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Transmembrane helix: simple or complex.  


Transmembrane helical segments (TMs) can be classified into two groups of so-called 'simple' and 'complex' TMs. Whereas the first group represents mere hydrophobic anchors with an overrepresentation of aliphatic hydrophobic residues that are likely attributed to convergent evolution in many cases, the complex ones embody ancestral information and tend to have structural and functional roles beyond just membrane immersion. Hence, the sequence homology concept is not applicable on simple TMs. In practice, these simple TMs can attract statistically significant but evolutionarily unrelated hits during similarity searches (whether through BLAST- or HMM-based approaches). This is especially problematic for membrane proteins that contain both globular segments and TMs. As such, we have developed the transmembrane helix: simple or complex (TMSOC) webserver for the identification of simple and complex TMs. By masking simple TM segments in seed sequences prior to sequence similarity searches, the false-discovery rate decreases without sacrificing sensitivity. Therefore, TMSOC is a novel and necessary sequence analytic tool for both the experimentalists and the computational biology community working on membrane proteins. It is freely accessible at or available for download. PMID:22564899

Wong, Wing-Cheong; Maurer-Stroh, Sebastian; Schneider, Georg; Eisenhaber, Frank



Membrane physical properties influence transmembrane helix formation  

E-print Network

Membrane physical properties influence transmembrane helix formation Francisco N. Barrera1 , Justin properties of the bilayer. These results suggest a physical role for helix-membrane interactions, containing hundreds of different lipid types (3) that impart specific physical properties to the bilayer


Structure of the archaeal Na+/H+ antiporter NhaP1 and functional role of transmembrane helix 1  

PubMed Central

We have determined the structure of the archaeal sodium/proton antiporter NhaP1 at 7 Å resolution by electron crystallography of 2D crystals. NhaP1 is a dimer in the membrane, with 13 membrane-spanning ?-helices per protomer, whereas the distantly related bacterial NhaA has 12. Dimer contacts in the two antiporters are very different, but the structure of a six-helix bundle at the tip of the protomer is conserved. The six-helix bundle of NhaA contains two partially unwound ?-helices thought to harbour the ion-translocation site, which is thus similar in NhaP1. A model of NhaP1 based on detailed sequence comparison and the NhaA structure was fitted to the 7 Å map. The additional N-terminal helix 1 of NhaP1, which appears to be an uncleaved signal sequence, is located near the dimer interface. Similar sequences are present in many eukaryotic homologues of NhaP1, including NHE1. Although fully folded and able to dimerize, NhaP1 constructs without helix 1 are inactive. Possible reasons are investigated and discussed. PMID:21151096

Goswami, Panchali; Paulino, Cristina; Hizlan, Dilem; Vonck, Janet; Yildiz, Ozkan; Kuhlbrandt, Werner



Structure of the Integrin ?IIb Transmembrane Segment*S?  

PubMed Central

Integrin cell-adhesion receptors transduce signals bidirectionally across the plasma membrane via the single-pass transmembrane segments of each ? and ? subunit. While the ?3 transmembrane segment consists of a linear 29-residue ?-helix, the structure of the ?IIb transmembrane segment reveals a linear 24-residue ?-helix (Ile-966 -Lys-989) followed by a backbone reversal that packs Phe-992-Phe-993 against the transmembrane helix. The length of the ?IIb transmembrane helix implies the absence of a significant transmembrane helix tilt in contrast to its partnering ?3 subunit. Sequence alignment shows Gly-991-Phe-993 to be fully conserved among all 18 human integrin ? subunits, suggesting that their unusual structural motif is prototypical for integrin ? subunits. The ?IIb transmembrane structure demonstrates a level of complexity within the membrane that is beyond simple transmembrane helices and forms the structural basis for assessing the extent of structural and topological rearrangements upon ?IIb-?3 association, i.e. integrin transmembrane signaling. PMID:18417472

Lau, Tong-Lay; Dua, Varun; Ulmer, Tobias S.



Molecular and structural transmembrane determinants critical for embedding claudin-5 into tight junctions reveal a distinct four-helix bundle arrangement.  


The mechanism of TJ (tight junction) assembly and the structure of TJ strand-forming Cldns (claudins) are unclear. To identify determinants of assembly of blood-brain barrier-related Cldn3 and Cldn5, chimaeric mutants were analysed by cellular reconstitution of TJ strands and live-cell imaging. On the basis of the rescue of mutants deficient for strand formation, we identified Cldn5 residues (Cys128, Ala132, Ile142, Ala163, Ile166 and Leu174) involved in Cldn folding and assembly. Experimental results were combined with structural bioinformatics approaches. Initially the experimentally validated previous model of the ECL2 (extracellular loop 2) of Cldn5 was extended to the flanking transmembrane segments (TM3/TM4). A coiled-coil interface probably caused by alternating small and large residues is supported by concomitant knob-into-hole interactions including Cldn5-specific residues identified in the present paper. To address arrangement of the TMs in a four-helix bundle, data from evolutionary sequence couplings and comparative modelling of intramolecular interfaces in the transmembrane region of Cldns led to a complete Cldn5 model. Our suggested Cldn subtype-specific intramolecular interfaces that are formed by conserved coiled-coil motifs and non-conserved residues in distinct TM positions were confirmed by the recently released crystal structure of Cldn15. The identified molecular and structural determinants essentially contribute to assembly of Cldns into TJ strands. PMID:25174580

Rossa, Jan; Protze, Jonas; Kern, Christian; Piontek, Anna; Günzel, Dorothee; Krause, Gerd; Piontek, Jörg



Sequence-Specific Dimerization of a Transmembrane Helix in Amphipol A8-35  

PubMed Central

As traditional detergents might destabilize or even denature membrane proteins, amphiphilic polymers have moved into the focus of membrane-protein research in recent years. Thus far, Amphipols are the best studied amphiphilic copolymers, having a hydrophilic backbone with short hydrophobic chains. However, since stabilizing as well as destabilizing effects of the Amphipol belt on the structure of membrane proteins have been described, we systematically analyze the impact of the most commonly used Amphipol A8-35 on the structure and stability of a well-defined transmembrane protein model, the glycophorin A transmembrane helix dimer. Amphipols are not able to directly extract proteins from their native membranes, and detergents are typically replaced by Amphipols only after protein extraction from membranes. As Amphipols form mixed micelles with detergents, a better understanding of Amphipol-detergent interactions is required. Therefore, we analyze the interaction of A8-35 with the anionic detergent sodium dodecyl sulfate and describe the impact of the mixed-micelle-like system on the stability of a transmembrane helix dimer. As A8-35 may highly stabilize and thereby rigidify a transmembrane protein structure, modest destabilization by controlled addition of detergents and formation of mixed micellar systems might be helpful to preserve the function of a membrane protein in Amphipol environments. PMID:25347769

Stangl, Michael; Unger, Sebastian; Keller, Sandro; Schneider, Dirk



Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.  

PubMed Central

Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins. Images Fig. 2 Fig. 4 PMID:8265602

Deber, C M; Khan, A R; Li, Z; Joensson, C; Glibowicka, M; Wang, J



TMpro web server and web service: transmembrane helix prediction through amino acid property analysis  

Microsoft Academic Search

Summary: TMpro is a transmembrane (TM) helix prediction algorithm that uses language processing methodology for TM segment identification. It is primarily based on the analysis of statistical distributions of properties of amino acids in transmembrane segments. This paper describes the availability of TMpro on the internet via a web interface. The key features of the interface are: (i) Output is

Madhavi Ganapathiraju; Christopher Jon Jursa; Hassan A. Karimi; Judith Klein-seetharaman



Structural studies of the putative helix 8 in the human ? 2 adrenergic receptor: an NMR study  

Microsoft Academic Search

The recently reported crystal structure of bovine rhodopsin revealed a cytoplasmic helix (helix 8) in addition to the seven transmembrane helices. This domain is roughly perpendicular to the transmembrane bundle in the presence of an interface and may be a loop-like structure in the absence of an interface. Several studies carried out on this domain suggested that it might act

M. Katragadda; M. W. Maciejewski; P. L. Yeagle



Second Transmembrane Helix (M2) and Long Range Coupling in Ca2+-ATPase.  


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

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



Effect of Detergents on the Association of the Glycophorin A Transmembrane Helix  

PubMed Central

We have examined the role of the environment on the interactions between transmembrane helices using, as a model system, the dimerization of the glycophorin A transmembrane helix. In this study we have focused on micellar environments and have examined a series of detergents that include a range of alkyl chain lengths, combined with ionic, zwitterionic, and nonionic headgroups. For each we have measured how the apparent equilibrium constant depends on the detergent concentration. In two detergents we also measured the thermal sensitivity of the equilibrium constant, from which we derive the van't Hoff enthalpy and entropy. We show that several simple models are inadequate for explaining our results; however, models that include the effect of detergent concentration on detergent binding are able to account for our measurements. Our analysis suggests that the effects of detergents on helix association are due to a pair of opposing effects: an enthalpic effect, which drives association as the detergent concentration is increased and which is sensitive to the chemical nature of the detergent headgroup, opposed by an entropic effect, which drives peptide dissociation as the detergent concentration is raised. Our results also indicate that the monomer-monomer interface is relatively hydrophilic and that association within detergent micelles is driven by the enthalpy change. The wide variations in glycophorin a dimmer, stability with the detergent used, together with the realization that this results from the balance between two opposing effects, suggests that detergents might be selected that drive association rather than dissociation of peptide dimers. PMID:14581210

Fisher, Lillian E.; Engelman, Donald M.; Sturgis, James N.



Structures of the transmembrane helices of the G-protein coupled receptor, rhodopsin.  


An hypothesis is tested that individual peptides corresponding to the transmembrane helices of the membrane protein, rhodopsin, would form helices in solution similar to those in the native protein. Peptides containing the sequences of helices 1, 4 and 5 of rhodopsin were synthesized. Two peptides, with overlapping sequences at their termini, were synthesized to cover each of the helices. The peptides from helix 1 and helix 4 were helical throughout most of their length. The N- and C-termini of all the peptides were disordered and proline caused opening of the helical structure in both helix 1 and helix 4. The peptides from helix 5 were helical in the middle segment of each peptide, with larger disordered regions in the N- and C-termini than for helices 1 and 4. These observations show that there is a strong helical propensity in the amino acid sequences corresponding to the transmembrane domain of this G-protein coupled receptor. In the case of the peptides from helix 4, it was possible to superimpose the structures of the overlapping sequences to produce a construct covering the whole of the sequence of helix 4 of rhodopsin. As similar superposition for the peptides from helix 1 also produced a construct, but somewhat less successfully because of the disordering in the region of sequence overlap. This latter problem was more severe for helix 5 and therefore a single peptide was synthesized for the entire sequence of this helix, and its structure determined. It proved to be helical throughout. Comparison of all these structures with the recent crystal structure of rhodopsin revealed that the peptide structures mimicked the structures seen in the whole protein. Thus similar studies of peptides may provide useful information on the secondary structure of other transmembrane proteins built around helical bundles. PMID:11454172

Katragadda, M; Chopra, A; Bennett, M; Alderfer, J L; Yeagle, P L; Albert, A D



Coupling of transmembrane helix orientation to membrane release of the juxtamembrane region in FGFR3.  


Activation of the protein tyrosine kinase receptors requires the coupling of ligand binding to a change in both the proximity and orientation of the single transmembrane (TM) helices of receptor monomers to allow transphosphorylation of the receptor kinase domain. We make use of peptides corresponding to the TM and juxtamembrane (JM) regions of the fibroblast growth factor receptor 3 to assess how mutations in the TM region (G380R and A391E), which lead to receptor activation, influence the orientation of the TM domain and interactions of the intracellular JM sequence with the membrane surface. On the basis of fluorescence and Fourier transform infrared spectroscopy, we find that both activating mutations change the TM helix tilt angle relative to the membrane normal and release the JM region from the membrane. These results suggest a general mechanism regarding how the TM-JM region functionally bridges the extracellular and intracellular regions for these receptors. PMID:25010350

Tamagaki, Hiroko; Furukawa, Yusuke; Yamaguchi, Ritsuko; Hojo, Hironobu; Aimoto, Saburo; Smith, Steven O; Sato, Takeshi



Specificity of helix packing in transmembrane dimer of the cell death factor BNIP3: a molecular modeling study.  


BNIP3 is a mitochondrial 19-kDa proapoptotic protein, a member of the Bcl-2 family. It has a single COOH-terminal transmembrane (TM) alpha-helical domain, which is required for membrane targeting, proapoptotic activity, hetero- and homo-dimerization in membrane. The role and the molecular details of association of TM helices of BNIP3 are yet to be established. Here, we present a molecular modeling study of helix interactions in its membrane domain. The approach combines Monte Carlo conformational search in an implicit hydrophobic slab followed by molecular dynamics simulations in a hydrated full-atom lipid bilayer. The former technique was used for exhaustive sampling of the peptides' conformational space and for generation of putative "native-like" structures of the dimer. The latter ones were taken as realistic starting points to assess stability and dynamic behavior of the complex in explicit lipid-water surrounding. As a result, several groups of tightly packed right-handed structures of the dimer were proposed. They have almost similar helix-helix interface, which includes the motif A(176)xxxG(180)xxxG(184) and agrees well with previous mutagenesis data and preliminary NMR analysis. Molecular dynamics simulations of these structures reveal perfect adaptation of most of them to heterogeneous membrane environment. A remarkable feature of the predicted dimeric structures is the occurrence of a cluster of H-bonded histidine 173 and serines 168 and 172 on the helix interface, near the N-terminus. Because of specific polar interactions between the monomers, this part of the dimer has no such dense packing as the C-terminal one, thus allowing penetration of water from the extramembrane side into the membrane interior. We propose that the ionization state of His(173) can mediate structural and dynamic properties of the dimer. This, in turn, may be related to pH-dependent proapoptotic activity of BNIP3, which is triggering on by acidosis appearing under hypoxic conditions. PMID:17600828

Vereshaga, Yana A; Volynsky, Pavel E; Pustovalova, Julia E; Nolde, Dmitry E; Arseniev, Alexander S; Efremov, Roman G



Mutations Define Cross-talk between the N-terminal Nucleotide-binding Domain and Transmembrane Helix-2 of the Yeast Multidrug Transporter Pdr5  

PubMed Central

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable Km(ATP) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a noncompetitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport. PMID:18842589

Sauna, Zuben E.; Bohn, Sherry Supernavage; Rutledge, Robert; Dougherty, Michael P.; Cronin, Susan; May, Leopold; Xia, Di; Ambudkar, Suresh V.; Golin, John



Automated method for modeling seven-helix transmembrane receptors from experimental data.  

