Science.gov

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. Structural Transitions of Transmembrane Helix 6 in the Formation of Metarhodopsin I

    PubMed Central

    Eilers, Markus; Goncalves, Joseph A.; Ahuja, Shivani; Kirkup, Colleen; Hirshfeld, Amiram; Simmerling, Carlos; Reeves, Philip J.; Sheves, Mordechai; Smith, Steven O.

    2012-01-01

    Absorption of light by the visual pigment rhodopsin triggers a rapid cis-trans photoisomerization of its retinal chromophore and a series of conformational changes in both the retinal and protein. The largest structural change is an outward tilt of transmembrane helix H6 that increases the separation of the intracellular ends of H6 and H3, and opens up the G-protein binding site. In the dark state of rhodopsin, Glu247 at the intracellular end of H6 forms a salt bridge with Arg135 on H3 to tether H6 in an inactive conformation. The Arg135-Glu247 interaction is broken in the active state of the receptor, and Arg135 is then stabilized by interactions with Tyr223, Met257 and Tyr306 on helices H5, H6 and H7, respectively. To address the mechanism of H6 motion, solid-state NMR measurements are undertaken of Metarhodopsin I (Meta I), the intermediate preceding the active Metarhodopsin II (Meta II) state of the receptor. 13C NMR dipolar recoupling measurements reveal an interhelical contact of 13Cζ-Arg135 with 13Cε-Met257 in Meta I, but not with 13Cζ-Tyr223 or 13Cζ-Tyr306. These observations suggest that helix H6 has rotated in the formation of Meta I, but that structural changes involving helices H5 and H7 have not yet occurred. Together, our results provide insights into the sequence of events leading up to the outward motion of H6, a hallmark of G protein-coupled receptor activation. PMID:22564141

  3. NMR-based approach to measure the free energy of transmembrane helix-helix interactions.

    PubMed

    Mineev, Konstantin S; Lesovoy, Dmitry M; Usmanova, Dinara R; Goncharuk, Sergey A; Shulepko, Mikhail A; Lyukmanova, Ekaterina N; Kirpichnikov, Mikhail P; Bocharov, Eduard V; Arseniev, Alexander S

    2014-01-01

    Knowledge of the energetic parameters of transmembrane helix-helix interactions is necessary for the establishment of a structure-energy relationship for α-helical membrane domains. A number of techniques have been developed to measure the free energies of dimerization and oligomerization of transmembrane α-helices, and all of these have their advantages and drawbacks. In this study we propose a methodology to determine the magnitudes of the free energy of interactions between transmembrane helices in detergent micelles. The suggested approach employs solution nuclear magnetic resonance (NMR) spectroscopy to determine the population of the oligomeric states of the transmembrane domains and introduces a new formalism to describe the oligomerization equilibrium, which is based on the assumption that both the dimerization of the transmembrane domains and the dissociation of the dimer can occur only upon the collision of detergent micelles. The technique has three major advantages compared with other existing approaches: it may be used to analyze both weak and relatively strong dimerization/oligomerization processes, it works well for the analysis of complex equilibria, e.g. when monomer, dimer and high-order oligomer populations are simultaneously present in the solution, and it can simultaneously yield both structural and energetic characteristics of the helix-helix interaction under study. The proposed methodology was applied to investigate the oligomerization process of transmembrane domains of fibroblast growth factor receptor 3 (FGFR3) and vascular endothelium growth factor receptor 2 (VEGFR2), and allowed the measurement of the free energy of dimerization of both of these objects. In addition the proposed method was able to describe the multi-state oligomerization process of the VEGFR2 transmembrane domain. PMID:24036227

  4. 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

  5. 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

  6. Assignment of oriented sample NMR resonances from a three transmembrane helix protein.

    PubMed

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

    2014-03-01

    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 orientations 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

  7. Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2.

    PubMed

    Volz, Pierre; Krause, Nils; Balke, Jens; Schneider, Constantin; Walter, Maria; Schneider, Franziska; Schlesinger, Ramona; Alexiev, Ulrike

    2016-08-12

    A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics. PMID:27268055

  8. 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

  9. Site-Directed Mutations and the Polymorphic Variant Ala160Thr in the Human Thromboxane Receptor Uncover a Structural Role for Transmembrane Helix 4

    PubMed Central

    Chakraborty, Raja; Pydi, Sai Prasad; Gleim, Scott; Dakshinamurti, Shyamala; Hwa, John; Chelikani, Prashen

    2012-01-01

    The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala1604.53 is highly conserved as is Gly1644.57. Here we target Ala1604.53 and Gly1644.57 in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala1604.53 and Gly1644.57 in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions. PMID:22272267

  10. Site-directed mutations and the polymorphic variant Ala160Thr in the human thromboxane receptor uncover a structural role for transmembrane helix 4.

    PubMed

    Chakraborty, Raja; Pydi, Sai Prasad; Gleim, Scott; Dakshinamurti, Shyamala; Hwa, John; Chelikani, Prashen

    2012-01-01

    The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala160(4.53) is highly conserved as is Gly164(4.57). Here we target Ala160(4.53) and Gly164(4.57) in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala160(4.53) and Gly164(4.57) in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions. PMID:22272267

  11. Terminal residue hydrophobicity modulates transmembrane helix-helix interactions.

    PubMed

    Ng, Derek P; Deber, Charles M

    2014-06-17

    Central to the formation of tertiary structure in membrane protein folding is the presence of amino acid sequence motifs (such as "small-XXX-small" segments) in the TM segments that promote interaction-compatible surfaces through which the TM α-helices interact. Here, we sought to elucidate additional factors that may work in tandem to dictate the ultimate interaction fate of TM-embedded segments. In this context, we used proteolipid protein (PLP), the major protein from central nervous system myelin for which mutant-dependent non-native oligomerization has been implicated in neurological disorders, to explore the specific effects of TM boundary residues (the membrane entry and exit points), keying on the secondary structure and self-association of peptides corresponding to the PLP TM2 α-helix (wild-type sequence ⁶⁶AFQYVIYGTASFFFLYGALLLAEGF⁹⁰). Using gel electrophoresis, circular dichroism, and Förster resonance energy transfer in the membrane-mimetic detergent sodium dodecyl sulfate (SDS), we found that mutation of F90 to residues such as A, I, L, or V maintains the onset of TM2-TM2 dimerization, whereas mutation to E, G, Q, N, S, or T abrogates dimer formation. We attribute this sensitivity to changes in local hydrophobicity, viz., a decrease in hydrophobicity reduces local lipid-peptide interactions, which in turn disrupts peptide α-helicity and hence the effectiveness of an incipient interaction-compatible surface. Our results show that the secondary structure and oligomeric state of PLP TM2 Lys-tagged peptides are significantly modulated by the specific nature of their C-terminal boundary residue, thus providing insight as to how point mutations, particularly where they produce disease states, can compromise the folding process. PMID:24857611

  12. 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

  13. 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

  14. 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

  15. Detergent Properties Influence the Stability of the Glycophorin A Transmembrane Helix Dimer in Lysophosphatidylcholine Micelles

    PubMed Central

    Stangl, Michael; Veerappan, Anbazhagan; Kroeger, Anja; Vogel, Peter; Schneider, Dirk

    2012-01-01

    Detergents might affect membrane protein structures by promoting intramolecular interactions that are different from those found in native membrane bilayers, and fine-tuning detergent properties can be crucial for obtaining structural information of intact and functional transmembrane proteins. To systematically investigate the influence of the detergent concentration and acyl-chain length on the stability of a transmembrane protein structure, the stability of the human glycophorin A transmembrane helix dimer has been analyzed in lyso-phosphatidylcholine micelles of different acyl-chain length. While our results indicate that the transmembrane protein is destabilized in detergents with increasing chain-length, the diameter of the hydrophobic micelle core was found to be less crucial. Thus, hydrophobic mismatch appears to be less important in detergent micelles than in lipid bilayers and individual detergent molecules appear to be able to stretch within a micelle to match the hydrophobic thickness of the peptide. However, the stability of the GpA TM helix dimer linearly depends on the aggregation number of the lyso-PC detergents, indicating that not only is the chemistry of the detergent headgroup and acyl-chain region central for classifying a detergent as harsh or mild, but the detergent aggregation number might also be important. PMID:23260047

  16. 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

  17. 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

  18. Modulating Transmembrane α-Helix Interactions through pH-Sensitive Boundary Residues.

    PubMed

    Ng, Derek P; Deber, Charles M

    2016-08-01

    Changes in pH can alter the structure and activity of proteins and may be used by the cell to control molecular function. This coupling can also be used in non-native applications through the design of pH-sensitive biomolecules. For example, the pH (low) insertion peptide (pHLIP) can spontaneously insert into a lipid bilayer when the pH decreases. We have previously shown that the α-helicity and helix-helix interactions of the TM2 α-helix of the proteolipid protein (PLP) are sensitive to the local hydrophobicity at its C-terminus. Given that there is an ionizable residue (Glu-88) at the C-terminus of this transmembrane (TM) segment, we hypothesized that changing the ionization state of this residue through pH may alter the local hydrophobicity of the peptide enough to affect both its secondary structure and helix-helix interactions. To examine this phenomenon, we synthesized peptide analogues of the PLP TM2 α-helix (wild-type sequence (66)AFQYVIYGTASFFFLYGALLLAEGF(90)). Using circular dichroism and Förster resonance energy transfer in the membrane-mimetic detergent sodium dodecyl sulfate, we found that a decrease in pH increases both peptide α-helicity and the extent of self-association. This pH-dependent effect is due specifically to the presence of Glu-88 at the C-terminus. Additional experiments in which Phe-90 was mutated to residues of varying hydrophobicities indicated that the strength of this effect is dependent on the local hydrophobicity near Glu-88. Our results have implications for the design of TM peptide switches and improve our understanding of how membrane protein structure and activity can be regulated through local molecular environmental changes. PMID:27434090

  19. 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

  20. 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…

  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. 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

  3. 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

  4. Left-Handed Dimer of EphA2 Transmembrane Domain: Helix Packing Diversity among Receptor Tyrosine Kinases

    PubMed Central

    Bocharov, Eduard V.; Mayzel, Maxim L.; Volynsky, Pavel E.; Mineev, Konstantin S.; Tkach, Elena N.; Ermolyuk, Yaroslav S.; Schulga, Alexey A.; Efremov, Roman G.; Arseniev, Alexander S.

    2010-01-01

    Abstract The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands control a diverse array of cell-cell interactions in the developing and adult organisms. During signal transduction across plasma membrane, Eph receptors, like other receptor tyrosine kinases, are involved in lateral dimerization and subsequent oligomerization presumably with proper assembly of their single-span transmembrane domains. Spatial structure of dimeric transmembrane domain of EphA2 receptor embedded into lipid bicelle was obtained by solution NMR, showing a left-handed parallel packing of the transmembrane helices (535–559)2. The helices interact through the extended heptad repeat motif L535X3G539X2A542X3V546X2L549 assisted by intermolecular stacking interactions of aromatic rings of (FF557)2, whereas the characteristic tandem GG4-like motif A536X3G540X3G544 is not used, enabling another mode of helix-helix association. Importantly, a similar motif AX3GX3G as was found is responsible for right-handed dimerization of transmembrane domain of the EphA1 receptor. These findings serve as an instructive example of the diversity of transmembrane domain formation within the same family of protein kinases and seem to favor the assumption that the so-called rotation-coupled activation mechanism may take place during the Eph receptor signaling. A possible role of membrane lipid rafts in relation to Eph transmembrane domain oligomerization and Eph signal transduction was also discussed. PMID:20197042

  5. Measuring Transmembrane Helix Interaction Strengths in Lipid Bilayers Using Steric Trapping

    PubMed Central

    Hong, Heedeok; Chang, Yu-Chu; Bowie, James U.

    2016-01-01

    We have developed a method to measure strong transmembrane (TM) helix interaction affinities in lipid bilayers that are difficult to measure by traditional dilution methods. The method, called steric trapping, couples dissociation of biotinylated TM helices to a competitive binding by monovalent streptavidin (mSA), so that dissociation is driven by the affinity of mSA for biotin and mSA concentration. By adjusting the binding affinity of mSA through mutation, the method can obtain dissociation constants of TM helix dimers (Kd,dimer) over a range of six orders of magnitudes. The Kd,dimer limit of measurable target interaction is extended 3–4 orders of magnitude lower than possible by dilution methods. Thus, steric trapping opens up new opportunities to study the folding and assembly of α-helical membrane proteins in lipid bilayer environments. Here we provide detailed methods for applying steric trapping to a TM helix dimer. PMID:23975771

  6. 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

  7. 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

  8. 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.

  9. Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry

    PubMed Central

    Trujillo, Carolina; Taylor-Parker, Julian; Harrison, Robert; Murphy, John R.

    2014-01-01

    The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol. PMID:20398220

  10. Structural and dynamic study of the transmembrane domain of the amyloid precursor protein.

    PubMed

    Nadezhdin, K D; Bocharova, O V; Bocharov, E V; Arseniev, A S

    2011-01-01

    Alzheimer's disease affects people all over the world, regardless of nationality, gender or social status. An adequate study of the disease requires essential understanding of the molecular fundamentals of the pathogenesis. The amyloid β-peptide, which forms amyloid plaques in the brain of people with Alzheimer's disease, is the product of sequential cleavage of a single-span membrane amyloid precursor protein (APP). More than half of the APP mutations found to be associated with familial forms of Alzheimer's disease are located in its transmembrane domain. The pathogenic mutations presumably affect the structural-dynamic properties of the APP transmembrane domain by changing its conformational stability and/or lateral dimerization. In the present study, the structure and dynamics of the recombinant peptide corresponding to the APP fragment, Gln686-Lys726, which comprises the APP transmembrane domain with an adjacent N-terminal juxtamembrane sequence, were determined in the membrane mimetic environment composed of detergent micelles using NMR spectroscopy. The structure obtained in dodecylphosphocholine micelles consists of two α-helices: a short surface-associated juxtamembrane helix (Lys687-Asp694) and a long transmembrane helix (Gly700-Leu723), both connected via a mobile loop region. A minor bend of the transmembrane α-helix is observed near the paired residues Gly708-Gly709. A cholesterol-binding hydrophobic cavity is apparently formed under the loop region, where the juxtamembrane α-helix comes into contact with the membrane surface near the N-terminus of the transmembrane α-helix. PMID:22649674

  11. Structural Basis of p75 Transmembrane Domain Dimerization.

    PubMed

    Nadezhdin, Kirill D; García-Carpio, Irmina; Goncharuk, Sergey A; Mineev, Konstantin S; Arseniev, Alexander S; Vilar, Marçal

    2016-06-01

    Dimerization of single span transmembrane receptors underlies their mechanism of activation. p75 neurotrophin receptor plays an important role in the nervous system, but the understanding of p75 activation mechanism is still incomplete. The transmembrane (TM) domain of p75 stabilizes the receptor dimers through a disulfide bond, essential for the NGF signaling. Here we solved by NMR the three-dimensional structure of the p75-TM-WT and the functionally inactive p75-TM-C257A dimers. Upon reconstitution in lipid micelles, p75-TM-WT forms the disulfide-linked dimers spontaneously. Under reducing conditions, p75-TM-WT is in a monomer-dimer equilibrium with the Cys(257) residue located on the dimer interface. In contrast, p75-TM-C257A forms dimers through the AXXXG motif on the opposite face of the α-helix. Biochemical and cross-linking experiments indicate that AXXXG motif is not on the dimer interface of p75-TM-WT, suggesting that the conformation of p75-TM-C257A may be not functionally relevant. However, rather than mediating p75 homodimerization, mutagenesis of the AXXXG motif reveals its functional role in the regulated intramembrane proteolysis of p75 catalyzed by the γ-secretase complex. Our structural data provide an insight into the key role of the Cys(257) in stabilization of the weak transmembrane dimer in a conformation required for the NGF signaling. PMID:27056327

  12. The Gramicidin A Transmembrane Channel: A Proposed π(L,D) Helix

    PubMed Central

    Urry, D. W.

    1971-01-01

    A lipophilic, left-handed helical structure is proposed for gramicidin A in which the C-O bonds alternately point toward the amino and carboxyl ends; it is a hybrid of the 4.314 and 4.416 helices. The C-O groups pointing toward the carboxyl end form part of 16-membered hydrogen-bonded rings, whereas the C-O moieties pointing toward the amino end form 14-membered hydrogenbonded rings. The proposed structure is based on conformational analysis combined with requirements for the gramicidin A transmembrane channel. Two helices combine to form the channel. The alternating C-O directions allow hydrogen-bonded dimerization by the unique possibilities of head-to-head and tail-to-tail attachment. The formyl group at the amino end allows for a favorable head-to-head attachment with no loss of structural continuity. Unpublished studies. by M. C. Goodall on the lipid bilayer conductance of deformyl gramicidin A strongly argue for head-to-head attachment. Such hydrogen-bonded association is not possible with previously described helices, as the C-O groups all point in the same direction. In relation to possible π(L,D) helices in mammalian systems, it should be noted that glycines would fill the role of D residues. The conformation can undergo ion-induced relaxations, which provide approximate tetrahedral coordination for the ion, with facile shifting of coordinations. The ready exchange of coordinations provides the mechanism for movement of the ion along the channel. Conceivably, such transmembrane channels could have application as models for ion transport across biological membranes—an application which may be as great as, or greater than, that of carriers such as valinomycin and nonactin. Specifically, biogenic amines and drugs containing aromatic groups could control access to the channel by interactions with the two tryptophan residues at the ethanolamine end and with the negative region provided by the three oxygens. Images PMID:5276779

  13. Progesterone modulation of transmembrane helix-helix interactions between the α-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane

    PubMed Central

    2010-01-01

    Background Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the α-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the α1-subunit. Results Preparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020) successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT) and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME), can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the α1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT. Conclusion We propose that progesterone binding to the first external loop of the α1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions. PMID:20500835

  14. A conserved motif in transmembrane helix 1 of diphtheria toxin mediates catalytic domain delivery to the cytosol

    PubMed Central

    Ratts, Ryan; Trujillo, Carolina; Bharti, Ajit; vanderSpek, Johanna; Harrison, Robert; Murphy, John R.

    2005-01-01

    A 10-aa motif in transmembrane helix 1 of diphtheria toxin that is conserved in anthrax edema factor, anthrax lethal factor, and botulinum neurotoxin serotypes A, C, and D was identified by blast, clustal w, and meme computational analysis. Using the diphtheria toxin-related fusion protein toxin DAB389IL-2, we demonstrate that introduction of the L221E mutation into a highly conserved residue within this motif results in a nontoxic catalytic domain translocation deficient phenotype. To further probe the function of this motif in the process by which the catalytic domain is delivered from the lumen of early endosomes to the cytosol, we constructed a gene encoding a portion of diphtheria toxin transmembrane helix 1, T1, which carries the motif and is expressed from a CMV promoter. We then isolated stable transfectants of Hut102/6TG cells that express the T1 peptide, Hut102/6TG-T1. In contrast to the parental cell line, Hut102/6TG-T1 cells are ca. 104-fold more resistant to the fusion protein toxin. This resistance is completely reversed by coexpression of small interfering RNA directed against the gene encoding the T1 peptide in Hut102/6TG-T1 cells. We further demonstrate by GST-DT140-271 pull-down experiments in the presence and absence of synthetic T1 peptides the specific binding of coatomer protein complex subunit β to this region of the diphtheria toxin transmembrane domain. PMID:16230620

  15. 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

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

    PubMed Central

    Holt, Andrea; Rougier, Léa; Réat, Valérie; 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 to any transmembrane helical peptide. The influence of the experimental data set on the solution space is discussed, as are potential sources of error. PMID:20441750

  17. Notch Transmembrane Domain: Secondary Structure and Topology

    PubMed Central

    2016-01-01

    The Notch signaling pathway is critical in development, neuronal maintenance, and hematopoiesis. An obligate step in the activation of this pathway is cleavage of its transmembrane (TM) domain by γ-secretase. While the soluble domains have been extensively studied, little has been done to characterize its TM and flanking juxtamembrane (JM) segments. Here, we present the results of nuclear magnetic resonance (NMR) studies of the human Notch1 TM/JM domain. The TM domain is largely α-helical. While the flanking JM segments do not adopt regular secondary structure, they interact with the membrane surface, suggesting membrane interactions may play a role in modulating its cleavage by γ-secretase and subsequent NOTCH signaling function. PMID:26023825

  18. Lanthanide-binding helix-turn-helix peptides: Solution structure of a designed metallonuclease

    PubMed Central

    Welch, Joel T.; Kearney, William R.; Franklin, Sonya J.

    2003-01-01

    A designed lanthanide-binding chimeric peptide based on the strikingly similar geometries of the EF-hand and helix-turn-helix (HTH) motifs was investigated by NMR and CD spectroscopy and found to retain the same overall solution structure of the parental motifs. CD spectroscopy showed that the 33-mer peptide P3W folds on binding lanthanides, with an increase in α-helicity from 20% in the absence of metal to 38% and 35% in the presence of excess Eu(III) and La(III) ions, respectively. The conditional binding affinities of P3W for La(III) (5.9 ± 0.3 μM) and for Eu(III) (6.2 ± 0.3 μM) (pH 7.8, 5 mM Tris) were determined by tryptophan fluorescence titration. The La(III) complex of peptide P3, which differs from P3W by only one Trp-to-His substitution, has much less signal dispersion in the proton NMR spectra than LaP3W, indicating that the Trp residue is a critical hydrophobic anchor for maintaining a well-folded helix-turn-helix structure. A chemical-shift index analysis indicates the metallopeptide has a helix-loop-helix secondary structure. A structure calculated by using nuclear Overhauser effect and other NMR constraints reveals that P3W not only has a tightly folded metal-binding loop but also retains the α−α corner supersecondary structure of the parental motifs. Although the solution structure is undefined at both the N and C termini, the NMR structure confirms the successful incorporation of a metal-binding loop into a HTH sequence. PMID:12644701

  19. Interdomain communication in calcium pump as revealed in the crystal structures with transmembrane inhibitors

    PubMed Central

    Takahashi, Mihoko; Kondou, Youhei; Toyoshima, Chikashi

    2007-01-01

    Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum is an ATP-driven Ca2+ pump consisting of three cytoplasmic domains and 10 transmembrane helices. In the absence of Ca2+, the three cytoplasmic domains gather to form a compact headpiece, but the ATPase is unstable without an inhibitor. Here we describe the crystal structures of Ca2+-ATPase in the absence of Ca2+ stabilized with cyclopiazonic acid alone and in combination with other inhibitors. Cyclopiazonic acid is located in the transmembrane region of the protein near the cytoplasmic surface. The binding site partially overlaps with that of 2,5-di-tert-butyl-1,4-dihydroxybenzene but is separate from that of thapsigargin. The overall structure is significantly different from that stabilized with thapsigargin: The cytoplasmic headpiece is more upright, and the transmembrane helices M1–M4 are rearranged. Cyclopiazonic acid primarily alters the position of the M1′ helix and thereby M2 and M4 and then M5. Because M5 is integrated into the phosphorylation domain, the whole cytoplasmic headpiece moves. These structural changes show how an event in the transmembrane domain can be transmitted to the cytoplasmic domain despite flexible links between them. They also reveal that Ca2+-ATPase has considerable plasticity even when fixed by a transmembrane inhibitor, presumably to accommodate thermal fluctuations. PMID:17389383

  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. 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

  2. Predictions of Tertiary Structures of α-Helical Membrane Proteins by Replica-Exchange Method with Consideration of Helix Deformations

    NASA Astrophysics Data System (ADS)

    Urano, Ryo; Kokubo, Hironori; Okamoto, Yuko

    2015-08-01

    We propose an improved prediction method of the tertiary structures of α-helical membrane proteins based on the replica-exchange method by taking into account helix deformations. Our method has wide applications because transmembrane helices of native membrane proteins are often distorted. In order to test the effectiveness of the present method, we applied it to the structure predictions of glycophorin A and phospholamban. The results were in good agreement with experiments.

  3. All-Atom Structural Models of the Transmembrane Domains of Insulin and Type 1 Insulin-Like Growth Factor Receptors

    PubMed Central

    Mohammadiarani, Hossein; Vashisth, Harish

    2016-01-01

    The receptor tyrosine kinase superfamily comprises many cell-surface receptors including the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) that are constitutively homodimeric transmembrane glycoproteins. Therefore, these receptors require ligand-triggered domain rearrangements rather than receptor dimerization for activation. Specifically, binding of peptide ligands to receptor ectodomains transduces signals across the transmembrane domains for trans-autophosphorylation in cytoplasmic kinase domains. The molecular details of these processes are poorly understood in part due to the absence of structures of full-length receptors. Using MD simulations and enhanced conformational sampling algorithms, we present all-atom structural models of peptides containing 51 residues from the transmembrane and juxtamembrane regions of IR and IGF1R. In our models, the transmembrane regions of both receptors adopt helical conformations with kinks at Pro961 (IR) and Pro941 (IGF1R), but the C-terminal residues corresponding to the juxtamembrane region of each receptor adopt unfolded and flexible conformations in IR as opposed to a helix in IGF1R. We also observe that the N-terminal residues in IR form a kinked-helix sitting at the membrane–solvent interface, while homologous residues in IGF1R are unfolded and flexible. These conformational differences result in a larger tilt-angle of the membrane-embedded helix in IGF1R in comparison to IR to compensate for interactions with water molecules at the membrane–solvent interfaces. Our metastable/stable states for the transmembrane domain of IR, observed in a lipid bilayer, are consistent with a known NMR structure of this domain determined in detergent micelles, and similar states in IGF1R are consistent with a previously reported model of the dimerized transmembrane domains of IGF1R. Our all-atom structural models suggest potentially unique structural organization of kinase domains in each receptor. PMID

  4. 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

  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. The accessory helix of complexin functions by stabilizing central helix secondary structure

    PubMed Central

    Radoff, Daniel T; Dong, Yongming; Snead, David; Bai, Jihong; Eliezer, David; Dittman, Jeremy S

    2014-01-01

    The presynaptic protein complexin (CPX) is a critical regulator of synaptic vesicle fusion, but the mechanisms underlying its regulatory effects are not well understood. Its highly conserved central helix (CH) directly binds the ternary SNARE complex and is required for all known CPX functions. The adjacent accessory helix (AH) is not conserved despite also playing an important role in CPX function, and numerous models for its mechanism have been proposed. We examined the impact of AH mutations and chimeras on CPX function in vivo and in vitro using C. elegans. The mouse AH fully restored function when substituted into worm CPX suggesting its mechanism is evolutionarily conserved. CPX inhibitory function was impaired when helix propagation into the CH was disrupted whereas replacing the AH with a non-native helical sequence restored CPX function. We propose that the AH operates by stabilizing CH secondary structure rather than through protein or lipid interactions. DOI: http://dx.doi.org/10.7554/eLife.04553.001 PMID:25383924

  7. 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

  8. 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

  9. Analysis of Trafficking, Stability and Function of Human Connexin 26 Gap Junction Channels with Deafness-Causing Mutations in the Fourth Transmembrane Helix

    PubMed Central

    Ambrosi, Cinzia; Walker, Amy E.; DePriest, Adam D.; Cone, Angela C.; Lu, Connie; Badger, John; Skerrett, I. Martha; Sosinsky, Gina E.

    2013-01-01

    Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased

  10. Structural and thermodynamic basis of proline-induced transmembrane complex stabilization

    PubMed Central

    Schmidt, Thomas; Situ, Alan J.; Ulmer, Tobias S.

    2016-01-01

    In membrane proteins, proline-mediated helix kinks are indispensable for the tight packing of transmembrane (TM) helices. However, kinks invariably affect numerous interhelical interactions, questioning the acceptance of proline substitutions and evolutionary origin of kinks. Here, we present the structural and thermodynamic basis of proline-induced integrin αIIbβ3 TM complex stabilization to understand the introduction of proline kinks in membrane proteins. In phospholipid bicelles, the A711P substitution in the center of the β3 TM helix changes the direction of adjacent helix segments to form a 35 ± 2° angle and predominantly repacks the segment in the inner membrane leaflet due to a swivel movement. This swivel repacks hydrophobic and electrostatic interhelical contacts within intracellular lipids, resulting in an overall TM complex stabilization of −0.82 ± 0.01 kcal/mol. Thus, proline substitutions can directly stabilize membrane proteins and such substitutions are proposed to follow the structural template of integrin αIIbβ3(A711P). PMID:27436065

  11. 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

  12. Evolution of the α-Subunit of Na/K-ATPase from Paramecium to Homo sapiens: Invariance of Transmembrane Helix Topology.

    PubMed

    Morrill, Gene A; Kostellow, Adele B; Liu, Lijun; Gupta, Raj K; Askari, Amir

    2016-05-01

    Na/K-ATPase is a key plasma membrane enzyme involved in cell signaling, volume regulation, and maintenance of electrochemical gradients. The α-subunit, central to these functions, belongs to a large family of P-type ATPases. Differences in transmembrane (TM) helix topology, sequence homology, helix-helix contacts, cell signaling, and protein domains of Na/K-ATPase α-subunit were compared in fungi (Beauveria), unicellular organisms (Paramecia), primitive multicellular organisms (Hydra), and vertebrates (Xenopus, Homo sapiens), and correlated with evolution of physiological functions in the α-subunit. All α-subunits are of similar length, with groupings of four and six helices in the N- and C-terminal regions, respectively. Minimal homology was seen for protein domain patterns in Paramecium and Hydra, with high correlation between Hydra and vertebrates. Paramecium α-subunits display extensive disorder, with minimal helix contacts. Increases in helix contacts in Hydra approached vertebrates. Protein motifs known to be associated with membrane lipid rafts and cell signaling reveal significant positional shifts between Paramecium and Hydra vulgaris, indicating that regional membrane fluidity changes occur during evolution. Putative steroid binding sites overlapping TM-3 occurred in all species. Sites associated with G-protein-receptor stimulation occur both in vertebrates and amphibia but not in Hydra or Paramecia. The C-terminus moiety "KETYY," necessary for the Na(+) activation of pump phosphorylation, is not present in unicellular species indicating the absence of classical Na(+)/K(+)-pumps. The basic protein topology evolved earliest, followed by increases in protein domains and ordered helical arrays, correlated with appearance of α-subunit regions known to involve cell signaling, membrane recycling, and ion channel formation. PMID:26961431

  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

  14. A possible family of B-like triple helix structures: comparison with the Arnott A-like triple helix.

    PubMed

    Ouali, M; Letellier, R; Adnet, F; Liquier, J; Sun, J S; Lavery, R; Taillandier, E

    1993-03-01

    Recent experimental studies of the structure of triple helices show that their conformation in solution differs from the A-like structure derived from diffraction data on triple helix fibers by Arnott and co-workers. Here we show by means of molecular modeling that a family of triple helix structures may exist with similar conformational energies, but with a variety of sugar puckers. The characteristics of these putative triple helices are analyzed for three different base sequences: (T.AxT)n, (C.GxC+)n, and alternating (C.GxC+/T.AxT)n. In the case of (C.GxC+)n triple helix, infrared and Raman spectra have been obtained and clearly reveal the existence of both N- and S-type sugars in solution. The molecular mechanics calculations allow us to propose a stereochemically reasonable model for this triple helix, in good agreement with the vibrational spectroscopy results. PMID:8448168

  15. 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

  16. Serine 85 in transmembrane helix 2 of short-wavelength visual pigments interacts with the retinylidene Schiff base counterion.

    PubMed

    Dukkipati, A; Vought, B W; Singh, D; Birge, R R; Knox, B E

    2001-12-18

    Short-wavelength cone visual pigments (SWS1) are responsible for detecting light from 350 to 430 nm. Models of this class of pigment suggest that TM2 has extensive contacts with the retinal binding pocket and stabilizes interhelical interactions. The role of TM2 in the structure-function of the Xenopus SWS1 (VCOP, lambda(max) = 427 nm) pigment was studied by replacement of the helix with that of bovine rhodopsin and also by mutagenesis of highly conserved residues. The TM2 chimera and G78D, F79L, M81E, P88T, V89S, and F90V mutants did not produce any significant spectral shift of the dark state or their primary photointermediate formed upon illumination at cryogenic temperatures. The mutant G77R (responsible for human tritanopia) was completely defective in folding, while C82A and F87T bound retinal at reduced levels. The position S85 was crucial for obtaining the appropriate spectroscopic properties of VCOP. S85A and S85T did not bind retinal. S85D bound retinal and had a wild-type dark state at room temperature and a red-shifted dark state at 45 K and formed an altered primary photointermediate. S85C absorbed maximally at 390 nm at neutral pH and at 365 nm at pH >7.5. The S85C dark state was red shifted by 20 nm at 45 K and formed an altered primary photointermediate. These data suggest that S85 is involved in a hydrogen bond with the protonated retinylidene Schiff base counterion in both the dark state and the primary photointermediate. PMID:11735392

  17. Tunable Control of Polyproline Helix (PPII) Structure via Aromatic Electronic Effects: An Electronic Switch of Polyproline Helix

    PubMed Central

    2015-01-01

    Aromatic rings exhibit defined interactions via the unique aromatic π face. Aromatic amino acids interact favorably with proline residues via both the hydrophobic effect and aromatic–proline interactions, C−H/π interactions between the aromatic π face and proline ring C–H bonds. The canonical aromatic amino acids Trp, Tyr, and Phe strongly disfavor a polyproline helix (PPII) when they are present in proline-rich sequences because of the large populations of cis amide bonds induced by favorable aromatic–proline interactions (aromatic–cis-proline and proline–cis-proline–aromatic interactions). We demonstrate the ability to tune polyproline helix conformation and cis–trans isomerism in proline-rich sequences using aromatic electronic effects. Electron-rich aromatic residues strongly disfavor polyproline helix and exhibit large populations of cis amide bonds, while electron-poor aromatic residues exhibit small populations of cis amide bonds and favor polyproline helix. 4-Aminophenylalanine is a pH-dependent electronic switch of polyproline helix, with cis amide bonds favored as the electron-donating amine, but trans amide bonds and polyproline helix preferred as the electron-withdrawing ammonium. Peptides with block proline–aromatic PPXPPXPPXPP sequences exhibited electronically switchable pH-dependent structures. Electron-poor aromatic amino acids provide special capabilities to integrate aromatic residues into polyproline helices and to serve as the basis of aromatic electronic switches to change structure. PMID:25075447

  18. Coupling H(+) transport to rotary catalysis in F-type ATP synthases: structure and organization of the transmembrane rotary motor.

    PubMed

    Fillingame, R H; Jiang, W; Dmitriev, O Y

    2000-01-01

    H(+)-transporting F(1)F(o)-type ATP synthases utilize a transmembrane H(+) potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating beta subunits of the extramembranous F(1) sector of the enzyme, synthesis being driven by rotation of the gamma subunit in the center of the F(1) molecule between the alternating catalytic sites. The H(+) electrochemical potential is thought to drive gamma subunit rotation by first coupling H(+) transport to rotation of an oligomeric rotor of c subunits within the transmembrane F(o) sector. The gamma subunit is forced to turn with the c(12) oligomeric rotor as a result of connections between subunit c and the gamma and epsilon subunits of F(1). In this essay, we will review recent studies on the Escherichia coli F(o) sector. The monomeric structure of subunit c, determined by nuclear magnetic resonance (NMR), is discussed first and used as a basis for the rest of the review. A model for the structural organization of the c(12) oligomer in F(o), deduced from extensive cross-linking studies and by molecular modeling, is then described. The interactions between the the a(1)b(2) 'stator' subcomplex of F(o) and the c(12) oligomer are then considered. A functional interaction between transmembrane helix 4 of subunit a (aTMH-4) and transmembrane helix 2 of subunit c (cTMH-2) during the proton-release step from Asp61 on cTMH-2 is suggested. Current a-c cross-linking data can only be explained by helix-helix swiveling or rotation during the proton transfer steps. A model that mechanically links helix rotation within a single subunit c to the incremental 30 degrees rotation of the c(12) oligomer is proposed. In the final section, the structural interactions between the surface residues of the c(12) oligomer and subunits epsilon and gamma are considered. A molecular model for the binding of subunit epsilon between the exposed, polar surfaces of two subunits c in the oligomer is proposed on the basis of cross

  19. Substrate specificity of rhomboid intramembrane proteases is governed by helix-breaking residues in the substrate transmembrane domain.

