of freedom whose harmonic fluctuations can be considered the initial steps of the DNA melting pathway. The basepair step elasticity contains a weaker, but detectable, entropic contribution in the roll, tilt, and rise degrees of freedom. To extend the validity of our results to the temperature interval beyond the standard melting transition relevant to extremophiles, we estimate the effects of superhelical stress on the stability of the basepair steps, as computed from the Benham model. We predict that although the average twist decreases with temperature invitro, the stabilizing external torque invivo results in an increase of ?1/bp (or a superhelical density of ???+0.03) in the interval 0100C. In the final step, we show that the experimentally observed apparent bending persistence length of torsionally unconstrained DNA can be calculated from a hybrid model that accounts for the softening of the double helix and the presence of transient denaturation bubbles. Although the latter dominate the behavior close to the melting transition, the inclusion of helix softening is important around standard physiological temperatures. PMID:24138866

  5. The Enhancer of split [E(spl)] locus of Drosophila encodes seven independent helix-loop-helix proteins.

    PubMed Central

    Delidakis, C; Artavanis-Tsakonas, S

    1992-01-01

    The E(spl) locus is thought to participate in a cell interaction mechanism that controls the choice of many cell fates during Drosophila development, including the segregation of neural precursors. Previous studies have demonstrated that E(spl) is defined by two groups of closely related transcripts, (i) a cluster of three transcripts encoding proteins bearing a helix-loop-helix (HLH) motif and (ii) a single-copy gene encoding a nuclear protein containing repeated motifs first identified in the beta subunit of guanine nucleotide-binding proteins. Both groups interact genetically with the Notch locus, which codes for a transmembrane protein. We report the structure of four additional HLH-encoding genes that reside in the E(spl) complex and provide evidence that we have now identified all the remaining members of the E(spl) HLH cluster. Images PMID:1528887

  6. Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex.

    PubMed

    Wiedemann, Christoph; Szambowska, Anna; Häfner, Sabine; Ohlenschläger, Oliver; Gührs, Karl-Heinz; Görlach, Matthias

    2015-03-11

    The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical 'wings' of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain. PMID:25712103

  7. Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

    PubMed Central

    Wiedemann, Christoph; Szambowska, Anna; Häfner, Sabine; Ohlenschläger, Oliver; Gührs, Karl-Heinz; Görlach, Matthias

    2015-01-01

    The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain. PMID:25712103

  8. Evolutionary-guided de novo structure prediction of self-associated transmembrane helical proteins with near-atomic accuracy

    NASA Astrophysics Data System (ADS)

    Wang, Y.; Barth, P.

    2015-05-01

    How specific protein associations regulate the function of membrane receptors remains poorly understood. Conformational flexibility currently hinders the structure determination of several classes of membrane receptors and associated oligomers. Here we develop EFDOCK-TM, a general method to predict self-associated transmembrane protein helical (TMH) structures from sequence guided by co-evolutionary information. We show that accurate intermolecular contacts can be identified using a combination of protein sequence covariation and TMH binding surfaces predicted from sequence. When applied to diverse TMH oligomers, including receptors characterized in multiple conformational and functional states, the method reaches unprecedented near-atomic accuracy for most targets. Blind predictions of structurally uncharacterized receptor tyrosine kinase TMH oligomers provide a plausible hypothesis on the molecular mechanisms of disease-associated point mutations and binding surfaces for the rational design of selective inhibitors. The method sets the stage for uncovering novel determinants of molecular recognition and signalling in single-spanning eukaryotic membrane receptors.

  9. Evolutionary-guided de novo structure prediction of self-associated transmembrane helical proteins with near-atomic accuracy

    PubMed Central

    Wang, Y.; Barth, P.

    2016-01-01

    How specific protein associations regulate the function of membrane receptors remains poorly understood. Conformational flexibility currently hinders the structure determination of several classes of membrane receptors and associated oligomers. Here we develop EFDOCK-TM, a general method to predict self-associated transmembrane protein helical (TMH) structures from sequence guided by co-evolutionary information. We show that accurate intermolecular contacts can be identified using a combination of protein sequence covariation and TMH binding surfaces predicted from sequence. When applied to diverse TMH oligomers, including receptors characterized in multiple conformational and functional states, the method reaches unprecedented near-atomic accuracy for most targets. Blind predictions of structurally uncharacterized receptor tyrosine kinase TMH oligomers provide a plausible hypothesis on the molecular mechanisms of disease-associated point mutations and binding surfaces for the rational design of selective inhibitors. The method sets the stage for uncovering novel determinants of molecular recognition and signalling in single-spanning eukaryotic membrane receptors. PMID:25995083

  10. Leukocyte common antigen-related phosphatase (LRP) gene structure: Conservation of the genomic organization of transmembrane protein tyrosine phosphatases

    SciTech Connect

    Wong, E.C.C.; Mullersman, J.E.; Thomas, M.L. )

    1993-07-01

    The leukocyte common antigen-related protein tyrosine phosphatase (LRP) is a widely expressed transmembrane glycoprotein thought to be involved in cell growth and differentiation. Similar to most other transmembrane protein tyrosine phosphatases, LRP contains two tandem cytoplasmic phosphatase domains. To understand further the regulation and evolution of LRP, the authors have isolated and characterized mouse [lambda] genomic clones. Thirteen genomic clones could be divided into two non-overlapping clusters. The first cluster contained the transcription initiation site and the exon encoding most of the 5[prime] untranslated region. The second cluster contained the remaining exons encoding the protein and the 3[prime] untranslated region. The gene consists of 22 exons spanning over 75 kb. The distance between exon 1 and exon 2 is at least 25 kb. Characterization of the 5[prime] ends of LRP mRNA by S1 nuclease protection identifies putative initiation start sites within a G/C-rich region. The upstream region does not contain a TATA box. Comparison of the LRP gene structure to the mammalian protein tyrosine phosphatase gene, CD45, shows striking similarities in size and genomic organization. 29 refs., 5 figs., 1 tab.

  11. Localization phenomena in a DNA double-helix structure: A twisted ladder model

    NASA Astrophysics Data System (ADS)

    Kundu, Sourav; Karmakar, S. N.

    2014-03-01

    In this work we propose a model for the DNA double helix within the tight-binding framework that incorporates the helicity of the molecules. We have studied the localization properties of three DNA sequences, the periodic poly(dG)-poly(dC) and poly(dA)-poly(dT) sequences and the random ATGC sequence (where A is adenine, T is thymine, G is guanine, and C is cytosine), all of which are coupled to the backbone with random site energies representing the environmental fluctuations. We observe that due to the helicity of DNA, electron transport is greatly enhanced and there exists an almost disorder-strength-independent critical value of the hopping integral, which accounts for the helicity of DNA, for which the electronic states become maximally extended. We have also investigated the effect of backbone energetics on the transmission and I-V characteristics of DNA.

  12. Crystal structure of decameric fructose-6-phosphate aldolase from Escherichia coli reveals inter-subunit helix swapping as a structural basis for assembly differences in the transaldolase family.

    PubMed

    Thorell, Stina; Schürmann, Melanie; Sprenger, Georg A; Schneider, Gunter

    2002-05-24

    Fructose-6-phosphate aldolase from Escherichia coli is a member of a small enzyme subfamily (MipB/TalC family) that belongs to the class I aldolases. The three-dimensional structure of this enzyme has been determined at 1.93 A resolution by single isomorphous replacement and tenfold non-crystallographic symmetry averaging and refined to an R-factor of 19.9% (R(free) 21.3%). The subunit folds into an alpha/beta barrel, with the catalytic lysine residue on barrel strand beta 4. It is very similar in overall structure to that of bacterial and mammalian transaldolases, although more compact due to extensive deletions of additional secondary structural elements. The enzyme forms a decamer of identical subunits with point group symmetry 52. Five subunits are arranged as a pentamer, and two ring-like pentamers pack like a doughnut to form the decamer. A major interaction within the pentamer is through the C-terminal helix from one monomer, which runs across the active site of the neighbouring subunit. In classical transaldolases, this helix folds back and covers the active site of the same subunit and is involved in dimer formation. The inter-subunit helix swapping appears to be a major determinant for the formation of pentamers rather than dimers while at the same time preserving importing interactions of this helix with the active site of the enzyme. The active site lysine residue is covalently modified, by forming a carbinolamine with glyceraldehyde from the crystallisation mixture. The catalytic machinery is very similar to that of transaldolase, which together with the overall structural similarity suggests that enzymes of the MipB/TALC subfamily are evolutionary related to the transaldolase family. PMID:12051943

  13. A tyrosine-containing motif mediates ER retention of CD3-epsilon and adopts a helix-turn structure.

    PubMed Central

    Mallabiabarrena, A; Jimnez, M A; Rico, M; Alarcn, B

    1995-01-01

    The CD3-epsilon endoplasmic reticulum (ER) retention motif has been characterized by mutagenesis and NMR spectroscopy. Tyr177, Leu180 and Arg183 are involved in ER retention. The motif forms an elongated alpha-helix in which the tyrosine and leucine residues are closely apposed, followed by a beta I' turn that places Arg183 in the vicinity of Leu180. The structure formed by Tyr177 and the leucine in position +3 is reminiscent of the beta-turn structure adopted by tyrosine-containing endocytosis signals. Moreover, substitution of the transferrin receptor (TfR) internalization sequence by the CD3-epsilon motif still allowed the rapid internalization of the TfR and, conversely, the chimeric protein resulting from the substitution of the CD3-epsilon motif by the endocytosis signal of the low density lipoprotein receptor was ER located. These data support the idea of a functional homology between the two types of signal. Images PMID:7774584

  14. Structure and computational analysis of a novel protein with metallopeptidase-like and circularly permuted winged-helix-turn-helix domains reveals a possible role in modified polysaccharide biosynthesis

    PubMed Central

    2014-01-01

    Background CA_C2195 from Clostridium acetobutylicum is a protein of unknown function. Sequence analysis predicted that part of the protein contained a metallopeptidase-related domain. There are over 200 homologs of similar size in large sequence databases such as UniProt, with pairwise sequence identities in the range of ~40-60%. CA_C2195 was chosen for crystal structure determination for structure-based function annotation of novel protein sequence space. Results The structure confirmed that CA_C2195 contained an N-terminal metallopeptidase-like domain. The structure revealed two extra domains: an α+β domain inserted in the metallopeptidase-like domain and a C-terminal circularly permuted winged-helix-turn-helix domain. Conclusions Based on our sequence and structural analyses using the crystal structure of CA_C2195 we provide a view into the possible functions of the protein. From contextual information from gene-neighborhood analysis, we propose that rather than being a peptidase, CA_C2195 and its homologs might play a role in biosynthesis of a modified cell-surface carbohydrate in conjunction with several sugar-modification enzymes. These results provide the groundwork for the experimental verification of the function. PMID:24646163

  15. Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors.

    PubMed

    Forman, Stuart A; Chiara, David C; Miller, Keith W

    2015-09-01

    General anesthetics are a heterogeneous group of small amphiphilic ligands that interact weakly at multiple allosteric sites on many pentameric ligand gated ion channels (pLGICs), resulting in either inhibition, potentiation of channel activity, or both. Allosteric principles imply that modulator sites must change configuration and ligand affinity during receptor state transitions. Thus, general anesthetics and related compounds are useful both as state-dependent probes of receptor structure and as potentially selective modulators of pLGIC functions. This review focuses on general anesthetic sites in nicotinic acetylcholine receptors, which were among the first anesthetic-sensitive pLGIC experimental models studied, with particular focus on sites formed by transmembrane domain elements. Structural models place many of these sites at interfaces between two or more pLGIC transmembrane helices both within subunits and between adjacent subunits, and between transmembrane helices and either lipids (the lipid-protein interface) or water (i.e. the ion channel). A single general anesthetic may bind at multiple allosteric sites in pLGICs, producing a net effect of either inhibition (e.g. blocking the ion channel) or enhanced channel gating (e.g. inter-subunit sites). Other general anesthetic sites identified by photolabeling or crystallography are tentatively linked to functional effects, including intra-subunit helix bundle sites and the lipid-protein interface. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25316107

  16. Rigid multibody simulation of a helix-like structure: the dynamics of bacterial adhesion pili.

    PubMed

    Zakrisson, Johan; Wiklund, Krister; Servin, Martin; Axner, Ove; Lacoursière, Claude; Andersson, Magnus

    2015-07-01

    We present a coarse-grained rigid multibody model of a subunit assembled helix-like polymer, e.g., adhesion pili expressed by bacteria, that is capable of describing the polymer's force-extension response. With building blocks representing individual subunits, the model appropriately describes the complex behavior of pili expressed by the gram-negative uropathogenic Escherichia coli bacteria under the action of an external force. Numerical simulations show that the dynamics of the model, which include the effects of both unwinding and rewinding, are in good quantitative agreement with the characteristic force-extension response as observed experimentally for type 1 and P pili. By tuning the model, it is also possible to reproduce the force-extension response in the presence of anti-shaft antibodies, which dramatically changes the mechanical properties. Thus, the model and results in this work give enhanced understanding of how a pilus unwinds under the action of external forces and provide a new perspective of the complex bacterial adhesion processes. PMID:25851543

  17. Disordered regions in transmembrane proteins.

    PubMed

    Tusnády, Gábor E; Dobson, László; Tompa, Peter

    2015-11-01

    The functions of transmembrane proteins in living cells are widespread; they range from various transport processes to energy production, from cell-cell adhesion to communication. Structurally, they are highly ordered in their membrane-spanning regions, but may contain disordered regions in the cytosolic and extra-cytosolic parts. In this study, we have investigated the disordered regions in transmembrane proteins by a stringent definition of disordered residues on the currently available largest experimental dataset, and show a significant correlation between the spatial distributions of positively charged residues and disordered regions. This finding suggests a new role of disordered regions in transmembrane proteins by providing structural flexibility for stabilizing interactions with negatively charged head groups of the lipid molecules. We also find a preference of structural disorder in the terminal--as opposed to loop--regions in transmembrane proteins, and survey the respective functions involved in recruiting other proteins or mediating allosteric signaling effects. Finally, we critically compare disorder prediction methods on our transmembrane protein set. While there are no major differences between these methods using the usual statistics, such as per residue accuracies, Matthew's correlation coefficients, etc.; substantial differences can be found regarding the spatial distribution of the predicted disordered regions. We conclude that a predictor optimized for transmembrane proteins would be of high value to the field of structural disorder. PMID:26275590

  18. Uniformity, ideality, and hydrogen bonds in transmembrane alpha-helices.

    PubMed Central

    Kim, Sanguk; Cross, Timothy A

    2002-01-01

    Protein environments substantially influence the balance of molecular interactions that generate structural stability. Transmembrane helices exist in the relatively uniform low dielectric interstices of the lipid bilayer, largely devoid of water and with a very hydrophobic distribution of amino acid residues. Here, through an analysis of bacteriorhodopsin crystal structures and the transmembrane helix structure from M2 protein of influenza A, some helices are shown to be exceptionally uniform in hydrogen bond geometry, peptide plane tilt angle, and backbone torsion angles. Evidence from both the x-ray crystal structures and solid-state NMR structure suggests that the intramolecular backbone hydrogen bonds are shorter than their counterparts in water-soluble proteins. Moreover, the geometry is consistent with a dominance of electrostatic versus covalent contributions to these bonds. A comparison of structure as a function of resolution shows that as the structures become better characterized the helices become much more uniform, suggesting that there is a possibility that many more uniform helices will be observed, even among the moderate resolution membrane protein structures that are currently in the Protein Data Bank that do not show such features. PMID:12324426

  19. Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

    2015-05-01

    Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels.

  20. Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy.

    PubMed

    Maity, Sourav; Mazzolini, Monica; Arcangeletti, Manuel; Valbuena, Alejandro; Fabris, Paolo; Lazzarino, Marco; Torre, Vincent

    2015-01-01

    Cyclic nucleotide-gated (CNG) channels are activated by binding of cyclic nucleotides. Although structural studies have identified the channel pore and selectivity filter, conformation changes associated with gating remain poorly understood. Here we combine single-molecule force spectroscopy (SMFS) with mutagenesis, bioinformatics and electrophysiology to study conformational changes associated with gating. By expressing functional channels with SMFS fingerprints in Xenopus laevis oocytes, we were able to investigate gating of CNGA1 in a physiological-like membrane. Force spectra determined that the S4 transmembrane domain is mechanically coupled to S5 in the closed state, but S3 in the open state. We also show there are multiple pathways for the unfolding of the transmembrane domains, probably caused by a different degree of α-helix folding. This approach demonstrates that CNG transmembrane domains have dynamic structure and establishes SMFS as a tool for probing conformational change in ion channels. PMID:25963832

  1. Structural basis of typhoid: Salmonella typhi type IVb pilin (PiLS) and cystic fibrosis transmembrane conductance regulator interaction

    SciTech Connect

    Balakrishna, A.M.; Saxena, A.; Mok, H. Y.-K.; Swaminathan, K.

    2009-11-01

    The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein ({Delta}PilS), which makes the pilus, was determined at 1.9 {angstrom} resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of {Delta}PilS and a target CFTR peptide, determined at 1.8 {angstrom}, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

  2. Structural Basis of Typhoid: Salmonella typhi Type IVb pilin (PilS) and Cystic Fibrosis Transmembrane Conductance Regulatory Interaction

    SciTech Connect

    Balakrishna, A.; Saxena, A; Mok, H; Swaminathan, K

    2009-01-01

    The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (PilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of PilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

  3. Structural basis of typhod: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

    SciTech Connect

    Balakrishna, A.; Saxena, A; Mok, H; Swaminathan, K

    2009-01-01

    The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (PilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of PilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

  4. The structure of Plasmodium vivax phosphatidylethanolamine-binding protein suggests a functional motif containing a left-handed helix

    SciTech Connect

    Arakaki, Tracy; Neely, Helen; Boni, Erica; Mueller, Natasha; Buckner, Frederick S.; Van Voorhis, Wesley C.; Lauricella, Angela; DeTitta, George; Luft, Joseph; Hol, Wim G. J.; Merritt, Ethan A.

    2007-03-01

    The crystal structure of a phosphatidylethanolamine-binding protein from P. vivax, a homolog of Raf-kinase inhibitor protein (RKIP), has been solved to a resolution of 1.3 Å. The inferred interaction surface near the anion-binding site is found to include a distinctive left-handed α-helix. The structure of a putative Raf kinase inhibitor protein (RKIP) homolog from the eukaryotic parasite Plasmodium vivax has been studied to a resolution of 1.3 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protozoan protein is topologically similar to previously studied members of the phosphatidylethanolamine-binding protein (PEBP) sequence family, but exhibits a distinctive left-handed α-helical region at one side of the canonical phospholipid-binding site. Re-examination of previously determined PEBP structures suggests that the P. vivax protein and yeast carboxypeptidase Y inhibitor may represent a structurally distinct subfamily of the diverse PEBP-sequence family.

  5. Structural Disorder of the CD3ζ Transmembrane Domain Studied with 2D IR Spectroscopy and Molecular Dynamics Simulations

    PubMed Central

    Mukherjee, Prabuddha; Kass, Itamar; Arkin, Isaiah T.; Zanni, Martin T.

    2009-01-01

    In a recently reported study [Mukherjee, et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3528], we used 2D IR spectroscopy and 1-13C═18O isotope labeling to measure the vibrational dynamics of 11 amide I modes in the CD3ζ transmembrane domain. We found that the homogeneous line widths and population relaxation times were all nearly identical, but that the amount of inhomogeneous broadening correlated with the position of the amide group inside the membrane. In this study, we use molecular dynamics simulations to investigate the structural and dynamical origins of these experimental observations. We use two models to convert the simulations to frequency trajectories from which the mean frequencies, standard deviations, frequency correlation functions, and 2D IR spectra are calculated. Model 1 correlates the hydrogen-bond length to the amide I frequency, whereas model 2 uses an ab initio-based electrostatic model. We find that the structural distributions of the peptidic groups and their environment are reflected in the vibrational dynamics of the amide I modes. Environmental forces from the water and lipid headgroups partially denature the helices, shifting the infrared frequencies and creating larger inhomogeneous distributions for residues near the ends. The least inhomogeneously broadened residues are those located in the middle of the membrane where environmental electrostatic forces are weakest and the helices are most ordered. Comparison of the simulations to experiment confirms that the amide I modes near the C-terminal are larger than at the N-terminal because of the asymmetric structure of the peptide bundle in the membrane. The comparison also reveals that residues at a kink in the α-helices have broader line widths than more helical parts of the peptide because the peptide backbone at the kink exhibits a larger amount of structural disorder. Taken together, the simulations and experiments reveal that infrared line shapes are sensitive probes of membrane protein structural and environmental heterogeneity. PMID:17134238

  6. [Preparation of Transmembrane Fragments Growth Hormone Receptor GHR in a Cell-Free Expression System for Structural Studies].

    PubMed

    Bocharova, O V; Kuzmichev, P K; Urban, A S; Goncharuk, S A; Bocharov, E V; Arsenyev, A S

    2015-01-01

    Growth hormone somatotropin and its membrane receptor GHR, belonging to a superfamily of the type I receptors possessing tyrosine kinase activity, are involved in the intercellular signal transduction cascade and regulate a number of important physiological and pathological processes in humans. Binding with somatotropin triggers a transition of GHR between two alternative dimer states, resulting in an allosteric activation of JAK2 tyrosine kinase in the cell cytoplasm. Transmembrane domain of GHR directly involved in this complex conformational transition. It has presumably two dimerization interfaces corresponding to the "unliganded" and the active state of GHR. In order to study the molecular basis of biochemical signal transduction mechanism across the cell membrane, we have developed an efficient cell-free production system of a TM fragment of GHR, which contains its TM domain flanked by functionally important juxtamembrane regions (GHRtm residues 254-298). The developed system allows to obtain -1 mg per 1 ml of reaction mixture of 13C- and 15N-isotope-labeled protein for structural and dynamic studies of the GHR TM domain dimerization in the membrane-mimicking medium by high-resolution heteronuclear NMR spectroscopy. PMID:27125024

  7. Global force-torque phase diagram for the DNA double helix: Structural transitions, triple points, and collapsed plectonemes

    NASA Astrophysics Data System (ADS)

    Marko, John F.; Neukirch, Sébastien

    2013-12-01

    We present a free energy model for structural transitions of the DNA double helix driven by tensile and torsional stress. Our model is coarse grained and is based on semiflexible polymer descriptions of B-DNA, underwound L-DNA, and highly overwound P-DNA. The statistical-mechanical model of plectonemic supercoiling previously developed for B-DNA is applied to semiflexible polymer models of P- and L-DNA to obtain a model of DNA structural transitions in quantitative accord with experiment. We identify two distinct plectonemic states, one "inflated" by electrostatic repulsion and thermal fluctuations and the other "collapsed," with the two double helices inside the supercoils driven to close contact. We find that supercoiled B and L are stable only in the inflated form, while supercoiled P is always collapsed. We also predict the behavior and experimental signatures of highly underwound "Q"-DNA, the left-handed analog of P-DNA; as for P, supercoiled Q is always collapsed. Overstretched "S"-DNA and strand-separated "stress-melted" DNA are also included in our model, allowing prediction of a global phase diagram for forces up to 1000 pN and torques between ±60 pN nm, or, in terms of linking number density, from σ =-5 to +3.

  8. Structural basis for recognition of breast and colon cancer epitopes Tn antigen and Forssman disaccharide by Helix pomatia lectin.

    PubMed

    Lescar, Julien; Sanchez, Jean-Frederic; Audfray, Aymeric; Coll, Jean-Luc; Breton, Christelle; Mitchell, Edward P; Imberty, Anne

    2007-10-01

    Helix pomatia agglutinin (HPA) is a lectin that has been used extensively in histopathology, since its binding to tissue sections from breast and colon cancers is correlated with the worst prognosis for the patients. The lectin recognizes alpha-d-N-acetylgalactosamine (alphaGalNAc) containing epitopes which are only present in cancer cell lines having a high likelihood to undergo metastasis, such as the HT29 cancer colon cell line. Several breast cancer cell lines have also been shown to be labeled, although IGROV1, an ovarian cancer cell line, is not. Inhibition studies, using GalNAc monosaccharides, are reported here, showing that the labeling is dependent upon the presence of carbohydrate epitopes. The crystal structures of the lectin complexed with two GalNAc containing epitopes associated with cancer, the Tn (alphaGalNAc-Ser) and Forssman (alphaGalNAc1-3GalNAc) antigens, show the lectin's specificity for GalNAc is due to a particular network of hydrogen bonds. A histidine residue makes hydrophobic contact with the aglycon, rationalizing the preference for GalNAc bearing an additional sugar or amino acid in the alpha position. These structures provide the molecular basis for the use of HPA in metastasis research. PMID:17652409

  9. Solution structure of an ABC collagen heterotrimer reveals a single-register helix stabilized by electrostatic interactions.

    PubMed

    Fallas, Jorge A; Gauba, Varun; Hartgerink, Jeffrey D

    2009-09-25

    Collagen, known for its structural role in tissues and also for its participation in the regulation of homeostatic and pathological processes in mammals, is assembled from triple helices that can be either homotrimers or heterotrimers. High resolution structural information for natural collagens has been difficult to obtain because of their size and the heterogeneity of their native environment. For this reason, peptides that self-assemble into collagen-like triple helices are used to gain insight into the structure, stability, and biochemistry of this important protein family. Although many of the most common collagens in humans are heterotrimers, almost all studies of collagen helices have been on homotrimers. Here we report the first structure of a collagen heterotrimer. Our structure, obtained by solution NMR, highlights the role of electrostatic interactions as stabilizing factors within the triple helical folding motif. This addresses an issue that has been actively researched because of the predominance of charged residues in the collagen family. We also find that it is possible to selectively form a collagen heterotrimer with a well defined composition and register of the peptide chains within the helix, based on information encoded solely in the collagenous domain. Globular domains are implicated in determining the composition of several collagen types, but it is unclear what their role in controlling register may be. We show that is possible to design peptides that not only selectively choose a composition but also a specific register without the assistance of other protein constructs. This mechanism may be used in nature as well. PMID:19625247

  10. Solution Structure of an ABC Collagen Heterotrimer Reveals a Single-register Helix Stabilized by Electrostatic Interactions*

    PubMed Central

    Fallas, Jorge A.; Gauba, Varun; Hartgerink, Jeffrey D.

    2009-01-01

    Collagen, known for its structural role in tissues and also for its participation in the regulation of homeostatic and pathological processes in mammals, is assembled from triple helices that can be either homotrimers or heterotrimers. High resolution structural information for natural collagens has been difficult to obtain because of their size and the heterogeneity of their native environment. For this reason, peptides that self-assemble into collagen-like triple helices are used to gain insight into the structure, stability, and biochemistry of this important protein family. Although many of the most common collagens in humans are heterotrimers, almost all studies of collagen helices have been on homotrimers. Here we report the first structure of a collagen heterotrimer. Our structure, obtained by solution NMR, highlights the role of electrostatic interactions as stabilizing factors within the triple helical folding motif. This addresses an issue that has been actively researched because of the predominance of charged residues in the collagen family. We also find that it is possible to selectively form a collagen heterotrimer with a well defined composition and register of the peptide chains within the helix, based on information encoded solely in the collagenous domain. Globular domains are implicated in determining the composition of several collagen types, but it is unclear what their role in controlling register may be. We show that is possible to design peptides that not only selectively choose a composition but also a specific register without the assistance of other protein constructs. This mechanism may be used in nature as well. PMID:19625247

  11. The dimeric transmembrane domain of prolyl dipeptidase DPP-IV contributes to its quaternary structure and enzymatic activities

    PubMed Central

    Chung, Kuei-Min; Cheng, Jai-Hong; Suen, Ching-Shu; Huang, Chih-Hsiang; Tsai, Cheng-Han; Huang, Li-Hao; Chen, Yi-Rong; Wang, Andrew H-J; Jiaang, Weir-Torn; Hwang, Ming-Jing; Chen, Xin

    2010-01-01

    Dipeptidyl peptidase IV (DPP-IV) is a drug target in the treatment of human type II diabetes. It is a type II membrane protein with a single transmembrane domain (TMD) anchoring the extracellular catalytic domain to the membrane. DPP-IV is active as a dimer, with two dimer interacting surfaces located extracellularly. In this study, we demonstrate that the TM of DPP-IV promotes DPP-IV dimerization and rescues monomeric DPP-IV mutants into partial dimers, which is specific and irreplaceable by TMs of other type II membrane proteins. By bioluminescence resonance energy transfer (BRET) and peptide electrophoresis, we found that the TM domain of DPP-IV is dimerized in mammalian cells and in vitro. The TM dimer interaction is very stable, based on our results with TM site-directed mutagenesis. None of the mutations, including the introduction of two prolines, resulted in their complete disruption to monomers. However, these TM proline mutations result in a significant reduction of DPP-IV enzymatic activity, comparable to what is found with mutations near the active site. A systematic analysis of TM structures deposited in the Protein Data Bank showed that prolines in the TM generally produce much bigger kinking angles than occur in nonproline-containing TMs. Thus, the proline-dependent reduction in enzyme activity may result from propagated conformational changes from the TM to the extracellular active site. Our results demonstrate that TM dimerization and conformation contribute significantly to the structure and activity of DPP-IV. Optimal enzymatic activity of DPP-IV requires an optimal interaction of all three dimer interfaces, including its TM. PMID:20572019

  12. Polysaccharide hydrogels with tunable stiffness and provasculogenic properties via α-helix to β-sheet switch in secondary structure

    PubMed Central

    Forget, Aurelien; Christensen, Jon; Lüdeke, Steffen; Kohler, Esther; Tobias, Simon; Matloubi, Maziar; Thomann, Ralf; Shastri, V. Prasad

    2013-01-01

    Mechanical aspects of the cellular environment can influence cell function, and in this context hydrogels can serve as an instructive matrix. Here we report that physicochemical properties of hydrogels derived from polysaccharides (agarose, κ-carrageenan) having an α-helical backbone can be tailored by inducing a switch in the secondary structure from α-helix to β-sheet through carboxylation. This enables the gel modulus to be tuned over four orders of magnitude (G′ 6 Pa–3.6 × 104 Pa) independently of polymer concentration and molecular weight. Using carboxylated agarose gels as a screening platform, we demonstrate that soft-carboxylated agarose provides a unique environment for the polarization of endothelial cells in the presence of soluble and bound signals, which notably does not occur in fibrin and collagen gels. Furthermore, endothelial cells organize into freestanding lumens over 100 μm in length. The finding that a biomaterial can modulate soluble and bound signals provides impetus for exploring mechanobiology paradigms in regenerative therapies. PMID:23886665

  13. Structures of two Arabidopsis thaliana major latex proteins represent novel helix-grip folds

    SciTech Connect

    Lytle, Betsy L.; Song, Jikui; de la Cruz, Norberto B.; Peterson, Francis C.; Johnson, Kenneth A.; Bingman, Craig A.; Phillips, Jr., George N.; Volkman, Brian F.

    2009-06-02

    Here we report the first structures of two major latex proteins (MLPs) which display unique structural differences from the canonical Bet v 1 fold described earlier. MLP28 (SwissProt/TrEMBL ID Q9SSK9), the product of gene At1g70830.1, and the At1g24000.1 gene product (Swiss- Prot/TrEMBL ID P0C0B0), proteins which share 32% sequence identity, were independently selected as foldspace targets by the Center for Eukaryotic Structural Genomics. The structure of a single domain (residues 17-173) of MLP28 was solved by NMR spectroscopy, while the full-length At1g24000.1 structure was determined by X-ray crystallography. MLP28 displays greater than 30% sequence identity to at least eight MLPs from other species. For example, the MLP28 sequence shares 64% identity to peach Pp-MLP119 and 55% identity to cucumber Csf2.20 In contrast, the At1g24000.1 sequence is highly divergent (see Fig. 1), containing a gap of 33 amino acids when compared with all other known MLPs. Even when the gap is excluded, the sequence identity with MLPs from other species is less than 30%. Unlike some of the MLPs from other species, none of the A. thaliana MLPs have been characterized biochemically. We show by NMR chemical shift mapping that At1g24000.1 binds progesterone, demonstrating that despite its sequence dissimilarity, the hydrophobic binding pocket is conserved and, therefore, may play a role in its biological function and that of the MLP family in general.

  14. Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk

    SciTech Connect

    Yuan, Ping; Swanson, Kurt A.; Leser, George P.; Paterson, Reay G.; Lamb, Robert A.; Jardetzky, Theodore S.

    2014-10-02

    The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HN interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.

  15. An Algorithm for Protein Helix Assignment Using Helix Geometry

    PubMed Central

    Cao, Chen; Xu, Shutan; Wang, Lincong

    2015-01-01

    Helices are one of the most common and were among the earliest recognized secondary structure elements in proteins. The assignment of helices in a protein underlies the analysis of its structure and function. Though the mathematical expression for a helical curve is simple, no previous assignment programs have used a genuine helical curve as a model for helix assignment. In this paper we present a two-step assignment algorithm. The first step searches for a series of bona fide helical curves each one best fits the coordinates of four successive backbone Cα atoms. The second step uses the best fit helical curves as input to make helix assignment. The application to the protein structures in the PDB (protein data bank) proves that the algorithm is able to assign accurately not only regular α-helix but also 310 and π helices as well as their left-handed versions. One salient feature of the algorithm is that the assigned helices are structurally more uniform than those by the previous programs. The structural uniformity should be useful for protein structure classification and prediction while the accurate assignment of a helix to a particular type underlies structure-function relationship in proteins. PMID:26132394

  16. Viral fusion protein transmembrane domain adopts β-strand structure to facilitate membrane topological changes for virus–cell fusion

    PubMed Central

    Yao, Hongwei; Lee, Michelle W.; Waring, Alan J.; Wong, Gerard C. L.; Hong, Mei

    2015-01-01

    The C-terminal transmembrane domain (TMD) of viral fusion proteins such as HIV gp41 and influenza hemagglutinin (HA) is traditionally viewed as a passive α-helical anchor of the protein to the virus envelope during its merger with the cell membrane. The conformation, dynamics, and lipid interaction of these fusion protein TMDs have so far eluded high-resolution structure characterization because of their highly hydrophobic nature. Using magic-angle-spinning solid-state NMR spectroscopy, we show that the TMD of the parainfluenza virus 5 (PIV5) fusion protein adopts lipid-dependent conformations and interactions with the membrane and water. In phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, the TMD is predominantly α-helical, but in phosphatidylethanolamine (PE) membranes, the TMD changes significantly to the β-strand conformation. Measured order parameters indicate that the strand segments are immobilized and thus oligomerized. 31P NMR spectra and small-angle X-ray scattering (SAXS) data show that this β-strand–rich conformation converts the PE membrane to a bicontinuous cubic phase, which is rich in negative Gaussian curvature that is characteristic of hemifusion intermediates and fusion pores. 1H-31P 2D correlation spectra and 2H spectra show that the PE membrane with or without the TMD is much less hydrated than PC and PG membranes, suggesting that the TMD works with the natural dehydration tendency of PE to facilitate membrane merger. These results suggest a new viral-fusion model in which the TMD actively promotes membrane topological changes during fusion using the β-strand as the fusogenic conformation. PMID:26283363

  17. Functional and pharmacological induced structural changes of the cystic fibrosis transmembrane conductance regulator in the membrane solved using SAXS.

    PubMed

    Baroni, Debora; Zegarra-Moran, Olga; Moran, Oscar

    2015-04-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to the ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. To investigate the conformation of the CFTR in the membrane, we applied the small-angle x-ray scattering (SAXS) technique on microsomal membranes extracted from NIH/3T3 cells permanentely transfected with wild-type (WT) CFTR and with CFTR carrying the ΔF508 mutation. The electronic density profile of the membranes was calculated from the SAXS data, assuming the lipid bilayer electronic density to be composed by a series of Gaussian shells. The data indicate that membranes in the microsome vesicles, that contain mostly endoplasmic reticulum membranes, are oriented in the outside-out conformation. Phosphorylation does not change significantly the electronic density profile, while dephosphorylation produces a significant modification in the inner side of the profile. Thus, we conclude that the CFTR and its associated protein complex in microsomes are mostly phosphorylated. The electronic density profile of the ΔF508-CFTR microsomes is completely different from WT, suggesting a different assemblage of the proteins in the membranes. Low-temperature treatment of cells rescues the ΔF508-CFTR protein, resulting in a conformation that resembles the WT. Differently, treatment with the corrector VX-809 modifies the electronic profile of ΔF508-CFTR membrane, but does not recover completely the WT conformation. To our knowledge, this is the first report of a direct physical measurement of the structure of membranes containing CFTR in its native environment and in different functional and pharmacological conditions. PMID:25274064

  18. Viral fusion protein transmembrane domain adopts β-strand structure to facilitate membrane topological changes for virus-cell fusion.

    PubMed

    Yao, Hongwei; Lee, Michelle W; Waring, Alan J; Wong, Gerard C L; Hong, Mei

    2015-09-01

    The C-terminal transmembrane domain (TMD) of viral fusion proteins such as HIV gp41 and influenza hemagglutinin (HA) is traditionally viewed as a passive α-helical anchor of the protein to the virus envelope during its merger with the cell membrane. The conformation, dynamics, and lipid interaction of these fusion protein TMDs have so far eluded high-resolution structure characterization because of their highly hydrophobic nature. Using magic-angle-spinning solid-state NMR spectroscopy, we show that the TMD of the parainfluenza virus 5 (PIV5) fusion protein adopts lipid-dependent conformations and interactions with the membrane and water. In phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, the TMD is predominantly α-helical, but in phosphatidylethanolamine (PE) membranes, the TMD changes significantly to the β-strand conformation. Measured order parameters indicate that the strand segments are immobilized and thus oligomerized. (31)P NMR spectra and small-angle X-ray scattering (SAXS) data show that this β-strand-rich conformation converts the PE membrane to a bicontinuous cubic phase, which is rich in negative Gaussian curvature that is characteristic of hemifusion intermediates and fusion pores. (1)H-(31)P 2D correlation spectra and (2)H spectra show that the PE membrane with or without the TMD is much less hydrated than PC and PG membranes, suggesting that the TMD works with the natural dehydration tendency of PE to facilitate membrane merger. These results suggest a new viral-fusion model in which the TMD actively promotes membrane topological changes during fusion using the β-strand as the fusogenic conformation. PMID:26283363

  19. Structural and Dynamic Study of the Tetramerization Region of Non-Erythroid α-Spectrin: A Frayed Helix Revealed by Site-Directed Spin Labeling EPR†

    PubMed Central

    Li, Qufei; Fung, L. W.-M.

    2009-01-01

    The N-terminal region of α-spectrin is responsible for its association with β-spectrin in a heterodimer to form functional tetramers. Non-erythroid alpha spectrin (αII-spectrin) has significantly higher association affinity with β-spectrin than the homologous erythroid alpha spectrin (αI-spectrin). We have previously determined the solution structure of the N-terminal region of αI-spectrin by NMR methods, but currently no structural information is available for αII-spectrin. We have now used cysteine scanning, spin labeling EPR and isothermal titration calorimetry (ITC) methods to study the tetramerization region of αII-spectrin. EPR data clearly showed that, in αII-spectrin, the first 9 N-terminal residues were unstructured, followed by an irregular helix (Helix C′), frayed at the N-terminal end, but rigid at the C-terminal end, which merges into the putative triple helical structural domain. The region corresponding to the important unstructured junction region linking Helix C′ to the first structural domain in αI-spectrin was clearly structured. Based on the published model for aligning Helices A′, B′ and C′, important interactions amongst residues in Helix C′ of αI/II-spectrin and Helices A′ and B′ of βI/II-spectrin are identified, suggesting similar coiled coil helical bundling for spectrin I and II in forming tetramers. The differences in affinities are likely due to the differences in the conformation of the junction regions. Equilibrium dissociation constants of spin labeled αII and βI complexes from ITC measurements indicate that residues 15, 19, 37 and 40 are functionally important residues in αII-spectrin. Interestingly, all four corresponding homologous residues in αI-spectrin (residues 24, 28, 46 and 49) have been reported to be clinically significant residues involved in hematological diseases. PMID:19072330

  20. Three-dimensional structure of P-glycoprotein: the transmembrane regions adopt an asymmetric configuration in the nucleotide-bound state.

    PubMed

    Rosenberg, Mark F; Callaghan, Richard; Modok, Szabolcs; Higgins, Christopher F; Ford, Robert C

    2005-01-28

    Multidrug resistance of cancer cells and pathogens is a serious clinical problem. A major factor contributing to drug resistance in cancer is the over-expression of P-glycoprotein, a plasma membrane ATP-binding cassette (ABC) drug efflux pump. Three-dimensional structural data with a resolution limit of approximately 8 A have been obtained from two-dimensional crystals of P-glycoprotein trapped in the nucleotide-bound state. Each of the two transmembrane domains of P-glycoprotein consists of six long alpha-helical segments. Five of the alpha-helices from each transmembrane domain are related by a pseudo-2-fold symmetry, whereas the sixth breaks the symmetry. The two alpha-helices positioned closest to the (pseudo-) symmetry axis at the center of the molecule appear to be kinked. A large loop of density at the extracellular surface of the transporter is likely to correspond to the glycosylated first extracellular loop, whereas two globular densities at the cytoplasmic side correspond to the hydrophilic, nucleotide-binding domains. This is the first three-dimensional structure for an intact eukaryotic ABC transporter. Comparison with the structures of two prokaryotic ABC transporters suggests significant differences in the packing of the transmembrane alpha-helices within this protein family. PMID:15485807

  1. Structure and function of transmembrane segment XII in osmosensor and osmoprotectant transporter ProP of Escherichia coli.