PubMed Central

A rule-based automated method is presented for modeling the structures of the seven transmembrane helices of G-protein-coupled receptors. The structures are generated by using a simulated annealing Monte Carlo procedure that positions and orients rigid helices to satisfy structural restraints. The restraints are derived from analysis of experimental information from biophysical studies on native and mutant proteins, from analysis of the sequences of related proteins, and from theoretical considerations of protein structure. Calculations are presented for two systems. The method was validated through calculations using appropriate experimental information for bacteriorhodopsin, which produced a model structure with a root mean square (rms) deviation of 1.87 A from the structure determined by electron microscopy. Calculations are also presented using experimental and theoretical information available for bovine rhodopsin to assign the helices to a projection density map and to produce a model of bovine rhodopsin that can be used as a template for modeling other G-protein-coupled receptors. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 8 FIGURE 11 PMID:8599649

Herzyk, P; Hubbard, R E



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

PubMed Central

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 ?-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 ?1-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 ?1-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 ?1-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



TMM@: a web application for the analysis of transmembrane helix mobility  

Microsoft Academic Search

BACKGROUND: To understand the mechanism by which a protein transmits a signal through the cell membrane, an understanding of the flexibility of its transmembrane (TM) region is essential. Normal Mode Analysis (NMA) has become the method of choice to investigate the slowest motions in macromolecular systems. It has been widely used to study transmembrane channels and pumps. It relies on

Lars Skjaerven; Inge Jonassen; Nathalie Reuter



Cysteine Scanning Mutagenesis of Transmembrane Helix 3 of a Brain Glutamate Transporter Reveals Two Conformationally Sensitive Positions*  

PubMed Central

Glutamate transporters in the brain remove the neurotransmitter from the synapse by cotransport with three sodium ions into the surrounding cells. Recent structural work on an archaeal homolog suggests that, during substrate translocation, the transport domain, including the peripheral transmembrane helix 3 (TM3), moves relative to the trimerization domain in an elevator-like process. Moreover, two TM3 residues have been proposed to form part of a transient Na3? site, and another, Tyr-124, appears close to both Na3? and Na1. To obtain independent evidence for the role of TM3 in glutamate transport, each of its 31 amino acid residues from the glial GLT-1 transporter was individually mutated to cysteine. Except for six mutants, substantial transport activity was detected. Aqueous accessibility of the introduced cysteines was probed with membrane-permeant and membrane-impermeant sulfhydryl reagents under a variety of conditions. Transport of six single cysteine mutants, all located on the intracellular side of TM3, was affected by membrane-permeant sulfhydryl reagents. However, only at two positions could ligands modulate the reactivity. A120C reactivity was diminished under conditions expected to favor the outward-facing conformation of the transporter. Sulfhydryl modification of Y124C by 2-aminoethyl methanethiosulfonate, but not by N-ethylmaleimide, was fully protected in the presence of sodium. Our data are consistent with the idea that TM3 moves during transport. Moreover, computational modeling indicated that electrostatic repulsion between the positive charge introduced at position 124 and the sodium ions bound at Na3? and Na1 underlies the protection by sodium. PMID:23188832

Silverstein, Nechama; Crisman, Thomas J.; Forrest, Lucy R.; Kanner, Baruch I.



The development of a model of Alpha helix formation for transmembrane peptides  

E-print Network

. Peptides, once synthesized, are characterized by MALDI mass spectrometry and HPLC and purified peptides are studied using circular dichroism (CD) spectroscopy to determine the a-helicity. Initial results suggest that the transmembrane environment...

Funk, Geoffrey Alexander



The Backbone Dynamics of the Amyloid Precursor Protein Transmembrane Helix Provides a Rationale for the Sequential Cleavage Mechanism of ?-Secretase  

PubMed Central

The etiology of Alzheimer’s disease depends on the relative abundance of different amyloid-? (A?) peptide species. These peptides are produced by sequential proteolytic cleavage within the transmembrane helix of the 99 residue C-terminal fragment of the amyloid precursor protein (C99) by the intramembrane protease ?-secretase. Intramembrane proteolysis is thought to require local unfolding of the substrate helix, which has been proposed to be cleaved as a homodimer. Here, we investigated the backbone dynamics of the substrate helix. Amide exchange experiments of monomeric recombinant C99 and of synthetic transmembrane domain peptides reveal that the N-terminal Gly-rich homodimerization domain exchanges much faster than the C-terminal cleavage region. MD simulations corroborate the differential backbone dynamics, indicate a bending motion at a di-glycine motif connecting dimerization and cleavage regions, and detect significantly different H-bond stabilities at the initial cleavage sites. Our results are consistent with the following hypotheses about cleavage of the substrate. First, the GlyGly hinge may precisely position the substrate within ?-secretase such that its catalytic center must start proteolysis at the known initial cleavage sites. Second, the ratio of cleavage products formed by subsequent sequential proteolysis could be influenced by differential extents of solvation and by the stabilities of H-bonds at alternate initial sites. Third, the flexibility of the Gly-rich domain may facilitate substrate movement within the enzyme during sequential proteolysis. Fourth, dimerization may affect substrate processing by decreasing the dynamics of the dimerization region and by increasing that of the C-terminal part of the cleavage region. PMID:23265086

Pester, Oxana; Barrett, Paul J.; Hornburg, Daniel; Hornburg, Philipp; Probstle, Rasmus; Widmaier, Simon; Kutzner, Christoph; Durrbaum, Milena; Kapurniotu, Aphrodite; Sanders, Charles R.; Scharnagl, Christina; Langosch, Dieter



Methods Mol Biol . Author manuscript 3D structural models of transmembrane proteins  

E-print Network

Methods Mol Biol . Author manuscript Page /1 9 3D structural models of transmembrane proteins: Alexandre De Brevern Abstract Summary Transmembrane proteins transmembrane protein structures are nowadays available. The building of structural models of transmembrane

Paris-Sud XI, Université de


Translocation of molecules into cells by pH-dependent insertion of a transmembrane helix  

E-print Network

in the cytoplasm, including peptide nucleic acids, a cyclic peptide (phalloidin), and organic compounds. Because control, or cell regulation. drug delivery peptide nucleic acid delivery tumors membrane transport helix, 2006 We have previously observed the spontaneous, pH-dependent insertion of a water-soluble peptide


Contribution of the M1 Transmembrane Helix and Pre-M1 Region to Positive Allosteric Modulation and Gating of N-Methyl-d-Aspartate Receptors  

PubMed Central

N-methyl-d-aspartate (NMDA) receptors are glutamate-gated ion channels whose function is critical for normal excitatory synaptic transmission in the brain and whose dysfunction has been implicated in several neurologic conditions. NMDA receptor function is subject to extensive allosteric regulation both by endogenous compounds and by exogenous small molecules. Elucidating the structural determinants and mechanism of action by which allosteric regulators control gating will enhance our understanding of NMDA receptor activation and facilitate the development of novel therapeutics. Here, we investigated the structural determinants for (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (CIQ), a GluN2C/2D-selective positive allosteric modulator. We show that CIQ does not bind to the amino-terminal domain of the NMDA receptor and does not share structural determinants with modulators acting at the agonist-binding domain dimer interface or ion channel pore. Rather, we identified critical determinants of CIQ modulation in the region near the first transmembrane helix of GluN2D, including in a putative pre-M1 cuff helix that may influence channel gating. We also show that mutations within the GluN2D pre-M1 region alter open probability of the NMDA receptor. These results suggest a novel site of action for potentiation of NMDA receptors by small molecules and implicate the pre-M1 region in NMDA receptor gating. PMID:23455314

Traynelis, Stephen F.



NMR Structure of the Transmembrane Domain of the n-Acetylcholine Receptor ?2 Subunit  

PubMed Central

SUMMARY Nicotinic acetylcholine receptors (nAChRs) are involved in fast synaptic transmission in the central and peripheral nervous system. Among the many different types of subunits in nAChRs, the ?2 subunit often combines with the ?4 subunit to form ?4?2 pentameric channels, the most abundant subtype of nAChRs in the brain. Besides computational predictions, there is limited experimental data available on the structure of the ?2 subunit. Using high-resolution NMR spectroscopy, we solved the structure of the entire transmembrane domain (TM1234) of the ?2 subunit. We found that TM1234 formed a four-helix bundle in the absence of the extracellular and intracellular domains. The structure exhibited many similarities to those previously determined for the Torpedo nAChR and the bacterial ion channel GLIC. We also assessed the influence of the fourth transmembrane helix (TM4) on the rest of the domain. Although secondary structures and tertiary arrangements were similar, the addition of TM4 caused dramatic changes in TM3 dynamics and subtle changes in TM1 and TM2. Taken together, this study suggests that the structures of the transmembrane domains of these proteins are largely shaped by determinants inherent in their sequence, but their dynamics may be sensitive to modulation by tertiary and quaternary contacts. PMID:20441771

Bondarenko, Vasyl; Tillman, Tommy; Xu, Yan; Tang, Pei



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

PubMed Central

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

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



Structural studies of the putative helix 8 in the human beta(2) adrenergic receptor: an NMR study.  


The recently reported crystal structure of bovine rhodopsin revealed a cytoplasmic helix (helix 8) in addition to the seven transmembrane helices. This domain is roughly perpendicular to the transmembrane bundle in the presence of an interface and may be a loop-like structure in the absence of an interface. Several studies carried out on this domain suggested that it might act as a conformational switch between the inactive and activated states of this G-protein coupled receptor (GPCR). These results raised the question whether helix 8 may be an important feature of other GPCRs as well. To explore this question, we determined the structure of a peptide representing the putative helix 8 domain in another receptor that belongs to the rhodopsin family of GPCRs, the human beta(2) adrenergic receptor (hbeta(2)AR), using two-dimensional (1)H nuclear magnetic resonance (NMR). The key results from this structural study are that the putative helix 8 domain is helical in detergent and in DMSO while in water this region is disordered; the conformation is therefore dependent upon the environment. Comparison of data from five GPCRs suggests that these observations may be generally important for GPCR structure and function. PMID:15157609

Katragadda, M; Maciejewski, M W; Yeagle, P L



Prediction, Refinement and Persistency of Transmembrane Helix Dimers in Lipid Bilayers using Implicit and Explicit Solvent/Lipid Representations: Microsecond Molecular Dynamics Simulations of ErbB1/B2 and EphA1  

PubMed Central

All-atom simulations are carried out on ErbB1/B2 and EphA1 transmembrane helix dimers in lipid bilayers starting from their solution/DMPC bicelle NMR structures. Over the course of microsecond trajectories, the structures remain in close proximity to the initial configuration and satisfy the great majority of experimental tertiary contact restraints. These results further validate CHARMM protein/lipid force fields and simulation protocols on Anton. Separately, dimer conformations are generated using replica exchange in conjunction with an implicit solvent and lipid representation. The implicit model requires further improvement, and this study investigates whether lengthy all-atom molecular dynamics simulations can alleviate the shortcomings of the initial conditions. The simulations correct many of the deficiencies. For example excessive helix twisting is eliminated over a period of hundreds of nanoseconds. The helix tilt, crossing angles and dimer contacts approximate those of the NMR derived structure, although the detailed contact surface remains off-set for one of two helices in both systems. Hence, even microsecond simulations are not long enough for extensive helix rotations. The alternate structures can be rationalized with reference to interaction motifs and may represent still sought after receptor states that are important in ErbB1/B2 and EphA1 signaling. PMID:23042146

Zhang, Liqun; Sodt, Alexander J.; Venable, Richard M.; Pastor, Richard W.; Buck, Matthias



Structural elucidation of transmembrane transporter protein bilitranslocase: conformational analysis of the second transmembrane region TM2 by molecular dynamics and NMR spectroscopy.  


Membrane proteins represent about a third of the gene products in most organisms, as revealed by the genome sequencing projects. They account for up to two thirds of known drugable targets, which emphasizes their critical pharmaceutical importance. Here we present a study on bilitranslocase (BTL) (TCDB 2.A.65), a membrane protein primarily involved in the transport of bilirubin from blood to liver cells. Bilitranslocase has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its 3D structure. However, at present, only a limited knowledge is available beyond the primary structure of BTL. It has been recently confirmed experimentally that one of the four computationally predicted transmembrane segments of bilitranslocase, TM3, has a helical structure with hydrophilic amino acid residues oriented towards one side, which is typical for transmembrane domains of membrane proteins. In this study we confirmed by the use of multidimensional NMR spectroscopy that the second transmembrane segment, TM2, also appears in a form of ?-helix. The stability of this polypeptide chain was verified by molecular dynamics (MD) simulation in dipalmitoyl phosphatidyl choline (DPPC) and in sodium dodecyl sulfate (SDS) micelles. The two ?-helices, TM2 corroborated in this study, and TM3 confirmed in our previous investigation, provide reasonable building blocks of a potential transmembrane channel for transport of bilirubin and small hydrophilic molecules, including pharmaceutically active compounds. PMID:23774522

Roy Choudhury, Amrita; Perdih, Andrej; Zuperl, Spela; Sikorska, Emilia; Solmajer, Tom; Jurga, Stefan; Zhukov, Igor; Novi?, Marjana



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

PubMed Central

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

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



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

PubMed Central

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 tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure. PMID:23967136

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



Contact-Induced Structure Transformation in Transmembrane Prion Propagation  

E-print Network

Contact-Induced Structure Transformation in Transmembrane Prion Propagation D.-M. Ou, C.-C. Chen on recent experimental evidences of the transmission of prion diseases due to a particular transmem- brane prions, we conclude that cooperative dimerization may play an important role in the pathological

Chen, Chi-Ming


Loop Diuretic and Ion-binding Residues Revealed by Scanning Mutagenesis of Transmembrane Helix 3 (TM3) of Na-K-Cl Cotransporter (NKCC1)*  

PubMed Central

The Na-K-Cl cotransporter (NKCC) plays central roles in cellular chloride homeostasis and in epithelial salt transport, but to date little is known about the mechanism by which the transporter moves ions across the membrane. We examined the functional role of transmembrane helix 3 (TM3) in NKCC1 using cysteine- and tryptophan-scanning mutagenesis and analyzed our results in the context of a structural homology model based on an alignment of NKCC1 with other amino acid polyamine organocation superfamily members, AdiC and ApcT. Mutations of residues along one face of TM3 (Tyr-383, Met-382, Ala-379, Asn-376, Ala-375, Phe-372, Gly-369, and Ile-368) had large effects on translocation rate, apparent ion affinities, and loop diuretic affinity, consistent with a proposed role of TM3 in the translocation pathway. The prediction that Met-382 is part of an extracellular gate that closes to form an occluded state is strongly supported by conformational sensitivity of this residue to 2-(trimethylammonium)ethyl methanethiosulfonate, and the bumetanide insensitivity of M382W is consistent with tryptophan blocking entry of bumetanide into the cavity. Substitution effects on residues at the intracellular end of TM3 suggest that this region is also involved in ion coordination and may be part of the translocation pathway in an inward-open conformation. Mutations of predicted pore residues had large effects on binding of bumetanide and furosemide, consistent with the hypothesis that loop diuretic drugs bind within the translocation cavity. The results presented here strongly support predictions of homology models of NKCC1 and demonstrate important roles for TM3 residues in ion translocation and loop diuretic inhibition. PMID:22437837

Somasekharan, Suma; Tanis, Jessica; Forbush, Biff



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

NASA Astrophysics Data System (ADS)

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

Harrington, Susan E.; Ben-Tal, Nir


Structural insight into the transmembrane domain and the juxtamembrane region of the erythropoietin receptor in micelles.  