    PubMed

    Urban, Sinisa; Freeman, Matthew

    2003-06-01

    Rhomboid intramembrane proteases initiate cell signaling during Drosophila development and Providencia bacterial growth by cleaving transmembrane ligand precursors. We have determined how specificity is achieved: Drosophila Rhomboid-1 is a site-specific protease that recognizes its substrate Spitz by a small region of the Spitz transmembrane domain (TMD). This substrate motif is necessary and sufficient for cleavage and is composed of residues known to disrupt helices. Rhomboids from diverse organisms including bacteria and vertebrates recognize the same substrate motif, suggesting that they use a universal targeting strategy. We used this information to search for other rhomboid substrates and identified a family of adhesion proteins from the human parasite Toxoplasma gondii, the TMDs of which were efficient substrates for rhomboid proteases. Intramembrane cleavage of these proteins is required for host cell invasion. These results provide an explanation of how rhomboid proteases achieve specificity, and allow some rhomboid substrates to be predicted from sequence information. PMID:12820957

  20. Identification of transmembrane helix 1 (TM1) surfaces important for EnvZ dimerisation and signal output.

    PubMed

    Heininger, Annika; Yusuf, Rahmi; Lawrence, Robert J; Draheim, Roger R

    2016-08-01

    The Escherichia coli sensor kinase EnvZ modulates porin expression in response to various stimuli, including extracellular osmolarity, the presence of procaine and interaction with an accessory protein, MzrA. Two major outer membrane porins, OmpF and OmpC, act as passive diffusion-limited pores that allow compounds, including certain classes of antibiotics such as β-lactams and fluoroquinolones, to enter the bacterial cell. Even though the mechanisms by which EnvZ detects and processes the presence of various stimuli are a fundamental component of microbial physiology, they are not yet fully understood. Here, we assess the role of TM1 during signal transduction in response to the presence of extracellular osmolarity. Various mechanisms of transmembrane communication have been proposed including rotation of individual helices within the transmembrane domain, dynamic movement of the membrane-distal portion of the cytoplasmic domain and regulated intra-protein unfolding. To assess these possibilities, we have created a library of single-Cys-containing EnvZ proteins in order to facilitate sulfhydryl-reactivity experimentation. Our results demonstrate that the major TM1-TM1' interface falls along a single surface consisting of residue positions 19, 23, 26, 30 and 34. In addition, we show that Cys substitutions within the N- and C-terminal regions of TM1 result in drastic changes to EnvZ signal output. Finally, we demonstrate that core residues within TM1 are responsible for both TM1 dimerisation and maintenance of steady-state signal output. Overall, our results suggest that no major rearrangement of the TM1-TM1' interface occurs during transmembrane communication in response to extracellular osmolarity. We conclude by discussing these results within the frameworks of several proposed models for transmembrane communication. PMID:27155567

  1. Molecular dynamics simulation of human serum paraoxonase 1 in DPPC bilayer reveals a critical role of transmembrane helix H1 for HDL association.

    PubMed

    Patra, Mahesh Chandra; Rath, Surya Narayan; Pradhan, Sukanta Kumar; Maharana, Jitendra; De, Sachinandan

    2014-01-01

    Serum paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-bound mammalian enzyme exhibiting antiatherosclerotic activity. Despite years of research, an accurate model for the binding interaction between PON1 and HDL has not been established. However, it is reported that anchoring of PON1 to HDL is mainly governed by an N-terminal alpha helix H1 and another short helix H2. Here, we studied the molecular association of full-length human PON1 (huPON1) with a HDL-mimetic dipalmitoylphosphatidylcholine (DPPC) bilayer using homology modeling and molecular dynamics simulations. Our results indicate that H1 is the highly dynamic part of huPON1, showing clockwise rotation of up to 30° within the DPPC bilayer. However, without phospholipid molecules, H1 experiences helical distortions, illustrating an incompatible HDL-anchoring conformation. Snorkeling interactions of K3, R18, and R27 together with aromatic locks formed by Y187, Y190, W194, and W202 are highly essential for anchoring of huPON1 to HDL's surface. Molecular mechanics/Poisson-Boltzmann solvent-accessible surface area (MM/PBSA) binding free energy calculation revealed that H1 displays greater binding affinity towards lipid molecules compared with H2 and H3, suggesting that H1 is the most probable HDL-binding domain of PON1. Binding free energy decomposition showed that K3, R18, and R27 interact with polar headgroups of DPPC membrane through electrostatic interaction. Moreover, Y187, Y190, W194, and W202 interact with DPPC lipids mainly through van der Waals interaction. Taken together, these results show that the transmembrane helix H1 along with the interfacial positively charged and aromatic resides were crucial for PON1's association with HDL particle. The current study will be useful towards understanding the antiatherosclerotic and bioscavenging properties of this promiscuous enzyme. PMID:24297451

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

    PubMed Central

    Monzel, Christian; Unden, Gottfried

    2015-01-01

    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

  3. 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

  4. Triple helix structures: sequence dependence, flexibility and mismatch effects.

    PubMed

    Sun, J S; Mergny, J L; Lavery, R; Montenay-Garestier, T; Hélène, C

    1991-12-01

    By means of molecular modelling, electrostatic interactions are shown to play an important role in the sequence-dependent structure of triple helices formed by a homopyrimidine oligonucleotide bound to a homopurine. homopyrimidine sequence on DNA. This is caused by the presence of positive charges due to the protonation of cytosines in the Hoogsteen-bonded strand, required in order to form C.GxC+ triplets. Energetic and conformational characteristics of triple helices with different sequences are analyzed and discussed. The effects of duplex mismatches on the triple helix stability are investigated via thermal dissociation using UV absorption. PMID:1815635

  5. Backbone Structure of Transmembrane Domain IX of the Na+/Proline Transporter PutP of Escherichia coli

    PubMed Central

    Hilger, Daniel; Polyhach, Yevhen; Jung, Heinrich; Jeschke, Gunnar

    2009-01-01

    Abstract The backbone structure is determined by site-directed spin labeling, double electron electron resonance measurements of distances, and modeling in terms of a helix-loop-helix construct for a transmembrane domain that is supposed to line the translocation pathway in the 54.3 kDa Na+/proline symporter PutP of Escherichia coli. The conformational distribution of the spin labels is accounted for by a rotamer library. An ensemble of backbone models with a root mean-square deviation of less than 2 Å is obtained. These models exhibit a pronounced kink near residue T341, which is involved in substrate binding. The kink may be associated with a hinge that allows the protein to open and close an inwardly oriented cavity. PMID:19134477

  6. 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.

  7. 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

  8. 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

  9. Structural Heterogeneity in Transmembrane Amyloid Precursor Protein Homodimer Is a Consequence of Environmental Selection

    PubMed Central

    2015-01-01

    The 99 amino acid C-terminal fragment of amyloid precursor protein (C99), consisting of a single transmembrane (TM) helix, is known to form homodimers. Homodimers can be processed by γ-secretase to produce amyloid-β (Aβ) protein, which is implicated in Alzheimer’s disease (AD). While knowledge of the structure of C99 homodimers is of great importance, experimental NMR studies and simulations have produced varying structural models, including right-handed and left-handed coiled-coils. In order to investigate the structure of this critical protein complex, simulations of the C9915–55 homodimer in POPC membrane bilayer and DPC surfactant micelle environments were performed using a multiscale approach that blends atomistic and coarse-grained models. The C9915–55 homodimer adopts a dominant right-handed coiled-coil topology consisting of three characteristic structural states in a bilayer, only one of which is dominant in the micelle. Our structural study, which provides a self-consistent framework for understanding a number of experiments, shows that the energy landscape of the C99 homodimer supports a variety of slowly interconverting structural states. The relative importance of any given state can be modulated through environmental selection realized by altering the membrane or micelle characteristics. PMID:24926593

  10. Alpha-Helix folding in the presence of structural constraints.

    PubMed

    Ihalainen, Janne A; Paoli, Beatrice; Muff, Stefanie; Backus, Ellen H G; Bredenbeck, Jens; Woolley, G Andrew; Caflisch, Amedeo; Hamm, Peter

    2008-07-15

    We have investigated the site-specific folding kinetics of a photoswitchable cross-linked alpha-helical peptide by using single (13)C = (18)O isotope labeling together with time-resolved IR spectroscopy. We observe that the folding times differ from site to site by a factor of eight at low temperatures (6 degrees C), whereas at high temperatures (45 degrees C), the spread is considerably smaller. The trivial sum of the site signals coincides with the overall folding signal of the unlabeled peptide, and different sites fold in a noncooperative manner. Moreover, one of the sites exhibits a decrease of hydrogen bonding upon folding, implying that the unfolded state at low temperature is not unstructured. Molecular dynamics simulations at low temperature reveal a stretched-exponential behavior which originates from parallel folding routes that start from a kinetically partitioned unfolded ensemble. Different metastable structures (i.e., traps) in the unfolded ensemble have a different ratio of loop and helical content. Control simulations of the peptide at high temperature, as well as without the cross-linker at low temperature, show faster and simpler (i.e., single-exponential) folding kinetics. The experimental and simulation results together provide strong evidence that the rate-limiting step in formation of a structurally constrained alpha-helix is the escape from heterogeneous traps rather than the nucleation rate. This conclusion has important implications for an alpha-helical segment within a protein, rather than an isolated alpha-helix, because the cross-linker is a structural constraint similar to those present during the folding of a globular protein. PMID:18621686

  11. Multiscale simulations on conformational dynamics and membrane interactions of the non-structural 2 (NS2) transmembrane domain.

    PubMed

    Hung, Huynh Minh; Hang, Tran Dieu; Nguyen, Minh Tho

    2016-09-01

    Hepatitis C virus (HCV) is one of the most crucial global health issues, in which the HCV non-structural protein 2 (NS2), particularly its three transmembrane segments, plays a crucial role in HCV assembly. In this context, multiscale MD simulations have been applied to investigate the preferred orientation of transmembrane domain of NS2 protein (TNS2) in a POPC bilayer, structural stability and characteristic of intramembrane protein-lipid and protein-protein interaction. Our study indicates that NS2 protein adopts three trans-membrane segments with highly stable α-helix structure in a POPC bilayer and a short helical luminal segment. While the first and second TM segment involved in continuous helical domain, the third TM segment is however cleaved into two sub-segments with different tilt angles via a kink at L87G88. Salt bridges K81-E45, R32-PO4 and R43-PO4 are determined as the key factor to stabilize the structure of TM2 and TM3 which consist of charged residues located in the hydrophobic region of the membrane. PMID:27444387

  12. An Unusual Transmembrane Helix in the Endoplasmic Reticulum Ubiquitin Ligase Doa10 Modulates Degradation of Its Cognate E2 Enzyme*

    PubMed Central

    Kreft, Stefan G.; Hochstrasser, Mark

    2011-01-01

    In the endoplasmic reticulum (ER), nascent membrane and secreted proteins that are misfolded are retrotranslocated into the cytosol and degraded by the proteasome. For most ER-associated degradation (ERAD) substrates, ubiquitylation is essential for both their retrotranslocation and degradation. Yeast Doa10 is a polytopic membrane ubiquitin ligase (E3) that along with its cognate ubiquitin-conjugating enzymes (E2s), Ubc7 and the C-terminally membrane-anchored Ubc6, makes a major contribution to ER-associated degradation. Ubc6 is also a substrate of Doa10. One highly conserved Doa10 element, the uncharacterized ∼130-residue TEB4-Doa10 domain, includes three transmembrane helices (TMs). We find that the first of these, TM5, includes an absolutely conserved ΦPΦXXG motif that is required for Doa10 function, as well as highly conserved negatively charged glutamate and aspartate residues. The conservative exchange of the TM5 glutamate to aspartate (doa10-E633D) results in complete stabilization of Ubc6 but has little if any effect on other substrates. Unexpectedly, mutating the glutamate to glutamine (doa10-E633Q) specifically accelerates Ubc6 degradation by ∼5-fold. Other substrates are weakly stabilized in doa10-E633Q cells, consistent with reduced Ubc6 levels. Notably, catalytically inactive ubc6-C87A is degraded in doa10-E633Q but not wild-type cells, but an active version of Ubc6 is required in trans. Fusion of the Ubc6 TM to a soluble protein yields a protein that is degraded in a doa10-E633Q-dependent manner, whereas fusion of the C-terminal TM from an unrelated protein does not. These results suggest that the TEB4-Doa10 domain regulates Doa10 association with the Ubc6 membrane anchor, thereby controlling the degradation rate of the E2. PMID:21467040

  13. 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

  14. 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

  15. Structural Studies of the Transmembrane C-Terminal Domain of the Amyloid Precursor Protein (APP): Does APP Function as a Cholesterol Sensor?†,‡

    PubMed Central

    Beel, Andrew J.; Mobley, Charles K.; Kim, Hak Jun; Tian, Fang; Hadziselimovic, Arina; Jap, Bing; Prestegard, James H.; Sanders, Charles R.

    2008-01-01

    The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-β (Aβ) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by β-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an α-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of α-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Aβ production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein–protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by β- and γ-secretase and resulting amyloid-β production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake. PMID:18702528

  16. 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

  17. The cystic fibrosis transmembrane conductance regulator (CFTR): three-dimensional structure and localization of a channel gate.

    PubMed

    Rosenberg, Mark F; O'Ryan, Liam P; Hughes, Guy; Zhao, Zhefeng; Aleksandrov, Luba A; Riordan, John R; Ford, Robert C

    2011-12-01

    Cystic fibrosis affects about 1 in 2500 live births and involves loss of transmembrane chloride flux due to a lack of a membrane protein channel termed the cystic fibrosis transmembrane conductance regulator (CFTR). We have studied CFTR structure by electron crystallography. The data were compared with existing structures of other ATP-binding cassette transporters. The protein was crystallized in the outward facing state and resembled the well characterized Sav1866 transporter. We identified regions in the CFTR map, not accounted for by Sav1866, which were potential locations for the regulatory region as well as the channel gate. In this analysis, we were aided by the fact that the unit cell was composed of two molecules not related by crystallographic symmetry. We also identified regions in the fitted Sav1866 model that were missing from the map, hence regions that were either disordered in CFTR or differently organized compared with Sav1866. Apart from the N and C termini, this indicated that in CFTR, the cytoplasmic end of transmembrane helix 5/11 and its associated loop could be partly disordered (or alternatively located). PMID:21931164

  18. 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.

  19. Structural organization and interactions of transmembrane domains in tetraspanin proteins

    PubMed Central

    Kovalenko, Oleg V; Metcalf, Douglas G; DeGrado, William F; Hemler, Martin E

    2005-01-01

    Background Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed. Results Among 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)n. In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80. Conclusion Our results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that

  20. Transmembrane structure predictions with hydropathy index/charge two-dimensional trajectories of stochastic dynamical systems.

    PubMed

    Kaburagi, Takashi; Muramatsu, Daigo; Matsumoto, Takashi

    2007-06-01

    A novel algorithm is proposed for predicting transmembrane protein secondary structure from two-dimensional vector trajectories consisting of a hydropathy index and formal charge of a test amino acid sequence using stochastic dynamical system models. Two prediction problems are discussed. One is the prediction of transmembrane region counts; another is that of transmembrane regions, i.e. predicting whether or not each amino acid belongs to a transmembrane region. The prediction accuracies, using a collection of well-characterized transmembrane protein sequences and benchmarking sequences, suggest that the proposed algorithm performs reasonably well. An experiment was performed with a glutamate transporter homologue from Pyrococcus horikoshii. The predicted transmembrane regions of the five human glutamate transporter sequences and observations based on the computed likelihood are reported. PMID:17688311

  1. 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

  2. Structural constraints regulating triple helix formation by A-tracts.

    PubMed

    Sen, A; Gräslund, A

    2000-12-15

    The study concerns the propensity of triple helix formation by different DNA oligonucleotides containing long A-tracts with and without flanking GxC base pairs in order to probe the role of length of the A-tract and the flanking sequences. From nuclear magnetic resonance (NMR) studies of imino proton spectra and circular dichroism (CD) spectroscopy of samples composed of potential triplex forming strand sequences in correct stoichiometries, we have concluded that 8-mer A-tracts flanked by GxC base pairs exert significant steric hindrance to triple helix formation. When as much as 50 mM Mg2+ was added, no triple helix formation was observed in these samples. In contrast, open-ended 8-mer A-tracts formed triplex with the corresponding two T8 strands under relatively mild ionic conditions (100 mM Na+). Moreover, the shorter the length of the A-tract, the less is the hindrance to form a triple helix. PMID:11152277

  3. Helix capping.

    PubMed Central

    Aurora, R.; Rose, G. D.

    1998-01-01

    Helix-capping motifs are specific patterns of hydrogen bonding and hydrophobic interactions found at or near the ends of helices in both proteins and peptides. In an alpha-helix, the first four >N-H groups and last four >C=O groups necessarily lack intrahelical hydrogen bonds. Instead, such groups are often capped by alternative hydrogen bond partners. This review enlarges our earlier hypothesis (Presta LG, Rose GD. 1988. Helix signals in proteins. Science 240:1632-1641) to include hydrophobic capping. A hydrophobic interaction that straddles the helix terminus is always associated with hydrogen-bonded capping. From a global survey among proteins of known structure, seven distinct capping motifs are identified-three at the helix N-terminus and four at the C-terminus. The consensus sequence patterns of these seven motifs, together with results from simple molecular modeling, are used to formulate useful rules of thumb for helix termination. Finally, we examine the role of helix capping as a bridge linking the conformation of secondary structure to supersecondary structure. PMID:9514257

  4. 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

  5. Structure of a C-terminal [alpha]-helix cap in a synthetic peptide

    SciTech Connect

    Zhou, H.X.; Kallenbach, N.R. ); Lyu, P.C.; Wemmer, D.E. )

    1994-02-09

    We report here a novel C-terminal capping structure in a peptide helix, in which the NH of the side chain of asparagine forms an H-bond with the helix main chain CO four residues away. The backbone forms a local 3[sub 10] helix at the C-terminus, with the side chain contributing an additional H-bonded loop. This structure reveals formation of H-bonds by the side chain and main chain of a single residue that serve as a fundamental signal at the C-terminus of helices. The structure formed in this way blocks continuation of the [alpha]helix, hence providing a stronger C-termination signal than Pro 19, as seen in the relative CD values. 12 refs., 2 figs., 1 tab.

  6. Ptuba: a tool for the visualization of helix surfaces in proteins.

    PubMed

    Lopera, Jaime Arce; Sturgis, James N; Duneau, Jean-Pierre

    2005-01-01

    Projection of transmembrane helices using a Uniform B-spline Algorithm is a tool for the visualization of interactions between helices in membrane proteins. It allows the user to generate projections of 3D helices, no matter what their deviations from a canonical helix might be. When associated with adapted coloring schemes it facilitates the comprehension of helix-helix interactions. Examples of transmembrane proteins were chosen to illustrate the advantages that this method provides. In the glycophorin A dimer we can easily appreciate the structural features behind homodimerisation. Using the structure of the fumarate reductase we analyze the contact surfaces inside a helical bundle and thanks to structures from a molecular dynamics simulation we see how modifications in structure and electrostatics relate to their interaction. We propose the use of this tool as an aid to the visualization and analysis of transmembrane helix surfaces and properties. PMID:15670951

  7. pH-dependent structural changes of helix 69 from Escherichia coli 23S ribosomal RNA

    PubMed Central

    Abeysirigunawardena, Sanjaya C.; Chow, Christine S.

    2008-01-01

    Helix 69 in 23S rRNA is a region in the ribosome that participates in a considerable number of RNA–RNA and RNA–protein interactions. Conformational flexibility is essential for such a region to interact and accommodate protein factors at different stages of protein biosynthesis. In this study, pH-dependent structural and stability changes were observed for helix 69 through a variety of spectroscopic techniques, such as circular dichroism spectroscopy, UV melting, and nuclear magnetic resonance spectroscopy. In Escherichia coli 23S rRNA, helix 69 contains pseudouridine residues at positions 1911, 1915, and 1917. The presence of these pseudouridines was found to be essential for the pH-induced conformational changes. Some of the pH-dependent changes appear to be localized to the loop region of helix 69, emphasizing the importance of the highly conserved nature of residues in this region. PMID:18268024

  8. Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region.

    PubMed

    Gupta, J; Evagelidis, A; Hanrahan, J W; Linsdell, P

    2001-06-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 membrane-spanning regions which are presumed to form the transmembrane pore. Although a number of findings have suggested that the sixth transmembrane region plays a key role in forming the pore and determining its functional properties, the role of other transmembrane regions is currently not well established. Here we assess the functional importance of the twelfth transmembrane region, which occupies a homologous position in the carboxy terminal half of the CFTR molecule to that of the sixth transmembrane region in the amino terminal half. Five residues in potentially important regions of the twelfth transmembrane region were mutated individually to alanines, and the function of the mutant channels was examined using patch clamp recording following expression in mammalian cell lines. Three of the five mutations significantly weakened block of unitary Cl(-) currents by SCN(-), implying a partial disruption of anion binding within the pore. Two of these mutations also caused a large reduction in the steady-state channel mean open probability, suggesting a role for the twelfth transmembrane region in channel gating. However, in direct contrast to analogous mutations in the sixth transmembrane region, all mutants studied here had negligible effects on the anion selectivity and unitary Cl(-) conductance of the channel. The relatively minor effects of these five mutations on channel permeation properties suggests that, despite their symmetrical positions within the CFTR protein, the sixth and twelfth transmembrane regions make highly asymmetric contributions to the functional properties of the pore. PMID:11380256

  9. 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.

  10. The discovery of the -helix and -sheet, the principal structural features of proteins

    NASA Astrophysics Data System (ADS)

    Eisenberg, David

    2003-09-01

    PNAS papers by Linus Pauling, Robert Corey, and Herman Branson in the spring of 1951 proposed the -helix and the -sheet, now known to form the backbones of tens of thousands of proteins. They deduced these fundamental building blocks from properties of small molecules, known both from crystal structures and from Pauling's resonance theory of chemical bonding that predicted planar peptide groups. Earlier attempts by others to build models for protein helices had failed both by including nonplanar peptides and by insisting on helices with an integral number of units per turn. In major respects, the Pauling-Corey-Branson models were astoundingly correct, including bond lengths that were not surpassed in accuracy for >40 years. However, they did not consider the hand of the helix or the possibility of bent sheets. They also proposed structures and functions that have not been found, including the -helix.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. Membrane-dependent effects of a cytoplasmic helix on the structure and drug binding of the influenza virus M2 protein.

    PubMed

    Cady, Sarah; Wang, Tuo; Hong, Mei

    2011-08-01

    The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22-46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45-62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21-61). (13)C-(2)H distance measurements of (13)C-labeled protein and (2)H-labeled amantadine showed that in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers, the first equivalent of drug bound S31 inside the M2(21-61) pore, similar to the behavior of M2 transmembrane peptide (M2TM) in DMPC bilayers. The nonspecific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21-61) is S31 in the TM pore. Interestingly, when M2(21-61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, whereas M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state but did not rescue drug binding to M2(21-61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helix to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of

  16. Structures and Energetics of Four Adjacent G·U Pairs That Stabilize an RNA Helix.

    PubMed

    Gu, Xiaobo; Mooers, Blaine H M; Thomas, Leonard M; Malone, Joshua; Harris, Steven; Schroeder, Susan J

    2015-10-22

    Consecutive G·U base pairs inside RNA helices can be destabilizing, while those at the ends of helices are thermodynamically stabilizing. To determine if this paradox could be explained by differences in base stacking, we determined the high-resolution (1.32 Å) crystal structure of (5'-GGUGGCUGUU-3')2 and studied three sequences with four consecutive terminal G·U pairs by NMR spectroscopy. In the crystal structure of (5'-GGUGGCUGUU-3')2, the helix is overwound but retains the overall features of A-form RNA. The penultimate base steps at each end of the helix have high base overlap and contribute to the unexpectedly favorable energetic contribution for the 5'-GU-3'/3'-UG-5' motif in this helix position. The balance of base stacking and helical twist contributes to the positional dependence of G·U pair stabilities. The energetic stabilities and similarity to A-form RNA helices suggest that consecutive G·U pairs would be recognized by RNA helix binding proteins, such as Dicer and Ago. Thus, these results will aid future searches for target sites of small RNAs in gene regulation. PMID:26425937

  17. Disulfide crosslinks to probe the structure and flexibility of a designed four-helix bundle protein.

    PubMed Central

    Regan, L.; Rockwell, A.; Wasserman, Z.; DeGrado, W.

    1994-01-01

    The introduction of disulfide crosslinks is a generally useful method by which to identify regions of a protein that are close together in space. Here we describe the use of disulfide crosslinks to investigate the structure and flexibility of a family of designed 4-helix bundle proteins. The results of these analyses lend support to our working model of the proteins' structure and suggest that the proteins have limited main-chain flexibility. PMID:7756995

  18. A photon-free approach to transmembrane protein structure determination.

    PubMed

    Soto, Cinque S; Hannigan, Brett T; DeGrado, William F

    2011-12-01

    The structures of membrane proteins are generally solved using samples dissolved in micelles, bicelles, or occasionally phospholipid bilayers using X-ray diffraction or magnetic resonance. Because these are less than perfect mimics of true biological membranes, the structures are often confirmed by evaluating the effects of mutations on the properties of the protein in their native cellular environments. Low-resolution structures are also sometimes generated from the results of site-directed mutagenesis when other structural data are incomplete or not available. Here, we describe a rapid and automated approach to determine structures from data on site-directed mutants for the special case of homo-oligomeric helical bundles. The method uses as input an experimental profile of the effects of mutations on some property of the protein. This profile is then interpreted by assuming that positions that have large effects on structure/function when mutated project toward the center of the oligomeric bundle. Model bundles are generated, and correlation analysis is used to score which structures have inter-subunit C(β) distances between adjoining monomers that best correlate with the experimental profile. These structures are then clustered and refined using energy-based minimization methods. For a set of 10 homo-oligomeric TM protein structures ranging from dimers to pentamers, we show that our method predicts structures to within 1-2 Å backbone RMSD relative to X-ray and NMR structures. This level of agreement approaches the precision of NMR structures solved in different membrane mimetics. PMID:22024595

  19. Stereochemical punctuation marks in protein structures: glycine and proline containing helix stop signals.

    PubMed

    Gunasekaran, K; Nagarajaram, H A; Ramakrishnan, C; Balaram, P

    1998-02-01

    An analysis on the nature of alpha-helix stop signals has been carried out, using a dataset of 1057 helices identified from 250 high resolution (structures. The backbone dihedral angles (phi, psi) of the terminating residue (T) were found to cluster either in the left-handed helical region (alphaL: phi=20 degrees to 125 degrees and psi=-45 degrees to 90 degrees ; 469 helices (44%)) or in the extended region (E: phi=-180 degrees to -30 degrees and psi=60 degrees to 180 degrees and -180 degrees to -150 degrees ; 459 helices (43%)) of the Ramachandran map. These two broad categories of helix stop signals, alphaL and E-terminated helices, were further examined for sequence preferences. Gly residues were found to have an overwhelming preference to occur as the "alphaL-terminator (T)" resulting in the classical Schellman motif, with a strong preference for hydrophobic residues at position T-4 and T+1. In the case of E-terminated helices His, Asn, Leu and Phe were found to occur with high propensity at position T. Quite remarkably Pro residues, with single exception, were absent at position T, but had the highest propensity at position T+1. Examination of the frequencies of hydrophobic (h) and polar (p) residues at positions flanking Gly/Pro permitted delineation of exclusive patterns and predictive rules for Gly-terminated helices and Pro-terminated helices. The analysis reveals that Pro residues flanked by polar amino acids have a very strong tendency to terminate helices. Examination of a segment ranging from T-4 to T+3 appeared to be necessary to determine whether helix termination or continuation occur at Gly residues. The two types of helix termination (alphaL, E) signals also differed dramatically in their solvent accessibility. Gly and Pro residues at helix termini appeared to be strongly conserved in homologous sequences. PMID:9480777

  20. Predicting loop–helix tertiary structural contacts in RNA pseudoknots

    PubMed Central

    Cao, Song; Giedroc, David P.; Chen, Shi-Jie

    2010-01-01

    Tertiary interactions between loops and helical stems play critical roles in the biological function of many RNA pseudoknots. However, quantitative predictions for RNA tertiary interactions remain elusive. Here we report a statistical mechanical model for the prediction of noncanonical loop–stem base-pairing interactions in RNA pseudoknots. Central to the model is the evaluation of the conformational entropy for the pseudoknotted folds with defined loop–stem tertiary structural contacts. We develop an RNA virtual bond-based conformational model (Vfold model), which permits a rigorous computation of the conformational entropy for a given fold that contains loop–stem tertiary contacts. With the entropy parameters predicted from the Vfold model and the energy parameters for the tertiary contacts as inserted parameters, we can then predict the RNA folding thermodynamics, from which we can extract the tertiary contact thermodynamic parameters from theory–experimental comparisons. These comparisons reveal a contact enthalpy (ΔH) of −14 kcal/mol and a contact entropy (ΔS) of −38 cal/mol/K for a protonated C+•(G–C) base triple at pH 7.0, and (ΔH = −7 kcal/mol, ΔS = −19 cal/mol/K) for an unprotonated base triple. Tests of the model for a series of pseudoknots show good theory–experiment agreement. Based on the extracted energy parameters for the tertiary structural contacts, the model enables predictions for the structure, stability, and folding pathways for RNA pseudoknots with known or postulated loop–stem tertiary contacts from the nucleotide sequence alone. PMID:20100813

  1. kPROT: a knowledge-based scale for the propensity of residue orientation in transmembrane segments. Application to membrane protein structure prediction.

    PubMed

    Pilpel, Y; Ben-Tal, N; Lancet, D

    1999-12-10

    Modeling of integral membrane proteins and the prediction of their functional sites requires the identification of transmembrane (TM) segments and the determination of their angular orientations. Hydrophobicity scales predict accurately the location of TM helices, but are less accurate in computing angular disposition. Estimating lipid-exposure propensities of the residues from statistics of solved membrane protein structures has the disadvantage of relying on relatively few proteins. As an alternative, we propose here a scale of knowledge-based Propensities for Residue Orientation in Transmembrane segments (kPROT), derived from the analysis of more than 5000 non-redundant protein sequences. We assume that residues that tend to be exposed to the membrane are more frequent in TM segments of single-span proteins, while residues that prefer to be buried in the transmembrane bundle interior are present mainly in multi-span TMs. The kPROT value for each residue is thus defined as the logarithm of the ratio of its proportions in single and multiple TM spans. The scale is refined further by defining it for three discrete sections of the TM segment; namely, extracellular, central, and intracellular. The capacity of the kPROT scale to predict angular helical orientation was compared to that of alternative methods in a benchmark test, using a diversity of multi-span alpha-helical transmembrane proteins with a solved 3D structure. kPROT yielded an average angular error of 41 degrees, significantly lower than that of alternative scales (62 degrees -68 degrees ). The new scale thus provides a useful general tool for modeling and prediction of functional residues in membrane proteins. A WWW server (http://bioinfo.weizmann.ac.il/kPROT) is available for automatic helix orientation prediction with kPROT. PMID:10588897

  2. 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.

  3. 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.

  4. Biological insertion of computationally designed short transmembrane segments.

    PubMed

    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

  5. 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

  6. Structural insights into triglyceride storage mediated by fat storage-inducing transmembrane (FIT) protein 2.

    PubMed

    Gross, David A; Snapp, Erik L; Silver, David L

    2010-01-01

    Fat storage-Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2) belong to a unique family of evolutionarily conserved proteins localized to the endoplasmic reticulum that are involved in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence homology with known proteins and no structural information is available to inform on the mechanism by which FIT proteins function. Here, we present the experimentally-solved topological models for FIT1 and FIT2 using N-glycosylation site mapping and indirect immunofluorescence techniques. These methods indicate that both proteins have six-transmembrane-domains with both N- and C-termini localized to the cytosol. Utilizing this model for structure-function analysis, we identified and characterized a gain-of-function mutant of FIT2 (FLL(157-9)AAA) in transmembrane domain 4 that markedly augmented the total number and mean size of lipid droplets. Using limited-trypsin proteolysis we determined that the FLL(157-9)AAA mutant has enhanced trypsin cleavage at K86 relative to wild-type FIT2, indicating a conformational change. Taken together, these studies indicate that FIT2 is a 6 transmembrane domain-containing protein whose conformation likely regulates its activity in mediating lipid droplet formation. PMID:20520733

  7. Dimeric Structure of the Transmembrane Domain of Glycophorin A in Lipidic and Detergent Environments

    PubMed Central

    Mineev, K.S.; Bocharov, E.V.; Volynsky, P.E.; Goncharuk, M.V.; Tkach, E.N.; Ermolyuk, Ya.S.; Schulga, A.A.; Chupin, V.V.; Maslennikov, I.V.; Efremov, R.G.; Arseniev, A.S.

    2011-01-01

    Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions between transmembrane α-helices by the example of dimerizing the transmembrane domain of the bitopic protein glycophorin A. However, a number of peculiarities place lipid bicelles closer to natural lipid bilayers in terms of their physical properties. PMID:22649687

  8. Helix induction by dirhodium: access to biocompatible metallopeptides with defined secondary structure.

    PubMed

    Zaykov, Alexander N; Popp, Brian V; Ball, Zachary T

    2010-06-11

    The use of carboxylate side chains to induce peptide helicity upon binding to dirhodium centers is examined through experimental and computational approaches. Dirhodium binding efficiently stabilizes alpha helicity or induces alpha helicity in otherwise unstructured peptides for peptides that contain carboxylate side chains with i, i+4 spacing. Helix induction is furthermore possible for sequences with i, i+3 carboxylate spacing, though in this case the length of the side chains is crucial: ligating to longer glutamate side chains is strongly helix inducing, whereas ligating the shorter aspartate side chains destabilizes the helical structure. Further studies demonstrate that a dirhodium metallopeptide complex persists for hours in cellular media and exhibits low toxicity toward mammalian cells, enabling exploitation of these metallopeptides for biological applications. PMID:20411535

  9. 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

  10. Characteristic Structural Parameters for the γ-Peptide 14-Helix:Importance of Subunit Preorganization

    PubMed Central

    Guo, Li; Zhang, Weicheng; Reidenbach, Andrew G.; Giuliano, Michael W.; Guzei, Ilia A.; Spenser, Lara C.; Gellman, Samuel H.