    PubMed

    Liu, Feng; Culham, Doreen E; Vernikovska, Yaroslava I; Keates, Robert A B; Boggs, Joan M; Wood, Janet M

    2007-05-15

    Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain and linked to TM XII, form an intermolecular, homodimeric alpha-helical coiled-coil that tunes the transporter's response to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged, Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with Oregon Green Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except for V429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior of ProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydrated polar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formed when ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TM XII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C rendered ProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at high osmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lower osmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XII required a higher osmolality to activate. They included elements of a GXXXG motif that are predicted to form the interface of TM XII with TM VII. These studies define the position of ProP TM XII within the membrane, further support the predicted structure of ProP, reveal the dimerization interface, and show that the structure of TM XII influences the osmolality at which ProP activates. PMID:17441691

  2. Dimerization of the EphA1 receptor tyrosine kinase transmembrane domain: Insights into the mechanism of receptor activation.

    PubMed

    Chavent, Matthieu; Chetwynd, Alan P; Stansfeld, Phillip J; Sansom, Mark S P

    2014-10-28

    EphA1 is a receptor tyrosine kinase (RTK) that plays a key role in developmental processes, including guidance of the migration of axons and cells in the nervous system. EphA1, in common with other RTKs, contains an N-terminal extracellular domain, a single transmembrane (TM) α-helix, and a C-terminal intracellular kinase domain. The TM helix forms a dimer, as seen in recent NMR studies. We have modeled the EphA1 TM dimer using a multiscale approach combining coarse-grain (CG) and atomistic molecular dynamics (MD) simulations. The one-dimensional potential of mean force (PMF) for this system, based on interhelix separation, has been calculated using CG MD simulations. This provides a view of the free energy landscape for helix-helix interactions of the TM dimer in a lipid bilayer. The resulting PMF profiles suggest two states, consistent with a rotation-coupled activation mechanism. The more stable state corresponds to a right-handed helix dimer interacting via an N-terminal glycine zipper motif, consistent with a recent NMR structure (2K1K). A second metastable state corresponds to a structure in which the glycine zipper motif is not involved. Analysis of unrestrained CG MD simulations based on representative models from the PMF calculations or on the NMR structure reveals possible pathways of interconversion between these two states, involving helix rotations about their long axes. This suggests that the interaction of TM helices in EphA1 dimers may be intrinsically dynamic. This provides a potential mechanism for signaling whereby extracellular events drive a shift in the repopulation of the underlying TM helix dimer energy landscape. PMID:25286141

  3. Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

    SciTech Connect

    Storrs, R.W.

    1992-08-01

    Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by [sup 31]P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 [Angstrom] of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an [alpha]-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

  4. Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

    SciTech Connect

    Storrs, R.W.

    1992-08-01

    Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by {sup 31}P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 {Angstrom} of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an {alpha}-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

  5. Crystal structure of an RNA helix recognized by a zinc-finger protein: an 18-bp duplex at 1.6 A resolution.

    PubMed Central

    Lima, Susana; Hildenbrand, Jayne; Korostelev, Andrei; Hattman, Stanley; Li, Hong

    2002-01-01

    The crystal structure of the 19-mer RNA, 5'-GAAUGCCUGCGAGCAUCCC-3' has been determined from X-ray diffraction data to 1.6 A resolution by the multiwavelength anomalous diffraction method from crystals containing a brominated uridine. In the crystal, this RNA forms an 18-mer self-complementary double helix with the 19th nucleotide flipped out of the helix. This helix contains most of the target stem recognized by the bacteriophage Mu Com protein (control of mom), which activates translation of an unusual DNA modification enzyme, Mom. The 19-mer duplex, which contains one A.C mismatch and one A.C/G.U tandem wobble pair, was shown to bind to the Com protein by native gel electrophoresis shift assay. Comparison of the geometries and base stacking properties between Watson-Crick base pairs and the mismatches in the crystal structure suggest that both hydrogen bonding and base stacking are important for stabilizing these mismatched base pairs, and that the unusual geometry adopted by the A.C mismatch may reveal a unique structural motif required for the function of Com. PMID:12166647

  6. Cell-free expression of the APP transmembrane fragments with Alzheimer's disease mutations using algal amino acid mixture for structural NMR studies.

    PubMed

    Bocharova, Olga V; Urban, Anatoly S; Nadezhdin, Kirill D; Bocharov, Eduard V; Arseniev, Alexander S

    2016-07-01

    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. PMID:27071311

  7. Structure of the 5th transmembrane segment of the Na,K-ATPase alpha subunit: a cysteine-scanning mutagenesis study.

    PubMed

    Guennoun, S; Horisberger, J D

    2000-09-29

    To study the structure of the pathway of cations across the Na, K-ATPase, we applied the substituted cysteine accessibility method to the putative 5th transmembrane segment of the alpha subunit of the Na,K-ATPase of the toad Bufo marinus. Only the most extracellular amino acid position (A(796)) was accessible from the extracellular side in the native Na,K-pump. After treatment with palytoxin, six other positions (Y(778), L(780), S(782), P(785), E(786) and L(791)), distributed along the whole length of the segment, became readily accessible to a small-size methanethiosulfonate compound (2-aminoethyl methanethiosulfonate). The accessible residues are not located on the same side of an alpha-helical model but the pattern of reactivity would rather suggest a beta-sheet structure for the inner half of the putative transmembrane segment. These results demonstrate the contribution of the 5th transmembrane segment to the palytoxin-induced channel and indicate which amino acid positions are exposed to the pore of this channel. PMID:11018538

  8. Disulfide cross-linking studies of the transmembrane regions of the aspartate sensory receptor of Escherichia coli.

    PubMed Central

    Lynch, B A; Koshland, D E

    1991-01-01

    The Escherichia coli aspartate receptor, a dimer of identical subunits, has two transmembrane regions (TM1, residues 7-30; TM2, residues 189-212) of 24 residues each. To study the relative placement and orientation of the regions, cysteine residues were introduced individually into the center of each: at positions 17, 18, and 19 in TM1; and at positions 198, 199, 200, and 201 in TM2. Based on the patterns of disulfide cross-linking observed between subunits in the mutant receptors, there appears to be close contact between the TM1 and TM1' regions at the dimer interface but no such direct interaction between the TM2 and TM2' regions. The cross-linking results are consistent with an alpha-helical structure extending across the transmembrane region up through at least residue 36, which lies on the periplasmic side of TM1. The ability of an 18-18' cross-linked dimer to transmit an aspartate-induced transmembrane signal is also supportive of such an extended helix. The changes in relative rates of disulfide cross-linking provide experimental evidence of a conformational change transmitted through the transmembrane domain during signaling. Once formed, disulfides between the transmembrane regions are unusually resistant to reduction by low molecular weight thiols in the presence of denaturants like SDS. These targeted disulfide cross-links can be used to reveal structural and dynamic aspects of protein function. Images PMID:1660136

  9. Solution structure of a DNA double helix incorporating four consecutive non-Watson-Crick base-pairs.

    PubMed

    Chou, S H; Chin, K H

    2001-09-28

    A series of DNA 21-mers containing a variety of the 4 x 4 internal loop sequence 5'-CAAG-3'/3'-ACGT-5' were studied using nuclear magnetic resonance (NMR) methodology and distance geometry (DG)/molecular dynamics (MD) approaches. Such oligomers exhibit excellent resolution in the NMR spectra and reveal many unusual NOEs (nuclear Overhauser effect) that allow for the detailed characterization of a DNA hairpin incorporating a track of four different non-Watson-Crick base-pairs in the stem. These include a wobble C.A base-pair, a sheared A.C base-pair, a sheared A.G base-pair, and a wobble G.T base-pair. Significantly different twisting angles were observed between the base-pairs in internal loop that results with excellent intra-strand and inter-strand base stacking within the four consecutive mismatches and the surrounding canonical base-pairs. This explains why it melts at 52 degrees C even though five out of ten base-pairs in the stem adopt non-Watson-Crick pairs. However, the 4 x 4 internal loop still fits into a B-DNA double helix very well without significant change in the backbone torsion angles; only zeta torsion angles between the tandem sheared base-pairs are changed to a great extent from the gauche(-) domain to the trans domain to accommodate the cross-strand base stacking in the internal loop. The observation that several consecutive non-canonical base-pairs can stably co-exist with Watson-Crick base-pairs greatly increases the limited repertoire of irregular DNA folds and reveals the possibility for unusual structural formation in the functionally important genomic regions that have potential to become single-stranded. PMID:11575931

  10. The domain structure of talin: Residues 1815–1973 form a five-helix bundle containing a cryptic vinculin-binding site

    PubMed Central

    Goult, Benjamin T.; Gingras, Alexandre R.; Bate, Neil; Barsukov, Igor L.; Critchley, David R.; Roberts, Gordon C.K.

    2010-01-01

    Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. Its rod region consists of a series of helical bundles. Here we show that residues 1815–1973 form a 5-helix bundle, with a topology unique to talin which is optimally suited for formation of a long rod such as talin. This is much more stable than the 4-helix (1843–1973) domain described earlier and as a result its vinculin binding sequence is inaccessible to vinculin at room temperature, with implications for the overall mechanism of the talin-vinculin interaction. Structured summary MINT-7722300, MINT-7760951: Talin-1 (uniprotkb:P26039) and Vinculin (uniprotkb:P12003) bind (MI:0407) by molecular sieving (MI:0071) PMID:20399778

  11. Effect of secondary structure on the potential of mean force for poly-L-lysine in the alpha-Helix and beta-sheet conformations

    SciTech Connect

    Grigsby, J.J.; Blanch, H.W.; Prausnitz, J.M.

    2001-10-30

    Because poly-L-lysine (PLL) can exist in the {alpha}-helix or {beta}-sheet conformation depending on solution preparation and solution conditions, PLL is a suitable candidate to probe the dependence of protein interactions on secondary structure. The osmotic second virial coefficient and weight-average molecular weight are reported from low-angle laser-light scattering measurements for PLL as a function of NaCl concentration, pH, and {alpha}-helix or {beta}-sheet content. Interactions between PLL molecules become more attractive as salt concentration increases due to screening of PLL charge by salt ions and at low salt concentration become more attractive as pH increases due to decreased net charge on PLL. The experimental results show that interactions are stronger for the {beta}-sheet conformation than for the {alpha}-helix conformation. A spherically-symmetric model for the potential of mean force is used to account for specific interactions not described by DLVO theory and to show how differences in secondary structure affect PLL interactions.

  12. A Trigger Residue for Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor

    PubMed Central

    Kitanovic, Smiljka; Ames, Peter

    2015-01-01

    ABSTRACT The transmembrane Tsr protein of Escherichia coli mediates chemotactic responses to environmental serine gradients. Serine binds to the periplasmic domain of the homodimeric Tsr molecule, promoting a small inward displacement of one transmembrane helix (TM2). TM2 piston displacements, in turn, modulate the structural stability of the Tsr-HAMP domain on the cytoplasmic side of the membrane to control the autophosphorylation activity of the signaling CheA kinase bound to the membrane-distal cytoplasmic tip of Tsr. A five-residue control cable segment connects TM2 to the AS1 helix of HAMP and transmits stimulus and sensory adaptation signals between them. To explore the possible role of control cable helicity in transmembrane signaling by Tsr, we characterized the signaling properties of mutant receptors with various control cable alterations. An all-alanine control cable shifted Tsr output toward the kinase-on state, whereas an all-glycine control cable prevented Tsr from reaching either a fully on or fully off output state. Restoration of the native isoleucine (I214) in these synthetic control cables largely alleviated their signaling defects. Single amino acid replacements at Tsr-I214 shifted output toward the kinase-off (L, N, H, and R) or kinase-on (A and G) states, whereas other control cable residues tolerated most amino acid replacements with little change in signaling behavior. These findings indicate that changes in control cable helicity might mediate transitions between the kinase-on and kinase-off states during transmembrane signaling by chemoreceptors. Moreover, the Tsr-I214 side chain plays a key role, possibly through interaction with the membrane interfacial environment, in triggering signaling changes in response to TM2 piston displacements. IMPORTANCE The Tsr protein of E. coli mediates chemotactic responses to environmental serine gradients. Stimulus signals from the Tsr periplasmic sensing domain reach its cytoplasmic kinase control domain through piston displacements of a membrane-spanning helix and an adjoining five-residue control cable segment. We characterized the signaling properties of Tsr variants to elucidate the transmembrane signaling role of the control cable, an element present in many microbial sensory proteins. Both the kinase-on and kinase-off output states of Tsr depended on control cable helicity, but only one residue, I214, was critical for triggering responses to attractant inputs. These findings suggest that signal transmission in Tsr involves modulation of control cable helicity through interaction of the I214 side chain with the cytoplasmic membrane. PMID:26013490

  13. Structural Determinants of Nitroxide Motion in Spin-labeled Proteins: Tertiary Contact and Solvent-inaccessible Sties in Helix G of T4 Lysozyme

    SciTech Connect

    Guo,Z.; Cascio, D.; Hideg, K.; Kalai, T.; Hubbell, W.

    2007-01-01

    A nitroxide side chain (R1) has been substituted at single sites along a helix-turn-helix motif in T4 lysozyme (residues 114-135). Together with previously published data, the new sites reported complete a continuous scan through the motif. Mutants with R1 at sites 115 and 118 were selected for crystallographic analysis to identify the structural origins of the corresponding two-component EPR spectra. At 115, R1 is shown to occupy two rotamers in the room temperature crystal structure, one of which has not been previously reported. The two components in the EPR spectrum apparently arise from differential interactions of the two rotamers with the surrounding structure, the most important of which is a hydrophobic interaction of the nitroxide ring. Interestingly, the crystal structure at 100 K reveals a single rotamer, emphasizing the possibility of rotamer selection in low-temperature crystal structures. Residue 118 is at a solvent-inaccessible site in the protein core, and the structure of 118R1, the first reported for the R1 side chain at a buried site, reveals how the side chain is accommodated in an overpacked core.

  14. Characterization of α-helix structures in polypeptides, revealed by 13CO⋯H- 15N hydrogen bond lengths determined by 13C REDOR NMR

    NASA Astrophysics Data System (ADS)

    Kimura, S.; Naito, A.; Saitô, H.; Ogawa, K.; Shoji, A.

    2001-05-01

    We have determined accurate 13C- 15N interatomic distances of mono-dispersed sequential oligopeptides, [1- 13C]Leu 8-, [ 15N]Ala 12-labeled (Phe-Leu-Ala) 6, and a variety of its derivatives in which Leu, at the residue11, was replaced by Ala, Phe, Gly or D-Ala, by means of rotational echo double resonance (REDOR) experiments. In order to minimize a plausible contribution from the peak intensities of natural abundant signals from amino-acid residues other than labeled, 13C NMR signals from such contributions were subtracted from the full echo amplitude of the doubly labeled spectra as reference. We have determined the interatomic distance as 4.5±0.1 Å for five kinds of sample examined without any significant deviation among them. Because [1- 13C]Leu 8 and [ 15N]Ala 12 form CO⋯HN hydrogen bond in the case of α-helical structure, this finding evidently showed the presence of α-helix and is consistent with the data from the conformation-dependent 13C NMR chemical shifts of corresponding 13C NMR signals. It turned out, therefore, that characterization of the α-helix structure based on REDOR experiment is straightforward and practical, if careful setting of experimental conditions is achieved to arrive at the meaningful data. It is also proved that no correction from the REDOR effect from the neighboring chains is necessary as far as α-helix structures are concerned, because fully and 30% labeled samples gave rise to identical REDOR results. Further, it is concluded that unusual displacement of 15N chemical shifts present in the sequential oligopeptide should be ascribed to a contribution other than any modified α-helical structures.

  15. Remeasuring the double helix

    SciTech Connect

    Mathew-Fenn, Rebecca S.; Das, Rhiju; Harbury, Pehr A.B.

    2008-10-20

    DNA is thought to behave as a stiff elastic rod with respect to the ubiquitous mechanical deformations inherent to its biology. To test this model at short DNA lengths, we measured the mean and variance of end-to-end length for a series of DNA double helices in solution, using small-angle x-ray scattering interference between gold nanocrystal labels. In the absence of applied tension, DNA is at least one order of magnitude softer than measured by single-molecule stretching experiments. Further, the data rule out the conventional elastic rod model. The variance in end-to-end length follows a quadratic dependence on the number of base pairs rather than the expected linear dependence, indicating that DNA stretching is cooperative over more than two turns of the DNA double helix. Our observations support the idea of long-range allosteric communication through DNA structure.

  16. Measuring the Double Helix

    SciTech Connect

    Mathew-Fenn, R.S.; Das, R.; Harbury, P.A.B.

    2009-05-26

    DNA is thought to behave as a stiff elastic rod with respect to the ubiquitous mechanical deformations inherent to its biology. To test this model at short DNA lengths, we measured the mean and variance of end-to-end length for a series of DNA double helices in solution, using small-angle x-ray scattering interference between gold nanocrystal labels. In the absence of applied tension, DNA is at least one order of magnitude softer than measured by single-molecule stretching experiments. Further, the data rule out the conventional elastic rod model. The variance in end-to-end length follows a quadratic dependence on the number of base pairs rather than the expected linear dependence, indicating that DNA stretching is cooperative over more than two turns of the DNA double helix. Our observations support the idea of long-range allosteric communication through DNA structure.

  17. The Infrared Helix

    NASA Technical Reports Server (NTRS)

    2006-01-01

    The Helix nebula exhibits complex structure on the smallest visible scales. It is composed of gaseous shells and disks puffed out by a dying sun-like star.

    In this new image from NASA's Spitzer Space Telescope, 'cometary knots' show blue-green heads caused by excitation of their molecular material from shocks or ultraviolet radiation. The tails of the cometary knots appear redder due to being shielded from the central star's ultraviolet radiation and wind by the heads of the knots.

    This image was captured by the telescope's infrared array camera. The false color composite depicts wavelengths of 3.6 microns (blue), 4.5 microns (green), and 8.0 microns (red). The color saturation has been increased to intensify hues.

  18. Dimerization of the EphA1 Receptor Tyrosine Kinase Transmembrane Domain: Insights into the Mechanism of Receptor Activation

    PubMed Central

    2014-01-01

    EphA1 is a receptor tyrosine kinase (RTK) that plays a key role in developmental processes, including guidance of the migration of axons and cells in the nervous system. EphA1, in common with other RTKs, contains an N-terminal extracellular domain, a single transmembrane (TM) α-helix, and a C-terminal intracellular kinase domain. The TM helix forms a dimer, as seen in recent NMR studies. We have modeled the EphA1 TM dimer using a multiscale approach combining coarse-grain (CG) and atomistic molecular dynamics (MD) simulations. The one-dimensional potential of mean force (PMF) for this system, based on interhelix separation, has been calculated using CG MD simulations. This provides a view of the free energy landscape for helix–helix interactions of the TM dimer in a lipid bilayer. The resulting PMF profiles suggest two states, consistent with a rotation-coupled activation mechanism. The more stable state corresponds to a right-handed helix dimer interacting via an N-terminal glycine zipper motif, consistent with a recent NMR structure (2K1K). A second metastable state corresponds to a structure in which the glycine zipper motif is not involved. Analysis of unrestrained CG MD simulations based on representative models from the PMF calculations or on the NMR structure reveals possible pathways of interconversion between these two states, involving helix rotations about their long axes. This suggests that the interaction of TM helices in EphA1 dimers may be intrinsically dynamic. This provides a potential mechanism for signaling whereby extracellular events drive a shift in the repopulation of the underlying TM helix dimer energy landscape. PMID:25286141

  19. Structural and functional interactions between six-transmembrane μ-opioid receptors and β2-adrenoreceptors modulate opioid signaling

    PubMed Central

    Samoshkin, Alexander; Convertino, Marino; Viet, Chi T.; Wieskopf, Jeffrey S.; Kambur, Oleg; Marcovitz, Jaclyn; Patel, Pinkal; Stone, Laura S.; Kalso, Eija; Mogil, Jeffrey S.; Schmidt, Brian L.; Maixner, William; Dokholyan, Nikolay V.; Diatchenko, Luda

    2015-01-01

    The primary molecular target for clinically used opioids is the μ-opioid receptor (MOR). Besides the major seven-transmembrane (7TM) receptors, the MOR gene codes for alternatively spliced six-transmembrane (6TM) isoforms, the biological and clinical significance of which remains unclear. Here, we show that the otherwise exclusively intracellular localized 6TM-MOR translocates to the plasma membrane upon coexpression with β2-adrenergic receptors (β2-ARs) through an interaction with the fifth and sixth helices of β2-AR. Coexpression of the two receptors in BE(2)-C neuroblastoma cells potentiates calcium responses to a 6TM-MOR ligand, and this calcium response is completely blocked by a selective β2-antagonist in BE(2)-C cells, and in trigeminal and dorsal root ganglia. Co-administration of 6TM-MOR and β2-AR ligands leads to substantial analgesic synergy and completely reverses opioid-induced hyperalgesia in rodent behavioral models. Together, our results provide evidence that the heterodimerization of 6TM-MOR with β2-AR underlies a molecular mechanism for 6TM cellular signaling, presenting a unique functional responses to opioids. This signaling pathway may contribute to the hyperalgesic effects of opioids that can be efficiently blocked by β2-AR antagonists, providing a new avenue for opioid therapy. PMID:26657998

  20. Rosalind Franklin and the Double Helix

    NASA Astrophysics Data System (ADS)

    Elkin, Lynne Osman

    2003-03-01

    Although she made essential contributions toward elucidating the structure of DNA, Rosalind Franklin is known to many only as seen through the distorting lens of James Watson's book, The Double Helix.

  1. A Sidekick for Membrane Simulations: Automated Ensemble Molecular Dynamics Simulations of Transmembrane Helices

    PubMed Central

    Hall, Benjamin A; Halim, Khairul Abd; Buyan, Amanda; Emmanouil, Beatrice; Sansom, Mark S P

    2016-01-01

    The interactions of transmembrane (TM) α-helices with the phospholipid membrane and with one another are central to understanding the structure and stability of integral membrane proteins. These interactions may be analysed via coarse-grained molecular dynamics (CGMD) simulations. To obtain statistically meaningful analysis of TM helix interactions, large (N ca. 100) ensembles of CGMD simulations are needed. To facilitate the running and analysis of such ensembles of simulations we have developed Sidekick, an automated pipeline software for performing high throughput CGMD simulations of α-helical peptides in lipid bilayer membranes. Through an end-to-end approach, which takes as input a helix sequence and outputs analytical metrics derived from CGMD simulations, we are able to predict the orientation and likelihood of insertion into a lipid bilayer of a given helix of family of helix sequences. We illustrate this software via analysis of insertion into a membrane of short hydrophobic TM helices containing a single cationic arginine residue positioned at different positions along the length of the helix. From analysis of these ensembles of simulations we estimate apparent energy barriers to insertion which are comparable to experimentally determined values. In a second application we use CGMD simulations to examine self-assembly of dimers of TM helices from the ErbB1 receptor tyrosine kinase, and analyse the numbers of simulation repeats necessary to obtain convergence of simple descriptors of the mode of packing of the two helices within a dimer. Our approach offers proof-of-principle platform for the further employment of automation in large ensemble CGMD simulations of membrane proteins. PMID:26580541

  2. Modified helix-like instability structure on imploding z-pinch liners that are pre-imposed with a uniform axial magnetic field

    SciTech Connect

    Awe, T. J. Jennings, C. A.; McBride, R. D.; Cuneo, M. E.; Lamppa, D. C.; Martin, M. R.; Rovang, D. C.; Sinars, D. B.; Slutz, S. A.; Owen, A. C.; Gomez, M. R.; Hansen, S. B.; Herrmann, M. C.; Jones, M. C.; McKenney, J. L.; Robertson, G. K.; Rochau, G. A.; Savage, M. E.; Stygar, W. A.; Tomlinson, K.; and others

    2014-05-15

    Recent experiments at the Sandia National Laboratories Z Facility have, for the first time, studied the implosion dynamics of magnetized liner inertial fusion (MagLIF) style liners that were pre-imposed with a uniform axial magnetic field. As reported [T. J. Awe et al., Phys. Rev. Lett. 111, 235005 (2013)] when premagnetized with a 7 or 10 T axial field, these liners developed 3D-helix-like hydrodynamic instabilities; such instabilities starkly contrast with the azimuthally correlated magneto-Rayleigh-Taylor (MRT) instabilities that have been consistently observed in many earlier non-premagnetized experiments. The helical structure persisted throughout the implosion, even though the azimuthal drive field greatly exceeded the expected axial field at the liner's outer wall for all but the earliest stages of the experiment. Whether this modified instability structure has practical importance for magneto-inertial fusion concepts depends primarily on whether the modified instability structure is more stable than standard azimuthally correlated MRT instabilities. In this manuscript, we discuss the evolution of the helix-like instability observed on premagnetized liners. While a first principles explanation of this observation remains elusive, recent 3D simulations suggest that if a small amplitude helical perturbation can be seeded on the liner's outer surface, no further influence from the axial field is required for the instability to grow.

  3. Modified helix-like instability structure on imploding z-pinch liners that are pre-imposed with a uniform axial magnetic fielda)

    NASA Astrophysics Data System (ADS)

    Awe, T. J.; Jennings, C. A.; McBride, R. D.; Cuneo, M. E.; Lamppa, D. C.; Martin, M. R.; Rovang, D. C.; Sinars, D. B.; Slutz, S. A.; Owen, A. C.; Tomlinson, K.; Gomez, M. R.; Hansen, S. B.; Herrmann, M. C.; Jones, M. C.; McKenney, J. L.; Robertson, G. K.; Rochau, G. A.; Savage, M. E.; Schroen, D. G.; Stygar, W. A.

    2014-05-01

    Recent experiments at the Sandia National Laboratories Z Facility have, for the first time, studied the implosion dynamics of magnetized liner inertial fusion (MagLIF) style liners that were pre-imposed with a uniform axial magnetic field. As reported [T. J. Awe et al., Phys. Rev. Lett. 111, 235005 (2013)] when premagnetized with a 7 or 10 T axial field, these liners developed 3D-helix-like hydrodynamic instabilities; such instabilities starkly contrast with the azimuthally correlated magneto-Rayleigh-Taylor (MRT) instabilities that have been consistently observed in many earlier non-premagnetized experiments. The helical structure persisted throughout the implosion, even though the azimuthal drive field greatly exceeded the expected axial field at the liner's outer wall for all but the earliest stages of the experiment. Whether this modified instability structure has practical importance for magneto-inertial fusion concepts depends primarily on whether the modified instability structure is more stable than standard azimuthally correlated MRT instabilities. In this manuscript, we discuss the evolution of the helix-like instability observed on premagnetized liners. While a first principles explanation of this observation remains elusive, recent 3D simulations suggest that if a small amplitude helical perturbation can be seeded on the liner's outer surface, no further influence from the axial field is required for the instability to grow.

  4. A Gly-zipper motif mediates homodimerization of the transmembrane domain of the mitochondrial kinase ADCK3.

    PubMed

    Khadria, Ambalika S; Mueller, Benjamin K; Stefely, Jonathan A; Tan, Chin Huat; Pagliarini, David J; Senes, Alessandro

    2014-10-01

    Interactions between ?-helices within the hydrophobic environment of lipid bilayers are integral to the folding and function of transmembrane proteins; however, the major forces that mediate these interactions remain debated, and our ability to predict these interactions is still largely untested. We recently demonstrated that the frequent transmembrane association motif GASright, the GxxxG-containing fold of the glycophorin A dimer, is optimal for the formation of extended networks of C?-H hydrogen bonds, supporting the hypothesis that these bonds are major contributors to association. We also found that optimization of C?-H hydrogen bonding and interhelical packing is sufficient to computationally predict the structure of known GASright dimers at near atomic level. Here, we demonstrate that this computational method can be used to characterize the structure of a protein not previously known to dimerize, by predicting and validating the transmembrane dimer of ADCK3, a mitochondrial kinase. ADCK3 is involved in the biosynthesis of the redox active lipid, ubiquinone, and human ADCK3 mutations cause a cerebellar ataxia associated with ubiquinone deficiency, but the biochemical functions of ADCK3 remain largely undefined. Our experimental analyses show that the transmembrane helix of ADCK3 oligomerizes, with an interface based on an extended Gly-zipper motif, as predicted by our models. The data provide strong evidence for the hypothesis that optimization of C?-H hydrogen bonding is an important factor in the association of transmembrane helices. This work also provides a structural foundation for investigating the role of transmembrane association in regulating the biological activity of ADCK3. PMID:25216398

  5. Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

    PubMed Central

    Sarker, Muzaddid; de Antueno, Roberto; Langelaan, David N.; Parmar, Hiren B.; Shin, Kyungsoo; Rainey, Jan K.; Duncan, Roy

    2015-01-01

    Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation. PMID:26061049

  6. Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA.

    PubMed

    Bartley, Laura E; Zhuang, Xiaowei; Das, Rhiju; Chu, Steven; Herschlag, Daniel

    2003-05-16

    Docking of the P1 duplex into the pre-folded core of the Tetrahymena group I ribozyme exemplifies the formation of tertiary interactions in the context of a complex, structured RNA. We have applied Phi-analysis to P1 docking, which compares the effects of modifications on the rate constant for docking (k(dock)) with the effects on the docking equilibrium (K(dock)). To accomplish this we used a single molecule fluorescence resonance energy transfer assay that allows direct determination of the rate constants for formation of thermodynamically favorable, as well as unfavorable, states. Modification of the eight groups of the P1 duplex that make tertiary interactions with the core and changes in solution conditions decrease K(dock) up to 500-fold, whereas k(dock) changes by structure formation event and suggest that large, highly structured RNAs may have local regions that are misordered. PMID:12729738

  7. Human ?,?-enoyl-CoA isomerase, type 2: a structural enzymology study on the catalytic role of its ACBP domain and helix-10.

    PubMed

    Onwukwe, Goodluck U; Kursula, Petri; Koski, M Kristian; Schmitz, Werner; Wierenga, Rik K

    2015-02-01

    The catalytic domain of the trimeric human ?(3),?(2)-enoyl-CoA isomerase, type 2 (HsECI2), has the typical crotonase fold. In the active site of this fold two main chain NH groups form an oxyanion hole for binding the thioester oxygen of the 3E- or 3Z-enoyl-CoA substrate molecules. A catalytic glutamate is essential for the proton transfer between the substrate C2 and C4 atoms for forming the product 2E-enoyl-CoA, which is a key intermediate in the ?-oxidation pathway. The active site is covered by the C-terminal helix-10. In HsECI2, the isomerase domain is extended at its N terminus by an acyl-CoA binding protein (ACBP) domain. Small angle X-ray scattering analysis of HsECI2 shows that the ACBP domain protrudes out of the central isomerase trimer. X-ray crystallography of the isomerase domain trimer identifies the active site geometry. A tunnel, shaped by loop-2 and extending from the catalytic site to bulk solvent, suggests a likely mode of binding of the fatty acyl chains. Calorimetry data show that the separately expressed ACBP and isomerase domains bind tightly to fatty acyl-CoA molecules. The truncated isomerase variant (without ACBP domain) has significant enoyl-CoA isomerase activity; however, the full-length isomerase is more efficient. Structural enzymological studies of helix-10 variants show the importance of this helix for efficient catalysis. Its hydrophobic side chains, together with residues from loop-2 and loop-4, complete a hydrophobic cluster that covers the active site, thereby fixing the thioester moiety in a mode of binding competent for efficient catalysis. PMID:25515061

  8. Helical hairpin structure of influenza hemagglutinin fusion peptide stabilized by charge-dipole interactions between the N-terminal amino group and the second helix.

    PubMed

    Lorieau, Justin L; Louis, John M; Bax, Ad

    2011-03-01

    The fusion domain of the influenza coat protein hemagglutinin HA2, bound to dodecyl phosphocholine micelles, was recently shown to adopt a structure consisting of two antiparallel α-helices, packed in an exceptionally tight hairpin configuration. Four interhelical H(α) to C═O aliphatic H-bonds were identified as factors stabilizing this fold. Here, we report evidence for an additional stabilizing force: a strong charge-dipole interaction between the N-terminal Gly(1) amino group and the dipole moment of helix 2. pH titration of the amino-terminal (15)N resonance, using a methylene-TROSY-based 3D NMR experiment, and observation of Gly(1 13)C' show a strongly elevated pK = 8.8, considerably higher than expected for an N-terminal amino group in a lipophilic environment. Chemical shifts of three C-terminal carbonyl carbons of helix 2 titrate with the protonation state of Gly(1)-N, indicative of a close proximity between the N-terminal amino group and the axis of helix 2, providing an optimal charge-dipole stabilization of the antiparallel hairpin fold. pK values of the side-chain carboxylate groups of Glu(11) and Asp(19) are higher by about 1 and 0.5 unit, respectively, than commonly seen for solvent-exposed side chains in water-soluble proteins, indicative of dielectric constants of ε = ∼30 (Glu(11)) and ∼60 (Asp(19)), placing these groups in the headgroup region of the phospholipid micelle. PMID:21319795

  9. Contributions of the C-terminal helix to the structural stability of a hyperthermophilic Fe-superoxide dismutase (TcSOD).

    PubMed

    Wang, Sha; Yan, Yong-Bin; Dong, Zhi-Yang

    2009-12-01

    Hyperthermophilic superoxide dismutases (SODs) are of particular interest due to their potential industrial importance and scientific merit in studying the molecular mechanisms of protein folding and stability. Compared to the mesophilic SODs, the hyperthermostable Fe-SODs (TcSOD and ApSOD) have an extended C-terminal helix, which forms an additional ion-pairing network. In this research, the role of the extended C-terminus in the structural stability of TcSOD was studied by investigating the properties of two deletion mutants. The results indicated that the ion-pairing network at the C-terminus had limited contributions to the stability of TcSOD against heat- and GdnHCl-induced inactivation. The intactness of the C-terminal helix had dissimilar impact on the two stages of TcSOD unfolding induced by guanidinium chloride. The mutations slightly decreased the Gibbs free energy of the dissociation of the tetrameric enzymes, while greatly affected the stability of the molten globule-like intermediate. These results suggested that the additional ion-pairing network mainly enhanced the structural stability of TcSOD by stabilizing the monomers. PMID:20054483

  10. Protein secondary structures (alpha-helix and beta-sheet) at a cellular level and protein fractions in relation to rumen degradation behaviours of protein: a new approach.

    PubMed

    Yu, Peiqiang

    2005-11-01

    Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the alpha-helix and beta-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of beta-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution ( approximately 10 microm). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of alpha-helixes and beta-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P<0.05) the percentage of alpha-helixes (from 47.1 % to 36.1 %: S-FTIR absorption intensity), increased the percentage of beta-sheets (from 37.2 % to 49.8 %: S-FTIR absorption intensity) and reduced the alpha-helix to beta-sheet ratio (from 0.3 to 0.7) in the golden flaxseeds, which indicated a negative effect of the roasting on protein values, utilisation and bioavailability. These results were proved by the Cornell Net Carbohydrate Protein System in situ animal trial, which also revealed that roasting increased the amount of protein bound to lignin, and well as of the Maillard reaction protein (both of which are poorly used by ruminants), and increased the level of indigestible and undegradable protein in ruminants. The present results demonstrate the potential of highly spatially resolved synchrotron-based infrared microspectroscopy to locate 'pure' protein in feed tissues, and reveal protein secondary structures and digestive behaviour, making a significant step forward in and an important contribution to protein nutritional research. Further study is needed to determine the sensitivities of protein secondary structures to various heat-processing conditions, and to quantify the relationship between protein secondary structures and the nutrient availability and digestive behaviour of various protein sources. Information from the present study arising from the synchrotron-based IR probing of the protein secondary structures of protein sources at the cellular level will be valuable as a guide to maintaining protein quality and predicting digestive behaviours. PMID:16277766

  11. Protein Secondary Structures (alpha-helix and beta-sheet) at a Cellular Levle and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    SciTech Connect

    Yu,P.

    2007-01-01

    Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the {alpha}-helix and {beta}-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of {beta}-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution ({approx}10 {mu}m). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of {alpha}-helixes and {beta}-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of {alpha}-helixes (from 47.1% to 36.1%: S-FTIR absorption intensity), increased the percentage of {beta}-sheets (from 37.2% to 49.8%: S-FTIR absorption intensity) and reduced the {alpha}-helix to {beta}-sheet ratio (from 0.3 to 0.7) in the golden flaxseeds, which indicated a negative effect of the roasting on protein values, utilisation and bioavailability. These results were proved by the Cornell Net Carbohydrate Protein System in situ animal trial, which also revealed that roasting increased the amount of protein bound to lignin, and well as of the Maillard reaction protein (both of which are poorly used by ruminants), and increased the level of indigestible and undegradable protein in ruminants. The present results demonstrate the potential of highly spatially resolved synchrotron-based infrared microspectroscopy to locate 'pure' protein in feed tissues, and reveal protein secondary structures and digestive behaviour, making a significant step forward in and an important contribution to protein nutritional research. Further study is needed to determine the sensitivities of protein secondary structures to various heat-processing conditions, and to quantify the relationship between protein secondary structures and the nutrient availability and digestive behaviour of various protein sources. Information from the present study arising from the synchrotron-based IR probing of the protein secondary structures of protein sources at the cellular level will be valuable as a guide to maintaining protein quality and predicting digestive behaviours.

  12. Crystal structure of the N-terminal region of human Ash2L shows a winged-helix motif involved in DNA binding

    SciTech Connect

    Chen, Yong; Wan, Bingbing; Wang, Kevin C.; Cao, Fang; Yang, Yuting; Protacio, Angeline; Dou, Yali; Chang, Howard Y.; Lei, Ming

    2011-09-06

    Ash2L is a core component of the MLL family histone methyltransferases and has an important role in regulating the methylation of histone H3 on lysine 4. Here, we report the crystal structure of the N-terminal domain of Ash2L and reveal a new function of Ash2L. The structure shows that Ash2L contains an atypical PHD finger that does not have histone tail-binding activity. Unexpectedly, the structure shows a previously unrecognized winged-helix motif that directly binds to DNA. The DNA-binding-deficient mutants of Ash2L reduced Ash2L localization to the HOX locus. Strikingly, a single mutation in Ash2L{sub WH} (K131A) breaks the chromatin domain boundary, suggesting that Ash2L also has a role in chromosome demarcation.

  13. The crystal structure of GXGD membrane protease FlaK

    SciTech Connect

    Hu, Jian; Xue, Yi; Lee, Sangwon; Ha, Ya

    2011-09-20

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  14. The Crystal Structure of GXGD Membrane Protease FlaK

    SciTech Connect

    J Hu; Y Xue; S Lee; Y Ha

    2011-12-31

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  15. Revisting the Double Helix

    SciTech Connect

    Ha, Taekjip

    2010-12-08

    Properties of DNA double helix have been studied for over 60 years. Yet as more sensitive tools become available, fundamental assumptions in our understanding of these properties are being challenged. One such question is over the flexibility of DNA. Looping or bending of DNA on short length scales is essential for many cellular processes but it is highly controversial exactly how flexible the DNA is. Using a new, single-molecule based method, we found that DNA of lengths as short as 50 base pairs can form a circle more than 108 times faster than theoretical predictions. Another question concerns the physical principles governing the reversible, helix-coil transitions of DNA between the double helix and single strands. Using porous nanocontainers, we found that the rate of double helix formation shows an abrupt 100 fold change depending on whether there are 7 or more contiguous base pairs or not.

  16. A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2)

    PubMed Central

    Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

    2016-01-01

    Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. PMID:26797119

  17. Structure of the unique SEFIR domain from human interleukin 17 receptor A reveals a composite ligand-binding site containing a conserved α-helix for Act1 binding and IL-17 signaling

    SciTech Connect

    Zhang, Bing; Liu, Caini; Qian, Wen; Han, Yue; Li, Xiaoxia; Deng, Junpeng

    2014-05-01

    Crystal structure of the SEFIR domain from human IL-17 receptor A provides new insights into IL-17 signaling. Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand βC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC′{sub ins}) and a flexible loop (CC′). The DD′ loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Å to accommodate the αCC′{sub ins} helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling.

  18. Alternative packing of EGFR transmembrane domain suggests that protein-lipid interactions underlie signal conduction across membrane.

    PubMed

    Bocharov, Eduard V; Lesovoy, Dmitry M; Pavlov, Konstantin V; Pustovalova, Yulia E; Bocharova, Olga V; Arseniev, Alexander S

    2016-06-01

    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. PMID:26903218

  19. A Helix Replacement Mechanism Directs Metavinculin Functions

    SciTech Connect

    Rangarajan, Erumbi S.; Lee, Jun Hyuck; Yogesha, S.D.; Izard, Tina

    2010-10-11

    Cells require distinct adhesion complexes to form contacts with their neighbors or the extracellular matrix, and vinculin links these complexes to the actin cytoskeleton. Metavinculin, an isoform of vinculin that harbors a unique 68-residue insert in its tail domain, has distinct actin bundling and oligomerization properties and plays essential roles in muscle development and homeostasis. Moreover, patients with sporadic or familial mutations in the metavinculin-specific insert invariably develop fatal cardiomyopathies. Here we report the high resolution crystal structure of the metavinculin tail domain, as well as the crystal structures of full-length human native metavinculin (1,134 residues) and of the full-length cardiomyopathy-associated {Delta}Leu954 metavinculin deletion mutant. These structures reveal that an {alpha}-helix (H1{prime}) and extended coil of the metavinculin insert replace {alpha}-helix H1 and its preceding extended coil found in the N-terminal region of the vinculin tail domain to form a new five-helix bundle tail domain. Further, biochemical analyses demonstrate that this helix replacement directs the distinct actin bundling and oligomerization properties of metavinculin. Finally, the cardiomyopathy associated {Delta}Leu954 and Arg975Trp metavinculin mutants reside on the replaced extended coil and the H1{prime} {alpha}-helix, respectively. Thus, a helix replacement mechanism directs metavinculin's unique functions.