Erythropoietin receptor (EpoR) dimerization is an important step in erythrocyte formation. Its transmembrane domain (TMD) and juxtamembrane (JM) region are essential for signal transduction across the membrane. A construct compassing residues S212-P259 and containing the TMD and JM region of the human EpoR was purified and reconstituted in detergent micelles. The solution structure of the construct was determined in dodecylphosphocholine (DPC) micelles by solution NMR spectroscopy. Structural and dynamic studies demonstrated that the TMD and JM region are an ?-helix in DPC micelles, whereas residues S212-D224 at the N-terminus of the construct are not structured. The JM region is a helix that contains a hydrophobic patch formed by conserved hydrophobic residues (L253, I257, and W258). Nuclear Overhauser effect analysis, fluorescence spectroscopy, and paramagnetic relaxation enhancement experiments suggested that the JM region is exposed to the solvent. The structures of the TMD and JM region of the mouse EpoR were similar to those of the human EpoR. PMID:25418301

Li, Qingxin; Wong, Ying Lei; Huang, Qiwei; Kang, CongBao



Probabilistic grammatical model for helix-helix contact site classification  

PubMed Central

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



The pore-lining regions in cytochrome c oxidases: A computational analysis of caveolin, cholesterol and transmembrane helix contributions to proton movement.  


Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain. CcO catalyzes a four electron reduction of O2 to water at a catalytic site formed by high-spin heme (a3) and copper atoms (CuB). While it is recognized that proton movement is coupled to oxygen reduction, the proton channel(s) have not been well defined. Using computational methods developed to study protein topology, membrane channels and 3D packing arrangements within transmembrane (TM) helix arrays, we find that subunit-1 (COX-1), subunit-2 (COX-2) and subunit-3 (COX-3) contribute 139, 46 and 25 residues, respectively, to channel formation between the mitochondrial matrix and intermembrane space. Nine of 12 TM helices in COX-1, both helices in COX-2 and 5 of the 6 TM helices in COX-3 are pore-lining regions (possible channel formers). Heme a3 and the CuB sites (as well as the CuA center of COX-2) are located within the channel that includes TM-6, TM-7, TM-10 and TM-11 of COX-1 and are associated with multiple cholesterol and caveolin-binding (CB) motifs. Sequence analysis identifies five CB motifs within COX-1, two within COX-2 and four within COX-3; each caveolin containing a pore-lining helix C-terminal to a TM helix-turn-helix. Channel formation involves interaction between multiple pore-lining regions within protein subunits and/or dimers. PoreWalker analysis lends support to the D-channel model of proton translocation. Under physiological conditions, caveolins may introduce channel formers juxtaposed to those in COX-1, COX-2 and COX-3, which together with cholesterol may form channel(s) essential for proton translocation through the inner mitochondrial membrane. PMID:25037006

Morrill, Gene A; Kostellow, Adele B; Gupta, Raj K



Novel Insights into CB1 Cannabinoid Receptor Signaling: A Key Interaction Identified between the Extracellular-3 Loop and Transmembrane Helix 2S?  

PubMed Central

Activation of the cannabinoid CB1 receptor (CB1) is modulated by aspartate residue D2.63176 in transmembrane helix (TMH) 2. Interestingly, D2.63 does not affect the affinity for ligand binding at the CB1 receptor. Studies in class A G protein-coupled receptors have suggested an ionic interaction between residues of TMH2 and 7. In this report, modeling studies identified residue K373 in the extracellular-3 (EC-3) loop in charged interactions with D2.63. We investigated this possibility by performing reciprocal mutations and biochemical studies. D2.63176A, K373A, D2.63176A-K373A, and the reciprocal mutant with the interacting residues juxtaposed D2.63176K-K373D were characterized using radioligand binding and guanosine 5?-3-O-(thio)triphosphate functional assays. None of the mutations resulted in a significant change in the binding affinity of N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A) or (?)-3cis -[2-hydroxyl-4-(1,1-dimethyl-heptyl)phenyl]-trans-4-[3-hydroxyl-propyl] cyclohexan-1-ol (CP55,940). Modeling studies indicated that binding-site interactions and energies of interaction for CP55,940 were similar between wild-type and mutant receptors. However, the signaling of CP55,940, and (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)-methanone mesylate (WIN55,212-2) was impaired at the D2.63176A-K373A and the single-alanine mutants. In contrast, the reciprocal D2.63176K-K373D mutant regained function for both CP55,940 and WIN55,212-2. Computational results indicate that the D2.63176-K373 ionic interaction strongly influences the conformation(s) of the EC-3 loop, providing a structure-based rationale for the importance of the EC-3 loop to signal transduction in CB1. The putative ionic interaction results in the EC-3 loop pulling over the top (extracellular side) of the receptor; this EC-3 loop conformation may serve protective and mechanistic roles. These results suggest that the ionic interaction between D2.63176 and K373 is important for CB1 signal transduction. PMID:23426954

Marcu, Jahan; Shore, Derek M.; Kapur, Ankur; Trznadel, Megan; Makriyannis, Alexandros; Reggio, Patricia H.



Secondary structure, orientation, and oligomerization of phospholemman, a cardiac transmembrane protein.  


Human phospholemman (PLM) is a 72-residue protein, which is expressed at high density in the cardiac plasma membrane and in various other tissues. It forms ion channels selective for K+, Cl-, and taurine in lipid bilayers and colocalizes with the Na+/K+-ATPase and the Na+/Ca2+-exchanger, which may suggest a role in the regulation of cell volume. Here we present the first structural data based on synthetic peptides representing the transmembrane domain of PLM. Perfluoro-octaneoate-PAGE of reconstituted proteoliposomes containing PLM reveals a tetrameric homo-oligomerization. Infrared spectroscopy of proteoliposomes shows that the PLM peptide is completely alpha-helical, even beyond the hydrophobic core residues. Hydrogen/deuterium exchange experiments reveal that a core of 20-22 residues is not accessible to water, thus embedded in the lipid membrane. The maximum helix tilt is 17 degrees +/- 2 degrees obtained by attenuated total reflection infrared spectroscopy. Thus, our data support the idea of ion channel formation by the PLM transmembrane domain. PMID:16597826

Beevers, Andrew J; Kukol, Andreas



Structure-based prediction reveals capping motifs that inhibit beta-helix aggregation  

E-print Network

The parallel beta-helix is a geometrically regular fold commonly found in the proteomes of bacteria, viruses, fungi, archaea, and some vertebrates. beta-helix structure has been observed in monomeric units of some aggregated ...

Bryan, Allen W.


Effects of water flow on transmembrane ionic currents in neurons of Helix pomatia and in squid giant axons.  


1. The effects of water flow through the membrane produced by an osmotic gradient on the ionic currents in Helix neurons and in squid giant axons were studied. 2. Outward water flow had a marked effect on the ionic currents. 3. Cell volume diminution in hypertonic solution was accompanied by a decrease in the number of functioning ionic channels in the neurons. 4. Decrease of the tonicity of the external 10(-8) M TTX-containing solution leads to a transient recovery of the action potentials of the squid. PMID:2452049

Ayrapetyan, S N; Rychkov, G Y; Suleymanyan, M A



Functional importance of polar and charged amino acid residues in transmembrane helix 14 of multidrug resistance protein 1 (MRP1/ABCC1): identification of an aspartate residue critical for conversion from a high to low affinity substrate binding state.  


Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions. We previously demonstrated that Glu1089 in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance. We have now assessed the functional importance of all polar and charged amino acids in this TM helix. Asn1100, Ser1097, and Lys1092, which are all predicted to be on the same face of the helix as to Glu1089, are involved in determining the substrate specificity of the protein. Notably, elimination of the positively charged side chain of Lys1092, increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16). In addition, mutations S1097A and N1100A selectively decreased transport of 17beta-estradiol 17-(beta-d-glucuronide) (E217betaG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity. In contrast, mutations of Asp1084 that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E217betaG and LTC4, despite the fact that LTC4 binding was unaffected. We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2. Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp1084 may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs. PMID:12954620

Zhang, Da-Wei; Gu, Hong-Mei; Situ, Donna; Haimeur, Anass; Cole, Susan P C; Deeley, Roger G



Open-Channel Structures of the Human Glycine Receptor ?1 Full-Length Transmembrane Domain  

PubMed Central

Summary Glycine receptors play a major role in mediating fast inhibitory neurotransmission in the spinal cord and brain stem, yet their high-resolution structures remain unsolved. We determined open-channel structures of the full-length transmembrane domain (TMD) of the human glycine receptor ?1-subunit (hGlyR-?1) using NMR and electron micrographs. The hGlyR-?1 TMD spontaneously forms pentameric Cl?-conducting channels, with structures sharing overall topology observed in crystal structures of homologous bacterial and nematode pentameric ligand-gated ion channels (pLGICs). However, the mammalian hGlyR-?1 structures present several unique features, including a shorter helix of the pore-lining TM2 with helical unwinding near the C-terminal end, a TM3 helical kink at A288 that partially overlaps with the homologous ivermectin-binding site in GluCl, and a highly dynamic segment between S267(15?) of TM2 and A288 that likely affects allosteric modulations of channel function. The NMR structures provide new templates for identifying potential drug targets in GlyRs and other mammalian pLGICs. PMID:23994010

Mowrey, David; Cui, Tanxing; Jia, Yuanyuan; Ma, Dejian; Makhov, Alexander M.; Zhang, Peijun; Tang, Pei; Xu, Yan



Structural heterogeneity in transmembrane amyloid precursor protein homodimer is a consequence of environmental selection.  


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 ?-secretase to produce amyloid-? (A?) 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 C99(15-55) homodimer in POPC membrane bilayer and DPC surfactant micelle environments were performed using a multiscale approach that blends atomistic and coarse-grained models. The C99(15-55) 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

Dominguez, Laura; Foster, Leigh; Meredith, Stephen C; Straub, John E; Thirumalai, D



PackHelix: a tool for helix-sheet packing during protein structure prediction  

PubMed Central

The three-dimensional structure of a protein is organized around the packing of its secondary structure elements. Predicting the topology and constructing the geometry of structural motifs involving ?-helices and/or ?-strands are therefore key steps for accurate prediction of protein structure. While many efforts have focused on how to pack helices and on how to sample exhaustively the topologies and geometries of multiple strands forming a ?-sheet in a protein, there has been little progress on generating native-like packing of helices on sheets. We describe a method that can generate the packing of multiple helices on a given ?-sheet for ??? sandwich type protein folds. This method mines the results of a statistical analysis of the conformations of ??2 motifs in protein structures to provide input values for the geometric attributes of the packing of a helix on a sheet. It then proceeds with a geometric builder that generates multiple arrangements of the helices on the sheet of interest by sampling through these values and performing consistency checks that guarantee proper loop geometry between the helices and the strands, minimal number of collisions between the helices, and proper formation of a hydrophobic core. The method is implemented as a module of ProteinShop. Our results show that it produces structures that are within 4–6 Å RMSD of the native one, regardless of the number of helices that need to be packed, though this number may increase if the protein has several helices between two consecutive strands in the sequence that pack on the sheet formed by these two strands. PMID:21905109

Hu, Chengcheng; Koehl, Patrice; Max, Nelson



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

SciTech Connect

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

Horne, W. Seth; Price, Joshua L.; Keck, James L.; Gellman, Samuel H. (UW-MED)



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

PubMed Central

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-? (A?) 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 ?-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 ?-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 ?-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for A? 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 ?- and ?-secretase and resulting amyloid-? 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

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



A helix-to-coil transition at the ?-cut site in the transmembrane dimer of the amyloid precursor protein is required for proteolysis  

Microsoft Academic Search

Processing of amyloid precursor protein (APP) by gamma-secretase is the last step in the formation of the Abeta peptides associated Alzheimer's disease. Solid-state NMR spectroscopy is used to establish the structural features of the transmembrane (TM) and juxtamembrane (JM) domains of APP that facilitate proteolysis. Using peptides corresponding to the APP TM and JM regions (residues 618-660), we show that

Takeshi Sato; Tzu-Chun Tang; Gabriella Reubins; Jeffrey Z. Fei; Taiki Fujimoto; Pascal Kienlen-Campard; Stefan N. Constantinescu; Jean-Noel Octave; Saburo Aimoto; Steven O. Smith



Structural Plasticity in Self-Assembling Transmembrane ?-Sheets  

PubMed Central

Here we test the hypothesis that membrane-spanning ?-sheets can exhibit structural plasticity in membranes due to their ability to shift hydrogen-bonding patterns. Transmembrane ?-sheet forming peptides of the sequence AcWLn, where n = 5, 6, or 7, which range from 21 to 27 Å in maximum length, were incorporated into bilayers made of phosphatidylcholine lipids with saturated acyl chains containing 14, 16, or 18 carbons, which are 36–50 Å in thickness. The effect of the peptide ?-sheets on fluid- and gel-phase bilayers were studied with differential scanning calorimetry and circular dichroism spectroscopy. We show that AcWL5 forms a stable, peptide-rich gel phase in all three lipids. The whole family of AcWLn peptides appears to form similarly stable, nonmembrane-disrupting ?-sheets in all bilayer phases and thicknesses. Bilayers containing up to 20 mol % peptide, which is the maximum concentration tested, formed gel phases with melting temperatures that were equal to, or slightly higher than, the pure lipid transitions. Given the range of peptide lengths and bilayer thicknesses tested, these experiments show that the AcWLn family of membrane-inserted ?-sheets exhibit remarkable structural plasticity in membranes. PMID:21843473

Bishop, Christopher M.; Wimley, William C.



Effect of Plated Metal Film on the Performance of Helix TWT Slow-Wave Structure  

Microsoft Academic Search

The effect of the plated metal film on the heat dissipation capability and cold-test characteristics of the helix slow-wave structure (SWS) have been studied in several experimental tests and computer simulations. The thermal conduction and the power losses of the helix SWS were improved by using a plated helix. A brazed SWS with end-plated rods exhibits excellent heat dissipation capability.

Yong Han; Yan-Wen Liu; Yao-Gen Ding; Pu-Kun Liu



Analysis of Helix Slow Wave Structure for High Efficiency Space TWT  

NASA Astrophysics Data System (ADS)

This paper describes the analysis of helix slow-wave structure (SWS) for a high efficiency space traveling wave tube that is carried out using Ansoft HFSS and CST microwave studio, which is a 3D electromagnetic field simulators. Two approaches of simulating the dispersion and impedance characteristics of the helix slow wave structure have been discussed and compared with measured results. The dispersion characteristic gives the information about axial propagation constant (Beta). Which in turn yields the phase velocity at a particular frequency. The dispersion and impedance characteristics can be used in finding the pertinent design parameters of the helix slow-wave structure. Therefore a new trend has been initiated at CEERI to use Ansoft HFSS code to analysis of the helix slow wave structure in its real environment. The analysis of the helix SWS for Ku-band 140W space TWT has been carried out and compared with experimental results, and found is close agreement.

Alaria, Mukesh Kumar; Bera, A.; Sinha, A. K.; Srivastava, V.