    2012-01-01

    We report crystallographic data for a set of homologous γ-peptides that contain a Boc-protected residue derived from the flexible gabapentin monomer at the N-terminus and cyclically constrained γ-residues at all other positions. The crystallized γ-peptides range in length from 3 to 7 residues. Previously only one atomic-resolution structure had been available for a short γ-peptide 14-helix. The new data provided here allow derivation of characteristic parameters for the γ-peptide 14-helix, and establish guidelines for characterizing 14-helical folding in solution via 2D NMR. In addition, the results suggest that the substitution pattern of a γ-residue has a profound effect on the propensity for 14-helical folding. PMID:21567680

  11. Globular structures of a helix-coil copolymer: self-consistent treatment.

    PubMed

    Nowak, C; Rostiashvili, V G; Vilgis, T A

    2007-01-21

    A self-consistent-field theory was developed in the grand canonical ensemble formulation to study transitions in a helix-coil multiblock globule. Helical and coil parts are treated as stiff rods and self-avoiding walks of variable lengths correspondingly. The resulting field theory takes, in addition to the conventional Zimm-Bragg, [J. Chem. Phys. 31, 526 (1959)] parameters, also three-dimensional interaction terms into account. The appropriate differential equations which determine the self-consistent fields were solved numerically with finite element method. Three different phase states are found: open chain, amorphous globule, and nematic liquid-crystalline (LC) globule. The LC-globule formation is driven by the interplay between the hydrophobic helical segment attraction and the anisotropic globule surface energy of an entropic nature. The full phase diagram of the helix-coil copolymer was calculated and thoroughly discussed. The suggested theory shows a clear interplay between secondary and tertiary structures in globular homopolypeptides. PMID:17249898

  12. Structural features and stability of an RNA triple helix in solution.

    PubMed Central

    Holland, J A; Hoffman, D W

    1996-01-01

    A 30 nt RNA with a sequence designed to form an intramolecular triple helix was analyzed by one-and two-dimensional NMR spectroscopy and UV absorption measurements. NMR data show that the RNA contains seven pyrimidine-purine-pyrimidine base triples stabilized by Watson-Crick and Hoogsteen interactions. The temperature dependence of the imino proton resonances, as well as UV absorption data, indicate that the triple helix is highly stable at acidic pH, melting in a single sharp transition centered at 62 degrees C at pH 4.3. The Watson-Crick and Hoogsteen pairings are disrupted simultaneously upon melting. The NMR data are consistent with a structural model where the Watson-Crick paired strands form an A-helix. Results of model building, guided by NMR data, suggest a possible hydrogen bond between the 2' hydroxyl proton of the Hoogsteen strand and a phosphate oxygen of the purine strand. The structural model is discussed in terms of its ability to account for some of the differences in stability reported for RNA and DNA triple helices and provides insight into features that are likely to be important in the design of RNA binding compounds. PMID:8759020

  13. Structural dynamics of a single-stranded RNA–helix junction using NMR

    PubMed Central

    Eichhorn, Catherine D.; Al-Hashimi, Hashim M.

    2014-01-01

    Many regulatory RNAs contain long single strands (ssRNA) that adjoin secondary structural elements. Here, we use NMR spectroscopy to study the dynamic properties of a 12-nucleotide (nt) ssRNA tail derived from the prequeuosine riboswitch linked to the 3′ end of a 48-nt hairpin. Analysis of chemical shifts, NOE connectivity, 13C spin relaxation, and residual dipolar coupling data suggests that the first two residues (A25 and U26) in the ssRNA tail stack onto the adjacent helix and assume an ordered conformation. The following U26-A27 step marks the beginning of an A6-tract and forms an acute pivot point for substantial motions within the tail, which increase toward the terminal end. Despite substantial internal motions, the ssRNA tail adopts, on average, an A-form helical conformation that is coaxial with the helix. Our results reveal a surprising degree of structural and dynamic complexity at the ssRNA–helix junction, which involves a fine balance between order and disorder that may facilitate efficient pseudoknot formation on ligand recognition. PMID:24742933

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. Unwinding of the uniform lying helix structure in cholesteric liquid crystals next to a spatially uniform aligning surface.

    PubMed

    Salter, Patrick S; Carbone, Giovanni; Jewell, Sharon A; Elston, Steve J; Raynes, Peter

    2009-10-01

    The symmetry of the cholesteric uniform lying helix (ULH) structure, where the helix axis is aligned in a single direction parallel to the device substrates, is not compatible with a uniform surface alignment and an unwinding of the helical structure is expected at the interface. Fluorescence confocal polarizing microscopy experiments are performed on the interface between a bulk ULH and a uniform aligning surface (for both planar and homeotropic alignments). The results are analyzed in the framework of a finite difference numerical simulation based on the Frank elastic distortion, to determine relevant director structures. An optical model is introduced to predict three-dimensional fluorescence profiles for the structures. Comparison of experimental and theoretical results shows that the equilibrium structure of the system involves a continuous unwinding of the helix close to the surface. PMID:19905325

  20. Structure of FitAB from Neisseria gonorrhoeae bound to DNA reveals a tetramer of toxin-antitoxin heterodimers containing pin domains and ribbon-helix-helix motifs.

    PubMed

    Mattison, Kirsten; Wilbur, J Scott; So, Magdalene; Brennan, Richard G

    2006-12-01

    Neisseria gonorrhoeae is a sexually transmitted pathogen that initiates infections in humans by adhering to the mucosal epithelium of the urogenital tract. The bacterium then enters the apical region of the cell and traffics across the cell to exit into the subepithelial matrix. Mutations in the fast intracellular trafficking (fitAB) locus cause the bacteria to transit a polarized epithelial monolayer more quickly than the wild-type parent and to replicate within cells at an accelerated rate. Here, we describe the crystal structure of the toxin-antitoxin heterodimer, FitAB, bound to a high affinity 36-bp DNA fragment from the fitAB promoter. FitA, the antitoxin, binds DNA through its ribbon-helix-helix motif and is tethered to FitB, the toxin, to form a heterodimer by the insertion of a four turn alpha-helix into an extensive FitB hydrophobic pocket. FitB is composed of a PIN (PilT N terminus) domain, with a central, twisted, 5-stranded parallel beta-sheet that is open on one side and flanked by five alpha-helices. FitB in the context of the FitAB complex does not display nuclease activity against tested PIN substrates. The FitAB complex points to the mechanism by which antitoxins with RHH motifs can block the activity of toxins with PIN domains. Interactions between two FitB molecules result in the formation of a tetramer of FitAB heterodimers, which binds to the 36-bp DNA fragment and provides an explanation for how FitB enhances the DNA binding affinity of FitA. PMID:16982615

  1. A Divalent Ion Is Crucial in the Structure and Dominant-Negative Function of ID Proteins, a Class of Helix-Loop-Helix Transcription Regulators

    PubMed Central

    Palasingam, Paaventhan; Kolatkar, Prasanna R.

    2012-01-01

    Inhibitors of DNA binding and differentiation (ID) proteins, a dominant-negative group of helix-loop-helix (HLH) transcription regulators, are well-characterized key players in cellular fate determination during development in mammals as well as Drosophila. Although not oncogenes themselves, their upregulation by various oncogenic proteins (such as Ras, Myc) and their inhibitory effects on cell cycle proteins (such as pRb) hint at their possible roles in tumorigenesis. Furthermore, their potency as inhibitors of cellular differentiation, through their heterodimerization with subsequent inactivation of the ubiquitous E proteins, suggest possible novel roles in engineering induced pluripotent stem cells (iPSCs). We present the high-resolution 2.1Å crystal structure of ID2 (HLH domain), coupled with novel biochemical insights in the presence of a divalent ion, possibly calcium (Ca2+), in the loop of ID proteins, which appear to be crucial for the structure and activity of ID proteins. These new insights will pave the way for new rational drug designs, in addition to current synthetic peptide options, against this potent player in tumorigenesis as well as more efficient ways for stem cells reprogramming. PMID:23119064

  2. 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

  3. Structure and dynamics of one-dimensional ionic solutions in biological transmembrane channels.

    PubMed Central

    Skerra, A; Brickmann, J

    1987-01-01

    The structure and dynamics of solvated alkali metal cations in transmembrane channels are treated using the molecular dynamics simulation technique. The simulations are based on a modified Fischer-Brickmann model (Fischer, W., and J. Brickmann, 1983, Biophys. Chem., 18:323-337) for gramicidin A-type channels. The trajectories of all particles in the channel as well as two-dimensional pair correlation functions are analyzed. It is found from the analysis of the stationary simulation state that one-dimensional solvation complexes are formed and that the number of water molecules in the channel varies for different alkali metal cations. PMID:2440485

  4. 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

  5. A note on crystal packing and global helix structure in short A-DNA duplexes.

    PubMed

    Heinemann, U

    1991-02-01

    A simple relation exists between the packing density in crystals of short A-DNA duplexes and their global double-helical structure. The volume per nucleotide pair shows a linear inverse correlation with the mean displacement of base pairs from the best straight helix axis. The mean displacement is a measure of major groove depth and varies between -3.3 A and -4.9 A in A-form oligonucleotides analysed in the crystalline state. Since the mean displacement of base pairs from the helix axis determines other helical parameters such as base-pair longitudinal slide, its correlation with crystal packing is of considerable interest. The displacement-packing correlation is very clear for octamer duplexes which crystallize in three different lattices. Longer A-helical fragments sometimes deviate from the rule. It may be speculated whether A-form duplexes not completing a full helical turn are especially prone to distortions due to packing in crystals or arising from intermolecular contacts in solution. PMID:2059340

  6. 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

  7. Structure and mechanism of a eukaryotic transmembrane ascorbate-dependent oxidoreductase

    PubMed Central

    Lu, Peilong; Ma, Dan; Yan, Chuangye; Gong, Xinqi; Du, Mingjian; Shi, Yigong

    2014-01-01

    Vitamin C, also known as ascorbate, is required in numerous essential metabolic reactions in eukaryotes. The eukaryotic ascorbate-dependent oxidoreductase cytochrome b561 (Cyt b561), a family of highly conserved transmembrane enzymes, plays an important role in ascorbate recycling and iron absorption. Although Cyt b561 was identified four decades ago, its atomic structure and functional mechanism remain largely unknown. Here, we report the high-resolution crystal structures of cytochrome b561 from Arabidopsis thaliana in both substrate-free and substrate-bound states. Cyt b561 forms a homodimer, with each protomer consisting of six transmembrane helices and two heme groups. The negatively charged substrate ascorbate, or monodehydroascorbate, is enclosed in a positively charged pocket on either side of the membrane. Two highly conserved amino acids, Lys81 and His106, play an essential role in substrate recognition and catalysis. Our structural and biochemical analyses allow the proposition of a general electron transfer mechanism for members of the Cyt b561 family. PMID:24449903

  8. 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.

  9. 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

  10. 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.

  11. 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

  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. Analysis of local helix bending in crystal structures of DNA oligonucleotides and DNA-protein complexes.

    PubMed Central

    Young, M A; Ravishanker, G; Beveridge, D L; Berman, H M

    1995-01-01

    Sequence-dependent bending of the helical axes in 112 oligonucleotide duplex crystal structures resident in the Nucleic Acid Database have been analyzed and compared with the use of bending dials, a computer graphics tool. Our analysis includes structures of both A and B forms of DNA and considers both uncomplexed forms of the double helix as well as those bound to drugs and proteins. The patterns in bending preferences in the crystal structures are analyzed by base pair steps, and emerging trends are noted. Analysis of the 66 B-form structures in the Nucleic Acid Database indicates that uniform trends within all pyrimidine-purine and purine-pyrimidine steps are not necessarily observed but are found particularly at CG and GC steps of dodecamers. The results support the idea that AA steps are relatively straight and that larger roll bends occur at or near the junctions of these A-tracts with their flanking sequences. The data on 16 available crystal structures of protein-DNA complexes indicate that the majority of the DNA bends induced via protein binding are sharp localized kinks. The analysis of the 30 available A-form DNA structures indicates that these structures are also bent and show a definitive preference for bending into the deep major groove over the shallow minor groove. PMID:7647248

  14. 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

  15. The 5 A projection structure of the transmembrane domain of the mannitol transporter enzyme II.

    PubMed

    Koning, R I; Keegstra, W; Oostergetel, G T; Schuurman-Wolters, G; Robillard, G T; Brisson, A

    1999-04-16

    The uptake of mannitol in Escherichia coli is controlled by the phosphoenolpyruvate dependent phosphotransferase system. Enzyme II mannitol (EIIMtl) is part of the phosphotransferase system and consists of three covalently bound domains. IICMtl, the integral membrane domain of EIIMtl, is responsible for mannitol transport across the cytoplasmic membrane. In order to understand this molecular process, two-dimensional crystals of IICMtl were grown by reconstitution into lipid bilayers and their structure was investigated by cryo-electron crystallography. The IICMtl crystals obey p22121 symmetry and have a unit cell of 125 Ax65 A, gamma=90 degrees. A projection structure was determined at 5 A resolution using both electron images and electron diffractograms. The unit cell contains two IICMtl dimers with a size of about 40 Ax90 A, which are oriented up and down in the crystal. Each monomer exhibits six domains of high density which most likely correspond to transmembrane alpha-helices and cytoplasmic loops. PMID:10222194

  16. Three-dimensional reconstruction of human cystic fibrosis transmembrane conductance regulator chloride channel revealed an ellipsoidal structure with orifices beneath the putative transmembrane domain.

    PubMed

    Mio, Kazuhiro; Ogura, Toshihiko; Mio, Muneyo; Shimizu, Hiroyasu; Hwang, Tzyh-Chang; Sato, Chikara; Sohma, Yoshiro

    2008-10-31

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to an ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three steps: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. The stoichiometry of the protein was analyzed using various biochemical approaches, including chemical cross-linking, blue-native PAGE, size exclusion chromatography, and electron microscopy (EM) observation of antibody-decorated CFTR. All these data support a dimeric assembly of CFTR. Using 5,039 automatically selected particles from negatively stained EM images, the three-dimensional structure of CFTR was reconstructed at 2-nm resolution assuming a 2-fold symmetry. CFTR, presumably in a closed state, was shown to be an ellipsoidal particle with dimensions of 120 x 106 x 162 A. It comprises a small dome-shaped extracellular and membrane-spanning domain and a large cytoplasmic domain with orifices beneath the putative transmembrane domain. EM observation of CFTR.anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain. PMID:18723516

  17. Three-dimensional Reconstruction of Human Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Revealed an Ellipsoidal Structure with Orifices beneath the Putative Transmembrane Domain*

    PubMed Central

    Mio, Kazuhiro; Ogura, Toshihiko; Mio, Muneyo; Shimizu, Hiroyasu; Hwang, Tzyh-Chang; Sato, Chikara; Sohma, Yoshiro

    2008-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to an ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three steps: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. The stoichiometry of the protein was analyzed using various biochemical approaches, including chemical cross-linking, blue-native PAGE, size exclusion chromatography, and electron microscopy (EM) observation of antibody-decorated CFTR. All these data support a dimeric assembly of CFTR. Using 5,039 automatically selected particles from negatively stained EM images, the three-dimensional structure of CFTR was reconstructed at 2-nm resolution assuming a 2-fold symmetry. CFTR, presumably in a closed state, was shown to be an ellipsoidal particle with dimensions of 120 × 106 × 162Å. It comprises a small dome-shaped extracellular and membrane-spanning domain and a large cytoplasmic domain with orifices beneath the putative transmembrane domain. EM observation of CFTR·anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain. PMID:18723516

  18. Structural model of the transmembrane Fo rotary sector of H+-transporting ATP synthase derived by solution NMR and intersubunit cross-linking in situ.

    PubMed

    Fillingame, Robert H; Dmitriev, Oleg Y

    2002-10-11

    H(+)-transporting, F(1)F(o)-type ATP synthases utilize a transmembrane H(+) potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating beta subunits of the extramembranous F(1) sector of the enzyme, synthesis being driven by rotation of the gamma subunit in the center of the F(1) molecule between the alternating catalytic sites. The H(+) electrochemical potential is thought to drive gamma subunit rotation by first coupling H(+) transport to rotation of an oligomeric rotor of c subunits within the transmembrane F(o) sector. The gamma subunit is forced to turn with the c-oligomeric rotor due to connections between subunit c and the gamma and epsilon subunits of F(1). In this essay we will review recent studies on the Escherichia coli F(o) sector. The monomeric structure of subunit c, determined by NMR, shows that subunit c folds in a helical hairpin with the proton carrying Asp(61) centered in the second transmembrane helix (TMH). A model for the structural organization of the c(10) oligomer in F(o) was deduced from extensive cross-linking studies and by molecular modeling. The model indicates that the H(+)-carrying carboxyl of subunit c is occluded between neighboring subunits of the c(10) oligomer and that two c subunits pack in a "front-to-back" manner to form the H(+) (cation) binding site. In order for protons to gain access to Asp(61) during the protonation/deprotonation cycle, we propose that the outer, Asp(61)-bearing TMH-2s of the c-ring and TMHs from subunits composing the inlet and outlet channels must turn relative to each other, and that the swiveling motion associated with Asp(61) protonation/deprotonation drives the rotation of the c-ring. The NMR structures of wild-type subunit c differs according to the protonation state of Asp(61). The idea that the conformational state of subunit c changes during the catalytic cycle is supported by the cross-linking evidence in situ, and two recent NMR structures of functional

  19. Bis-Histidine-Coordinated Hemes in Four-Helix Bundles: How the Geometry of the Bundle Controls the Axial Imidazole Plane Orientations in Transmembrane Cytochromes of Mitochondrial Complexes II and III and Related Proteins

    PubMed Central

    Berry, Edward A.; Walker, F. Ann

    2009-01-01

    Early investigation of the EPR spectra of bis-histidine-coordinated membrane-bound ferriheme proteins led to the description of a spectral signal that had only one resolved feature. These became known as “highly anisotropic low-spin” (HALS) or “large gmax” ferriheme centers. Extensive work with small-molecule model heme complexes showed that this spectroscopic signature occurs in bis-imidazole ferrihemes in which the planes of the imidazole ligands are nearly perpendicular, Δϕ = 57–90°. In the last decade protein crystallographic studies have revealed the atomic structures of a number of examples of bis-histidine heme proteins. A frequent characteristic of these large gmax ferrihemes in membrane-bound proteins is the occurrence of the heme within a four-helix bundle with a left-handed twist. The histidine ligands occur at the same level on two diametrically opposed helices of the bundle. These ligands have the same side chain conformation and ligate heme iron on the bundle axis, resulting in a quasi-2-fold symmetric structure. The two non-ligand-bearing helices also obey this symmetry, and have a conserved small residue, usually glycine, where the edge of the heme ring makes contact with the helix backbones. In many cases this small residue is preceded by a threonine or serine residue whose side chain hydroxyl oxygen acts as a hydrogen-bond acceptor from the Nδ1 atom of the heme-ligating histidine. The Δϕ angle is thus determined by the common histidine side-chain conformation and the crossing angle of the ligand-bearing helices, in some cases constrained by H-bonds to the Ser/Thr residues on the non-ligand-bearing helices. PMID:18418633

  20. Artificial Diiron Enzymes with a De Novo Designed Four-Helix Bundle Structure

    PubMed Central

    Chino, Marco; Maglio, Ornella; Nastri, Flavia; Pavone, Vincenzo; DeGrado, William F.

    2016-01-01

    A single polypeptide chain may provide an astronomical number of conformers. Nature selected only a trivial number of them through evolution, composing an alphabet of scaffolds, that can afford the complete set of chemical reactions needed to support life. These structural templates are so stable that they allow several mutations without disruption of the global folding, even having the ability to bind several exogenous cofactors. With this perspective, metal cofactors play a crucial role in the regulation and catalysis of several processes. Nature is able to modulate the chemistry of metals, adopting only a few ligands and slightly different geometries. Several scaffolds and metal-binding motifs are representing the focus of intense interest in the literature. This review discusses the widespread four-helix bundle fold, adopted as a scaffold for metal binding sites in the context of de novo protein design to obtain basic biochemical components for biosensing or catalysis. In particular, we describe the rational refinement of structure/function in diiron–oxo protein models from the due ferri (DF) family. The DF proteins were developed by us through an iterative process of design and rigorous characterization, which has allowed a shift from structural to functional models. The examples reported herein demonstrate the importance of the synergic application of de novo design methods as well as spectroscopic and structural characterization to optimize the catalytic performance of artificial enzymes.

  1. The molecular structure of the left-handed supra-molecular helix of eukaryotic polyribosomes

    NASA Astrophysics Data System (ADS)

    Myasnikov, Alexander G.; Afonina, Zhanna A.; Ménétret, Jean-François; Shirokov, Vladimir A.; Spirin, Alexander S.; Klaholz, Bruno P.

    2014-11-01

    During protein synthesis, several ribosomes bind to a single messenger RNA (mRNA) forming large macromolecular assemblies called polyribosomes. Here we report the detailed molecular structure of a 100 MDa eukaryotic poly-ribosome complex derived from cryo electron tomography, sub-tomogram averaging and pseudo-atomic modelling by crystal structure fitting. The structure allowed the visualization of the three functional parts of the polysome assembly, the central core region that forms a rather compact left-handed supra-molecular helix, and the more open regions that harbour the initiation and termination sites at either ends. The helical region forms a continuous mRNA channel where the mRNA strand bridges neighbouring exit and entry sites of the ribosomes and prevents mRNA looping between ribosomes. This structure provides unprecedented insights into protein- and RNA-mediated inter-ribosome contacts that involve conserved sites through 40S subunits and long protruding RNA expansion segments, suggesting a role in stabilizing the overall polyribosomal assembly.

  2. CAPS-DB: a structural classification of helix-capping motifs

    PubMed Central

    Segura, Joan; Oliva, Baldomero; Fernandez-Fuentes, Narcis

    2012-01-01

    The regions of the polypeptide chain immediately preceding or following an α-helix are known as Nt- and Ct cappings, respectively. Cappings play a central role stabilizing α-helices due to lack of intrahelical hydrogen bonds in the first and last turn. Sequence patterns of amino acid type preferences have been derived for cappings but the structural motifs associated to them are still unclassified. CAPS-DB is a database of clusters of structural patterns of different capping types. The clustering algorithm is based in the geometry and the (ϕ–ψ)-space conformation of these regions. CAPS-DB is a relational database that allows the user to search, browse, inspect and retrieve structural data associated to cappings. The contents of CAPS-DB might be of interest to a wide range of scientist covering different areas such as protein design and engineering, structural biology and bioinformatics. The database is accessible at: http://www.bioinsilico.org/CAPSDB. PMID:22021380

  3. 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.

  4. 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

  5. Architecture of the cystic fibrosis transmembrane conductance regulator protein and structural changes associated with phosphorylation and nucleotide binding.

    PubMed

    Zhang, Liang; Aleksandrov, Luba A; Zhao, Zhefeng; Birtley, James R; Riordan, John R; Ford, Robert C

    2009-09-01

    We describe biochemical and structural studies of the isolated cystic fibrosis transmembrane conductance regulator (CFTR) protein. Using electron cryomicroscopy, low resolution three-dimensional structures have been obtained for the non-phosphorylated protein in the absence of nucleotide and for the phosphorylated protein with ATP. In the latter state, the cytosolic nucleotide-binding domains move closer together, forming a more compact packing arrangement. Associated with this is a reorganization within the cylindrical transmembrane domains, consistent with a shift from an inward-facing to outward-facing configuration. A region of density in the non-phosphorylated protein that extends from the bottom of the cytosolic regions up to the transmembrane domains is hypothesised to represent the unique regulatory region of CFTR. These data offer insights into the architecture of this ATP-binding cassette protein, and shed light on the global motions associated with nucleotide binding and priming of the chloride channel via phosphorylation of the regulatory region. PMID:19524678

  6. Structural Variation and Uniformity among Tetraloop-Receptor Interactions and Other Loop-Helix Interactions in RNA Crystal Structures

    PubMed Central

    Wu, Li; Chai, Dinggeng; Fraser, Marie E.; Zimmerly, Steven

    2012-01-01

    Tetraloop-receptor interactions are prevalent structural units in RNAs, and include the GAAA/11-nt and GNRA-minor groove interactions. In this study, we have compiled a set of 78 nonredundant loop-helix interactions from X-ray crystal structures, and examined them for the extent of their sequence and structural variation. Of the 78 interactions in the set, only four were classical GAAA/11-nt motifs, while over half (48) were GNRA-minor groove interactions. The GNRA-minor groove interactions were not a homogeneous set, but were divided into five subclasses. The most predominant subclass is characterized by two triple base pair interactions in the minor groove, flanked by two ribose zipper contacts. This geometry may be considered the “standard” GNRA-minor groove interaction, while the other four subclasses are alternative ways to form interfaces between a minor groove and tetraloop. The remaining 26 structures in the set of 78 have loops interacting with mostly idiosyncratic receptors. Among the entire set, a number of sequence-structure correlations can be identified, which may be used as initial hypotheses in predicting three-dimensional structures from primary sequences. Conversely, other sequence patterns are not predictive; for example, GAAA loop sequences and GG/CC receptors bind to each other with three distinct geometries. Finally, we observe an example of structural evolution in group II introns, in which loop-receptor motifs are substituted for each other while maintaining the larger three-dimensional geometry. Overall, the study gives a more complete view of RNA loop-helix interactions that exist in nature. PMID:23152878

  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. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. Oligomerization state and supramolecular structure of the HIV-1 Vpu protein transmembrane segment in phospholipid bilayers

    PubMed Central

    Lu, Jun-Xia; Sharpe, Simon; Ghirlando, Rodolfo; Yau, Wai-Ming; Tycko, Robert

    2010-01-01

    HIV-1 Vpu is an 81-residue protein with a single N-terminal transmembrane (TM) helical segment that is involved in the release of new virions from host cell membranes. Vpu and its TM segment form ion channels in phospholipid bilayers, presumably by oligomerization of TM helices into a pore-like structure. We describe measurements that provide new constraints on the oligomerization state and supramolecular structure of residues 1–40 of Vpu (Vpu1–40), including analytical ultracentrifugation measurements to investigate oligomerization in detergent micelles, photo-induced crosslinking experiments to investigate oligomerization in bilayers, and solid-state nuclear magnetic resonance measurements to obtain constraints on intermolecular contacts between and orientations of TM helices in bilayers. From these data, we develop molecular models for Vpu TM oligomers. The data indicate that a variety of oligomers coexist in phospholipid bilayers, so that a unique supramolecular structure can not be defined. Nonetheless, since oligomers of various sizes have similar intermolecular contacts and orientations, molecular models developed from our data are most likely representative of Vpu TM oligomers that exist in host cell membranes. PMID:20669237

  14. Functional Implications of the Human T-Lymphotropic Virus Type 1 Transmembrane Glycoprotein Helical Hairpin Structure

    PubMed Central

    Maerz, Anne L.; Center, Rob J.; Kemp, Bruce E.; Kobe, Bostjan; Poumbourios, Pantelis

    2000-01-01

    Retrovirus entry into cells follows receptor binding by the surface-exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein. PMID:10864675

  15. Structure, stability, and thermodynamics of a short intermolecular purine-purine-pyrimidine triple helix

    SciTech Connect

    Pilch, D.S.; Shafer, R.H. ); Levenson, C. )

    1991-06-25

    The authors have investigated the structure and physical chemistry of the d(C{sub 3}T{sub 4}C{sub 3}){center dot}2(d(G{sub 3}A{sub 4}G{sub 3})) triple helix by polyacrylamide gel electrophoresis (PAGE), {sup 1}H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl{sub 2} at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur{center dot}pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the puring strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 C, depending on the DNA concentration. This marked enhancement in stability, coupled with the lack of an acidic pH requirement, suggests that pur-pur-pyr triplexes are appealing choices for use in applications involving oligonucleotide targeting of duplex DNA in vitro and in vivo.

  16. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

    PubMed Central

    Rosenberg, Mark F.; O'Ryan, Liam P.; Hughes, Guy; Zhao, Zhefeng; Aleksandrov, Luba A.; Riordan, John R.; Ford, Robert C.

    2011-01-01

    Cystic fibrosis affects about 1 in 2500 live births and involves loss of transmembrane chloride flux due to a lack of a membrane protein channel termed the cystic fibrosis transmembrane conductance regulator (CFTR). We have studied CFTR structure by electron crystallography. The data were compared with existing structures of other ATP-binding cassette transporters. The protein was crystallized in the outward facing state and resembled the well characterized Sav1866 transporter. We identified regions in the CFTR map, not accounted for by Sav1866, which were potential locations for the regulatory region as well as the channel gate. In this analysis, we were aided by the fact that the unit cell was composed of two molecules not related by crystallographic symmetry. We also identified regions in the fitted Sav1866 model that were missing from the map, hence regions that were either disordered in CFTR or differently organized compared with Sav1866. Apart from the N and C termini, this indicated that in CFTR, the cytoplasmic end of transmembrane helix 5/11 and its associated loop could be partly disordered (or alternatively located). PMID:21931164

  17. Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure

    PubMed Central

    Moraga-Cid, Gustavo; Sauguet, Ludovic; Huon, Christèle; Malherbe, Laurie; Girard-Blanc, Christine; Petres, Stéphane; Murail, Samuel; Taly, Antoine; Baaden, Marc; Delarue, Marc; Corringer, Pierre-Jean

    2015-01-01

    The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor. PMID:25730860

  18. Structure, attachment properties, and ecological importance of the attachment system of English ivy (Hedera helix)

    PubMed Central

    Melzer, Björn; Seidel, Robin; Steinbrecher, Tina; Speck, Thomas

    2012-01-01

    Root climbers such as English ivy (Hedera helix) rely on specialized adventitious roots for attachment, enabling the plants to climb on a wide range of natural and artificial substrates. Despite their importance for the climbing habit, the biomechanical properties of these specialized adventitious roots compared with standard roots and their performance in the attachment to different host species or inert substrates have not been studied. Here organs and tissues involved in the attachment are characterized and their significance in regard to a broader functional and ecological aspect is discussed. Depending on the substrate, the root clusters show different types of failure modes at various frequencies, demonstrating the close interaction between the climber and its substrates. With a Young’s Modulus of 109.2 MPa, the attachment roots are relatively stiff for non-woody roots. The central cylinders of the attachment roots show a high tensile strength of 38 MPa and a very high extensibility of 34%. In host trees naturally co-distributed with English ivy, a ‘balanced’ occurrence of failure of the attachment system of the climber and the bark of the host is found, suggesting a co-evolution of climber and host. Maximum loads of root clusters normalized by the number of roots match those of individually tested attachment roots. In comparison with most subterranean roots the properties and structure of the attachment roots of English ivy show distinct differences. There exist similarities to the properties found for roots of Galium aparine, suggesting a trend in not fully self-supporting plants towards a higher extensibility. PMID:21914660

  19. The structural properties of the transmembrane segment of the integral membrane protein phospholamban utilizing (13)C CPMAS, (2)H, and REDOR solid-state NMR spectroscopy.

    PubMed

    Karp, Ethan S; Tiburu, Elvis K; Abu-Baker, Shadi; Lorigan, Gary A

    2006-06-01

    Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca(2+) regulator. (13)C cross-polarization magic angle spinning spectra of (13)C carbonyl-labeled Leu39 of TM-PLB exhibited two peaks in a pure 1-palmitoyl-2-oleoyl-phosphocholine (POPC) bilayer, each due to a different structural conformation of phospholamban as characterized by the corresponding (13)C chemical shift. The addition of a negatively charged phospholipid (1-palmitoyl-2-oleoylphosphatidylglycerol (POPG)) to the POPC bilayer stabilized TM-PLB to an alpha-helical conformation as monitored by an enhancement of the alpha-helical carbonyl (13)C resonance in the corresponding NMR spectrum. (13)C-(15)N REDOR solid-state NMR spectroscopic experiments revealed the distance between the (13)C carbonyl carbon of Leu39 and the (15)N amide nitrogen of Leu42 to be 4.2+/-0.2A indicating an alpha-helical conformation of TM-PLB with a slight deviation from an ideal 3.6 amino acid per turn helix. Finally, the quadrupolar splittings of three (2)H labeled leucines (Leu28, Leu39, and Leu51) incorporated in mechanically aligned DOPE/DOPC bilayers yielded an 11 degrees +/-5 degrees tilt of TM-PLB with respect to the bilayer normal. In addition to elucidating valuable TM-PLB secondary structure information, the solid-state NMR spectroscopic data indicates that the type of phospholipids and the water content play a crucial role in the secondary structure and folding of TM-PLB in a phospholipid bilayer. PMID:16839519

  20. 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.

  1. Transmembrane Domain Interactions and Residue Proline 378 Are Essential for Proper Structure, Especially Disulfide Bond Formation, in the Human Vitamin K-Dependent γ-Glutamyl Carboxylase†

    PubMed Central

    Tie, Jian-Ke; Zheng, Mei-Yan; Hsiao, Kuang-Ling N.; Perera, Lalith; Stafford, Darrel W.; Straight, David L.

    2009-01-01

    We used recombinant techniques to create a two-chain form (residues 1–345 and residues 346–758) of the vitamin K-dependent γ-glutamyl carboxylase, a glycoprotein located in the endoplasmic reticulum containing five transmembrane domains. The two-chain carboxylase had carboxylase and epoxidase activities similar to those of one-chain carboxylase. In addition, it had normal affinity for the propeptide of factor IX. We employed this molecule to investigate formation of the one disulfide bond in carboxylase, the transmembrane structure of carboxylase, and the potential interactions among the carboxylase’s transmembrane domains. Our results indicate that the two peptides of the two-chain carboxylase are joined by a disulfide bond. Proline 378 is important for the structure necessary for disulfide formation. Results with the P378L carboxylase indicate that noncovalent bonds maintain the two-chain structure even when the disulfide bond is disrupted. As we had previously proposed, the fifth transmembrane domain of carboxylase is the last and only transmembrane domain in the C-terminal peptide of the two-chain carboxylase. We show that the noncovalent association between the two chains of carboxylase involves an interaction between the fifth transmembrane domain and the second transmembrane domain. Results of a homology model of transmembrane domains 2 and 5 suggest that not only do these two domains associate but that transmembrane domain 2 may interact with another transmembrane domain. This latter interaction may be mediated at least in part by a motif of glycine residues in the second transmembrane domain. PMID:18498174

  2. transFold: a web server for predicting the structure and residue contacts of transmembrane beta-barrels

    PubMed Central

    Waldispühl, J.; Berger, Bonnie; Clote, Peter; Steyaert, Jean-Marc

    2006-01-01

    Transmembrane β-barrel (TMB) proteins are embedded in the outer membrane of Gram-negative bacteria, mitochondria and chloroplasts. The cellular location and functional diversity of β-barrel outer membrane proteins makes them an important protein class. At the present time, very few non-homologous TMB structures have been determined by X-ray diffraction because of the experimental difficulty encountered in crystallizing transmembrane (TM) proteins. The transFold web server uses pairwise inter-strand residue statistical potentials derived from globular (non-outer-membrane) proteins to predict the supersecondary structure of TMB. Unlike all previous approaches, transFold does not use machine learning methods such as hidden Markov models or neural networks; instead, transFold employs multi-tape S-attribute grammars to describe all potential conformations, and then applies dynamic programming to determine the global minimum energy supersecondary structure. The transFold web server not only predicts secondary structure and TMB topology, but is the only method which additionally predicts the side-chain orientation of transmembrane β-strand residues, inter-strand residue contacts and TM β-strand inclination with respect to the membrane. The program transFold currently outperforms all other methods for accuracy of β-barrel structure prediction. Available at . PMID:16844989

  3. 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

  4. 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

  5. Understanding self-assembled amphiphilic peptide supramolecular structures from primary structure helix propensity.

    PubMed

    Baumann, Martina K; Textor, Marcus; Reimhult, Erik

    2008-08-01

    Small amphiphilic peptides are attractive building blocks to design biocompatible supramolecular structures via self-assembly, with applications in, for example, drug delivery, tissue engineering, and nanotemplating. We address the influence of systematical changes in the amino acid sequence of such peptides on the self-assembled macromolecular structures. For cationic-head surfactant-like eight-residue peptides, the apolar tail amino acids were chosen to systematically vary the propensity to form an alpha-helical secondary structure while conserving the overall hydrophobicity of the sequence. Characterization of the supramolecular structures indicates that for short peptides a beta-sheet secondary structure correlates with ribbonlike assemblies while random-coil and alpha-helical secondary structures correlate with assembly of rods. PMID:18597507

  6. A single aldehyde group can serve as a structural element for recognition by transmembrane protein CD36.

    PubMed

    Tsuzuki, Satoshi; Amitsuka, Takahiko; Okahashi, Tatsuya; Kozai, Yuki; Matsumura, Shigenobu; Inoue, Kazuo; Fushiki, Tohru

    2016-07-01

    Transmembrane protein CD36 is considered to bind its distinct ligands such as long-chain fatty acids primarily by recognizing their terminal carboxyl moiety. In this study, we provide evidence that long-chain fatty aldehydes, such as oleic aldehyde, can be recognized by CD36. We suggest that a single aldehyde group may also serve as one of the structural elements recognizable by CD36. PMID:26923548

  7. 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

  8. 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

  9. Interactions of H562 in the S5 Helix with T618 and S621 in the Pore Helix Are Important Determinants of hERG1 Potassium Channel Structure and Function

    PubMed Central

    Lees-Miller, James P.; Subbotina, Julia O.; Guo, Jiqing; Yarov-Yarovoy, Vladimir; Noskov, Sergei Y.; Duff, Henry J.