  20. SU-E-T-241: Monte Carlo Simulation Study About the Prediction of Proton-Induced DNA Strand Breakage On the Double Helix Structure

    SciTech Connect

    Shin, J; Park, S; Jeong, J; Jeong, C; Lim, Y; Lee, S; SHIN, D; Incerti, S

    2014-06-01

    Purpose: In particle therapy and radiobiology, the investigation of mechanisms leading to the death of target cancer cells induced by ionising radiation is an active field of research. Recently, several studies based on Monte Carlo simulation codes have been initiated in order to simulate physical interactions of ionising particles at cellular scale and in DNA. Geant4-DNA is the one of them; it is an extension of the general purpose Geant4 Monte Carlo simulation toolkit for the simulation of physical interactions at sub-micrometre scale. In this study, we present Geant4-DNA Monte Carlo simulations for the prediction of DNA strand breakage using a geometrical modelling of DNA structure. Methods: For the simulation of DNA strand breakage, we developed a specific DNA geometrical structure. This structure consists of DNA components, such as the deoxynucleotide pairs, the DNA double helix, the nucleosomes and the chromatin fibre. Each component is made of water because the cross sections models currently available in Geant4-DNA for protons apply to liquid water only. Also, at the macroscopic-scale, protons were generated with various energies available for proton therapy at the National Cancer Center, obtained using validated proton beam simulations developed in previous studies. These multi-scale simulations were combined for the validation of Geant4-DNA in radiobiology. Results: In the double helix structure, the deposited energy in a strand allowed to determine direct DNA damage from physical interaction. In other words, the amount of dose and frequency of damage in microscopic geometries was related to direct radiobiological effect. Conclusion: In this report, we calculated the frequency of DNA strand breakage using Geant4- DNA physics processes for liquid water. This study is now on-going in order to develop geometries which use realistic DNA material, instead of liquid water. This will be tested as soon as cross sections for DNA material become available in Geant4-DNA.

  1. Nuclear Magnetic Resonance Structure Revealed that the Human Polyomavirus JC Virus Agnoprotein Contains an α-Helix Encompassing the Leu/Ile/Phe-Rich Domain

    PubMed Central

    Coric, Pascale; Saribas, A. Sami; Abou-Gharbia, Magid; Childers, Wayne; White, Martyn K.

    2014-01-01

    ABSTRACT Agnoprotein is a small multifunctional regulatory protein required for sustaining the productive replication of JC virus (JCV). It is a mostly cytoplasmic protein localizing in the perinuclear area and forms highly stable dimers/oligomers through a Leu/Ile/Phe-rich domain. There have been no three-dimensional structural data available for agnoprotein due to difficulties associated with the dynamic conversion from monomers to oligomers. Here, we report the first nuclear magnetic resonance (NMR) structure of a synthetic agnoprotein peptide spanning amino acids Thr17 to Glu55 where Lys23 to Phe39 encompassing the Leu/Ile/Phe-rich domain forms an amphipathic α-helix. On the basis of these structural data, a number of Ala substitution mutations were made to investigate the role of the α-helix in the structure and function of agnoprotein. Single L29A and L36A mutations exhibited a significant negative effect on both protein stability and viral replication, whereas the L32A mutation did not. In addition, the L29A mutant displayed a highly nuclear localization pattern, in contrast to the pattern for the wild type (WT). Interestingly, a triple mutant, the L29A+L32A+L36A mutant, yielded no detectable agnoprotein expression, and the replication of this JCV mutant was significantly reduced, suggesting that Leu29 and Leu36 are located at the dimer interface, contributing to the structure and stability of agnoprotein. Two other single mutations, L33A and E34A, did not perturb agnoprotein stability as drastically as that observed with the L29A and L36A mutations, but they negatively affected viral replication, suggesting that the role of these residues is functional rather than structural. Thus, the agnoprotein dimerization domain can be targeted for the development of novel drugs active against JCV infection. IMPORTANCE Agnoprotein is a small regulatory protein of JC virus (JCV) and is required for the successful completion of the viral replication cycle. It forms highly stable dimers and oligomers through its hydrophobic (Leu/Ile/Phe-rich) domain, which has been shown to play essential roles in the stability and function of the protein. In this work, the Leu/Ile/Phe-rich domain has been further characterized by NMR studies using an agnoprotein peptide spanning amino acids T17 to Q54. Those studies revealed that the dimerization domain of the protein forms an amphipathic α-helix. Subsequent NMR structure-based mutational analysis of the region highlighted the critical importance of certain amino acids within the α-helix for the stability and function of agnoprotein. In conclusion, this study provides a solid foundation for developing effective therapeutic approaches against the dimerization domain of the protein to inhibit its critical roles in JCV infection. PMID:24672035

  2. Structure Determination of a Membrane Protein with Two Trans-membrane Helices in Aligned Phospholipid Bicelles by Solid-state NMR Spectroscopy

    PubMed Central

    De Angelis, Anna A.; Howell, Stanley C.; Nevzorov, Alexander A.; Opella, Stanley J.

    2011-01-01

    The structure of the membrane protein MerFt was determined in magnetically aligned phospholipid bicelles by solid-state NMR spectroscopy. With two trans-membrane helices and a 10-residue inter-helical loop, this truncated construct of the mercury transport membrane protein MerF has sufficient structural complexity to demonstrate the feasibility of determining the structures of polytopic membrane proteins in their native phospholipid bilayer environment under physiological conditions. PISEMA, SAMMY, and other double-resonance experiments were applied to uniformly and selectively 15N labeled samples to resolve and assign the backbone amide resonances, and to measure the associated 15N chemical shift and 1H-15N heteronuclear dipolar coupling frequencies as orientation constraints for structure calculations. 1H/13C/15N triple-resonance experiments were applied to selectively 13C′ and 15N labeled samples to complete the resonance assignments, especially for residues in the non-helical regions of the protein. A single resonance is observed for each labeled site in one- and two-dimensional spectra. Therefore, each residue has a unique conformation, and all protein molecules in the sample have the same three-dimensional structure and are oriented identically in planar phospholipid bilayers. Combined with the absence of significant intensity near the isotropic resonance frequency, this demonstrates that the entire protein, including the loop and terminal regions, has a well-defined, stable structure in phospholipid bilayers. PMID:16967977

  3. The Atlastin C-terminal Tail Is an Amphipathic Helix That Perturbs the Bilayer Structure during Endoplasmic Reticulum Homotypic Fusion

    PubMed Central

    Faust, Joseph E.; Desai, Tanvi; Verma, Avani; Ulengin, Idil; Sun, Tzu-Lin; Moss, Tyler J.; Betancourt-Solis, Miguel A.; Huang, Huey W.; Lee, Tina; McNew, James A.

    2015-01-01

    Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel ? helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion. PMID:25555915

  4. Structural changes in single membranes in response to an applied transmembrane electric potential revealed by time-resolved neutron/X-ray interferometry

    PubMed Central

    Tronin, A.; Chen, C-H.; Gupta, S.; Worcester, D.; Lauter, V.; Strzalka, J.; Kuzmenko, I.; Blasie, J. K.

    2013-01-01

    The profile structure of a hybrid lipid bilayer, tethered to the surface of an inorganic substrate and fully hydrated with a bulk aqueous medium in an electrochemical cell, was investigated as a function of the applied transbilayer electric potential via time-resolved neutron reflectivity, enhanced by interferometry. Significant, and fully reversible structural changes were observed in the distal half (with respect to the substrate surface) of the hybrid bilayer comprised of a zwitterionic phospholipid in response to a +100mV potential with respect to 0mV. These arise presumably due to reorientation of the electric dipole present in the polar headgroup of the phospholipid and its resulting effect on the thickness of the phospholipid’s hydrocarbon chain layer within the hybrid bilayer’s profile structure. The profile structure of the voltage-sensor domain from a voltage-gated ion channel protein within a phospholipid bilayer membrane, tethered to the surface of an inorganic substrate and fully hydrated with a bulk aqueous medium in an electrochemical cell, was also investigated as a function of the applied transmembrane electric potential via time-resolved X-ray reflectivity, enhanced by interferometry. Significant, fully-reversible, and different structural changes in the protein were detected in response to ±100mV potentials with respect to 0mV. The approach employed is that typical of transient spectroscopy, shown here to be applicable to both neutron and X-ray reflectivity of thin films. PMID:24222930

  5. Helix mimetics: Recent developments.

    PubMed

    Wilson, Andrew J

    2015-10-01

    The development of protein-protein interaction (PPIs) inhibitors represents a challenging goal in chemical biology and drug discovery. PPIs are problematic targets because they involve large surfaces with less well defined features and recognition motifs that are less amenable to conventional experimental and computational ligand discovery methodologies. α-Helix mediated PPIs represent a sub group with a clearly defined interface and thus may be more amenable to the development of generic ligand discovery methods. Indeed, this is borne out in numerous studies using peptides covalently constrained into a helical conformation resulting in improvement of myriad biophysical and cellular properties. It is however desirable to have small molecule alternatives: a helix mimetic (proteomimetic) is a generic small molecule scaffold that projects functional groups in a similar spatial orientation so as to mimic the presentation of key amino acid side chains from the helix that mediates the PPI. The first true example of a helix mimetic was described over a decade ago however this approach has not yet been elaborated to the extent that it receives similar levels of attention to constrained peptides. This review explores recent significant developments in the area of small molecule α-helix mimetics and provides a critical overview of success stories, potential limitations of the approach, and areas for future development. PMID:26048776

  6. Nonaxiality in infrared dichroic ratios of polytopic transmembrane proteins.

    PubMed Central

    Marsh, D

    1998-01-01

    In polytopic alpha-helical transmembrane proteins, the distribution of amide vibrational transition moments can be nonaxial, if the helix axes are tilted relative to the symmetry axis of the helix bundle. The infrared dichroic ratios from oriented samples then contain nonaxial terms and, in the most general case, require a second-order parameter for the axis of the helix bundle. The extent of nonaxiality depends on the summation over the individual amide transition moments along the helix. Because this is strongly oscillatory, with a 3.6-residue periodicity, complete axial symmetry is not achieved rapidly on progressive summation. Expressions for the contributions of residual nonaxiality to the dichroic ratios are derived. A similar situation arises for oligomers of transmembrane beta-barrel proteins, e.g., the porin trimer. In this case, the extent of nonaxiality depends not only on the number of residues in the beta-barrel, but also on the tilt of the beta-strands relative to the barrel axis and the characteristic dimensions of a beta-sheet, which together determine the axial periodicity. The nonaxial contributions to the dichroic ratios of beta-barrel oligomers are also derived. Estimates are given of the likely size of the nonaxial contributions for the different alpha-helical and beta-sheet systems. PMID:9649392

  7. Elongation factor G-induced structural change in helix 34 of 16S rRNA related to translocation on the ribosome.

    PubMed Central

    Matassova, A B; Rodnina, M V; Wintermeyer, W

    2001-01-01

    During the translocation step of the elongation cycle, two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. The movement is catalyzed by the binding of elongation factor G (EF-G) and driven by GTP hydrolysis. Here we study structural changes of the ribosome related to EF-G binding and translocation by monitoring the accessibility of ribosomal RNA (rRNA) for chemical modification by dimethyl sulfate or cleavage by hydroxyl radicals generated by Fe(II)-EDTA. In the state of the ribosome that is formed upon binding of EF-G but before the movement of the tRNAs takes place, residues 1054,1196, and 1201 in helix 34 in 16S rRNA are strongly protected. The protections depend on EF-G binding, but do not require GTP hydrolysis, and are lost upon translocation. Mutants of EF-G, which are active in ribosome binding and GTP hydrolysis but impaired in translocation, do not bring about the protections. According to cryo-electron microscopy (Stark et al., Cell, 2000, 100:301-309), there is no contact of EF-G with the protected residues of helix 34 in the pretranslocation state, suggesting that the observed protections are due to an induced conformational change. Thus, the present results indicate that EF-G binding to the pretranslocation ribosome induces a structural change of the head of the 30S subunit that is essential for subsequent tRNA-mRNA movement in translocation. PMID:11780642

  8. Double-helix stellarator

    SciTech Connect

    Moroz, P.E.

    1997-09-01

    A new stellarator configuration, the Double-Helix Stellarator (DHS), is introduced. This novel configuration features a double-helix center post as the only helical element of the stellarator coil system. The DHS configuration has many unique characteristics. One of them is the extreme low plasma aspect ratio, A {approx} 1--1.2. Other advantages include a high enclosed volume, appreciable rotational transform, and a possibility of extreme-high-{beta} MHD equilibria. Moreover, the DHS features improved transport characteristics caused by the absence of the magnetic field ripple on the outboard of the torus. Compactness, simplicity and modularity of the coil system add to the DHS advantages for fusion applications.

  9. Structural details (kinks and non-α conformations) in transmembrane helices are intrahelically determined and can be predicted by sequence pattern descriptors

    PubMed Central

    Rigoutsos, Isidore; Riek, Peter; Graham, Robert M.; Novotny, Jiri

    2003-01-01

    One of the promising methods of protein structure prediction involves the use of amino acid sequence-derived patterns. Here we report on the creation of non-degenerate motif descriptors derived through data mining of training sets of residues taken from the transmembrane-spanning segments of polytopic proteins. These residues correspond to short regions in which there is a deviation from the regular α-helical character (i.e. π-helices, 310-helices and kinks). A ‘search engine’ derived from these motif descriptors correctly identifies, and discriminates amongst instances of the above ‘non-canonical’ helical motifs contained in the SwissProt/TrEMBL database of protein primary structures. Our results suggest that deviations from α-helicity are encoded locally in sequence patterns only about 7–9 residues long and can be determined in silico directly from the amino acid sequence. Delineation of such variations in helical habit is critical to understanding the complex structure–function relationships of polytopic proteins and for drug discovery. The success of our current methodology foretells development of similar prediction tools capable of identifying other structural motifs from sequence alone. The method described here has been implemented and is available on the World Wide Web at http://cbcsrv.watson.ibm.com/Ttkw.html. PMID:12888523

  10. Probing the Transmembrane Structure and Topology of Microsomal Cytochrome-P450 by Solid-State NMR on Temperature-Resistant Bicelles

    PubMed Central

    Yamamoto, Kazutoshi; Gildenberg, Melissa; Ahuja, Shivani; Im, Sang-Choul; Pearcy, Paige; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2013-01-01

    Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochromeP4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal. PMID:23989972

  11. Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor

    SciTech Connect

    Bahl, C.; Morisseau, C; Bomberger, J; Stanton, B; Hammock, B; O' Toole, G; Madden, D

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other {alpha}/{beta} hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-{angstrom} resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of {alpha}/{beta} hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.

  12. Structural Determinants of Nitroxide Motion in Spin-Labeled Proteins: Solvent-Exposed Sites in Helix B of T4 Lysozyme

    SciTech Connect

    Guo,Z.; Cascio, D.; Hideg, K.; Hubbell, W.

    2008-01-01

    Site-directed spin labeling provides a means for exploring structure and dynamics in proteins. To interpret the complex EPR spectra that often arise, it is necessary to characterize the rotamers of the spin-labeled side chain and the interactions they make with the local environment in proteins of known structure. For this purpose, crystal structures have been determined for T4 lysozyme bearing a nitroxide side chain (R1) at the solvent-exposed helical sites 41 and 44 in the B helix. These sites are of particular interest in that the corresponding EPR spectra reveal two dynamic states of R1, one of which is relatively immobilized suggesting interactions of the nitroxide with the environment. The crystal structures together with the effect of mutagenesis of nearest neighbors on the motion of R1 suggest intrahelical interactions of 41R1 with the i + 4 residue and of 44R1 with the i + 1 residue. Such interactions appear to be specific to particular rotamers of the R1 side chain.

  13. A small nonerythropoietic helix B surface peptide based upon erythropoietin structure is cardioprotective against ischemic myocardial damage.

    PubMed

    Ahmet, Ismayil; Tae, Hyun-Jin; Juhaszova, Magdalena; Riordon, Daniel R; Boheler, Kenneth R; Sollott, Steven J; Brines, Michael; Cerami, Anthony; Lakatta, Edward G; Talan, Mark I

    2011-01-01

    Strong cardioprotective properties of erythropoietin (EPO) reported over the last 10 years have been difficult to translate to clinical applications for ischemic cardioprotection owing to undesirable parallel activation of erythropoiesis and thrombogenesis. A pyroglutamate helix B surface peptide (pHBP), recently engineered to include only a part of the EPO molecule that does not bind to EPO receptor and thus, is not erythropoietic, retains tissue protective properties of EPO. Here we compared the ability of pHBP and EPO to protect cardiac myocytes from oxidative stress in vitro and cardiac tissue from ischemic damage in vivo. HBP, similar to EPO, increased the reactive oxygen species (ROS) threshold for induction of the mitochondrial permeability transition by 40%. In an experimental model of myocardial infarction induced by permanent ligation of a coronary artery in rats, a single bolus injection of 60 μg/kg of pHBP immediately after coronary ligation, similar to EPO, reduced apoptosis in the myocardial area at risk, examined 24 h later, by 80% and inflammation by 34%. Myocardial infarction (MI) measured 24 h after coronary ligation was similarly reduced by 50% in both pHBP- and EPO-treated rats. Two wks after surgery, left ventricular remodeling (ventricular dilation) and functional decline (fall in ejection fraction) assessed by echocardiography were significantly and similarly attenuated in pHBP- and EPO-treated rats, and MI size was reduced by 25%. The effect was retained during the 6-wk follow-up. A single bolus injection of pHBP immediately after coronary ligation was effective in reduction of MI size in a dose as low as 1 μg/kg, but was ineffective at a 60 μg/kg dose if administered 24 h after MI induction. We conclude that pHBP is equally cardioprotective with EPO and deserves further consideration as a safer alternative to rhEPO in the search for therapeutic options to reduce myocardial damage following blockade of the coronary circulation. PMID:21170473

  14. Double Helix Revisited.

    ERIC Educational Resources Information Center

    Glickstein, Neil M.

    1995-01-01

    Describes the use of James Watson's book, "The Double Helix," as a multidisciplinary way of introducing students to actual science; the scientific method; dilemmas encountered in the world of research; and the rich setting of personalities, politics, and history in post-World War II Europe. (MKR)

  15. Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain

    NASA Technical Reports Server (NTRS)

    Curran, A. C.; Hwang, I.; Corbin, J.; Martinez, S.; Rayle, D.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.

  16. Role of GxxxG Motifs in Transmembrane Domain Interactions.

    PubMed

    Teese, Mark G; Langosch, Dieter

    2015-08-25

    Transmembrane (TM) helices of integral membrane proteins can facilitate strong and specific noncovalent protein-protein interactions. Mutagenesis and structural analyses have revealed numerous examples in which the interaction between TM helices of single-pass membrane proteins is dependent on a GxxxG or (small)xxx(small) motif. It is therefore tempting to use the presence of these simple motifs as an indicator of TM helix interactions. In this Current Topic review, we point out that these motifs are quite common, with more than 50% of single-pass TM domains containing a (small)xxx(small) motif. However, the actual interaction strength of motif-containing helices depends strongly on sequence context and membrane properties. In addition, recent studies have revealed several GxxxG-containing TM domains that interact via alternative interfaces involving hydrophobic, polar, aromatic, or even ionizable residues that do not form recognizable motifs. In multipass membrane proteins, GxxxG motifs can be important for protein folding, and not just oligomerization. Our current knowledge thus suggests that the presence of a GxxxG motif alone is a weak predictor of protein dimerization in the membrane. PMID:26244771

  17. A left-hand beta-helix revealed by the crystal structure of a carbonic anhydrase from the archaeon Methanosarcina thermophila.

    PubMed Central

    Kisker, C; Schindelin, H; Alber, B E; Ferry, J G; Rees, D C

    1996-01-01

    A carbonic anhydrase from the thermophilic archaeon Methanosarcina thermophila that exhibits no significant sequence similarity to known carbonic anhydrases has recently been characterized. Here we present the structure of this enzyme, which adopts a left-handed parallel beta-helix fold. This fold is of particular interest since it contains only left-handed crossover connections between the parallel beta-strands, which so far have been observed very infrequently. The active form of the enzyme is a trimer with three zinc-containing active sites, each located at the interface between two monomers. While the arrangement of active site groups differs between this enzyme and the carbonic anhydrases from higher vertebrates, there are structural similarities in the zinc coordination environment, suggestive of convergent evolution dictated by the chemical requirements for catalysis of the same reaction. Based on sequence similarities, the structure of this enzyme is the prototype of a new class of carbonic anhydrases with representatives in all three phylogenetic domains of life. Images PMID:8665839

  18. Intra-helical salt-bridge and helix destabilizing residues within the same helical turn: Role of functionally important loop E half-helix in channel regulation of major intrinsic proteins.

    PubMed

    Verma, Ravi Kumar; Prabh, Neel Duti; Sankararamakrishnan, Ramasubbu

    2015-06-01

    The superfamily of major intrinsic proteins (MIPs) includes aquaporin (AQP) and aquaglyceroporin (AQGP) and it is involved in the transport of water and neutral solutes across the membrane. Diverse MIP sequences adopt a unique hour-glass fold with six transmembrane helices (TM1 to TM6) and two half-helices (LB and LE). Loop E contains one of the two conserved NPA motifs and contributes two residues to the aromatic/arginine selectivity filter. Function and regulation of majority of MIP channels are not yet characterized. We have analyzed the loop E region of 1468 MIP sequences and their structural models from six different organism groups. They can be phylogenetically clustered into AQGPs, AQPs, plant MIPs and other MIPs. The LE half-helix in all AQGPs contains an intra-helical salt-bridge and helix-breaking residues Gly/Pro within the same helical turn. All non-AQGPs lack this salt-bridge but have the helix destabilizing Gly and/or Pro in the same positions. However, the segment connecting LE half-helix and TM6 is longer by 10-15 residues in AQGPs compared to all non-AQGPs. We speculate that this longer loop in AQGPs and the LE half-helix of non-AQGPs will be relatively more flexible and this could be functionally important. Molecular dynamics simulations on glycerol-specific GlpF, water-transporting AQP1, its mutant and a fungal AQP channel confirm these predictions. Thus two distinct regions of loop E, one in AQGPs and the other in non-AQGPs, seem to be capable of modulating the transport. These regions can also act in conjunction with other extracellular residues/segments to regulate MIP channel transport. PMID:25797519

  19. Binding of the protein kinase PKR to RNAs with secondary structure defects: role of the tandem A-G mismatch and noncontiguous helixes.

    PubMed

    Bevilacqua, P C; George, C X; Samuel, C E; Cech, T R

    1998-05-01

    The human interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an antiviral agent that is activated by long stretches of dsRNA. PKR can also be activated or repressed by a series of cellular and viral RNAs containing non-Watson-Crick motifs. PKR has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). In vitro selection experiments were carried out to search for RNAs capable of binding to a truncated version of PKR containing the dsRBD. RNA ligands were selected by binding to His6-tagged proteins and chromatography on nickel(II) nitrilotriacetic acid agarose. A series of RNAs was selected that bind either similar to or tighter than a model dsRNA stem loop. Examination of these RNAs by a variety of methods, including sequence comparison, free-energy minimization, structure mapping, boundary experiments, site-directed mutagenesis, and footprinting, revealed protein-binding sites composed of noncontiguous helices. In addition, selected RNAs contained tandem A-G mismatches (5'AG3'/3'GA5'), yet bound to the truncated protein with affinities similar to duplexes containing only Watson-Crick base pairs. The NMR structure of the tandem A-G mismatch in an RNA helix (rGGCAGGCC)2 reveals a global A-form helix with minor perturbations at the mismatch [Wu, M., SantaLucia, J., Jr., and Turner, D. H. (1997) Biochemistry 36, 4449-4460]. This supports the notion that dsRBM-containing proteins can bind to RNAs with secondary structure defects as long as the RNA has an overall A-form geometry. In addition, selected RNAs are able to activate or repress wild-type PKR autophosphorylation as well as its phosphorylation of protein synthesis initiation factor eIF-2, suggesting full-length PKR can bind to and be regulated by RNAs containing a tandem A-G mismatch. PMID:9572845

  20. Characterization of a helix-loop-helix (EF hand) motif of silver hake parvalbumin isoform B.

    PubMed Central

    Revett, S. P.; King, G.; Shabanowitz, J.; Hunt, D. F.; Hartman, K. L.; Laue, T. M.; Nelson, D. J.

    1997-01-01

    Parvalbumins are a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. It is suspected that these proteins evolved via intragene duplication from a single EF-hand. Silver hake parvalbumin (SHPV) consists of three EF-type helix-loop-helix regions, two of which have the ability to bind calcium. The three helix-loop-helix motifs are designated AB, CD, and EF, respectively. In this study, native silver hake parvalbumin isoform B (SHPV-B) has been sequenced by mass spectrometry. The sequence indicates that this parvalbumin is a beta-lineage parvalbumin. SHPV-B was cleaved into two major fragments, consisting of the ABCD and EF regions of the native protein. The 33-amino acid EF fragment (residues 76-108), containing one of the calcium ion binding sites in native SHPV-B, has been isolated and studied for its structural characteristics, ability to bind divalent and trivalent cations, and for its propensity to undergo metal ion-induced self-association. The presence of Ca2+ does not induce significant secondary structure in the EF fragment. However, NMR and CD results indicate significant secondary structure promotion in the EF fragment in the presence of the higher charge-density trivalent cations. Sedimentation equilibrium analysis results show that the EF fragment exists in a monomer-dimer equilibrium when complexed with La3+. PMID:9385642

  1. Detection of drug-induced conformational change of a transmembrane protein in lipid bilayers using site-directed spin labeling

    PubMed Central

    Thomaston, Jessica L; Nguyen, Phuong A; Brown, Emily C; Upshur, Mary Alice; Wang, Jun; DeGrado, William F; Howard, Kathleen P

    2013-01-01

    As a target of antiviral drugs, the influenza A M2 protein has been the focus of numerous structural studies and has been extensively explored as a model ion channel. In this study, we capitalize on the expanding body of high-resolution structural data available for the M2 protein to design and interpret site-directed spin-labeling electron paramagnetic resonance spectroscopy experiments on drug-induced conformational changes of the M2 protein embedded in lipid bilayers. We obtained data in the presence of adamantane drugs for two different M2 constructs (M2TM 22–46 and M2TMC 23–60). M2TM peptides were spin labeled at the N-terminal end of the transmembrane domain. M2TMC peptides were spin labeled site specifically at cysteine residues substituted for amino acids within the transmembrane domain (L36, I39, I42, and L43) and the C-terminal amphipathic helix (L46, F47, F48, C50, I51, Y52, R53, F54, F55, and E56). Addition of adamantane drugs brought about significant changes in measured electron paramagnetic resonance spectroscopy environmental parameters consistent with narrowing of the transmembrane channel pore and closer packing of the C-terminal amphipathic helices. PMID:23139077

  2. Structural design and synthesis of arylalkynyl amide-type peroxisome proliferator-activated receptor γ (PPARγ)-selective antagonists based on the helix12-folding inhibition hypothesis.

    PubMed

    Ohashi, Masao; Gamo, Kanae; Tanaka, Yuta; Waki, Minoru; Beniyama, Yoko; Matsuno, Kenji; Wada, Jun; Tenta, Masafumi; Eguchi, Jun; Makishima, Makoto; Matsuura, Nobuyasu; Oyama, Takuji; Miyachi, Hiroyuki

    2015-01-27

    Peroxisome proliferator-activated receptor γ (PPARγ) antagonists are candidates for treatment of type 2 diabetes, obesity and osteoporosis. However, few rational design strategies are currently available. Here, we utilized the helix12 (H12)-folding inhibition hypothesis, in combination with our previously determined X-ray crystal structure of PPARγ agonist MEKT-21 (6) complexed with the PPARγ ligand-binding domain, to design and develop a potent phenylalkynyl amide-type PPARγ antagonist 9i, focusing initially on pinpoint structural modification of the propanoic acid moiety of 6. Since 9i retained very weak, but distinct, PPARγ agonist activity, we next modified the distal benzene ring of 9i, aiming to delete the residual PPARγ agonist activity while retaining the antagonist activity. Introduction of a chlorine atom at the 2-position of the distal benzene ring afforded 9p, which exhibited potent, PPARγ-selective full antagonist activity without detectable agonist activity. We found that 9p stabilized the corepressor-PPARγ complex and suppressed basal PPARγ activity. This compound showed anti-adipogenesis activity at the cellular level. This agonist-antagonist switching concept based on the H12-folding inhibition hypothesis should also be applicable for designing other classes of PPARγ full antagonists. PMID:25461311

  3. CHUK, a conserved helix-loop-helix ubiquitous kinase, maps to human chromosome 10 and mouse chromosome 19

    SciTech Connect

    Mock, B.A.; McBride, O.W.; Kozak, C.A.

    1995-05-20

    Helix-loop-helix proteins contain stretches of DNA that encode two amphipathic {alpha}-helices joined by a loop structure and are involved in protein dimerization and transcriptional regulation essential to a variety of cellular processes. CHUK, a newly described conserved helix-loop-helix ubiquitous kinase, was mapped by somatic cell hybrid analyses to human Chr 10q24-q25. Chuk and a related sequence, Chuk-rs1, were mapped to mouse chromosomes 19 and 16, respectively, by a combination of somatic cell hybrid, recombinant inbred, and backcross analyses. 17 refs., 2 figs., 1 tab.

  4. Impact of the [delta]F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure

    SciTech Connect

    Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B.; Hunt, John F.; Emtage, Spencer

    2010-07-19

    Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

  5. Synaptobrevin Transmembrane Domain Dimerization Studied by Multiscale Molecular Dynamics Simulations.

    PubMed

    Han, Jing; Pluhackova, Kristyna; Wassenaar, Tsjerk A; Bckmann, Rainer A

    2015-08-18

    Synaptic vesicle fusion requires assembly of the SNARE complex composed of SNAP-25, syntaxin-1, and synaptobrevin-2 (sybII) proteins. The SNARE proteins found in vesicle membranes have previously been shown to dimerize via transmembrane (TM) domain interactions. While syntaxin homodimerization is supposed to promote the transition from hemifusion to complete fusion, the role of synaptobrevin's TM domain association in the fusion process remains poorly understood. Here, we combined coarse-grained and atomistic simulations to model the homodimerization of the sybII transmembrane domain and of selected TM mutants. The wild-type helix is shown to form a stable, right-handed dimer with the most populated helix-helix interface, including key residues predicted in a previous mutagenesis study. In addition, two alternative binding interfaces were discovered, which are essential to explain the experimentally observed higher-order oligomerization of sybII. In contrast, only one dimerization interface was found for a fusion-inactive poly-Leu mutant. Moreover, the association kinetics found for this mutant is lower as compared to the wild-type. These differences in dimerization between the wild-type and the poly-Leu mutant are suggested to be responsible for the reported differences in fusogenic activity between these peptides. This study provides molecular insight into the role of TM sequence specificity for peptide aggregation in membranes. PMID:26287628

  6. Lasing thresholds of helical photonic structures with different positions of a single light-amplifying helix turn

    SciTech Connect

    Blinov, L M; Palto, S P

    2013-09-30

    Numerical simulation is used to assess the lasing threshold of helical structures of cholesteric liquid crystals (CLCs) in which only one turn amplifies light. This turn is located either in the centre of symmetric structures of various sizes or in an arbitrary place in asymmetric structures of preset size. In all cases, we find singularities in light amplification by a one-dimensional CLC structure for the most important band-edge modes (m1, m2 and m3) and plot the threshold gain coefficient k{sub th} against the position of the amplifying turn. For the symmetric structures, the lasing threshold of the m1 mode is shown to vary linearly with the inverse of the square of the cavity length. Moreover, modes with a lower density of photonic states (DOS) in the cavity may have a lower lasing threshold. This can be accounted for by the dependence of the density of photonic states on the position of the amplifying turn and, accordingly, by the nonuniform electromagnetic field intensity distribution along the cavity for different modes. In the asymmetric structures, the same field energy distribution is responsible for a correlation between k{sub th} and DOS curves. (lasers)

  7. Tailored fibro-porous structure of electrospun polyurethane membranes, their size-dependent properties and trans-membrane glucose diffusion

    PubMed Central

    Wang, Ning; Burugapalli, Krishna; Song, Wenhui; Halls, Justin; Moussy, Francis; Zheng, Yudong; Ma, Yanxuan; Wu, Zhentao; Li, Kang

    2012-01-01

    The aim of this study was to develop polyurethane (PU) based fibro-porous membranes and to investigate the size-effect of hierarchical porous structure on permeability and surface properties of the developed electrospun membranes. Non-woven Selectophore™ PU membranes having tailored fibre diameters, pore sizes, and thickness were spun using electrospinning, and their chemical, physical and glucose permeability properties were characterised. Solvents, solution concentration, applied voltage, flow rate and distance to collector, each were systematically investigated, and electrospinning conditions for tailoring fibre diameters were identified. Membranes having average fibre diameters – 347, 738 and 1102 nm were characterized, revealing average pore sizes of 800, 870 and 1060 nm and pore volumes of 44, 63 and 68% respectively. Hydrophobicity increased with increasing fibre diameter and porosity. Effective diffusion coefficients for glucose transport across the electrospun membranes varied as a function of thickness and porosity, indicating high flux rates for mass transport. Electrospun PU membranes having significantly high pore volumes, extensively interconnected porosity and tailorable properties compared to conventional solvent cast membranes can find applications as coatings for sensors requiring analyte exchange. PMID:23170040

  8. A Novel Topology of Proline-rich Transmembrane Protein 2 (PRRT2): HINTS FOR AN INTRACELLULAR FUNCTION AT THE SYNAPSE.

    PubMed

    Rossi, Pia; Sterlini, Bruno; Castroflorio, Enrico; Marte, Antonella; Onofri, Franco; Valtorta, Flavia; Maragliano, Luca; Corradi, Anna; Benfenati, Fabio

    2016-03-18

    Proline-rich transmembrane protein 2 (PRRT2) has been identified as the single causative gene for a group of paroxysmal syndromes of infancy, including epilepsy, paroxysmal movement disorders, and migraine. On the basis of topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the two-transmembrane domain topology with a large N terminus and short C terminus oriented toward the outside of the cell. Because PRRT2 plays a role at the synapse, it is important to confirm the exact orientation of its N and C termini with respect to the plasma membrane to get clues regarding its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein in which only the second hydrophobic segment spans the plasma membrane, whereas the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space but is localized intracellularly, and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we show that PRRT2 interacts with the Src homology 3 domain-bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to the clarification of the role of PRRT2 at the synapse and the understanding of pathogenic mechanisms on the basis of PRRT2-related neurological disorders. PMID:26797119

  9. The crystal structure of Z-(Aib)10 -OH at 0.65 Å resolution: three complete turns of 310 -helix.

    PubMed

    Gessmann, Renate; Brückner, Hans; Petratos, Kyriacos

    2016-02-01

    The synthetic peptide Z-(Aib)10 -OH was crystallized from hot methanol by slow evaporation. The crystal used for data collection reflected synchrotron radiation to sub-atomic resolution, where the bonding electron density becomes visible between the non-hydrogen atoms. Crystals belong to the centrosymmetric space group P 1¯. Both molecules in the asymmetric unit form regular 310 -helices. All residues in each molecule possess the same handedness, which is in contrast to all other crystal structure determined to date of longer Aib-homopeptides. These other peptides are C-terminal protected by OtBu or OMe. In these cases, because of the missing ability of the C-terminal protection group to form a hydrogen bond to the residue i-3, the sense of the helix is reversed in the last residue. Here, the C-terminal OH-groups form hydrogen bonds to the residues i-3, in part mediated by water molecules. This makes Z-(Aib)10 -OH an Aib-homopeptide with three complete 310 -helical turns in spite of the shorter length it has compared with Z-(Aib)11 -OtBu, the only homopeptide to date with three complete turns. PMID:26680663

  10. Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban.

    PubMed Central

    Ludlam, C F; Arkin, I T; Liu, X M; Rothman, M S; Rath, P; Aimoto, S; Smith, S O; Engelman, D M; Rothschild, K J

    1996-01-01

    Phospholamban is a 52-amino acid residue membrane protein that regulates Ca(2+)-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca(2+)-selective ion channel. We have used polarized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLB. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm-1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidycholine bilayer membranes is predominantly alpha-helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are alpha-helical, with an axial orientation that is approximately 30 degrees relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope-labeled hPLB films, our results strongly support models of PLB that predict the existence of an alpha-helical hydrophobic region spanning the membrane domain. PMID:8785331

  11. The transmembrane domains of the bacterial cell division proteins FtsB and FtsL form a stable high-order oligomer.

    PubMed

    Khadria, Ambalika S; Senes, Alessandro

    2013-10-29

    FtsB and FtsL are two essential integral membrane proteins of the bacterial division complex or "divisome", both characterized by a single transmembrane helix and a juxtamembrane coiled coil domain. The two domains are important for the association of FtsB and FtsL, a key event for their recruitment to the divisome, which in turn allows the recruitment of the late divisomal components to the Z-ring and subsequent completion of the division process. Here we present a biophysical analysis performed in vitro that shows that the transmembrane domains of FtsB and FtsL associate strongly in isolation. Using Förster resonance energy transfer, we have measured the oligomerization of fluorophore-labeled transmembrane domains of FtsB and FtsL in both detergent and lipid. The data indicate that the transmembrane helices are likely a major contributor to the stability of the FtsB-FtsL complex. Our analyses show that FtsB and FtsL form a 1:1 higher-order oligomeric complex, possibly a tetramer. This finding suggests that the FtsB-FtsL complex is capable of multivalent binding to FtsQ and other divisome components, a hypothesis that is consistent with the possibility that the FtsB-FtsL complex has a structural role in the stabilization of the Z-ring. PMID:24083359

  12. A distinct three-helix centipede toxin SSD609 inhibits Iks channels by interacting with the KCNE1 auxiliary subunit

    PubMed Central

    Sun, Peibei; Wu, Fangming; Wen, Ming; Yang, Xingwang; Wang, Chenyang; Li, Yiming; He, Shufang; Zhang, Longhua; Zhang, Yun; Tian, Changlin

    2015-01-01

    KCNE1 is a single-span transmembrane auxiliary protein that modulates the voltage-gated potassium channel KCNQ1. The KCNQ1/KCNE1 complex in cardiomyocytes exhibited slow activated potassium (Iks) currents. Recently, a novel 47-residue polypeptide toxin SSD609 was purified from Scolopendra subspinipes dehaani venom and showed Iks current inhibition. Here, chemically synthesized SSD609 was shown to exert Iks inhibition in extracted guinea pig cardiomyocytes and KCNQ1/KCNE1 current attenuation in CHO cells. The K+ current attenuation of SSD609 showed decent selectivity among different auxiliary subunits. Solution nuclear magnetic resonance analysis of SSD609 revealed a distinctive three-helix conformation that was stabilized by a new disulfide bonding pattern as well as segregated surface charge distribution. Structure-activity studies demonstrated that negatively charged Glu19 in the amphipathic extracellular helix of KCNE1 was the key residue that interacted with SSD609. The distinctive three-helix centipede toxin SSD609 is known to be the first polypeptide toxin acting on channel auxiliary subunit KCNE1, which suggests a new type of pharmacological regulation for ion channels in cardiomyocytes. PMID:26307551

  13. Structure and Mutagenesis of the Parainfluenza Virus 5 Hemagglutinin-Neuraminidase Stalk Domain Reveals a Four-Helix Bundle and the Role of the Stalk in Fusion Promotion

    SciTech Connect

    Bose, Sayantan; Welch, Brett D.; Kors, Christopher A.; Yuan, Ping; Jardetzky, Theodore S.; Lamb, Robert A.

    2014-10-02

    Paramyxovirus entry into cells requires the fusion protein (F) and a receptor binding protein (hemagglutinin-neuraminidase [HN], H, or G). The multifunctional HN protein of some paramyxoviruses, besides functioning as the receptor (sialic acid) binding protein (hemagglutinin activity) and the receptor-destroying protein (neuraminidase activity), enhances F activity, presumably by lowering the activation energy required for F to mediate fusion of viral and cellular membranes. Before or upon receptor binding by the HN globular head, F is believed to interact with the HN stalk. Unfortunately, until recently none of the receptor binding protein crystal structures have shown electron density for the stalk domain. Parainfluenza virus 5 (PIV5) HN exists as a noncovalent dimer-of-dimers on the surface of cells, linked by a single disulfide bond in the stalk. Here we present the crystal structure of the PIV5-HN stalk domain at a resolution of 2.65 {angstrom}, revealing a four-helix bundle (4HB) with an upper (N-terminal) straight region and a lower (C-terminal) supercoiled part. The hydrophobic core residues are a mix of an 11-mer repeat and a 3- to 4-heptad repeat. To functionally characterize the role of the HN stalk in F interactions and fusion, we designed mutants along the PIV5-HN stalk that are N-glycosylated to physically disrupt F-HN interactions. By extensive study of receptor binding, neuraminidase activity, oligomerization, and fusion-promoting functions of the mutant proteins, we found a correlation between the position of the N-glycosylation mutants on the stalk structure and their neuraminidase activities as well as their abilities to promote fusion.

  14. Annular Anionic Lipids Stabilize the Integrin αIIbβ3 Transmembrane Complex*

    PubMed Central

    Schmidt, Thomas; Suk, Jae-Eun; Ye, Feng; Situ, Alan J.; Mazumder, Parichita; Ginsberg, Mark H.; Ulmer, Tobias S.

    2015-01-01

    Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbβ3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg995)-β3(Asp723) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg995) contacts with β3(Asp723) but paradoxically do not diminish the contribution of αIIb(Arg995)-β3(Asp723) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbβ3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact. PMID:25632962

  15. Thermal and Structural Analysis of Co-axial Coupler used in High Power Helix Traveling-Wave Tube

    NASA Astrophysics Data System (ADS)

    Gahlaut, Vishant; Alvi, Parvez Ahmad; Ghosh, Sanjay Kumar

    2014-07-01

    In traveling-wave tubes (TWTs), coaxial couplers or window assemblies are used for coupling milliwatt (mW) to hundreds of watts of average power. For the proper transformation of impedance of interaction structure to the standard connector, coaxial couplers are suitably modeled as multi-section coaxial transformer. Due to high average power propagation, center conductor of coupler gets heated and the dimensions of multi-section coupler get deformed from its cold condition which causes impedance mismatch and increase of thermal load. Due to impedance mismatch, reflection of RF signal occurs from the couplers causing oscillation and finally leads of destruction of the TWT. This paper presents the thermal and structural analysis of coaxial coupler to quantify the temperature distribution at different regions, deformation of cold dimensions and stress due to material property of window disc.