Functional Identification of Close Proximity Amino Acid Side Chains within the Transmembrane-Spanning Helixes of the P2X2 Receptor  

PubMed Central

The transition from the closed to open state greatly alters the intra- and inter-subunit interactions of the P2X receptor (P2XR). The interactions that occur in the transmembrane domain of the P2X2R remain unclear. We used substituted cysteine mutagenesis disulfide mapping to identify pairs of residues that are in close proximity within the transmembrane domain of rP2X2R and compared our results to the predicted positions of these amino acids obtained from a rat P2X2R homology model of the available open and closed zebrafish P2X4R structures. Alternations in channel function were measured as a change in the ATP-gated current before and after exposure to dithiothreitol. Thirty-six pairs of double mutants of rP2X2R expressed in HEK293 cells produced normal functioning channels. Thirty-five pairs of these mutants did not exhibit a functionally detectable disulfide bond. The double mutant H33C/S345C formed redox-dependent cross-links in the absence of ATP. Dithiothreitol ruptured the disulfide bond of H33C/S345C and induced a 2 to 3-fold increase in current. The EC50 for H33C/S345C before dithiothreitol treatment was ?2-fold higher than that after dithiothreitol treatment. Dithiothreitol reduced the EC50 to wild-type levels. Furthermore, expression of trimeric concatamer receptors with Cys mutations at some but not all six positions showed that the more disulfide bond formation sites within the concatamer, the greater current potentiation after dithiothreitol incubation. Immunoblot analysis of H33C/S345C revealed one monomer band under nonreducing conditions strongly suggesting that disulfide bonds are formed within single subunits (intra-subunit) and not between two subunits (inter-subunit). Taken together, these data indicate that His33 and Ser345 are proximal to each other across an intra-subunit interface. The relative movement between the first transmembrane and the second transmembrane in the intra-subunit is likely important for transmitting the action of ATP binding to the opening of the channel. PMID:23936459

Liang, Xin; Xu, Huijuan; Li, Caiyue; Yin, Shikui; Xu, Tingting; Liu, Jinsong; Li, Zhiyuan



Correlation of the structure of the transmembrane domain of the neu oncogene-encoded p185 protein with its function.  


The human homologue of the neu oncogene is frequently found in human tumors. Certain amino acid substitutions at position 664 in the transmembrane domain of the neu oncogene-encoded p185 protein product are known to cause malignant transformation of cells. Using conformational energy analysis based on ECEPP (empirical conformational energies for polypeptides program), we have previously determined the preferred three-dimensional structures for the transmembrane domain of the p185 protein with a transforming (glutamic acid) and a nontransforming (valine) substitution at the critical position 664 and found that the global minimum-energy conformation of this region in the nontransforming protein contains a sharp bend, whereas the global minimum-energy conformation for this region from the transforming protein is entirely alpha-helical. We now demonstrate that this result holds for other known nontransforming (glycine, histidine, tyrosine, and lysine) and transforming (glutamine) substitutions at position 664. Furthermore, a simple statistical thermodynamic analysis of the results indicates that approximately 85% of each of the nontransforming sequences exist with the bend at positions 664 and 665, while approximately 90% of each of the transforming sequences exist as an alpha-helix. About 9% of the nontransforming sequences exist as the alpha-helix. These results suggest that if the intracellular concentration of the normal protein is increased at least 10-fold, thereby increasing the alpha-helical form by this factor, cell transformation should result. This conclusion is directly supported by genetic experiments in which this level of overexpression of the normal protein was achieved with attendant cell transformation. PMID:1978329

Brandt-Rauf, P W; Rackovsky, S; Pincus, M R



Systems and Methods for Predicting the Structure and Function of Multipass Transmembrane Proteins.  

National Technical Information Service (NTIS)

The invention provides computer-implemented methods and apparatus implementing a hierarchical protocol using multiscale molecular dynamics and molecular modeling methods to predict the structure of transmembrane proteins such as G-Protein Coupled Receptor...

N. Vaidehi, R. J. Trabanino, S. E. Hall, W. Floriano, W. A. Goddard



Analyzing oligomerization of individual transmembrane helices and of entire membrane proteins in E. coli: A hitchhiker's guide to GALLEX.  


Genetic systems, which allow monitoring interactions of individual transmembrane ?-helices within the cytoplasmic membrane of the bacterium Escherichia coli, are now widely used to probe the structural biology and energetics of helix-helix interactions and the consequences of mutations. In contrast to other systems, the GALLEX system allows studying homo- as well as heterooligomerization of individual transmembrane ?-helices, and even enables estimation of the energetics of helix-helix interactions within a biological membrane. Given that many polytopic membrane proteins form oligomers within membranes, the GALLEX system represents a unique and powerful approach to monitor formation and stability of oligomeric complexes of polytopic membrane proteins in vivo. PMID:22987358

Cymer, Florian; Sanders, Charles R; Schneider, Dirk



A Crystal Structure of an Oligoproline PPII-Helix, at Last.  


The first crystal structure of an oligoproline adopting an all-trans polyproline II (PPII) helix is presented. The high-resolution structure provides detailed insight into the dimensions and conformational properties of oligoprolines that are important for, e.g., their use as "molecular rulers" and "molecular scaffolds". The structure also showed that the amides interact with each other within a PPII helix and that water is not necessary for PPII helicity. PMID:25368901

Wilhelm, Patrick; Lewandowski, Bartosz; Trapp, Nils; Wennemers, Helma



A graph-theoretic approach for classification and structure prediction of transmembrane ?-barrel proteins  

PubMed Central

Background Transmembrane ?-barrel proteins are a special class of transmembrane proteins which play several key roles in human body and diseases. Due to experimental difficulties, the number of transmembrane ?-barrel proteins with known structures is very small. Over the years, a number of learning-based methods have been introduced for recognition and structure prediction of transmembrane ?-barrel proteins. Most of these methods emphasize on homology search rather than any biological or chemical basis. Results We present a novel graph-theoretic model for classification and structure prediction of transmembrane ?-barrel proteins. This model folds proteins based on energy minimization rather than a homology search, avoiding any assumption on availability of training dataset. The ab initio model presented in this paper is the first method to allow for permutations in the structure of transmembrane proteins and provides more structural information than any known algorithm. The model is also able to recognize ?-barrels by assessing the pseudo free energy. We assess the structure prediction on 41 proteins gathered from existing databases on experimentally validated transmembrane ?-barrel proteins. We show that our approach is quite accurate with over 90% F-score on strands and over 74% F-score on residues. The results are comparable to other algorithms suggesting that our pseudo-energy model is close to the actual physical model. We test our classification approach and show that it is able to reject ?-helical bundles with 100% accuracy and ?-barrel lipocalins with 97% accuracy. Conclusions We show that it is possible to design models for classification and structure prediction for transmembrane ?-barrel proteins which do not depend essentially on training sets but on combinatorial properties of the structures to be proved. These models are fairly accurate, robust and can be run very efficiently on PC-like computers. Such models are useful for the genome screening. PMID:22537300



Atomic Structure of a Short alpha-Helix Stabilized by a Main Chain Hydrogen-Bond Surrogate  

SciTech Connect

Herein we report the 1.15 Angstroms X-ray crystal structure of a short {alpha}-helix in which the N-terminal i and i + 4 main chain hydrogen bond is replaced with a carbon-carbon bond. The structure shows that the hydrogen-bond surrogate (HBS) derived a-helix truly resembles the structure of canonical a-helices and provides unequivocal support for our helix nucleation strategy.

Liu,J.; Wang, D.; Zheng, Q.; Lu, M.; Arora, P.



Translocation of molecules into cells by pH-dependent insertion of a transmembrane helix Yana K. Reshetnyak, Oleg A. Andreev, Ursula Lehnert, and Donald M. Engelman  

E-print Network

be released by reduc- tion in the cytoplasm, including peptide nucleic acids, a cyclic peptide (phalloidin imaging, genetic control, or cell regulation. drug delivery peptide nucleic acid delivery tumors membrane-soluble peptide to form a helix across lipid bilayers [Hunt, J. F., Rath, P., Rothschild, K. J. & Engelman, D. M

Rhode Island, University of


The close-packed triple helix as a possible new structural motif for collagen  

E-print Network

The one-dimensional problem of selecting the triple helix with the highest volume fraction is solved and hence the condition for a helix to be close-packed is obtained. The close-packed triple helix is shown to have a pitch angle of $v_{CP} =43.3 ^\\circ$. Contrary to the conventional notion, we suggest that close packing form the underlying principle behind the structure of collagen, and the implications of this suggestion are considered. Further, it is shown that the unique zero-twist structure with no strain-twist coupling is practically identical to the close-packed triple helix. Some of the difficulties for the current understanding of the structure of collagen are reviewed: The ambiguity in assigning crystal structures for collagen-like peptides, and the failure to satisfactorily calculate circular dichroism spectra. Further, the proposed new geometrical structure for collagen is better packed than both the 10/3 and the 7/2 structure. A feature of the suggested collagen structure is the existence of a central channel with negatively charged walls. We find support for this structural feature in some of the early x-ray diffraction data of collagen. The central channel of the structure suggests the possibility of a one-dimensional proton lattice. This geometry can explain the observed magic angle effect seen in NMR studies of collagen. The central channel also offers the possibility of ion transport and may cast new light on various biological and physical phenomena, including biomineralization.

Jakob Bohr; Kasper Olsen



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

NASA Astrophysics Data System (ADS)

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.

Eisenberg, David



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

PubMed Central

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

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



The close-packed triple helix as a possible new structural motif for collagen  

E-print Network

The one-dimensional problem of selecting the triple helix with the highest volume fraction is solved and hence the condition for a helix to be close-packed is obtained. The close-packed triple helix is shown to have a pitch angle of $v_{CP} =43.3 ^\\circ$. Contrary to the conventional notion, we suggest that close packing form the underlying principle behind the structure of collagen, and the implications of this suggestion are considered. Further, it is shown that the unique zero-twist structure with no strain-twist coupling is practically identical to the close-packed triple helix. Some of the difficulties for the current understanding of the structure of collagen are reviewed: The ambiguity in assigning crystal structures for collagen-like peptides, and the failure to satisfactorily calculate circular dichroism spectra. Further, the proposed new geometrical structure for collagen is better packed than both the 10/3 and the 7/2 structure. A feature of the suggested collagen structure is the existence of a ce...

Bohr, Jakob



Backbone Structure of the Amantadine-Blocked Trans-Membrane Domain M2 Proton Channel from Influenza A Virus  

E-print Network

Backbone Structure of the Amantadine-Blocked Trans-Membrane Domain M2 Proton Channel from Influenza proton channel of the Influenza A virus. Here, we present a structure of the M2 trans-membrane domain. INTRODUCTION Influenza is a worldwide epidemic that causes substantial morbidity and mortality. Of the three

Bertram, Richard


Amelogenin Supramolecular Assembly in Nanospheres Defined by a Complex Helix-Coil-PPII Helix 3D-Structure  

PubMed Central

Tooth enamel, the hardest material in the human body, is formed within a self-assembled matrix consisting mostly of amelogenin proteins. Here we have determined the complete mouse amelogenin structure under physiological conditions and defined interactions between individual domains. NMR spectroscopy revealed four major amelogenin structural motifs, including an N-terminal assembly of four ?-helical segments (S9-V19, T21-P33, Y39-W45, V53-Q56), an elongated random coil region interrupted by two 310 helices (?P60-Q117), an extended proline-rich PPII-helical region (P118-L165), and a charged hydrophilic C-terminus (L165-D180). HSQC experiments demonstrated ipsilateral interactions between terminal domains of individual amelogenin molecules, i.e. N-terminal interactions with corresponding N-termini and C-terminal interactions with corresponding C-termini, while the central random coil domain did not engage in interactions. Our HSQC spectra of the full-length amelogenin central domain region completely overlapped with spectra of the monomeric Amel-M fragment, suggesting that the central amelogenin coil region did not involve in assembly, even in assembled nanospheres. This finding was confirmed by analytical ultracentrifugation experiments. We conclude that under conditions resembling those found in the developing enamel protein matrix, amelogenin molecules form complex 3D-structures with N-terminal ?-helix-like segments and C-terminal PPII-helices, which self-assemble through ipsilateral interactions at the N-terminus of the molecule. PMID:21984897

Zhang, Xu; Ramirez, Benjamin E.; Liao, Xiubei; Diekwisch, Thomas G. H.



Degeneracy and Inversion of Band Structure for Wigner Crystals on a Toroidal Helix  

E-print Network

We explore the formation of Wigner crystals for charged particles on a toroidal helix. Focusing on certain commensurate cases we show that the ground state undergoes a pitchfork bifurcation from the totally symmetric polygonic to a zig-zag-like configuration with increasing radius of the helix. Remarkably, we find that for a specific value of the helix radius, below the bifurcation point, the vibrational frequency spectrum collapses to a single frequency. This allows for an essentially independent small-amplitude motion of the individual particles and consequently localized excitations can propagate in time without significant spreading. Increasing the radius beyond the degeneracy point, the band structure is inverted, with the out-of-phase oscillation mode becoming lower in frequency than the mode corresponding to the center of mass motion.

Zampetaki, A V; Schmelcher, P



Transformation between -helix and -sheet structures of one and two polyglutamine peptides in explicit water  

E-print Network

Transformation between -helix and -sheet structures of one and two polyglutamine peptides-Jung Chen , Hisashi Okumura , Chin-Kun Hu§ April 19, 2014 Abstract Aggregation of polyglutamine peptides. However it is not clear how polyglutamine peptides form the -sheets and aggregate. To understand


Binding of MgtR, a Salmonella transmembrane regulatory peptide, to MgtC, a Mycobacterium tuberculosis virulence factor: a structural study.  


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 homologous to its S. Typhi 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. Typhi 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

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



A Secondary Structural Transition in the C-helix Promotes Gating of Cyclic Nucleotide-regulated Ion Channels*  

PubMed Central

Cyclic nucleotide-regulated ion channels bind second messengers like cAMP to a C-terminal domain, consisting of a ?-roll, followed by two ?-helices (B- and C-helices). We monitored the cAMP-dependent changes in the structure of the C-helix of a C-terminal fragment of HCN2 channels using transition metal ion FRET between fluorophores on the C-helix and metal ions bound between histidine pairs on the same helix. cAMP induced a change in the dimensions of the C-helix and an increase in the metal binding affinity of the histidine pair. cAMP also caused an increase in the distance between a fluorophore on the C-helix and metal ions bound to the B-helix. Stabilizing the C-helix of intact CNGA1 channels by metal binding to a pair of histidines promoted channel opening. These data suggest that ordering of the C-helix is part of the gating conformational change in cyclic nucleotide-regulated channels. PMID:23525108

Puljung, Michael C.; Zagotta, William N.



Structural Investigation of the Transmembrane Domain of KCNE1 in Proteoliposomes.  