    2009-01-01

    hERG1 is a member of the cyclic nucleotide binding domain family of K+ channels. Alignment of cyclic nucleotide binding domain channels revealed an evolutionary conserved sequence HwX(A/G)C in the S5 domain. We reasoned that histidine 562 in hERG1 could play an important structure-function role. To explore this role, we created in silica models of the hERG1 pore domain based on the KvAP crystal structure with Rosetta-membrane modeling and molecular-dynamics simulations. Simulations indicate that the H562 residue in the S5 helix spans the gap between the S5 helix and the pore helix, stabilizing the pore domain, and that mutation at the H562 residue leads to a disruption of the hydrogen bonding to T618 and S621, resulting in distortion of the selectivity filter. Analysis of the simulated point mutations at positions 562/618/621 showed that the reciprocal double mutations H562W/T618I would partially restore the orientation of the 562 residue. Matching hydrophobic interactions between mutated W562 residue and I618 partially compensate for the disrupted hydrogen bonding. Complementary in vitro electrophysiological studies confirmed the results of the molecular-dynamics simulations on single mutations at positions 562, 618, and 621. Experimentally, mutations of the H562 to tryptophan produced a functional channel, but with slowed deactivation and shifted V1/2 of activation. Furthermore, the double mutation T618I/H562W rescued the defects seen in activation, deactivation, and potassium selectivity seen with the H562W mutation. In conclusion, interactions between H562 in the S5 helix and amino acids in the pore helix are important determinants of hERG1 potassium channel function, as confirmed by theory and experiment. PMID:19413965

  10. Structural studies of Helix aspersa agglutinin complexed with GalNAc: A lectin that serves as a diagnostic tool.

    PubMed

    Pietrzyk, Agnieszka J; Bujacz, Anna; Mak, Paweł; Potempa, Barbara; Niedziela, Tomasz

    2015-11-01

    Lectins belong to a differentiated group of proteins known to possess sugar-binding properties. Due to this fact, they are interesting research targets in medical diagnostics. Helix aspersa agglutinin (HAA) is a lectin that recognizes the epitopes containing α-d-N-acetylgalactosamine (GalNAc), which is present at the surface of metastatic cancer cells. Although several reports have already described the use of HAA as a diagnostic tool, this protein was not characterized on the molecular level. Here, we present for the first time the structural information about lectin isolated from mucus of Helix aspersa (garden snail). The amino acid sequence of this agglutinin was determined by Edman degradation and tertiary as well as quaternary structure by X-ray crystallography. The high resolution crystal structure (1.38Å) and MALDI-TOF mass spectrometry analysis provide the detailed information about a large part of the HAA natural glycan chain. The topology of the GalNAc binding cleft and interaction with lectin are very well defined in the structure and fully confirmed by STD HSQC NMR spectroscopy. Together, this provides structural clues regarding HAA specificity and opens possibilities to rational modifications of this important diagnostic tool. PMID:26416237

  11. Structure Change from β-Strand and Turn to α-Helix in Histone H2A-H2B Induced by DNA Damage Response.

    PubMed

    Izumi, Yudai; Fujii, Kentaro; Wien, Frank; Houée-Lévin, Chantal; Lacombe, Sandrine; Salado-Leza, Daniela; Porcel, Erika; Masoud, Rawand; Yamamoto, Satoshi; Réfrégiers, Matthieu; Hervé du Penhoat, Marie-Anne; Yokoya, Akinari

    2016-07-12

    Using synchrotron radiation-based circular dichroism spectroscopy, we found that the DNA damage response induces an increase of α-helix structure and a decrease of β-strand and turn structures in histone H2A-H2B extracted from x-irradiated human HeLa cells. The structural alterations correspond to the assumption that an average of eight amino acid residues form new α-helix structures at 310 K. We propose the structural transition from β-strand and turn structures to an α-helix structure in H2A-H2B as a novel, to our knowledge, process involved in the DNA damage response. PMID:27410735

  12. The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins.

    PubMed Central

    Oberer, Monika; Zangger, Klaus; Prytulla, Stefan; Keller, Walter

    2002-01-01

    NMR and CD spectroscopy have been used to characterize, both structurally and dynamically, the 82-amino-acid ParD protein of the post-segregational killing module of the broad-host-range plasmid RP4/RK2. ParD occurs as a dimer in solution and exercises two different control functions; an autoregulatory function by binding to its own promoter P(parDE) and a plasmid-stabilizing function by inhibiting ParE toxicity in cells that express ParD and ParE. Analysis of the secondary structure based on the chemical-shift indices, sequential nuclear Overhauser enhancements (NOEs) and (3)J(Halpha-NH) scalar coupling constants showed that the N-terminal domain of ParD consists of a short beta-ribbon followed by three alpha-helices, demonstrating that ParD contains a ribbon-helix-helix fold, a DNA-binding motif found in a family of small prokaryotic repressors. (15)N longitudinal (T(1)) and transverse (T(2)) relaxation measurements and hetero nuclear NOEs showed that ParD is divided into two separate domains, a well-ordered N-terminal domain and a very flexible C-terminal domain. An increase in secondary structure was observed upon addition of trifluoroethanol, suggested to result from the formation of structured stretches in the C-terminal part of the protein. This is the first experimental evidence that the DNA-binding domain of ParD belongs to the ribbon-helix-helix fold family, and this structural motif is proposed to be present in functionally similar antidote proteins. PMID:11743881

  13. Crystal structures of SIRT3 reveal that the α2-α3 loop and α3-helix affect the interaction with long-chain acyl lysine.

    PubMed

    Gai, Wei; Li, He; Jiang, Hualiang; Long, Yaqiu; Liu, Dongxiang

    2016-09-01

    SIRT1-7 play important roles in many biological processes and age-related diseases. In addition to a NAD(+) -dependent deacetylase activity, they can catalyze several other reactions, including the hydrolysis of long-chain fatty acyl lysine. To study the binding modes of sirtuins to long-chain acyl lysines, we solved the crystal structures of SIRT3 bound to either a H3K9-myristoylated- or a H3K9-palmitoylated peptide. Interaction of SIRT3 with the palmitoyl group led to unfolding of the α3-helix. The myristoyl and palmitoyl groups bind to the C-pocket and an allosteric site near the α3-helix, respectively. We found that the residues preceding the α3-helix determine the size of the C-pocket. The flexibility of the α2-α3 loop and the plasticity of the α3-helix affect the interaction with long-chain acyl lysine. PMID:27501476

  14. 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

  15. Cu**+ Transporting ATPases: Structure of the Two Transmembrane Cu**+ Transport Sites

    SciTech Connect

    Gonzalez-Guerrero, M.; Eren, E.; Rawat, S.; Stemmler, T.L.; Arguello, J.M.

    2009-05-18

    Cu{sup +}-ATPases drive metal efflux from the cell cytoplasm. Paramount to this function is the binding of Cu{sup +} within the transmembrane region and its coupled translocation across the permeability barrier. Here, we describe the two transmembrane Cu{sup +} transport sites present in Archaeoglobus fulgidus CopA. Both sites can be independently loaded with Cu{sup +}. However, their simultaneous occupation is associated with enzyme turnover. Site I is constituted by two Cys in transmembrane segment (TM) 6 and a Tyr in TM7. An Asn in TM7 and Met and Ser in TM8 form Site II. Single site x-ray spectroscopic analysis indicates a trigonal coordination in both sites. This architecture is distinct from that observed in Cu{sup +}-trafficking chaperones and classical cuproproteins. The high affinity of these sites for Cu{sup +} (Site I K{sub {alpha}} = 1.3 fM{sup -1}, Site II K{sub {alpha}} = 1.1 fM{sup -1}), in conjunction with reversible direct Cu{sup +} transfer from chaperones, points to a transport mechanism where backward release of free Cu{sup +} to the cytoplasm is largely prevented.

  16. Mutational analysis of the control cable that mediates transmembrane signaling in the Escherichia coli serine chemoreceptor.

    PubMed

    Kitanovic, Smiljka; Ames, Peter; Parkinson, John S

    2011-10-01

    During transmembrane signaling by Escherichia coli Tsr, changes in ligand occupancy in the periplasmic serine-binding domain promote asymmetric motions in a four-helix transmembrane bundle. Piston displacements of the signaling TM2 helix in turn modulate the HAMP bundle on the cytoplasmic side of the membrane to control receptor output signals to the flagellar motors. A five-residue control cable joins TM2 to the HAMP AS1 helix and mediates conformational interactions between them. To explore control cable structural features important for signal transmission, we constructed and characterized all possible single amino acid replacements at the Tsr control cable residues. Only a few lesions abolished Tsr function, indicating that the chemical nature and size of the control cable side chains are not individually critical for signal control. Charged replacements at I214 mimicked the signaling consequences of attractant or repellent stimuli, most likely through aberrant structural interactions of the mutant side chains with the membrane interfacial environment. Prolines at residues 214 to 217 also caused signaling defects, suggesting that the control cable has helical character. However, proline did not disrupt function at G213, the first control cable residue, which might serve as a structural transition between the TM2 and AS1 helix registers. Hydrophobic amino acids at S217, the last control cable residue, produced attractant-mimic effects, most likely by contributing to packing interactions within the HAMP bundle. These results suggest a helix extension mechanism of Tsr transmembrane signaling in which TM2 piston motions influence HAMP stability by modulating the helicity of the control cable segment. PMID:21803986

  17. 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.

  18. The position of the Gly-xxx-Gly motif in transmembrane segments modulates dimer affinity.

    PubMed

    Johnson, Rachel M; Rath, Arianna; Deber, Charles M

    2006-12-01

    Although the intrinsic low solubility of membrane proteins presents challenges to their high-resolution structure determination, insight into the amino acid sequence features and forces that stabilize their folds has been provided through study of sequence-dependent helix-helix interactions between single transmembrane (TM) helices. While the stability of helix-helix partnerships mediated by the Gly-xxx-Gly (GG4) motif is known to be generally modulated by distal interfacial residues, it has not been established whether the position of this motif, with respect to the ends of a given TM segment, affects dimer affinity. Here we examine the relationship between motif position and affinity in the homodimers of 2 single-spanning membrane protein TM sequences: glycophorin A (GpA) and bacteriophage M13 coat protein (MCP). Using the TOXCAT assay for dimer affinity on a series of GpA and MCP TM segments that have been modified with either 4 Leu residues at each end or with 8 Leu residues at the N-terminal end, we show that in each protein, centrally located GG4 motifs are capable of stronger helix-helix interactions than those proximal to TM helix ends, even when surrounding interfacial residues are maintained. The relative importance of GG4 motifs in stabilizing helix-helix interactions therefore must be considered not only in its specific residue context but also in terms of the location of the interactive surface relative to the N and C termini of alpha-helical TM segments. PMID:17215886

  19. Structure of USP7 catalytic domain and three Ubl-domains reveals a connector α-helix with regulatory role.

    PubMed

    Kim, Robbert Q; van Dijk, Willem J; Sixma, Titia K

    2016-07-01

    Ubiquitin conjugation is an important signal in cellular pathways, changing the fate of a target protein, by degradation, relocalisation or complex formation. These signals are balanced by deubiquitinating enzymes (DUBs), which antagonize ubiquitination of specific protein substrates. Because ubiquitination pathways are critically important, DUB activity is often carefully controlled. USP7 is a highly abundant DUB with numerous targets that plays complex roles in diverse pathways, including DNA regulation, p53 stress response and endosomal protein recycling. Full-length USP7 switches between an inactive and an active state, tuned by the positioning of 5 Ubl folds in the C-terminal HUBL domain. The active state requires interaction between the last two Ubls (USP7(45)) and the catalytic domain (USP7(CD)), and this can be promoted by allosteric interaction from the first 3 Ubl domains of USP7 (USP7(123)) interacting with GMPS. Here we study the transition between USP7 states. We provide a crystal structure of USP7(CD123) and show that CD and Ubl123 are connected via an extended charged alpha helix. Mutational analysis is used to determine whether the charge and rigidity of this 'connector helix' are important for full USP7 activity. PMID:27183903

  20. New insights into human prostacyclin receptor structure and function through natural and synthetic mutations of transmembrane charged residues

    PubMed Central

    Stitham, J; Arehart, E; Gleim, S R; Li, N; Douville, K; Hwa, J

    2007-01-01

    Background and purpose: The human prostacyclin receptor (hIP), a G-protein coupled receptor (GPCR) expressed mainly on platelets and vascular smooth muscle cells, plays important protective roles in the cardiovascular system. We hypothesized that significant insights could be gained into the structure and function of the hIP through mutagenesis of its energetically unfavourably located transmembrane charged residues. Experimental approach: Within its putative transmembrane helices fourteen hydrophilic residues, both unique and conserved across GPCRs, were systematically mutated to assess for effects on receptor structure and function. Key results: Mutations of ten of the fourteen charged residues to alanine exhibited defective binding and/or activation. Key potential interactions were identified between 6 core residues; E1163.49-R1173.50 (salt bridge TMIII), D2747.35-R2797.40 (salt bridge TMVII), and D602.50-D2887.49 (H-bond network TMII-TMVII). Further detailed investigation of E1163.49 (TMIII) with mutation to a glutamine showed a 2.6-fold increase in agonist-independent basal activity. This increase in activity accounts for a proportion (∼13%) of full agonist induced activation. We further characterized two novel naturally occurring human mutations, R772.33C and R2797.40C recently identified in a 1455 human genomic DNA sample screen. The R772.33C variant appeared to exclusively affect expression, while the R2797.40C variant, exhibited considerable deficiencies in both agonist binding and activation. Conclusions and implications: Transmembrane charged residues play important roles in maintaining the hIP binding pocket and ensuring normal activation. The critical nature of these charged residues and the presence of naturally occurring mutations have important implications in the rational design of prostacyclin agonists for treating cardiovascular disease. PMID:17704830

  1. 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

  2. Correlating structure, dynamics, and function in transmembrane segment VII of the Na+/H+ exchanger isoform 1.

    PubMed

    Reddy, Tyler; Li, Xiuju; Fliegel, Larry; Sykes, Brian D; Rainey, Jan K

    2010-02-01

    We place (15)N nuclear magnetic resonance relaxation analysis and functional mutagenesis studies in the context of our previous structural and mutagenesis work to correlate structure, dynamics and function for the seventh transmembrane segment of the human Na(+)/H(+) exchanger isoform 1. Although G261-S263 was previously identified as an interruption point in the helical structure of this isolated transmembrane peptide in dodecylphosphocholine micelles, and rapid conformational exchange was implicated in the NOE measurements, the six (15)N labelled residues examined in this study all have similar dynamics on the ps-ns time scale. A mathematical model incorporating chemical exchange is the best fit for residues G261, L264, and A268. This implies that a segment of residues from G261 to A268 samples different conformations on the mus-ms time scale. Chemical exchange on an intermediate time scale is consistent with an alternating-access cycle where E262 is bent away from the cytosol during proton translocation by the exchanger. The functional importance of chemical exchange at G261-A268 is corroborated by the abrogated activity of the full-length exchanger with the bulky and restricting Ile substitutions F260I, G261I, E262I, S263I, and A268I. PMID:19595986

  3. A New LxxxA Motif in the Transmembrane Helix3 of Maize Aquaporins Belonging to the Plasma Membrane Intrinsic Protein PIP2 Group Is Required for Their Trafficking to the Plasma Membrane1[W][OPEN

    PubMed Central

    Chevalier, Adrien S.; Bienert, Gerd Patrick; Chaumont, François

    2014-01-01

    Aquaporins play important roles in maintaining plant water status under challenging environments. The regulation of aquaporin density in cell membranes is essential to control transcellular water flows. This work focuses on the maize (Zea mays) plasma membrane intrinsic protein (ZmPIP) aquaporin subfamily, which is divided into two sequence-related groups (ZmPIP1s and ZmPIP2s). When expressed alone in mesophyll protoplasts, ZmPIP2s are efficiently targeted to the plasma membrane, whereas ZmPIP1s are retained in the endoplasmic reticulum (ER). A protein domain-swapping approach was utilized to demonstrate that the transmembrane domain3 (TM3), together with the previously identified N-terminal ER export diacidic motif, account for the differential localization of these proteins. In addition to protoplasts, leaf epidermal cells transiently transformed by biolistic particle delivery were used to confirm and refine these results. By generating artificial proteins consisting of a single transmembrane domain, we demonstrated that the TM3 of ZmPIP1;2 or ZmPIP2;5 discriminates between ER and plasma membrane localization, respectively. More specifically, a new LxxxA motif in the TM3 of ZmPIP2;5, which is highly conserved in plant PIP2s, was shown to regulate its anterograde routing along the secretory pathway, particularly its export from the ER. PMID:24989232

  4. 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.

  5. Similar Structures to the E-to-H Helix Unit in the Globin-Like Fold are Found in Other Helical Folds

    PubMed Central

    Matsuoka, Masanari; Fujita, Aoi; Kawai, Yosuke; Kikuchi, Takeshi

    2014-01-01

    A protein in the globin-like fold contains six alpha-helices, A, B, E, F, G and H. Among them, the E-to-H helix unit (E, F, G and H helices) forms a compact structure. In this study, we searched similar structures to the E-to-H helix of leghomoglobin in the whole protein structure space using the Dali program. Several similar structures were found in other helical folds, such as KaiA/RbsU domain and Type III secretion system domain. These observations suggest that the E-to-H helix unit may be a common subunit in the whole protein 3D structure space. In addition, the common conserved hydrophobic residues were found among the similar structures to the E-to-H helix unit. Hydrophobic interactions between the conserved residues may stabilize the 3D structures of the unit. We also predicted the possible compact regions of the units using the average distance method. PMID:24970216

  6. Singlet Oxygen Attack on Guanine: Reactivity and Structural Signature within the B-DNA Helix.

    PubMed

    Dumont, Elise; Grüber, Raymond; Bignon, Emmanuelle; Morell, Christophe; Aranda, Juan; Ravanat, Jean-Luc; Tuñón, Iñaki

    2016-08-22

    Oxidatively generated DNA lesions are numerous and versatile, and have been the subject of intensive research since the discovery of 8-oxoguanine in 1984. Even for this prototypical lesion, the precise mechanism of formation remains elusive due to the inherent difficulties in characterizing high-energy intermediates. We have probed the stability of the guanine endoperoxide in B-DNA as a key intermediate and determined a unique activation free energy of around 6 kcal mol(-1) for the formation of the first C-O covalent bond upon the attack of singlet molecular oxygen ((1) O2 ) on the central guanine of a solvated 13 base-pair poly(dG-dC), described by means of quantum mechanics/molecular mechanics (QM/MM) simulations. The B-helix remains stable upon oxidation in spite of the bulky character of the guanine endoperoxide. Our modeling study has revealed the nature of the versatile (1) O2 attack in terms of free energy and shows a sensitivity to electrostatics and solvation as it involves a charge-separated intermediate. PMID:27440482

  7. 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

  8. Structure of amantadine-bound M2 transmembrane peptide of influenza A in lipid bilayers from magic-angle-spinning solid-state NMR: the role of Ser31 in amantadine binding.

    PubMed

    Cady, Sarah D; Mishanina, Tatiana V; Hong, Mei

    2009-01-30

    The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine, whose effectiveness has been abolished by a single-site mutation of Ser31 to Asn in the transmembrane domain of the protein. Recent high-resolution structures of the M2 transmembrane domain obtained from detergent-solubilized protein in solution and crystal environments gave conflicting drug binding sites. We present magic-angle-spinning solid-state NMR results of Ser31 and a number of other residues in the M2 transmembrane peptide (M2TMP) bound to lipid bilayers. Comparison of the spectra of the membrane-bound apo and complexed M2TMP indicates that Ser31 is the site of the largest chemical shift perturbation by amantadine. The chemical shift constraints lead to a monomer structure with a small kink of the helical axis at Gly34. A tetramer model is then constructed using the helix tilt angle and several interhelical distances previously measured on unoriented bilayer samples. This tetramer model differs from the solution and crystal structures in terms of the openness of the N-terminus of the channel, the constriction at Ser31, and the side-chain conformations of Trp41, a residue important for channel gating. Moreover, the tetramer model suggests that Ser31 may interact with amantadine amine via hydrogen bonding. While the apo and drug-bound M2TMP have similar average structures, the complexed peptide has much narrower linewidths at physiological temperature, indicating drug-induced changes of the protein dynamics in the membrane. Further, at low temperature, several residues show narrower lines in the complexed peptide than the apo peptide, indicating that amantadine binding reduces the conformational heterogeneity of specific residues. The differences of the current solid-state NMR structure of the bilayer-bound M2TMP from the detergent-based M2 structures suggest that the M2 conformation is sensitive to the environment, and care must be taken when interpreting

  9. The use of mould-templated surface structures for high-quality uniform-lying-helix liquid-crystal alignment

    NASA Astrophysics Data System (ADS)

    Outram, B. I.; Elston, S. J.; Tuffin, R.; Siemianowski, S.; Snow, B.

    2013-06-01

    The chiral-flexoelectrooptic effect in a Uniform Lying Helix (ULH) configuration provides a sub-millisecond in-plane rotation of the optic axis with the application of a transverse field. This enables displays with a wide viewing angle without costly in-plane-type electrodes. The salient challenge is one of alignment of the ULH, which is not topologically compatible with uniform alignment surface treatments. Here, we create a micro-grooved surface structure with features on the micron scale by using a replica-moulding technique. When the cell is assembled, the micro-grooves create channels, and using surface-energy considerations, we explain how and show experimentally that the channels align a cholesteric material in the ULH geometry with the helicoidal axis oriented parallel to the channels. The resultant alignment provides a high level of contrast between crossed polarizers and exhibits an electrooptic response with a switching time of the order of tens of microseconds.

  10. Synergistic transmembrane alignment of the antimicrobial heterodimer PGLa/magainin.

    PubMed

    Tremouilhac, Pierre; Strandberg, Erik; Wadhwani, Parvesh; Ulrich, Anne S

    2006-10-27

    The antimicrobial activity of amphipathic alpha-helical peptides is usually attributed to the formation of pores in bacterial membranes, but direct structural information about such a membrane-bound state is sparse. Solid state (2)H-NMR has previously shown that the antimicrobial peptide PGLa undergoes a concentration-dependent realignment from a surface-bound S-state to a tilted T-state. The corresponding change in helix tilt angle from 98 to 125 degrees was interpreted as the formation of PGLa/magainin heterodimers residing on the bilayer surface. Under no conditions so far, has an upright membrane-inserted I-state been observed in which a transmembrane helix alignment would be expected. Here, we have demonstrated that PGLa is able to assume such an I-state in a 1:1 mixture with magainin 2 at a peptide-to-lipid ratio as low as 1:100 in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol model membranes. This (2)H-NMR analysis is based on seven orientational constraints from Ala-3,3,3-d(3) substituted in a non-perturbing manner for four native Ala residues as well as two Ile and one Gly. The observed helix tilt of 158 degrees is rationalized by the formation of heterodimers. This structurally synergistic effect between the two related peptides from the skin of Xenopus laevis correlates very well with their known functional synergistic mode of action. To our knowledge, this example of PGLa is the first case where an alpha-helical antimicrobial peptide is directly shown to assume a transmembrane state that is compatible with the postulated toroidal wormhole pore structure. PMID:16877761

  11. Solution Structure and DNA-binding Properties of the Winged Helix Domain of the Meiotic Recombination HOP2 Protein*

    PubMed Central

    Moktan, Hem; Guiraldelli, Michel F.; Eyster, Craig A.; Zhao, Weixing; Lee, Chih-Ying; Mather, Timothy; Camerini-Otero, R. Daniel; Sung, Patrick; Zhou, Donghua H.; Pezza, Roberto J.

    2014-01-01

    The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination. PMID:24711446

  12. Solution Structure of an Alternate Conformation of Helix 27 from Escherichia Coli 16S rRNA†

    PubMed Central

    Spano, Meredith Newby; Walter, Nils G.

    2011-01-01

    Helix (H)27 of 16S ribosomal (r)RNA from Escherichia coli was dubbed the “switch helix” when mutagenesis suggested that two alternative base pair registers may have distinct functional roles in the bacterial ribosome. Although more recent genetic analyses suggest that H27 conformational switching is not required for translation, previous solution studies demonstrated that the isolated E. coli H27 can dynamically convert between the 885 and 888 conformations. Here, we have solved the NMR solution structure of a locked 888 conformation. NOE and residual dipolar coupling restraints reveal an architecture that markedly differs from that of the 885 conformation found in crystal structures of the bacterial ribosome. In place of the loop E motif that characterizes the 885 conformer and that the 888 conformer cannot adopt, we find evidence for an asymmetrical A-rich internal loop stabilized by stacking interactions among the unpaired A’s. Comparison of the isolated H27 888 solution structure with the 885 crystal structure within the context of the ribosome suggests a difference in overall length of H27 that presents one plausible reason for the absence of H27 conformational switching within the sterically confining ribosome. PMID:21442607

  13. 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.

  14. Transmembrane Signaling by the Aspartate Receptor: Engineered Disulfides Reveal Static Regions of the Subunit Interface†

    PubMed Central

    Chervitz, Stephen A.; Lin, Christina M.; Falke, Joseph J.

    2010-01-01

    Ligand binding to the periplasmic domain of the transmembrane aspartate receptor generates an intramolecular conformational change which spans the bilayer and ultimately signals the cytoplasmic CheA histidine kinase, thereby triggering chemotaxis. The receptor is a homodimer stabilized by the interface between its two identical subunits: the present study investigates the role of the periplasmic and transmembrane regions of this interface in the mechanism of transmembrane signaling. Free cysteines and disulfide bonds are engineered into selected interfacial positions, and the resulting effects on the transmembrane signal are assayed by monitoring in vitro regulation of kinase activity. Three of the 14 engineered cysteine pairs examined, as well as six of the 14 engineered disulfides, cause perturbations of the interface structure which essentially destroy transmembrane regulation of the kinase. The remaining 11 cysteine pairs, and eight engineered disulfides covalently linking the two subunits at locations spanning positions 18–75, are observed to retain significant transmembrane kinase regulation. The eight functional disulfides positively identify adjacent faces of the two N-terminal helices in the native receptor dimer and indicate that large regions of the periplasmic and transmembrane subunit interface remain effectively static during the transmembrane signal. The results are consistent with a model in which the subunit interface plays a structural role, while the second membrane-spanning helix transmits the ligand-induced signal across the bilayer to the kinase binding domain. The effects of engineered cysteines and disulfides on receptor methylation in vitro are also measured, enabling direct comparison of the in vitro methylation and phosphorylation assays. PMID:7626643

  15. Recent advances in helix-coil theory.

    PubMed

    Doig, Andrew J

    2002-12-10

    Peptide helices in solution form a complex mixture of all helix, all coil or, most frequently, central helices with frayed coil ends. In order to interpret experiments on helical peptides and make theoretical predictions on helices, it is therefore essential to use a helix-coil theory that takes account of this equilibrium. The original Zimm-Bragg and Lifson-Roig helix-coil theories have been greatly extended in the last 10 years to include additional interactions. These include preferences for the N-cap, N1, N2, N3 and C-cap positions, capping motifs, helix dipoles, side chain interactions and 3(10)-helix formation. These have been applied to determine energies for these preferences from experimental data and to predict the helix contents of peptides. This review discusses these newly recognised structural features of helices and how they have been included in helix-coil models. PMID:12488008

  16. Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

    PubMed Central

    Kobe, Bostjan; Center, Rob J.; Kemp, Bruce E.; Poumbourios, Pantelis

    1999-01-01

    Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Å resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation. PMID:10200260

  17. Detecting pore-lining regions in transmembrane protein sequences

    PubMed Central

    2012-01-01

    Background Alpha-helical transmembrane channel and transporter proteins play vital roles in a diverse range of essential biological processes and are crucial in facilitating the passage of ions and molecules across the lipid bilayer. However, the experimental difficulties associated with obtaining high quality crystals has led to their significant under-representation in structural databases. Computational methods that can identify structural features from sequence alone are therefore of high importance. Results We present a method capable of automatically identifying pore-lining regions in transmembrane proteins from sequence information alone, which can then be used to determine the pore stoichiometry. By labelling pore-lining residues in crystal structures using geometric criteria, we have trained a support vector machine classifier to predict the likelihood of a transmembrane helix being involved in pore formation. Results from testing this approach under stringent cross-validation indicate that prediction accuracy of 72% is possible, while a support vector regression model is able to predict the number of subunits participating in the pore with 62% accuracy. Conclusion To our knowledge, this is the first tool capable of identifying pore-lining regions in proteins and we present the results of applying it to a data set of sequences with available crystal structures. Our method provides a way to characterise pores in transmembrane proteins and may even provide a starting point for discovering novel routes of therapeutic intervention in a number of important diseases. This software is freely available as source code from: http://bioinf.cs.ucl.ac.uk/downloads/memsat-svm/. PMID:22805427

  18. Structural plasticity in the eight-helix fold of a trematode haemoglobin.

    PubMed

    Milani, Mario; Pesce, Alessandra; Dewilde, Sylvia; Ascenzi, Paolo; Moens, Luc; Bolognesi, Martino

    2002-04-01

    The three-dimensional structure of recombinant haemoglobin from the trematode Paramphistomum epiclitum, displaying the highest oxygen affinity so far observed for (non)vertebrate haemoglobins, has previously been determined at 1.17 A resolution (orthorhombic space group P2(1)2(1)2(1)). In the present communication, the three-dimensional structure of wild-type P. epiclitum haemoglobin is reported at 1.85 A resolution in a monoclinic crystal form (R factor = 16.1%, R(free) = 22.0%). Comparison of P. epiclitum (recombinant versus wild-type ferric Hb) structures in the two crystal forms shows structural differences in the haem proximal and distal sites which have not been reported for other known haemoglobin structures previously. PMID:11914507

  19. 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

  20. 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.

  1. A model for the [C+-GxC]n triple helix derived from observation of the C+-GxC base triplet in a crystal structure.

    PubMed

    Nunn, C M; Trent, J O; Neidle, S

    1997-10-13

    A molecular modelling study on the [C+-GxC]n triple helix is reported. We have observed the C+-GxC base triplet in the crystal structure of an oligonucleotide-drug complex, between the minor-groove drug netropsin and the decanucleotide d(CGCAATTGCG)2. The complex was crystallised at pH 7.0, but the crystal structure, at a resolution of 2.4 A, shows that a terminal cytosine has become protonated and participates in a parallel C+-GxC base triplet. The structure of this triplet and its associated sugar-phosphate backbones have been energy-refined and then used to generate a triple helix. This has characteristics of the B-type family of DNA structures for two strands, with the third, the C+ strand, having backbone conformations closer to the A family. PMID:9369239

  2. 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

  3. The importance of helix P1 stability for structural pre-organization and ligand binding affinity of the adenine riboswitch aptamer domain

    PubMed Central

    Nozinovic, Senada; Reining, Anke; Kim, Yong-Boum; Noeske, Jonas; Schlepckow, Kai; Wöhnert, Jens; Schwalbe, Harald

    2014-01-01

    We report here an in-depth characterization of the aptamer domain of the transcriptional adenine-sensing riboswitch (pbuE) by NMR and fluorescence spectroscopy. By NMR studies, the structure of two aptamer sequences with different lengths of the helix P1, the central element involved in riboswitch conformational switching, was characterized. Hydrogen-bond interactions could be mapped at nucleotide resolution providing information about secondary and tertiary structure, structure homogeneity and dynamics. Our study reveals that the elongation of helix P1 has pronounced effects not only on the local but on the global structure of the apo aptamer domain. The structural differences induced by stabilizing helix P1 were found to be linked to changes of the ligand binding affinity as revealed from analysis of kinetic and thermodynamic data obtained from stopped-flow fluorescence studies. The results provide new insight into the sequence-dependent fine tuning of the structure and function of purine-sensing riboswitches. PMID:24921630

  4. Structural Determinants of Binding the Seven-transmembrane Domain of the Glucagon-like Peptide-1 Receptor (GLP-1R).

    PubMed

    Yang, Dehua; de Graaf, Chris; Yang, Linlin; Song, Gaojie; Dai, Antao; Cai, Xiaoqing; Feng, Yang; Reedtz-Runge, Steffen; Hanson, Michael A; Yang, Huaiyu; Jiang, Hualiang; Stevens, Raymond C; Wang, Ming-Wei

    2016-06-17

    The glucagon-like peptide-1 receptor (GLP-1R) belongs to the secretin-like (class B) family of G protein-coupled receptors. Members of the class B family are distinguished by their large extracellular domain, which works cooperatively with the canonical seven-transmembrane (7TM) helical domain to signal in response to binding of various peptide hormones. We have combined structure-based site-specific mutational studies with molecular dynamics simulations of a full-length model of GLP-1R bound to multiple peptide ligand variants. Despite the high sequence similarity between GLP-1R and its closest structural homologue, the glucagon receptor (GCGR), nearly half of the 62 stably expressed mutants affected GLP-1R in a different manner than the corresponding mutants in GCGR. The molecular dynamics simulations of wild-type and mutant GLP-1R·ligand complexes provided molecular insights into GLP-1R-specific recognition mechanisms for the N terminus of GLP-1 by residues in the 7TM pocket and explained how glucagon-mimicking GLP-1 mutants restored binding affinity for (GCGR-mimicking) GLP-1R mutants. Structural analysis of the simulations suggested that peptide ligand binding mode variations in the 7TM binding pocket are facilitated by movement of the extracellular domain relative to the 7TM bundle. These differences in binding modes may account for the pharmacological differences between GLP-1 peptide variants. PMID:27059958

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Sensitivity of immune response quality to influenza helix 190 antigen structure displayed on a modular virus-like particle.

    PubMed

    Anggraeni, Melisa R; Connors, Natalie K; Wu, Yang; Chuan, Yap P; Lua, Linda H L; Middelberg, Anton P J

    2013-09-13

    Biomolecular engineering enables synthesis of improved proteins through synergistic fusion of modules from unrelated biomolecules. Modularization of peptide antigen from an unrelated pathogen for presentation on a modular virus-like particle (VLP) represents a new and promising approach to synthesize safe and efficacious vaccines. Addressing a key knowledge gap in modular VLP engineering, this study investigates the underlying fundamentals affecting the ability of induced antibodies to recognize the native pathogen. Specifically, this quality of immune response is correlated to the peptide antigen module structure. We modularized a helical peptide antigen element, helix 190 (H190) from the influenza hemagglutinin (HA) receptor binding region, for presentation on murine polyomavirus VLP, using two strategies aimed to promote H190 helicity on the VLP. In the first strategy, H190 was flanked by GCN4 structure-promoting elements within the antigen module; in the second, dual H190 copies were arrayed as tandem repeats in the module. Molecular dynamics simulation predicted that tandem repeat arraying would minimize secondary structural deviation of modularized H190 from its native conformation. In vivo testing supported this finding, showing that although both modularization strategies conferred high H190-specific immunogenicity, tandem repeat arraying of H190 led to a strikingly higher immune response quality, as measured by ability to generate antibodies recognizing a recombinant HA domain and split influenza virion. These findings provide new insights into the rational engineering of VLP vaccines, and could ultimately enable safe and efficacious vaccine design as an alternative to conventional approaches necessitating pathogen cultivation. PMID:23845811

  10. 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.

  11. Mycobacterium tuberculosis Rv0899 adopts a mixed α/β structure and does not form a transmembrane β-barrel

    PubMed Central

    Teriete, Peter; Yao, Yong; Kolodzik, Adrian; Yu, Jinghua; Song, Houhui; Niederweis, Michael; Marassi, Francesca M.