  16. PPARγ helix 12 exhibits an antagonist conformation.

    PubMed

    Fratev, Filip

    2016-03-23

    Although the peroxisome proliferator-activated receptor γ (PPARγ) is one of the most studied nuclear receptors (NR), it is still unknown whether its activation helix (helix 12, H12) could exhibit an antagonist conformation as previously demonstrated for most of the NRs. The high H12 flexibility in the apo PPARγ form and the lack of appropriate antagonist ligands complicate the structural and dynamics description by most of the experimental techniques. Based on intensive (≈12 μs) accelerated molecular dynamics (aMD) simulations together with metadynamics and conventional MD runs, we reveal that H12 could exist in an antagonist conformation. This H12 state and the well-known agonist configuration have virtually identical free energy. Notably, significant deviations in the H12 conformations are detected in a homodimer. In chain A the activation helix is stabilized only in a full agonist conformation whereas in chain B, due to agonist to antagonist state exchanges, H12 is oriented toward helix 4. In summary, the results provide an explanation of the observed asymmetry in most of the PPARγ homodimer crystal structures. They also suggest selection guidance for protein moieties and structure candidates that would best serve as potential ligand binding sites to achieve a stable antagonist form of the receptor. PMID:26975214

  17. Efflux by Small Multidrug Resistance Proteins Is Inhibited by Membrane-interactive Helix-stapled Peptides*

    PubMed Central

    Bellmann-Sickert, Kathrin; Stone, Tracy A.; Poulsen, Bradley E.; Deber, Charles M.

    2015-01-01

    Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues 90GLALIVAGV98); and (ii) a peptide comprised of the full TM4(85–105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88–100), and then “staple” this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-88VVGLXLIZXGVVV100-KKK-NH2 (X = 2-(4′-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn95 peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88–100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes. PMID:25425644

  18. Accurate prediction of helix interactions and residue contacts in membrane proteins.

    PubMed

    Hönigschmid, Peter; Frishman, Dmitrij

    2016-04-01

    Accurate prediction of intra-molecular interactions from amino acid sequence is an important pre-requisite for obtaining high-quality protein models. Over the recent years, remarkable progress in this area has been achieved through the application of novel co-variation algorithms, which eliminate transitive evolutionary connections between residues. In this work we present a new contact prediction method for α-helical transmembrane proteins, MemConP, in which evolutionary couplings are combined with a machine learning approach. MemConP achieves a substantially improved accuracy (precision: 56.0%, recall: 17.5%, MCC: 0.288) compared to the use of either machine learning or co-evolution methods alone. The method also achieves 91.4% precision, 42.1% recall and a MCC of 0.490 in predicting helix-helix interactions based on predicted contacts. The approach was trained and rigorously benchmarked by cross-validation and independent testing on up-to-date non-redundant datasets of 90 and 30 experimental three dimensional structures, respectively. MemConP is a standalone tool that can be downloaded together with the associated training data from http://webclu.bio.wzw.tum.de/MemConP. PMID:26851352

  19. Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of Bordetella adenylate cyclase toxin.

    PubMed

    Wald, Tomas; Osickova, Adriana; Masin, Jiri; Liskova, Petra M; Petry-Podgorska, Inga; Matousek, Tomas; Sebo, Peter; Osicka, Radim

    2016-04-01

    Adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) of the whooping cough agent Bordetella pertussis penetrates phagocytes expressing the integrin complement receptor 3 (CR3, CD11b/CD18, αMβ2 or Mac-1). CyaA translocates its adenylate cyclase (AC) enzyme domain into cell cytosol and catalyzes unregulated conversion of ATP to cAMP, thereby subverting cellular signaling. In parallel, CyaA forms small cation-selective membrane pores that permeabilize cells for potassium efflux, contributing to cytotoxicity of CyaA and eventually provoking colloid-osmotic cell lysis. To investigate whether the single-pass α-helical transmembrane segments of CR3 subunits CD11b and CD18 do directly participate in AC domain translocation and/or pore formation by the toxin, we expressed in CHO cells variants of CR3 that contained artificial transmembrane segments, or lacked the transmembrane segment(s) at all. The results demonstrate that the transmembrane segments of CR3 are not directly involved in the cytotoxic activities of CyaA but serve for maintaining CR3 in a conformation that is required for efficient toxin binding and action. PMID:26802078

  20. OVERVIEW OF HELIX HOUSE NO. 2 (S87), WITH ANTENNA TOWERS, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    OVERVIEW OF HELIX HOUSE NO. 2 (S-87), WITH ANTENNA TOWERS, HELIX HOUSE NO. 1 (S-3) AND TRANSMITTER BLDG. (S-2) AT REAR, LOOKING WEST SOUTHWEST. - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

  1. NMR structures of the second transmembrane domain of the human glycine receptor alpha(1) subunit: model of pore architecture and channel gating.

    PubMed Central

    Tang, Pei; Mandal, Pravat K; Xu, Yan

    2002-01-01

    Glycine receptors (GlyR) are the primary inhibitory receptors in the spinal cord and belong to a superfamily of ligand-gated ion channels (LGICs) that are extremely sensitive to low-affinity neurological agents such as general anesthetics and alcohols. The high-resolution pore architecture and the gating mechanism of this superfamily, however, remain unclear. The pore-lining second transmembrane (TM2) segments of the GlyR alpha(1) subunit are unique in that they form functional homopentameric channels with conductance characteristics nearly identical to those of an authentic receptor (Opella, S. J., J. Gesell, A. R. Valente, F. M. Marassi, M. Oblatt-Montal, W. Sun, A. F. Montiel, and M. Montal. 1997. Chemtracts Biochem. Mol. Biol. 10:153-174). Using NMR and circular dichroism (CD), we determined the high-resolution structures of the TM2 segment of human alpha(1) GlyR and an anesthetic-insensitive mutant (S267Y) in dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) micelles. The NMR structures showed right-handed alpha-helices without kinks. A well-defined hydrophilic path, composed of side chains of G2', T6', T10', Q14', and S18', runs along the helical surfaces at an angle approximately 10-20 degrees relative to the long axis of the helices. The side-chain arrangement of the NMR-derived structures and the energy minimization of a homopentameric TM2 channel in a fully hydrated DMPC membrane using large-scale computation suggest a model of pore architecture in which simultaneous tilting movements of entire TM2 helices by a mere 10 degrees may be sufficient to account for the channel gating. The model also suggests that additional residues accessible from within the pore include L3', T7', T13', and G17'. A similar pore architecture and gating mechanism may apply to other channels in the same superfamily, including GABA(A), nACh, and 5-HT(3) receptors. PMID:12080117

  2. The domain structure of talin: residues 1815-1973 form a five-helix bundle containing a cryptic vinculin-binding site.

    PubMed

    Goult, Benjamin T; Gingras, Alexandre R; Bate, Neil; Barsukov, Igor L; Critchley, David R; Roberts, Gordon C K

    2010-06-01

    Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. Its rod region consists of a series of helical bundles. Here we show that residues 1815-1973 form a 5-helix bundle, with a topology unique to talin which is optimally suited for formation of a long rod such as talin. This is much more stable than the 4-helix (1843-1973) domain described earlier and as a result its vinculin binding sequence is inaccessible to vinculin at room temperature, with implications for the overall mechanism of the talin-vinculin interaction. PMID:20399778

  3. Helix control in polymers

    PubMed Central

    Totsingan, Filbert; Jain, Vipul; Green, Mark M.

    2012-01-01

    The helix is a critical conformation exhibited by biological macromolecules and plays a key role in fundamental biological processes. Biological helical polymers exist in a single helical sense arising from the chiral effect of their primary units—for example, DNA and proteins adopt predominantly a right-handed helix conformation in response to the asymmetric conformational propensity of D-sugars and L-amino acids, respectively. In using these homochiral systems, nature blocks our observations of some fascinating aspects of the cooperativity in helical systems, although when useful for a specific purpose, “wrong” enantiomers may be incorporated in specific places. In synthetic helical systems, on the contrary, incorporation of non-racemic chirality is an additional burden, and the findings discussed in this review show that this burden may be considerably alleviated by taking advantage of the amplification of chirality, in which small chiral influences lead to large consequences. Peptide nucleic acid (PNA), which is a non-chiral synthetic DNA mimic, shows a cooperative response to a small chiral effect induced by a chiral amino acid, which is limited, however, due to the highly flexible nature of this oligomeric chimera. The lack of internal stereochemical bias is an important factor which makes PNA an ideal system to understand some cooperative features that are not directly accessible from DNA. PMID:22772039

  4. Energetic Frustration of Apomyoglobin Folding: Role of the B Helix

    PubMed Central

    Nishimura, Chiaki; Dyson, H. Jane; Wright, Peter E.

    2010-01-01

    Apomyoglobin folds by a sequential mechanism in which the A, G, and H helix regions undergo rapid collapse to form a compact intermediate onto which the central portion of the B helix subsequently docks. To investigate the factors that frustrate folding, we have made mutations in the N-terminus of the B helix to stabilize helical structure (in the mutant G23A/G25A) and to promote native-like hydrophobic packing interactions with helix G (in the mutant H24L/H119F). The kinetic and equilibrium intermediates of G23A/G25A and H24L/H119F were studied by hydrogen exchange pulse labeling and interrupted hydrogen/deuterium exchange combined with NMR. For both mutants, stabilization of helical structure in the N-terminal region of the B-helix is confirmed by increased exchange protection in the equilibrium molten globule states near pH 4. Increased protection is also observed in the G-H turn region in the G23A/G25A mutant, suggesting that stabilization of the B-helix facilitates native-like interactions with the C-terminal region of helix G. These interactions are further enhanced in H24L/H119F. The kinetic burst phase intermediates of both mutants show increased protection, relative to wild type protein, of amides in the N-terminus of the B helix and in part of the E helix. Stabilization of the E helix in the intermediate is attributed to direct interactions between E helix residues and the newly stabilized N-terminus of helix B. Stabilization of native packing between the B and G helices in H24L/H119F also favors formation of native-like interactions in the GH turn and between the G and H helices in the ensemble of burst phase intermediates. We conclude that instability at the N-terminus of the B helix of apomyoglobin contributes to the energetic frustration of folding by preventing docking and stabilization of the E helix. PMID:20043917

  5. Influence of Proline on the Thermostability of the Active Site and Membrane Arrangement of Transmembrane Proteins

    PubMed Central

    Perálvarez-Marín, Alex; Lórenz-Fonfría, Victor A.; Simón-Vázquez, Rosana; Gomariz, Maria; Meseguer, Inmaculada; Querol, Enric; Padrós, Esteve

    2008-01-01

    Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro50, Pro91, and Pro186 in the native membrane environment. This information has been complemented with the analysis of the respective crystallographic structures by the FoldX force field. Differential scanning calorimetry allowed us to determine distorted membrane arrangement for P50A and P186A. The protein stability was severely affected for P186A and P91A. In the case of Pro91, a single point mutation is capable of strongly slowing down the conformational diffusion along the denaturation coordinate, becoming a barrier-free downhill process above 371 K. Temperature derivative spectroscopy, applied for first time to study thermal stability of proteins, has been used to monitor the stability of the active site of bacteriorhodopsin. The mutation of Pro91 and Pro186 showed the most striking effects on the retinal binding pocket. These residues are the Pro in closer contact to the active site (activation energies for retinal release of 60.1 and 76.8 kcal/mol, respectively, compared to 115.8 kcal/mol for WT). FoldX analysis of the protein crystal structures indicates that the Pro-to-Ala mutations have both local and long-range effects on the structural stability of residues involved in the architecture of the protein and the active site and in the proton pumping function. Thus, this study provides a complete overview of the substitution effect of helix-embedded prolines in the thermodynamic and dynamic stability of a membrane protein, also related to its structure and function. PMID:18658225

  6. Crystallizing Transmembrane Peptides in Lipidic Mesophases

    SciTech Connect

    Höfer, Nicole; Aragão, David; Caffrey, Martin

    2011-09-28

    Structure determination of membrane proteins by crystallographic means has been facilitated by crystallization in lipidic mesophases. It has been suggested, however, that this so-called in meso method, as originally implemented, would not apply to small protein targets having {le}4 transmembrane crossings. In our study, the hypothesis that the inherent flexibility of the mesophase would enable crystallogenesis of small proteins was tested using a transmembrane pentadecapeptide, linear gramicidin, which produced structure-grade crystals. This result suggests that the in meso method should be considered as a viable means for high-resolution structure determination of integral membrane peptides, many of which are predicted to be coded for in the human genome.

  7. Into the Eye of the Helix

    NASA Astrophysics Data System (ADS)

    2009-02-01

    A deep new image of the magnificent Helix planetary nebula has been obtained using the Wide Field Imager at ESO's La Silla Observatory. The image shows a rich background of distant galaxies, usually not seen in other images of this object. ESO PR Photo 07a/09 The Helix Nebula ESO PR Video 06a/09 Helix Nebula Zoom-in ESO PR Video 06b/09 Pan over the Helix Nebula ESO PR Video 06c/09 Zoom and pan over the Helix Nebula The Helix Nebula, NGC 7293, lies about 700 light-years away in the constellation of Aquarius (the Water Bearer). It is one of the closest and most spectacular examples of a planetary nebula. These exotic objects have nothing to do with planets, but are the final blooming of Sun-like stars before their retirement as white dwarfs. Shells of gas are blown off from a star's surface, often in intricate and beautiful patterns, and shine under the harsh ultraviolet radiation from the faint, but very hot, central star. The main ring of the Helix Nebula is about two light-years across or half the distance between the Sun and its closest stellar neighbour. Despite being photographically very spectacular the Helix is hard to see visually as its light is thinly spread over a large area of sky and the history of its discovery is rather obscure. It first appears in a list of new objects compiled by the German astronomer Karl Ludwig Harding in 1824. The name Helix comes from the rough corkscrew shape seen in the earlier photographs. Although the Helix looks very much like a doughnut, studies have shown that it possibly consists of at least two separate discs with outer rings and filaments. The brighter inner disc seems to be expanding at about 100 000 km/h and to have taken about 12 000 years to have formed. Because the Helix is relatively close -- it covers an area of the sky about a quarter of the full Moon -- it can be studied in much greater detail than most other planetary nebulae and has been found to have an unexpected and complex structure. All around the inside of the ring are small blobs, known as "cometary knots", with faint tails extending away from the central star. They look remarkably like droplets of liquid running down a sheet of glass. Although they look tiny, each knot is about as large as our Solar System. These knots have been extensively studied, both with the ESO Very Large Telescope and with the NASA/ESA Hubble Space Telescope, but remain only partially understood. A careful look at the central part of this object reveals not only the knots, but also many remote galaxies seen right through the thinly spread glowing gas. Some of these seem to be gathered in separate galaxy groups scattered over various parts of the image.

  8. Converting One-Face α-Helix Mimetics into Amphiphilic α-Helix Mimetics as Potent Inhibitors of Protein-Protein Interactions.

    PubMed

    Lee, Ji Hoon; Oh, Misook; Kim, Hyun Soo; Lee, Huisun; Im, Wonpil; Lim, Hyun-Suk

    2016-01-11

    Many biologically active α-helical peptides adopt amphiphilic helical structures that contain hydrophobic residues on one side and hydrophilic residues on the other side. Therefore, α-helix mimetics capable of mimicking such amphiphilic helical peptides should possess higher binding affinity and specificity to target proteins. Here we describe an efficient method for generating amphiphilic α-helix mimetics. One-face α-helix mimetics having hydrophobic side chains on one side was readily converted into amphiphilic α-helix mimetics by introducing appropriate charged residues on the opposite side. We also demonstrate that such two-face amphiphilic α-helix mimetics indeed show remarkably improved binding affinity to a target protein, compared to one-face hydrophobic α-helix mimetics. We believe that generating a large combinatorial library of these amphiphilic α-helix mimetics can be valuable for rapid discovery of highly potent and specific modulators of protein-protein interactions. PMID:26651509

  9. Structure/function studies of human immunodeficiency virus type 1 reverse transcriptase. Alanine scanning mutagenesis of an alpha-helix in the thumb subdomain.

    PubMed

    Beard, W A; Stahl, S J; Kim, H R; Bebenek, K; Kumar, A; Strub, M P; Becerra, S P; Kunkel, T A; Wilson, S H

    1994-11-11

    Human immunodeficiency virus type 1 reverse transcriptase has subunits of 66 and 51 kDa (p66 and p51, respectively). Structural studies indicate that each subunit consists of common subdomains. The polymerase domain of p66 forms a nucleic acid binding cleft, and, by analogy with a right hand, the subdomains are referred to as fingers, palm, and thumb (Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Residues 257-266 correspond to a highly conserved region of primary structure among retroviral pol genes. Crystallographic evidence indicates that these residues are in the thumb subdomain and form part of an alpha-helix (alpha H), which interacts with DNA (Jacobo-Molina, A., Ding, J., Nanni, R. G., Clark, A. D., Jr., Lu, X., Tantillo, C., Williams, R. L., Kamer, G., Ferris, A. L., Clark, P., Hizi, A., Hughes, S. H., and Arnold, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6320-6324). To define the role of this region during catalytic cycling, we performed systematic site-directed mutagenesis from position 253 through position 271 by changing each residue, one by one, to alanine. Each mutant protein was expressed and purified, and their substrate-specific activities were surveyed. The results are consistent with alpha H (residues 255-268) of p66 interacting with the template and/or primer strand. The core of alpha H appears to play an important role in template-primer binding (residues Gln-258, Gly-262, and Trp-266), and in protein-protein interactions (residues Val-261 and Leu-264). The periodicity of the effects observed suggest that a segment of one face of alpha H interacts with the template-primer. The lower fidelity observed with alanine mutants of Gly-262 and Trp-266 correlated with an over 200-fold increase in the dissociation rate constant for template-primer relative to wild type enzyme and suggests that enzyme-DNA interactions in the template-primer stem are important fidelity determinants. PMID:7525566

  10. STRUCTURE/FUNCTION MAPPING OF AMINO ACIDS IN THE N-TERMINAL ZINC FINGER OF THE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 NUCLEOCAPSID PROTEIN: RESIDUES RESPONSIBLE FOR NUCLEIC ACID HELIX DESTABILIZING ACTIVITY*

    PubMed Central

    Narayanan, Nirupama; Gorelick, Robert J.; DeStefano, Jeffrey J.

    2016-01-01

    The nucleocapsid protein (NC) of HIV-1 is 55 amino acids in length and possesses two CCHC-type zinc fingers. Finger one (N-terminal) contributes significantly more to helix destabilizing activity than finger two (C-terminal). Five amino acids differ between the two zinc fingers. To determine at the amino acid level the reason for the apparent distinction between the fingers, each different residue in finger one was incrementally replaced by the one at the corresponding location in finger two. Mutants were analyzed in annealing assays with unstructured and structured substrates. Three groupings emerged: (1) those similar to wild type levels (N17K, A25M), (2) those with diminished activity (I24Q, N27D), and (3) mutant F16W which had substantially greater helix destabilizing activity than wild type. Unlike I24Q and the other mutants, N27D was defective in DNA binding. Only I24Q and N27D showed reduced strand transfer in in vitro assays. Double and triple mutants F16W/I24Q, F16W/N27D, and F16W/I24Q/N27D all showed defects in DNA binding, strand transfer, and helix destabilization, suggesting that the I24Q and N27D mutations have a “dominant negative” effect and abolish the positive influence of F16W. Results show that amino acid differences at positions 24 and 27 contribute significantly to finger one’s helix destabilizing activity. PMID:17029416

  11. A deterministic algorithm for constrained enumeration of transmembrane protein folds.

    SciTech Connect

    Brown, William Michael; Young, Malin M.; Sale, Kenneth L.; Faulon, Jean-Loup Michel; Schoeniger, Joseph S.

    2004-07-01

    A deterministic algorithm for enumeration of transmembrane protein folds is presented. Using a set of sparse pairwise atomic distance constraints (such as those obtained from chemical cross-linking, FRET, or dipolar EPR experiments), the algorithm performs an exhaustive search of secondary structure element packing conformations distributed throughout the entire conformational space. The end result is a set of distinct protein conformations, which can be scored and refined as part of a process designed for computational elucidation of transmembrane protein structures.

  12. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

    PubMed Central

    Corradi, Valentina; Vergani, Paola; Tieleman, D. Peter

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily. CFTR controls the flow of anions through the apical membrane of epithelia. Dysfunctional CFTR causes the common lethal genetic disease cystic fibrosis. Transitions between open and closed states of CFTR are regulated by ATP binding and hydrolysis on the cytosolic nucleotide binding domains, which are coupled with the transmembrane (TM) domains forming the pathway for anion permeation. Lack of structural data hampers a global understanding of CFTR and thus the development of “rational” approaches directly targeting defective CFTR. In this work, we explored possible conformational states of the CFTR gating cycle by means of homology modeling. As templates, we used structures of homologous ABC transporters, namely TM(287–288), ABC-B10, McjD, and Sav1866. In the light of published experimental results, structural analysis of the transmembrane cavity suggests that the TM(287–288)-based CFTR model could correspond to a commonly occupied closed state, whereas the McjD-based model could represent an open state. The models capture the important role played by Phe-337 as a filter/gating residue and provide structural information on the conformational transition from closed to open channel. PMID:26229102

  13. SuperBiHelix method for predicting the pleiotropic ensemble of G-protein–coupled receptor conformations

    PubMed Central

    Bray, Jenelle K.; Abrol, Ravinder; Goddard, William A.; Trzaskowski, Bartosz; Scott, Caitlin E.

    2014-01-01

    There is overwhelming evidence that G-protein–coupled receptors (GPCRs) exhibit several distinct low-energy conformations, each of which might favor binding to different ligands and/or lead to different downstream functions. Understanding the function of such proteins requires knowledge of the ensemble of low-energy configurations that might play a role in this pleiotropic functionality. We earlier reported the BiHelix method for efficiently sampling the (12)7 = 35 million conformations resulting from 30° rotations about the axis (η) of all seven transmembrane helices (TMHs), showing that the experimental structure is reliably selected as the best conformation from this ensemble. However, various GPCRs differ sufficiently in the tilts of the TMHs that this method need not predict the optimum conformation starting from any other template. In this paper, we introduce the SuperBiHelix method in which the tilt angles (θ, φ) are optimized simultaneously with rotations (η) efficiently enough that it is practical and sufficient to sample (5 × 3 × 5)7 = 13 trillion configurations. This method can correctly identify the optimum structure of a GPCR starting with the template from a different GPCR. We have validated this method by predicting known crystal structure conformations starting from the template of a different protein structure. We find that the SuperBiHelix conformational ensemble includes the higher energy conformations associated with the active protein in addition to those associated with the more stable inactive protein. This methodology was then applied to design and experimentally confirm structures of three mutants of the CB1 cannabinoid receptor associated with different functions. PMID:24344284

  14. Salvador-Warts-Hippo pathway in a developmental checkpoint monitoring Helix-Loop-Helix proteins

    PubMed Central

    Wang, Lan-Hsin; Baker, Nicholas E.

    2014-01-01

    The E-proteins and Id-proteins are, respectively, the positive and negative heterodimer partners for the basic-helix-loop-helix protein family, and as such contribute to a remarkably large number of cell fate decisions. E-proteins and Id-proteins also function to inhibit or promote cell proliferation and cancer. Using a genetic modifier screen in Drosophila, we show that the Id-protein Extramacrochaetae enables growth by suppressing activation of the Salvador-Warts-Hippo pathway of tumor suppressors, activation that requires transcriptional activation of the expanded gene by the E-protein Daughterless. Daughterless protein binds to an intronic enhancer in the expanded gene, both activating the SWH pathway independently of the transmembrane protein Crumbs, and bypassing the negative feedback regulation that targets the same expanded enhancer. Thus the Salvador-Warts-Hippo pathway has a cell-autonomous function to prevent inappropriate differentiation due to transcription factor imbalance, and monitors the intrinsic developmental status of progenitor cells, distinct from any responses to cell-cell interactions. PMID:25579975

  15. Direct folding simulation of a long helix in explicit water

    NASA Astrophysics Data System (ADS)

    Gao, Ya; Lu, Xiaoliang; Duan, Lili; Zhang, Dawei; Mei, Ye; Zhang, John Z. H.

    2013-05-01

    A recently proposed Polarizable Hydrogen Bond (PHB) method has been employed to simulate the folding of a 53 amino acid helix (PDB ID 2KHK) in explicit water. Under PHB simulation, starting from a fully extended structure, the peptide folds into the native state as confirmed by measured time evolutions of radius of gyration, root mean square deviation (RMSD), and native hydrogen bond. Free energy and cluster analysis show that the folded helix is thermally stable under the PHB model. Comparison of simulation results under, respectively, PHB and standard nonpolarizable force field demonstrates that polarization is critical for stable folding of this long α-helix.

  16. Incorporation of transmembrane peptides from the vacuolar H(+)-ATPase in phospholipid membranes: spin-label electron paramagnetic resonance and polarized infrared spectroscopy.

    PubMed

    Kóta, Zoltán; Páli, Tibor; Dixon, Neil; Kee, Terry P; Harrison, Michael A; Findlay, John B C; Finbow, Malcolm E; Marsh, Derek

    2008-03-25

    Peptides were designed that are based on candidate transmembrane sequences of the V o-sector from the vacuolar H (+)-ATPase of Saccharomyces cerevisiae. Spin-label EPR studies of lipid-protein interactions were used to characterize the state of oligomerization, and polarized IR spectroscopy was used to determine the secondary structure and orientation, of these peptides in lipid bilayer membranes. Peptides corresponding to the second and fourth transmembrane domains (TM2 and TM4) of proteolipid subunit c (Vma3p) and of the putative seventh transmembrane domain (TM7) of subunit a (Vph1p) are wholly, or predominantly, alpha-helical in membranes of dioleoyl phosphatidylcholine. All three peptides self-assemble into oligomers of different sizes, in which the helices are differently inclined with respect to the membrane normal. The coassembly of rotor (Vma3p TM4) and stator (Vph1p TM7) peptides, which respectively contain the glutamate and arginine residues essential to proton transport by the rotary ATPase mechanism, is demonstrated from changes in the lipid interaction stoichiometry and helix orientation. Concanamycin, a potent V-ATPase inhibitor, and a 5-(2-indolyl)-2,4-pentadienoyl inhibitor that exhibits selectivity for the osteoclast subtype, interact with the membrane-incorporated Vma3p TM4 peptide, as evidenced by changes in helix orientation; concanamycin additionally interacts with Vph1p TM7, suggesting that both stator and rotor elements contribute to the inhibitor site within the membrane. Comparison of the peptide behavior in lipid bilayers is made with membranous subunit c assemblies of the 16-kDa proteolipid from Nephrops norvegicus, which can substitute functionally for Vma3p in S. cerevisiae. PMID:18307317

  17. A distinct three-helix centipede toxin SSD609 inhibits I(ks) channels by interacting with the KCNE1 auxiliary subunit.

    PubMed

    Sun, Peibei; Wu, Fangming; Wen, Ming; Yang, Xingwang; Wang, Chenyang; Li, Yiming; He, Shufang; Zhang, Longhua; Zhang, Yun; Tian, Changlin

    2015-01-01

    KCNE1 is a single-span transmembrane auxiliary protein that modulates the voltage-gated potassium channel KCNQ1. The KCNQ1/KCNE1 complex in cardiomyocytes exhibited slow activated potassium (I(ks)) currents. Recently, a novel 47-residue polypeptide toxin SSD609 was purified from Scolopendra subspinipes dehaani venom and showed I(ks) current inhibition. Here, chemically synthesized SSD609 was shown to exert I(ks) inhibition in extracted guinea pig cardiomyocytes and KCNQ1/KCNE1 current attenuation in CHO cells. The K(+) current attenuation of SSD609 showed decent selectivity among different auxiliary subunits. Solution nuclear magnetic resonance analysis of SSD609 revealed a distinctive three-helix conformation that was stabilized by a new disulfide bonding pattern as well as segregated surface charge distribution. Structure-activity studies demonstrated that negatively charged Glu19 in the amphipathic extracellular helix of KCNE1 was the key residue that interacted with SSD609. The distinctive three-helix centipede toxin SSD609 is known to be the first polypeptide toxin acting on channel auxiliary subunit KCNE1, which suggests a new type of pharmacological regulation for ion channels in cardiomyocytes. PMID:26307551

  18. The transmembrane domain 6 of vacuolar H(+)-pyrophosphatase mediates protein targeting and proton transport.

    PubMed

    Pan, Yih-Jiuan; Lee, Chien-Hsien; Hsu, Shen-Hsing; Huang, Yun-Tzu; Lee, Ching-Hung; Liu, Tseng-Huang; Chen, Yen-Wei; Lin, Shih-Ming; Pan, Rong-Long

    2011-01-01

    Vacuolar H(+)-pyrophosphatase (V-PPase; EC 3.6.1.1) plays a significant role in the maintenance of the pH in cytoplasm and vacuoles via proton translocation from the cytosol to the vacuolar lumen at the expense of PP(i) hydrolysis. The topology of V-PPase as predicted by TopPred II suggests that the catalytic site is putatively located in loop e and exposed to the cytosol. The adjacent transmembrane domain 6 (TM6) is highly conserved and believed to participate in the catalytic function and conformational stability of V-PPase. In this study, alanine-scanning mutagenesis along TM6 of the mung bean V-PPase was carried out to identify its structural and functional role. Mutants Y299A, A306S and L317A exhibited gross impairment in both PP(i) hydrolysis and proton translocation. Meanwhile, mutations at L307 and N318 completely abolished the targeting of the enzyme, causing broad cytosolic localization and implicating a possible role of these residues in protein translocation. The location of these amino acid residues was on the same side of the helix wheel, suggesting their involvement in maintaining the stability of enzyme conformation. G297A, E301A and A305S mutants showed declines in proton translocation but not in PP(i) hydrolysis, consequently resulting in decreases in the coupling efficiency. These amino acid residues cluster at one face of the helix wheel, indicating their direct/indirect participation in proton translocation. Taken together, these data indicate that TM6 is crucial to vacuolar H(+)-pyrophosphatase, probably mediating protein targeting, proton transport, and the maintenance of enzyme structure. PMID:20937245

  19. A general method for determining helix packing in membrane proteins in situ: Helices I and II are close to helix VII in the lactose permease of Escherichia?coli

    PubMed Central

    Wu, Jianhua; Kaback, H.?Ronald

    1996-01-01

    It was previously shown that coexpression of the lactose permease of Escherichia coli in two contiguous fragments leads to functional complementation. We demonstrate here that site-directed thiol crosslinking of coexpressed permease fragments can be used to determine helix proximity in situ without the necessity of purifying the permease. After coexpression of the six N-terminal (N6) and six C-terminal (C6) transmembrane helices, each with a single Cys residue, crosslinking was carried out in native membranes and assessed by the mobility of anti-C-terminal-reactive polypeptides on immunoblots. A Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 28 or 29 (helix I), but not with a Cys residue at position 27, which is on the opposite face of helix I, thereby indicating that the face of helix I containing Pro-28 and Phe-29 is close to helix VII. Similarly, a Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 52 or 53 (helix II), but not with a Cys residue at position 54. Furthermore, low-efficiency crosslinking is observed between a Cys residue at position 52 or 53 and a Cys residue at position 361 (helix XI). The results indicate that helix VII lies in close proximity to both helices I and II and that helix II is also close to helix XI. The method should be applicable to a number of different polytopic membrane proteins. PMID:8962080

  20. Conformational and dynamics simulation study of antimicrobial peptide hedistin-heterogeneity of its helix-turn-helix motif.

    PubMed

    Xu, Guohua; Wu, Min; Wang, Lin; Zhang, Xu; Cao, Shufen; Liu, Maili; Cui, Yanfang

    2009-12-01

    Hedistin is an antimicrobial peptide isolated from the coelomocytes of Nereis diversicolor, possessing activity against a large spectrum of bacteria including the methicillin resistant Staphylococcus aureus and Vibrio alginolyticus. The three-dimensional structure of hedistin in both aqueous solution and deuterated dodecylphosphocholine (DPC) micelles was examined using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques. And, the early events of the antibacterial process of hedistin were simulated using palmitoyl-oleoyl-phophatidylcholine (POPC) lipid bilayers and molecular dynamics (MD) simulation methods. Hedistin lacks secondary structure in aqueous solution, however, in DPC micelles, it features with a heterogeneous helix-turn-helix moiety and exhibits obvious amphipathic nature. The turn region (residues Val9-Thr12) in the moiety is a four-residue hinge, lying in between the first N-terminal alpha-helix (residues Leu5-Lys8) and the second alpha-helix (residues Val13-Ala17) regions and causing an approximately 120 degrees angle between the axes of the two helices. The segmental and nonlinear nature of hedistin structure is referred to as the heterogeneity of its helix-turn-helix motif which was found to be corresponding to a kind of discrete dynamics behavior, herein coined as its dynamical heterogeneity, at the early stage (0-50 ns) of the MD simulations. That is, the first helix segment, prior to (at 310 K) or following (at 363 K) the second helix, binds to the lipid head-group region and subsequently permeates into the hydrophobic lipid tail region, and the hinge is the last portion entering the lipid environment. This result implies that hedistin may adopt a "carpet" model action when disrupting bacterial membrane. PMID:19819221

  1. A classification of basic helix-loop-helix transcription factors of soybean.

    PubMed

    Hudson, Karen A; Hudson, Matthew E

    2015-01-01

    The complete genome sequence of soybean allows an unprecedented opportunity for the discovery of the genes controlling important traits. In particular, the potential functions of regulatory genes are a priority for analysis. The basic helix-loop-helix (bHLH) family of transcription factors is known to be involved in controlling a wide range of systems critical for crop adaptation and quality, including photosynthesis, light signalling, pigment biosynthesis, and seed pod development. Using a hidden Markov model search algorithm, 319 genes with basic helix-loop-helix transcription factor domains were identified within the soybean genome sequence. These were classified with respect to their predicted DNA binding potential, intron/exon structure, and the phylogeny of the bHLH domain. Evidence is presented that the vast majority (281) of these 319 soybean bHLH genes are expressed at the mRNA level. Of these soybean bHLH genes, 67% were found to exist in two or more homeologous copies. This dataset provides a framework for future studies on bHLH gene function in soybean. The challenge for future research remains to define functions for the bHLH factors encoded in the soybean genome, which may allow greater flexibility for genetic selection of growth and environmental adaptation in this widely grown crop. PMID:25763382

  2. Correlation of biological activity with computationally derived structural features from transmembrane hetero-dimers of HIV-1 Vpu with host factors.

    PubMed

    Li, Li-Hua; Fischer, Wolfgang B

    2014-04-01

    Vpu is an 81 amino acid type I integral membrane protein encoded by human immunodeficiency virus type 1 (HIV-1). It is identified to support viral release by potentially forming ion and substrate conducting channels and by modulating the function of host factors. The focus is on the interaction of the transmembrane domains of Vpu with those of host factors using a combination of molecular dynamics simulations and docking approach. Binding poses and adopted tilt angles of the dimers are analyzed and correlated with experimentally derived activity data from literature. Vpu activity is driven by dimerization with the host protein via its alanine rim Ala-8/11/15/19. Tight binding is shown by an almost parallel alignment of the helices in the dimers. Less parallel alignment is proposed to correlate with lower activity. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking. PMID:24036078

  3. Polyproline and Triple Helix Motifs in Host-Pathogen Recognition

    PubMed Central

    Berisio, Rita; Vitagliano, Luigi

    2012-01-01

    Secondary structure elements often mediate protein-protein interactions. Despite their low abundance in folded proteins, polyproline II (PPII) and its variant, the triple helix, are frequently involved in protein-protein interactions, likely due to their peculiar propensity to be solvent-exposed. We here review the role of PPII and triple helix in mediating host-pathogen interactions, with a particular emphasis to the structural aspects of these processes. After a brief description of the basic structural features of these elements, examples of host-pathogen interactions involving these motifs are illustrated. Literature data suggest that the role played by PPII motif in these processes is twofold. Indeed, PPII regions may directly mediate interactions between proteins of the host and the pathogen. Alternatively, PPII may act as structural spacers needed for the correct positioning of the elements needed for adhesion and infectivity. Recent investigations have highlighted that collagen triple helix is also a common target for bacterial adhesins. Although structural data on complexes between adhesins and collagen models are rather limited, experimental and theoretical studies have unveiled some interesting clues of the recognition process. Interestingly, very recent data show that not only is the triple helix used by pathogens as a target in the host-pathogen interaction but it may also act as a bait in these processes since bacterial proteins containing triple helix regions have been shown to interact with host proteins. As both PPII and triple helix expose several main chain non-satisfied hydrogen bond acceptors and donors, both elements are highly solvated. The preservation of the solvation state of both PPII and triple helix upon protein-protein interaction is an emerging aspect that will be here thoroughly discussed. PMID:23305370

  4. Triple helix purification and sequencing

    DOEpatents

    Wang, R.; Smith, L.M.; Tong, X.E.

    1995-03-28

    Disclosed herein are methods, kits, and equipment for purifying single stranded circular DNA and then using the DNA for DNA sequencing purposes. Templates are provided with an insert having a hybridization region. An elongated oligonucleotide has two regions that are complementary to the insert and the oligo is bound to a magnetic anchor. The oligo hybridizes to the insert on two sides to form a stable triple helix complex. The anchor can then be used to drag the template out of solution using a magnet. The system can purify sequencing templates, and if desired the triple helix complex can be opened up to a double helix so that the oligonucleotide will act as a primer for further DNA synthesis. 4 figures.

  5. Triple helix purification and sequencing

    DOEpatents

    Wang, Renfeng; Smith, Lloyd M.; Tong, Xinchun E.

    1995-01-01

    Disclosed herein are methods, kits, and equipment for purifying single stranded circular DNA and then using the DNA for DNA sequencing purposes. Templates are provided with an insert having a hybridization region. An elongated oligonucleotide has two regions that are complementary to the insert and the oligo is bound to a magnetic anchor. The oligo hybridizes to the insert on two sides to form a stable triple helix complex. The anchor can then be used to drag the template out of solution using a magnet. The system can purify sequencing templates, and if desired the triple helix complex can be opened up to a double helix so that the oligonucleotide will act as a primer for further DNA synthesis.

  6. Misplaced helix slows down ultrafast pressure-jump protein folding.

    PubMed

    Prigozhin, Maxim B; Liu, Yanxin; Wirth, Anna Jean; Kapoor, Shobhna; Winter, Roland; Schulten, Klaus; Gruebele, Martin

    2013-05-14

    Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6-85, from 3 μs to 5 ms after a 1,200-bar P-drop. In addition to a microsecond phase, we observe a slower 1.4-ms phase during refolding to the native state. Unlike temperature denaturation, pressure denaturation produces a highly reversible helix-coil-rich state. This difference highlights the importance of the denatured initial condition in folding experiments and leads us to assign a compact nonnative helical trap as the reason for slower P-jump-induced refolding. To complement the experiments, we performed over 50 μs of all-atom molecular dynamics P-drop refolding simulations with four different force fields. Two of the force fields yield compact nonnative states with misplaced α-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of λ*YA contains mainly residual helix and little β-sheet; following a fast P-drop, at least some λ*YA forms misplaced helical structure within microseconds. We hypothesize that nonnative helix at helix-turn interfaces traps the protein in compact nonnative conformations. These traps delay the folding of at least some of the population for 1.4 ms en route to the native state. Based on molecular dynamics, we predict specific mutations at the helix-turn interfaces that should speed up refolding from the pressure-denatured state, if this hypothesis is correct. PMID:23620522

  7. Misplaced helix slows down ultrafast pressure-jump protein folding

    PubMed Central

    Prigozhin, Maxim B.; Liu, Yanxin; Wirth, Anna Jean; Kapoor, Shobhna; Winter, Roland; Schulten, Klaus; Gruebele, Martin

    2013-01-01

    Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6–85, from 3 μs to 5 ms after a 1,200-bar P-drop. In addition to a microsecond phase, we observe a slower 1.4-ms phase during refolding to the native state. Unlike temperature denaturation, pressure denaturation produces a highly reversible helix-coil-rich state. This difference highlights the importance of the denatured initial condition in folding experiments and leads us to assign a compact nonnative helical trap as the reason for slower P-jump–induced refolding. To complement the experiments, we performed over 50 μs of all-atom molecular dynamics P-drop refolding simulations with four different force fields. Two of the force fields yield compact nonnative states with misplaced α-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of λ*YA contains mainly residual helix and little β-sheet; following a fast P-drop, at least some λ*YA forms misplaced helical structure within microseconds. We hypothesize that nonnative helix at helix-turn interfaces traps the protein in compact nonnative conformations. These traps delay the folding of at least some of the population for 1.4 ms en route to the native state. Based on molecular dynamics, we predict specific mutations at the helix-turn interfaces that should speed up refolding from the pressure-denatured state, if this hypothesis is correct. PMID:23620522

  8. Characterization of Disease-Associated Mutations in Human Transmembrane Proteins

    PubMed Central

    Molnár, János; Szakács, Gergely; Tusnády, Gábor E.