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

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



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

PubMed Central

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

Schlenker, Casey; Goel, Anupam; Tripet, Brian P.; Menon, Smita; Willi, Taylor; Dlakic, Mensur; Young, Mark J.; Lawrence, C Martin; Copie, Valerie



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


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

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



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

PubMed Central

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

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



Structure prediction of the dimeric neu\\/ErbB-2 transmembrane domain from multi-nanosecond molecular dynamics simulations  

Microsoft Academic Search

Dimerization of the neu\\/ErbB-2 receptor tyrosine kinase is a necessary but not a sufficient step for signaling. Despite the\\u000a efforts expended to identify the molecular interactions responsible for receptor-receptor contacts and particularly those\\u000a involving the transmembrane domain, structural details are still unknown. In this work, molecular dynamics simulations of\\u000a the helical transmembrane domain (TM) of neu and ErbB-2 receptors are

Nicolas Sajot; Monique Genest



Structure determination of the seven-helical transmembrane receptor sensory rhodopsin II by solution NMR spectroscopy  

PubMed Central

Seven-helical membrane proteins represent a challenge for structural biology. Here, we report the first NMR structure determination of a detergent-solubilized seven-helical transmembrane (7TM) protein, the phototaxis receptor sensory rhodopsin II (pSRII) from Natronomonas pharaonis, as a proof of principle. The overall quality of the structure ensemble is extremely good (backbone root mean squared deviation of 0.48 Å) and agrees well with previously determined X-ray structures. Furthermore, measurements in more native-like small phospholipid bicelles indicate that the protein structure is the same as in detergent micelles, suggesting that environment specific effects are minimal when using mild detergents. We use our case study as a platform to discuss the feasibility of similar solution NMR studies for other 7TM proteins including members of the family of G protein-coupled receptors (GPCRs). PMID:20512150

Gautier, Antoine; Mott, Helen R.; Bostock, Mark J.; Kirkpatrick, John P.; Nietlispach, Daniel



Homology Modeling and Molecular Dynamics Simulations of Transmembrane Domain Structure of Human Neuronal Nicotinic Acetylcholine Receptor  

Microsoft Academic Search

A three-dimensional model of the transmembrane domain of a neuronal-type nicotinic acetylcholine receptor (nAChR), (?4)2(?2)3, was constructed from a homology structure of the muscle-type nAChR recently determined by cryo-electron microscopy. The neuronal channel model was embedded in a fully hydrated DMPC lipid bilayer, and molecular-dynamics simulations were performed for 5ns. A comparative analysis of the neuronal- versus muscle-type nAChR models

Alexander C. Saladino; Yan Xu; Pei Tang



Structure of a Double Transmembrane Fragment of a G-Protein-Coupled Receptor in Micelles  

PubMed Central

Abstract The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for ?-factor of Saccharomyces cerevisiae, was studied in LPPG micelles with the use of solution NMR spectroscopy. The fragment Ste2p(G31-T110) (TM1-TM2) consisted of 19 residues from the N-terminal domain, the first TM helix (TM1), the first cytoplasmic loop, the second TM helix (TM2), and seven residues from the first extracellular loop. Multidimensional NMR experiments on [15N], [15N, 13C], [15N, 13C, 2H]-labeled TM1-TM2 and on protein fragments selectively labeled at specific amino acid residues or protonated at selected methyl groups resulted in >95% assignment of backbone and side-chain nuclei. The NMR investigation revealed the secondary structure of specific residues of TM1-TM2. TALOS constraints and NOE connectivities were used to calculate a structure for TM1-TM2 that was highlighted by the presence of three ?-helices encompassing residues 39–47, 49–72, and 80–103, with higher flexibility around the internal Arg58 site of TM1. RMSD values of individually superimposed helical segments 39–47, 49–72, and 80–103 were 0.25 ± 0.10 Å, 0.40 ± 0.13 Å, and 0.57 ± 0.19 Å, respectively. Several long-range interhelical connectivities supported the folding of TM1-TM2 into a tertiary structure typified by a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G56VRSG60 region. 15N-relaxation and hydrogen-deuterium exchange data support a stable fold for the TM parts of TM1-TM2, whereas the solvent-exposed segments are more flexible. The NMR structure is consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor. PMID:19383463

Neumoin, Alexey; Cohen, Leah S.; Arshava, Boris; Tantry, Subramanyam; Becker, Jeffrey M.; Zerbe, Oliver; Naider, Fred



Free Energy and Structure of Helix-forming Peptides: A Theoretical Investigation  

NASA Astrophysics Data System (ADS)

This thesis focuses on the structure and free energy of helical secondary structures of short peptides in a variety of experimental settings. Specifically, the formation of alpha-, pi- and 310-helices was investigated using large-scale classical molecular dynamics simulations with state-of-the-art force fields. In addition, the recently developed Adaptively Biased Molecular Dynamics (ABMD) and Steered Molecular Dynamics (SMD) methods were used to calculate the corresponding free energies. The most important results are as follows. For the examined peptide homopolymers, the observed minima on the free energy landscapes (based on suitable collective variables such as the radius of gyration, number of hydrogen bonds, and handedness) were associated with alpha-helices and "globular" or "knot-like" configurations only. No evidence was found to indicate that 310- or pi-helices represent equilibrium structures for these systems. In addition, the free energy landscape of short peptide chains formed by mixing two different amino acids were also examined. These results too indicate that the alpha-helix is only equilibrium helical secondary structure, and that the mixing of different amino acids does not result in the introduction of any significant new minima into the free energy landscapes. These results are in agreement with experimental observations insofar as these indicate that helical structural motifs are primary based on alpha-helices, with 310- and pi-helices being observed only rarely. Although pi- and 310-helices represent nonequilibrium structures, we were still able to estimate their free energies by means of SMD simulations. The helical secondary structure of the examined polypeptide chains is due to the formation of hydrogen bonds. However, there are other mechanisms that may allow for the additional stabilization of these structures. Specifically, in the so-called AK-(4,7) protein, the possible presence of disulfide bonds connecting cysteine residues may significantly alter the free energy landscapes and therefore the stability of different helical structures. We therefore examined this issue with ABMD simulations. However, our results show that while the free energy landscapes are indeed significantly altered only the formation of alpha-helices is favored as a secondary structural motif. Since all the results indicate that alpha-helix formation dominates, it is natural to think in terms of an alpha-helix forming propensity for different amino acids. To address this question, we carried out an extensive residue-by-residue population analysis of different amino acid guests in an alanine-based host setting. Such an analysis allows us to rank the different amino acid guests based on whether they increased or decreased the population in the alpha-helix region of the corresponding Ramachandran plots. Our ranking of the different guest amino acids is in reasonable correspondence with the experimental results, although some differences are observed. Finally, using a four-beads coarse-grained model were have investigated the stability of GA88 and GB88 proteins, which are quite similar in terms of their amino acid sequence, by means of 10mus simulations. The results indicate that while the three alpha-helix bundle of the GA88 protein remains stable, the 2beta--alpha--2beta configuration of the GB88 protein does not: the latter rapidly converts to a structure consisting mostly of helices similar to the GA88 protein design. These results indicate that this particular four-bead coarse-grained model is not able to properly grasp the dynamics of the beta-sheet secondary structure and overstabilizes the corresponding helical content.

Karpusenka, Vadzim


Structure of transmembrane pore induced by Bax-derived peptide: Evidence for lipidic pores  

PubMed Central

The structures of transmembrane pores formed by a large family of pore-forming proteins and peptides are unknown. These proteins, whose secondary structures are predominantly ?-helical segments, and many peptides form pores in membranes without a crystallizable protein assembly, contrary to the family of ?-pore-forming proteins, which form crystallizable ?-barrel pores. Nevertheless, a protein-induced pore in membranes is commonly assumed to be a protein channel. Here, we show a type of peptide-induced pore that is not framed by a peptide structure. Peptide-induced pores in multiple bilayers were long-range correlated into a periodically ordered lattice and analyzed by X-ray diffraction. We found the pores induced by Bax-derived helical peptides were at least partially framed by a lipid monolayer. Evidence suggests that the formation of such lipidic pores is a major mechanism for ?-pore-forming proteins, including apoptosis-regulator Bax. PMID:18987313

Qian, Shuo; Wang, Wangchen; Yang, Lin; Huang, Huey W.



PolyprOnline: polyproline helix II and secondary structure assignment database.  


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

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



PolyprOnline: polyproline helix II and secondary structure assignment database  

PubMed Central

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

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



Helix coupling  


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.

Ginell, W.S.



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

PubMed Central

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

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



Structure of the Dense Molecular Gas in the Helix Nebula: Large Scale Mapping of HCO^{+}  

NASA Astrophysics Data System (ADS)

The Helix Nebula, NGC 7293, has been mapped in HCO^{+} J = 1 ? 0 emission with a 70'' spatial resolution (1.68 km s^{-1} velocity resolution) using the Arizona Radio Observatory (ARO) 12 m telescope on Kitt Peak. Over 200 individual positions covering an area of roughly 1000 × 800'' were observed down to a 3? noise level of ˜20 mK. HCO^{+} was detected at over three-quarters of the positions, with the majority of spectra showing multiple velocity components indicative of a complex kinematic structure. The column density of HCO^{+} across the Helix ranges from N_{tot} ˜ 1.4 × 10^{11} to ˜2.4 × 10^{12} cm^{-2}, with an average N_{tot} ˜ 4 × 10^{11} cm^{-2}. The HCO^{+} distribution, which traces gas with densities ˜10^{5} cm^{-3}, is similar to that of CO and H_{2} , with certain point symmetries and red- and blue-shifted regions common in all three molecules; these data are also consistent with observations of atomic lines. The HCO^{+} emission appears to trace two ring-like structures emanating from the central star: one is blue-shifted and the other red-shifted with respect to the star, and possibly trace the edges of a bipolar outflow.

Zeigler, N. R.; Ziurys, L. M.; Zack, L. N.



Self-organization of amphiphilic macromolecules with local helix structure in concentrated solutions  

NASA Astrophysics Data System (ADS)

Concentrated solutions of amphiphilic macromolecules with local helical structure were studied by means of molecular dynamic simulations. It is shown that in poor solvent the macromolecules are assembled into wire-like aggregates having complex core-shell structure. The core consists of a hydrophobic backbone of the chains which intertwine around each other. It is protected by the shell of hydrophilic side groups. In racemic mixture of right-hand and left-hand helix macromolecules the wire-like complex is a chain of braid bundles of macromolecules with the same chirality stacking at their ends. The average number of macromolecules in the wire cross-section is close to that of separate bundles observed in dilute solutions of such macromolecules. The effects described here could serve as a simple model of self-organization in solutions of macromolecules with local helical structure.

Glagolev, M. K.; Vasilevskaya, V. V.; Khokhlov, A. R.



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

SciTech Connect

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.

Jankowski, S.A. [Univ. of Michigan, Ann Arbor, MI (United States)] [Univ. of Michigan, Ann Arbor, MI (United States); De Jong, P. [Roswell Park Cancer Institute, Buffalo, NY (United States)] [Roswell Park Cancer Institute, Buffalo, NY (United States); Meltzer, P.S. [National Institutes of Health, Bethesda, MD (United States)] [National Institutes of Health, Bethesda, MD (United States)



Predicting three-dimensional structures of transmembrane domains of ?-barrel membrane proteins.  


?-Barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They are important for pore formation, membrane anchoring, and enzyme activity. These proteins are also often responsible for bacterial virulence. Due to difficulties in experimental structure determination, they are sparsely represented in the protein structure databank. We have developed a computational method for predicting structures of the transmembrane (TM) domains of ?-barrel membrane proteins. Based on physical principles, our method can predict structures of the TM domain of ?-barrel membrane proteins of novel topology, including those from eukaryotic mitochondria. Our method is based on a model of physical interactions, a discrete conformational state space, an empirical potential function, as well as a model to account for interstrand loop entropy. We are able to construct three-dimensional atomic structure of the TM domains from sequences for a set of 23 nonhomologous proteins (resolution 1.8-3.0 Å). The median rmsd of TM domains containing 75-222 residues between predicted and measured structures is 3.9 Å for main chain atoms. In addition, stability determinants and protein-protein interaction sites can be predicted. Such predictions on eukaryotic mitochondria outer membrane protein Tom40 and VDAC are confirmed by independent mutagenesis and chemical cross-linking studies. These results suggest that our model captures key components of the organization principles of ?-barrel membrane protein assembly. PMID:22148174

Naveed, Hammad; Xu, Yun; Jackups, Ronald; Liang, Jie



NMR Structures of the Transmembrane Domains of the ?4?2 nAChR  

PubMed Central

The ?4?2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the ?4?2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the ?4 and ?2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography–multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of ?4 and ?2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na+ flux assay revealed that ?4?2 formed Na+ permeable channels in lipid vesicles. Efflux of Na+ through the ?4?2 channels reduced intra-vesicle Sodium Green™ fluorescence in a time-dependent manner that was not observed in vesicles without incorporating ?4?2. The study provides the structural insight into the TM domains of the ?4?2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the ?4?2 nAChR and for designing new therapeutic modulators. PMID:22361591

Bondarenko, Vasyl; Mowrey, David; Tillman, Tommy; Cui, Tanxing; Liu, Lu Tian; Xu, Yan; Tang, Pei



Assessing the local structural quality of transmembrane protein models using statistical potentials (QMEANBrane)  

PubMed Central

Motivation: Membrane proteins are an important class of biological macromolecules involved in many cellular key processes including signalling and transport. They account for one third of genes in the human genome and >50% of current drug targets. Despite their importance, experimental structural data are sparse, resulting in high expectations for computational modelling tools to help fill this gap. However, as many empirical methods have been trained on experimental structural data, which is biased towards soluble globular proteins, their accuracy for transmembrane proteins is often limited. Results: We developed a local model quality estimation method for membrane proteins (‘QMEANBrane’) by combining statistical potentials trained on membrane protein structures with a per-residue weighting scheme. The increasing number of available experimental membrane protein structures allowed us to train membrane-specific statistical potentials that approach statistical saturation. We show that reliable local quality estimation of membrane protein models is possible, thereby extending local quality estimation to these biologically relevant molecules. Availability and implementation: Source code and datasets are available on request. Contact: Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:25161240

Studer, Gabriel; Biasini, Marco; Schwede, Torsten



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.  


Early investigation of the electron paramagnetic resonance 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" or "large g(max)" 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, deltaphi = 57-90 degrees. 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 g(max) 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-twofold 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 N(delta1) atom of the heme-ligating histidine. The deltaphi 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 hydrogen bonds to the serine/threonine residues on the non-ligand-bearing helices. PMID:18418633

Berry, Edward A; Walker, F Ann



Field-induced chirality in the helix structure of Ho/Y multilayers  

NASA Astrophysics Data System (ADS)

We study the net chirality in the spin helix structure of Ho/Y multilayers induced by an in-plane applied magnetic field. The lifting of degeneracy of the chiral symmetry was revealed by means of polarized neutron reflectometry. Three samples of different thicknesses of the Ho and Y layers were grown by molecular-beam epitaxy. The chiral states are degenerated upon zero field cooling below the critical temperature TN=115±3 K. The chirality parameter ? rises during the field cooling procedure in the field range from 0 to 1 T and saturates at a value of 0.12±0.01. The chirality appears stepwise below TN and depends weakly on temperature. The phenomenon is interpreted in terms of the Dzyaloshinskii-Moriya interaction appearing at the interface between the Ho and Y layers.

Tarnavich, V. V.; Lott, D.; Mattauch, S.; Oleshkevych, A.; Kapaklis, V.; Grigoriev, S. V.



Structural insight into the transmembrane segments 3 and 4 of the hERG potassium channel.  