    2010-01-01

    The membrane protein, Rv0899 (OmpATb), from M. tuberculosis, has been proposed to act as an outer membrane porin and to contribute to the bacterium's adaptation to the acidic environment of the phagosome during infection. The gene is restricted to pathogenic mycobacteria and, thus, is an attractive candidate for the development of anti-TB chemotherapy. The 326-residue protein contains three domains: an N-terminal domain (residues 1-72) which includes a sequence of 20 hydrophobic amino acids that is required for membrane translocation; a central B domain (residues 73-200) with homology to the conserved putative lipid-binding BON (bacterial OsmY and nodulation) superfamily; and a C domain (residues 201-326) with homology to the OmpA-C-like superfamily of periplasmic peptidoglycan-binding sequences, found in several types of bacterial membrane proteins, including in the C-terminus of the E. coli outer membrane protein OmpA. We have characterized the structure and dynamics of the B and C domains, and have determined the three-dimensional structure of the B domain. Rv0899 does not form a transmembrane β-barrel. Residues 73 to 326 form a mixed α/β globular structure, encompassing two independently folded modules corresponding to the B and C domains connected by a flexible linker. The B domain folds with three parallel/antiparallel α-helices packed against six parallel/antiparallel β-strands which form a flat β-sheet. The core is hydrophobic while the exterior is polar and predominantly acidic. The structure of a BON homology domain is revealed here for the first time. In light of this unexpected structure, it is hard to reconcile an outer membrane porin activity with the central domain of the protein. The structure of the B domain and the overall architecture of the protein suggest alternative modes of membrane association. PMID:20199110

  12. Triple-helix DNA structural studies using a Love wave acoustic biosensor.

    PubMed

    Papadakis, George; Tsortos, Achilleas; Gizeli, Electra

    2009-12-15

    The development of sensors for detecting the conformation of surface-attached molecules is an emerging field with significance in the pharmaceutical industry and in drug design. In this work, triplex-forming oligos (TFOs), a separate class of non-natural DNA bending agents that can affect the mechanical properties of DNA through the formation of triple-helical structures of specific conformation and/or flexibility, are used as a model system in combination with an acoustic biosensor to determine molecular geometrical features. In practice, the degree of bending of a specific DNA target caused by a particular TFO was evaluated by measuring the ratio of acoustic energy change over phase change observed during the binding of pre-formed triplex DNA molecules to the device surface. The DNA bending angle derived via acoustic measurements is in excellent agreement with previously reported values using molecular biology techniques. The reported acoustic technique appears quite appealing for the biophysical study of DNA molecules providing rapid qualitative and quantitative information, at the same time holding promise to be developed as a high-throughput method for the evaluation of DNA conformational changes. PMID:19748772

  13. 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.

  14. 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.

  15. 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

  16. NMR Dynamics of Transmembrane and Intracellular Domains of p75NTR in Lipid-Protein Nanodiscs.

    PubMed

    Mineev, Konstantin S; Goncharuk, Sergey A; Kuzmichev, Pavel K; Vilar, Marçal; Arseniev, Alexander S

    2015-08-18

    P75NTR is a type I integral membrane protein that plays a key role in neurotrophin signaling. However, structural data for the receptor in various functional states are sparse and controversial. In this work, we studied the spatial structure and mobility of the transmembrane and intracellular parts of p75NTR, incorporated into lipid-protein nanodiscs of various sizes and compositions, by solution NMR spectroscopy. Our data reveal a high level of flexibility and disorder in the juxtamembrane chopper domain of p75NTR, which results in the motions of the receptor death domain being uncoupled from the motions of the transmembrane helix. Moreover, none of the intracellular domains of p75NTR demonstrated a propensity to interact with the membrane or to self-associate under the experimental conditions. The obtained data are discussed in the context of the receptor activation mechanism. PMID:26287629

  17. [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

  18. Inhibiting nucleation of amyloid structure in a huntingtin fragment by targeting α-helix rich oligomeric intermediates

    PubMed Central

    Mishra, Rakesh; Jayaraman, Murali; Roland, Bartholomew P.; Landrum, Elizabeth; Fullam, Timothy; Kodali, Ravindra; Thakur, Ashwani K.; Arduini, Irene; Wetzel, Ronald

    2011-01-01

    Although oligomeric intermediates are transiently formed in almost all known amyloid assembly reactions, their mechanistic roles are poorly understood. Recently we demonstrated a critical role for the 17 amino acid N-terminal segment (httNT) of huntingtin (htt) in oligomer-mediated amyloid assembly of htt N-terminal fragments. In this mechanism, the httNT segment forms the α-helix rich core of the oligomers, leaving most or all of each polyglutamine (polyQ) segment disordered and solvent-exposed. Nucleation of amyloid structure occurs within this local high concentration of disordered polyQ. Here we demonstrate the kinetic importance of httNT self-assembly by describing inhibitory httNT-containing peptides that appear to work by targeting nucleation within the oligomer fraction. These molecules inhibit amyloid nucleation by forming mixed oligomers with the httNT domains of polyQ-containing htt N-terminal fragments. In one class of inhibitor, nucleation is passively suppressed due to the reduced local concentration of polyQ within the mixed oligomer. In the other class, nucleation is actively suppressed by a proline-rich polyQ segment covalently attached to httNT. Studies with D-amino acid and scrambled sequence versions of httNT suggest that inhibition activity is strongly linked to the propensity of inhibitory peptides to make amphipathic α-helices. HttNT derivatives with C-terminal cell penetrating peptide segments, also exhibit excellent inhibitory activity. The httNT-based peptides described here, especially those with protease-resistant D-amino acids and/or with cell penetrating sequences, may prove useful as lead therapeutics for inhibiting nucleation of amyloid formation in Huntington’s disease. PMID:22178478

  19. Structural Elements in the Transmembrane and Cytoplasmic Domains of the Metal Transporter SLC30A10 Are Required for Its Manganese Efflux Activity.

    PubMed

    Zogzas, Charles E; Aschner, Michael; Mukhopadhyay, Somshuvra

    2016-07-29

    Homozygous mutations in SLC30A10 lead to the development of familial manganese-induced parkinsonism. We previously demonstrated that SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its trafficking and efflux activity. Interestingly, other transporters in the SLC30 family mediate zinc efflux. Determining the mechanisms that allow SLC30A10 to transport manganese, which are unclear, is essential to understand its role in parkinsonism. Here, we generated a predicted structure of SLC30A10, based on the structure of the bacterial zinc transporter YiiP, and performed functional studies. In YiiP, side chains of residues Asp-45 and Asp-49 in the second and His-153 and Asp-157 in the fifth transmembrane segments coordinate zinc and are required for transport. In SLC30A10, the corresponding residues are Asn-43 and Asp-47 in the second and His-244 and Asp-248 in the fifth transmembrane segments. Surprisingly, although alanine substitution of Asp-248 abolished manganese efflux, that of Asn-43 and Asp-47 did not. Instead, side chains of charged or polar residues adjacent to Asp-248 in the first (Glu-25) or fourth (Asn-127) transmembrane segments were required. Further analyses revealed that residues His-333 and His-350 in the cytoplasmic C-terminal domain were required for full activity. However, the C-terminal domain failed to transfer manganese transport capability to a related zinc transporter. Overall, our results indicate that residues in the transmembrane and C-terminal domains together confer optimal manganese transport capability to SLC30A10 and suggest that the mechanism of ion coordination in the transmembrane domain of SLC30A10 may be substantially different from that in YiiP/other SLC30 proteins. PMID:27307044

  20. Mechanism of helix induction in poly(4-carboxyphenyl isocyanide) with chiral amines and memory of the macromolecular helicity and its helical structures.

    PubMed

    Hase, Yoko; Nagai, Kanji; Iida, Hiroki; Maeda, Katsuhiro; Ochi, Noriaki; Sawabe, Kyoichi; Sakajiri, Koichi; Okoshi, Kento; Yashima, Eiji

    2009-08-01

    An optically inactive poly(4-carboxyphenyl isocyanide) (poly-1-H) changed its structure into the prevailing, one-handed helical structure upon complexation with optically active amines in dimethylsulfoxide (DMSO) and water, and the complexes show a characteristic induced circular dichroism in the polymer backbone region. Moreover, the macromolecular helicity induced in water and aqueous organic solutions containing more than 50 vol % water could be "memorized" even after complete removal of the chiral amines (h-poly-1b-H), while that induced in DMSO and DMSO-water mixtures containing less than 30 vol % water could not maintain the optical activity after removal of the chiral amines (poly-1a-H). We now report fully detailed studies of the helix induction mechanism with chiral amines and the memory of the macromolecular helicity in water and a DMSO-water mixture by various spectroscopic measurements, theoretical calculations, and persistence length measurements together with X-ray diffraction (XRD) measurements. From the spectroscopic results, such as circular dichroism (CD), absorption, IR, vibrational CD, and NMR of poly-1a-H, h-poly-1b-H, and original poly-1-H, we concluded that the specific configurational isomerization around the C horizontal lineN double bonds occurs during the helicity induction process in each solvent. In order to obtain the structural information, XRD measurements were done on the uniaxially oriented films of the corresponding methyl esters (poly-1-Me, poly-1a-Me, and h-poly-1b-Me) prepared from their liquid crystalline polymer solutions. On the basis of the XRD analyses, the most plausible helical structure of poly-1a-Me was proposed to be a 9-unit/5-turn helix with two monomer units as a repeating unit, and that of h-poly-1b-Me was proposed to be a 10-unit/3-turn helix consisting of one repeating monomer unit. The density functional theory calculations of poly(phenyl isocyanide), a model polymer of h-poly-1b-Me, afforded a 7-unit/2-turn

  1. 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.

  2. Hemolytic lectin CEL-III heptamerizes via a large structural transition from α-helices to a β-barrel during the transmembrane pore formation process.

    PubMed

    Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2014-05-01

    CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer. PMID:24652284

  3. Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process*

    PubMed Central

    Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2014-01-01

    CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer. PMID:24652284

  4. Deletion of the N- or C-Terminal Helix of Apolipophorin III To Create a Four-Helix Bundle Protein.

    PubMed

    Dwivedi, Pankaj; Rodriguez, Johana; Ibe, Nnejiuwa U; Weers, Paul M M

    2016-07-01

    Apolipophorin III (apoLp-III) is an exchangeable apolipoprotein found in insects and plays an important function in lipid transport. The protein has an unusual five-helix bundle architecture, deviating from the common four-helix bundle motif. To understand the role of the additional helix in apoLp-III, the N-terminal or C-terminal helix was deleted to create a putative four-helix bundle protein. While the protein lacking helix-1 could be expressed in bacteria albeit at reduced yields, apoLp-III lacking helix-5 could not be produced. Mutational analysis by truncating helix-5 showed that a minimum segment of approximately one-third of the C-terminal helix is required for protein expression. The variant lacking helix-5 was produced by inserting a methionine residue between helix-4 and -5; subsequent cyanogenbromide cleavage generated the four-helix variant. Both N- and C-terminal helix deletion variants displayed significantly reduced helical content, protein stability, and tertiary structure. Despite the significantly altered structure, the variants were still fully functional. The rate of dimyristoylphosphatidylcholine vesicle solubilization was enhanced 4-5-fold compared to the wild-type protein, and the deletion variants were effective in binding to lipolyzed low density lipoprotein thereby preventing lipoprotein aggregation. These results show that the additional helix of apoLp-III is not essential for lipid binding but is required for proper folding to keep the protein into a stable conformation. PMID:27280697

  5. v-SNARE transmembrane domains function as catalysts for vesicle fusion.

    PubMed

    Dhara, Madhurima; Yarzagaray, Antonio; Makke, Mazen; Schindeldecker, Barbara; Schwarz, Yvonne; Shaaban, Ahmed; Sharma, Satyan; Böckmann, Rainer A; Lindau, Manfred; Mohrmann, Ralf; Bruns, Dieter

    2016-01-01

    Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca(2+)-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle's outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion. PMID:27343350

  6. Structure and Dynamics of the Membrane-Bound Form of Pf1 Coat Protein: Implications of Structural Rearrangement for Virus Assembly

    PubMed Central

    Park, Sang Ho; Marassi, Francesca M.; Black, David; Opella, Stanley J.

    2010-01-01

    The three-dimensional structure of the membrane-bound form of the major coat protein of Pf1 bacteriophage was determined in phospholipid bilayers using orientation restraints derived from both solid-state and solution NMR experiments. In contrast to previous structures determined solely in detergent micelles, the structure in bilayers contains information about the spatial arrangement of the protein within the membrane, and thus provides insights to the bacteriophage assembly process from membrane-inserted to bacteriophage-associated protein. Comparisons between the membrane-bound form of the coat protein and the previously determined structural form found in filamentous bacteriophage particles demonstrate that it undergoes a significant structural rearrangement during the membrane-mediated virus assembly process. The rotation of the transmembrane helix (Q16–A46) around its long axis changes dramatically (by 160°) to obtain the proper alignment for packing in the virus particles. Furthermore, the N-terminal amphipathic helix (V2–G17) tilts away from the membrane surface and becomes parallel with the transmembrane helix to form one nearly continuous long helix. The spectra obtained in glass-aligned planar lipid bilayers, magnetically aligned lipid bilayers (bicelles), and isotropic lipid bicelles reflect the effects of backbone motions and enable the backbone dynamics of the N-terminal helix to be characterized. Only resonances from the mobile N-terminal helix and the C-terminus (A46) are observed in the solution NMR spectra of the protein in isotropic q > 1 bicelles, whereas only resonances from the immobile transmembrane helix are observed in the solid-state 1H/15N-separated local field spectra in magnetically aligned bicelles. The N-terminal helix and the hinge that connects it to the transmembrane helix are significantly more dynamic than the rest of the protein, thus facilitating structural rearrangement during bacteriophage assembly. PMID:20816058

  7. 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

  8. Multiwalled ice helixes and ice nanotubes

    PubMed Central

    Bai, Jaeil; Wang, Jun; Zeng, X. C.

    2006-01-01

    We report six phases of high-density nano-ice predicted to form within carbon nanotubes (CNTs) at high pressure. High-density nano-ice self-assembled within smaller-diameter CNT (17,0) exhibits a double-walled helical structure where the outer wall consists of four double-stranded helixes, which resemble a DNA double helix, and the inner wall is a quadruple-stranded helix. Four other double-walled nano-ices, self-assembled respectively in two larger-diameter CNTs (20,0 and 22,0), display tubular structure. Within CNT (24,0), the confined water can freeze spontaneously into a triple-walled helical nano-ice where the outer wall is an 18-stranded helix and the middle and inner walls are hextuple-stranded helixes. PMID:17170136

  9. Slow alpha helix formation during folding of a membrane protein.

    PubMed

    Riley, M L; Wallace, B A; Flitsch, S L; Booth, P J

    1997-01-01

    Very little is known about the folding of proteins within biological membranes. A "two-stage" model has been proposed on thermodynamic grounds for the folding of alpha helical, integral membrane proteins, the first stage of which involves formation of transmembrane alpha helices that are proposed to behave as autonomous folding domains. Here, we investigate alpha helix formation in bacteriorhodopsin and present a time-resolved circular dichroism study of the slow in vitro folding of this protein. We show that, although some of the protein's alpha helices form early, a significant part of the protein's secondary structure appears to form late in the folding process. Over 30 amino acids, equivalent to at least one of bacteriorhodopsin's seven transmembrane segments, slowly fold from disordered structures to alpha helices with an apparent rate constant of about 0.012 s-1 at pH 6 or 0.0077 s-1 at pH 8. This is a rate-limiting step in protein folding, which is dependent on the pH and the composition of the lipid bilayer. PMID:8993333

  10. Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation.

    PubMed

    Bai, Yonghong; Li, Min; Hwang, Tzyh-Chang

    2010-09-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily that functions as a chloride channel. Previous work has suggested that the external side of the sixth transmembrane segment (TM6) plays an important role in governing chloride permeation, but the function of the internal side remains relatively obscure. Here, on a cysless background, we performed cysteine-scanning mutagenesis and modification to screen the entire TM6 with intracellularly applied thiol-specific methanethiosulfonate reagents. Single-channel amplitude was reduced in seven cysteine-substituted mutants, suggesting a role of these residues in maintaining the pore structure for normal ion permeation. The reactivity pattern of differently charged reagents suggests that the cytoplasmic part of TM6 assumes a secondary structure of an alpha helix, and that reactive sites (341, 344, 345, 348, 352, and 353) reside in two neighboring faces of the helix. Although, as expected, modification by negatively charged reagents inhibits anion permeation, interestingly, modification by positively charged reagents of cysteine thiolates on one face (344, 348, and 352) of the helix affects gating. For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. These results corroborate the idea that a helix rotation of TM6, which has been proposed to be part of the molecular motions during transport cycles in other ABC transporters, is associated with gating of the CFTR pore. PMID:20805575

  11. Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation

    PubMed Central

    Bai, Yonghong; Li, Min

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the adenosine triphosphate–binding cassette (ABC) transporter superfamily that functions as a chloride channel. Previous work has suggested that the external side of the sixth transmembrane segment (TM6) plays an important role in governing chloride permeation, but the function of the internal side remains relatively obscure. Here, on a cysless background, we performed cysteine-scanning mutagenesis and modification to screen the entire TM6 with intracellularly applied thiol-specific methanethiosulfonate reagents. Single-channel amplitude was reduced in seven cysteine-substituted mutants, suggesting a role of these residues in maintaining the pore structure for normal ion permeation. The reactivity pattern of differently charged reagents suggests that the cytoplasmic part of TM6 assumes a secondary structure of an α helix, and that reactive sites (341, 344, 345, 348, 352, and 353) reside in two neighboring faces of the helix. Although, as expected, modification by negatively charged reagents inhibits anion permeation, interestingly, modification by positively charged reagents of cysteine thiolates on one face (344, 348, and 352) of the helix affects gating. For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. These results corroborate the idea that a helix rotation of TM6, which has been proposed to be part of the molecular motions during transport cycles in other ABC transporters, is associated with gating of the CFTR pore. PMID:20805575

  12. 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

  13. 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

  14. Structural waters in the minor and major grooves of DNA--a major factor governing structural adjustments of the A-T mini-helix.

    PubMed

    Zubatiuk, Tetiana; Shishkin, Oleg; Gorb, Leonid; Hovorun, Dmytro; Leszczynski, Jerzy

    2015-01-15

    The role of microhydration in structural adjustments of the AT-tract in B-DNA was studied at the B97-D/def2-SV(P) level. The (dA:dT)5 complexes with 10 water molecules in minor and 15 water molecules in major grooves were studied. The obtained network of hydrogen bonds revealed the dependence between the groove width and the types of water patterns. In the minor groove, the following patterns were observed: interstrand one-water bridges similar to that of the Dickerson "water spine" and interstrand two-water bridges. The network of structural waters in the major groove is more diverse than that in the minor groove, which agrees with crystallographic data. As the major groove is wider, it is enriched by water molecules forming two- and three-water bridges. Results suggest the nucleobase-water interactions in both grooves prevent AT-tract twisting and its "collapse" along the minor groove. Whereby, a helix structure with narrow minor and wide major grooves is formed. The structural waters affect the polynucleotide conformation so that it becomes similar to poly(dA)·poly(dT) in fibers and acquires features of the A-tracts in DNA in solution. We suggest that formation of specific water patterns in both grooves is the factor responsible for stabilization of A-tracts with a narrowed minor groove, leading in turn to their strong intrinsic bending in DNA. PMID:25495126

  15. 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.

  16. 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

  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. 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.

  19. 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

  20. Helix-helix interfaces and ligand binding.

    PubMed

    Kurochkina, Natalya; Choekyi, Tsering

    2011-08-21

    Helix-helix parallel interfaces can be characterized by certain combinations of amino acids, which repeatedly occur at core positions a and d (leucine zipper nomenclature) in homologous and nonhomologous proteins and influence interhelical angles. Applied for the prediction of interhelical angles in glutathione S-transferase, intracellular chloride channel and annexin molecules from various sources, correct results were achieved in 58 out of 62 proteins. Interhelical angles are found to correlate with the conformation of the glutathione S-transferase ligands glutathione, s-hexylglutathione, glutathione sulfonic acid, and glutathione-s-dinitrobenzene. PMID:21620863

  1. The Nuclear Magnetic Resonance of CCCC RNA Reveals a Right-Handed Helix, and Revised Parameters for AMBER Force Field Torsions Improve Structural Predictions from Molecular Dynamics

    PubMed Central

    2013-01-01

    The sequence dependence of RNA energetics is important for predicting RNA structure. Hairpins with Cn loops are consistently less stable than hairpins with other loops, which suggests the structure of Cn regions could be unusual in the “unfolded” state. For example, previous nuclear magnetic resonance (NMR) evidence suggested that polycytidylic acid forms a left-handed helix. In this study, UV melting experiments show that the hairpin formed by r(5′GGACCCCCGUCC) is less stable than r(5′GGACUUUUGUCC). NMR spectra for single-stranded C4 oligonucleotide, mimicking the unfolded hairpin loop, are consistent with a right-handed A-form-like helix. Comparisons between NMR spectra and molecular dynamics (MD) simulations suggest that recent reparametrizations, parm99χ_YIL and parm99TOR, of the AMBER parm99 force field improve the agreement between structural features for C4 determined by NMR and predicted by MD. Evidently, the force field revisions to parm99 improve the modeling of RNA energetics and therefore structure. PMID:23286901

  2. 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.

  3. Transmembrane Communication: General Principles and Lessons from the Structure and Function of the M2 Proton Channel, K+ Channels, and Integrin Receptors

    PubMed Central

    Grigoryan, Gevorg; Moore, David T.; DeGrado, William F.

    2013-01-01

    Signal transduction across biological membranes is central to life. This process generally happens through communication between different domains and hierarchical coupling of information. Here, we review structural and thermodynamic principles behind transmembrane (TM) signal transduction and discuss common themes. Communication between signaling domains can be understood in terms of thermodynamic and kinetic principles, and complex signaling patterns can arise from simple wiring of thermodynamically coupled domains. We relate this to functions of several signal transduction systems: the M2 proton channel from influenza A virus, potassium channels, integrin receptors, and bacterial kinases. We also discuss key features in the structural rearrangements responsible for signal transduction in these systems. PMID:21548783

  4. 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.

  5. Spontaneous assembly of double-stranded helicates from oligobipyridine ligands and copper(I) cations: structure of an inorganic double helix.

    PubMed Central

    Lehn, J M; Rigault, A; Siegel, J; Harrowfield, J; Chevrier, B; Moras, D

    1987-01-01

    Two oligobipyridine ligands containing two and three 2,2'-bipyridine subunits separated by 2-oxapropylene bridges have been synthesized and some of their complexation properties with metal ions have been investigated. In particular, with copper(I) they form, respectively, a dinuclear and a trinuclear complex containing two ligand molecules and two or three Cu(I) ions. In view of the pseudotetrahedral coordination geometry of Cu(I) X bis(bipyridine) sites and of NMR data indicating that the present complexes are chiral, one may assign to these dinuclear and trinuclear species a double-helical structure in which two molecular strands are wrapped around two or three Cu(I) ions, which hold them together. These complexes may thus be termed "double-stranded helicates." Determination of the crystal structure of the trinuclear species has confirmed that it is indeed an inorganic double helix, possessing characteristic features (helical parameters, stacking of bipyridine bases) reminiscent of the DNA double helix. This spontaneous formation of an organized structure by oligobipyridine ligands and suitable metal ions opens ways to the design and study of self-assembling systems presenting cooperativity and regulation features. Various further developments may be envisaged along organic, inorganic, and biochemical lines. PMID:3472223

  6. 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

  7. 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

  8. Probing and improving student's understanding of protein α-helix structure using targeted assessment and classroom interventions in collaboration with a faculty community of practice.

    PubMed

    Loertscher, Jennifer; Villafañe, Sachel M; Lewis, Jennifer E; Minderhout, Vicky

    2014-01-01

    The increasing availability of concept inventories and other assessment tools in the molecular life sciences provides instructors with myriad avenues to probe student understanding. For example, although molecular visualization is central to the study of biochemistry, a growing body of evidence suggests that students have substantial limitations in their ability to recognize and interpret basic features of biological macromolecules. In this study, a pre/posttest administered to students at diverse institutions nationwide revealed a robust incorrect idea about the location of the amino acid side chains in the protein α-helix structure. Because this incorrect idea was present even after a semester of biochemistry instruction at a range of institutions, an intervention was necessary. A community of expert biochemistry instructors collaborated to design two active learning classroom activities that systematically examine α-helix structure and function. Several participating faculty used one or both of the activities in their classrooms and some improvement of student understanding of this concept was observed. This study provides a model of how a community of instructors can work together using assessment data to inform targeted changes in instruction with the goal of improving student understanding of fundamental concepts. PMID:24599610

  9. 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

  10. 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.

  11. The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel.

    PubMed

    Meng, Xuan-Yu; Liu, Shengtang; Cui, Meng; Zhou, Ruhong; Logothetis, Diomedes E

    2016-01-01

    Inwardly rectifying K(+) (Kir) channels, serving as natural molecular nanomachines, transport potassium ions across the plasma membrane of the cell. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter (SF), the inner helix bundle crossing (HBC), and the cytosolic G loop) may serve as gates to control ion permeation. Our previous molecular dynamics simulations based on the crystal structure of a Kir3.1 chimera revealed the possible gating mechanism of the G loop gate. Here, we introduced a proline mutation in the inner helix and obtained a channel model of the open HBC gate. The open HBC gate reaches 0.6 nm in diameter, which allows partial hydrated K(+) ions to pass through. During the gating process, both the transmembrane helices TM1 and TM2 cooperatively rotate in a counterclockwise direction (viewed from the extracellular side) with the aid of the phospholipid PIP2. Only when all the transmembrane helices adopt a counterclockwise rotation, the HBC gate can be stabilized in the open state. We estimate that introduction of the proline mutation decreases the energy required to open the HBC gate by about 1.4 kcal/mol (ΔΔG). PMID:27439597

  12. The Molecular Mechanism of Opening the Helix Bundle Crossing (HBC) Gate of a Kir Channel

    PubMed Central

    Meng, Xuan-Yu; Liu, Shengtang; Cui, Meng; Zhou, Ruhong; Logothetis, Diomedes E.

    2016-01-01

    Inwardly rectifying K+ (Kir) channels, serving as natural molecular nanomachines, transport potassium ions across the plasma membrane of the cell. Along the ion permeation pathway, three relatively narrow regions (the selectivity filter (SF), the inner helix bundle crossing (HBC), and the cytosolic G loop) may serve as gates to control ion permeation. Our previous molecular dynamics simulations based on the crystal structure of a Kir3.1 chimera revealed the possible gating mechanism of the G loop gate. Here, we introduced a proline mutation in the inner helix and obtained a channel model of the open HBC gate. The open HBC gate reaches 0.6 nm in diameter, which allows partial hydrated K+ ions to pass through. During the gating process, both the transmembrane helices TM1 and TM2 cooperatively rotate in a counterclockwise direction (viewed from the extracellular side) with the aid of the phospholipid PIP2. Only when all the transmembrane helices adopt a counterclockwise rotation, the HBC gate can be stabilized in the open state. We estimate that introduction of the proline mutation decreases the energy required to open the HBC gate by about 1.4 kcal/mol (ΔΔG). PMID:27439597

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. Current-voltage characteristics of seven-helix proteins from a cubic array of amino acids

    NASA Astrophysics Data System (ADS)

    Alfinito, Eleonora; Reggiani, Lino

    2016-06-01

    The electrical properties of a set of seven-helix transmembrane proteins, whose space arrangement [three-dimensional (3D) structure] is known, are investigated by using regular arrays of the amino acids. These structures, specifically cubes, have topological features similar to those shown by the chosen proteins. The theoretical results show a good agreement between the predicted current-voltage characteristics obtained from a cubic array and those obtained from a detailed 3D structure. The agreement is confirmed by available experiments on bacteriorhodopsin. Furthermore, all the analyzed proteins are found to share the same critical behavior of the voltage-dependent conductance and of its variance. In particular, the cubic arrangement evidences a short plateau of the excess conductance and its variance at high voltages. The results of the present investigation show the possibility to predict the I -V characteristics of a multiple-protein sample even in the absence of detailed knowledge of the proteins' 3D structure.

  19. 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

  20. 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.

  1. 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.

  2. Solution NMR studies reveal the location of the second transmembrane domain of the human sigma-1 receptor

    PubMed Central

    Ortega-Roldan, Jose Luis; Ossa, Felipe; Amin, Nader T.; Schnell, Jason R.

    2015-01-01

    The sigma-1 receptor (S1R) is a ligand-regulated membrane chaperone protein associated with endoplasmic reticulum stress response, and modulation of ion channel activities at the plasma membrane. We report here a solution NMR study of a S1R construct (S1R(Δ35)) in which only the first transmembrane domain and the eight-residue N-terminus have been removed. The second transmembrane helix is found to be composed of residues 91–107, which corresponds to the first steroid binding domain-like region. The cytosolic domain is found to contain three helices, and the secondary structure and backbone dynamics of the chaperone domain are consistent with that determined previously for the chaperone domain alone. The position of TM2 provides a framework for ongoing studies of S1R ligand binding and oligomerisation. PMID:25647032

  3. Structure of a Ca2+/CaM:Kv7.4 (KCNQ4) B helix complex provides insight into M-current modulation

    PubMed Central

    Xu, Qiang; Chang, Aram; Tolia, Alexandra; Minor, Daniel L.

    2012-01-01

    Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage-gated potassium channels. Channels from this family produce neuronal M-currents and cardiac and auditory IKS currents, and harbor mutations that cause arrhythmias, epilepsy, and deafness. Despite extensive functional characterization, biochemical and structural details of the interaction between CaM and the channel have remained elusive. Here, we show that both apo-CaM and Ca2+/CaM bind to the C-terminal tail of the neuronal channel Kv7.4 (KCNQ4), which is involved both hearing and mechanosensation. Interactions between apo-CaM and the Kv7.4 tail involve two C-terminal tail segments, known as the A and B segments, whereas the interaction between Ca2+/CaM and the Kv7.4 C-terminal tail requires only the B segment. Biochemical studies show that the calcium dependence of the CaM:B segment interaction is conserved in all Kv7 subtypes. X-ray crystallographic determination of the structure of the Ca2+/CaM:Kv7.4 B segment complex shows that Ca2+/CaM wraps around the Kv7.4 B segment, which forms an α-helix, in an antiparallel orientation that embodies a variation of the classic 1-14 Ca2+/CaM interaction motif. Taken together with the context of prior studies, our data suggest a model for modulation of neuronal Kv7 channels involving a calcium-dependent conformational switch from an apo-CaM form that bridges the A and B segments to a Ca2+/CaM form bound to the B-helix. The structure presented here also provides a context for a number of disease causing mutations and for further dissection of the mechanisms by which CaM controls Kv7 function. PMID:23178170

  4. 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

  5. Super D-helix

    NASA Astrophysics Data System (ADS)

    Cho, Jin-Ho; Oh, Phillial

    2001-11-01

    We study the ``Myers effect'' for a bunch of D1-branes with type-IIB superstrings moving in one direction along the branes. We show that the ``blown-up'' configuration is a helical D1-brane, which is self-supported from collapse by the axial momentum flow. The tilting angle of the helix is determined by the number of D1-branes. The radius of the helix is stabilized to a certain value depending on the number of D1-branes and the momentum carried by type-IIB superstrings. This helix is actually a T-dual version of the supertube recently found as the ``blown-up'' configuration of a bunch of type-IIA superstrings carrying a D0-brane charge. It is shown that the helical D1 configuration preserves one-quarter of the supersymmetry of the type-IIB Minkowski vacuum.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. Facile transition between 3(10)- and alpha-helix: structures of 8-, 9-, and 10-residue peptides containing the -(Leu-Aib-Ala)2-Phe-Aib- fragment.

    PubMed Central

    Karle, I. L.; Flippen-Anderson, J. L.; Gurunath, R.; Balaram, P.

    1994-01-01

    A structural transition from a 3(10)-helix to an alpha-helix has been characterized at high resolution for an octapeptide segment located in 3 different sequences. Three synthetic peptides, decapeptide (A) Boc-Aib-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, nonapeptide (B) Boc-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, and octapeptide (C) Boc-(Leu-Aib-Ala)2-Phe-Aib-OMe, are completely helical in their respective crystals. At 0.9 A resolution, R factors for A, B, and C are 8.3%, 5.4%, and 7.3%, respectively. The octapeptide and nonapeptide form ideal 3(10)-helices with average torsional angles phi(N-C alpha) and psi(C alpha-C') of -57 degrees, -26 degrees C and -60 degrees, -27 degrees for B. The 10-residue peptide (A) begins as a 3(10)-helix and abruptly changes to an alpha-helix at carbonyl O(3), which is the acceptor for both a 4-->1 hydrogen bond with N(6)H and a 5-->1 hydrogen with N(7)H, even though the last 8 residues have the same sequence in all 3 peptides. The average phi, psi angles in the decapeptide are -58 degrees, -28 degrees for residues 1-3 and -63 degrees, -41 degrees for residues 4-10. The packing of helices in the crystals does not provide any obvious reason for the transition in helix type. Fourier transform infrared studies in the solid state also provide evidence for a 3(10)- to alpha-helix transition with the amide I band appearing at 1,656-1,657 cm-1 in the 9- and 10-residue peptides, whereas in shorter sequences the band is observed at 1,667 cm-1. PMID:7833814

  12. 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

  13. [Quaternion-based Characterization of Protein α Helix].

    PubMed

    Xu, Yonghong; Chu, Zefei

    2016-02-01

    This paper proposes a method based on quaternion for characterization a helix of proteins. The method defines the parameter called Quaternion Helix Axis Spherical Distance (QHASD) on the basis of mapping protein Cα frames' helical axis onto a unit sphere, and uses QHASD to characterize the a helix of the protein secondary structure. Application of this method has been verified based on the PDBselect database, with an a helix characterization accuracy of 91.7%. This method possesses significant advantages of high detection accuracy, low computation and clear geometric significance. PMID:27382757

  14. 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

  15. THE FIRST TRANSMEMBRANE REGION OF THE BETA CHAIN STABILIZES THE TETRAMERIC FcεRI COMPLEX

    PubMed Central

    Singleton, Theresa E.; Platzer, Barbara; Dehlink, Eleonora; Fiebiger, Edda

    2009-01-01

    The family of activating immune receptors stabilizes via the 3-helix assembly principle. A charged basic transmembrane residue interacts with two charged acidic transmembrane residues and forms a 3-helix interface to stabilize receptor complexes in the lipid bilayer. One family member, the high affinity receptor for IgE, FcεRI, is a key regulator of immediate allergic responses. Tetrameric FcεRI consists of the IgE-binding α-chain, the multimembrane spanning β-chain and a dimer of the γ-subunit (FcεRγ). Comparative analysis of these seven transmembrane regions indicates that FcεRI does not meet the charge requirements for the 3-helix assembly mechanism. We performed alanine mutagenesis to show that the only basic amino acid in the transmembrane regions, βK97, is not involved in FcεRI stabilization or surface up-regulation, a hallmark function of the β-chain. Even a βK97E mutant is functional despite four negatively charged acidic amino acids in the transmembrane regions. Using truncation mutants, we demonstrate that the first uncharged transmembrane domain of the β-chain contains the interface for receptor stabilization. In vitro translation experiments depict the first transmembrane region as the internal signal peptide of the β-chain. We also show that this β-chain domain can function as a cleavable signal peptide when used as a leader peptide for a Type I protein. Our results provide evidence that tetrameric FcεRI does not assemble according to the 3-helix assembly principle. We conclude that receptors formed with multispanning proteins use different mechanisms of shielding transmembrane charged amino acids. PMID:19406478

  16. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2013-08-01

    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.

  18. 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

  19. 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.

  20. 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 P1. 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

  1. FMRFamide receptors of Helix aspersa

    SciTech Connect

    Payza, K.