    2016-01-01

    Transmembrane protein coding genes are commonly associated with human diseases. We characterized disease causing mutations and natural polymorphisms in transmembrane proteins by mapping missense genetic variations from the UniProt database on the transmembrane protein topology listed in the Human Transmembrane Proteome database. We found characteristic differences in the spectrum of amino acid changes within transmembrane regions: in the case of disease associated mutations the non-polar to non-polar and non-polar to charged amino acid changes are equally frequent. In contrast, in the case of natural polymorphisms non-polar to charged amino acid changes are rare while non-polar to non-polar changes are common. The majority of disease associated mutations result in glycine to arginine and leucine to proline substitutions. Mutations to positively charged amino acids are more common in the center of the lipid bilayer, where they cause more severe structural and functional anomalies. Our analysis contributes to the better understanding of the effect of disease associated mutations in transmembrane proteins, which can help prioritize genetic variations in personal genomic investigations. PMID:26986070

  9. Analysis of Human Dopamine D3 Receptor Quaternary Structure*

    PubMed Central

    Marsango, Sara; Caltabiano, Gianluigi; Pou, Chantevy; Varela Liste, María José; Milligan, Graeme

    2015-01-01

    The dopamine D3 receptor is a class A, rhodopsin-like G protein-coupled receptor that can form dimers and/or higher order oligomers. However, the molecular basis for production of these complexes is not well defined. Using combinations of molecular modeling, site-directed mutagenesis, and homogenous time-resolved FRET, the interfaces that allow dopamine D3 receptor monomers to interact were defined and used to describe likely quaternary arrangements of the receptor. These were then compared with published crystal structures of dimeric β1-adrenoreceptor, μ-opioid, and CXCR4 receptors. The data indicate important contributions of residues from within each of transmembrane domains I, II, IV, V, VI, and VII as well as the intracellular helix VIII in the formation of D3-D3 receptor interfaces within homo-oligomers and are consistent with the D3 receptor adopting a β1-adrenoreceptor-like quaternary arrangement. Specifically, results suggest that D3 protomers can interact with each other via at least two distinct interfaces: the first one comprising residues from transmembrane domains I and II along with those from helix VIII and a second one involving transmembrane domains IV and V. Moreover, rather than existing only as distinct dimeric species, the results are consistent with the D3 receptor also assuming a quaternary structure in which two transmembrane domain I-II-helix VIII dimers interact to form a ”rhombic” tetramer via an interface involving residues from transmembrane domains VI and VII. In addition, the results also provide insights into the potential contribution of molecules of cholesterol to the overall organization and potential stability of the D3 receptor and possibly other GPCR quaternary structures. PMID:25931118

  10. Analysis of Human Dopamine D3 Receptor Quaternary Structure.

    PubMed

    Marsango, Sara; Caltabiano, Gianluigi; Pou, Chantevy; Varela Liste, María José; Milligan, Graeme

    2015-06-12

    The dopamine D3 receptor is a class A, rhodopsin-like G protein-coupled receptor that can form dimers and/or higher order oligomers. However, the molecular basis for production of these complexes is not well defined. Using combinations of molecular modeling, site-directed mutagenesis, and homogenous time-resolved FRET, the interfaces that allow dopamine D3 receptor monomers to interact were defined and used to describe likely quaternary arrangements of the receptor. These were then compared with published crystal structures of dimeric β1-adrenoreceptor, μ-opioid, and CXCR4 receptors. The data indicate important contributions of residues from within each of transmembrane domains I, II, IV, V, VI, and VII as well as the intracellular helix VIII in the formation of D3-D3 receptor interfaces within homo-oligomers and are consistent with the D3 receptor adopting a β1-adrenoreceptor-like quaternary arrangement. Specifically, results suggest that D3 protomers can interact with each other via at least two distinct interfaces: the first one comprising residues from transmembrane domains I and II along with those from helix VIII and a second one involving transmembrane domains IV and V. Moreover, rather than existing only as distinct dimeric species, the results are consistent with the D3 receptor also assuming a quaternary structure in which two transmembrane domain I-II-helix VIII dimers interact to form a "rhombic" tetramer via an interface involving residues from transmembrane domains VI and VII. In addition, the results also provide insights into the potential contribution of molecules of cholesterol to the overall organization and potential stability of the D3 receptor and possibly other GPCR quaternary structures. PMID:25931118

  11. The Other Double Helix--The Fascinating Chemistry of Starch

    NASA Astrophysics Data System (ADS)

    Hancock, Robert D.; Tarbet, Bryon J.

    2000-08-01

    Current textbooks deal only briefly with the chemistry of starch. A short review with 21 references is presented, describing the structure of starch and indicating the double helix structure of A-type and B-type starch. The structure of the starch granule is examined, pointing out the existence of growth rings of alternating crystalline and noncrystalline starch, with growing amylopectin molecules extending from the hilum (point of origin) to the surface of the starch granule. The swelling of starch granules in water, above the gelatinization temperature of about 60 °C, is discussed. The process of gelatinization involves unraveling of the starch helix and a manyfold increase in volume of the starch granule as water is imbibed and bound to the unraveled starch polymer by hydrogen bonding. Baking bread or pastries causes unraveling of the starch helix, and the process by which these products become stale corresponds primarily to the re-forming of the starch helix. The importance of this phenomenon in food science is discussed. The absorption of nonpolar linear molecules such as I2, or linear nonpolar portions of molecules such as n-butanol or fats and phospholipids, by the C-type helix of starch is examined. The way in which starch is structurally modified to retard staling is discussed in relation to food technology.

  12. CERAMIDE-RICH PLATFORMS IN TRANSMEMBRANE SIGNALING

    PubMed Central

    Stancevic, Branka; Kolesnick, Richard

    2015-01-01

    Recent evidence suggests that ceramide regulates stress signaling via reorganization of the plasma membrane. The focus of this review will be to discuss the mechanism by which acid sphingomyelinase (ASMase)-generated ceramide initiates transmembrane signaling in the plasma membrane exoplasmic leaflet. In particular, we review the unique biophysical properties of ceramide that render it proficient in formation of signaling domains termed ceramide-rich platforms (CRPs), and the role of CRPs in the pathophysiology of various diseases. The biomedical significance of CRPs makes these structures an attractive therapeutic target. PMID:20178791

  13. A Solid-State NMR Index of Helical Membrane Protein Structure and Topology

    NASA Astrophysics Data System (ADS)

    Marassi, Francesca M.; Opella, Stanley J.

    2000-05-01

    The secondary structure and topology of membrane proteins can be described by inspection of two-dimensional 1H-15N dipolar coupling/15N chemical shift polarization inversion spin exchange at the magic angle spectra obtained from uniformly 15N-labeled samples in oriented bilayers. The characteristic wheel-like patterns of resonances observed in these spectra reflect helical wheel projections of residues in both transmembrane and in-plane helices and hence provide direct indices of the secondary structure and topology of membrane proteins in phospholipid bilayers. We refer to these patterns as PISA (polarity index slant angle) wheels. The transmembrane helix of the M2 peptide corresponding to the pore-lining segment of the acetylcholine receptor and the membrane surface helix of the antibiotic peptide magainin are used as examples.

  14. Modeling Transmembrane Domain Dimers/Trimers of Plexin Receptors: Implications for Mechanisms of Signal Transmission across the Membrane

    PubMed Central

    Zhang, Liqun; Polyansky, Anton; Buck, Matthias

    2015-01-01

    Single-pass transmembrane (TM) receptors transmit signals across lipid bilayers by helix association or by configurational changes within preformed dimers. The structure determination for such TM regions is challenging and has mostly been accomplished by NMR spectroscopy. Recently, the computational prediction of TM dimer structures is becoming recognized for providing models, including alternate conformational states, which are important for receptor regulation. Here we pursued a strategy to predict helix oligomers that is based on packing considerations (using the PREDDIMER webserver) and is followed by a refinement of structures, utilizing microsecond all-atom molecular dynamics simulations. We applied this method to plexin TM receptors, a family of 9 human proteins, involved in the regulation of cell guidance and motility. The predicted models show that, overall, the preferences identified by PREDDIMER are preserved in the unrestrained simulations and that TM structures are likely to be diverse across the plexin family. Plexin-B1 and –B3 TM helices are regular and tend to associate, whereas plexin-A1, -A2, –A3, -A4, -C1 and –D1 contain sequence elements, such as poly-Glycine or aromatic residues that distort helix conformation and association. Plexin-B2 does not form stable dimers due to the presence of TM prolines. No experimental structural information on the TM region is available for these proteins, except for plexin-C1 dimeric and plexin-B1 – trimeric structures inferred from X-ray crystal structures of the intracellular regions. Plexin-B1 TM trimers utilize Ser and Thr sidechains for interhelical contacts. We also modeled the juxta-membrane (JM) region of plexin-C1 and plexin-B1 and show that it synergizes with the TM structures. The structure and dynamics of the JM region and TM-JM junction provide determinants for the distance and distribution of the intracellular domains, and for their binding partners relative to the membrane. The structures suggest experimental tests and will be useful for the interpretation of future studies. PMID:25837709

  15. Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker

    PubMed Central

    Jeong, Woo Hyeon; Lee, Haerim; Song, Dong Hyun; Eom, Jae-Hoon; Kim, Sun Chang; Lee, Hee-Seung; Lee, Hayyoung; Lee, Jie-Oh

    2016-01-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a ‘fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures. PMID:26980593

  16. Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker

    NASA Astrophysics Data System (ADS)

    Jeong, Woo Hyeon; Lee, Haerim; Song, Dong Hyun; Eom, Jae-Hoon; Kim, Sun Chang; Lee, Hee-Seung; Lee, Hayyoung; Lee, Jie-Oh

    2016-03-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a `fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures.

  17. Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker.

    PubMed

    Jeong, Woo Hyeon; Lee, Haerim; Song, Dong Hyun; Eom, Jae-Hoon; Kim, Sun Chang; Lee, Hee-Seung; Lee, Hayyoung; Lee, Jie-Oh

    2016-01-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a 'fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures. PMID:26980593

  18. Strong dimerization of wild-type ErbB2/Neu transmembrane domain and the oncogenic Val664Glu mutant in mammalian plasma membranes

    PubMed Central

    Placone, Jesse; He, Lijuan; Del Piccolo, Nuala; Hristova, Kalina

    2014-01-01

    Here, we study the homodimerization of the transmembrane domain of Neu, as well as an oncogenic mutant (V664E), in vesicles derived from the plasma membrane of mammalian cells. For the characterization, we use a Förster resonance energy transfer (FRET)-based method termed Quantitative Imaging-FRET (QI-FRET), which yields the donor and acceptor concentrations in addition to the FRET efficiencies in individual plasma membrane-derived vesicles. Our results demonstrate that both the wild-type and the mutant are 100% dimeric, suggesting that the Neu TM helix dimerizes more efficiently than other RTK TM domains in mammalian membranes. Furthermore, the data suggest that the V664E mutation causes a very small, but statistically significant change in dimer structure. PMID:24631664

  19. Conformational changes induced by a single amino acid substitution in the trans-membrane domain of Vpu: Implications for HIV-1 susceptibility to channel blocking drugs

    PubMed Central

    Park, Sang Ho; Opella, Stanley J.

    2007-01-01

    The channel-forming trans-membrane domain of Vpu (Vpu TM) from HIV-1 is known to enhance virion release from the infected cells and is a potential target for ion-channel blockers. The substitution of alanine at position 18 by a histidine (A18H) has been shown to render HIV-1 infections susceptible to rimantadine, a channel blocker of M2 protein from the influenza virus. In order to describe the influence of the mutation on the structure and rimantadine susceptibility of Vpu, we determined the structure of A18H Vpu TM, and compared it to those of wild-type Vpu TM and M2 TM. Both isotropic and orientationally dependent NMR frequencies of the backbone amide resonance of His18 were perturbed by rimantadine, and those of Ile15 and Trp22 were also affected, suggesting that His18 is the key residue for rimantadine binding and that residues located on the same face of the TM helix are also involved. A18H Vpu TM has an ideal, straight α-helix spanning residues 6–27 with an average tilt angle of 41° in C14 phospholipid bicelles, indicating that the tilt angle is increased by 11° compared to that of wild-type Vpu TM. The longer helix formed by the A18H mutation has a larger tilt angle to compensate for the hydrophobic mismatch with the length of the phospholipids in the bilayer. These results demonstrate that the local change of the primary structure plays an important role in secondary and tertiary structures of Vpu TM in lipid bilayers and affects its ability to interact with channel blockers. PMID:17766368

  20. Crown ether helical peptides are preferentially inserted in lipid bilayers as a transmembrane ion channels.

    PubMed

    Savoie, Jean-Daniel; Otis, François; Bürck, Jochen; Ulrich, Anne S; Voyer, Normand

    2015-07-01

    Oriented circular dichroism was used to study the alignment crown ether-modified peptides. The influence of different N- and C-functionalities was assessed using at variable peptide:lipid ratios from 1:20 to 1:200. Neither the functionalities nor the concentration had any major effect on the orientation. The alignment of the 21-mer peptides was also examined with lipid membranes of different bilayer thickness. The use of synchrotron radiation as light source allowed the study of peptide:lipid molar ratios from 1:20 to 1:1000. For all conditions studied, the peptides were found to be predominantly incorporated as a transmembrane helix into the membrane, especially at low peptide concentration, but started to aggregate on the membrane surface at higher peptide:lipid ratios. The structural information on the preferred trans-bilayer alignment of the crown ether functional groups explains their ion conductivity and is useful for the further development of membrane-active nanochemotherapeutics. PMID:25753314

  1. Single-spanning transmembrane domains in cell growth and cell-cell interactions

    PubMed Central

    Sawma, Paul; Duneau, Jean-Pierre; Khao, Jonathan; Hénin, Jélerôme; Bagnard, Dominique; Sturgis, James

    2010-01-01

    As a whole, integral membrane proteins represent about one third of sequenced genomes, and more than 50% of currently available drugs target membrane proteins, often cell surface receptors. Some membrane protein classes, with a defined number of transmembrane (TM) helices, are receiving much attention because of their great functional and pharmacological importance, such as G protein-coupled receptors possessing 7 TM segments. Although they represent roughly half of all membrane proteins, bitopic proteins (with only 1 TM helix) have so far been less well characterized. Though they include many essential families of receptors, such as adhesion molecules and receptor tyrosine kinases, many of which are excellent targets for biopharmaceuticals (peptides, antibodies, et al.). A growing body of evidence suggests a major role for interactions between TM domains of these receptors in signaling, through homo and heteromeric associations, conformational changes, assembly of signaling platforms, etc. Significantly, mutations within single domains are frequent in human disease, such as cancer or developmental disorders. This review attempts to give an overview of current knowledge about these interactions, from structural data to therapeutic perspectives, focusing on bitopic proteins involved in cell signaling. PMID:20543559

  2. Predicting transmembrane beta-barrels in proteomes

    PubMed Central

    Bigelow, Henry R.; Petrey, Donald S.; Liu, Jinfeng; Przybylski, Dariusz; Rost, Burkhard

    2004-01-01

    Very few methods address the problem of predicting beta-barrel membrane proteins directly from sequence. One reason is that only very few high-resolution structures for transmembrane beta-barrel (TMB) proteins have been determined thus far. Here we introduced the design, statistics and results of a novel profile-based hidden Markov model for the prediction and discrimination of TMBs. The method carefully attempts to avoid over-fitting the sparse experimental data. While our model training and scoring procedures were very similar to a recently published work, the architecture and structure-based labelling were significantly different. In particular, we introduced a new definition of beta- hairpin motifs, explicit state modelling of transmembrane strands, and a log-odds whole-protein discrimination score. The resulting method reached an overall four-state (up-, down-strand, periplasmic-, outer-loop) accuracy as high as 86%. Furthermore, accurately discriminated TMB from non-TMB proteins (45% coverage at 100% accuracy). This high precision enabled the application to 72 entirely sequenced Gram-negative bacteria. We found over 164 previously uncharacterized TMB proteins at high confidence. Database searches did not implicate any of these proteins with membranes. We challenge that the vast majority of our 164 predictions will eventually be verified experimentally. All proteome predictions and the PROFtmb prediction method are available at http://www.rostlab.org/services/PROFtmb/. PMID:15141026

  3. Circular dichroism, molecular modeling, and serology indicate that the structural basis of antigenic variation in foot-and-mouth disease virus is [alpha]-helix formation

    SciTech Connect

    France, L.L.; Piatti, P.G.; Newman, J.F.E.; Brown, F. ); Toth, I.; Gibbons, W.A. )

    1994-08-30

    Seven antigenic variants obtained from a single field isolate of foot-and-mouth disease virus, serotype A12, differ only at residues 148 and 153 in the immunodominant loop of viral protein VP1. Synthetic peptides corresponding to the region 141-160 are highly immunogenic. UV circular dichroism shows that (i) in aqueous solution of the peptides are nearly identical, but in 100% trifluoroethanol they display helix-forming properties which correlate well with their serological crossreactivities for anti-peptide sera, and (ii) these properties are insensitive to substitutions at position 153, except for proline, but are highly sensitive to substitutions at position 148. This pattern can be explained by the effects of these substitutions on the amphiphilic character and positions of helices postulated in the region 146-156. Molecular models indicate that residues 147, 148, 150, 151, 153-155, and 157 are most likely to interact with residues of the antibody paratopes. The data are consistent with the existence of an inverse [gamma]-turn around Pro-153, and a [beta]-turn at the cell-attachment site at residues 145-147. 31 refs., 5 figs.

  4. Transmembrane Segment II of NhaA Na+/H+ Antiporter Lines the Cation Passage, and Asp65 Is Critical for pH Activation of the Antiporter*

    PubMed Central

    Herz, Katia; Rimon, Abraham; Olkhova, Elena; Kozachkov, Lena; Padan, Etana

    2010-01-01

    The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 5.58.5), many questions related to the active state of NhaA have remained elusive. Our experimental results at physiological pH and computational analyses reveal that amino acid residues in transmembrane segment II contribute to the cation pathway of NhaA and its pH regulation: 1) transmembrane segment II is a highly conserved helix and the conserved amino acid residues are located on one side of the helix facing either the cytoplasmic or periplasmic funnels of NhaA structure. 2) Cys replacements of the conserved residues and measuring their antiporter activity in everted membrane vesicles showed that D65C, L67C, E78C, and E82C increased the apparent Km to Na+ and Li+ and changed the pH response of the antiporter. 3) Introduced Cys replacements, L60C, N64C, F71C, F72C, and E78C, were significantly alkylated by [14C]N-ethylmaleimide implying the presence of water-filled cavities in NhaA. 4) Several Cys replacements were modified by MTSES and/or MTSET, membrane impermeant, negatively and positively charged reagents, respectively, that could reach Cys replacements from the periplasm only via water-filled funnel(s). Remarkably, the reactivity of D65C to MTSES increased with increasing pH and chemical modification by MTSES but not by MTSET, decreased the apparent Km of the antiporter at pH 7.5 (10-fold) but not at pH 8.5, implying the importance of Asp65 negative charge for pH activation of the antiporter. PMID:19923224

  5. Biosynthesis and purification of a hydrophobic peptide from transmembrane domains of G-protein-coupled CB2 receptor.

    PubMed

    Zheng, H; Zhao, J; Wang, S; Lin, C-M; Chen, T; Jones, D H; Ma, C; Opella, S; Xie, X-Q

    2005-04-01

    A major challenge for the structural study of the seven-transmembrane G-protein-coupled receptors is to obtain a sufficient amount of purified protein at the milligram level, which is required for either nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography. In order to develop a high-yield and cost-effective method, and also to obtain preliminary structural information for the computer modeling of the three-dimensional receptor structural model, a highly hydrophobic peptide from human cannabinoid subtype 2 receptor CB2(65-101), was chosen to develop high-yield membrane protein expression and purification methods. The peptide included the second transmembrane helix with the associated loop regions of the CB2 receptor. It was over-expressed in Escherichia coli, with a modified TrpDelta LE1413 (TrpLE) leading fusion sequence and a nine-histidine tag, and was then separated and purified from the tag in a preparative scale. An experimental protocol for the chemical cleavage of membrane protein fragment was developed using cyanogen bromide to remove the TrpLE tag from the hydrophobic fusion protein. In addition, protein uniformly labeled with isotopic 15N was obtained by expression in 15N-enriched minimum media. The developed and optimized preparation scheme of expression, cleavage, and purification provided a sufficient amount of peptide for NMR structure analysis and other biophysical studies that will be reported elsewhere. The process of fusion protein cleavage following purification was monitored by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), and the final sample was validated by MS and circular dichroism experiments. PMID:15813893

  6. Conformational Flexibility in the Transmembrane Protein TSPO.

    PubMed

    Jaremko, Łukasz; Jaremko, Mariusz; Giller, Karin; Becker, Stefan; Zweckstetter, Markus

    2015-11-01

    The translocator protein (TSPO) is an integral membrane protein that interacts with a wide variety of endogenous ligands, such as cholesterol and porphyrins, and is also the target for several small molecules with substantial in vivo efficacy. When complexed with the TSPO-specific radioligand (R)-PK11195, TSPO folds into a rigid five-helix bundle. However, little is known about the structure and dynamics of TSPO in the absence of high-affinity ligands. By means of NMR spectroscopy, we show that TSPO exchanges between multiple conformations in the absence of (R)-PK11195. Extensive motions on time scales from pico- to microseconds occur all along the primary sequence of the protein, leading to a loss of stable tertiary interactions and local unfolding of the helical structure in the vicinity of the ligand-binding site. The flexible nature of TSPO highlights the importance of conformational plasticity in integral membrane proteins. PMID:26394723

  7. Specificity of RNA–RNA helix recognition

    PubMed Central

    Battle, Daniel J.; Doudna, Jennifer A.

    2002-01-01

    Functional RNAs often form compact structures characterized by closely packed helices. Crystallographic analysis of several large RNAs revealed a prevalent interaction in which unpaired adenosine residues dock into the minor groove of a receptor helix. This A-minor motif, potentially the most important element responsible for global RNA architecture, has also been suggested to contribute to the fidelity of protein synthesis by discriminating against near-cognate tRNAs on the ribosome. The specificity of A-minor interactions is fundamental to RNA tertiary structure formation, as well as to their proposed role in translational accuracy. To investigate A-minor motif specificity, we analyzed mutations in an A-minor interaction within the Tetrahymena group I self-splicing intron. Thermodynamic and x-ray crystallographic results show that the A-minor interaction strongly prefers canonical base pairs over base mismatches in the receptor helix, enabling RNA interhelical packing through specific recognition of Watson–Crick minor groove geometry. PMID:12189204

  8. Integrin ?IIb?3 Transmembrane Domain Separation Mediates Bi-Directional Signaling across the Plasma Membrane

    PubMed Central

    Hu, Ping; Luo, Bing-Hao

    2015-01-01

    Integrins play an essential role in hemostasis, thrombosis, and cell migration, and they transmit bidirectional signals. Transmembrane/cytoplasmic domains are hypothesized to associate in the resting integrins; whereas, ligand binding and intracellular activating signals induce transmembrane domain separation. However, how this conformational change affects integrin outside-in signaling and whether the ? subunit cytoplasmic domain is important for this signaling remain elusive. Using Chinese Hamster Ovary (CHO) cells that stably expressed different integrin ?IIb?3 constructs, we discovered that an ?IIb cytoplasmic domain truncation led to integrin activation but not defective outside-in signaling. In contrast, preventing transmembrane domain separation abolished both inside-out and outside-in signaling regardless of removing the ?IIb cytoplasmic tail. Truncation of the ?IIb cytoplasmic tail did not obviously affect adhesion-induced outside-in signaling. Our research revealed that transmembrane domain separation is a downstream conformational change after the cytoplasmic domain dissociation in inside-out activation and indispensable for ligand-induced outside-in signaling. The result implicates that the ? TM helix rearrangement after dissociation is essential for integrin transmembrane signaling. Furthermore, we discovered that the PI3K/Akt pathway is not essential for cell spreading but spreading-induced Erk1/2 activation is PI3K dependent implicating requirement of the kinase for cell survival in outside-in signaling. PMID:25617834

  9. A GxxxG-like motif within HIV-1 fusion peptide is critical to its immunosuppressant activity, structure, and interaction with the transmembrane domain of the T-cell receptor.

    PubMed

    Faingold, Omri; Cohen, Tomer; Shai, Yechiel

    2012-09-28

    To thrive in the human body, HIV fuses to its target cell and evades the immune response via several mechanisms. The fusion cascade is initiated by the fusion peptide (FP), which is located at the N-terminal of gp41, the transmembrane protein of HIV. Recently, it has been shown that the HIV-1 FP, particularly its 5-13 amino acid region (FP(5-13)), suppresses T-cell activation and interacts with the transmembrane domain (TMD) of the T-cell receptor (TCR) complex. Specific amino acid motifs often contribute to such interactions in TMDs of membrane proteins. Using bioinformatics and experimental studies, we report on a GxxxG-like motif (AxxxG), which is conserved in the FP throughout different clades and strains of HIV-1. Biological activity studies and FTIR spectroscopy revealed that HIV FP(5-13)-derived peptides, in which the motif was altered either by randomization or by a single amino acid shift, lost their immunosuppressive activity concomitant with a loss of the β-sheet structure in a membranous environment. Furthermore, fluorescence studies revealed that the inactive mutants lost their ability to interact with their target site, namely, the TMD of TCRα, designated CP. Importantly, lipotechoic acid activated macrophages (lacking TCR) were not affected by FP, further demonstrating the specificity of the immunosuppressant activity of CP. Finally, although the AxxxG WT and the GxxxG analog both associated with the CP and immunosuppressed T-cells, the AxxxG WT but not the GxxxG analog induced lipid mixing. Overall, the data support an important role for the AxxxG motif in the function of FP and might explain the natural selection of the AxxxG motif rather than the classical GxxxG motif in FP. PMID:22872636

  10. Probing α-310 Transitions in a Voltage-Sensing S4 Helix

    PubMed Central

    Kubota, Tomoya; Lacroix, Jérôme J.; Bezanilla, Francisco; Correa, Ana M.

    2014-01-01

    The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 310 conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 310 conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 310 conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-310 helical conversions of the S4 in Ci-VSP associated with voltage activation. PMID:25185547

  11. An extended CCR5-ECL2 peptide forms a helix that binds HIV-1 gp120 through non-specific hydrophobic interactions

    PubMed Central

    Kessler, Naama; Arshava, Boris; Naider, Fred; Scherf, Tali; Anglister, Jacob

    2015-01-01

    The chemokine receptor CCR5 serves as a co-receptor for the Human Immunodefficiency Virus type-1, HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing the ECL1 and ECL3 were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study we determined that in aqueous buffer the segment Q188-Q194 in an elongated ECL2 peptide (R168 to K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two dimensional Saturation Transfer Difference NMR spectroscopy and dynamic filtering studies revealed the involvement of Y187, F189, W190 and F193 of the helical segment, in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule and therefore, could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors. PMID:25703038

  12. VIEW OF HELIX HOUSE NO. 2 (S87), WITH ANTENNA TOWER ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW OF HELIX HOUSE NO. 2 (S-87), WITH ANTENNA TOWER CABLE SUPPORT IN FOREGROUND, LOOKING SOUTHEAST. - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

  13. VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S87), ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S-87), LOOKING WEST (with scale stick). - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

  14. VIEW OF SOUTH ELEVATION OF HELIX HOUSE NO. 2 (S87) ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW OF SOUTH ELEVATION OF HELIX HOUSE NO. 2 (S-87) SHOWING MAIN ENTRY DOOR, LOOKING NORTH NORTHWEST. - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

  15. VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S87), ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S-87), LOOKING WEST (without scale stick). - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

  16. Transmembrane domain oligomerization propensity determined by ToxR assay.

    PubMed

    Joce, Catherine; Wiener, Alyssa; Yin, Hang

    2011-01-01

    The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action. One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers. Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association. Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve, direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays. The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1). In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay. The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where β-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for β-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by β-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2). PMID:21654625

  17. Artificial metalloenzymes derived from three-helix bundles

    PubMed Central

    Tebo, Alison G.; Pecoraro, Vincent L.

    2015-01-01

    Three-helix bundles and coiled-coil motifs are well-established de novo designed scaffolds that have been investigated for their metal-binding and catalytic properties. Satisfaction of the primary coordination sphere for a given metal is sufficient to introduce catalytic activity and a given structure may catalyze different reactions dependent on the identity of the incorporated metal. Here we describe recent contributions in the de novo design of metalloenzymes based on three-helix bundles and coiled-coil motifs, focusing on non-heme systems for hydrolytic and redox chemistry. PMID:25579452

  18. Artificial metalloenzymes derived from three-helix bundles.

    PubMed

    Tebo, Alison G; Pecoraro, Vincent L

    2015-04-01

    Three-helix bundles and coiled-coil motifs are well-established de novo designed scaffolds that have been investigated for their metal-binding and catalytic properties. Satisfaction of the primary coordination sphere for a given metal is sufficient to introduce catalytic activity and a given structure may catalyze different reactions dependent on the identity of the incorporated metal. Here we describe recent contributions in the de novo design of metalloenzymes based on three-helix bundles and coiled-coil motifs, focusing on non-heme systems for hydrolytic and redox chemistry. PMID:25579452

  19. Helix 8 Plays a Crucial Role in Bradykinin B2 Receptor Trafficking and Signaling

    PubMed Central

    Feierler, Jens; Wirth, Markus; Welte, Benjamin; Schüssler, Steffen; Jochum, Marianne; Faussner, Alexander

    2011-01-01

    Upon activation the human bradykinin B2 receptor (B2R) acts as guanine nucleotide exchange factor for the G proteins Gq/11 and Gi. Thereafter, it gets phosphorylated by G protein-coupled receptor kinases (GRKs) and recruits β-arrestins, which block further G protein activation and promote B2R internalization via clathrin-coated pits. As for most G protein-coupled receptors of family A, an intracellular helix 8 after transmembrane domain 7 is also predicted for the B2R. We show here that disruption of helix 8 in the B2R by either C-terminal truncation or just by mutation of a central amino acid (Lys-315) to a helix-breaking proline resulted in strong reduction of surface expression. Interestingly, this malfunction could be overcome by the addition of the membrane-permeable B2R antagonist JSM10292, suggesting that helix 8 has a general role for conformational stabilization that can be accounted for by an appropriate antagonist. Intriguingly, an intact helix 8, but not the C terminus with its phosphorylation sites, was indispensable for receptor sequestration and for interaction of the B2R with GRK2/3 and β-arrestin2 as shown by co-immunoprecipitation. Recruitment of β-arrestin1, however, required the presence of the C terminus. Taken together, our results demonstrate that helix 8 of the B2R plays a crucial role not only in efficient trafficking to the plasma membrane or the activation of G proteins but also for the interaction of the B2R with GRK2/3 and β-arrestins. Additional data obtained with chimera of B2R with other G protein-coupled receptors of family A suggest that helix 8 might have similar functions in other GPCRs as well. PMID:22016392

  20. Folding and insertion thermodynamics of the transmembrane WALP peptide.

    PubMed

    Bereau, Tristan; Bennett, W F Drew; Pfaendtner, Jim; Deserno, Markus; Karttunen, Mikko

    2015-12-28

    The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA)n (L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural properties have been illuminated in a large number of experimental and simulation studies. In this combined coarse-grained and atomistic simulation study, we probe the thermodynamics of a single WALP peptide, focusing on both the insertion across the water-membrane interface, as well as folding in both water and a membrane. The potential of mean force characterizing the peptide's insertion into the membrane shows qualitatively similar behavior across peptides and three force fields. However, the Martini force field exhibits a pronounced secondary minimum for an adsorbed interfacial state, which may even become the global minimum-in contrast to both atomistic simulations and the alternative PLUM force field. Even though the two coarse-grained models reproduce the free energy of insertion of individual amino acids side chains, they both underestimate its corresponding value for the full peptide (as compared with atomistic simulations), hinting at cooperative physics beyond the residue level. Folding of WALP in the two environments indicates the helix as the most stable structure, though with different relative stabilities and chain-length dependence. PMID:26723612

  1. Folding and insertion thermodynamics of the transmembrane WALP peptide

    NASA Astrophysics Data System (ADS)

    Bereau, Tristan; Bennett, W. F. Drew; Pfaendtner, Jim; Deserno, Markus; Karttunen, Mikko

    2015-12-01

    The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA)n (L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural properties have been illuminated in a large number of experimental and simulation studies. In this combined coarse-grained and atomistic simulation study, we probe the thermodynamics of a single WALP peptide, focusing on both the insertion across the water-membrane interface, as well as folding in both water and a membrane. The potential of mean force characterizing the peptide's insertion into the membrane shows qualitatively similar behavior across peptides and three force fields. However, the Martini force field exhibits a pronounced secondary minimum for an adsorbed interfacial state, which may even become the global minimum—in contrast to both atomistic simulations and the alternative PLUM force field. Even though the two coarse-grained models reproduce the free energy of insertion of individual amino acids side chains, they both underestimate its corresponding value for the full peptide (as compared with atomistic simulations), hinting at cooperative physics beyond the residue level. Folding of WALP in the two environments indicates the helix as the most stable structure, though with different relative stabilities and chain-length dependence.

  2. Folding simulations of gramicidin A into the beta-helix conformations: Simulated annealing molecular dynamics study.

    PubMed

    Mori, Takaharu; Okamoto, Yuko

    2009-10-28

    Gramicidin A is a linear hydrophobic 15-residue peptide which consists of alternating D- and L-amino acids and forms a unique tertiary structure, called the beta(6.3)-helix, to act as a cation-selective ion channel in the natural conditions. In order to investigate the intrinsic ability of the gramicidin A monomer to form secondary structures, we performed the folding simulation of gramicidin A using a simulated annealing molecular dynamics (MD) method in vacuum mimicking the low-dielectric, homogeneous membrane environment. The initial conformation was a fully extended one. From the 200 different MD runs, we obtained a right-handed beta(4.4)-helix as the lowest-potential-energy structure, and left-handed beta(4.4)-helix, right-handed and left-handed beta(6.3)-helix as local-minimum energy states. These results are in accord with those of the experiments of gramicidin A in homogeneous organic solvent. Our simulations showed a slight right-hand sense in the lower-energy conformations and a quite beta-sheet-forming tendency throughout almost the entire sequence. In order to examine the stability of the obtained right-handed beta(6.3)-helix and beta(4.4)-helix structures in more realistic membrane environment, we have also performed all-atom MD simulations in explicit water, ion, and lipid molecules, starting from these beta-helix structures. The results suggested that beta(6.3)-helix is more stable than beta(4.4)-helix in the inhomogeneous, explicit membrane environment, where the pore water and the hydrogen bonds between Trp side-chains and lipid-head groups have a role to further stabilize the beta(6.3)-helix conformation. PMID:19894978

  3. Transmembrane protein sorting driven by membrane curvature

    NASA Astrophysics Data System (ADS)

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-11-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  4. Transmembrane protein sorting driven by membrane curvature

    PubMed Central

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-01-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization. PMID:26522943

  5. Surfactant Bilayers Maintain Transmembrane Protein Activity

    PubMed Central

    Rayan, Gamal; Adrien, Vladimir; Reffay, Myriam; Picard, Martin; Ducruix, Arnaud; Schmutz, Marc; Urbach, Wladimir; Taulier, Nicolas

    2014-01-01

    In vitro studies of membrane proteins are of interest only if their structure and function are significantly preserved. One approach is to insert them into the lipid bilayers of highly viscous cubic phases rendering the insertion and manipulation of proteins difficult. Less viscous lipid sponge phases are sometimes used, but their relatively narrow domain of existence can be easily disrupted by protein insertion. We present here a sponge phase consisting of nonionic surfactant bilayers. Its extended domain of existence and its low viscosity allow easy insertion and manipulation of membrane proteins. We show for the first time, to our knowledge, that transmembrane proteins, such as bacteriorhodopsin, sarcoplasmic reticulum Ca2+ATPase (SERCA1a), and its associated enzymes, are fully active in a surfactant phase. PMID:25185548

  6. Surfactant bilayers maintain transmembrane protein activity.

    PubMed

    Rayan, Gamal; Adrien, Vladimir; Reffay, Myriam; Picard, Martin; Ducruix, Arnaud; Schmutz, Marc; Urbach, Wladimir; Taulier, Nicolas

    2014-09-01

    In vitro studies of membrane proteins are of interest only if their structure and function are significantly preserved. One approach is to insert them into the lipid bilayers of highly viscous cubic phases rendering the insertion and manipulation of proteins difficult. Less viscous lipid sponge phases are sometimes used, but their relatively narrow domain of existence can be easily disrupted by protein insertion. We present here a sponge phase consisting of nonionic surfactant bilayers. Its extended domain of existence and its low viscosity allow easy insertion and manipulation of membrane proteins. We show for the first time, to our knowledge, that transmembrane proteins, such as bacteriorhodopsin, sarcoplasmic reticulum Ca(2+)ATPase (SERCA1a), and its associated enzymes, are fully active in a surfactant phase. PMID:25185548

  7. An Autonomously Reciprocating Transmembrane Nanoactuator.

    PubMed

    Watson, Matthew A; Cockroft, Scott L

    2016-01-22

    Biological molecular machines operate far from equilibrium by coupling chemical potential to repeated cycles of dissipative nanomechanical motion. This principle has been exploited in supramolecular systems that exhibit true machine behavior in solution and on surfaces. However, designed membrane-spanning assemblies developed to date have been limited to simple switches or stochastic shuttles, and true machine behavior has remained elusive. Herein, we present a transmembrane nanoactuator that turns over chemical fuel to drive autonomous reciprocating (back-and-forth) nanomechanical motion. Ratcheted reciprocating motion of a DNA/PEG copolymer threaded through a single α-hemolysin pore was induced by a combination of DNA strand displacement processes and enzyme-catalyzed reactions. Ion-current recordings revealed saw-tooth patterns, indicating that the assemblies operated in autonomous, asymmetric cycles of conformational change at rates of up to one cycle per minute. PMID:26661295

  8. HELIX: The High Energy Light Isotope Experiment

    NASA Astrophysics Data System (ADS)

    Nutter, Scott

    This is the lead proposal for a new suborbital program, HELIX (High-Energy Light Isotope eXperiment), designed to make measurements of the isotopic composition of light cosmic-ray nuclei from ~200 MeV/nuc to ~10 GeV/nuc. Past measurements of this kind have provided profound insights into the nature and origin of cosmic rays, revealing, for instance, information on acceleration and confinement time scales, and exposing some conspicuous discrepancies between solar and cosmic-ray abundances. The most detailed information currently available comes from the ACE/CRIS mission, but is restricted to energies below a few 100 MeV/nuc. HELIX aims at extending this energy range by over an order of magnitude, where, in most cases, no measurements of any kind exist, and where relativistic time dilation affects the apparent lifetime of radioactive clock nuclei. The HELIX measurements will provide essential information for understanding the propagation history of cosmic rays in the galaxy. This is crucial for properly interpreting several intriguing anomalies reported in recent cosmic-ray measurements, pertaining to the energy spectra of protons, helium, and heavier nuclei, and to the anomalous rise in the positron fraction at higher energy. HELIX employs a high-precision magnet spectrometer to provide measurements which are not achievable by any current or planned instrument. The superconducting magnet originally used for the HEAT payload in five successful high-altitude flights will be combined with state-of-the-art detectors to measure the charge, time-of-flight, magnetic rigidity, and velocity of cosmic-ray particles with high precision. The instrumentation includes plastic scintillators, silicon-strip detectors repurposed from Fermilab's CDF detector, a high-performance gas drift chamber, and a ring-imaging Cherenkov counter employing aerogel radiators and silicon photomultipliers. To reduce cost and technical risk, the HELIX program will be structured in two stages. The first stage, which is the subject of this proposal, will focus on the design and construction of the main HELIX instrument, and the measurement of key light isotope ratios from ~200 MeV/n to ~3GeV/n. A future stage 2 will build on this work, incorporating evolutionary enhancements to the instrumentation to extend the energy reach into the challenging ~10 GeV/n range. The stage 1 instrument achieves a maximum detectable rigidity of ~800GV and charge range from Z=1 to Z=10. The high field of the HEAT magnet will make it possible to reach the required mass resolution dm/m = 2.5% over the energy range of concern with very small systematic limitations due to multiple Coulomb scattering in the thin tracker. This is a decisive advantage over the current AMS-02 instrument which employs a permanent magnet with an average field ~7 times smaller than that of HELIX. The primary scientific goals of the full HELIX program are: - a high-statistics measurement of the 10Be/9Be 'clock ratio' to ~10 GeV/nuc - a high-statistics measurement of the 3He/4He ratio to ~12GeV/nuc - the first measurements of 22Ne/20Ne above 1 GeV/nuc - the first measurements of 7Li/6Li, and 10B/11B above 1 GeV//nuc A number of secondary goals will also be pursued including the measurement of several other isotopic and elemental abundance ratios and fluxes, as well as the primary Helium flux to ~125 GeV/nuc, where there is currently disagreement between AMS-02 and PAMELA on the shape of the energy spectrum. The proposed work will be conducted by a team of US scientists and engineers with extensive experience in cosmic-ray observations on balloons or in space, and, specifically in magnet spectroscopy, with the SMILI, P-BAR, and HEAT programs. Significant participation from a crew of more than 10 graduate and undergraduate students forms an important educational element of the program.

  9. Improved detection of helix-turn-helix DNA-binding motifs in protein sequences.

    PubMed Central

    Dodd, I B; Egan, J B

    1990-01-01

    We present an update of our method for systematic detection and evaluation of potential helix-turn-helix DNA-binding motifs in protein sequences [Dodd, I. and Egan, J. B. (1987) J. Mol. Biol. 194, 557-564]. The new method is considerably more powerful, detecting approximately 50% more likely helix-turn-helix sequences without an increase in false predictions. This improvement is due almost entirely to the use of a much larger reference set of 91 presumed helix-turn-helix sequences. The scoring matrix derived from this reference set has been calibrated against a large protein sequence database so that the score obtained by a sequence can be used to give a practical estimation of the probability that the sequence is a helix-turn-helix motif. PMID:2402433

  10. Identification of a cluster of residues in transmembrane segment 6 of domain III of the cockroach sodium channel essential for the action of pyrethroid insecticides.