The hERG (human ether-a-go-go related gene) potassium channel is a voltage-gated potassium channel containing an N-terminal domain, a voltage-sensor domain, a pore domain and a C-terminal domain. The transmembrane segment 4 (S4) is important for sensing changes of membrane potentials through positively charge residues. A construct containing partial S2-S3 linker, S3, S4 and the S4-S5 linker of the hERG channel was purified into detergent micelles. This construct exhibits good quality NMR spectrum when it was purified in lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Structural study showed that S3 contains two short helices with a negatively charged surface. The S4 and S4-S5 linker adopt helical structures. The six positively charged residues in S4 localize at different sides, suggesting that they may have different functions in channel gating. Relaxation studies indicated that S3 is more flexible than S4. The boundaries of S3-S4 and S4-S4-S5 linker were identified. Our results provided structural information of the S3 and S4, which will be helpful to understand their roles in channel gating. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. PMID:25331429

Li, Qingxin; Wong, Ying Lei; Ng, Hui Qi; Gayen, Shovanlal; Kang, CongBao



Structural Studies of Amphiphilic 4-Helix Bundle Peptides Incorporating Designed Extended Chromophores for Nonlinear Optical Biomolecular Materials  

SciTech Connect

Extended conjugated chromophores containing (porphinato)zinc components that exhibit large optical polarizabilities and hyperpolarizabiliites are incorporated into amphiphilic 4-helix bundle peptides via specific axial histidyl ligation of the metal. The bundle's designed amphiphilicity enables vectorial orientation of the chromophore/peptide complex in macroscopic monolayer ensembles. The 4-helix bundle structure is maintained upon incorporation of two different chromophores at stoichiometries of 1-2 per bundle. The axial ligation site appears to effectively control the position of the chromophore along the length of the bundle.

Strzalka,J.; Xu, T.; Tronin, A.; Wu, S.; Miloradovic, I.; Kuzmenko, I.; Gog, T.; Therien, M.; Blasie, K.



Observation of quadrupole helix chirality and its domain structure in DyFe3(BO3)4  

NASA Astrophysics Data System (ADS)

Resonant X-ray diffraction (RXD) uses X-rays in the vicinity of a specific atomic absorption edge and is a powerful technique for studying symmetry breaking by motifs of various multipole moments, such as electric monopoles (charge), magnetic dipoles (spin) and electric quadrupoles (orbital). Using circularly polarized X-rays, this technique has been developed to verify symmetry breaking effects arising from chirality, the asymmetry of an object upon its mirroring. Chirality plays a crucial role in the emergence of functionalities such as optical rotatory power and multiferroicity. Here we apply spatially resolved RXD to reveal the helix chirality of Dy 4f electric quadrupole orientations and its domain structure in DyFe3(BO3)4, which shows a reversible phase transition into an enantiomorphic space-group pair. The present study provides evidence for a helix chiral motif of quadrupole moments developed in crystallographic helix chirality.

Usui, T.; Tanaka, Y.; Nakajima, H.; Taguchi, M.; Chainani, A.; Oura, M.; Shin, S.; Katayama, N.; Sawa, H.; Wakabayashi, Y.; Kimura, T.



Sequence similarity between Photosystems I and II. Identification of a Photosystem I reaction center transmembrane helix that is similar to transmembrane helix IV of the D2 subunit of Photosystem II and the M subunit of the non-sulfur purple and flexible green bacteria  

Microsoft Academic Search

There are basic structural similarities between plant PS II and bacterial RCs of the Chloroflexaceae and Rhodospirillaceae. These RCs are referred to as PS II-type RCs. A similar relationship of PS I RC to PS II-type RCs has not been established. Although plant PS I and PS II RCs show structural and functional differences, they also share similarities. Therefore, the

Maurice M. Margulies



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

PubMed Central

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

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



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

SciTech Connect

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.

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



Impact of bilayer lipid composition on the structure and topology of the transmembrane amyloid precursor C99 protein.  


C99 (also known as ?-CTF) is the 99 residue transmembrane C-terminal domain (residues 672-770) of the amyloid precursor protein and is the immediate precursor of the amyloid-? (A?) polypeptides. To test the dependence of the C99 structure on the composition of the host model membranes, NMR studies of C99 were conducted both in anionic lyso-myristoylphosphatidylglycerol (LMPG) micelles and in a series of five zwitterionic bicelle compositions involving phosphatidylcholine and sphingomyelin in which the acyl chain lengths of these lipid components varied from 14 to 24 carbons. Some of these mixtures are reported for the first time in this work and should be of broad utility in membrane protein research. The site-specific backbone (15)N and (1)H chemical shifts for C99 in LMPG and in all five bicelle mixtures were seen to be remarkably similar, indicating little dependence of the backbone structure of C99 on the composition of the host model membrane. However, the length of the transmembrane span was seen to vary in a manner that alters the positioning of the ?-secretase cleavage sites with respect to the center of the bilayer. This observation may contribute to the known dependency of the A?42-to-A?40 production ratio on both membrane thickness and the length of the C99 transmembrane domain. PMID:24564538

Song, Yuanli; Mittendorf, Kathleen F; Lu, Zhenwei; Sanders, Charles R



Structural models of the transmembrane region of voltage-gated and other K+ channels in open, closed, and inactivated conformations.  


A large collaborative, multidisciplinary effort involving many research laboratories continues which uses indirect methods of molecular biology and membrane biophysics to analyze the three-dimensional structures and functional mechanisms of K+ channels. This work also extends to the distant relatives of these channels, including the voltage-gated Na+ and Ca2+ channels. The role that our group plays in this process is to combine the information gained from experimental studies with molecular modeling techniques to generate atomic-scale structural models of these proteins. The modeling process involves three stages which are summarized as: (I) prediction of the channel sequence transmembrane topology, including the functionality and secondary structure of the segments; (II) prediction of the relative positions of the transmembrane segments, and (III) filling in all atoms of the amino acid residues, with conformations for energetically stabilized interactions. Both physiochemical and evolutionary principles (including sequence homology analysis) are used to guide the development. In addition to testing the steric and energetic feasibilities of different structural hypotheses, the models provide guidance for the design of new experiments. Structural modeling also serves to "fill in the gaps" of experimental data, such as predicting additional residue interactions and conformational changes responsible for functional processes. The modeling process is currently at the stage that experimental studies have definitely confirmed most of our earlier predictions about the transmembrane topology and functionality of different segments. Additionally, this report describes the detailed, three-dimensional models we have developed for the entire transmembrane region and important functional sites of the voltage-gated Shaker K+ channel in the open, closed, and inactivated conformations (including the ion-selective pore and voltage-sensor regions). As part of this effort, we also describe how our development of structural models for many of the other major K+ channel families aids in determining common structural motifs. As an example, we also present a detailed model of the smaller, bacterial K+ channel from Streptomyces lividans. Finally, we discuss strategies for using newly developed experimental methods for determining the structures and analyzing the functions of these channel proteins. PMID:9615442

Durell, S R; Hao, Y; Guy, H R



Comparison Analysis of Primary Ligand Binding Sites in Seven-Helix Membrane Proteins  

PubMed Central

Seven-helix transmembrane proteins, including the G-protein coupled receptors, mediate a broad range of fundamental cellular activities through binding to a wide range of ligands. Understanding the structural basis for the ligand-binding selectivity of these proteins is of significance to their structure-based drug design. Comparison analysis of proteins’ ligand binding sites provides a useful way to study their structure-activity relationships. Various computational methods have been developed for the binding site comparison of soluble proteins. In this work, we applied this approach to the analysis of the primary ligand-binding sites of 92 seven-helix transmembrane proteins. Results of the studies confirmed that the binding site of bacterial rhodopsins is indeed different from all G-protein coupled receptors. In the latter group, further comparison of the binding sites indicated a group of residues that could be responsible for ligand-binding selectivity and important for structure-based drug design. Further, unexpected binding site dissimilarities were observed among adrenergic and adenosine receptors, suggesting that the percentage of the overall sequence identity between a target protein and a template protein alone is not sufficient for selecting the best template for homology modeling of seven-helix membrane proteins. These results provided novel insight into the structural basis of ligand-binding selectivity of seven-helix membrane proteins and are of practical use to the computational modeling of these proteins. PMID:20672377

Pabuwal, Vagmita; Li, Zhijun



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

NASA Technical Reports Server (NTRS)

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.

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



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

SciTech Connect

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.

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



An open-pore structure of the mechanosensitive channel MscL derived by determining transmembrane domain interactions upon gating  

PubMed Central

Mechanosensation, the ability to detect mechanical forces, underlies the senses of hearing, balance, touch, and pain, as well as renal and cardiovascular regulation. Although the sensors are thought to be channels, relatively little is known about eukaryotic mechanosensitive channels or their molecular mechanisms. Thus, because of its tractable nature, a bacterial mechanosensitive channel that serves as an in vivo osmotic “emergency release valve,” MscL, has become a paradigm of how a mechanosensitive channel can sense and respond to membrane tension. Here, we have determined the structural rearrangements and interactions between transmembrane domains of MscL that occur upon gating. We utilize an electrostatic repulsion test: If two residues approach upon gating we predicted that substituting like-charges at those sites would inhibit gating. The in vivo growth and viability and in vitro vesicular flux and electrophysiological data all support the hypothesis that residues G26 and I92 directly interact upon gating. The resulting model predicted other interacting residues. One of these sets, V23 and I96, was confirmed to truly interact upon gating by disulfide trapping as well as the electrostatic repulsion test. Together, the data strongly suggest a model for structural transitions and residue-residue proximities that occur upon MscL gating.—Li, Y., Wray, R., Eaton, C., Blount, P. An open-pore structure of the mechanosensitive channel MscL derived by determining transmembrane domain interactions upon gating. PMID:19261722

Li, Yuezhou; Wray, Robin; Eaton, Christina; Blount, Paul



Structure-Activity Relationships in Tripodal Transmembrane Anion Transporters: The Effect of Fluorination  

PubMed Central

A series of easy-to-make fluorinated tripodal anion transporters containing urea and thiourea groups have been prepared and their anion transport properties studied. Vesicle anion transport assays using ion-selective electrodes show that this class of compound is capable of transporting chloride through a lipid bilayer via a variety of mechanisms, including chloride/H+ cotransport and chloride/nitrate, chloride/bicarbonate, and to a lesser extent an unusual chloride/sulfate antiport process. Calculations indicate that increasing the degree of fluorination of the tripodal transmembrane transporters increases the lipophilicity of the transporter and this is shown to be the major contributing factor in the superior transport activity of the fluorinated compounds, with a maximum transport rate achieved for clog P = 8. The most active transporter 5 contained a urea functionality appended with a 3,5-bis(trifluoromethyl)phenyl group and was able to mediate transmembrane chloride transport at receptor to lipid ratios as low as 1:250000. Proton NMR titration and single crystal X-ray diffraction revealed the ability of the tripodal receptors to bind different anions with varying affinities in a 1:1 or 2:1 stoichiometry in solution and in the solid state. We also provide evidence that the most potent anion transporters are able to induce apoptosis in human cancer cells by using a selection of in vitro viability and fluorescence assays. PMID:21846096



Structure-activity relationships in tripodal transmembrane anion transporters: the effect of fluorination.  


A series of easy-to-make fluorinated tripodal anion transporters containing urea and thiourea groups have been prepared and their anion transport properties studied. Vesicle anion transport assays using ion-selective electrodes show that this class of compound is capable of transporting chloride through a lipid bilayer via a variety of mechanisms, including chloride/H(+) cotransport and chloride/nitrate, chloride/bicarbonate, and to a lesser extent an unusual chloride/sulfate antiport process. Calculations indicate that increasing the degree of fluorination of the tripodal transmembrane transporters increases the lipophilicity of the transporter and this is shown to be the major contributing factor in the superior transport activity of the fluorinated compounds, with a maximum transport rate achieved for clog P = 8. The most active transporter 5 contained a urea functionality appended with a 3,5-bis(trifluoromethyl)phenyl group and was able to mediate transmembrane chloride transport at receptor to lipid ratios as low as 1:250000. Proton NMR titration and single crystal X-ray diffraction revealed the ability of the tripodal receptors to bind different anions with varying affinities in a 1:1 or 2:1 stoichiometry in solution and in the solid state. We also provide evidence that the most potent anion transporters are able to induce apoptosis in human cancer cells by using a selection of in vitro viability and fluorescence assays. PMID:21846096

Busschaert, Nathalie; Wenzel, Marco; Light, Mark E; Iglesias-Hernández, Paulina; Pérez-Tomás, Ricardo; Gale, Philip A



Structural characteristics of zebrafish orthologs of adaptor molecules that associate with transmembrane immune receptors  

PubMed Central

Transmembrane bound receptors comprised of extracellular immunoglobulin (Ig) or lectin domains play integral roles in a large number of immune functions including inhibitory and activating responses. The function of many of the activating receptors requires a physical interaction with an adaptor protein possessing a cytoplasmic regulatory motif. The partnering of an activating receptor with an adaptor protein relies on complementary charged residues in the two transmembrane domains. The mammalian natural killer (NK) and Fc receptors (FcR) represent two of many receptor families, which possess activating receptors that partner with adaptor proteins for signaling. Zebrafish represent a powerful experimental model for understanding developmental regulation at early stages of embryogenesis and for efficiently generating transgenic animals. In an effort to understand developmental aspects of immune receptor function, we have accessed the partially annotated zebrafish genome to identify six different adaptor molecules: Dap10, Dap12, Cd3?, Cd3?-like, FcR? and FcR?-like that are homologous to those effecting immune function in mammals. Their genomic organizations have been characterized, cDNA transcripts have been recovered, phylogenetic relationships have been defined and their cell lineage-specific expression patterns have been established. PMID:17719728

Yoder, Jeffrey A.; Orcutt, Timothy M.; Traver, David; Litman, Gary W.



Predictions of tertiary stuctures of $\\alpha$-helical membrane proteins by replica-exchange method with consideration of helix deformations  

E-print Network

We propose an improved prediction method of the tertiary structures of $\\alpha$-helical membrane proteins based on the replica-exchange method by taking into account helix deformations. Our method allows 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 accord with experiments.

Urano, Ryo; Okamoto, Yuko



Yummy Gummy Double Helix  

NSDL National Science Digital Library

In this activity, learners make their own edible DNA double helix out of candy and find out about the shape of DNA. This activity is a fun and tasty way to learn about the basics of DNA structure and complementary base pairs.

Institute, Wellcome T.



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 membrane.  


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

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



Synergistic transmembrane alignment of the antimicrobial heterodimer PGLa/magainin.  


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

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



Effect of polarization on the stability of a helix dimer  

NASA Astrophysics Data System (ADS)

Molecular dynamics (MD) simulations have been carried out to study helix-helix interaction using both standard AMBER and polarized force fields. Comparison of the two simulations shows that electrostatic polarization of intra-protein hydrogen bonds plays a significant role in stabilizing the structure of helix dimer. This stabilizing effect is clearly demonstrated by examining the monomer structure, helix crossing angle and stability of backbone hydrogen bonds under AMBER and PPC. Since reliable prediction of protein-protein structure is a significant challenge, the current study should help shed light on the importance of electrostatic polarization of protein in helix-helix interaction and helix bundle structures.

Wang, Xing Y.; Zhang, John Z. H.