    1988-01-01

    A receptor binding assay and an isolated heart bioassay were used to identify and characterize the FMRFamide receptors in Helix. In the heart bioassay, FMRFamide increased myocardial contraction force. A potent FMRFamide analog, desaminoTyr-Phe-norLeu-arg-Phe-amide (daYFnLRFamide), was used as a radioiodinated receptor ligand. The high affinity binding of {sup 125}I-daYFnLRFamide at 0{degree}C to Helix brain membranes was reversible, saturable, pH-dependent and specific, with a K{sub D} of 13-14 nM. A lower affinity (245 nM) site was also observed. Radioligand binding sites were also identified in the heart, male reproductive organs and digestive organs. The structure-activity relations (SAR) of cardiostimulation correlated with the specificity of {sup 125}I-daYFnLRFamide binding to brain and heart receptors. The SAR were similar to those of other molluscan FMRFamide bioassays, except that they showed a marked preference for some analogs with blocked amino-terminals.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Structure of Leishmania donovani coronin coiled coil domain reveals an antiparallel 4 helix bundle with inherent asymmetry.

    PubMed

    Nayak, Ashok Ranjan; Karade, Sharanbasappa Shrimant; Srivastava, Vijay Kumar; Rana, Ajay Kumar; Gupta, C M; Sahasrabuddhe, Amogh A; Pratap, J Venkatesh

    2016-07-01

    Coiled coils are ubiquitous structural motifs that serve as a platform for protein-protein interactions and play a central role in myriad physiological processes. Though the formation of a coiled coil requires only the presence of suitably spaced hydrophobic residues, sequence specificities have also been associated with specific oligomeric states. RhXXhE is one such sequence motif, associated with parallel trimers, found in coronins and other proteins. Coronin, present in all eukaryotes, is an actin-associated protein involved in regulating actin turnover. Most eukaryotic coronins possess the RhXXhE trimerization motif. However, a unique feature of parasitic kinetoplastid coronin is that the positions of R and E are swapped within their coiled coil domain, but were still expected to form trimers. To understand the role of swapped motif in oligomeric specificity, we determined the X-ray crystal structure of Leishmania donovani coronin coiled coil domain (LdCoroCC) at 2.2Å, which surprisingly, reveals an anti-parallel tetramer assembly. Small angle X-ray scattering studies and chemical crosslinking confirm the tetramer in solution and is consistent with the oligomerization observed in the full length protein. Structural analyses reveal that LdCoroCC possesses an inherent asymmetry, in that one of the helices of the bundle is axially shifted with respect to the other three. The analysis also identifies steric reasons that cause this asymmetry. The bundle adapts an extended a-d-e core packing, the e residue being polar (with an exception) which results in a thermostable bundle with polar and apolar interfaces, unlike the existing a-d-e core antiparallel homotetramers with apolar core. Functional implications of the anti-parallel association in kinetoplastids are discussed. PMID:26940672

  7. 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

  8. Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications.

    PubMed

    Gentzsch, Martina; Aleksandrov, Andrei; Aleksandrov, Luba; Riordan, John R

    2002-09-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) contains two nucleotide-binding domains (NBDs) or ATP-binding cassettes (ABCs) that characterize a large family of membrane transporters. Although the three-dimensional structures of these domains from several ABC proteins have been determined, this is not the case for CFTR, and hence the domains are defined simply on the basis of sequence alignment. The functional C-terminal boundary of NBD1 of CFTR was located by analysis of chloride channel function [Chan, Csanady, Seto-Young, Nairn and Gadsby (2000) J. Gen. Physiol. 116, 163-180]. However, the boundary between the C-terminal end of NBD2 and sequences further downstream in the whole protein, that are important for its cellular localization and endocytotic turnover, has not been defined. We have now done this by assaying the influence of progressive C-terminal truncations on photolabelling of NBD2 by 8-azido-ATP, which reflects hydrolysis, as well as binding, at that domain, and on NBD2-dependent channel gating itself. The boundary defined in this way is between residues 1420 and 1424, which corresponds to the final beta-strand in aligned NBDs whose structures have been determined. Utilization of this information should facilitate the generation of monodisperse NBD2 polypeptides for structural analysis, which until now has not been possible. The established boundary includes within NBD2 a hydrophobic patch of four residues (1413-1416) previously shown to be essential for CFTR maturation and stability [Gentzsch and Riordan (2001) J. Biol. Chem. 276, 1291-1298]. This hydrophobic cluster is conserved in most ABC proteins, and on alignment with ones of known structure constitutes the penultimate beta-strand of the domain which is likely to participate in essential structure-stabilizing beta-sheet formation. PMID:12020354

  9. Functional analysis of the C-terminal boundary of the second nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator and structural implications.

    PubMed Central

    Gentzsch, Martina; Aleksandrov, Andrei; Aleksandrov, Luba; Riordan, John R

    2002-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) contains two nucleotide-binding domains (NBDs) or ATP-binding cassettes (ABCs) that characterize a large family of membrane transporters. Although the three-dimensional structures of these domains from several ABC proteins have been determined, this is not the case for CFTR, and hence the domains are defined simply on the basis of sequence alignment. The functional C-terminal boundary of NBD1 of CFTR was located by analysis of chloride channel function [Chan, Csanady, Seto-Young, Nairn and Gadsby (2000) J. Gen. Physiol. 116, 163-180]. However, the boundary between the C-terminal end of NBD2 and sequences further downstream in the whole protein, that are important for its cellular localization and endocytotic turnover, has not been defined. We have now done this by assaying the influence of progressive C-terminal truncations on photolabelling of NBD2 by 8-azido-ATP, which reflects hydrolysis, as well as binding, at that domain, and on NBD2-dependent channel gating itself. The boundary defined in this way is between residues 1420 and 1424, which corresponds to the final beta-strand in aligned NBDs whose structures have been determined. Utilization of this information should facilitate the generation of monodisperse NBD2 polypeptides for structural analysis, which until now has not been possible. The established boundary includes within NBD2 a hydrophobic patch of four residues (1413-1416) previously shown to be essential for CFTR maturation and stability [Gentzsch and Riordan (2001) J. Biol. Chem. 276, 1291-1298]. This hydrophobic cluster is conserved in most ABC proteins, and on alignment with ones of known structure constitutes the penultimate beta-strand of the domain which is likely to participate in essential structure-stabilizing beta-sheet formation. PMID:12020354

  10. A Conserved Ectodomain-Transmembrane Domain Linker Motif Tunes the Allosteric Regulation of Cell Surface Receptors.

    PubMed

    Schmidt, Thomas; Ye, Feng; Situ, Alan J; An, Woojin; Ginsberg, Mark H; Ulmer, Tobias S

    2016-08-19

    In many families of cell surface receptors, a single transmembrane (TM) α-helix separates ecto- and cytosolic domains. A defined coupling of ecto- and TM domains must be essential to allosteric receptor regulation but remains little understood. Here, we characterize the linker structure, dynamics, and resulting ecto-TM domain coupling of integrin αIIb in model constructs and relate it to other integrin α subunits by mutagenesis. Cellular integrin activation assays subsequently validate the findings in intact receptors. Our results indicate a flexible yet carefully tuned ecto-TM coupling that modulates the signaling threshold of integrin receptors. Interestingly, a proline at the N-terminal TM helix border, termed NBP, is critical to linker flexibility in integrins. NBP is further predicted in 21% of human single-pass TM proteins and validated in cytokine receptors by the TM domain structure of the cytokine receptor common subunit β and its P441A-substituted variant. Thus, NBP is a conserved uncoupling motif of the ecto-TM domain transition and the degree of ecto-TM domain coupling represents an important parameter in the allosteric regulation of diverse cell surface receptors. PMID:27365391

  11. Canonical Azimuthal Rotations and Flanking Residues Constrain the Orientation of Transmembrane Helices

    PubMed Central

    Sánchez-Muñoz, Orlando L.; Strandberg, Erik; Esteban-Martín, E.; Grage, Stephan L.; Ulrich, Anne S.; Salgado, Jesús

    2013-01-01

    In biological membranes the alignment of embedded proteins provides crucial structural information. The transmembrane (TM) parts have well-defined secondary structures, in most cases α-helices and their orientation is given by a tilt angle and an azimuthal rotation angle around the main axis. The tilt angle is readily visualized and has been found to be functionally relevant. However, there exist no general concepts on the corresponding azimuthal rotation. Here, we show that TM helices prefer discrete rotation angles. They arise from a combination of intrinsic properties of the helix geometry plus the influence of the position and type of flanking residues at both ends of the hydrophobic core. The helical geometry gives rise to canonical azimuthal angles for which the side chains of residues from the two ends of the TM helix tend to have maximum or minimum immersion within the membrane. This affects the preferential position of residues that fall near hydrophobic/polar interfaces of the membrane, depending on their hydrophobicity and capacity to form specific anchoring interactions. On this basis, we can explain the orientation and dynamics of TM helices and make accurate predictions, which correspond well to the experimental values of several model peptides (including dimers), and TM segments of polytopic membrane proteins. PMID:23561527

  12. Signal transmission through the CXC chemokine receptor 4 (CXCR4) transmembrane helices.

    PubMed

    Wescott, Melanie P; Kufareva, Irina; Paes, Cheryl; Goodman, Jason R; Thaker, Yana; Puffer, Bridget A; Berdougo, Eli; Rucker, Joseph B; Handel, Tracy M; Doranz, Benjamin J

    2016-08-30

    The atomic-level mechanisms by which G protein-coupled receptors (GPCRs) transmit extracellular ligand binding events through their transmembrane helices to activate intracellular G proteins remain unclear. Using a comprehensive library of mutations covering all 352 residues of the GPCR CXC chemokine receptor 4 (CXCR4), we identified 41 amino acids that are required for signaling induced by the chemokine ligand CXCL12 (stromal cell-derived factor 1). CXCR4 variants with each of these mutations do not signal properly but remain folded, based on receptor surface trafficking, reactivity to conformationally sensitive monoclonal antibodies, and ligand binding. When visualized on the structure of CXCR4, the majority of these residues form a continuous intramolecular signaling chain through the transmembrane helices; this chain connects chemokine binding residues on the extracellular side of CXCR4 to G protein-coupling residues on its intracellular side. Integrated into a cohesive model of signal transmission, these CXCR4 residues cluster into five functional groups that mediate (i) chemokine engagement, (ii) signal initiation, (iii) signal propagation, (iv) microswitch activation, and (v) G protein coupling. Propagation of the signal passes through a "hydrophobic bridge" on helix VI that coordinates with nearly every known GPCR signaling motif. Our results agree with known conserved mechanisms of GPCR activation and significantly expand on understanding the structural principles of CXCR4 signaling. PMID:27543332

  13. 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

  14. 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

  15. 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.

  16. 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 (N cyt/C exo 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. 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

  18. Transmembrane allosteric coupling of the gates in a potassium channel

    PubMed Central

    Wylie, Benjamin J.; Bhate, Manasi P.; McDermott, Ann E.

    2014-01-01

    It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98–103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening. PMID:24344306

  19. 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

  20. v-SNARE transmembrane domains function as catalysts for vesicle fusion

    PubMed Central

    Dhara, Madhurima; Yarzagaray, Antonio; Makke, Mazen; Schindeldecker, Barbara; Schwarz, Yvonne; Shaaban, Ahmed; Sharma, Satyan; Böckmann, Rainer A; Lindau, Manfred

    2016-01-01

    Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca2+-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle’s outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion. DOI: http://dx.doi.org/10.7554/eLife.17571.001 PMID:27343350

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. Effective harmonic approach to helix proteins

    NASA Astrophysics Data System (ADS)

    Choi, Sung Nam Peter

    Recent laser-induced experiments have shown that on the time scale of long range tertiary contact formation, the kinetics of the helix-coil interconversion are very rapid (1). The notion that secondary structures form first before long range tertiary contacts are made, suggests that the helix-coil interconversion is one key in understanding the protein folding problem. We developed a method of generating an alpha-helix conformation of any residue using AMBER 4.0, the Protein Data Bank (PDB), and a series of pattern matching translation and rotation operations. We have chosen to study Alanine for its highly non polar nature, and Glutamine for its highly polar characteristic. Using the information obtained from the above method, analysis of vibrational lattice dynamics are carried out on polymers Poly-alpha-L alanine (PLA) and Poly-alpha-L glutamic acid (PLGA). The lattice dynamics method called Modified Self- consistent Harmonic Approach theory (MSHA) was used in carrying out the analysis of the PLA and PLGA helix-coil transition. MSHA was originally developed for the DNA molecule by Prohofsky et al. In this model, the molecule is considered as a one dimensional, repeating unit cell of an infinite helix. The lattice was modeled with harmonic force constants. Anharmonicity of hydrogen bond force constants at different temperatures are introduced along with the effects of water on the polar PLGA and the non polar PLA. Using MSHA, we calculated the opening bond probabilities of PLGA at different temperatures. The experimentally observed helix to coil relaxation time τ* of PLGA was inversely compared to our results with a correlation coefficient of 0.8424 (5, 13, 58). These experiments were done at temperatures ranging from 295 K to 310 K. Our calculation also yielded critical melting temperatures of Tc=317 K for PLGA and Tc=347 K for PLA. The calculated acoustic compressional velocities were 4.78 km/s and 4.84 km/s for PLA and PLGA respectively. Our calculation

  7. 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

  8. 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, α(M)β(2) 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

  9. 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.

  10. The double helix and the 'wronged heroine'.

    PubMed

    Maddox, Brenda

    2003-01-23

    In 1962, James Watson, Francis Crick and Maurice Wilkins received the Nobel prize for the discovery of the structure of DNA. Notably absent from the podium was Rosalind Franklin, whose X-ray photographs of DNA contributed directly to the discovery of the double helix. Franklin's premature death, combined with misogynist treatment by the male scientific establishment, cast her as a feminist icon. This myth overshadowed her intellectual strength and independence both as a scientist and as an individual. PMID:12540909

  11. Marginally hydrophobic transmembrane α-helices shaping membrane protein folding

    PubMed Central

    De Marothy, Minttu T; Elofsson, Arne

    2015-01-01

    Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices—implying a dynamic relationship between membrane proteins and their environment. PMID:25970811

  12. Structural determinants of human proton-coupled folate transporter oligomerization: role of GXXXG motifs and identification of oligomeric interfaces at transmembrane domains 3 and 6

    PubMed Central

    Wilson, Mike R.; Kugel, Sita; Huang, Jenny; Wilson, Lucas J.; Wloszczynski, Patrick A.; Ye, Jun; Matherly, Larry H.; Hou, Zhanjun

    2016-01-01

    The human proton-coupled folate transporter (hPCFT) is expressed in solid tumours and is active at pHs characterizing the tumour microenvironment. Recent attention focused on exploiting hPCFT for targeting solid tumours with novel cytotoxic anti-folates. hPCFT has 12 transmembrane domains (TMDs) and forms homo-oligomers with functional significance. The hPCFT primary sequence includes GXXXG motifs in TMD2 (G93XXXG97) and TMD4 (G155XXXG159). To investigate roles of these motifs in hPCFT function, stability and surface expression, we mutated glycine to leucine to generate single or multiple substitution mutants. Only the G93L and G159L mutants preserved substantial [3H]methotrexate (Mtx) transport when expressed in hPCFT-null (R1-11) HeLa cells. Transport activity of the glycine-to-leucine mutants correlated with surface hPCFT by surface biotinylation and confocal microscopy with ECFP*-tagged hPCFTs, suggesting a role for GXXXG in hPCFT stability and intracellular trafficking. When co-expressed in R1-11 cells, haemagglutinin-tagged glycine-to-leucine mutants and His10-tagged wild-type (WT) hPCFT co-associated on nickel affinity columns, suggesting that the GXXXG motifs are not directly involved in hPCFT oligomerization. This was substantiated by in situ FRET experiments with co-expressed ECFP*- and YFP-tagged hPCFT. Molecular modelling of dimeric hPCFT structures showed juxtaposed TMDs 2, 3, 4 and 6 as potential structural interfaces between monomers. hPCFT cysteine insertion mutants in TMD3 (Q136C and L137C) and TMD6 (W213C, L214C, L224C, A227C, F228C, F230C and G231C) were expressed in R1-11 cells and cross-linked with 1,6-hexanediyl bismethanethiosulfonate, confirming TMD juxtapositions. Altogether, our results imply that TMDs 3 and 6 provide critical interfaces for formation of hPCFT oligomers, which might be facilitated by the GXXXG motifs in TMD2 and TMD4. PMID:25877470

  13. 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.

  14. The role of transmembrane span 2 in the structure and function of subunit a of the ATP synthase from Escherichia coli.

    PubMed

    DeLeon-Rangel, Jessica; Zhang, Di; Vik, Steven B

    2003-10-01

    The importance of the second transmembrane span of subunit a of the ATP synthase from Escherichia coli has been established by two approaches. First, biochemical analysis of five cysteine-substitution mutants, four of which were previously constructed for labeling experiments, revealed that only D119C, found within the second transmembrane span, was deleterious to ATP synthase function. This mutant had a greatly reduced growth yield, indicating inefficient ATP synthesis, but it retained a significant level of ATP-driven proton translocation and sensitivity to N,N(')-dicyclohexyl-carbodiimide, indicating more robust function in the direction of ATP hydrolysis. Second, the entire second transmembrane span was probed by alanine-insertion mutagenesis at six different positions, from residues 98 to 122. Insertions at the central four positions from residues 107 to 117 resulted in the inability to grow on succinate minimal medium, although normal levels of membrane-bound ATPase activity and significant levels of subunit a were detected. Double mutants were constructed with a mutation that permits cross-linking to the b subunit. Cross-linked products in the mutant K74C/114iA were seen, indicating no major disruption of the a-b interface due to the insertion at 114. Analysis of the K74C/110iA double mutant indicated that K74C is a partial suppressor of 110iA. In summary, the results support a model in which the amino-terminal, cytoplasmic end of the second transmembrane span has close contact with subunit b, while the carboxy-terminal, periplasmic end is important for proton translocation. PMID:13679083

  15. Transmembrane heme delivery systems

    PubMed Central

    Goldman, Barry S.; Beck, David L.; Monika, Elizabeth M.; Kranz, Robert G.

    1998-01-01

    Heme proteins play pivotal roles in a wealth of biological processes. Despite this, the molecular mechanisms by which heme traverses bilayer membranes for use in biosynthetic reactions are unknown. The biosynthesis of c-type cytochromes requires that heme is transported to the bacterial periplasm or mitochondrial intermembrane space where it is covalently ligated to two reduced cysteinyl residues of the apocytochrome. Results herein suggest that a family of integral membrane proteins in prokaryotes, protozoans, and plants act as transmembrane heme delivery systems for the biogenesis of c-type cytochromes. The complete topology of a representative from each of the three subfamilies was experimentally determined. Key histidinyl residues and a conserved tryptophan-rich region (designated the WWD domain) are positioned at the site of cytochrome c assembly for all three subfamilies. These histidinyl residues were shown to be essential for function in one of the subfamilies, an ABC transporter encoded by helABCD. We believe that a directed heme delivery pathway is vital for the synthesis of cytochromes c, whereby heme iron is protected from oxidation via ligation to histidinyl residues within the delivery proteins. PMID:9560218

  16. 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.

  17. 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

  18. 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

  19. Translocation of α-helix chains through a nanopore

    NASA Astrophysics Data System (ADS)

    Yang, Zhiyong; Li, Shiben; Zhang, Linxi; ur Rehman, Ateeq; Liang, Haojun

    2010-10-01

    The translocation of α-helix chains through a nanopore is studied through Langevin dynamics simulations. The α-helix chains exhibit several different characteristics about their average translocation times and the α-helix structures when they transport through the nanopores under the driving forces. First, the relationship between average translocation times τ and the chain length N satisfies the scaling law, τ ˜Nα, and the scaling exponent α depends on the driving force f for the small forces while it is close to the Flory exponent (ν) in the other force regions. For the chains with given chain lengths, it is observed that the dependence of the average translocation times can be expressed as τ ˜f-1/2 for the small forces while can be described as τ ˜f in the large force regions. Second, for the large driving force, the average number of α-helix structures Nh decreases first and then increases in the translocation process. The average waiting time of each bead, especially of the first bead, is also dependent on the driving forces. Furthermore, an elasticity spring model is presented to reasonably explain the change of the α-helix number during the translocation and its elasticity can be locally damaged by the large driving forces. Our results demonstrate the unique behaviors of α-helix chains transporting through the pores, which can enrich our insights into and knowledge on biopolymers transporting through membranes.

  20. A Classification of Basic Helix-Loop-Helix Transcription Factors of Soybean

    PubMed Central

    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

  1. Helix Nucleation by the Smallest Known α-Helix in Water.

    PubMed

    Hoang, Huy N; Driver, Russell W; Beyer, Renée L; Hill, Timothy A; D de Araujo, Aline; Plisson, Fabien; Harrison, Rosemary S; Goedecke, Lena; Shepherd, Nicholas E; Fairlie, David P

    2016-07-11

    Cyclic pentapeptides (e.g. Ac-(cyclo-1,5)-[KAXAD]-NH2 ; X=Ala, 1; Arg, 2) in water adopt one α-helical turn defined by three hydrogen bonds. NMR structure analysis reveals a slight distortion from α-helicity at the C-terminal aspartate caused by torsional restraints imposed by the K(i)-D(i+4) lactam bridge. To investigate this effect on helix nucleation, the more water-soluble 2 was appended to N-, C-, or both termini of a palindromic peptide ARAARAARA (≤5 % helicity), resulting in 67, 92, or 100 % relative α-helicity, as calculated from CD spectra. From the C-terminus of peptides, 2 can nucleate at least six α-helical turns. From the N-terminus, imperfect alignment of the Asp5 backbone amide in 2 reduces helix nucleation, but is corrected by a second unit of 2 separated by 0-9 residues from the first. These cyclic peptides are extremely versatile helix nucleators that can be placed anywhere in 5-25 residue peptides, which correspond to most helix lengths in protein-protein interactions. PMID:27226426

  2. The infrared dichroism of transmembrane helical polypeptides.

    PubMed Central

    Axelsen, P H; Kaufman, B K; McElhaney, R N; Lewis, R N

    1995-01-01

    Polarized attenuated total internal reflectance techniques were applied to study the infrared dichroism of the amide I transition moment in two membrane-bound peptides that are known to form oriented transmembrane helices: gramicidin A in a supported phospholipid monolayer and Ac-Lys2-Leu24-Lys2-amide (L24) in oriented multibilayers. These studies were performed to test the ability of these techniques to determine the orientation of these peptides, to verify the value of optical parameters used to calculate electric field strengths, to examine the common assumptions regarding the amide I transition moment orientation, and to ascertain the effect of surface imperfections on molecular disorder. The two peptides exhibit marked differences in the shape and frequency of their amide I absorption bands. Yet both peptides are highly ordered and oriented with their helical axes perpendicular to the membrane surface. In the alpha-helix formed by L24, there is evidence for a mode with type E1 symmetry contributing to amide I, and the amide I transition moment must be more closely aligned with the peptide C=O (< 34 degrees) than earlier studies have suggested. These results indicate that long-standing assumptions about the orientation of amide I in a peptide require some revision, but that in general, infrared spectroscopy yields reliable information about the orientation of membrane-bound helical peptides. Images FIGURE 1 PMID:8599683

  3. 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.

  4. 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.

  5. The structure of GlpF, a glycerol conducting channel.

    PubMed

    Fu, Dax; Libson, Andrew; Stroud, Robert

    2002-01-01

    The passage of water or small neutral solutes across the cell membrane in animals, plants and bacteria is facilitated by a family of homologous membrane channels, variously known as aquaporins though perhaps more correctly as aquaglyceroporins. The glycerol facilitator (GlpF) is a 28 kDa aquaglyceroporin that catalyses transmembrane diffusion of glycerol and certain linear polyhydric alcohols in Escherichia coli. X-ray crystallographic analysis of GlpF to 2.2 A resolution revealed an alpha-barrel structure, surrounded by six full-length transmembrane helices and two half-spanning helices that are joined head-to-head in the middle of the membrane. These helices are arranged to a quasi twofold manner relative to the central membrane plane, where highly conserved residues make helix-to-helix contacts that stabilize the relative position and orientation of the helices in the structure. This sequence-structure correlation suggests that the evolutionary divergence of aquaporins and aquaglyceroporins is constrained by a conserved structural framework within which specialized function may be developed. Three glycerol molecules were resolved in the central channel through the GlpF monomer, thereby defining a transmembrane channel for glycerol permeation. The structure of glycerol GlpF complex provides insight into the chemical basis for transmembrane selective permeability. PMID:12027015

  6. The Transmembrane Domain of CEACAM1-4S Is a Determinant of Anchorage Independent Growth and Tumorigenicity

    PubMed Central

    Lawson, Erica L.; Mills, David R.; Brilliant, Kate E.; Hixson, Douglas C.

    2012-01-01

    CEACAM1 is a multifunctional Ig-like cell adhesion molecule expressed by epithelial cells in many organs. CEACAM1-4L and CEACAM1-4S, two isoforms produced by differential splicing, are predominant in rat liver. Previous work has shown that downregulation of both isoforms occurs in rat hepatocellular carcinomas. Here, we have isolated an anchorage dependent clone, designated 253T-NT that does not express detectable levels of CEACAM1. Stable transfection of 253-NT cells with a wild type CEACAM1-4S expression vector induced an anchorage independent growth in vitro and a tumorigenic phenotype in vivo. These phenotypes were used as quantifiable end points to examine the functionality of the CEACAM1-4S transmembrane domain. Examination of the CEACAM1 transmembrane domain showed N-terminal GXXXG dimerization sequences and C-terminal tyrosine residues shown in related studies to stabilize transmembrane domain helix-helix interactions. To examine the effects of transmembrane domain mutations, 253-NT cells were transfected with transmembrane domain mutants carrying glycine to leucine or tyrosine to valine substitutions. Results showed that mutation of transmembrane tyrosine residues greatly enhanced growth in vitro and in vivo. Mutation of transmembrane dimerization motifs, in contrast, significantly reduced anchorage independent growth and tumorigenicity. 253-NT cells expressing CEACAM1-4S with both glycine to leucine and tyrosine to valine mutations displayed the growth-enhanced phenotype of tyrosine mutants. The dramatic effect of transmembrane domain mutations constitutes strong evidence that the transmembrane domain is an important determinant of CEACAM1-4S functionality and most likely by other proteins with transmembrane domains containing dimerization sequences and/or C-terminal tyrosine residues. PMID:22235309

  7. Lipid solvation effects contribute to the affinity of Gly-xxx-Gly motif-mediated helix-helix interactions.

    PubMed

    Johnson, Rachel M; Rath, Arianna; Melnyk, Roman A; Deber, Charles M

    2006-07-18

    Interactions between transmembrane helices are mediated by the concave Gly-xxx-Gly motif surface. Whether Gly residues per se are sufficient for selection of this motif has not been established. Here, we used the in vivo TOXCAT assay to measure the relative affinities of all 18 combinations of Gly, Ala, and Ser "small-xxx-small" mutations in glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP) homodimers. Affinity values were compared with the accessibility to a methylene-sized probe of the total surface area of each helix monomer as a measure of solvation by membrane components. A strong inverse correlation was found between nonpolar-group lipid accessibility and dimer affinity (R = 0.75 for GpA, p = 0.013, and R = 0.81 for MCP, p = 0.004), suggesting that lipid as a poor membrane protein solvent, conceptually analogous to water in soluble protein folding, can contribute to dimer stability and help to define helix-helix interfaces. PMID:16834324

  8. Transmembrane aromatic amino acid distribution in P-glycoprotein. A functional role in broad substrate specificity.

    PubMed

    Pawagi, A B; Wang, J; Silverman, M; Reithmeier, R A; Deber, C M

    1994-01-14

    Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity. In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues. Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore. In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces. Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding. The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization. PMID:7904655

  9. 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

  10. 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.

  11. Helix/Coil Nucleation: A Local Response to Global Demands

    PubMed Central

    Vorov, Oleg K.; Livesay, Dennis R.; Jacobs, Donald J.

    2009-01-01

    Abstract A complete description of protein structure and function must include a proper treatment of mechanisms that lead to cooperativity. The helix/coil transition serves as a simple example of a cooperative folding process, commonly described by a nucleation-propagation mechanism. The prevalent view is that coil structure must first form a short segment of helix in a localized region despite paying a free energy cost (nucleation). Afterward, helical structure propagates outward from the nucleation site. Both processes entail enthalpy-entropy compensation that derives from the loss in conformational entropy on helix formation with concomitant gain in favorable interactions. Nucleation-propagation models inherently assume that cooperativity arises from a sequential series of local events. An alternative distance constraint model asserts there is a direct link between available degrees of freedom and cooperativity through the nonadditivity in conformational entropy. That is, helix nucleation is a concerted manifestation of rigidity propagating through atomic structure. The link between network rigidity and nonadditivity of conformational entropy is shown in this study by solving the distance constraint model using a simple global constraint counting approximation. Cooperativity arises from competition between excess and deficiency in available degrees of freedom in the coil and helix states respectively. PMID:19948130

  12. Free Energy Landscape of Lipid Interactions with Regulatory Binding Sites on the Transmembrane Domain of the EGF Receptor

    PubMed Central

    2016-01-01

    Lipid molecules can bind to specific sites on integral membrane proteins, modulating their structure and function. We have undertaken coarse-grained simulations to calculate free energy profiles for glycolipids and phospholipids interacting with modulatory sites on the transmembrane helix dimer of the EGF receptor within a lipid bilayer environment. We identify lipid interaction sites at each end of the transmembrane domain and compute interaction free energy profiles for lipids with these sites. Interaction free energies ranged from ca. −40 to −4 kJ/mol for different lipid species. Those lipids (glycolipid GM3 and phosphoinositide PIP2) known to modulate EGFR function exhibit the strongest binding to interaction sites on the EGFR, and we are able to reproduce the preference for interaction with GM3 over other glycolipids suggested by experiment. Mutation of amino acid residues essential for EGFR function reduce the binding free energy of these key lipid species. The residues interacting with the lipids in the simulations are in agreement with those suggested by experimental (mutational) studies. This approach provides a generalizable tool for characterizing the interactions of lipids that bind to specific sites on integral membrane proteins. PMID:27109430

  13. High power, high frequency helix TWT's

    NASA Astrophysics Data System (ADS)

    Sloley, H. J.; Willard, J.; Paatz, S. R.; Keat, M. J.

    The design and performance characteristics of a 34-GHz pulse tube capable of 75 W peak power output at 30 percent duty cycle and a broadband CW tube are presented. Particular attention is given to the engineering problems encountered during the development of the tubes, including the suppression of backward wave oscillation, the design of electron guns for small-diameter high-current beams, and the thermal capability of small helix structures. The discussion also covers the effects of various design parameters and choice of engineering materials on the ultimate practical limit of power and gain at the operating frequencies. Measurements are presented for advanced experimental tubes.

  14. Effects of the oncogenic V(664)E mutation on membrane insertion, structure, and sequence-dependent interactions of the Neu transmembrane domain in micelles and model membranes: an integrated biophysical and simulation study.

    PubMed

    Beevers, Andrew J; Nash, Anthony; Salazar-Cancino, Martha; Scott, David J; Notman, Rebecca; Dixon, Ann M

    2012-03-27

    Receptor tyrosine kinases bind ligands such as cytokines, hormones, and growth factors and regulate key cellular processes, including cell division. They are also implicated in the development of many types of cancer. One such example is the Neu receptor tyrosine kinase found in rats (homologous to the human ErbB2 protein), which can undergo a valine to glutamic acid (V(664)E) mutation at the center of its α-helical transmembrane domain. This substitution results in receptor activation and oncogenesis. The molecular basis of this dramatic change in behavior upon introduction of the V(664)E mutation has been difficult to pin down, with conflicting results reported in the literature. Here we report the first quantitative, thermodynamic analysis of dimerization and biophysical characterization of the rat Neu transmembrane domain and several mutants in a range of chemical environments. These data have allowed us to identify the effects of the V(664)E mutation in the isolated TM domain with respect to protein-protein and protein-lipid interactions, membrane insertion, and secondary structure. We also report the results from a 100 ns atomistic molecular dynamics simulation of the Neu transmembrane domain in a model membrane bilayer (dipalmitoylphosphatidylcholine). The results from simulation and experiment are in close agreement and suggest that, in the model systems investigated, the V(664)E mutation leads to a weakening of the TM dimer and a change in sequence-dependent interactions. These results are contrary to recent results obtained in mammalian membranes, and the implications of this are discussed. PMID:22385253

  15. 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

  16. 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

  17. 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.

  18. 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

  19. Functional analysis of the transmembrane domain in paramyxovirus F protein-mediated membrane fusion

    PubMed Central

    Bissonnette, Mei Lin Z.; Donald, Jason E.; DeGrado, William F.; Jardetzky, Theodore S.; Lamb, Robert A.

    2009-01-01

    To enter cells enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 (PIV5) mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the PIV5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion. PMID:19121325

  20. 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.

  1. Molecular Mechanism of Holin Transmembrane Domain I in Pore Formation and Bacterial Cell Death.

    PubMed

    Lella, Muralikrishna; Kamilla, Soumya; Jain, Vikas; Mahalakshmi, Radhakrishnan

    2016-04-15

    Bacterial cell lysis during bacteriophage infection is timed by perfect orchestration between components of the holin-endolysin cassette. In bacteria, progressively accumulating holin in the inner membrane, retained in its inactive form by antiholin, is triggered into active hole formation, resulting in the canonical host cell lysis. However, the molecular mechanism of regulation and physical basis of pore formation in the mycobacterial cell membrane by D29 mycobacteriophage holin, particularly in the nonexistence of a known antiholin, is poorly understood. In this study, we report, for the first time, the use of fluorescence resonance transfer measurements to demonstrate that the first transmembrane domain (TM1) of D29 holin undergoes a helix ↔ β-hairpin conformational interconversion. We validate that this structural malleability is mediated by a centrally positioned proline and is responsible for controlled TM1 self-association in membrana, in the presence of a proton gradient across the lipid membrane. We demonstrate that TM1 is sufficient for bacterial growth inhibition. The biological effect of D29 holin structural alteration is presented as a holin self-regulatory mechanism, and its implications are discussed in the context of holin function. PMID:26701742

  2. Membrane-inserted conformation of transmembrane domain 4 of divalent-metal transporter.

    PubMed Central

    Li, Hongyan; Li, Fei; Sun, Hongzhe; Qian, Zhong Ming

    2003-01-01

    Divalent-metal transporter 1 (DMT1) is involved in the intestinal iron absorption and in iron transport in the transferrin cycle. It transports metal ions at low pH ( approximately 5.5), but not at high pH (7.4), and the transport is a proton-coupled process. Previously it has been shown that transmembrane domain 4 (TM4) is crucial for the function of this protein. Here we provide the first direct experimental evidence for secondary-structural features and membrane insertions of a 24-residue peptide, corresponding to TM4 of DMT1 (DMTI-TM4), in various membrane-mimicking environments by the combined use of CD and NMR spectroscopies. The peptide mainly adopts an alpha-helical structure in trifluoroethanol, SDS and dodecylphosphocholine micelles, and dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol small unilamellar vesicles. It has been demonstrated from both Halpha secondary shifts and nuclear-Overhauser-enhancement (NOE) connectivities that the peptide is well folded into an alpha-helix from Val(8) to Lys(23) in SDS micelles at pH 4.0, whereas the N-terminus is highly flexible. The alpha-helical content estimated from NMR data is in agreement with that extracted from CD simulations. The highest helicity was observed in the anionic phospholipids [1,2-dimyristoyl- sn -glycero-3-[phospho-rac -(1-glycerol)

  3. Structural model for the protein-translocating element of the twin-arginine transport system

    PubMed Central

    Rodriguez, Fernanda; Rouse, Sarah L.; Tait, Claudia E.; Harmer, Jeffrey; De Riso, Antonio; Timmel, Christiane R.; Sansom, Mark S. P.; Berks, Ben C.; Schnell, Jason R.