    PubMed

    Du, Yuzhe; Lee, Jung-Eun; Nomura, Yoshiko; Zhang, Tianxiang; Zhorov, Boris S; Dong, Ke

    2009-04-15

    A phenylalanine residue (Phe1519) in the sixth transmembrane segment of domain III (IIIS6) of the cockroach BgNa(v) sodium channel is required for the binding and action of pyrethroids. However, whether or not other residues in IIIS6 participate in the action of pyrethroids remains to be determined. In the present study, we conducted a systematic analysis of 20 residues in IIIS6 of the BgNa(v) channel using alanine-scanning mutagenesis. Our results show that alanine substitutions of four residues, Ile1514, Gly1516, Phe1518 and Asn1522, altered sodium channel sensitivity to pyrethroid insecticides. Whereas the G1516A, F1518A and N1522A substitutions diminished sodium channel sensitivity to all seven pyrethroids examined, including four type I (lacking the alpha-cyano group at the phenoxybenzyl alcohol) and three type II (containing the alpha-cyano group) pyrethroids, the I1514A substitution enhanced sodium channel sensitivity to four type I and type II pyrethroids that contain the phenoxybenzyl alcohol only. We also show that alanine/lysine substitutions of Leu1521 and Ser1517 affected the action of BTX (batrachotoxin), but not pyrethroids. In the Kv1.2-based homology model of the open sodium channel, side chains of Ile1514, Phe1518 and Asn1522 are exposed towards helix IIS5 and linker IIS4-IIS5, which contain previously identified pyrethroid-interacting residues, whereas Ser1517 and Leu1521 face the inner pore where the BTX receptor is located. Thus the present study provides further evidence for structural models in which pyrethroids bind to the lipid-exposed interface formed by helices IIIS6, IIS5 and linker helix IIS4-IIS5, whereas BTX binds to the pore-exposed side of the IIIS6 helix. PMID:19154185

  11. Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding.

    PubMed

    Reichart, Timothy M; Baksh, Michael M; Rhee, Jin-Kyu; Fiedler, Jason D; Sligar, Stephen G; Finn, M G; Zwick, Michael B; Dawson, Philip E

    2016-02-01

    The membrane-proximal external region (MPER) of HIV gp41 is an established target of antibodies that neutralize a broad range of HIV isolates. To evaluate the role of the transmembrane (TM) domain, synthetic MPER-derived peptides were incorporated into lipid nanoparticles using natural and designed TM domains, and antibody affinity was measured using immobilized and solution-based techniques. Peptides incorporating the native HIV TM domain exhibit significantly stronger interactions with neutralizing antibodies than peptides with a monomeric TM domain. Furthermore, a peptide with a trimeric, three-helix bundle TM domain recapitulates the binding profile of the native sequence. These studies suggest that neutralizing antibodies can bind the MPER when the TM domain is a three-helix bundle and this presentation could influence the binding of neutralizing antibodies to the virus. Lipid-bilayer presentation of viral antigens in Nanodiscs is a new platform for evaluating neutralizing antibodies. PMID:26799917

  12. Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal α-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

    PubMed Central

    Morgan, Rhodri M. L.; Hernández-Ramírez, Laura C.; Trivellin, Giampaolo; Zhou, Lihong; Roe, S. Mark; Korbonits, Márta; Prodromou, Chrisostomos

    2012-01-01

    Mutations of the aryl hydrocarbon receptor interacting protein (AIP) have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal α-7 helix (Cα-7h) mutations, R304* (nonsense mutation), R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the Cα-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the Cα-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE) of TOMM20. PMID:23300914

  13. Phasing and structure of bestrophin-1: a case study in the use of heavy-atom cluster compounds with multi-subunit transmembrane proteins

    PubMed Central

    Kane Dickson, Veronica

    2016-01-01

    The purification and three-dimensional crystallization of membrane proteins are commonly affected by a cumulation of pathologies that are less prevalent in their soluble counterparts. This may include severe anisotropy, poor spot shape, poor to moderate-resolution diffraction, crystal twinning, translational pseudo-symmetry and poor uptake of heavy atoms for derivatization. Such challenges must be circumvented by adaptations in the approach to crystallization and/or phasing. Here, an example of a protein that exhibited all of the above-mentioned complications is presented. Bestrophin-1 is a eukaryotic calcium-activated chloride channel, the structure of which was recently determined in complex with monoclonal antibody fragments using SAD phasing with tantalum bromide clusters (Ta6Br12·Br2). Some of the obstacles to obtaining improved diffraction and phasing for this particular channel are discussed, as well as the approach and adaptations that were key to determining the structure. PMID:26960119

  14. Phasing and structure of bestrophin-1: a case study in the use of heavy-atom cluster compounds with multi-subunit transmembrane proteins.

    PubMed

    Kane Dickson, Veronica

    2016-03-01

    The purification and three-dimensional crystallization of membrane proteins are commonly affected by a cumulation of pathologies that are less prevalent in their soluble counterparts. This may include severe anisotropy, poor spot shape, poor to moderate-resolution diffraction, crystal twinning, translational pseudo-symmetry and poor uptake of heavy atoms for derivatization. Such challenges must be circumvented by adaptations in the approach to crystallization and/or phasing. Here, an example of a protein that exhibited all of the above-mentioned complications is presented. Bestrophin-1 is a eukaryotic calcium-activated chloride channel, the structure of which was recently determined in complex with monoclonal antibody fragments using SAD phasing with tantalum bromide clusters (Ta6Br12·Br2). Some of the obstacles to obtaining improved diffraction and phasing for this particular channel are discussed, as well as the approach and adaptations that were key to determining the structure. PMID:26960119

  15. ¹³C- and ¹H-detection under fast MAS for the study of poorly available proteins: application to sub-milligram quantities of a 7 trans-membrane protein.

    PubMed

    Dannatt, Hugh R W; Taylor, Garrick F; Varga, Krisztina; Higman, Victoria A; Pfeil, Marc-Philipp; Asilmovska, Lubica; Judge, Peter J; Watts, Anthony

    2015-05-01

    We demonstrate that (13)C-detected spectra recorded using fast (60 kHz) magic angle spinning on sub-milligram (<10 μmol) quantities of a protonated 7 trans-membrane helix protein (bacteriorhodopsin) in its native lipid environment are comparable in sensitivity and resolution to those recorded using 15-fold larger sample volumes with conventional solid state NMR methodology. We demonstrate the utility of proton-detected measurements which yield narrow (1)H linewidths under these conditions, and that no structural alterations are observed. We propose that these methods will prove useful to gain structural information on membrane proteins with poor availability, which can be studied in their native lipid environments. PMID:25701262

  16. Dispersion relations for a plasma-filled helix-loaded-waveguide

    SciTech Connect

    Makowski, M.A.; Hooper, E.B.; Stallard, B.W.

    1994-01-01

    The propagation of waves on bounded, magnetized plasma columns arises in connection with a variety of applications. To this end dispersion relations axe developed for a variety of multi-region circularly symmetric configurations. These include, a sheath helix in free space, a plasma column in free space, a plasma filled conducting tube, a plasma filled sheath-helix in free space, a sheath helix within a conducting cylinder, a plasma filled sheath-helix within a conducting cylinder, and a plasma column within a sheath-helix contained within a conducting cylinder. The latter configuration is of the most interest for whistler wave excitation for plasma thruster applications, since it includes the effect of a vacuum region separating the plasma column from the helical excitation structure.

  17. First principles design of a core bioenergetic transmembrane electron-transfer protein.

    PubMed

    Goparaju, Geetha; Fry, Bryan A; Chobot, Sarah E; Wiedman, Gregory; Moser, Christopher C; Leslie Dutton, P; Discher, Bohdana M

    2016-05-01

    Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics - the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. PMID:26672896

  18. Lessons from Free Energy Simulations of δ-Opioid Receptor Homodimers Involving the Fourth Transmembrane Helix†

    PubMed Central

    2010-01-01

    Several G protein-coupled receptors (GPCRs), including opioid receptors δOR, μOR, and κOR, have been reported to form stable dimers or oligomers in lipid bilayers and cell membranes. This notion has been recently challenged by imaging data supporting a transient nature of GPCR association. Here we use umbrella sampling reconstructed free energies of δOR homodimers involving the fourth transmembrane helix to predict their association constant. The results of these simulations, combined with estimates of diffusion-limited association rates, suggest a short lifetime for δOR homodimers in the membrane, in agreement with recent trends. PMID:20617813

  19. Reversible dimerization of acid-denatured ACBP controlled by helix A4.

    PubMed

    Fieber, Wolfgang; Kragelund, Birthe B; Meldal, Morten; Poulsen, Flemming M

    2005-02-01

    The peptide segment corresponding to helix A4 in acyl-coenzyme-A-binding protein (ACBP) is an exceptionally stable helix in the denatured state of the protein as well as in its isolated form. Circular dichroism spectroscopy showed an alpha-helix content in the helix A4 peptide (HA4) of 45%, and under denaturing conditions at pH 2.3, helix conformations are still populated in 24% of the ensemble of molecules. The structure of HA4 at atomic resolution was assessed using nuclear magnetic resonance (NMR) spectroscopy. Long-range NOEs between remote residues at opposite peptide ends suggested the formation of an antiparallel homodimer, and the resulting structure was treated as the minimum higher-order structure. The dimerization property of helix A4 is maintained in the full-length protein under denaturing conditions. NMR diffusion studies and concentration-dependent experiments on ACBP at low pH proved the formation of dimers and revealed a cooperative stabilization of helix A4 in this process. This emphasizes its special role in the structure formation in the denatured state of ACBP. No dimers are formed in the presence of guanidine hydrochloride, which underlines the fundamental difference between the nature of these two denatured states. PMID:15683223

  20. Conformational dynamics of a seven transmembrane helical protein Anabaena Sensory Rhodopsin probed by solid-state NMR.

    PubMed

    Good, Daryl B; Wang, Shenlin; Ward, Meaghan E; Struppe, Jochem; Brown, Leonid S; Lewandowski, Józef R; Ladizhansky, Vladimir

    2014-02-19

    The ability to detect and characterize molecular motions represents one of the unique strengths of nuclear magnetic resonance (NMR) spectroscopy. In this study, we report solid-state NMR site-specific measurements of the dipolar order parameters and (15)N rotating frame spin-lattice (R1ρ) relaxation rates in a seven transmembrane helical protein Anabaena Sensory Rhodopsin reconstituted in lipids. The magnitudes of the observed order parameters indicate that both the well-defined transmembrane regions and the less structured intramembrane loops undergo restricted submicrosecond time scale motions. In contrast, the R1ρ rates, which were measured under fast magic angle spinning conditions, vary by an order of magnitude between the TM and exposed regions and suggest the presence of intermediate time scale motions. Using a simple model, which assumes a single exponential autocorrelation function, we estimated the time scales of dominant stochastic motions to be on the order of low tens of nanoseconds for most residues within the TM helices and tens to hundreds of nanoseconds for the extracellular B-C and F-G loops. These relatively slow time scales could be attributed to collective anisotropic motions. We used the 3D Gaussian axial fluctuations model to estimate amplitudes, directions, and time scales of overall motions for helices and the extracellular B-C and F-G loops. Within this model, the TM helices A,B,C,D,E,F undergo rigid body motions on a time scale of tens of nanoseconds, while the time scale for the seventh helix G approaches 100 ns. Similar time scales of roughly 100-200 ns are estimated for the B-C and F-G loops. PMID:24467417

  1. The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

    SciTech Connect

    Hollier, Mark J.; Dimmock, Nigel J. . E-mail: n.j.dimmock@warwick.ac.uk

    2005-07-05

    In addition to the major ectodomain, the gp41 transmembrane glycoprotein of HIV-1 is now known to have a minor ectodomain that is part of the long C-terminal tail. Both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. However, data have so far been biologically based, and derived solely from T cell line-adapted (TCLA), B clade viruses. Here we have carried out sequence and theoretically based structural analyses of 357 gp41 C-terminal sequences of mainly primary isolates of HIV-1 clades A, B, C, and D. Data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, {beta}-sheet, membrane-spanning domains (MSDs). This means that the first (N-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. However, we suggest that only a minority of cell-associated gp41 molecules - those destined for incorporation into virions - has 3 MSDs and the minor ectodomain. Most intracellular gp41 has the conventional single MSD, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. The gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize HIV-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell.

  2. Crystal Structure of the Interferon Gamma Receptor Alpha Chain from Chicken Reveals an Undetected Extra Helix Compared with the Human Counterparts

    PubMed Central

    Ping, Zhiguang; Qi, Jianxun; Sun, Yanling; Lu, Guangwen; Shi, Yi; Wang, Xiaojia

    2014-01-01

    Interferon gamma (IFN-γ) is an important cytokine that induces antiviral, antiproliferative, and immunomodulatory effects on target cells, and is also crucial in the early defense against intracellular parasites, such as Listeria monocytogenes and Toxoplasma gondii. The biological activity of IFN-γ relies upon the formation of a complex with its 2 receptors, the interferon gamma alpha chain (IFNGR1) and beta chain (IFNGR2), which are type II cytokine receptors. Structural models of ligand–receptor interaction and complex structure of chicken IFNs with their receptors have remained elusive. Here we report the first structure of Gallus gallus (chicken) IFNGR1 (chIFNGR1) at 2.0 Å by molecule replacement according to the structure of selenomethionine substituted chIFNGR1. The structural comparison reveals its structural similarities with other class II cytokine receptors, despite divergent primary sequences. We further investigate the ligand–receptor interaction properties of chicken IFN-γ (chIFN-γ) and chIFNGR1 using size-exclusion chromatography and surface plasmon resonance techniques. These data aid in the understanding of the interaction of chicken (avian) IFN-γ with its receptors and its signal transduction. PMID:24283193

  3. Lightweight S-band helix antenna

    NASA Technical Reports Server (NTRS)

    Cribb, H. E.

    1970-01-01

    Pyrotechnically operated S-band helical antenna is developed in which helix is deployed subsequent to antenna placement. Antenna is small, lightweight, and novel in that deployable helix is used in place of fixed dish or horn. It can be designed to cover L- and X-band frequencies.

  4. Computational approaches to detect allosteric pathways in transmembrane molecular machines.

    PubMed

    Stolzenberg, Sebastian; Michino, Mayako; LeVine, Michael V; Weinstein, Harel; Shi, Lei

    2016-07-01

    Many of the functions of transmembrane proteins involved in signal processing and transduction across the cell membrane are determined by allosteric couplings that propagate the functional effects well beyond the original site of activation. Data gathered from breakthroughs in biochemistry, crystallography, and single molecule fluorescence have established a rich basis of information for the study of molecular mechanisms in the allosteric couplings of such transmembrane proteins. The mechanistic details of these couplings, many of which have therapeutic implications, however, have only become accessible in synergy with molecular modeling and simulations. Here, we review some recent computational approaches that analyze allosteric coupling networks (ACNs) in transmembrane proteins, and in particular the recently developed Protein Interaction Analyzer (PIA) designed to study ACNs in the structural ensembles sampled by molecular dynamics simulations. The power of these computational approaches in interrogating the functional mechanisms of transmembrane proteins is illustrated with selected examples of recent experimental and computational studies pursued synergistically in the investigation of secondary active transporters and GPCRs. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26806157

  5. PredβTM: A Novel β-Transmembrane Region Prediction Algorithm

    PubMed Central

    Roy Choudhury, Amrita; Novič, Marjana

    2015-01-01

    Predicting the transmembrane regions is an important aspect of understanding the structures and architecture of different β-barrel membrane proteins. Despite significant efforts, currently available β-transmembrane region predictors are still limited in terms of prediction accuracy, especially in precision. Here, we describe PredβTM, a transmembrane region prediction algorithm for β-barrel proteins. Using amino acid pair frequency information in known β-transmembrane protein sequences, we have trained a support vector machine classifier to predict β-transmembrane segments. Position-specific amino acid preference data is incorporated in the final prediction. The predictor does not incorporate evolutionary profile information explicitly, but is based on sequence patterns generated implicitly by encoding the protein segments using amino acid adjacency matrix. With a benchmark set of 35 β-transmembrane proteins, PredβTM shows a sensitivity and precision of 83.71% and 72.98%, respectively. The segment overlap score is 82.19%. In comparison with other state-of-art methods, PredβTM provides a higher precision and segment overlap without compromising with sensitivity. Further, we applied PredβTM to analyze the β-barrel membrane proteins without defined transmembrane regions and the uncharacterized protein sequences in eight bacterial genomes and predict possible β-transmembrane proteins. PredβTM can be freely accessed on the web at http://transpred.ki.si/. PMID:26694538

  6. Structural snapshots of full-length Jak1, a transmembrane gp130/IL-6/IL-6Rα cytokine receptor complex, and the receptor-Jak1 holocomplex

    PubMed Central

    Lupardus, Patrick J.; Skiniotis, Georgios; Rice, Amanda J.; Thomas, Christoph; Fischer, Suzanne; Walz, Thomas; Garcia, K. Christopher

    2011-01-01

    Summary The shared cytokine receptor gp130 signals as a homodimer or heterodimer through activation of Janus kinases (Jaks) associated with the receptor intracellular domains. Here we reconstitute, in parts and whole, the full-length gp130 homodimer in complex with the cytokine interleukin-6 (IL-6), its alpha receptor (IL-6Rα) and Jak1, for electron microscopy imaging. We find that the full-length gp130 homodimer complex has intimate interactions between the trans- and juxtamembrane segments of the two receptors, appearing to rigidify the connection between the extra- and intracellular regions. 2D averages and 3D reconstructions of full-length Jak1 reveal a three-lobed structure comprised of FERM-SH2, pseudokinase and kinase modules possessing extensive inter-segmental flexibility that likely facilitates allosteric activation. Single-particle imaging of the gp130/IL-6/IL-6Rα/Jak1 holocomplex shows Jak1 associated with the membrane proximal intracellular regions of gp130, abutting the would-be inner leaflet of the cell membrane. Jak1 association with gp130 is enhanced by the presence of a membrane environment. PMID:21220115

  7. Cooperative transmembrane penetration of nanoparticles.

    PubMed

    Zhang, Haizhen; Ji, Qiuju; Huang, Changjin; Zhang, Sulin; Yuan, Bing; Yang, Kai; Ma, Yu-qiang

    2015-01-01

    Physical penetration of lipid bilayer membranes presents an alternative pathway for cellular delivery of nanoparticles (NPs) besides endocytosis. NPs delivered through this pathway could reach the cytoplasm, thereby opening the possibility of organelle-specific targeting. Herein we perform dissipative particle dynamics simulations to elucidate the transmembrane penetration mechanisms of multiple NPs. Our simulations demonstrate that NPs' translocation proceeds in a cooperative manner, where the interplay of the quantity and surface chemistry of the NPs regulates the translocation efficiency. For NPs with hydrophilic surfaces, the increase of particle quantity facilitates penetration, while for NPs with partly or totally hydrophobic surfaces, the opposite highly possibly holds. Moreover, a set of interesting cooperative ways, such as aggregation, aggregation-dispersion, and aggregation-dispersion-reaggregation of the NPs, are observed during the penetration process. We find that the penetration behaviors of multiple NPs are mostly dominated by the changes of the NP-membrane force components in the membrane plane direction, in addition to that in the penetration direction, suggesting a different interaction mechanism between the multiple NPs and the membrane compared with the one-NP case. These results provide a fundamental understanding in the underlying mechanisms of cooperative penetration of NPs, and shed light on the NP-based drug and gene delivery. PMID:26013284

  8. Mutational analysis of trans-membrane helices M3, M4, M5 and M7 of the fast-twitch Ca2+-ATPase.

    PubMed Central

    Adams, P; East, J M; Lee, A G; O'Connor, C D

    1998-01-01

    Mutational analysis of trans-membrane helices M3, M4, M5 and M7 of the Ca2+-ATPase revealed a novel phenotypic variant, M4 [Y295A (the one-letter symbols are used for amino acid residues throughout)], displaying an increased affinity for Pi and decreased affinity for MgATP, while retaining the ability to translocate Ca2+ ions across the endoplasmic reticulum membrane. The properties of this mutant suggest that the E1-E2 equilibrium is shifted towards E2, and indicate a key role for this aromatic residue (Y295) at the end of trans-membrane helix M4. A mutant containing three amino acid residue substitutions at the end of the seventh trans-membrane helix, M7 (F834A, F835A, T837F), showed a complete loss of ATPase activity and a reduced ability to phosphorylate with Pi, although MgATP-initiated phosphorylation was unaffected. The observation that single mutations in this cluster of residues had no effect on Ca2+ transport suggests that correct anchoring of the helix at the lipid-water interface by these aromatic residues is important in the functioning of the ATPase. Mutation of polar residues in helix M3 did not affect inhibition of the ATPase by thapsigargin, thapsivillosin A or t-butyl hydroquinone, suggesting that hydrogen-bonding partners for the essential -OH groups on these inhibitors lie elsewhere in the ATPase. PMID:9742222

  9. The Origins of Transmembrane Ion Channels

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Wilson, Michael A.

    2012-01-01

    Even though membrane proteins that mediate transport of ions and small molecules across cell walls are among the largest and least understood biopolymers in contemporary cells, it is still possible to shed light on their origins and early evolution. The central observation is that transmembrane portions of most ion channels are simply bundles of -helices. By combining results of experimental and computer simulation studies on synthetic models and natural channels, mostly of non-genomic origin, we show that the emergence of -helical channels was protobiologically plausible, and did not require highly specific amino acid sequences. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. Specifically, we explain how the antiamoebin channels, which are made of identical helices, 16 amino acids in length, achieve efficiency comparable to that of highly evolved channels. We further show that antiamoebin channels are extremely flexible, compared to modern, genetically coded channels. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire stable rigid structures and mechanisms for precise regulation rather than improve efficiency. In general, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

  10. Partitivirus Structure Reveals a 120-Subunit, Helix-Rich Capsid with Distinctive Surface Arches Formed by Quasisymmetric Coat-Protein Dimers

    PubMed Central

    Ochoa, Wendy F.; Havens, Wendy M.; Sinkovits, Robert S.; Nibert, Max L.; Ghabrial, Said A.; Baker, Timothy S.

    2008-01-01

    SUMMARY Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3-Å resolution. The capsid, ~350 Å in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is in turn formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of α-helices and consistent with secondary-structure predictions for the coat protein. This is the first three-dimensional structure of a virus from the family Partitiviridae and exhibits both similarities to and differences from the so-called “T=2” capsids of other dsRNA viruses. PMID:18462682

  11. Helix Dipole Movement and Conformational Variability Contribute to Allosteric GDP Release in G[alpha] Subunits

    SciTech Connect

    Preininger, Anita M.; Funk, Michael A.; Oldham, William M.; Meier, Scott M.; Johnston, Christopher A.; Adhikary, Suraj; Kimple, Adam J.; Siderovski, David P.; Hamm, Heidi E.; Iverson, Tina M.

    2009-06-01

    Heterotrimeric G proteins (Galphabetagamma) transmit signals from activated G protein-coupled receptors (GPCRs) to downstream effectors through a guanine nucleotide signaling cycle. Numerous studies indicate that the carboxy-terminal alpha5 helix of Galpha subunits participates in Galpha-receptor binding, and previous EPR studies suggest this receptor-mediated interaction induces a rotation and translation of the alpha5 helix of the Galpha subunit [Oldham, W. M., et al. (2006) Nat. Struct. Mol. Biol. 13, 772-777]. On the basis of this result, an engineered disulfide bond was designed to constrain the alpha5 helix of Galpha(i1) into its EPR-measured receptor-associated conformation through the introduction of cysteines at position 56 in the alpha1 helix and position 333 in the alpha5 helix (I56C/Q333C Galpha(i1)). A functional mimetic of the EPR-measured alpha5 helix dipole movement upon receptor association was additionally created by introduction of a positive charge at the amino terminus of this helix, D328R Galpha(i1). Both proteins exhibit a dramatically elevated level of basal nucleotide exchange. The 2.9 A resolution crystal structure of I56C/Q333C Galpha(i1) in complex with GDP-AlF(4)(-) reveals the shift of the alpha5 helix toward the guanine nucleotide binding site that is anticipated by EPR measurements. The structure of the I56C/Q333C Galpha(i1) subunit further revealed altered positions for the switch regions and throughout the Galpha(i1) subunit, accompanied by significantly elevated crystallographic temperature factors. Combined with previous evidence in the literature, the structural analysis supports the critical role of electrostatics of the alpha5 helix dipole and overall conformational variability during nucleotide release.

  12. Consequences of Stabilizing the Natively Disordered F helix for the Folding Pathway of Apomyoglobin

    PubMed Central

    Nishimura, Chiaki; Dyson, H. Jane; Wright, Peter E.

    2011-01-01

    The F helix region of sperm whale apomyoglobin (apoMb) is disordered, undergoing conformational fluctuations between a folded helical conformation and one or more locally unfolded states. In order to examine the effects of F helix stabilization on the folding pathway of apoMb, we have introduced mutations to augment intrinsic helical structure in the F helix of the kinetic folding intermediate and to increase its propensity to fold early in the pathway, using predictions based on plots of the average area buried upon folding (AABUF) derived from the primary sequence. Two mutant proteins were prepared, a double mutant P88K/S92K (F2) and a quadruple mutant, P88K/A90L/S92K/A94L (F4). Whereas the AABUF for F2 predicts that the F helix will not fold early in the pathway, the F helix in F4 shows a significantly increased AABUF and is therefore predicted to fold early. Protection of amide protons by formation of hydrogen-bonded helical structure during the early folding events has been analyzed by pH-pulse labeling. Consistent with the AABUF prediction, many of the F helix residues for F4 are significantly protected in the kinetic intermediate but are not protected in the F2 mutant. F4 folds via a kinetically trapped burst phase intermediate that contains stabilized secondary structure in the A, B, F, G, and H helix regions. Rapid folding of the F helix stabilizes the central core of the misfolded intermediate and inhibits translocation of the H helix back to its native position, thereby decreasing the overall folding rate. PMID:21640124

  13. The role of glycine (residue 89) in the central helix of EF-hand protein troponin-C exposed following amino-terminal alpha-helix deletion.

    PubMed

    Ding, X L; Akella, A B; Su, H; Gulati, J

    1994-11-01

    Because an N-terminal alpha-helical (N-helix) arm and a KGK-triplet (residues 88KGK90) in the central helix of troponin-C (TnC) are missing in calmodulin, several recent studies have attempted to elucidate the structure-function correlations of these units. Presently, with a family of genetically manipulated derivatives especially developed for this study and tested on permeabilized isolated single skeletal muscle fiber segments, we explored the specificities of the amino acid residues within the N-helix and the KGK-triplet in TnC. Noticeably, the amino acid compositions vary between the N-helices of the cardiac and skeletal TnC isoforms. On the other hand, the KGK-triplet is located similarly in both TnC isoforms. We previously indicated that deletion of the N-helix (mutant delta Nt) diminishes the tension obtained on activation with maximal calcium, but the contractile function is revived by the superimposed deletion of the 88KGK90-triplet (mutant delta Nt delta KGK; see Gulati J, Babu A, Su H, Zhang YF, 1993, J Biol Chem 268:11685-11690). Using this functional test, we find that replacement of Gly-89 with a Leu or an Ala could also overcome the contractile defect associated with N-helix deletion. On the other hand, replacement of the skeletal TnC N-helix with cardiac type N-helix was unable to restore contractile function. The findings indicate a destabilizing influence of Gly-89 residue in skeletal TnC and suggest that the N-terminal arm in normal TnC serves to moderate this effect. Moreover, specificity of the N-helix between cardiac and skeletal TnCs raises the possibility that resultant structural disparities are also important for the functional distinctions of the TnC isoforms. PMID:7703855

  14. The first transmembrane domain (TM1) of ?2-subunit binds to the transmembrane domain S1 of ?-subunit in BK potassium channels

    PubMed Central

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming ?-subunit is associated to an auxiliary ?-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in ?-subunits that are important for the physical association with the ?-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the ?2-subunit are dispensable for association with the ?-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane proteinprotein interactions demonstrated for the first time that TM1 of the ?2-subunit physically binds to the transmembrane S1 domain of the ?-subunit. PMID:22710124

  15. Hydroxyproline Ring Pucker Causes Frustration of Helix Parameters in the Collagen Triple Helix

    NASA Astrophysics Data System (ADS)

    Ying Chow, W.; Bihan, Dominique; Forman, Chris J.; Slatter, David A.; Reid, David G.; Wales, David J.; Farndale, Richard W.; Duer, Melinda J.

    2015-07-01

    Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix.

  16. Hydroxyproline Ring Pucker Causes Frustration of Helix Parameters in the Collagen Triple Helix

    PubMed Central

    Ying Chow, W.; Bihan, Dominique; Forman, Chris J.; Slatter, David A.; Reid, David G.; Wales, David J.; Farndale, Richard W.; Duer, Melinda J.

    2015-01-01

    Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations. Here we use a combination of solid-state NMR spectroscopy and potential energy landscape modelling of synthetic triple-helical collagen peptides to understand this effect. We show that hydroxylation of the Yaa proline causes the Xaa proline ring conformation to become metastable, which in turn confers flexibility on the triple helix. PMID:26220399

  17. Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1

    SciTech Connect

    Desprez, P.; Hara, E.; Bissell, M.J.

    1995-06-01

    Cell proliferation and differentiation are precisely coordinated during the development and maturation of the mammary gland, and this balance invariably is disrupted during carcinogenesis. Little is known about the cell-specific transcription factors that regulate these processes in the mammary gland. The mouse mammary epithelial cell line SCp2 grows well under standard culture conditions but arrests growth, forms alveolus-like structures, and expresses {beta}-casein, a differentiation marker, 4 to 5 days after exposure to basement membrane and lactogenic hormones (differentiation signals). The authors show that this differentiation entails a marked decline in the expression of Id-1, a helix-loop-helix (HLH) protein that inactivates basic HLH transcription factors in other cell types. SCp2 cells stably transfected with an Id-1 expression vector grew more rapidly than control cells under standard conditions, but in response to differentiation signals, they lost three-dimensional organization, invaded the basement membrane, and then resumed growth. SCp2 cells expressing an Id-1 antisense vector grew more slowly than controls; in response to differentiation signals, they remained stably growth arrested and fully differentiated, as did control cells. The authors suggest that Id-1 renders cells refractory to differentiation signals and receptive to growth signals by inactivating one or more basic HLH proteins that coordinate growth and differentiation in the mammary epithelium. 53 refs., 6 figs.

  18. Nucleobase-Modified PNA Suppresses Translation by Forming a Triple Helix with a Hairpin Structure in mRNA In?Vitro and in Cells.

    PubMed

    Endoh, Tamaki; Hnedzko, Dziyana; Rozners, Eriks; Sugimoto, Naoki

    2016-01-01

    Compounds that bind specifically to double-stranded regions of RNA have potential as regulators of structure-based RNA function; however, sequence-selective recognition of double-stranded RNA is challenging. The modification of peptide nucleic acid (PNA) with unnatural nucleobases enables the formation of PNA-RNA triplexes. Herein, we demonstrate that a 9-mer PNA forms a sequence-specific PNA-RNA triplex with a dissociation constant of less than 1?nm at physiological pH. The triplex formed within the 5' untranslated region of an mRNA reduces the protein expression levels both in?vitro and in cells. A single triplet mismatch destabilizes the complex, and in this case, no translation suppression is observed. The triplex-forming PNAs are unique and potent compounds that hold promise as inhibitors of cellular functions that are controlled by double-stranded RNAs, such as RNA interference, RNA editing, and RNA localization mediated by protein-RNA interactions. PMID:26473504

  19. Structure of an atypical FeoB G-domain reveals a putative domain-swapped dimer

    PubMed Central

    Deshpande, Chandrika N.; McGrath, Aaron P.; Font, Josep; Guilfoyle, Amy P.; Maher, Megan J.; Jormakka, Mika

    2013-01-01

    FeoB is a transmembrane protein involved in ferrous iron uptake in prokaryotic organisms. FeoB comprises a cytoplasmic soluble domain termed NFeoB and a C-terminal polytopic transmembrane domain. Recent structures of NFeoB have revealed two structural subdomains: a canonical GTPase domain and a five-helix helical domain. The GTPase domain hydrolyses GTP to GDP through a well characterized mechanism, a process which is required for Fe2+ transport. In contrast, the precise role of the helical domain has not yet been fully determined. Here, the structure of the cytoplasmic domain of FeoB from Gallionella capsiferriformans is reported. Unlike recent structures of NFeoB, the G. capsiferriformans NFeoB structure is highly unusual in that it does not contain a helical domain. The crystal structures of both apo and GDP-bound protein forms a domain-swapped dimer. PMID:23545645

  20. Structure of an atypical FeoB G-domain reveals a putative domain-swapped dimer.

    PubMed

    Deshpande, Chandrika N; McGrath, Aaron P; Font, Josep; Guilfoyle, Amy P; Maher, Megan J; Jormakka, Mika

    2013-04-01

    FeoB is a transmembrane protein involved in ferrous iron uptake in prokaryotic organisms. FeoB comprises a cytoplasmic soluble domain termed NFeoB and a C-terminal polytopic transmembrane domain. Recent structures of NFeoB have revealed two structural subdomains: a canonical GTPase domain and a five-helix helical domain. The GTPase domain hydrolyses GTP to GDP through a well characterized mechanism, a process which is required for Fe(2+) transport. In contrast, the precise role of the helical domain has not yet been fully determined. Here, the structure of the cytoplasmic domain of FeoB from Gallionella capsiferriformans is reported. Unlike recent structures of NFeoB, the G. capsiferriformans NFeoB structure is highly unusual in that it does not contain a helical domain. The crystal structures of both apo and GDP-bound protein forms a domain-swapped dimer. PMID:23545645

  1. The Mtr4 ratchet helix and arch domain both function to promote RNA unwinding.

    PubMed

    Taylor, Lacy L; Jackson, Ryan N; Rexhepaj, Megi; King, Alejandra Klauer; Lott, Lindsey K; van Hoof, Ambro; Johnson, Sean J

    2014-12-16

    Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3'-5' turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The 'ratchet helix' is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates. PMID:25414331

  2. A supramolecular helix that disregards chirality

    NASA Astrophysics Data System (ADS)

    Roche, Cécile; Sun, Hao-Jan; Leowanawat, Pawaret; Araoka, Fumito; Partridge, Benjamin E.; Peterca, Mihai; Wilson, Daniela A.; Prendergast, Margaret E.; Heiney, Paul A.; Graf, Robert; Spiess, Hans W.; Zeng, Xiangbing; Ungar, Goran; Percec, Virgil

    2016-01-01

    The functions of complex crystalline systems derived from supramolecular biological and non-biological assemblies typically emerge from homochiral programmed primary structures via first principles involving secondary, tertiary and quaternary structures. In contrast, heterochiral and racemic compounds yield disordered crystals, amorphous solids or liquids. Here, we report the self-assembly of perylene bisimide derivatives in a supramolecular helix that in turn self-organizes in columnar hexagonal crystalline domains regardless of the enantiomeric purity of the perylene bisimide. We show that both homochiral and racemic perylene bisimide compounds, including a mixture of 21 diastereomers that cannot be deracemized at the molecular level, self-organize to form single-handed helical assemblies with identical single-crystal-like order. We propose that this high crystalline order is generated via a cogwheel mechanism that disregards the chirality of the self-assembling building blocks. We anticipate that this mechanism will facilitate access to previously inaccessible complex crystalline systems from racemic and homochiral building blocks.

  3. A supramolecular helix that disregards chirality.

    PubMed

    Roche, Cécile; Sun, Hao-Jan; Leowanawat, Pawaret; Araoka, Fumito; Partridge, Benjamin E; Peterca, Mihai; Wilson, Daniela A; Prendergast, Margaret E; Heiney, Paul A; Graf, Robert; Spiess, Hans W; Zeng, Xiangbing; Ungar, Goran; Percec, Virgil

    2016-01-01

    The functions of complex crystalline systems derived from supramolecular biological and non-biological assemblies typically emerge from homochiral programmed primary structures via first principles involving secondary, tertiary and quaternary structures. In contrast, heterochiral and racemic compounds yield disordered crystals, amorphous solids or liquids. Here, we report the self-assembly of perylene bisimide derivatives in a supramolecular helix that in turn self-organizes in columnar hexagonal crystalline domains regardless of the enantiomeric purity of the perylene bisimide. We show that both homochiral and racemic perylene bisimide compounds, including a mixture of 21 diastereomers that cannot be deracemized at the molecular level, self-organize to form single-handed helical assemblies with identical single-crystal-like order. We propose that this high crystalline order is generated via a cogwheel mechanism that disregards the chirality of the self-assembling building blocks. We anticipate that this mechanism will facilitate access to previously inaccessible complex crystalline systems from racemic and homochiral building blocks. PMID:26673268

  4. Structures of SemiSWEET transporters in two distinct conformations

    PubMed Central

    Fan, Chao; Chen, Li-Qing; Xu, Sophia; Perry, Kay; Frommer, Wolf B.; Feng, Liang

    2015-01-01

    SemiSWEETs and SWEETs are mono- and disaccharide transporters present from Archaea to higher plants and humans1-3. SWEETs play crucial roles in cellular sugar efflux processes, i.e. phloem loading4, pollen nutrition5 and nectar secretion6. Their bacterial homologs, SemiSWEETs, are among the smallest known transporters1,3. Here we show SemiSWEET, consisting of a triple-helix-bundle (THB), forms a symmetric parallel dimer to create the translocation pathway. Two SemiSWEET isoforms were crystallized in apparent open and occluded states, indicating that SemiSWEETs/SWEETs are transporters that undergo rocking-type movements during the transport cycle. The topology of THB is similar to the basic building block in MFS transporters (GLUTs, SUTs), indicating that they may have evolved from an ancestral THB into a parallel configuration to produce 6/6+1 transmembrane-helix pores for SemiSWEETs/SWEETs, and an antiparallel configuration of 2×2 THBs to generate 12 transmembrane-helix pores for MFS transporters. Given the similarity of SemiSWEETs/SWEETs to PQ-loop amino acid transporters and mitochondrial MPC organic acid transporters, the structures characterized here may also be relevant for other MtN3 clan transporters7-9. PMID:25186729

  5. Comparison of design strategies for ?-helix backbone modification in a protein tertiary fold.

    PubMed

    Tavenor, Nathan A; Reinert, Zachary E; Lengyel, George A; Griffith, Brian D; Horne, W Seth

    2016-02-25

    We report here the comparison of five classes of unnatural amino acid building blocks for their ability to be accommodated into an ?-helix in a protein tertiary fold context. High-resolution structural characterization and analysis of folding thermodynamics yield new insights into the relationship between backbone composition and folding energetics in ?-helix mimetics and suggest refined design rules for engineering the backbones of natural sequences. PMID:26853882

  6. Structure of a bacterial homologue of vitamin K epoxide reductase

    SciTech Connect

    Li, Weikai; Schulman, Sol; Dutton, Rachel J.; Boyd, Dana; Beckwith, Jon; Rapoport, Tom A.

    2010-03-19

    Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain {gamma}-carboxylation of many blood coagulation factors. Here, we report the 3.6 {angstrom} crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.

  7. Triple Helix Formation in a Topologically Controlled DNA Nanosystem.

    PubMed

    Yamagata, Yutaro; Emura, Tomoko; Hidaka, Kumi; Sugiyama, Hiroshi; Endo, Masayuki

    2016-04-11

    In the present study, we demonstrate single-molecule imaging of triple helix formation in DNA nanostructures. The binding of the single-molecule third strand to double-stranded DNA in a DNA origami frame was examined using two different types of triplet base pairs. The target DNA strand and the third strand were incorporated into the DNA frame, and the binding of the third strand was controlled by the formation of Watson-Crick base pairing. Triple helix formation was monitored by observing the structural changes in the incorporated DNA strands. It was also examined using a photocaged third strand wherein the binding of the third strand was directly observed using high-speed atomic force microscopy during photoirradiation. We found that the binding of the third strand could be controlled by regulating duplex formation and the uncaging of the photocaged strands in the designed nanospace. PMID:26938310

  8. Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1.

    PubMed Central

    Desprez, P Y; Hara, E; Bissell, M J; Campisi, J

    1995-01-01

    Cell proliferation and differentiation are precisely coordinated during the development and maturation of the mammary gland, and this balance invariably is disrupted during carcinogenesis. Little is known about the cell-specific transcription factors that regulate these processes in the mammary gland. The mouse mammary epithelial cell line SCp2 grows well under standard culture conditions but arrests growth, forms alveolus-like structures, and expresses beta-casein, a differentiation marker, 4 to 5 days after exposure to basement membrane and lactogenic hormones (differentiation signals). We show that this differentiation entails a marked decline in the expression of Id-1, a helix-loop-helix (HLH) protein that inactivates basic HLH transcription factors in other cell types. SCp2 cells stably transfected with an Id-1 expression vector grew more rapidly than control cells under standard conditions, but in response to differentiation signals, they arrested growth and formed three-dimensional structures similar to those of control cells. Id-1-expressing cells did not, however, express beta-casein. Moreover, 8 to 10 days after receiving differentiation signals, they lost three-dimensional organization, invaded the basement membrane, and then resumed growth. SCp2 cells expressing an Id-1 antisense vector grew more slowly than controls; in response to differentiation signals, they remained stably growth arrested and fully differentiated, as did control cells. We suggest that Id-1 renders cells refractory to differentiation signals and receptive to growth signals by inactivating one or more basic HLH proteins that coordinate growth and differentiation in the mammary epithelium. PMID:7760836

  9. Uropathogenic E. coli adhesin-induced host cell receptor conformational changes: implications in transmembrane signaling transduction

    PubMed Central

    Wang, Huaibin; Min, Guangwei; Glockshuber, Rudi; Sun, Tung-Tien; Kong, Xiang-Peng

    2009-01-01

    Urinary tract infection (UTI) is the second most common infectious disease, and is caused predominantly by type 1-fimbriated uropathogenic E. coli (UPEC). UPEC initiates infection by attaching to uroplakin Ia, its urothelial surface receptor, via the FimH adhesins capping the distal end of its fimbriae. Uroplakin Ia, together with uroplakins Ib, II and IIIa, forms a 16 nm receptor complex that is assembled into hexagonally packed two-dimensional crystals (urothelial plaques) covering >90% of the urothelial apical surface. Recent studies indicate that FimH is the invasin of UPEC as its attachment to the urothelial surface can induce cellular signaling events including calcium elevation and the phosphorylation of the uroplakin IIIa cytoplasmic tail, leading to cytoskeletal rearrangements and bacterial invasion. However, it remains unknown how the binding of FimH to the uroplakin receptor triggers a signal that can be transmitted through the highly impermeable urothelial apical membrane. We show here by cryo-electron microscopy that FimH-binding to the extracellular domain of UPIa induces global conformational changes in the entire uroplakin receptor complex, including a coordinated movement of the tightly bundled transmembrane helices. This movement of the transmembrane helix bundles can cause a corresponding lateral translocation of the uroplakin cytoplasmic tails, which can be sufficient to trigger downstream signaling events. Our results suggest a novel pathogen-induced transmembrane signal transduction mechanism that plays a key role in the initial stages of UPEC invasion and receptor-mediated bacterial invasion in general. PMID:19577575

  10. Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry

    NASA Technical Reports Server (NTRS)

    Wang, N.; Ingber, D. E.