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

PubMed Central

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

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



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

PubMed Central

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

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



The transmembrane homotrimer of ADAM 1 in model lipid bilayers  

PubMed Central

Fertilin is a transmembrane protein heterodimer formed by the two subunits fertilin ? and fertilin ? that plays an important role in sperm–egg fusion. Fertilin ? and ? are members of the ADAM family, and contain each one transmembrane ?-helix, and are termed ADAM 1 and ADAM 2, respectively. ADAM 1 is the subunit that contains a putative fusion peptide, and we have explored the possibility that the transmembrane ?-helical domain of ADAM 1 forms homotrimers, in common with other viral fusion proteins. Although this peptide was found to form various homooligomers in SDS, the infrared dichroic data obtained with the isotopically labeled peptide at specific positions is consistent with the presence of only one species in DMPC or POPC lipid bilayers. Comparison of the experimental orientational data with molecular dynamics simulations performed with sequence homologues of ADAM 1 show that the species present in lipid bilayers is only consistent with an evolutionarily conserved homotrimeric model for which we provide a backbone structure. These results support a model where ADAM 1 forms homotrimers as a step to create a fusion active intermediate. PMID:17189481

Gan, Siok Wan; Xin, Lin; Torres, Jaume



Molecular Characterization of Helix-Loop-Helix Peptides  

Microsoft Academic Search

A class of regulators of eokaryotic gene expression contains a conserved amino acid sequence responsible for protein oligomerization and binding to DNA. This structure consists of an arginine- and lysine-rich basic region followed by a helix-loop-helix motif, which together mediate specific binding to DNA. Peptides were prepared that span this motif in the MyoD protein; in solution, they formed alpha-helical

Spencer J. Anthony-Cahill; Pamela A. Benfield; Robert Fairman; Zelda R. Wasserman; Stephen L. Brenner; Walter F. Stafford III; Christian Altenbach; Wayne L. Hubbell; William F. Degrado



Structural Model of the Anion Exchanger 1 (SLC4A1) and Identification of Transmembrane Segments Forming the Transport Site  

PubMed Central

The anion exchanger 1 (AE1), a member of bicarbonate transporter family SLC4, mediates an electroneutral chloride/bicarbonate exchange in physiological conditions. However, some point mutations in AE1 membrane-spanning domain convert the electroneutral anion exchanger into a Na+ and K+ conductance or induce a cation leak in a still functional anion exchanger. The molecular determinants that govern ion movement through this transporter are still unknown. The present study was intended to identify the ion translocation pathway within AE1. In the absence of a resolutive three-dimensional structure of AE1 membrane-spanning domain, in silico modeling combined with site-directed mutagenesis experiments was done. A structural model of AE1 membrane-spanning domain is proposed, and this model is based on the structure of a uracil-proton symporter. This model was used to design cysteine-scanning mutagenesis on transmembrane (TM) segments 3 and 5. By measuring AE1 anion exchange activity or cation leak, it is proposed that there is a unique transport site comprising TM3–5 and TM8 that should function as an anion exchanger and a cation leak. PMID:23846695

Barneaud-Rocca, Damien; Etchebest, Catherine; Guizouarn, Helene



The Influence of Hydrophobic Mismatch on Structure and Dynamics of Transmembrane Helices and Lipid Bilayers  

E-print Network

of hydrophobic mismatch on structure and dynamics of TM helices and lipid bilayers through molecular dynamics simulation of Gramicidin A (gA) channel in various lipid bilayers. The structure and dynamics of the gA channel as well as important lipid properties...

Kim, Taehoon



The discovery of the double helix structure of DNA, James Watson, 3D animation with basic narrationSite: DNA Interactive (  

NSDL National Science Digital Library

DNAi Location: Code>Finding the Structure>putting it together>The DNA double helix We knew if we just, even if we go up to the ceiling, we're building a tiny fraction of a molecule. Hundreds of millions of these base pairs in one molecule, all fitting into this wonderful symmetry, which we saw the morning of February 28, 1953."



The three-dimensional structure of a helix-less variant of intestinal fatty acid-binding protein.  

PubMed Central

Intestinal fatty acid-binding protein (I-FABP) is a cytosolic 15.1-kDa protein that appears to function in the intracellular transport and metabolic trafficking of fatty acids. It binds a single molecule of long-chain fatty acid in an enclosed cavity surrounded by two five-stranded antiparallel beta-sheets and a helix-turn-helix domain. To investigate the role of the helical domain, we engineered a variant of I-FABP by deleting 17 contiguous residues and inserting a Ser-Gly linker (Kim K et al., 1996, Biochemistry 35:7553-7558). This variant, termed delta17-SG, was remarkably stable, exhibited a high beta-sheet content and was able to bind fatty acids with some features characteristic of the wild-type protein. In the present study, we determined the structure of the delta17-SG/palmitate complex at atomic resolution using triple-resonance 3D NMR methods. Sequence-specific 1H, 13C, and 15N resonance assignments were established at pH 7.2 and 25 degrees C and used to define the consensus 1H/13C chemical shift-derived secondary structure. Subsequently, an iterative protocol was used to identify 2,544 NOE-derived interproton distance restraints and to calculate its tertiary structure using a unique distance geometry/simulated annealing algorithm. In spite of the sizable deletion, the delta17-SG structure exhibits a backbone conformation that is nearly superimposable with the beta-sheet domain of the wild-type protein. The selective deletion of the alpha-helical domain creates a very large opening that connects the interior ligand-binding cavity with exterior solvent. Unlike wild-type I-FABP, fatty acid dissociation from delta17-SG is structurally and kinetically unimpeded, and a protein conformational transition is not required. The delta17-SG variant of I-FABP is the only wild-type or engineered member of the intracellular lipid-binding protein family whose structure lacks alpha-helices. Thus, delta17-SG I-FABP constitutes a unique model system for investigating the role of the helical domain in ligand-protein recognition, protein stability and folding, lipid transfer mechanisms, and cellular function. PMID:9655337

Steele, R. A.; Emmert, D. A.; Kao, J.; Hodsdon, M. E.; Frieden, C.; Cistola, D. P.



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

Microsoft Academic Search

Solid-state NMR spectroscopic techniques were used to investigate the secondary structure of the transmembrane peptide phospholamban (TM-PLB), a sarcoplasmic Ca2+ regulator. 13C cross-polarization magic angle spinning spectra of 13C 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 13C chemical shift. The

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



Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin  

PubMed Central

The four R-spondin (Rspo) proteins are secreted agonists of Wnt signalling in vertebrates, functioning in embryogenesis and adult stem cell biology. Through ubiquitination and degradation of Wnt receptors, the transmembrane E3 ubiquitin ligase ZNRF3 and related RNF43 antagonize Wnt signalling. Rspo ligands have been reported to inhibit the ligase activity through direct interaction with ZNRF3 and RNF43. Here we report multiple crystal structures of the ZNRF3 ectodomain (ZNRF3ecto), a signalling-competent Furin1–Furin2 (Fu1–Fu2) fragment of Rspo2 (Rspo2Fu1–Fu2), and Rspo2Fu1–Fu2 in complex with ZNRF3ecto, or RNF43ecto. A prominent loop in Fu1 clamps into equivalent grooves in the ZNRF3ecto and RNF43ecto surface. Rspo binding enhances dimerization of ZNRF3ecto but not of RNF43ecto. Comparison of the four Rspo proteins, mutants and chimeras in biophysical and cellular assays shows that their signalling potency depends on their ability to recruit ZNRF3 or RNF43 via Fu1 into a complex with LGR receptors, which interact with Rspo via Fu2. PMID:24225776

Zebisch, Matthias; Xu, Yang; Krastev, Christos; MacDonald, Bryan T.; Chen, Maorong; Gilbert, Robert J. C.; He, Xi; Jones, E. Yvonne



From 1D helix to 0D loop: nitrite anion induced structural transformation associated with unexpected N-nitrosation of amine ligand.  


An infinite Ag(I) coordination 41-helical chain, [Ag(Hdpma)](NO3)2·H2O (1), was synthesized by the self-assembly of AgNO3 and di(3-pyridylmethyl)amine (dpma). Helix 1 is 5-fold interweaved and has a topological diamondoid-like net that is extended by ligand-unsupported helix-to-helix argentophilic interactions. Two identical diamondoid-like nets with opposite chiralities interpenetrate to form the whole 3D framework as a meso compound. Typical anion-exchange reactions cause a remarkable single-crystal-to-single-crystal (SCSC) structural transformation from the 1D helix 1 to the 0D molecular loop [Ag(dpma-NO)(NO2)]2 (2) (induced by the nitrite anion, NO2(-)) and a 1D molecular ladder [Ag(dpma)(H2O)](NO3) (induced by the fluoride anion, F(-)). Molecular loop 2 is an N-nitroso compound. This work is the first to present observations of nitrite-dominated in situ N-nitrosation of an amine ligand which accompanies SCSC structural transformation via an anion-exchange reaction. PMID:24849148

Wu, Jing-Yun; Liu, Yu-Chiao; Chao, Tzu-Ching



Stable interactions between the transmembrane domains of the adenosine A2A receptor  

PubMed Central

G-protein-coupled receptors (GPCRs) must properly insert and fold in the membrane to adopt a stable native structure and become biologically active. The interactions between transmembrane (TM) helices are believed to play a major role in these processes. Previous studies in our group showed that specific interactions between TM helices occur, leading to an increase in helical content, especially in weakly helical TM domains, suggesting that helix–helix interactions in addition to helix–lipid interactions facilitate helix formation. They also demonstrated that TM peptides interact in a similar fashion in micelles and lipid vesicles, as they exhibit relatively similar thermal stability and ?-helicity inserted in SDS micelles to that observed in liposomes. In this study, we perform an analysis of pairwise interactions between peptides corresponding to the seven TM domains of the human A2A receptor (A2AR). We used a combination of Förster resonance energy transfer (FRET) measurement and circular dichroism (CD) spectroscopy to detect and analyze these interactions in detergent micelles. We found that strong and specific interactions occur in only seven of the 28 possible peptide pairs. Furthermore, not all interactions, identified by FRET, lead to a change in helicity. Our results identify stabilizing contacts that are likely related to the stability of the receptor and that are consistent with what is known about the three-dimensional structure and stability of rhodopsin and the ?2 adrenergic receptor. PMID:18434504

Thevenin, Damien; Lazarova, Tzvetana



Low Resolution Structural Models of the Basic Helix-Loop-Helix Leucine Zipper Domain of Upstream Stimulatory Factor 1 and Its Complexes with DNA from Small Angle X-Ray Scattering Data  

PubMed Central

The upstream stimulatory factor 1 (USF1) belongs to the basic helix-loop-helix leucine zipper (b/HLH/Z) transcription factor family, recognizing the CACGTG DNA motive as a dimer and playing an important role in the regulation of transcription in a variety of cellular and viral promoters. In this study we investigate the USF1 b/HLH/Z domain and its complexes with DNA by small angle x-ray scattering. We present low resolution structural models of monomeric b/HLH/Z USF1 in the absence of DNA and USF1 dimeric (b/HLH/Z)2-DNA and tetrameric (b/HLH/Z)4-DNA2 complexes. The data reveal a concentration-dependent USF1 dimer (b/HLH/Z)2-DNA-tetramer (b/HLH/Z)4-DNA2 equilibrium. The ability of b/HLH/Z USF1 to form a tetrameric assembly on two distant DNA binding sites as a consequence of increased protein concentration suggest a USF1 concentration-dependant mechanism of transcription activation involving DNA loop formation. PMID:17827227

Lamber, Ekaterina P.; Wilmanns, Matthias; Svergun, Dmitri I.



Low resolution structural models of the basic helix-loop-helix leucine zipper domain of upstream stimulatory factor 1 and its complexes with DNA from small angle X-ray scattering data.  


The upstream stimulatory factor 1 (USF1) belongs to the basic helix-loop-helix leucine zipper (b/HLH/Z) transcription factor family, recognizing the CACGTG DNA motive as a dimer and playing an important role in the regulation of transcription in a variety of cellular and viral promoters. In this study we investigate the USF1 b/HLH/Z domain and its complexes with DNA by small angle x-ray scattering. We present low resolution structural models of monomeric b/HLH/Z USF1 in the absence of DNA and USF1 dimeric (b/HLH/Z)(2)-DNA and tetrameric (b/HLH/Z)(4)-DNA(2) complexes. The data reveal a concentration-dependent USF1 dimer (b/HLH/Z)(2)-DNA-tetramer (b/HLH/Z)(4)-DNA(2) equilibrium. The ability of b/HLH/Z USF1 to form a tetrameric assembly on two distant DNA binding sites as a consequence of increased protein concentration suggest a USF1 concentration-dependant mechanism of transcription activation involving DNA loop formation. PMID:17827227

Lamber, Ekaterina P; Wilmanns, Matthias; Svergun, Dmitri I



Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus.  


Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis. PMID:17098849

Paolocci, Francesco; Robbins, Mark P; Madeo, Laura; Arcioni, Sergio; Martens, Stefan; Damiani, Francesco



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


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) ?-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

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



Transmembrane Interactions in the Activation of the Neu Receptor Tyrosine Kinase †  

Microsoft Academic Search

The Neu receptor tyrosine kinase is constitutively activated by a single amino acid change in the transmembrane domain of the receptor. The mutation of Val664 to glutamate or glutamine induces receptor dimerization and autophosphorylation of the receptor's intracellular kinase domain. The ability of this single mutation to activate the receptor is sequence-dependent, suggesting that specific helix - helix interactions stabilize

Steven O. Smith; Charles Smith; Srinivasan Shekar; Olve Peersen; Martine Ziliox; Saburo Aimoto



Structural and Biochemical Analysis of DNA Helix Invasion by the Bacterial 8-Oxoguanine DNA Glycosylase MutM*  

PubMed Central

MutM is a bacterial DNA glycosylase that serves as the first line of defense against the highly mutagenic 8-oxoguanine (oxoG) lesion, catalyzing glycosidic bond cleavage of oxoG to initiate base excision DNA repair. Previous work has shown that MutM actively interrogates DNA for the presence of an intrahelical oxoG lesion. This interrogation process involves significant buckling and bending of the DNA to promote extrusion of oxoG from the duplex. Structural snapshots have revealed several different highly conserved residues that are prominently inserted into the duplex in the vicinity of the target oxoG before and after base extrusion has occurred. However, the roles of these helix-invading residues during the lesion recognition and base extrusion process remain unclear. In this study, we set out to probe the function of residues Phe114 and Met77 in oxoG recognition and repair. Here we report a detailed biochemical and structural characterization of MutM variants containing either a F114A or M77A mutation, both of which showed significant decreases in the efficiency of oxoG repair. These data reveal that Met77 plays an important role in stabilizing the lesion-extruded conformation of the DNA. Phe114, on the other hand, appears to destabilize the intrahelical state of the oxoG lesion, primarily by buckling the target base pair. We report the observation of a completely unexpected interaction state, in which the target base pair is ruptured but remains fully intrahelical; this structure vividly illustrates the disruptive influence of MutM on the target base pair. PMID:23404556

Sung, Rou-Jia; Zhang, Michael; Qi, Yan; Verdine, Gregory L.