    2013-01-01

    The twin-arginine translocase (Tat) carries out the remarkable process of translocating fully folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Tat is required for bacterial pathogenesis and for photosynthesis in plants. TatA, the protein-translocating element of the Tat system, is a small transmembrane protein that assembles into ring-like oligomers of variable size. We have determined a structural model of the Escherichia coli TatA complex in detergent solution by NMR. TatA assembly is mediated entirely by the transmembrane helix. The amphipathic helix extends outwards from the ring of transmembrane helices, permitting assembly of complexes with variable subunit numbers. Transmembrane residue Gln8 points inward, resulting in a short hydrophobic pore in the center of the complex. Simulations of the TatA complex in lipid bilayers indicate that the short transmembrane domain distorts the membrane. This finding suggests that TatA facilitates protein transport by sensitizing the membrane to transient rupture. PMID:23471988

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  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. A Membrane Proximal Helix in the Cytosolic Domain of the Human APP Interacting Protein LR11/SorLA Deforms Liposomes

    PubMed Central

    Gill, Richard L.; Wang, Xingsheng; Tian, Fang

    2014-01-01

    Over the last decade, compelling evidence has linked the development of Alzheimer’s disease (AD) to defective intracellular trafficking of the amyloid precursor protein (APP). Faulty APP trafficking results in an overproduction of Aβ peptides, which is generally agreed to be the primary cause of AD-related pathogenesis. LR11 (SorLA), a type I transmembrane sorting receptor, has emerged as a key regulator of APP trafficking and processing. It directly interacts with APP and diverts it away from amyloidogenic processing. The 54-residue cytosolic domain of LR11 is essential for its proper intracellular localization and trafficking which, in turn, determines the fate of APP. Here, we have found a surprising membrane-proximal amphipathic helix in the cytosolic domain of LR11. Moreover, a peptide corresponding to this region folds into an α-helical structure in the presence of liposomes and transforms liposomes to small vesicles and tubule-like particles. We postulate that this amphipathic helix may contribute to the dynamic remodeling of membrane structure and facilitate LR11 intracellular transport. PMID:24866012

  13. A membrane proximal helix in the cytosolic domain of the human APP interacting protein LR11/SorLA deforms liposomes.

    PubMed

    Gill, Richard L; Wang, Xingsheng; Tian, Fang

    2015-01-01

    Over the last decade, compelling evidence has linked the development of Alzheimer's disease (AD) to defective intracellular trafficking of the amyloid precursor protein (APP). Faulty APP trafficking results in an overproduction of Aβ peptides, which is generally agreed to be the primary cause of AD-related pathogenesis. LR11 (SorLA), a type I transmembrane sorting receptor, has emerged as a key regulator of APP trafficking and processing. It directly interacts with APP and diverts it away from amyloidogenic processing. The 54-residue cytosolic domain of LR11 is essential for its proper intracellular localization and trafficking which, in turn, determines the fate of APP. Here, we have found a surprising membrane-proximal amphipathic helix in the cytosolic domain of LR11. Moreover, a peptide corresponding to this region folds into an α-helical structure in the presence of liposomes and transforms liposomes to small vesicles and tubule-like particles. We postulate that this amphipathic helix may contribute to the dynamic remodeling of membrane structure and facilitate LR11 intracellular transport. PMID:24866012

  14. Folding simulations of gramicidin A into the β-helix conformations: Simulated annealing molecular dynamics study

    NASA Astrophysics Data System (ADS)

    Mori, Takaharu; Okamoto, Yuko

    2009-10-01

    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 β6.3-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 β4.4-helix as the lowest-potential-energy structure, and left-handed β4.4-helix, right-handed and left-handed β6.3-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 β-sheet-forming tendency throughout almost the entire sequence. In order to examine the stability of the obtained right-handed β6.3-helix and β4.4-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 β-helix structures. The results suggested that β6.3-helix is more stable than β4.4-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 β6.3-helix conformation.

  15. 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

  16. Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid–virus pSSVx: a novel peculiar member of the winged helix–turn–helix transcription factor family

    PubMed Central

    Contursi, Patrizia; Farina, Biancamaria; Pirone, Luciano; Fusco, Salvatore; Russo, Luigi; Bartolucci, Simonetta; Fattorusso, Roberto; Pedone, Emilia

    2014-01-01

    The hybrid plasmid–virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 amino acid protein, namely Stf76, that does not show significant sequence homology with any protein with known 3D structure. The recombinant protein recognizes specifically two DNA-binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. Circular dichroism, spectrofluorimetric, light scattering and isothermal titration calorimetry experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix–turn–helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76–DNA complex has been built using as template a structurally related homolog. PMID:24682827

  17. 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

  18. 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

  19. HELIX: The High Energy Light Isotope Experiment

    NASA Astrophysics Data System (ADS)

    Tarle, Gregory

    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

  20. 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

  1. 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.

  2. Recapitulating the α-helix: nonpeptidic, low-molecular-weight ligands for the modulation of helix-mediated protein-protein interactions.

    PubMed

    Lanning, Maryanna; Fletcher, Steven

    2013-12-01

    Protein-protein interactions play critical roles in a wide variety of biological processes, and their dysregulations contribute to the pathogenesis of several diseases, including cancer. Chemical entities that can abrogate aberrant protein-protein interactions may provide novel therapeutic agents. A large number of protein-protein interactions are mediated by protein secondary structure, the most commonly encountered form of which is the α-helix. Accordingly, over the last decade, there has been a flood of nonpeptidic small molecules that recapitulate the projection and chemical nature of key side chains of the canonical α-helix as a strategy to disrupt helix-mediated protein-protein interactions. In this review, we discuss recent advances (post 2006) in the design of synthetic α-helix mimetics, which include single-faced and two-faced/amphipathic structures, for the modulation of protein-protein interactions. PMID:24261892

  3. Proposal of a new hydrogen-bonding form to maintain curdlan triple helix.

    PubMed

    Miyoshi, Kentaro; Uezu, Kazuya; Sakurai, Kazuo; Shinkai, Seiji

    2004-06-01

    Curdlan and other beta-1,3-D-glucans form right-handed triple helices, and it has been believed that the intermolecular H-bond is present at the center of the helix to maintain the structure. In this H-bond model, three secondary OH groups form an inequilateral hexagonal shape perpendicular to the helix axis. This hexagonal form seems to be characteristic for beta-1,3-D-glucans and is widely accepted. We carried out MOPAC and ab initio calculations for the curdlan helix, and we propose a new intermolecular H-bonding model. In our model, the H-bonds are formed between the O2-atoms on different x-y planes along the curdlan helix, hence the H-bonds are not perpendicular to the helix axis. The new H-bonds are connected along the helix, traversing three curdlan chains to make a left-handed helix. Therefore, the H-bonding array leads to a reverse helix of the main chain. According to our MOPAC calculation, this model is more stable than the previous one. We believe that the continuous H-bonding array is stabilized by cooperative phenomena in the polymeric system. PMID:17191891

  4. X-ray structure of dopamine transporter elucidates antidepressant mechanism.

    PubMed

    Penmatsa, Aravind; Wang, Kevin H; Gouaux, Eric

    2013-11-01

    Antidepressants targeting Na(+)/Cl(-)-coupled neurotransmitter uptake define a key therapeutic strategy to treat clinical depression and neuropathic pain. However, identifying the molecular interactions that underlie the pharmacological activity of these transport inhibitors, and thus the mechanism by which the inhibitors lead to increased synaptic neurotransmitter levels, has proven elusive. Here we present the crystal structure of the Drosophila melanogaster dopamine transporter at 3.0 Å resolution bound to the tricyclic antidepressant nortriptyline. The transporter is locked in an outward-open conformation with nortriptyline wedged between transmembrane helices 1, 3, 6 and 8, blocking the transporter from binding substrate and from isomerizing to an inward-facing conformation. Although the overall structure of the dopamine transporter is similar to that of its prokaryotic relative LeuT, there are multiple distinctions, including a kink in transmembrane helix 12 halfway across the membrane bilayer, a latch-like carboxy-terminal helix that caps the cytoplasmic gate, and a cholesterol molecule wedged within a groove formed by transmembrane helices 1a, 5 and 7. Taken together, the dopamine transporter structure reveals the molecular basis for antidepressant action on sodium-coupled neurotransmitter symporters and elucidates critical elements of eukaryotic transporter structure and modulation by lipids, thus expanding our understanding of the mechanism and regulation of neurotransmitter uptake at chemical synapses. PMID:24037379

  5. 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

  6. 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.

  7. Folding and insertion thermodynamics of the transmembrane WALP peptide

    SciTech Connect

    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){sub 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.

  8. Type I Interferon Signaling Is Decoupled from Specific Receptor Orientation through Lenient Requirements of the Transmembrane Domain.

    PubMed

    Sharma, Nanaocha; Longjam, Geeta; Schreiber, Gideon

    2016-02-12

    Type I interferons serve as the first line of defense against pathogen invasion. Binding of IFNs to its receptors, IFNAR1 and IFNAR2, is leading to activation of the IFN response. To determine whether structural perturbations observed during binding are propagated to the cytoplasmic domain, multiple mutations were introduced into the transmembrane helix and its surroundings. Insertion of one to five alanine residues near either the N or C terminus of the transmembrane domain (TMD) likely promotes a rotation of 100° and a translation of 1.5 Å per added residue. Surprisingly, the added alanines had little effect on the binding affinity of IFN to the cell surface receptors, STAT phosphorylation, or gene induction. Similarly, substitution of the juxtamembrane residues of the TMD with alanines, or replacement of the TMD of IFNAR1 with that of IFNAR2, did not affect IFN binding or activity. Finally, only the addition of 10 serine residues (but not 2 or 4) between the extracellular domain of IFNAR1 and the TMD had some effect on signaling. Bioinformatic analysis shows a correlation between high sequence conservation of TMDs of cytokine receptors and the ability to transmit structural signals. Sequence conservation near the TMD of IFNAR1 is low, suggesting limited functional importance for this region. Our results suggest that IFN binding to the extracellular domains of IFNAR1 and IFNAR2 promotes proximity between the intracellular domains and that differential signaling is a function of duration of activation and affinity of binding rather than specific conformational changes transmitted from the outside to the inside of the cell. PMID:26679999

  9. Light signal transduction pathway from flavin chromophore to the J alpha helix of Arabidopsis phototropin1.

    PubMed

    Yamamoto, Atsushi; Iwata, Tatsuya; Sato, Yoshiaki; Matsuoka, Daisuke; Tokutomi, Satoru; Kandori, Hideki

    2009-04-01

    In the plant blue-light sensor phototropin, illumination of the chromophoric LOV domains causes activation of the serine/threonine kinase domain. Flavin mononucleotide (FMN) is a chromophore molecule in the two LOV domains (LOV1 and LOV2), but only LOV2 is responsible for kinase activation. Previous studies reported an important role of an additional helix connected to the C-terminal of LOV2 (Jalpha helix) for the function of phototropin; however, it remains unclear how the Jalpha helix affects light-induced structural changes in LOV2. In this study we compared light-induced protein structural changes of the LOV2 domain of Arabidopsis phot1 in the absence (LOV2-core) and presence (LOV2-Jalpha) of the Jalpha helix by Fourier-transform infrared spectroscopy. Prominent peaks were observed only in the amide-I region (1650 (-)/1625 (+) cm(-1)) of LOV2-Jalpha at physiological temperatures (>/=260 K), corresponding to structural perturbation of the alpha-helix. The peaks were diminished by point mutation of functionally important amino acids such as Phe-556 between FMN and the beta-sheet, Gln-575 being hydrogen-bonded with FMN, and Ile-608 on the Jalpha helix. We thus conclude that a light signal is relayed from FMN through these amino acids and eventually changes the interaction between LOV2-core and the Jalpha helix in Arabidopsis phot1. PMID:19348760

  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 Central

    Du, Yuzhe; Lee, Jung-Eun; Nomura, Yoshiko; Zhang, Tianxiang; Zhorov, Boris S.; Dong, Ke

    2011-01-01

    A phenylalanine residue (Phe1519) in the sixth transmembrane segment of domain III (IIIS6) of the cockroach BgNav 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 BgNav 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 α-cyano group at the phenoxybenzyl alcohol) and three type II (containing the α-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. 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

  12. 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.

  13. 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

  14. Helix dipole movement and conformational variability contribute to allosteric GDP release in Galphai subunits.

    PubMed

    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

    2009-03-31

    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. PMID:19222191

  15. 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.

  16. 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.

  17. 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

  18. Macrocyclization and labeling of helix-loop-helix peptide with intramolecular bis-thioether linkage.

    PubMed

    Nishihara, Toshio; Kitada, Hidekazu; Fujiwara, Daisuke; Fujii, Ikuo

    2016-11-01

    Conformationally constrained peptides have been developed as an inhibitor for protein-protein interactions (PPIs), and we have de novo designed cyclized helix-loop-helix (cHLH) peptide with a disulfide bond consisting of 40 amino acids to generate molecular-targeting peptides. However, synthesis of long peptides has sometimes resulted in low yield according to the respective amino acid sequences. Here we developed a method for efficient synthesis and labeling for cHLH peptides. First, we synthesized two peptide fragments and connected them by the copper-mediated alkyne and azide cycloaddition (CuAAC) reaction. Cyclization was performed by bis-thioether linkage using 1,3-dibromomethyl-5-propargyloxybenzene, and subsequently, the cHLH peptide was labeled with an azide-labeled probe. Finally, we designed and synthesized a peptide inhibitor for the p53-HDM2 interaction using a structure-guided design and successfully labeled it with a fluorescent probe or a functional peptide, respectively, by click chemistry. This macrocyclization and labeling method for cHLH peptide would facilitate the discovery of de novo bioactive ligands and therapeutic leads. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 415-421, 2016. PMID:26917088

  19. 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.

  20. Basic helix-loop-helix transcription factor gene family phylogenetics and nomenclature.

    PubMed

    Skinner, Michael K; Rawls, Alan; Wilson-Rawls, Jeanne; Roalson, Eric H

    2010-07-01

    A phylogenetic analysis of the basic helix-loop-helix (bHLH) gene superfamily was performed using seven different species (human, mouse, rat, worm, fly, yeast, and plant Arabidopsis) and involving over 600 bHLH genes (Stevens et al., 2008). All bHLH genes were identified in the genomes of the various species, including expressed sequence tags, and the entire coding sequence was used in the analysis. Nearly 15% of the gene family has been updated or added since the original publication. A super-tree involving six clades and all structural relationships was established and is now presented for four of the species. The wealth of functional data available for members of the bHLH gene superfamily provides us with the opportunity to use this exhaustive phylogenetic tree to predict potential functions of uncharacterized members of the family. This phylogenetic and genomic analysis of the bHLH gene family has revealed unique elements of the evolution and functional relationships of the different genes in the bHLH gene family. PMID:20219281

  1. The structure of aquaporin-1 at 4.5-A resolution reveals short alpha-helices in the center of the monomer.

    PubMed

    Mitsuoka, K; Murata, K; Walz, T; Hirai, T; Agre, P; Heymann, J B; Engel, A; Fujiyoshi, Y

    1999-12-01

    Aquaporin-1 is a water channel found in mammalian red blood cells that is responsible for high water permeability of its membrane. Our electron crystallographic analysis of the three-dimensional structure of aquaporin-1 at 4.5-A resolution confirms the previous finding that each subunit consists of a right-handed bundle of six highly tilted transmembrane helices that surround a central X-shaped structure. In our new potential map, the rod-like densities for the transmembrane helices show helically arranged protrusions, indicating the positions of side chains. Thus, in addition to the six transmembrane helices, observation of helically arranged side-chain densities allowed the identification of two short alpha-helices representing the two branches of the central X-shaped structure that extend to the extracellular and cytoplasmic membrane surfaces. The other two branches are believed to be loops connecting the short alpha-helix to a neighboring transmembrane helix. A pore found close to the center of the aquaporin-1 monomer is suggested to be the course of water flow with implications for the water selectivity. PMID:10600556

  2. 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

  3. 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; Dutton, P Leslie; 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

  4. Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).

    PubMed

    Wang, Wuyang; Linsdell, Paul

    2012-03-23

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway. PMID:22303012

  5. Alternating Access to the Transmembrane Domain of the ATP-binding Cassette Protein Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7)*

    PubMed Central

    Wang, Wuyang; Linsdell, Paul

    2012-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between “outwardly facing” and “inwardly facing” conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)2− ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)2−, access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional (“vestigial”) gates within the CFTR permeation pathway. PMID:22303012

  6. 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

  7. 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

  8. 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

  9. 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

  10. Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics.

    PubMed

    Moin, Syed M; Urban, Sinisa

    2012-01-01

    Rhomboid proteases reside within cellular membranes, but the advantage of this unusual environment is unclear. We discovered membrane immersion allows substrates to be identified in a fundamentally-different way, based initially upon exposing 'masked' conformational dynamics of transmembrane segments rather than sequence-specific binding. EPR and CD spectroscopy revealed that the membrane restrains rhomboid gate and substrate conformation to limit proteolysis. True substrates evolved intrinsically-unstable transmembrane helices that both become unstructured when not supported by the membrane, and facilitate partitioning into the hydrophilic, active-site environment. Accordingly, manipulating substrate and gate dynamics in living cells shifted cleavage sites in a manner incompatible with extended sequence binding, but correlated with a membrane-and-helix-exit propensity scale. Moreover, cleavage of diverse non-substrates was provoked by single-residue changes that destabilize transmembrane helices. Membrane immersion thus bestows rhomboid proteases with the ability to identify substrates primarily based on reading their intrinsic transmembrane dynamics.DOI:http://dx.doi.org/10.7554/eLife.00173.001. PMID:23150798

  11. 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.

  12. Wave patterns in α-helix proteins with interspine coupling

    NASA Astrophysics Data System (ADS)

    Mimshe Fewu, J. C.; Tabi, C. B.; Edongue, H.; Ekobena Fouda, H. P.; Kofané, T. C.

    2013-02-01

    Modulational instability is a direct way by which localized structures emerge in nonlinear systems. We investigate analytically, through the linear stability of plane wave solutions, the existence of localized structures in α-helix proteins with three spines. Through numerical simulations, trains of pulses are found and confirm our analytical predictions. The presence of higher-order interactions between adjacent spines tends to suppress the formed localized structures for erratic ones to emerge. These erratic structures are highly localized and rather reinforce the idea that the energy to be used in metabolic processes is rather confined to specific regions for its efficiency.

  13. 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

  14. 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

  15. 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.

  16. Enhancer of splitD, a dominant mutation of Drosophila, and its use in the study of functional domains of a helix-loop-helix protein.

    PubMed Central

    Tietze, K; Oellers, N; Knust, E

    1992-01-01

    Helix-loop-helix proteins play important roles in developmental processes, such as myogenesis, neurogenesis, and sex determination. The gene Enhancer of split [E(spl)] of Drosophila, a member of a gene complex that is involved in early neurogenesis, encodes a protein with a basic domain and a helix-loop-helix motif. We took advantage of a dominant mutation of this gene, E(spl)D, to define in vivo structural features of this protein for proper function. The mutation renders the otherwise recessive eye phenotype of spl dominant. By germ-line transformation of different in vitro mutagenized versions of the E(spl) gene, we could demonstrate that the basic domain of this helix-loop-helix protein is functional and necessary for expression of the dominant phenotype. These results are supported by in vitro DNA-binding assays, which showed that the basic domain is in fact necessary for DNA binding, despite the presence of a proline residue. Furthermore, we could show that the dominant enhancement of spl is caused by truncation of the E(SPL)D protein in combination with deletion of a putative regulatory element. Images PMID:1631102

  17. Structure in the channel forming domain of colicin E1 bound to membranes: the 402-424 sequence.

    PubMed Central

    Salwiński, L.; Hubbell, W. L.

    1999-01-01

    To explore the structure of the pore-forming fragment of colicin E1 in membranes, a series of 23 consecutive single cysteine substitution mutants was prepared in the sequence 402-424. Each mutant was reacted with a sulfhydryl-specific reagent to generate a nitroxide labeled side chain, and the mobility of the side chain and its accessibility to collision with paramagnetic reagents was determined from the electron paramagnetic resonance spectrum. Individual values of these quantities were used to identify tertiary contact sites and the nature of the surrounding solvent, while their periodic dependence on sequence position was used to identify secondary structure. In solution, the data revealed a regular helix of 11 residues in the region 406-416, consistent with helix IV of the crystal structure. Upon binding to negatively charged membranes at pH 4.0, helix IV apparently grows to a length of 19 residues, extending from 402-420. One face of the helix is solvated by the lipid bilayer, and the other by an environment of a polar nature. Surprisingly, a conserved charged pair, D408-R409, is located on the lipid-exposed face. Evidence is presented to suggest a transmembrane orientation of this new helix, although other topographies may exist in equilibrium. PMID:10091659

  18. 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

  19. 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

  20. The NC16A Domain of Collagen XVII Plays a Role in Triple Helix Assembly and Stability*

    PubMed Central

    Van den Bergh, Françoise; Fu, Chang-Ling; Olague-Marchan, Monica; Giudice, George J.

    2007-01-01

    Collagen XVII/BP180 is a transmembrane constituent of the epidermal anchoring complex. To study the role of its non-collagenous linker domain, NC16A, in protein assembly and stability, we analyzed the following recombinant proteins: the collagen XVII extracellular domain with or without NC16A, and a pair of truncated proteins comprising the COL15-NC15 stretch expressed with or without NC16A. All four proteins were found to exist as stable collagen triple helices; however, the two missing NC16A exhibited melting temperatures significantly lower than their NC16A-containing counterparts. Protein refolding experiments revealed that the rate of triple helix assembly of the collagen model peptide GPP10 is greatly increased by the addition of an upstream NC16A domain. In summary, the NC16A linker domain of collagen XVII exhibits a positive effect on both the rate of assembly and the stability of the adjoining collagen structure. PMID:17045967

  1. Recent Progresses in Studying Helix-Helix Interactions in Proteins by Incorporating the Wenxiang Diagram into the NMR Spectroscopy.

    PubMed

    Zhou, Guo-Ping; Chen, Dong; Liao, Siming; Huang, Ri-Bo

    2016-01-01

    All residues in an alpha helix can be characterized and dispositioned on a 2D the wenxiang diagram, which possesses the following features: (1) the relative locations of the amino acids in the α-helix can be clearly displayed regardless how long it is; (2) direction of an alphahelix can be indicated; and (3) more information regarding each of the constituent amino acid residues in an alpha helix. Owing to its intuitionism and easy visibility, wenxiang diagrams have had an immense influence on our understanding of protein structure, protein-protein interactions, and the effect of helical structural stability on protein conformational transitions. In this review, we summarize two recent applications of wenxiang diagrams incorporating NMR spectroscopy in the researches of the coiled-coil protein interactions related to the regulation of contraction or relaxation states of vascular smooth muscle cells, and the effects of α-helical stability on the protein misfolding in prion disease, in hopes that the gained valuable information through these studies can stimulate more and more widely applications of wenxiang diagrams in structural biology. PMID:26286215

  2. Fluctuations of an Exposed π-Helix Involved in Lipoxygenase Substrate Recognition

    PubMed Central

    2014-01-01

    The second helix in lipoxygenases adapts to permit substrate access to the active site, but details of this process are varied and poorly understood. We therefore examined the dynamics of helix 2 in solutions of spin-labeled soybean lipoxygenase-1 and spin relaxation at 60 K of the spin-labels by catalytic iron. Helix 2 in soybean lipoxygenase structures is surface-exposed and contains one turn of π-helix, centrally located. A site-directed spin-label scan of 18 of the 21 helix 2 residues, and electron paramagnetic resonance, showed that the π-helical segment became unusually mobile, on a nanosecond time scale, under conditions favoring substrate binding (pH 9 and lipid addition), while segments before and after had relatively unchanged dynamics. Backbone dynamics of residues in the π-helical segment appeared to be correlated, at pH 9. Samples also were frozen to examine the polarity and proticity of the local environments, the effect of the local environment on intrinsic relaxation, and dipolar relaxation by two symmetries of catalytic iron. The average hyperfine tensor component, Azz, of four π-helix residues decreased by 1.75 G, with an increase in pH from 7 to 9, while it remained unaffected for nearby buried residues. Power saturation data suggested the change in polarity specific to the π-helix altered the intrinsic relaxation rates. Different symmetries of iron contributed to distance-dependent magnetic relaxation. We interpret these data to mean that a π-helix in the second helix of plant lipoxygenases is highly dynamic and is the site where lipid chains penetrate to inner helices that outline the substrate pocket. PMID:25036469

  3. 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.

  4. Role of the transmembrane domain in SNARE protein mediated membrane fusion: peptide nucleic acid/peptide model systems.

    PubMed

    Wehland, Jan-Dirk; Lygina, Antonina S; Kumar, Pawan; Guha, Samit; Hubrich, Barbara E; Jahn, Reinhard; Diederichsen, Ulf

    2016-08-16

    Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor proteins also known as SNAREs. The backbone of this essential process is the assembly of SNAREs from opposite membranes into tight four helix bundles forcing membranes in close proximity. With model systems resembling SNAREs with reduced complexity we aim to understand how these proteins work at the molecular level. Here, peptide nucleic acids (PNAs) are used as excellent candidates for mimicking the SNARE recognition motif by forming well-characterized duplex structures. Hybridization between complementary PNA strands anchored in liposomes through native transmembrane domains (TMDs) induces the merger of the outer leaflets of the participating vesicles but not of the inner leaflets. A series of PNA/peptide hybrids differing in the length of TMDs and charges at the C-terminal end is presented. Interestingly, mixing of both outer and inner leaflets is seen for TMDs containing an amide in place of the natural carboxylic acid at the C-terminal end. Charged side chains at the C-terminal end of the TMDs are shown to have a negative impact on the mixing of liposomes. The length of the TMDs is vital for fusion as with the use of shortened TMDs, fusion was completely prevented. PMID:27345759

  5. Structure-based Functional Study Reveals Multiple Roles of Transmembrane Segment IX and Loop VIII–IX in NhaA Na+/H+ Antiporter of Escherichia coli at Physiological pH*

    PubMed Central

    Tzubery, Tzvi; Rimon, Abraham; Padan, Etana

    2008-01-01

    The three-dimensional crystal structure of Escherichia coli NhaA determined at pH 4 provided the first structural insights into the mechanism of antiport and pH regulation of a Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 6.5–8.5), many questions pertaining to the active state of NhaA have remained open including the structural and physiological roles of helix IX and its loop VIII–IX. Here we studied this NhaA segment (Glu241–Phe267) by structure-based biochemical approaches at physiological pH. Cysteine-scanning mutagenesis identified new mutations affecting the pH dependence of NhaA, suggesting their contribution to the “pH sensor.” Furthermore mutation F267C reduced the H+/Na+ stoichiometry of the antiporter, and F267C/F344C inactivated the antiporter activity. Tests of accessibility to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide, a membrane-impermeant positively charged SH reagent with a width similar to the diameter of hydrated Na+, suggested that at physiological pH the cytoplasmic cation funnel is more accessible than at acidic pH. Assaying intermolecular cross-linking in situ between single Cys replacement mutants uncovered the NhaA dimer interface at the cytoplasmic side of the membrane; between Leu255 and the cytoplasm, many Cys replacements cross-link with various cross-linkers spanning different distances (10–18Å) implying a flexible interface. L255C formed intermolecular S–S bonds, cross-linked only with a 5-Å cross-linker, and when chemically modified caused an alkaline shift of 1 pH unit in the pH dependence of NhaA and a 6-fold increase in the apparent Km for Na+ of the exchange activity suggesting a rigid point in the dimer interface critical for NhaA activity and pH regulation. PMID:18387952

  6. Cooperative Transmembrane Penetration of Nanoparticles

    PubMed Central

    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

  7. In vitro dimerization of the bovine papillomavirus E5 protein transmembrane domain

    PubMed Central

    Oates, Joanne; Hicks, Matthew; Dafforn, Timothy R.; DiMaio, Daniel; Dixon, Ann M.

    2013-01-01

    The E5 protein from bovine papillomavirus is a type II membrane protein and the product of the smallest known oncogene. E5 causes tumor formation by binding and activating the platelet derived growth factor beta receptor (PDGFβR). In order to productively interact with the receptor it is thought that E5 binds as a dimer. However, wild-type E5 and various mutants have also been shown to form trimers, tetramers and even higher order oligomers. The residues in E5 that drive and stabilize a dimeric state are also still in question. At present, two different models for the E5 dimer exist in the literature, one symmetric and one asymmetric. There is universal agreement, however, that the transmembrane (TM) domain plays a vital role in stabilizing the functional oligomer, indeed mutation of various TM domain residues can abolish E5 function. In order to better resolve the role of the E5 TM domain in function, we have undertaken the first quantitative in vitro characterization of the E5 TM domain in detergent micelles and liposomes. Circular and linear dichroism analyses verify that the TM domain adopts a stable α-helical structure and is able to partition efficiently across lipid bilayers. SDS-PAGE and analytical ultracentrifugation confirm for the first time that the TM domain of E5 forms a strong dimer with a standard state free energy of dissociation of 5.0 kcal mol−1. We have used our new results to interpret existing models of E5 dimer formation and provide a direct link between TM helix interactions and E5 function. PMID:18672907

  8. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-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 α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-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 β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Phylogenetic analyses of vector mosquito basic helix-loop-helix transcription factors.

    PubMed

    Zhang, D B; Wang, Y; Liu, A K; Wang, X H; Dang, C W; Yao, Q; Chen, K P

    2013-10-01

    Basic helix-loop-helix (bHLH) transcription factors play critical roles in the regulation of a wide range of developmental processes in higher organisms and have been identified in more than 20 organisms. Mosquitoes are important vectors of certain human diseases. In this study, Aedes aegypti, Anopheles gambiae str. PEST and Culex quinquefasciatus genomes were found to encode 55, 55 and 57 bHLH genes, respectively. Further phylogenetic analyses and OrthoDB and Kyoto encyclopedia of genes and genomes orthology database searches led us to define orthology for all the identified mosquito bHLHs successfully. This provides useful information with which to update annotations to 40 Ae. aegypti, 55 An. gambiae and 38 C. quinquefasciatus bHLH genes in VectorBase. The mosquito lineage has more bHLH genes in the Atonal, neurogenin (Ngn) and Hes-related with YRPW motif (Hey) families than do other insect species, suggesting that mosquitoes have evolved to be more sensitive to vibration, light and chemicals. Mosquito bHLH genes generally have higher evolutionary rates than other insect species. However, no pervasive positive selection occurred in the evolution of insect bHLH genes. Only episodic positive selection was found to affect evolution of bHLH genes in 11 families. Besides, coding regions of several Ae. aegypti bHLH motifs have unusually long introns in which multiple copies of transposable elements have been identified. These data provide a solid basis for further studies on structures and functions of bHLH proteins in the regulation of mosquito development and for prevention and control of mosquito-mediated human diseases. PMID:23906262

  14. Cystic Fibrosis Transmembrane Conductance Regulator

    PubMed Central

    Smith, Stephen S.; Steinle, Erich D.; Meyerhoff, Mark E.; Dawson, David C.

    1999-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel exhibits lyotropic anion selectivity. Anions that are more readily dehydrated than Cl exhibit permeability ratios (PS/PCl) greater than unity and also bind more tightly in the channel. We compared the selectivity of CFTR to that of a synthetic anion-selective membrane [poly(vinyl chloride)–tridodecylmethylammonium chloride; PVC-TDMAC] for which the nature of the physical process that governs the anion-selective response is more readily apparent. The permeability and binding selectivity patterns of CFTR differed only by a multiplicative constant from that of the PVC-TDMAC membrane; and a continuum electrostatic model suggested that both patterns could be understood in terms of the differences in the relative stabilization of anions by water and the polarizable interior of the channel or synthetic membrane. The calculated energies of anion–channel interaction, derived from measurements of either permeability or binding, varied as a linear function of inverse ionic radius (1/r), as expected from a Born-type model of ion charging in a medium characterized by an effective dielectric constant of 19. The model predicts that large anions, like SCN, although they experience weaker interactions (relative to Cl) with water and also with the channel, are more permeant than Cl because anion–water energy is a steeper function of 1/r than is the anion–channel energy. These large anions also bind more tightly for the same reason: the reduced energy of hydration allows the net transfer energy (the well depth) to be more negative. This simple selectivity mechanism that governs permeability and binding acts to optimize the function of CFTR as a Cl filter. Anions that are smaller (more difficult to dehydrate) than Cl are energetically retarded from entering the channel, while the larger (more readily dehydrated) anions are retarded in their passage by “sticking” within the channel. PMID:10578016

  15. Spatial organization of transmembrane receptor signalling

    PubMed Central

    Bethani, Ioanna; Skånland, Sigrid S; Dikic, Ivan; Acker-Palmer, Amparo

    2010-01-01

    The spatial organization of transmembrane receptors is a critical step in signal transduction and receptor trafficking in cells. Transmembrane receptors engage in lateral homotypic and heterotypic cis-interactions as well as intercellular trans-interactions that result in the formation of signalling foci for the initiation of different signalling networks. Several aspects of ligand-induced receptor clustering and association with signalling proteins are also influenced by the lipid composition of membranes. Thus, lipid microdomains have a function in tuning the activity of many transmembrane receptors by positively or negatively affecting receptor clustering and signal transduction. We review the current knowledge about the functions of clustering of transmembrane receptors and lipid–protein interactions important for the spatial organization of signalling at the membrane. PMID:20717138

  16. 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

  17. Relativistic helix traveling wave tube amplifiers

    SciTech Connect

    Freund, H.P.; Vanderplaats, N.R.; Kodis, M.A. )

    1992-07-01

    A relativistic field theory of a helix traveling wave tube (TWT) is described for the case in which a thin annular beam propagates through a sheath helix enclosed within a loss-free wall. The theory is applied to the study of a TWT with an intense relativistic electron beam. The analysis implicitly includes beam space-charge effects and is valid for arbitrary azimuthal mode number, and the coupled-wave Pierce theory is recovered in the [ital near]-[ital resonant] limit. The results indicate that impressive gains and efficiencies are possible in this regime. In addition, the interaction is relatively insensitive to the effects of a beam energy spread.

  18. 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.

  19. Helix propensities of conformationally restricted amino acids. Non-natural substitutes for helix breaking proline and helix forming alanine.

    PubMed

    Alías, Miriam; Ayuso-Tejedor, Sara; Fernández-Recio, Juan; Cativiela, Carlos; Sancho, Javier

    2010-02-21

    Alpha helices are useful scaffolds to build biologically active peptides. The intrinsic stability of an alpha-helix is a key feature that can be successfully designed, and it is governed by the constituting amino acid residues. Their individual contributions to helix stability are given, according to Lifson-Roig theory, by their w parameters, which are known for all proteinogenic amino acids, but not for non-natural ones. On the other hand, non-natural, conformationally-restricted amino acids can be used to impart biochemical stability to peptides intended for in vivo administration. Efficient design of peptides based on these amino acids requires the previous determination of their w parameters. We begin here this task by determining the w parameters of two restricted analogs of alanine: (alpha-methyl)alanine and 1-aminocyclopropanecarboxylic acid. According to their w values (alpha-methyl)alanine is almost as good a helix forming residue as alanine, while 1-aminocyclopropanecarboxylic acid is, similarly to proline, a helix breaker. PMID:20135035

  20. TMRPres2D: high quality visual representation of transmembrane protein models.

    PubMed

    Spyropoulos, Ioannis C; Liakopoulos, Theodore D; Bagos, Pantelis G; Hamodrakas, Stavros J

    2004-11-22

    The 'TransMembrane protein Re-Presentation in 2-Dimensions' (TMRPres2D) tool, automates the creation of uniform, two-dimensional, high analysis graphical images/models of alpha-helical or beta-barrel transmembrane proteins. Protein sequence data and structural information may be acquired from public protein knowledge bases, emanate from prediction algorithms, or even be defined by the user. Several important biological and physical sequence attributes can be embedded in the graphical representation. PMID:15201184

  1. 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

  2. Heat conductivity of DNA double helix

    PubMed Central

    Savin, Alexander V.; Mazo, Mikhail A.; Kikot, Irina P.; Manevitch, Leonid I.; Onufriev, Alexey V.