    1995-01-01

    We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) beta 1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (beta 1, alpha V beta 3, alpha V, alpha 5, alpha 2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.

  11. A role for U2/U6 helix Ib in 5' splice site selection.

    PubMed Central

    Luukkonen, B G; Séraphin, B

    1998-01-01

    Selection of pre-mRNA splice sites is a highly accurate process involving many trans-acting factors. Recently, we described a role for U6 snRNA position G52 in selection of the first intron nucleotide (+1G). Because some U2 alleles suppress U6-G52 mutations, we investigated whether the corresponding U2 snRNA region also influenced 5' splice site selection. Our results demonstrate that U2 snRNAs mutated at position U23, but not adjacent nucleotides, specifically affect 5' splice site cleavage. Furthermore, all U2 position U23 mutations are synthetic lethal with the thermosensitive U6-G52U allele. Interestingly, the U2-U23C substitution has an unprecedented hyperaccurate splicing phenotype in which cleavage of introns with a +1G substitution is reduced, whereas the strain grows with wild-type kinetics. U2 position U23 forms the first base pair with U6 position A59 in U2/U6 helix Ib. Restoration of the helical structure suppresses 5' splice site cleavage defects, showing an important role for the helix Ib structure in 5' splice site selection. U2/U6 helix Ib and helix II have recently been described as being functionally redundant. This report demonstrates a unique role for helix Ib in 5' splice site selection that is not shared with helix II. PMID:9701283

  12. Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1.

    PubMed Central

    Slepkov, Emily R; Chow, Signy; Lemieux, M Joanne; Fliegel, Larry

    2004-01-01

    NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167-->Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE. PMID:14680478

  13. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD)

    PubMed Central

    2012-01-01

    Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are retained indicating that the helix B-Met loop-helix C is the minimal functional “domain” found to date for the matrixin family. Conclusions The helix B-Met loop-helix C folding conserved in metalloprotease metzincin super family is able to facilitate proteolytic catalysis for specific substrate and inhibitor recognition. The autolysis processing and producing 6 kDa mini MMP-7 is the smallest metalloprotease in living world. PMID:22642296

  14. Equilibrium Stability of Transmembrane Proteins : A Hard-Core Gas Problem.

    NASA Astrophysics Data System (ADS)

    Wahba, Karim; Schwab, David; Bruinsma, Robijn

    2008-03-01

    Hydropathy plots, a moving average of amino acid hydrophobicity over a sequence, can be used to predict potential protein structure, in particular transmembrane proteins. Traditionally transmembrane regions are identified by peaks above an empirical cutoff. We treat the transmembrane segments as a one-dimensional gas of hard rods in a correlated random energy landscape. At zero temperature, where the entropic contribution due to the loops is negligible, we calculate the density profile as a function of the chemical potential in the case of the original as well as randomly generated landscapes. The density profile exhibits plateaus indicating regions where a transmembrane segment has been established. For designed versus random sequences we explore the distribution of the sizes of these plateaus and attempt to infer characteristic features that may be interpreted in terms of the stability of the protein in its inserted state.

  15. Structure of a prokaryotic fumarate transporter reveals the architecture of the SLC26 family.

    PubMed

    Geertsma, Eric R; Chang, Yung-Ning; Shaik, Farooque R; Neldner, Yvonne; Pardon, Els; Steyaert, Jan; Dutzler, Raimund

    2015-10-01

    The SLC26 family of membrane proteins combines a variety of functions within a conserved molecular scaffold. Its members, besides coupled anion transporters and channels, include the motor protein Prestin, which confers electromotility to cochlear outer hair cells. To gain insight into the architecture of this protein family, we characterized the structure and function of SLC26Dg, a facilitator of proton-coupled fumarate symport, from the bacterium Deinococcus geothermalis. Its modular structure combines a transmembrane unit and a cytoplasmic STAS domain. The membrane-inserted domain consists of two intertwined inverted repeats of seven transmembrane segments each and resembles the fold of the unrelated transporter UraA. It shows an inward-facing, ligand-free conformation with a potential substrate-binding site at the interface between two helix termini at the center of the membrane. This structure defines the common framework for the diverse functional behavior of the SLC26 family. PMID:26367249

  16. Highly twisted double-helix carbon nanotube yarns.

    PubMed

    Shang, Yuanyuan; Li, Yibin; He, Xiaodong; Du, Shanyi; Zhang, Luhui; Shi, Enzheng; Wu, Shiting; Li, Zhen; Li, Peixu; Wei, Jinquan; Wang, Kunlin; Zhu, Hongwei; Wu, Dehai; Cao, Anyuan

    2013-02-26

    The strength and flexibility of carbon nanotubes (CNTs) allow them to be constructed into a variety of innovated architectures with fascinating properties. Here, we show that CNTs can be made into a highly twisted yarn-derived double-helix structure by a conventional twist-spinning process. The double-helix is a stable and hierarchical configuration consisting of two single-helical yarn segments, with controlled pitch and unique mechanical properties. While one of the yarn components breaks early under tension due to the highly twisted state, the second yarn produces much larger tensile strain and significantly prolongs the process until ultimate fracture. In addition, these elastic and conductive double-helix yarns show simultaneous and reversible resistance change in response to a wide range of input sources (mechanical, photo, and thermal) such as applied strains or stresses, light illumination, and environmental temperature. Our results indicate that it is possible to create higher-level, more complex architectures from CNT yarns and fabricate multifunctional nanomaterials with potential applications in many areas. PMID:23289799

  17. Structural Analysis of Cloned Plasma Membrane Proteins by Freeze-Fracture Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Eskandari, Sepehr; Wright, Ernest M.; Kreman, Mike; Starace, Dorine M.; Zampighi, Guido A.

    1998-09-01

    We have used freeze-fracture electron microscopy to examine the oligomeric structure and molecular asymmetry of integral plasma membrane proteins. Recombinant plasma membrane proteins were functionally expressed in Xenopus laevis oocytes, and the dimensions of their freeze-fracture particles were analyzed. To characterize the freeze-fracture particles, we compared the particle cross-sectional area of proteins with α -helical transmembrane domains (opsin, aquaporin 1, and a connexin) with their area obtained from existing maps calculated from two-dimensional crystals. We show that the cross-sectional area of the freeze-fracture particles corresponds to the area of the transmembrane domain of the protein, and that the protein cross-sectional area varies linearly with the number membrane-spanning helices. On average, each helix occupies 1.40 ± 0.03 nm2. By using this information, we examined members from three classes of plasma membrane proteins: two ion channels, the cystic fibrosis transmembrane conductance regulator and connexin 50 hemi-channel; a water channel, the major intrinsic protein (the aquaporin 0); and a cotransporter, the Na+/glucose cotransporter. Our results suggest that the cystic fibrosis transmembrane conductance regulator is a dimer containing 25 ± 2 transmembrane helices, connexin 50 is a hexamer containing 24 ± 3 helices, the major intrinsic protein is a tetramer containing 24 ± 3 helices, and the Na+/glucose cotransporter is an asymmetrical monomer containing 15 ± 2 helices.

  18. Gold helix photonic metamaterial as broadband circular polarizer.

    PubMed

    Gansel, Justyna K; Thiel, Michael; Rill, Michael S; Decker, Manuel; Bade, Klaus; Saile, Volker; von Freymann, Georg; Linden, Stefan; Wegener, Martin

    2009-09-18

    We investigated propagation of light through a uniaxial photonic metamaterial composed of three-dimensional gold helices arranged on a two-dimensional square lattice. These nanostructures are fabricated via an approach based on direct laser writing into a positive-tone photoresist followed by electrochemical deposition of gold. For propagation of light along the helix axis, the structure blocks the circular polarization with the same handedness as the helices, whereas it transmits the other, for a frequency range exceeding one octave. The structure is scalable to other frequency ranges and can be used as a compact broadband circular polarizer. PMID:19696310

  19. Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor

    PubMed Central

    Chantreau, Vanessa; Taddese, Bruck; Munier, Mathilde; Gourdin, Louis; Henrion, Daniel; Rodien, Patrice; Chabbert, Marie

    2015-01-01

    The thyrotropin receptor (TSHR) is a G protein-coupled receptor (GPCR) that is member of the leucine-rich repeat subfamily (LGR). In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM) 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors. PMID:26545118

  20. Epicyclic Twin-Helix Ionization Cooling Simulations

    SciTech Connect

    Vasiliy Morozov, Yaroslav Derbenev, A. Afanaciev, R.P. Johnson

    2011-04-01

    Parametric-resonance Ionization Cooling (PIC) is proposed as the final 6D cooling stage of a highluminosity muon collider. For the implementation of PIC, we earlier developed an epicyclic twin-helix channel with correlated behavior of the horizontal and vertical betatron motions and dispersion. We now insert absorber plates with short energy-recovering units located next to them at the appropriate locations in the twin-helix channel. We first demonstrate conventional ionization cooling in such a system with the optics uncorrelated. We then adjust the correlated optics state and induce a parametric resonance to study ionization cooling under the resonant condition.

  1. Light Stress-Induced One-Helix Protein of the Chlorophyll a/b-Binding Family Associated with Photosystem I1

    PubMed Central

    Andersson, Ulrica; Heddad, Mounia; Adamska, Iwona

    2003-01-01

    The superfamily of light-harvesting chlorophyll a/b-binding (Lhc) proteins in higher plants and green algae is composed of more than 20 different antenna proteins associated either with photosystem I (PSI) or photosystem II (PSII). Several distant relatives of this family with conserved chlorophyll-binding residues and proposed photoprotective functions are induced transiently under various stress conditions. Whereas “classical” Lhc proteins contain three-transmembrane α-helices, their distant relatives span the membrane with between one and four transmembrane segments. Here, we report the identification and isolation of a novel member of the Lhc family from Arabidopsis with one predicted transmembrane α-helix closely related to helix I of Lhc protein from PSI (Lhca4) that we named Ohp2 (for a second one-helix protein of Lhc family described from higher plants). We showed that the Ohp2 gene expression is triggered by light stress and that the Ohp2 transcript and protein accumulated in a light intensity-dependent manner. Other stress conditions did not up-regulate the expression of the Ohp2 gene. Localization studies revealed that Ohp2 is associated with PSI under low- or high-light conditions. Because all stress-induced Lhc relatives reported so far were found in PSII, we propose that the accumulation of Ohp2 might represent a novel photoprotective strategy induced within PSI in response to light stress. PMID:12805611

  2. Evolutionary analysis of the segment from helix 3 through helix 5 in vertebrate progesterone receptors.

    PubMed

    Baker, Michael E; Uh, Kayla Y

    2012-10-01

    The interaction between helix 3 and helix 5 in the human mineralocorticoid receptor [MR], progesterone receptor [PR] and glucocorticoid receptor [GR] influences their response to steroids. For the human PR, mutations at Gly-722 on helix 3 and Met-759 on helix 5 alter responses to progesterone. We analyzed the evolution of these two sites and the rest of a 59 residue segment containing helices 3, 4 and 5 in vertebrate PRs and found that a glycine corresponding to Gly-722 on helix 3 in human PR first appears in platypus, a monotreme. In lamprey, skates, fish, amphibians and birds, cysteine is found at this position in helix 3. This suggests that the cysteine to glycine replacement in helix 3 in the PR was important in the evolution of mammals. Interestingly, our analysis of the rest of the 59 residue segment finds 100% sequence conservation in almost all mammal PRs, substantial conservation in reptile and amphibian PRs and divergence of land vertebrate PR sequences from the fish PR sequences. The differences between fish and land vertebrate PRs may be important in the evolution of different biological progestins in fish and mammalian PR, as well as differences in susceptibility to environmental chemicals that disrupt PR-mediated physiology. PMID:22575083

  3. Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation

    SciTech Connect

    Yunus, Abdul S.; Jackson, Trent P.; Crisafi, Katherine; Burimski, Irina; Kilgore, Nicole R.; Zoumplis, Dorian; Allaway, Graham P.; Wild, Carl T.; Salzwedel, Karl

    2010-01-20

    Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 deg. C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes.

  4. Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation.

    PubMed

    Yunus, Abdul S; Jackson, Trent P; Crisafi, Katherine; Burimski, Irina; Kilgore, Nicole R; Zoumplis, Dorian; Allaway, Graham P; Wild, Carl T; Salzwedel, Karl

    2010-01-20

    Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 degrees C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes. PMID:19922971

  5. The C-Terminal RpoN Domain of sigma54 Forms an unpredictedHelix-Turn-Helix Motif Similar to domains of sigma70

    SciTech Connect

    Doucleff, Michaeleen; Malak, Lawrence T.; Pelton, Jeffrey G.; Wemmer, David E.

    2005-11-01

    The ''{delta}'' subunit of prokaryotic RNA-polymerase allows gene-specific transcription initiation. Two {sigma} families have been identified, {sigma}{sup 70} and {sigma}{sup 54}, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the {sigma}{sup 70}-type factors have been well characterized structurally by x-ray crystallography, no high-resolution structural information is available for the {sigma}{sup 54}-type factors. Here we present the NMR derived structure of the C-terminal domain of {sigma}{sup 54} from Aquifex aeolicus. This domain (Thr323 to Gly389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the {sigma}{sup 70}-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. This structure's homology with other DNA binding proteins, combined with previous biochemical data, suggest how the C-terminal domain of {sigma}{sup 54} binds to DNA.

  6. Rigidity of transmembrane proteins determines their cluster shape.

    PubMed

    Jafarinia, Hamidreza; Khoshnood, Atefeh; Jalali, Mir Abbas

    2016-01-01

    Protein aggregation in cell membrane is vital for the majority of biological functions. Recent experimental results suggest that transmembrane domains of proteins such as α-helices and β-sheets have different structural rigidities. We use molecular dynamics simulation of a coarse-grained model of protein-embedded lipid membranes to investigate the mechanisms of protein clustering. For a variety of protein concentrations, our simulations under thermal equilibrium conditions reveal that the structural rigidity of transmembrane domains dramatically affects interactions and changes the shape of the cluster. We have observed stable large aggregates even in the absence of hydrophobic mismatch, which has been previously proposed as the mechanism of protein aggregation. According to our results, semiflexible proteins aggregate to form two-dimensional clusters, while rigid proteins, by contrast, form one-dimensional string-like structures. By assuming two probable scenarios for the formation of a two-dimensional triangular structure, we calculate the lipid density around protein clusters and find that the difference in lipid distribution around rigid and semiflexible proteins determines the one- or two-dimensional nature of aggregates. It is found that lipids move faster around semiflexible proteins than rigid ones. The aggregation mechanism suggested in this paper can be tested by current state-of-the-art experimental facilities. PMID:26871097

  7. Rigidity of transmembrane proteins determines their cluster shape

    NASA Astrophysics Data System (ADS)

    Jafarinia, Hamidreza; Khoshnood, Atefeh; Jalali, Mir Abbas

    2016-01-01

    Protein aggregation in cell membrane is vital for the majority of biological functions. Recent experimental results suggest that transmembrane domains of proteins such as α -helices and β -sheets have different structural rigidities. We use molecular dynamics simulation of a coarse-grained model of protein-embedded lipid membranes to investigate the mechanisms of protein clustering. For a variety of protein concentrations, our simulations under thermal equilibrium conditions reveal that the structural rigidity of transmembrane domains dramatically affects interactions and changes the shape of the cluster. We have observed stable large aggregates even in the absence of hydrophobic mismatch, which has been previously proposed as the mechanism of protein aggregation. According to our results, semiflexible proteins aggregate to form two-dimensional clusters, while rigid proteins, by contrast, form one-dimensional string-like structures. By assuming two probable scenarios for the formation of a two-dimensional triangular structure, we calculate the lipid density around protein clusters and find that the difference in lipid distribution around rigid and semiflexible proteins determines the one- or two-dimensional nature of aggregates. It is found that lipids move faster around semiflexible proteins than rigid ones. The aggregation mechanism suggested in this paper can be tested by current state-of-the-art experimental facilities.

  8. Homologue Structure of the SLAC1 Anion Channel for Closing Stomata in Leaves

    SciTech Connect

    Y Chen; L Hu; M Punta; R Bruni; B Hillerich; B Kloss; B Rost; J Love; S Siegelbaum; W Hendrickson

    2011-12-31

    The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 {angstrom} resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

  9. Molecular modelling approaches for cystic fibrosis transmembrane conductance regulator studies.

    PubMed

    Odolczyk, Norbert; Zielenkiewicz, Piotr

    2014-07-01

    Cystic fibrosis (CF) is one of the most common genetic disorders, caused by loss of function mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. CFTR is a member of ATP-binding cassette (ABC) transporters superfamily and functions as an ATP-gated anion channel. This review summarises the vast majority of the efforts which utilised molecular modelling approaches to gain insight into the various aspects of CFTR protein, related to its structure, dynamic properties, function and interactions with other protein partners, or drug-like compounds, with emphasis to its relation to CF disease. PMID:24735712

  10. Structural Basis for the Trembler-J Phenotype of Charcot-Marie-Tooth Disease

    PubMed Central

    Sakakura, Masayoshi; Hadziselimovic, Arina; Wang, Zhen; Schey, Kevin L.; Sanders, Charles R.

    2011-01-01

    Summary Mutations in peripheral myelin protein 22 (PMP22) can result in the common peripheral neuropathy, Charcot-Marie-Tooth disease (CMTD). The Leu16Pro mutation in PMP22 results in misassembly of the protein, which causes the Trembler-J (TrJ) disease phenotype. Here we elucidate the structural defects present in a partially folded state of TrJ PMP22 that are decisive in promoting CMTD-causing misfolding. In this state transmembrane helices 2-4 (TM2-4) form a molten-globular bundle while transmembrane helix 1 (TM1) is dissociated from this bundle. The TrJ mutation was seen to profoundly disrupt the TM1 helix, resulting in increased backbone dynamics and changes in the tertiary interactions of TM1 with the PMP22 TM2-4 core in the folded state. Consequently, TM1 undergoes enhanced dissociation from the other transmembrane segments in TrJ PMP22, becoming available for recognition and sequestration by protein folding quality control, leading to loss of function and toxic accumulation of aggregates that results in CMTD. PMID:21827951

  11. Helix A Stabilization Precedes Amino-terminal Lobe Activation upon Calcium Binding to Calmodulin

    SciTech Connect

    Chen, Baowei; Lowry, David; Mayer, M. Uljana; Squier, Thomas C.

    2008-08-09

    The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)-resorufin (ReAsH), which upon binding to an engineered tetracysteine binding motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the 1H-15N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe (Kd = 0.36 ± 0.04 μM), which results in a reduction in the rate of ReAsH binding from 4900 M-1 sec-1 to 370 M-1 sec-1. In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe requires calcium-occupancy of amino-terminal sites (Kd = 18 ± 3 μM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain linker that release helix A, thereby facilitating the formation of calcium binding sites in the amino-terminal lobe and linked tertiary structural rearrangements to form a high-affinity binding cleft that can associate with target proteins.

  12. Histone Octamer Helical Tubes Suggest that an Internucleosomal Four-Helix Bundle Stabilizes the Chromatin Fiber

    PubMed Central

    Frouws, Timothy D.; Patterton, Hugh-G.; Sewell, Bryan T.

    2009-01-01

    Abstract A major question in chromatin involves the exact organization of nucleosomes within the 30-nm chromatin fiber and its structural determinants of assembly. Here we investigate the structure of histone octamer helical tubes via the method of iterative helical real-space reconstruction. Accurate placement of the x-ray structure of the histone octamer within the reconstructed density yields a pseudoatomic model for the entire helix, and allows precise identification of molecular interactions between neighboring octamers. One such interaction that would not be obscured by DNA in the nucleosome consists of a twofold symmetric four-helix bundle formed between pairs of H2B-α3 and H2B-αC helices of neighboring octamers. We believe that this interface can act as an internucleosomal four-helix bundle within the context of the chromatin fiber. The potential relevance of this interface in the folding of the 30-nm chromatin fiber is discussed. PMID:19383479

  13. Double helix quinine-based supergelator.

    PubMed

    Roszak, Kinga; Piasecka, Monika; Katrusiak, Andrzej; Kacprzak, Karol

    2016-02-01

    10,11-Didehydroquinine is a simple, low molecular weight supergelator which forms, in nonpolar media, stable chiral organogels composed of unique double-helix nano-sized fibers. A novel gelation mechanism involves a hydrogen bonding network formed by an acidic alkyne proton of the Cinchona gelator and the carbonyl group of ethyl acetate used as a solvent. PMID:26701368

  14. Conformational Diffusion and Helix Formation Kinetics

    SciTech Connect

    Hummer, Gerhard; Garcia, Angel E.; Garde, Shekhar

    2000-09-18

    The time, temperature, and sequence dependences of helix formation kinetics of fully atomistic peptide models in explicit solvent are described quantitatively by a diffusive search within the coil state with barrierless transitions into the helical state. Conformational diffusion leads to nonexponential kinetics and jump-width dependences in temperature jump experiments. (c) 2000 The American Physical Society.

  15. Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the Cys-Loop and M2–M3 Linker

    PubMed Central

    Jha, Archana; Cadugan, David J.; Purohit, Prasad; Auerbach, Anthony

    2007-01-01

    Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a “gate” in the transmembrane domain (TMD). We used Φ-value analysis to probe the relative timing of the gating motions of α-subunit residues located near the ECD–TMD interface. Mutation of four of the seven amino acids in the M2–M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant (Keq) by up to 10,000-fold (P272 > I274 > A270 > G275). The average Φ-value for the whole linker was ∼0.64. One interpretation of this result is that the gating motions of the M2–M3 linker are approximately synchronous with those of much of M2 (∼0.64), but occur after those of the transmitter binding site region (∼0.93) and loops 2 and 7 (∼0.77). We also examined mutants of six cys-loop residues (V132, T133, H134, F135, P136, and F137). Mutation of V132, H134, and F135 changed Keq by 2800-, 10-, and 18-fold, respectively, and with an average Φ-value of 0.74, similar to those of other cys-loop residues. Even though V132 and I274 are close, the energetic coupling between I and V mutants of these positions was small (≤0.51 kcal mol−1). The M2–M3 linker appears to be the key moving part that couples gating motions at the base of the ECD with those in TMD. These interactions are distributed along an ∼16-Å border and involve about a dozen residues. PMID:18040057

  16. [Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].

    PubMed

    Goncharuk, M V; Shul'ga, A A; Ermoliuk, Ia S; Tkach, E N; Goncharuk, S A; Pustovalova, Iu E; Mineev, K S; Bocharov, É V; Maslennikov, I V; Arsen'ev, A S; Kirpichnikov, M P

    2011-01-01

    A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy. PMID:22393787

  17. Ion fluxes through nanopores and transmembrane channels.

    PubMed

    Bordin, J R; Diehl, A; Barbosa, M C; Levin, Y

    2012-03-01

    We introduce an implicit solvent Molecular Dynamics approach for calculating ionic fluxes through narrow nanopores and transmembrane channels. The method relies on a dual-control-volume grand-canonical molecular dynamics (DCV-GCMD) simulation and the analytical solution for the electrostatic potential inside a cylindrical nanopore recently obtained by Levin [Europhys. Lett. 76, 163 (2006)]. The theory is used to calculate the ionic fluxes through an artificial transmembrane channel which mimics the antibacterial gramicidin A channel. Both current-voltage and current-concentration relations are calculated under various experimental conditions. We show that our results are comparable to the characteristics associated to the gramicidin A pore, especially the existence of two binding sites inside the pore and the observed saturation in the current-concentration profiles. PMID:22587130

  18. An Unfolding Story of Helical Transmembrane Proteins†

    PubMed Central

    Renthal, Robert

    2008-01-01

    Reversible unfolding of helical transmembrane proteins could provide valuable information about the free energy of interaction between transmembrane helices. Thermal unfolding experiments suggest that this process for integral membrane proteins is irreversible. Chemical unfolding has been accomplished with organic acids, but the unfolding or refolding pathways involve irreversible steps. Sodium dodecyl sulfate (SDS) has been used as a perturbant to study reversible unfolding and refolding kinetics. However, the interpretation of these experiments is not straightforward. It is shown that the results could be explained by SDS binding without substantial unfolding. Furthermore, the SDS perturbed state is unlikely to include all of the entropy terms involved in an unfolding process. Alternative directions for future research are suggested: fluorinated alcohols in homogeneous solvent systems; inverse micelles; and fragment association studies. PMID:17144649

  19. The double helix and immunology

    NASA Astrophysics Data System (ADS)

    Nossal, Gustav J. V.

    2003-01-01

    The immune system can recognize and produce antibodies to virtually any molecule in the Universe. This enormous diversity arises from the ingenious reshuffling of DNA sequences encoding components of the immune system. Immunology is an example of a field completely transformed during the past 50 years by the discovery of the structure of DNA and the emergence of DNA technologies that followed.

  20. Comparison of Class A and D G Protein-Coupled Receptors: Common Features in Structure and Activation†

    PubMed Central

    Eilers, Markus; Hornak, Viktor; Smith, Steven O.; Konopka, James B.

    2005-01-01

    All G protein-coupled receptors (GPCRs) share a common seven TM helix architecture and the ability to activate heterotrimeric G proteins. Nevertheless, these receptors have widely divergent sequences with no significant homology. We present a detailed structure–function comparison of the very divergent Class A and D receptors to address whether there is a common activation mechanism across the GPCR superfamily. The Class A and D receptors are represented by the vertebrate visual pigment rhodopsin and the yeast α-factor pheromone receptor Ste2, respectively. Conserved amino acids within each specific receptor class and amino acids where mutation alters receptor function were located in the structures of rhodopsin and Ste2 to assess whether there are functionally equivalent positions or regions within these receptors. We find several general similarities that are quite striking. First, strongly polar amino acids mediate helix interactions. Their mutation generally leads to loss of function or constitutive activity. Second, small and weakly polar amino acids facilitate tight helix packing. Third, proline is essential at similar positions in transmembrane helices 6 and 7 of both receptors. Mapping the specific location of the conserved amino acids and sites of constitutively active mutations identified conserved microdomains on transmembrane helices H3, H6, and H7, suggesting that there are underlying similarities in the mechanism of the widely divergent Class A and Class D receptors. PMID:15966721

  1. Helix-specific interactions induce condensation of guanosine four-stranded helices in concentrated salt solutions.

    PubMed

    Mariani, P; Ciuchi, F; Saturni, L

    1998-01-01

    Deoxyguanosine-5'-monophosphate in water self-associates into stable structures, which include liquid-crystalline hexagonal and cholesteric phases. The structural unit is a four-stranded helix, composed of stacked Hoogsteen-bonded guanosine quartets. By using the osmotic stress method, we recently measured the force between helices in KCl solutions up to 2 M. In addition to the long-range electrostatic force, a short-range hydration repulsive contribution was recognized. The hydration repulsion is exponential, and shows a decay length independent from the ionic strength of the solution. Here, we report that more concentrated KCl solutions cause condensation of the guanosine helix in a hexagonal phase with constant equilibrium separation of approximately 7 A between helix surfaces. Long-range attraction, which induces the self-assembly, and short-range repulsion, which prevents the contact between the helices, are implied. By using osmotic stress, the force needed to push helices closer from the spontaneously assumed position has been measured. The attractive force was then estimated as a difference between the net force and the repulsive contribution, revealing an exponential decay length about two times larger than that of the short-range repulsion. The agreement with the helix interaction theory introduced recently by Kornyshev and Leikin (Kornyshev, A. A., and S. Leikin, 1997. Theory of interaction between helical molecules. J. Phys. Chem. 107:3656-3674) suggests that the repulsive and attractive forces originate from helix-specific interactions. PMID:9449343

  2. Cytotoxic Helix-Rich Oligomer Formation by Melittin and Pancreatic Polypeptide

    PubMed Central

    Singh, Pradeep K.; Ghosh, Dhiman; Tewari, Debanjan; Mohite, Ganesh M.; Carvalho, Edmund; Jha, Narendra Nath; Jacob, Reeba S.; Sahay, Shruti; Banerjee, Rinti; Bera, Amal K.; Maji, Samir K.

    2015-01-01

    Conversion of amyloid fibrils by many peptides/proteins involves cytotoxic helix-rich oligomers. However, their toxicity and biophysical studies remain largely unknown due to their highly dynamic nature. To address this, we chose two helical peptides (melittin, Mel and pancreatic polypeptide, PP) and studied their aggregation and toxicity. Mel converted its random coil structure to oligomeric helical structure upon binding to heparin; however, PP remained as helix after oligomerization. Interestingly, similar to Parkinson’s associated α-synuclein (AS) oligomers, Mel and PP also showed tinctorial properties, higher hydrophobic surface exposure, cellular toxicity and membrane pore formation after oligomerization in the presence of heparin. We suggest that helix-rich oligomers with exposed hydrophobic surface are highly cytotoxic to cells irrespective of their disease association. Moreover as Mel and PP (in the presence of heparin) instantly self-assemble into stable helix-rich amyloidogenic oligomers; they could be represented as models for understanding the biophysical and cytotoxic properties of helix-rich intermediates in detail. PMID:25803428

  3. 50 Hz Electromagnetic Field Produced Changes in FTIR Spectroscopy Associated with Mitochondrial Transmembrane Potential Reduction in Neuronal-Like SH-SY5Y Cells

    PubMed Central

    Calabrò, Emanuele; Condello, Salvatore; Currò, Monica; Ferlazzo, Nadia; Vecchio, Mercurio; Caccamo, Daniela; Magazù, Salvatore; Ientile, Riccardo

    2013-01-01

    SH-SY5Y neuroblastoma cells were used as an experimental model to study the effects of 50 Hz electromagnetic field, in the range from 50 µT to 1.4 mT. Fourier transform infrared spectroscopy analysis evidenced a reduction in intensity of the amide A band and a slight increase of vibration bands at 2921 cm−1 and 2853 cm−1 corresponding to methylene groups. A further increase of the magnetic field intensity of exposure up to 0.8 mT and 1.4 mT produced a clear increase in intensity of CH2 vibration bands. Moreover, it has been observed some alterations in the amide I region, such as a shifted peak of the amide I band to a smaller wavenumber, probably due to protein conformational changes. These results suggested that exposure to extremely low electromagnetic fields influenced lipid components of cellular membrane and the N–H in-plane bending and C–N stretching vibrations of peptide linkages, modifying the secondary structures of α-helix and β-sheet contents and producing unfolding process in cell membrane proteins. The observed changes after exposure to 50 Hz electromagnetic field higher than 0.8 mT were associated with a significant reduction of cell viability and reduced mitochondrial transmembrane potential. PMID:23970948

  4. Structure of CrgA, a cell division structural and regulatory protein from Mycobacterium tuberculosis, in lipid bilayers

    PubMed Central

    Das, Nabanita; Dai, Jian; Hung, Ivan; Rajagopalan, Malini; Zhou, Huan-Xiang; Cross, Timothy A.

    2015-01-01

    The 93-residue transmembrane protein CrgA in Mycobacterium tuberculosis is a central component of the divisome, a large macromolecular machine responsible for cell division. Through interactions with multiple other components including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA, CrgA facilitates the recruitment of the proteins essential for peptidoglycan synthesis to the divisome and stabilizes the divisome. CrgA is predicted to have two transmembrane helices. Here, the structure of CrgA was determined in a liquid–crystalline lipid bilayer environment by solid-state NMR spectroscopy. Oriented-sample data yielded orientational restraints, whereas magic-angle spinning data yielded interhelical distance restraints. These data define a complete structure for the transmembrane domain and provide rich information on the conformational ensembles of the partially disordered N-terminal region and interhelical loop. The structure of the transmembrane domain was refined using restrained molecular dynamics simulations in an all-atom representation of the same lipid bilayer environment as in the NMR samples. The two transmembrane helices form a left-handed packing arrangement with a crossing angle of 24° at the conserved Gly39 residue. This helix pair exposes other conserved glycine and alanine residues to the fatty acyl environment, which are potential sites for binding CrgA’s partners such as CwsA and FtsQ. This approach combining oriented-sample and magic-angle spinning NMR spectroscopy in native-like lipid bilayers with restrained molecular dynamics simulations represents a powerful tool for structural characterization of not only isolated membrane proteins, but their complexes, such as those that form macromolecular machines. PMID:25548160

  5. Structure of CrgA, a cell division structural and regulatory protein from Mycobacterium tuberculosis, in lipid bilayers.

    PubMed

    Das, Nabanita; Dai, Jian; Hung, Ivan; Rajagopalan, Malini R; Rajagopalan, Malini R; Zhou, Huan-Xiang; Cross, Timothy A

    2015-01-13

    The 93-residue transmembrane protein CrgA in Mycobacterium tuberculosis is a central component of the divisome, a large macromolecular machine responsible for cell division. Through interactions with multiple other components including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA, CrgA facilitates the recruitment of the proteins essential for peptidoglycan synthesis to the divisome and stabilizes the divisome. CrgA is predicted to have two transmembrane helices. Here, the structure of CrgA was determined in a liquid-crystalline lipid bilayer environment by solid-state NMR spectroscopy. Oriented-sample data yielded orientational restraints, whereas magic-angle spinning data yielded interhelical distance restraints. These data define a complete structure for the transmembrane domain and provide rich information on the conformational ensembles of the partially disordered N-terminal region and interhelical loop. The structure of the transmembrane domain was refined using restrained molecular dynamics simulations in an all-atom representation of the same lipid bilayer environment as in the NMR samples. The two transmembrane helices form a left-handed packing arrangement with a crossing angle of 24° at the conserved Gly39 residue. This helix pair exposes other conserved glycine and alanine residues to the fatty acyl environment, which are potential sites for binding CrgA's partners such as CwsA and FtsQ. This approach combining oriented-sample and magic-angle spinning NMR spectroscopy in native-like lipid bilayers with restrained molecular dynamics simulations represents a powerful tool for structural characterization of not only isolated membrane proteins, but their complexes, such as those that form macromolecular machines. PMID:25548160

  6. Probing the non-native H helix translocation in apomyoglobin folding intermediates.

    PubMed

    Aoto, Phillip C; Nishimura, Chiaki; Dyson, H Jane; Wright, Peter E

    2014-06-17

    Apomyoglobin folds via sequential helical intermediates that are formed by rapid collapse of the A, B, G, and H helix regions. An equilibrium molten globule with a similar structure is formed near pH 4. Previous studies suggested that the folding intermediates are kinetically trapped states in which folding is impeded by non-native packing of the G and H helices. Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates. Förster resonance energy transfer measurements show that a 5-({2-[(acetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid acceptor coupled to K140C (helix H) is closer to Trp14 (helix A) in the equilibrium molten globule than in the native state, by a distance that is consistent with sliding of the H helix in an N-terminal direction by approximately one helical turn. Formation of an S108C-L135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register. By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate. The apomyoglobin folding landscape is highly rugged, with several energetic bottlenecks that frustrate folding; relief of any one of the major identified bottlenecks is insufficient to speed progression to the transition state. PMID:24857522

  7. Probing the Non-Native H Helix Translocation in Apomyoglobin Folding Intermediates

    PubMed Central

    2015-01-01

    Apomyoglobin folds via sequential helical intermediates that are formed by rapid collapse of the A, B, G, and H helix regions. An equilibrium molten globule with a similar structure is formed near pH 4. Previous studies suggested that the folding intermediates are kinetically trapped states in which folding is impeded by non-native packing of the G and H helices. Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates. Frster resonance energy transfer measurements show that a 5-({2-[(acetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid acceptor coupled to K140C (helix H) is closer to Trp14 (helix A) in the equilibrium molten globule than in the native state, by a distance that is consistent with sliding of the H helix in an N-terminal direction by approximately one helical turn. Formation of an S108CL135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register. By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate. The apomyoglobin folding landscape is highly rugged, with several energetic bottlenecks that frustrate folding; relief of any one of the major identified bottlenecks is insufficient to speed progression to the transition state. PMID:24857522

  8. Local protein structure and dynamics at kinked transmembrane α-helices of [1- 13C]Pro-labeled bacteriorhodopsin as revealed by site-directed solid-state 13C NMR

    NASA Astrophysics Data System (ADS)

    Tuzi, Satoru; Naito, Akira; Saitô, Hazime

    2003-06-01

    We have recorded 13C NMR spectra of [1- 13C]Pro-labeled bacteriorhodopsin (bR) and P50G, P91G, and P186A mutants under fully hydrated condition, by high-resolution solid-state NMR utilizing cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques. Seven well-resolved 13C NMR signals including a shoulder peak were distinguished by CP-MAS NMR, although only two signals were resolved by DD-MAS NMR. We assigned these 13C NMR signals among them to Pro50, 91 and 186 residues at the kinks in the inner part of the transmembrane α-helices, on the basis of compared peak-intensities between wild type bR and the above-mentioned site-directed mutants, together with aid of Mn 2+-induced suppression of peaks from residues located near at the surfaces due to accelerated spin-spin relaxation times. It turned out that these Pro 13C NMR signals of wild type were appreciably broadened at temperature below -40 °C as in [3- 13C]Ala-bR, as a result of superposition of a variety of frozen conformers of the transmembrane α-helices exhibiting dispersion of chemical shifts. This means that the dynamic behavior of bR as viewed from Pro residue is very similar to that of ordinary amino acid residues such as Ala, Val, Phe, etc. Further, it was found that no appreciable conformational change was noted for wild type bR within a temperature range between -20 and 35 °C at these kinked portions, although such change was noted at 35 °C for Y185F mutant which lacks interchain hydrogen bonding interaction as observed for wild type bR between the side-chains of Asp212 and Tyr185.

  9. Left handed β helix models for mammalian prion fibrils

    PubMed Central

    Kunes, Kay C; Clark, Scott C; Singh, Rajiv RP

    2008-01-01

    We propose models for in vitro grown mammalian prion protein fibrils based upon left handed beta helices formed both from the N-terminal and C-terminal regions of the proteinase resistant infectious prion core. The C-terminal threading onto a β-helical structure is almost uniquely determined by fixing the cysteine disulfide bond on a helix corner. In comparison to known left handed helical peptides, the resulting model structures have similar stability attributes including relatively low root mean square deviations in all atom molecular dynamics, substantial side-chain-to-side-chain hydrogen bonding, good volume packing fraction, and low hydrophilic/hydrophobic frustration. For the N-terminus, we propose a new threading of slightly more than two turns, which improves upon the above characteristics relative to existing three turn β-helical models. The N-terminal and C-terminal beta helices can be assembled into eight candidate models for the fibril repeat units, held together by large hinge (order 30 residues) domain swapping, with three amenable to fibril promoting domain swapping via a small (five residue) hinge on the N-terminal side. Small concentrations of the metastable C-terminal β helix in vivo might play a significant role in templating the infectious conformation and in enhancing conversion kinetics for inherited forms of the disease and explain resistance (for canines) involving hypothesized coupling to the methionine 129 sulfur known to play a role in human disease. PMID:19098440

  10. Role of the Lipid Environment in the Dimerization of Transmembrane Domains of Glycophorin A.

    PubMed

    Kuznetsov, A S; Volynsky, P E; Efremov, R G

    2015-01-01

    An efficient computational approach is developed to quantify the free energy of a spontaneous association of the ?-helices of proteins in the membrane environment. The approach is based on the numerical decomposition of the free energy profiles of the transmembrane (TM) helices into components corresponding to protein-protein, protein-lipid, and protein-water interactions. The method was tested for the TM segments of human glycophorin A (GpA) and two mutant forms, Gly83Ala and Thr87Val. It was shown that lipids make a significant negative contribution to the free energy of dimerization, while amino acid residues forming the interface of the helix-helix contact may be unfavorable in terms of free energy. The detailed balance between different energy contributions is highly dependent on the amino acid sequence of the TM protein segment. The results show the dominant role of the environment in the interaction of membrane proteins that is changing our notion of the driving force behind the spontaneous association of TM ?-helices. Adequate estimation of the contribution of the water-lipid environment thus becomes an extremely urgent task for a rational design of new molecules targeting bitopic membrane proteins, including receptor tyrosine kinases. PMID:26798499

  11. DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy

    PubMed Central

    Tubbs, Julie L.; Pegg, Anthony E.; Tainer, John A.