    2015-01-01

    Thermal conductivity of isolated single molecule DNA fragments is of importance for nanotechnology, but has not yet been measured experimentally. Theoretical estimates based on simplified (1D) models predict anomalously high thermal conductivity. To investigate thermal properties of single molecule DNA we have developed a 3D coarse-grained (CG) model that retains the realism of the full all-atom description, but is significantly more efficient. Within the proposed model each nucleotide is represented by 6 particles or grains; the grains interact via effective potentials inferred from classical molecular dynamics (MD) trajectories based on a well-established all-atom potential function. Comparisons of 10 ns long MD trajectories between the CG and the corresponding all-atom model show similar root-mean-square deviations from the canonical B-form DNA, and similar structural fluctuations. At the same time, the CG model is 10 to 100 times faster depending on the length of the DNA fragment in the simulation. Analysis of dispersion curves derived from the CG model yields longitudinal sound velocity and torsional stiffness in close agreement with existing experiments. The computational efficiency of the CG model makes it possible to calculate thermal conductivity of a single DNA molecule not yet available experimentally. For a uniform (polyG-polyC) DNA, the estimated conductivity coefficient is 0.3 W/mK which is half the value of thermal conductivity for water. This result is in stark contrast with estimates of thermal conductivity for simplified, effectively 1D chains (”beads on a spring”) that predict anomalous (infinite) thermal conductivity. Thus, full 3D character of DNA double-helix retained in the proposed model appears to be essential for describing its thermal properties at a single molecule level. PMID:26207085

  3. 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

  4. 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.

  5. Solution NMR Structure of Membrane-Integral Diacylglycerol Kinase

    PubMed Central

    Van Horn, Wade D.; Kim, Hak-Jun; Ellis, Charles D.; Hadziselimovic, Arina; Sulistijo, Endah S.; Karra, Murthy D.; Tian, Changlin; Sönnichsen, Frank D.; Sanders, Charles R.

    2009-01-01

    Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer using solution NMR. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK’s three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK’s lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis. PMID:19556511

  6. TCFL4: a gene at 17q21.1 encoding a putative basic helix-loop-helix leucine-zipper transcription factor.

    PubMed

    Bjerknes, M; Cheng, H

    1996-11-28

    TCFL4 (transcription factor like 4) is the HGMW-approved symbol for the gene of a widely expressed putative basic helix-loop-helix leucine-zipper (bHLH-zip) transcription factor which is located 3' to HSD17B1 (17-beta-hydroxysteroid dehydrogenase gene) at 17q21.1, centromeric to the BRCA1 (a gene implicated in familial breast cancer) locus. We report the human gene structure and the murine cDNA sequence of two variants, about 1.5 and 2.2 kb in size. The deduced protein is highly conserved between mouse and man. PMID:8973301

  7. Nonlinear analysis of helix traveling wave tubes

    SciTech Connect

    Freund, H.P.; Zaidman, E.G.; Vanderplaats, N.R.; Kodis, M.A.

    1994-12-31

    A nonlinear formulation of the interaction in a helix traveling wave tube (TWT) is presented. The formulation is intended to treat a wide class of helix TWTs including both emission-gated and multi-tone operation. The essential feature of each of these configurations is that multiple waves must be included in the formulation. As a result, a fully time-dependent analysis is required. The numerical procedure for this in a helix TWT is complicated by the fact that the radial profile of the field varies with frequency. This contrasts, for example, with the case of a smooth bore waveguide in which the radial profile for each TE{sub ln} or TM{sub ln} mode is invariant in frequency. Because of this, a complete self-consistent particle-in-cell (PIC) formulation must be three-dimensional. In order to circumvent the computational expense of a 3D PIC formulation, the authors adopt an approach in which the electromagnetic field is represented as a superposition of azimuthally symmetric modes in a vacuum sheath helix. The specific electron distributions are chosen to model either a continuous beam for the multi-tone TWT and a pulsed beam for the emission-gated TWT. Numerical results of the simulation for examples of interest to an emission-gated TWT experiment at NRL will be presented.

  8. High velocity knot in the Helix nebula

    SciTech Connect

    Meaburn, J.; Walsh, J.R.

    1980-01-01

    A high velocity (about 66 km/s) split feature about 15 arcseconds in extent has been detected in forbidden O II emission over a dark knot in the loop of the Helix nebula. This velocity splitting is much greater than the 20 km/s large scale splitting observed previously, and several mechanisms are proposed to account for this feature.

  9. 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.

  10. The Fifth Transmembrane Segment of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Its Anion Permeation Pathway.

    PubMed

    Zhang, Jingyao; Hwang, Tzyh-Chang

    2015-06-23

    Previous studies have identified several transmembrane segments (TMs), including TM1, TM3, TM6, TM9, TM11, and TM12, as pore-lining segments in cystic fibrosis transmembrane conductance regulator (CFTR), but the role of TM5 in pore construction remains controversial. In this study, we employed substituted cysteine accessibility methodology (SCAM) to screen the entire TM5 defined by the original topology model and its cytoplasmic extension in a Cysless background. We found six positions (A299, R303, N306, S307, F310, and F311) where engineered cysteines react to intracellular 2-sulfonatoethyl methanethiosulfonate (MTSES⁻). Quantification of the modification rate of engineered cysteines in the presence or absence of ATP suggests that these six residues are accessible in both the open and closed states. Whole-cell experiments with external MTSES⁻ identified only two positive positions (L323 and A326), resulting in a segment containing 11 consecutive amino acids, where substituted cysteines respond to neither internal nor external MTSES⁻, a unique feature not seen previously in CFTR's pore-lining segments. The observation that these positions are inaccessible to channel-permeant thiol-specific reagent [Au(CN)₂]⁻ suggests that this segment of TM5 between F311 and L323 is concealed from the pore by other TMs and/or lipid bilayers. In addition, our data support the idea that the positively charged arginine at position 303 poses a pure electrostatic action in determining the single-channel current amplitude of CFTR and the effect of an open-channel blocker glibencalmide. Collectively, we conclude that the cytoplasmic portion of CFTR's TM5 lines the pore. Our functional data are remarkably consistent with predicted structural arrangements of TM5 in some homology models of CFTR. PMID:26024338

  11. 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

  12. Repair and recombination induced by triple helix DNA.

    PubMed

    Chin, Joanna Y; Schleifman, Erica B; Glazer, Peter M

    2007-01-01

    Triple-helix DNA structures can form endogenously at mirror repeat polypurine/polypyrimidine sequences or by introduction of triplex-forming oligonucleotides (TFOs). Recent evidence suggests that triple helices are sources of genetic instability, and are subject to increased rates of mutagenesis and recruitment of repair factors. Indeed, observations using TFOs suggest that triple helices provoke a variety of biological processes which can be harnessed to modulate gene expression and induce heritable changes in targeted genes. This review surveys the biological applications of TFOs, with particular attention to their recombinogenic and mutagenic potential, and summarizes available evidence for the mechanism of triplex and triplex-associated repair. PMID:17485375

  13. Structural conservation among the rhodopsin-like and other G protein-coupled receptors

    PubMed Central

    Kinoshita, Mikitaka; Okada, Tetsuji

    2015-01-01

    Intramolecular remote coupling within the polypeptide backbones of membrane proteins is difficult to analyze owing to the limited structural information available at the atomic level. Nonetheless, recent progress in the crystallographic study of G protein-coupled receptors (GPCRs) has provided an unprecedented opportunity for understanding the sophisticated architecture of heptahelical transmembrane (7TM) bundles. These 7TM bundles can respond to a wide range of extracellular stimuli while retaining the common function of binding trimeric G proteins. Here we have systematically analyzed select sets of inactive-like 7TM bundles to highlight the structural conservation of the receptors, in terms of intramolecular Cα-Cα distances. Distances with the highest scores were found to be dominated by the intrahelical distances of helix III, regardless of the choice of bundles in the set, indicating that the intracellular half of this helix is highly conserved. Unexpectedly, the distances between the cytoplasmic side of helix I and the extracellular region of helix VI provided the largest contribution to the high score populations among the interhelical pairs in most of the selected sets, including class B, C and frizzled receptors. These findings are expected to be valuable in further studies of GPCRs with unknown structure and of other protein families. PMID:25775952

  14. Thermodynamics of helix-coil transitions studied by multicanonical algorithms

    SciTech Connect

    Okamoto, Yuko; Hansmann, U.H.E.

    1995-07-13

    Thermodynamics of helix-coil transitions in amino acid homo-oligomers are studied by the recently proposed multicanonical algorithms. Homo-oligomers of length 10 are considered for three characteristic amino acids, alanine (helix former), valine (helix indifferent), and glycine (helix breaker). For alanine other lengths (15 and 20) are also considered in order to examine the length dependence. From one multicanonical production run with completely random initial conformations, we have obtained the lowest-energy conformations and various thermodynamic quantities (average helicity, Zimm-Bragg s and {sigma} parameters, free energy differences between helix and coil states, etc.) as functions of temperature. The results confirm the fact that alanine is helix-forming, valine is helix-indifferent, and glycine is helix-breaking. 36 refs., 10 figs., 5 tabs.

  15. Basic amino-acid side chains regulate transmembrane integrin signalling.

    PubMed

    Kim, Chungho; Schmidt, Thomas; Cho, Eun-Gyung; Ye, Feng; Ulmer, Tobias S; Ginsberg, Mark H

    2012-01-12

    Side chains of Lys/Arg near transmembrane domain (TMD) membrane-water interfaces can 'snorkel', placing their positive charge near negatively charged phospholipid head groups; however, snorkelling's functional effects are obscure. Integrin β TMDs have such conserved basic amino acids. Here we use NMR spectroscopy to show that integrin β(3)(Lys 716) helps determine β(3) TMD topography. The α(ΙΙb)β(3) TMD structure indicates that precise β(3) TMD crossing angles enable the assembly of outer and inner membrane 'clasps' that hold the αβ TMD together to limit transmembrane signalling. Mutation of β(3)(Lys 716) caused dissociation of α(ΙΙb)β(3) TMDs and integrin activation. To confirm that altered topography of β(3)(Lys 716) mutants activated α(ΙΙb)β(3), we used directed evolution of β(3)(K716A) to identify substitutions restoring default state. Introduction of Pro(711) at the midpoint of β(3) TMD (A711P) increased α(ΙΙb)β(3) TMD association and inactivated integrin α(ΙΙb)β(3)(A711P,K716A). β(3)(Pro 711) introduced a TMD kink of 30 ± 1° precisely at the border of the outer and inner membrane clasps, thereby decoupling the tilt between these segments. Thus, widely occurring snorkelling residues in TMDs can help maintain TMD topography and membrane-embedding, thereby regulating transmembrane signalling. PMID:22178926

  16. 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.

  17. Splice isoform estrogen receptors as integral transmembrane proteins

    PubMed Central

    Kim, Kyung Hee; Toomre, Derek; Bender, Jeffrey R.

    2011-01-01

    In addition to enhancing or repressing transcription, steroid hormone receptors rapidly transduce kinase activation signals. On ligand engagement, an N-terminus–truncated splice isoform of estrogen receptor (ER) α, ER46, triggers membrane-initiated signals, resulting in endothelial nitric oxide synthase (eNOS) activation and endothelial NO production. The orientation of ER46 at the plasma membrane is incompletely defined. With the use of ecliptic pHluorin-fused ER46, total internal reflection fluorescence microscopy in live human endothelial cells illustrates that ER46 can topologically conform to a type I transmembrane protein structure. Mutation of isoleucine-386 at the center of ER46's transmembrane hydrophobic core prevents membrane spanning, obscures the N-terminal ectodomain, and effects a marked reduction in membrane-impermeant estrogen binding with diminished rapid eNOS activation and NO production, despite maintained genomic induction of an estrogen response element–luciferase reporter. Thus there exist pools of transmembrane steroid hormone receptors that are efficient signaling molecules and potential novel therapeutic targets. PMID:21937726

  18. Bidirectional Transformation of a Metamorphic Protein between the Water-Soluble and Transmembrane Native States.

    PubMed

    Tanaka, Koji; Caaveiro, Jose M M; Tsumoto, Kouhei

    2015-11-24

    The bidirectional transformation of a protein between its native water-soluble and integral transmembrane conformations is demonstrated for FraC, a hemolytic protein of the family of pore-forming toxins. In the presence of biological membranes, the water-soluble conformation of FraC undergoes a remarkable structural reorganization generating cytolytic transmembrane nanopores conducive to cell death. So far, the reverse transformation from the native transmembrane conformation to the native water-soluble conformation has not been reported. We describe the use of detergents with different physicochemical properties to achieve the spontaneous conversion of transmembrane pores of FraC back into the initial water-soluble state. Thermodynamic and kinetic stability data suggest that specific detergents cause an asymmetric change in the energy landscape of the protein, allowing the bidirectional transformation of a membrane protein. PMID:26544760

  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. 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.

  1. Designing Covalently Linked Heterodimeric Four-Helix Bundles.

    PubMed

    Chino, M; Leone, L; Maglio, O; Lombardi, A

    2016-01-01

    De novo design has proven a powerful methodology for understanding protein folding and function, and for mimicking or even bettering the properties of natural proteins. Extensive progress has been made in the design of helical bundles, simple structural motifs that can be nowadays designed with a high degree of precision. Among helical bundles, the four-helix bundle is widespread in nature, and is involved in numerous and fundamental processes. Representative examples are the carboxylate bridged diiron proteins, which perform a variety of different functions, ranging from reversible dioxygen binding to catalysis of dioxygen-dependent reactions, including epoxidation, desaturation, monohydroxylation, and radical formation. The "Due Ferri" (two-irons; DF) family of proteins is the result of a de novo design approach, aimed to reproduce in minimal four-helix bundle models the properties of the more complex natural diiron proteins, and to address how the amino acid sequence modulates their functions. The results so far obtained point out that asymmetric metal environments are essential to reprogram functions, and to achieve the specificity and selectivity of the natural enzymes. Here, we describe a design method that allows constructing asymmetric four-helix bundles through the covalent heterodimerization of two different α-helical harpins. In particular, starting from the homodimeric DF3 structure, we developed a protocol for covalently linking the two α2 monomers by using the Cu(I) catalyzed azide-alkyne cycloaddition. The protocol was then generalized, in order to include the construction of several linkers, in different protein positions. Our method is fast, low cost, and in principle can be applied to any couple of peptides/proteins we desire to link. PMID:27586346

  2. Different role of the Jalpha helix in the light-induced activation of the LOV2 domains in various phototropins.

    PubMed

    Koyama, Takayuki; Iwata, Tatsuya; Yamamoto, Atsushi; Sato, Yoshiaki; Matsuoka, Daisuke; Tokutomi, Satoru; Kandori, Hideki

    2009-08-18

    Phototropins (phot) are blue light receptors in plants which are involved in phototropism, stomatal opening, and chloroplast movements. Phototropin has two LOV domains (LOV1 and LOV2), and the LOV2 domain is responsible for activation of Ser/Thr kinase. There is an alpha-helix at the C-terminal side of the LOV2 domain, which is called the Jalpha helix. The functional importance of the Jalpha helix has been established for Arabidopsis phot1, where light-induced structural perturbation takes place in the Jalpha helix during the photocycle of LOV2 domains. However, the present FTIR study reports a different role of the Jalpha helix in light-induced signal transduction of LOV2 domains. Here we construct LOV2 domains with (LOV-Jalpha) and without (LOV-core) the Jalpha helix for Arabidopsis phot1 and phot2 and Adiantum neochrome 1 and compare their light-induced difference FTIR spectra. Light-induced protein structural changes differ significantly between LOV-Jalpha and LOV-core for Arabidopsis phot1 [Yamamoto, A., Iwata, T., Sato, Y., Matsuoka, D., Tokutomi, S., and Kandori, H. (2009) Biophys. J. 96, 2771-2778]. In contrast, the difference spectra are identical between LOV-Jalpha and LOV-core for Adiantum neochrome 1. In Arabidopsis phot2, the protein structural changes are intermediate between Arabidopsis phot1 and Adiantum neochrome 1. These results suggest that the conformational changes of the Jalpha helix and the interaction between the LOV-core and the Jalpha helix are different among phototropins. The role of the Jalpha helix for signal transduction in phototropins is discussed. PMID:19601589

  3. 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

  4. Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

    PubMed Central

    Seibold, Steve A.; Singh, Badri Nath; Zhang, Chunfen; Kireeva, Maria; Domecq, Céline; Bouchard, Annie; Nazione, Anthony M.; Feig, Michael; Cukier, Robert I.; Coulombe, Benoit; Kashlev, Mikhail; Hampsey, Michael; Burton, Zachary F.

    2010-01-01

    Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β’ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed. PMID:20478425

  5. Nonlocal helix formation is key to understanding S-adenosylmethionine-1 riboswitch function.

    PubMed

    Whitford, Paul C; Schug, Alexander; Saunders, John; Hennelly, Scott P; Onuchic, José N; Sanbonmatsu, Kevin Y

    2009-01-01

    Riboswitches are noncoding RNAs that regulate gene expression in response to changing concentrations of specific metabolites. Switching activity is affected by the interplay between the aptamer domain and expression platform of the riboswitch. The aptamer domain binds the metabolite, locking the riboswitch in a ligand-bound conformation. In absence of the metabolite, the expression platform forms an alternative secondary structure by sequestering the 3' end of a nonlocal helix called P1. We use all-atom structure-based simulations to characterize the folding, unfolding, and metabolite binding of the aptamer domain of the S-adenosylmethionine-1 (SAM-1) riboswitch. Our results suggest that folding of the nonlocal helix (P1) is rate-limiting in aptamer domain formation. Interestingly, SAM assists folding of the P1 helix by reducing the associated free energy barrier. Because the 3' end of the P1 helix is sequestered by an alternative helix in the absence of metabolites, this observed ligand-control of P1 formation provides a mechanistic explanation of expression platform regulation. PMID:19167285

  6. Vibrations of ordered counterions around left- and right-handed DNA double helixes

    NASA Astrophysics Data System (ADS)

    Perepelytsya, S. M.; Volkov, S. N.

    2013-06-01

    The vibrations of ordered counterions around right- and left-handed DNA double helix are studied. To determine the modes of DNA conformational vibrations the structure of the double helix with counterions is considered as ionic lattice (ion-phosphate lattice). Using the developed approach the frequencies and Raman intensities for right-handed B-form and left-handed Z-form of the double helix with Na+, K+, Rb+, Cs+, and Mg2+ counterions are calculated. The obtained frequencies of vibrations of internal structure elements of the double helix (<100 cm-1) weakly depend on counterion type. In contrast, the vibrations of the ion-phosphate lattice are determined by counterion mass and charge. The frequencies of ion-phosphate vibrations of alkali metal counterions decrease from 180 to 100 cm-1, while their Raman intensities increase as the counterion mass increases for the both B- and Z-DNA. In the case of Z-DNA new mode of ion-phosphate vibrations near 150 cm-1 is found. This mode is characterized by vibrations of Mg2+ counterions with respect to the phosphates of different strands of the double helix. Our results explain the experimental Raman spectra of Z-DNA.

  7. Single Tryptophan and Tyrosine Comparisons in the N-terminal and C-terminal Interface Regions of Transmembrane GWALP Peptides†

    PubMed Central

    Gleason, Nicholas J.; Greathouse, Denise V.; Grant, Christopher V.; Opella, Stanley J.; Koeppe, Roger E.

    2014-01-01

    Hydrophobic membrane-spanning helices often are flanked by interfacial aromatic or charged residues. In this paper we compare the consequences of single Trp → Tyr substitutions at each interface for the properties of a defined transmembrane helix, in the absence of charged residues. The choice of molecular framework is critical for these single-residue experiments, because the presence of “too many” aromatic residues (more than one at either membrane-water interface) introduces excess dynamic averaging of solid-state NMR observables. To this end, we compare the outcomes when changing W5 or W19, or both of them, to tyrosine in the well characterized transmembrane peptide acetyl-GGALW5(LA)6LW19LAGA-amide (“GWALP23”). By means of solid-state 2H and 15N NMR experiments, we find that Y19GW5ALP23 displays similar magnitudes of peptide helix tilt as Y5GW19ALP23 and responds similarly to changes in bilayer thickness, from DLPC to DMPC to DOPC. The presence of Y19 changes the azimuthal rotation angle ρ (about the helix axis) to a similar extent as Y5, but in the opposite direction. When tyrosines are substituted for both tryptophans to yield GY5, 19ALP23, the helix tilt angle is again of comparable magnitude, and furthermore the preferred azimuthal rotation angle ρ is relatively unchanged from that of GW5,19ALP23. The extent of dynamic averaging increases marginally when Tyr replaces Trp. Yet, importantly, all members of the peptide family having single Tyr or Trp residues near each interface exhibit only moderate and not highly extensive dynamic averaging. The results provide important benchmarks for evaluating conformational and dynamic control of membrane protein function. PMID:24111589

  8. Conformational Response of Influenza A M2 Transmembrane Domain to Amantadine Drug Binding at Low pH (pH 5.5).

    PubMed

    Georgieva, Elka R; Borbat, Peter P; Grushin, Kirill; Stoilova-McPhie, Svetla; Kulkarni, Nichita J; Liang, Zhichun; Freed, Jack H

    2016-01-01

    The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21-49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21-49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21-49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21-49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/L's ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5-8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD 21-49 through increased motional ordering. In contrast to wild-type M2TMD21-49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation found in certain

  9. Conformational Response of Influenza A M2 Transmembrane Domain to Amantadine Drug Binding at Low pH (pH 5.5)

    PubMed Central

    Georgieva, Elka R.; Borbat, Peter P.; Grushin, Kirill; Stoilova-McPhie, Svetla; Kulkarni, Nichita J.; Liang, Zhichun; Freed, Jack H.

    2016-01-01

    The M2 protein from influenza A plays important roles in its viral cycle. It contains a single transmembrane helix, which oligomerizes into a homotetrameric proton channel that conducts in the low-pH environment of the host-cell endosome and Golgi apparatus, leading to virion uncoating at an early stage of infection. We studied conformational rearrangements that occur in the M2 core transmembrane domain residing on the lipid bilayer, flanked by juxtamembrane residues (M2TMD21−49 fragment), upon its interaction with amantadine drug at pH 5.5 when M2 is conductive. We also tested the role of specific mutation and lipid chain length. Electron spin resonance (ESR) spectroscopy and electron microscopy were applied to M2TMD21−49, labeled at the residue L46C with either nitroxide spin-label or Nanogold® reagent, respectively. Electron microscopy confirmed that M2TMD21−49 reconstituted into DOPC/POPS at 1:10,000 peptide-to-lipid molar ratio (P/L) either with or without amantadine, is an admixture of monomers, dimers, and tetramers, confirming our model based on a dimer intermediate in the assembly of M2TMD21−49. As reported by double electron-electron resonance (DEER), in DOPC/POPS membranes amantadine shifts oligomer equilibrium to favor tetramers, as evidenced by an increase in DEER modulation depth for P/L's ranging from 1:18,000 to 1:160. Furthermore, amantadine binding shortens the inter-spin distances (for nitroxide labels) by 5–8 Å, indicating drug induced channel closure on the C-terminal side. No such effect was observed for the thinner membrane of DLPC/DLPS, emphasizing the role of bilayer thickness. The analysis of continuous wave (cw) ESR spectra of spin-labeled L46C residue provides additional support to a more compact helix bundle in amantadine-bound M2TMD 21−49 through increased motional ordering. In contrast to wild-type M2TMD21−49, the amantadine-bound form does not exhibit noticeable conformational changes in the case of G34A mutation

  10. 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

  11. Broadband circular polarizers constructed using helix-like chiral metamaterials

    NASA Astrophysics Data System (ADS)

    Ji, Ruonan; Wang, Shao-Wei; Liu, Xingxing; Chen, Xiaoshuang; Lu, Wei

    2016-08-01

    In this paper, one kind of helix-like chiral metamaterial which can be realized by multiple conventional lithography or electron beam lithographic techniques is proposed to have a broadband bianisotropic optical response analogous to helical metamaterials. On the basis of twisted metamaterials, via tailoring the relative orientation within the lattice, the anisotropy of arcs is converted into magneto-electric coupling of closely spaced arc pairs, which leads to a broad bianisotropic optical response. By connecting the adjacent upper and lower arcs, the coupling of metasurface pairs is transformed into the coupling of the three-dimensional inclusions, and provides a much broader and higher bianisotropic optical response. For only a four-layer helix-like metamaterial, the maximum extinction ratio can reach 19.7. The operation band is in the wavelength range of 4.69 μm to 8.98 μm with an average extinction ratio of 6.9. And the transmittance for selective polarization is above 0.8 in the entire operation band. Such a structure is a promising candidate for integratable and scalable broadband circular polarizers, especially it has great potential to act as a broadband circular micropolarizer in the field of the full-Stokes division of focal plane polarimeters.In this paper, one kind of helix-like chiral metamaterial which can be realized by multiple conventional lithography or electron beam lithographic techniques is proposed to have a broadband bianisotropic optical response analogous to helical metamaterials. On the basis of twisted metamaterials, via tailoring the relative orientation within the lattice, the anisotropy of arcs is converted into magneto-electric coupling of closely spaced arc pairs, which leads to a broad bianisotropic optical response. By connecting the adjacent upper and lower arcs, the coupling of metasurface pairs is transformed into the coupling of the three-dimensional inclusions, and provides a much broader and higher bianisotropic optical

  12. Folding Dynamics of an α Helix and a β Hairpin

    NASA Astrophysics Data System (ADS)

    Hofrichter, James

    1998-03-01

    What processes limit the rate at which proteins fold? In an effort to address this question we have begun to study the dynamics of the formation of loops, α helices and the minimal β structural element, a β hairpin, which must occur on the pathway from random coils to folded proteins. Because these processes occur on time scales of 10-5-10-9 seconds and experimental access to these time scales has been limited, the kinetics of these processes have not been extensively studied. The expectation is that a more complete understanding of the dynamics of these microprocesses will provide constraints on possible mechanisms for the overall folding of more complex structures. We have explored the kinetics of the helix-coil transition of a synthetic, 21-residue peptide: Ac-WAAAH^+(AAARA)_3A-NH2 and of the folding of a 16 residue β hairpin from protein G B1 using the nanosecond temperature jump technique. Both processes were studied by monitoring tryptophan fluorescence. In the helical peptide, the quantum yield of tryptophan decreases as a result of the interaction between tryptophan in position 1 with the protonated histidine in position 5. In the native conformation of the hairpin, it increases because it forms part of a hydrophobic cluster which stabilizes the native conformation (in a peptide in which a dansylated lysine is incorporated at the C-terminus the fluorescence is quenched). At 300 K, the relaxation time for the helix-coil transition is ~ 250 ns and that for the hairpin-coil transition is ~ 2.2 μs, about 10 times slower. The apparent activation energies are 6.8 kcal/mol for the helix and 10 kcal/mol for the hairpin. We have developed simple kinetic models for these processes which incorporate the sequence- and position-dependent properties known from equilibrium studies and the single-sequence approximation. These models provide a remarkably consistent picture of the dynamics, permitting us to extract information on both the microscopic rates for the c

  13. 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

  14. Phenylene vinylene macrocycles as artificial transmembrane transporters.

    PubMed

    Hu, Xinyu; Yu, Chao; D Okochi, Kenji; Jin, Yinghua; Liu, Zhenning; Zhang, Wei

    2016-04-30

    A series of rigid phenylene vinylene macrocycles and phenylene ethynylene macrocycles with various substituents have been investigated as transmembrane ion channels. The length and polarity of the substituents have a significant effect on the ion channel formation and the mass transport efficiency. Macrocycles with strong aggregation facilitate ion passage across lipid bilayers. PMID:27048875

  15. 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

  16. Structure of the membrane domain of respiratory complex I.

    PubMed

    Efremov, Rouslan G; Sazanov, Leonid A

    2011-08-25

    Complex I is the first and largest enzyme of the respiratory chain, coupling electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It has a central role in cellular energy production and has been implicated in many human neurodegenerative diseases. The L-shaped enzyme consists of hydrophilic and membrane domains. Previously, we determined the structure of the hydrophilic domain. Here we report the crystal structure of the Esherichia coli complex I membrane domain at 3.0 Å resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements. PMID:21822288

  17. 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.

  18. 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

  19. 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

  20. 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

  1. Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase.

    PubMed

    Van Horn, Wade D; Kim, Hak-Jun; Ellis, Charles D; Hadziselimovic, Arina; Sulistijo, Endah S; Karra, Murthy D; Tian, Changlin; Sönnichsen, Frank D; Sanders, Charles R

    2009-06-26

    Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer with the use of solution nuclear magnetic resonance. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK's three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK's lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis. PMID:19556511

  2. 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

  3. 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.

  4. Broadband circular polarizers constructed using helix-like chiral metamaterials.

    PubMed

    Ji, Ruonan; Wang, Shao-Wei; Liu, Xingxing; Chen, Xiaoshuang; Lu, Wei

    2016-08-01

    In this paper, one kind of helix-like chiral metamaterial which can be realized by multiple conventional lithography or electron beam lithographic techniques is proposed to have a broadband bianisotropic optical response analogous to helical metamaterials. On the basis of twisted metamaterials, via tailoring the relative orientation within the lattice, the anisotropy of arcs is converted into magneto-electric coupling of closely spaced arc pairs, which leads to a broad bianisotropic optical response. By connecting the adjacent upper and lower arcs, the coupling of metasurface pairs is transformed into the coupling of the three-dimensional inclusions, and provides a much broader and higher bianisotropic optical response. For only a four-layer helix-like metamaterial, the maximum extinction ratio can reach 19.7. The operation band is in the wavelength range of 4.69 μm to 8.98 μm with an average extinction ratio of 6.9. And the transmittance for selective polarization is above 0.8 in the entire operation band. Such a structure is a promising candidate for integratable and scalable broadband circular polarizers, especially it has great potential to act as a broadband circular micropolarizer in the field of the full-Stokes division of focal plane polarimeters. PMID:27352818

  5. A Cyclized Helix-Loop-Helix Peptide as a Molecular Scaffold for the Design of Inhibitors of Intracellular Protein-Protein Interactions by Epitope and Arginine Grafting.

    PubMed

    Fujiwara, Daisuke; Kitada, Hidekazu; Oguri, Masahiro; Nishihara, Toshio; Michigami, Masataka; Shiraishi, Kazunori; Yuba, Eiji; Nakase, Ikuhiko; Im, Haeri; Cho, Sunhee; Joung, Jong Young; Kodama, Seiji; Kono, Kenji; Ham, Sihyun; Fujii, Ikuo

    2016-08-26

    The design of inhibitors of intracellular protein-protein interactions (PPIs) remains a challenge in chemical biology and drug discovery. We propose a cyclized helix-loop-helix (cHLH) peptide as a scaffold for generating cell-permeable PPI inhibitors through bifunctional grafting: epitope grafting to provide binding activity, and arginine grafting to endow cell-permeability. To inhibit p53-HDM2 interactions, the p53 epitope was grafted onto the C-terminal helix and six Arg residues were grafted onto another helix. The designed peptide cHLHp53-R showed high inhibitory activity for this interaction, and computational analysis suggested a binding mode for HDM2. Confocal microscopy of cells treated with fluorescently labeled cHLHp53-R revealed cell membrane penetration and cytosolic localization. The peptide inhibited the growth of HCT116 and LnCap cancer cells. This strategy of bifunctional grafting onto a well-structured peptide scaffold could facilitate the generation of inhibitors for intracellular PPIs. PMID:27467415

  6. 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.

  7. Helix versus sheet formation in a small peptide

    NASA Astrophysics Data System (ADS)

    Peng, Yong; Hansmann, Ulrich H.

    2003-10-01

    Segments with the amino acid sequence EKAYLRT (glutamine-lysine-alanine-tyrosine-leucine-arginine-threonine) appear in naturally occurring proteins both in α-helices and β-sheets. For this reason, we have used this peptide to study how secondary structure formation in proteins depends on the local environment. Our data rely on multicanonical Monte Carlo simulations where the interactions among all atoms are taken into account. Results in gas phase are compared with that in an implicit solvent. We find that both the solvated molecule and EKAYLRT in gas phase form an α-helix when not interacting with other molecules. However, in the vicinity of a β-strand, the peptide forms a β-strand. Because of this change in secondary structure our peptide may provide a simple model for the α→β transition that is supposedly related to the outbreak of prion diseases and similar illnesses.

  8. Ion fluxes through nanopores and transmembrane channels

    NASA Astrophysics Data System (ADS)

    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.EULEEJ0295-507510.1209/epl/i2006-10240-4 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.

  9. Genome-Wide Analysis of Basic/Helix-Loop-Helix Transcription Factor Family in Rice and Arabidopsis1[W

    PubMed Central

    Li, Xiaoxing; Duan, Xuepeng; Jiang, Haixiong; Sun, Yujin; Tang, Yuanping; Yuan, Zheng; Guo, Jingkang; Liang, Wanqi; Chen, Liang; Yin, Jingyuan; Ma, Hong; Wang, Jian; Zhang, Dabing

    2006-01-01

    The basic/helix-loop-helix (bHLH) transcription factors and their homologs form a large family in plant and animal genomes. They are known to play important roles in the specification of tissue types in animals. On the other hand, few plant bHLH proteins have been studied functionally. Recent completion of whole genome sequences of model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) allows genome-wide analysis and comparison of the bHLH family in flowering plants. We have identified 167 bHLH genes in the rice genome, and their phylogenetic analysis indicates that they form well-supported clades, which are defined as subfamilies. In addition, sequence analysis of potential DNA-binding activity, the sequence motifs outside the bHLH domain, and the conservation of intron/exon structural patterns further support the evolutionary relationships among these proteins. The genome distribution of rice bHLH genes strongly supports the hypothesis that genome-wide and tandem duplication contributed to the expansion of the bHLH gene family, consistent with the birth-and-death theory of gene family evolution. Bioinformatics analysis suggests that rice bHLH proteins can potentially participate in a variety of combinatorial interactions, endowing them with the capacity to regulate a multitude of transcriptional programs. In addition, similar expression patterns suggest functional conservation between some rice bHLH genes and their close Arabidopsis homologs. PMID:16896230

  10. 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

  11. 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

  12. 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 α-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 α-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

  13. Role of the Lipid Environment in the Dimerization of Transmembrane Domains of Glycophorin A

    PubMed Central

    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 α-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 α-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

  14. Residues accessible in the binding-site crevice of transmembrane helix 6 of the CB2 cannabinoid receptor.

    PubMed

    Nebane, Ntsang M; Hurst, Dow P; Carrasquer, Carl A; Qiao, Zhuanhong; Reggio, Patricia H; Song, Zhao-Hui

    2008-12-30

    We have used the substituted-cysteine accessibility method (SCAM) to map the residues in the sixth membrane-spanning segment of the CB2 cannabinoid receptor that contribute to the surface of the water-accessible binding-site crevice. Using a background of the mutant C2.59S which is relatively insensitive to the methanethiosulfonate (MTS) reagents, we mutated to cysteine, one at a time, 34 consecutive residues in TMH6 of the CB2 receptor. These mutant receptors were then expressed in HEK293 cells. By incubating HEK293 cells stably transfected with CB2 receptors with the small, charged, hydrophilic, thiol-specific reagent methanethiosulfonate ethylammonium (MTSEA), [(3)H]CP55940 binding was significantly inhibited for six mutant receptors. All six of the mutants that reacted with MTSEA were protected from the reaction when pretreated with the cannabinoid agonist WIN55212-2, suggesting that MTSEA modification occurred within the binding crevice. Therefore, the side chains of the residues at these reactive loci (V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62) are on the water-accessible surface of the binding-site crevice. These residues are extracellular to the TMH6 CWXP hinge motif. The pattern of accessibility is consistent with a alpha-helical conformation for this segment of TMH6. Molecular modeling studies performed in the context of the CB2 model show that V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62 face into the CB2 binding pocket, further confirming our SCAM results. These results are similar to the accessibility patterns determined by SCAM studies of TMH6 in the opioid and dopamine D2 receptors. PMID:19053233

  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. 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