    2007-01-01

    O6-alkylguanine-DNA alkyltransferase (AGT) is a crucial target both for the prevention of cancer and for chemotherapy, since it repairs mutagenic lesions in DNA, and it limits the effectiveness of alkylating chemotherapies. AGT catalyzes the unique, single-step, direct damage reversal repair of O6-alkylguanines by selectively transferring the O6-alkyl adduct to an internal cysteine residue. Recent crystal structures of human AGT alone and in complex with substrate DNA reveal a two-domain a/β fold and a bound zinc ion. AGT uses its helix-turn-helix motif to bind substrate DNA via the minor groove. The alkylated guanine is then flipped out from the base stack into the AGT active site for repair by covalent transfer of the alkyl adduct to Cys145. An asparagine hinge (Asn137) couples the helix-turn-helix DNA binding and active site motifs. An arginine finger (Arg128) stabilizes the extrahelical DNA conformation. With this newly improved structural understanding of AGT and its interactions with biologically relevant substrates, we can now begin to unravel the role it plays in preserving genetic integrity and discover how it promotes resistance to anticancer therapies. PMID:17485252

  12. The mechanisms of HAMP-mediated signaling in transmembrane receptors.

    PubMed

    Ferris, Hedda U; Dunin-Horkawicz, Stanislaw; Mondéjar, Laura García; Hulko, Michael; Hantke, Klaus; Martin, Jörg; Schultz, Joachim E; Zeth, Kornelius; Lupas, Andrei N; Coles, Murray

    2011-03-01

    HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems. PMID:21397188

  13. A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome

    PubMed Central

    BÉLANGER, FRANÇOIS; THÉBERGE-JULIEN, GABRIEL; CUNNINGHAM, PHILIP R.; BRAKIER-GINGRAS, LÉA

    2005-01-01

    The conserved 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) interacts with helix 24 of 16S rRNA and also with helix 67 of 23S rRNA, forming the intersubunit bridge B2c, proximal to the decoding center. In previous studies, we investigated how the interaction between the 900 tetraloop and helix 24 participates in subunit association and translational fidelity. In the present study, we investigated whether the 900 tetraloop is involved in other undetected interactions with different regions of the Escherichia coli 16S rRNA. Using a genetic complementation approach, we selected mutations in 16S rRNA that compensate for a 900 tetraloop mutation, A900G, which severely impairs subunit association and translational fidelity. Mutations were randomly introduced in 16S rRNA, using either a mutagenic XL1-Red E. coli strain or an error-prone PCR strategy. Gain-offunction mutations were selected in vivo with a specialized ribosome system. Two mutations, the deletion of U12 and the U12C substitution, were thus independently selected in helix 1 of 16S rRNA. This helix is located in the vicinity of helix 27, but does not directly contact the 900 tetraloop in the crystal structures of the ribosome. Both mutations correct the subunit association and translational fidelity defects caused by the A900G mutation, revealing an unanticipated functional interaction between these two regions of 16S rRNA. PMID:15872184

  14. A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome.

    PubMed

    Bélanger, François; Théberge-Julien, Gabriel; Cunningham, Philip R; Brakier-Gingras, Léa

    2005-06-01

    The conserved 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) interacts with helix 24 of 16S rRNA and also with helix 67 of 23S rRNA, forming the intersubunit bridge B2c, proximal to the decoding center. In previous studies, we investigated how the interaction between the 900 tetraloop and helix 24 participates in subunit association and translational fidelity. In the present study, we investigated whether the 900 tetraloop is involved in other undetected interactions with different regions of the Escherichia coli 16S rRNA. Using a genetic complementation approach, we selected mutations in 16S rRNA that compensate for a 900 tetraloop mutation, A900G, which severely impairs subunit association and translational fidelity. Mutations were randomly introduced in 16S rRNA, using either a mutagenic XL1-Red E. coli strain or an error-prone PCR strategy. Gain-offunction mutations were selected in vivo with a specialized ribosome system. Two mutations, the deletion of U12 and the U12C substitution, were thus independently selected in helix 1 of 16S rRNA. This helix is located in the vicinity of helix 27, but does not directly contact the 900 tetraloop in the crystal structures of the ribosome. Both mutations correct the subunit association and translational fidelity defects caused by the A900G mutation, revealing an unanticipated functional interaction between these two regions of 16S rRNA. PMID:15872184

  15. Structure of an E. coli integral membrane sulfurtransferase and its structural transition upon SCN− binding defined by EPR-based hybrid method

    PubMed Central

    Ling, Shenglong; Wang, Wei; Yu, Lu; Peng, Junhui; Cai, Xiaoying; Xiong, Ying; Hayati, Zahra; Zhang, Longhua; Zhang, Zhiyong; Song, Likai; Tian, Changlin

    2016-01-01

    Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN−, which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane. PMID:26817826

  16. Structure of an E. coli integral membrane sulfurtransferase and its structural transition upon SCN- binding defined by EPR-based hybrid method

    NASA Astrophysics Data System (ADS)

    Ling, Shenglong; Wang, Wei; Yu, Lu; Peng, Junhui; Cai, Xiaoying; Xiong, Ying; Hayati, Zahra; Zhang, Longhua; Zhang, Zhiyong; Song, Likai; Tian, Changlin

    2016-01-01

    Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN-, which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane.

  17. Structure of an E. coli integral membrane sulfurtransferase and its structural transition upon SCN(-) binding defined by EPR-based hybrid method.

    PubMed

    Ling, Shenglong; Wang, Wei; Yu, Lu; Peng, Junhui; Cai, Xiaoying; Xiong, Ying; Hayati, Zahra; Zhang, Longhua; Zhang, Zhiyong; Song, Likai; Tian, Changlin

    2016-01-01

    Electron paramagnetic resonance (EPR)-based hybrid experimental and computational approaches were applied to determine the structure of a full-length E. coli integral membrane sulfurtransferase, dimeric YgaP, and its structural and dynamic changes upon ligand binding. The solution NMR structures of the YgaP transmembrane domain (TMD) and cytosolic catalytic rhodanese domain were reported recently, but the tertiary fold of full-length YgaP was not yet available. Here, systematic site-specific EPR analysis defined a helix-loop-helix secondary structure of the YagP-TMD monomers using mobility, accessibility and membrane immersion measurements. The tertiary folds of dimeric YgaP-TMD and full-length YgaP in detergent micelles were determined through inter- and intra-monomer distance mapping and rigid-body computation. Further EPR analysis demonstrated the tight packing of the two YgaP second transmembrane helices upon binding of the catalytic product SCN(-), which provides insight into the thiocyanate exportation mechanism of YgaP in the E. coli membrane. PMID:26817826

  18. Infrared dichroism from the X-ray structure of bacteriorhodopsin.

    PubMed Central

    Marsh, D; Páli, T

    2001-01-01

    A detailed comparison with the three-dimensional protein structure provides a stringent test of the models and parameters commonly used in determining the orientation of the alpha-helices from the linear dichroism of the infrared amide bands, particularly in membranes. The order parameters of the amide vibrational transition moments are calculated for the transmembrane alpha-helices of bacteriorhodopsin by using the crystal structure determined at a resolution of 1.55 A (PDB accession number 1C3W). The dependence on the angle delta(M) that the transition moment makes with the peptide carbonyl bond is fit by the expression ((3)/(2)S(alpha) cos(2) alpha)cos(2)(delta(M) + beta) - 1/2S(alpha), where S(alpha) (0.91) is the order parameter of the alpha-helices, alpha (13 degrees ) is the angle that the peptide plane makes with the helix axis, and beta (11 degrees ) is the angle that the peptide carbonyl bond makes with the projection of the helix axis on the peptide plane. This result is fully consistent with the model of nested axial distributions commonly used in interpreting infrared linear dichroism of proteins. Comparison with experimental infrared dichroic ratios for bacteriorhodopsin yields values of Theta(A) = 33 +/- 1 degree, Theta(I) = 39.5 +/- 1 degree, and Theta(II) = 70 +/- 2 degrees for the orientation of the transition moments of the amide A, amide I, and amide II bands, respectively, relative to the helix axis. These estimates are close to those found for model alpha-helical polypeptides, indicating that side-chain heterogeneity and slight helix imperfections are unlikely to affect the reliability of infrared measurements of helix orientations. PMID:11159403

  19. On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes.

    PubMed

    Cho, Hyundae D; Sood, Vanita D; Baker, David; Weiner, Alan M

    2008-07-01

    Archaeal class I CCA-adding enzymes use a ribonucleoprotein template to build and repair the universally conserved 3'-terminal CCA sequence of the acceptor stem of all tRNAs. A wealth of structural and biochemical data indicate that the Archaeoglobus fulgidus CCA-adding enzyme binds primarily to the tRNA acceptor stem through a long, highly conserved alpha-helix that lies nearly parallel to the acceptor stem and makes many contacts with its sugar-phosphate backbone. Although the geometry of this alpha-helix is nearly ideal in all available cocrystal structures, the helix contains a highly conserved, potentially helix-breaking proline or glycine near the N terminus. We performed a mutational analysis to dissect the role of this residue in CCA-addition activity. We found that the phylogenetically permissible P295G mutant and the phylogenetically absent P295T had little effect on CCA addition, whereas P295A and P295S progressively interfered with CCA addition (C74>C75>A76 addition). We also examined the effects of these mutations on tRNA binding and the kinetics of CCA addition, and performed a computational analysis using Rosetta Design to better understand the role of P295 in nucleotide transfer. Our data indicate that CCA-adding activity does not correlate with the stability of the pre-addition cocrystal structures visualized by X-ray crystallography. Rather, the data are consistent with a transient conformational change involving P295 of the tRNA-binding alpha-helix during or between one or more steps in CCA addition. PMID:18495940

  20. The Crystal Structures of Yeast Get3 Suggest a Mechanism for Tail-Anchored Protein Membrane Insertion

    SciTech Connect

    Hu, Junbin; Li, Jingzhi; Qian, Xinguo; Denic, Vlad; Sha, Bingdong

    2010-08-16

    Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a 'finger' domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an 'open' conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a 'closed' conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.

  1. Site-directed spin labeling and chemical crosslinking demonstrate that helix V is close to helices VII and VIII in the lactose permease of Escherichia coli.

    PubMed Central

    Wu, J; Voss, J; Hubbell, W L; Kaback, H R

    1996-01-01

    Site-directed chemical cleavage of lactose permease indicates that helix V is in close proximity to helices VII and VIII. To test this conclusion further, permease containing a biotin-acceptor domain and paired Cys residues at positions 148 (helix V) and 228 (helix VII), 148 and 226 (helix VII), or 148 and 275 (helix VIII) was affinity purified and labeled with a sulfhydryl-specific nitroxide spin label. Spin-spin interactions are observed with the 148/228 and 148/275 pairs, indicating close proximity between appropriate faces of helix V and helices VII and VIII. Little or no interaction is evident with the 148/226 pair, in all likelihood because position 226 is on the opposite face of helix VII from position 228. Broadening of the electron paramagnetic resonance spectra in the frozen state was used to estimate distance between the 148/228 and the 148/275 pairs. The nitroxides at positions 148 and 228 or 148 and 275 are within approximately 13-15 A. Finally, Cys residues at positions 148 and 228 are crosslinked by dibromobimane, a bifunctional crosslinker that is approximately 5 A. long, while no crosslinking is detected between Cys residues at positions 148 and 275 or 148 and 226. The results provide strong support for a structure in which helix V is in close proximity to both helices VII and VIII and is oriented in such a fashion that Cys-148 is closer to helix VII. Images Fig. 1 Fig. 6 PMID:8816762

  2. Rapid helix--coil transitions in the S-2 region of myosin.

    PubMed

    Tsong, T Y; Karr, T; Harrington, W F

    1979-03-01

    Temperature-jump studies on the long S-2 fragment (100,000 daltons) isolated from myosin show that this structure can undergo alpha-helix--random coil transitions in a time range approximating the cycle time of a crossbridge. Two relaxation times are observed after temperature jumps of 5 degrees C over the range 35--55 degrees C, one in the submillisecond (tau f) and the other in the millisecond (tau s) time ranges. Both processes exhibit maxima near the midpoint of the helix--coil transition (tm = 45 +/- 2 degrees C) as determined by optical rotation melt experiments. Similar results were observed for the low temperature transition (tm = 45 degrees C) of the myosin rod. Viscosity studies reveal that the S-2 particles has significant flexibility at physiological temperature. Results are considered in terms of the Huxley--Simmons and helix--coil transition models for force generation in muscle. PMID:286296

  3. Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage.

    PubMed Central

    Wu, J; Perrin, D M; Sigman, D S; Kaback, H R

    1995-01-01

    Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity chromatography and derivatized with 5-(alpha-bromoacetamido)-1,10-phenanthroline-copper [OP(Cu)]. Studies with purified, OP(Cu)-labeled Leu-330 --> Cys permease in dodecyl-beta-D-maltopyranoside demonstrate that after incubation in the presence of ascorbate, cleavage products of approximately 19 and 6-8 kDa are observed on immunoblots with anti-C-terminal antibody. Remarkably, the same cleavage products are observed with permease embedded in the native membrane. Comparison with the C-terminal half of the permease expressed independently as a standard indicates that the 19-kDa product results from cleavage near the cytoplasmic end of helix VII, whereas the 6- to 8-kDa fragment probably results from fragmentation near the cytoplasmic end of helix XI. Results are entirely consistent with a tertiary-structure model of the C-terminal half of the permease derived from earlier site-directed fluorescence and site-directed mutagenesis studies. Similar studies with OP(Cu)-labeled Cys-148 permease exhibit cleavage products at approximately 19 kDa and at 15-16 kDa. The larger fragment probably reflects cleavage at a site near the cytoplasmic end of helix VII, whereas the 15- to 16-kDa fragment is consistent with cleavage near the cytoplasmic end of helix VIII. When OP(Cu) is moved 100 degrees to position 149 (Val-149 --> Cys permease), a single product is observed at 19 kDa, suggesting fragmentation at the cytoplasmic end of helix VII. However, when the reagent is moved 100 degrees in the other direction to position 147 (Gly-147 --> Cys permease), cleavage is not observed. The results suggest that helix V is in close proximity to helices VII and VIII with position 148 in the interface between the helices, position 149 facing helix VII, and position 147 facing the lipid bilayer. Images Fig. 3 Fig. 4 PMID:7568098

  4. Yeast screens show aromatic residues at the end of the sixth helix anchor transient receptor potential channel gate

    PubMed Central

    Zhou, Xinliang; Su, Zhenwei; Anishkin, Andriy; Haynes, W. John; Friske, Eric M.; Loukin, Stephen H.; Kung, Ching; Saimi, Yoshiro

    2007-01-01

    Transient receptor potential (TRP) channels are first elements in sensing chemicals, heat, and force and are widespread among protists and fungi as well as animals. Despite their importance, the arrangement and roles of the amino acids that constitute the TRP channel gate are unknown. The yeast TRPY1 is activated in vivo by osmotically induced vacuolar membrane deformation and by cytoplasmic Ca2+. After a random mutagenesis, we isolated TRPY1 mutants that responded more strongly to mild osmotic upshocks. One such gain-of-function mutant has a Y458H substitution at the C terminus of the predicted sixth transmembrane helix. Direct patch-clamp examination of vacuolar membranes showed that Y458H channels were already active with little stimulus and showed marked flickers between the open and intraburst closed states. They remained responsive to membrane stretch force and to Ca2+, indicating primary defects in the gate region but not in the sensing of gating principles. None of the other 18 amino acid replacements engineered here showed normal channel kinetics except the two aromatic substitutions, Y458F and Y458W. The Y458 of TRPY1 has its aromatic counterpart in mammalian TRPM. Furthermore, conserved aromatics one α-helical turn downstream from this point are also found in animal TRPC, TRPN, TRPP, and TRPML, suggesting that gate anchoring with aromatics may be common among many TRP channels. The possible roles of aromatics at the end of the sixth transmembrane helix are discussed. PMID:17878311

  5. Mechanical coupling of the multiple structural elements of the large-conductance mechanosensitive channel during expansion

    PubMed Central

    Li, Jie; Guo, Jianli; Ou, Xiaomin; Zhang, Mingfeng; Li, Yuezhou; Liu, Zhenfeng

    2015-01-01

    The prokaryotic mechanosensitive channel of large conductance (MscL) is a pressure-relief valve protecting the cell from lysing during acute osmotic downshock. When the membrane is stretched, MscL responds to the increase of membrane tension and opens a nonselective pore to about 30 Å wide, exhibiting a large unitary conductance of ∼3 nS. A fundamental step toward understanding the gating mechanism of MscL is to decipher the molecular details of the conformational changes accompanying channel opening. By applying fusion-protein strategy and controlling detergent composition, we have solved the structures of an archaeal MscL homolog from Methanosarcina acetivorans trapped in the closed and expanded intermediate states. The comparative analysis of these two new structures reveals significant conformational rearrangements in the different domains of MscL. The large changes observed in the tilt angles of the two transmembrane helices (TM1 and TM2) fit well with the helix-pivoting model derived from the earlier geometric analyses based on the previous structures. Meanwhile, the periplasmic loop region transforms from a folded structure, containing an ω-shaped loop and a short β-hairpin, to an extended and partly disordered conformation during channel expansion. Moreover, a significant rotating and sliding of the N-terminal helix (N-helix) is coupled to the tilting movements of TM1 and TM2. The dynamic relationships between the N-helix and TM1/TM2 suggest that the N-helix serves as a membrane-anchored stopper that limits the tilts of TM1 and TM2 in the gating process. These results provide direct mechanistic insights into the highly coordinated movement of the different domains of the MscL channel when it expands. PMID:26261325

  6. Mechanical coupling of the multiple structural elements of the large-conductance mechanosensitive channel during expansion.

    PubMed

    Li, Jie; Guo, Jianli; Ou, Xiaomin; Zhang, Mingfeng; Li, Yuezhou; Liu, Zhenfeng

    2015-08-25

    The prokaryotic mechanosensitive channel of large conductance (MscL) is a pressure-relief valve protecting the cell from lysing during acute osmotic downshock. When the membrane is stretched, MscL responds to the increase of membrane tension and opens a nonselective pore to about 30 Å wide, exhibiting a large unitary conductance of ∼ 3 nS. A fundamental step toward understanding the gating mechanism of MscL is to decipher the molecular details of the conformational changes accompanying channel opening. By applying fusion-protein strategy and controlling detergent composition, we have solved the structures of an archaeal MscL homolog from Methanosarcina acetivorans trapped in the closed and expanded intermediate states. The comparative analysis of these two new structures reveals significant conformational rearrangements in the different domains of MscL. The large changes observed in the tilt angles of the two transmembrane helices (TM1 and TM2) fit well with the helix-pivoting model derived from the earlier geometric analyses based on the previous structures. Meanwhile, the periplasmic loop region transforms from a folded structure, containing an ω-shaped loop and a short β-hairpin, to an extended and partly disordered conformation during channel expansion. Moreover, a significant rotating and sliding of the N-terminal helix (N-helix) is coupled to the tilting movements of TM1 and TM2. The dynamic relationships between the N-helix and TM1/TM2 suggest that the N-helix serves as a membrane-anchored stopper that limits the tilts of TM1 and TM2 in the gating process. These results provide direct mechanistic insights into the highly coordinated movement of the different domains of the MscL channel when it expands. PMID:26261325

  7. Mutational analyses define helix organization and key residues of a bacterial membrane energy-transducing complex.

    PubMed

    Goemaere, Emilie L; Cascales, Eric; Lloubès, Roland

    2007-03-01

    In Gram-negative bacteria, many biological processes are coupled to inner membrane ion gradients. Ions transit at the interface of helices of integral membrane proteins, generating mechanical energy to drive energetic processes. To better understand how ions transit through these channels, we used a model system involved in two different processes, one of which depends on inner membrane energy. The Tol machinery of the Escherichia coli cell envelope is dedicated to maintaining outer membrane stability, a process driven by the proton-motive force. The Tol system is parasitized by bacterial toxins called colicins, which are imported through the outer membrane using an energy-independent process. Herein, we mutated TolQ and TolR transmembrane residues, and we analyzed the mutants for outer membrane stability, colicin import and protein complex formation. We identified residues involved in the assembly of the complex, and a new class of discriminative mutations that conferred outer membrane destabilization identical to a tol deletion mutant, but which remained fully sensitive to colicins. Further genetic approaches revealed transmembrane helix interactions and organization in the bilayer, and suggested that most of the discriminative residues are located in a putative aqueous ion channel. We discuss a model for the function of related bacterial molecular motors. PMID:17222427

  8. Heterogeneous H-bonding in a foldamer helix.

    PubMed

    Fisher, Brian F; Guo, Li; Dolinar, Brian S; Guzei, Ilia A; Gellman, Samuel H

    2015-05-27

    Structural characterization of new ?/?-peptide foldamers containing the cyclically constrained ?-amino acid I is described. Crystallographic and 2D NMR analysis shows that ? residue I promotes the formation of a 12/10-helical secondary structure in ?/?-peptides. This helix contains two different types of internal H-bond, and the data show that the 12-atom C?O(i) ? H-N(i+3) H-bond is more favorable than the 10-atom C?O(i) ? H-N(i-1) H-bond. Several foldamer helices featuring topologically distinct H-bonds have been discovered, but our findings are the first to show that such H-bonds may differ in their favorability. PMID:25974390

  9. The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy.

    PubMed

    Bechinger, B

    1999-12-15

    Linear peptide antibiotics have been isolated from amphibians, insects and humans and used as templates to design cheaper and more potent analogues for medical applications. Peptides such as cecropins or magainins are < or = 40 amino acids in length. Many of them have been prepared by solid-phase peptide synthesis with isotopic labels incorporated at selected sites. Structural analysis by solid-state NMR spectroscopy and other biophysical techniques indicates that these peptide antibiotics strongly interact with lipid membranes. In bilayer environments they exhibit amphipathic alpha-helical conformations and alignments of the helix axis parallel to the membrane surface. This contrasts the transmembrane orientations observed for alamethicin or gramicidin A. Models that have been proposed to explain the antibiotic and pore-forming activities of membrane-associated peptides, as well as other experimental results, include transmembrane helical bundles, wormholes, carpets, detergent-like effects or the in-plane diffusion of peptide-induced bilayer instabilities. PMID:10590307

  10. Evolution of vertebrate interferon inducible transmembrane proteins

    PubMed Central

    2012-01-01

    Background Interferon inducible transmembrane proteins (IFITMs) have diverse roles, including the control of cell proliferation, promotion of homotypic cell adhesion, protection against viral infection, promotion of bone matrix maturation and mineralisation, and mediating germ cell development. Most IFITMs have been well characterised in human and mouse but little published data exists for other animals. This study characterised IFITMs in two distantly related marsupial species, the Australian tammar wallaby and the South American grey short-tailed opossum, and analysed the phylogeny of the IFITM family in vertebrates. Results Five IFITM paralogues were identified in both the tammar and opossum. As in eutherians, most marsupial IFITM genes exist within a cluster, contain two exons and encode proteins with two transmembrane domains. Only two IFITM genes, IFITM5 and IFITM10, have orthologues in both marsupials and eutherians. IFITM5 arose in bony fish and IFITM10 in tetrapods. The bone-specific expression of IFITM5 appears to be restricted to therian mammals, suggesting that its specialised role in bone production is a recent adaptation specific to mammals. IFITM10 is the most highly conserved IFITM, sharing at least 85% amino acid identity between birds, reptiles and mammals and suggesting an important role for this presently uncharacterised protein. Conclusions Like eutherians, marsupials also have multiple IFITM genes that exist in a gene cluster. The differing expression patterns for many of the paralogues, together with poor sequence conservation between species, suggests that IFITM genes have acquired many different roles during vertebrate evolution. PMID:22537233

  11. The basic helix-loop-helix leucine zipper transcription factor Mitf is conserved in Drosophila and functions in eye development.

    PubMed Central

    Hallsson, Jón H; Haflidadóttir, Benedikta S; Stivers, Chad; Odenwald, Ward; Arnheiter, Heinz; Pignoni, Francesca; Steingrímsson, Eiríkur

    2004-01-01

    The MITF protein is a member of the MYC family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors and is most closely related to the TFE3, TFEC, and TFEB proteins. In the mouse, MITF is required for the development of several different cell types, including the retinal pigment epithelial (RPE) cells of the eye. In Mitf mutant mice, the presumptive RPE cells hyperproliferate, abnormally express the retinal transcriptional regulator Pax6, and form an ectopic neural retina. Here we report the structure of the Mitf gene in Drosophila and demonstrate expression during embryonic development and in the eye-antennal imaginal disc. In vitro, transcriptional regulation by Drosophila Mitf, like its mouse counterpart, is modified by the Eyeless (Drosophila Pax6) transcription factor. In vivo, targeted expression of wild-type or dominant-negative Drosophila Mitf results in developmental abnormalities reminiscent of Mitf function in mouse eye development. Our results suggest that the Mitf gene is the original member of the Mitf-Tfe subfamily of bHLH-Zip proteins and that its developmental function is at least partially conserved between vertebrates and invertebrates. These findings further support the common origin of the vertebrate and invertebrate eyes. PMID:15166150

  12. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

    PubMed

    Kang, Yanyong; Zhou, X Edward; Gao, Xiang; He, Yuanzheng; Liu, Wei; Ishchenko, Andrii; Barty, Anton; White, Thomas A; Yefanov, Oleksandr; Han, Gye Won; Xu, Qingping; de Waal, Parker W; Ke, Jiyuan; Tan, M H Eileen; Zhang, Chenghai; Moeller, Arne; West, Graham M; Pascal, Bruce D; Van Eps, Ned; Caro, Lydia N; Vishnivetskiy, Sergey A; Lee, Regina J; Suino-Powell, Kelly M; Gu, Xin; Pal, Kuntal; Ma, Jinming; Zhi, Xiaoyong; Boutet, Sébastien; Williams, Garth J; Messerschmidt, Marc; Gati, Cornelius; Zatsepin, Nadia A; Wang, Dingjie; James, Daniel; Basu, Shibom; Roy-Chowdhury, Shatabdi; Conrad, Chelsie E; Coe, Jesse; Liu, Haiguang; Lisova, Stella; Kupitz, Christopher; Grotjohann, Ingo; Fromme, Raimund; Jiang, Yi; Tan, Minjia; Yang, Huaiyu; Li, Jun; Wang, Meitian; Zheng, Zhong; Li, Dianfan; Howe, Nicole; Zhao, Yingming; Standfuss, Jörg; Diederichs, Kay; Dong, Yuhui; Potter, Clinton S; Carragher, Bridget; Caffrey, Martin; Jiang, Hualiang; Chapman, Henry N; Spence, John C H; Fromme, Petra; Weierstall, Uwe; Ernst, Oliver P; Katritch, Vsevolod; Gurevich, Vsevolod V; Griffin, Patrick R; Hubbell, Wayne L; Stevens, Raymond C; Cherezov, Vadim; Melcher, Karsten; Xu, H Eric

    2015-07-30

    G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. PMID:26200343

  13. Deletion mutations in N-terminal α1 helix render heat labile enterotoxin B subunit susceptible to degradation

    PubMed Central

    Alone, Pankaj V.; Malik, Gunjan; Krishnan, Anuja; Garg, Lalit C.

    2007-01-01

    Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is a heterohexameric protein consisting of an enzymatically active A subunit, LTA, and a carrier pentameric B subunit, LTB. It is clear from the crystal structure of LTB that the N-terminal α1 helix lies outside the core structure. However, the function of the N-terminal α1 helix of LTB is unknown. The present work was carried out to investigate the effect of site-directed mutagenesis of the α1 helix on LTB synthesis. Six amino acids (PQSITE) located at positions 2–7 from the N terminus, including 4 aa from the α1 helix, were deleted by site-directed mutagenesis. The deletion resulted in complete inhibition of LTB expression in E. coli when expressed along with its signal sequence. A single amino acid deletion within the α1 helix also resulted in loss of expression. However, a single amino acid deletion outside the α1 helix did not affect LTB synthesis. Mutant proteins, whose synthesis was not detected in vivo, could be successfully translated in vitro by using the coupled transcription–translation system. Immunoblot analysis, Northern blot analysis, and in vitro transcription–translation data collectively indicate that the lack of synthesis of the mutant proteins is caused by the immediate degradation of the expressed product by cellular proteases rather than by faulty translation of mutant LTB mRNA. Coexpression of the LTA could not rescue the degradation of LTB mutants. PMID:17911243

  14. The Structure of a Soluble Chemoreceptor Suggests a Mechanism for Propagating Conformational Signals

    SciTech Connect

    Pollard, Abiola M.; Bilwes, Alexandrine M.; Crane, Brian R.; Cornell

    2009-09-02

    Transmembrane chemoreceptors, also known as methyl-accepting chemotaxis proteins (MCPs), translate extracellular signals into intracellular responses in the bacterial chemotaxis system. MCP ligand binding domains control the activity of the CheA kinase, situated {approx}200 {angstrom} away, across the cytoplasmic membrane. The 2.17 {angstrom} resolution crystal structure of a Thermotoga maritima soluble receptor (Tm14) reveals distortions in its dimeric four-helix bundle that provide insight into the conformational states available to MCPs for propagating signals. A bulge in one helix generates asymmetry between subunits that displaces the kinase-interacting tip, which resides more than 100 {angstrom} away. The maximum bundle distortion maps to the adaptation region of transmembrane MCPs where reversible methylation of acidic residues tunes receptor activity. Minor alterations in coiled-coil packing geometry translate the bulge distortion to a >25 {angstrom} movement of the tip relative to the bundle stalks. The Tm14 structure discloses how alterations in local helical structure, which could be induced by changes in methylation state and/or by conformational signals from membrane proximal regions, can reposition a remote domain that interacts with the CheA kinase.

  15. An Accurate Model for Biomolecular Helices and Its Application to Helix Visualization

    PubMed Central

    Wang, Lincong; Qiao, Hui; Cao, Chen; Xu, Shutan; Zou, Shuxue

    2015-01-01

    Helices are the most abundant secondary structural elements in proteins and the structural forms assumed by double stranded DNAs (dsDNA). Though the mathematical expression for a helical curve is simple, none of the previous models for the biomolecular helices in either proteins or DNAs use a genuine helical curve, likely because of the complexity of fitting backbone atoms to helical curves. In this paper we model a helix as a series of different but all bona fide helical curves; each one best fits the coordinates of four consecutive backbone Cα atoms for a protein or P atoms for a DNA molecule. An implementation of the model demonstrates that it is more accurate than the previous ones for the description of the deviation of a helix from a standard helical curve. Furthermore, the accuracy of the model makes it possible to correlate deviations with structural and functional significance. When applied to helix visualization, the ribbon diagrams generated by the model are less choppy or have smaller side chain detachment than those by the previous visualization programs that typically model a helix as a series of low-degree splines. PMID:26126117

  16. Anchors Aweigh: Protein Traffic Mediated by Transmembrane Domains

    PubMed Central

    Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S.

    2013-01-01

    The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in the cytosolic domains, TMD sorting determinants are not conserved amino-acid sequences but physical properties such as length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport. PMID:23806646

  17. Predictive energy landscapes for folding α-helical transmembrane proteins

    PubMed Central

    Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.

    2014-01-01

    We explore the hypothesis that the folding landscapes of membrane proteins are funneled once the proteins’ topology within the membrane is established. We extend a protein folding model, the associative memory, water-mediated, structure, and energy model (AWSEM) by adding an implicit membrane potential and reoptimizing the force field to account for the differing nature of the interactions that stabilize proteins within lipid membranes, yielding a model that we call AWSEM-membrane. Once the protein topology is set in the membrane, hydrophobic attractions play a lesser role in finding the native structure, whereas polar–polar attractions are more important than for globular proteins. We examine both the quality of predictions made with AWSEM-membrane when accurate knowledge of the topology and secondary structure is available and the quality of predictions made without such knowledge, instead using bioinformatically inferred topology and secondary structure based on sequence alone. When no major errors are made by the bioinformatic methods used to assign the topology of the transmembrane helices, these two types of structure predictions yield roughly equivalent quality structures. Although the predictive energy landscape is transferable and not structure based, within the correct topological sector we find the landscape is indeed very funneled: Thermodynamic landscape analysis indicates that both the total potential energy and the contact energy decrease as native contacts are formed. Nevertheless the near symmetry of different helical packings with respect to native contact formation can result in multiple packings with nearly equal thermodynamic occupancy, especially at temperatures just below collapse. PMID:25030446

  18. Functional relevance of transmembrane domains in membrane fusion.

    PubMed

    Nikolaus, Jörg; Herrmann, Andreas

    2012-11-01

    Membrane fusion is ubiquitous in life. Fusion of biological membranes is mediated by specialized fusion proteins anchored to the bilayers destined to fuse. Here we describe these proteins as being instrumental in viral, intracellular and developmental fusion. Next, we review experimental and theoretical evidence that points to fusion in the different systems as following a common 'fusion through hemifusion' pathway. We also focus on the structure and dynamics of the transmembrane segment that anchors the fusion proteins to the bilayer, and its role in driving fusion. In particular, we highlight the influence of this single segment on the surrounding membrane lipids and on the overall shape of the membrane along the way to fusion. PMID:23109541

  19. Optimal bundling of transmembrane helices using sparse distance constraints

    PubMed Central

    Sale, Ken; Faulon, Jean-Loup; Gray, Genetha A.; Schoeniger, Joseph S.; Young, Malin M.

    2004-01-01

    We present a two-step approach to modeling the transmembrane spanning helical bundles of integral membrane proteins using only sparse distance constraints, such as those derived from chemical cross-linking, dipolar EPR and FRET experiments. In Step 1, using an algorithm, we developed, the conformational space of membrane protein folds matching a set of distance constraints is explored to provide initial structures for local conformational searches. In Step 2, these structures refined against a custom penalty function that incorporates both measures derived from statistical analysis of solved membrane protein structures and distance constraints obtained from experiments. We begin by describing the statistical analysis of the solved membrane protein structures from which the theoretical portion of the penalty function was derived. We then describe the penalty function, and, using a set of six test cases, demonstrate that it is capable of distinguishing helical bundles that are close to the native bundle from those that are far from the native bundle. Finally, using a set of only 27 distance constraints extracted from the literature, we show that our method successfully recovers the structure of dark-adapted rhodopsin to within 3.2 Å of the crystal structure. PMID:15340162

  20. Insulin Receptor Activation with Transmembrane Domain Ligands*

    PubMed Central

    Lee, Jongsoon; Miyazaki, Masaya; Romeo, Giulio R.; Shoelson, Steven E.

    2014-01-01

    Complementary surfaces are buried when peptide hormones, growth factors, or cytokines bind and activate cellular receptors. Although these extended surfaces provide high affinity and specificity to the interactions, they also present great challenges to the design of small molecules that might either mimic or antagonize the process. We show that the insulin receptor (IR) and downstream signals can be activated by targeting a site outside of its ligand-binding domain. A 24-residue peptide having the IR transmembrane (TM) domain sequence activates IR, but not related growth factor receptors, through specific interactions with the receptor TM domain. Like insulin-dependent activation, IR-TM requires that IR have a competent ATP-binding site and kinase activation loop. IR-TM also activates mutated receptors from patients with severe insulin resistance, which do not respond to insulin. These results show that IR can be activated through a pathway that bypasses its canonical ligand-binding domain. PMID:24867955

  1. Transmembrane topology of the mammalian KDEL receptor.

    PubMed Central

    Singh, P; Tang, B L; Wong, S H; Hong, W

    1993-01-01

    The mammalian KDEL receptor is an integral membrane protein with seven hydrophobic regions. Fusion proteins comprising a 37-kDa N-glycosylation reporter fused downstream of amino-terminal fragments of the KDEL receptor with varying numbers of hydrophobic regions were synthesized in an in vitro translation system containing canine pancreatic microsomes. The luminal or cytosolic orientation of the reporter, and hence of the hydrophilic region to which it is fused, was inferred from the presence or absence of glycosylation, which occurs only in the lumen of the microsomes. The cytosolic orientation of the N and C termini was also confirmed immunocytochemically. Our results suggest that the KDEL receptor is inserted into the membrane with only six transmembrane domains and that both the amino and carboxy termini are located in the cytoplasm. Images PMID:8413242

  2. Cystic fibrosis transmembrane regulator correctors and potentiators.

    PubMed

    Rowe, Steven M; Verkman, Alan S

    2013-07-01

    Cystic fibrosis (CF) is caused by loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) protein, a cAMP-regulated anion channel expressed primarily at the apical plasma membrane of secretory epithelia. Nearly 2000 mutations in the CFTR gene have been identified that cause disease by impairing its translation, cellular processing, and/or chloride channel gating. The fundamental premise of CFTR corrector and potentiator therapy for CF is that addressing the underlying defects in the cellular processing and chloride channel function of CF-causing mutant CFTR alleles will result in clinical benefit by addressing the basic defect underlying CF. Correctors are principally targeted at F508del cellular misprocessing, whereas potentiators are intended to restore cAMP-dependent chloride channel activity to mutant CFTRs at the cell surface. This article reviews the discovery of CFTR potentiators and correctors, what is known regarding their mechanistic basis, and encouraging results achieved in clinical testing. PMID:23818513

  3. Cystic Fibrosis Transmembrane Regulator Correctors and Potentiators

    PubMed Central

    Rowe, Steven M.; Verkman, Alan S.

    2013-01-01

    Cystic fibrosis (CF) is caused by loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) protein, a cAMP-regulated anion channel expressed primarily at the apical plasma membrane of secretory epithelia. Nearly 2000 mutations in the CFTR gene have been identified that cause disease by impairing its translation, cellular processing, and/or chloride channel gating. The fundamental premise of CFTR corrector and potentiator therapy for CF is that addressing the underlying defects in the cellular processing and chloride channel function of CF-causing mutant CFTR alleles will result in clinical benefit by addressing the basic defect underlying CF. Correctors are principally targeted at F508del cellular misprocessing, whereas potentiators are intended to restore cAMP-dependent chloride channel activity to mutant CFTRs at the cell surface. This article reviews the discovery of CFTR potentiators and correctors, what is known regarding their mechanistic basis, and encouraging results achieved in clinical testing. PMID:23818513

  4. Mechanical performance of PPy helix tube microactuator

    NASA Astrophysics Data System (ADS)

    Bahrami Samani, Mehrdad; Spinks, Geoffrey M.; Cook, Christopher

    2004-02-01

    Conducting polymer actuators with favourable properties such as linearity, high power density and compliance are of increasing demand in micro applications. These materials generate forces over two times larger than produced by mammalian skeletal muscles. They operate to convert electro chemical energy to mechanical stress and strain. On the other hand, the application of conducting polymers is limited by the lack of a full description of the relation between four essential parameters: stress, strain, voltage and current. In this paper, polypyrrole helix tube micro actuator mechanical characteristics are investigated. The electrolyte is propylene carbonate and the dopant is TBA. PF6. The experiments are both in isotonic and isometric conditions and the input parameters are both electrical and mechanical. A dual mode force and length control and potentiostat / galvanostat are utilized for this purpose. Ultimately, the viscoelastic behaviour of the actuator is presented in this paper by a standard stress relaxation test. The effect of electrical stimulus on mechanical parameters is also explored by cyclic voltametry at different scan rates to obtain the best understanding of the actuation mechanism. The results demonstrate that the linear viscoelastic model, which performed well on conducting polymer film actuators, has to be modified to explain the mechanical behaviour of PPy helix tube fibre micro actuators. Secondly, the changes in mechanical properties of PPy need to be considered when modelling electromechanical behaviour.

  5. Orbital dynamics in the Tevatron double helix

    SciTech Connect

    Michelotti, L.; Saritepe, S.

    1989-03-01

    A key feature of the Tevatron upgrade is the placement of proton and anti-proton bunches on the branches of a double helix which winds around the current closed orbit. Electrostatic separators will transfer the bunches on and off the double helix so that they experience head-on collisions only at the experimental areas, BO and DO, all other encounters occurring at large transverse separation. In this way the number of bunches, and the luminosity, can be increased without a proportional growth in the beam-beam tune shift. The scenario raises a number of beam dynamics issues, especially the consequences of sampling magnetic fields far from the magnets' center lines, and the effects of the long-range beam-beam interaction. This report presents the results of (admittedly incomplete) calculations and simulations done to date to explore: a Fermilab team have been studying, both experimentally and theoretically, but we shall not review those efforts here. The constraint of a page limit has forced us to bound this discussion rather stringently, but a more complete paper will be available as a Fermilab Technical Memo. 7 refs., 7 figs.

  6. Molecular mechanisms for generating transmembrane proton gradients

    PubMed Central

    Gunner, M.R.; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

    2013-01-01

    Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side. PMID:23507617

  7. Specificity of Transmembrane Protein Palmitoylation in Yeast

    PubMed Central

    González Montoro, Ayelén; Chumpen Ramirez, Sabrina; Quiroga, Rodrigo; Valdez Taubas, Javier

    2011-01-01

    Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs), characterized by the presence of a conserved 50- aminoacids domain called “Asp-His-His-Cys- Cysteine Rich Domain” (DHHC-CRD). There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether. Here we investigate the specificity of transmembrane protein palmitoylation in S. cerevisiae, which is carried out predominantly by two PATs, Swf1 and Pfa4. We show that palmitoylation of transmembrane substrates requires dedicated PATs, since other yeast PATs are mostly unable to perform Swf1 or Pfa4 functions, even when overexpressed. Furthermore, we find that Swf1 is highly specific for its substrates, as it is unable to substitute for other PATs. To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs) and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively. These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity. Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as expected for a highly specific enzymatic reaction. PMID:21383992

  8. Nonequilibrium dynamics of helix reorganization observed by transient 2D IR spectroscopy

    PubMed Central

    Tucker, Matthew J.; Abdo, Mohannad; Courter, Joel R.; Chen, Jianxin; Brown, Stephen P.; Smith, Amos B.; Hochstrasser, Robin M.

    2013-01-01

    The relaxation of helical structures very close to equilibrium is observed via transient 2D IR spectroscopy. An initial distribution of synthetically distorted helices having an unnatural bridge linking the 10th and 12th residues of an alanine-rich α-helix is released to evolve into the equilibrium distribution of α-helix conformations. The bridge constrains the structure to be sli