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

PubMed Central

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

Lynch, B A; Koshland, D E



A common motif organizes the structure of multi-helix loops in 16 S and 23 S ribosomal RNAs 1 1 Edited by K. Nagai  

Microsoft Academic Search

Phylogenetic and chemical probing data indicate that a modular RNA motif, common to loop E of eucaryotic 5 S ribosomal RNA (rRNA) and the ?-sarcin\\/ricin loop of 23 S rRNA, organizes the structure of multi-helix loops in 16 S and 23 S ribosomal RNAs. The motif occurs in the 3? domain of 16 S rRNA at positions 1345–1350\\/1372–1376 (Escherichia coli

Neocles B. Leontis; Eric Westhof



Energetics of [alpha]-helix formation in peptides and proteins  

E-print Network

This thesis focuses on the energetics of !-helix formation in peptides and proteins. The [alpha]-helix is the most prevalent type of secondary structure found in proteins, and has arguably dominated our thinking about ...

Schubert, Christian Reinhold



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

SciTech Connect

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.

Storrs, R.W.



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

SciTech Connect

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.

Storrs, R.W.



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


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

Papadakis, George; Tsortos, Achilleas; Gizeli, Electra



Molecular Architecture of a Sodium Channel S6 Helix  

PubMed Central

Voltage-gated sodium (NaV) channels are membrane proteins that consist of 24 transmembrane segments organized into four homologous domains and are essential for action potential generation and propagation. Although the S6 helices of NaV channels line the ion-conducting pore and participate in channel activation, their functional architecture is incompletely understood. Our recent studies show that a naturally occurring in-frame deletion mutation (Del-L955) of NaV1.7 channel, identified in individuals with a severe inherited pain syndrome (inherited erythromelalgia) causes a substantial hyperpolarizing shift of channel activation. Here we took advantage of this deletion mutation to understand the role of the S6 helix in the channel activation. Based on the recently published structure of a bacterial NaV channel (NaVAb), we modeled the WT and Del-L955 channel. Our structural model showed that Del-L955 twists the DII/S6 helix, shifting location and radial orientation of the activation gate residue (Phe960). Hypothesizing that these structural changes produce the shift of channel activation of Del-L955 channels, we restored a phenylalanine in wild-type orientation by mutating Ser961 (Del-L955/S961F), correcting activation by ?10 mV. Correction of the displaced Phe960 (F960S) together with introduction of the rescuing activation gate residue (S961F) produced an additional ?6-mV restoration of activation of the mutant channel. A simple point mutation in the absence of a twist (L955A) did not produce a radial shift and did not hyperpolarize activation. Our results demonstrate the functional importance of radial tuning of the sodium channel S6 helix for the channel activation. PMID:23536180

Yang, Yang; Estacion, Mark; Dib-Hajj, Sulayman D.; Waxman, Stephen G.



Transmembrane communication: general principles and lessons from the structure and function of the M2 proton channel, K? channels, and integrin receptors.  


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

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



Crystal Structure of an Ecotin-Collagenase Complex Suggests a Model for Recognition and Cleavage of the Collagen Triple Helix  

E-print Network

of the Collagen Triple Helix John J. Perona,,§ Christopher A. Tsu,,| Charles S. Craik,*,, and Robert J. Fletterick interactions in the cleft, with the known sequences at the cleavage site of type I collagen, suggests that the protease binding loop of ecotin adopts a conformation mimicking that of the cleaved strand of collagen

Craik, Charles S.


Membrane binding and insertion of the predicted transmembrane domain of human scramblase 1.  


Human phospholipid scramblase 1 (SCR) was originally described as an intrinsic membrane protein catalyzing transbilayer phospholipid transfer in the absence of ATP. More recently, a role as a nuclear transcription factor has been proposed for SCR, either in addition or alternatively to its capacity to facilitate phospholipid flip-flop. Uncertainties exist as well from the structural point of view. A predicted ?-helix (aa residues 288-306) located near the C-terminus has been alternatively proposed as a transmembrane domain, or as a protein core structural element. This paper explores the possibilities of the above helical segment as a transmembrane domain. To this aim two peptides were synthesized, one corresponding to the 19 ?-helical residues, and one containing both the helix and the subsequent 12-residues constituting the C-end of the protein. The interaction of these peptides with lipid monolayers and bilayers was tested with Langmuir balance surface pressure measurements, proteoliposome reconstitution and analysis, differential scanning calorimetry, tests of bilayer permeability, and fluorescence confocal microscopy. Bilayers of 28 different lipid compositions were examined in which lipid electric charge, bilayer fluidity and lateral heterogeneity (domain formation) were varied. All the results concur in supporting the idea that the 288-306 peptide of SCR becomes membrane inserted in the presence of lipid bilayers. Thus, the data are in agreement with the possibility of SCR as an integral membrane protein, without rejecting alternative cell locations. PMID:24099740

Posada, Itziar M D; Busto, Jon V; Goñi, Félix M; Alonso, Alicia



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

SciTech Connect

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.

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



Structural determinants of nitroxide motion in spin-labeled proteins: Tertiary contact and solvent-inaccessible sites in helix G of T4 lysozyme  

PubMed Central

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. PMID:17473014

Guo, Zhefeng; Cascio, Duilio; Hideg, Kalman; Kalai, Tamas; Hubbell, Wayne L.



Molecular dynamics simulations of the dimerization of transmembrane alpha-helices.  


Membrane proteins account for nearly a quarter of all genes, but their structure and function remain incompletely understood. Most membrane proteins have transmembrane (TM) domains made up of bundles of hydrophobic alpha-helices. The lateral association of TM helices within the lipid bilayer is a key stage in the folding of membrane proteins. It may also play a role in signaling across cell membranes. Dimerization of TM helices is a simple example of such lateral association. Molecular dynamics (MD) simulations have been used for over a decade to study membrane proteins in a lipid bilayer environment. However, direct atomistic (AT) MD simulation of self-assembly of a TM helix bundle remains challenging. AT-MD may be complemented by coarse-grained (CG) simulations, in which small numbers of atoms are grouped together into particles. In this Account, we demonstrate how CG-MD may be used to simulate formation of dimers of TM helices. We also show how a serial combination of CG and AT simulation provides a multiscale approach for generating and refining models of TM helix dimers. The glycophorin A (GpA) TM helix dimer represents a paradigm for helix-helix packing, mediated by a GxxxG sequence motif. It is well characterized experimentally and so is a good test case for evaluating computational methods. CG-MD simulations in which two separate TM helices are inserted in a lipid bilayer result in spontaneous formation of a right-handed GpA dimer, in agreement with NMR structures. CG-MD models were evaluated via comparison with data on destabilizing mutants of GpA. Such mutants increased the conformational flexibility and the dissociation constants of helix dimers. GpA dimers have been used to evaluate a multiscale approach: A CG model is converted to an AT model, which is used as the basis of an AT-MD simulation. Comparison of three AT-MD simulations of GpA, one starting from a CG model and two starting from NMR structures, leads to convergence to a common refined structure for the dimer. CG-MD self-assembly has also been used to model dimerization of the TM domain of the syndecan-2 receptor protein. This TM helix contains a GxxxG motif, which mediates right-handed helix packing comparable to that of the GxxxG motif in GpA. The multiscale approach has been applied to a more complex system, the heterodimeric alphaIIb/beta3 integrin TM helix dimer. In CG-MD, both right-handed and left-handed structures were formed. Subsequent AT-MD simulations showed that the right-handed structure was more stable, yielding a dimer in which the GxxxG motif of the alphaIIb TM helix packed against a hydrophobic surface of the beta3 helix in a manner comparable to that observed in two recent NMR studies. This work demonstrates that the multiscale simulation approach can be used to model simple membrane proteins. The method may be applied to more complex proteins, such as the influenza M2 channel protein. Future refinements, such as extending the multiscale approach to a wider range of scales (from CG through QM/MM simulations, for example), will expand the range of applications and the accuracy of the resultant models. PMID:20017540

Psachoulia, Emi; Marshall, David P; Sansom, Mark S P



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

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, parm99?_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



Rosalind Franklin and the Double Helix  

NASA Astrophysics Data System (ADS)

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.

Elkin, Lynne Osman



Common interruptions in the repeating tripeptide sequence of non-fibrillar collagens: Sequence analysis and structural studies on triple-helix peptide models  

PubMed Central

Interruptions in the repeating (Gly-X1-X2)n amino acid sequence pattern are found in the triple-helix domains of all non-fibrillar collagens, and perturbations to the triple-helix at such sites are likely to play a role in collagen higher order structure and function. This report defines the sequence features and structural consequences of the most common interruption, where one residue is missing in the tripeptide pattern, Gly-X1-X2-Gly-AA1-Gly-X1-X2, designated as G1G interruptions. Residues found within G1G interruptions are predominantly hydrophobic (70%), followed by a significant amount of charged residues (16%), and the Gly-X1-X2 triplets flanking the interruption are atypical. Studies on peptide models indicate the degree of destabilization is much greater when a Pro is in the interruption, GP, than when hydrophobic residues (GF, GY) are present, and a rigid Gly-Pro-Hyp tripeptide adjacent to the interruption leads to greater destabilization than a flexible Gly-Ala-Ala sequence. Modeling based on NMR data indicates the Phe residue within a GF interruption is located on the outside of the triple-helix. The G1G interruptions resemble a previously studied collagen interruption GPOGAAVMGPO, designated as a G4G type, in that both are destabilizing, but allow continuation of rod-like triple-helices and maintenance of the 1-residue stagger throughout the imperfection, with a loss of axial register of the superhelix on both sides. Both kinds of interruptions result in a highly localized perturbation in hydrogen bonding and dihedral angles, but the hydrophobic residue of a G4G interruption packs near the central axis of the superhelix while the hydrophobic residue of a G1G interruption is located on the triple-helix surface. The different structural consequences of G1G and G4G interruptions in the repeating tripeptide sequence pattern suggest a physical basis for their differential susceptibility to matrix metalloproteinases in type X collagen. PMID:18187152

Thiagarajan, Geetha; Li, Yingjie; Mohs, Angela; Strafaci, Christopher; Popiel, Magdalena; Baum, Jean; Brodsky, Barbara



Three-dimensional structure of a single filament in the Limulus acrosomal bundle: scruin binds to homologous helix-loop-beta motifs in actin  

PubMed Central

Frozen, hydrated acrosomal bundles from Limulus sperm were imaged with a 400 kV electron cryomicroscope. Segments of this long bundle can be studied as a P1 crystal with a unit cell containing an acrosomal filament with 28 actin and 28 scruin molecules in 13 helical turns. A novel computational procedure was developed to extract single columns of superimposed acrosomal filaments from the distinctive crystallographic view. Helical reconstruction was used to generate a three-dimensional structure of this computationally isolated acrosomal filament. The scruin molecule is organized into two domains which contact two actin subunits in different strands of the same actin filament. A correlation of Holmes' actin filament model to the density in our acrosomal filament map shows that actin subdomains 1, 2, and 3 match the model density closely. However, actin subdomain 4 matches rather poorly, suggesting that interactions with scruin may have altered actin conformation. Scruin makes extensive interactions with helix-loop-beta motifs in subdomain 3 of one actin subunit and in subdomain 1 of a consecutive actin subunit along the genetic filament helix. These two actin subdomains are structurally homologous and are closely spaced along the actin filament. Our model suggests that scruin, which is derived from a tandemly duplicated gene, has evolved to bind structurally homologous but non-identical positions across two consecutive actin subunits. PMID:8294517



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

NASA Astrophysics Data System (ADS)

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.

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



The T?R-I Pre-Helix Extension Is Structurally Ordered in the Unbound Form and Its Flanking Prolines Are Essential for Binding  

PubMed Central

Transforming growth factor ? isoforms (TGF-?) are among the most recently evolved members of a signaling superfamily with more than 30 members. TGF-? play vital roles in regulating cellular growth and differentiation, and they signal through a highly restricted subset of receptors known as TGF-? type I receptor (T?R-I) and TGF-? type II receptor (T?R-II). TGF-?'s specificity for T?R-I has been proposed to arise from its pre-helix extension, a five-residue loop that binds in the cleft between TGF-? and T?R-II. The structure and backbone dynamics of the unbound form of the T?R-I extracellular domain were determined using NMR to investigate the extension's role in binding. This showed that the unbound form is highly similar to the bound form in terms of both the ?-strand framework that defines the three-finger toxin fold and the extension and its characteristic cis-Ile54-Pro55 peptide bond. The NMR data further showed that the extension and two flanking 310 helices are rigid on the nanosecond-to-picosecond timescale. The functional significance of several residues within the extension was investigated by binding studies and reporter gene assays in cultured epithelial cells. These demonstrated that the pre-helix extension is essential for binding, with Pro55 and Pro59 each playing a major role. These findings suggest that the pre-helix extension and its flanking prolines evolved to endow the TGF-? signaling complex with its unique specificity, departing from the ancestral promiscuity of the bone morphogenetic protein subfamily, where the binding interface of the type I receptor is highly flexible. PMID:21821041

Zuniga, Jorge E.; Ilangovan, Udayar; Mahlawat, Pardeep; Hinck, Cynthia S.; Huang, Tao; Groppe, Jay C.; McEwen, Donald G.; Hinck, Andrew P.



The crystal structures of Lactococcus lactis MG1363 Dps proteins reveal the presence of an N-terminal helix that is required for DNA binding.  


Dps proteins play a major role in the protection of bacterial DNA from damage by reactive oxygen species. Previous studies have implicated the extended lysine-containing N-terminal regions of Dps subunits in DNA binding, but this part of the structure has not previously been observed crystallographically. Here the structures of two Dps proteins (DpsA and DpsB) from Lactococcus lactis MG1363 reveal for the first time the presence of an N-terminal alpha helix that extends from the core of the Dps subunit. Consequently, the N-terminal helices are displayed in parallel pairs on the exterior of the dodecameric Dps assemblies. Both DpsA and DpsB bind DNA. Deletion of the DpsA N-terminal helix impaired DNA binding. The N-terminal Lys residues of Escherichia coli Dps have been implicated in DNA binding. Replacement of the lactococcal DpsA Lys residues 9, 15 and 16 by Glu did not inhibit DNA binding. However, DNA binding was inhibited by EDTA, suggesting a role for cations in DNA binding. In contrast to E. coli, Bacillus brevis and Mycobacterium smegmatis Dps:DNA complexes, in which DNA interacts with crystalline Dps phases, L. lactis DNA:Dps complexes appeared as non-crystalline aggregates of protein and DNA in electron micrographs. PMID:16091047

Stillman, Timothy J; Upadhyay, Manisha; Norte, Valia A; Sedelnikova, Svetlana E; Carradus, Maria; Tzokov, Svetomir; Bullough, Per A; Shearman, Claire A; Gasson, Michael J; Williams, Colin H; Artymiuk, Peter J; Green, Jeffrey



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

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




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

SciTech Connect

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.

Chen, Yong; Wan, Bingbing; Wang, Kevin C.; Cao, Fang; Yang, Yuting; Protacio, Angeline; Dou, Yali; Chang, Howard Y.; Lei, Ming (Michigan-Med); (HHMI)



Response of GWALP Transmembrane Peptides to Changes in the Tryptophan Anchor Positions†  

PubMed Central

While the interfacial partitioning of charged or aromatic anchor residues may determine the preferred orientations of transmembrane peptide helices, the dependence of helix orientation on anchor residue position is not well understood. When anchor residue locations are changed systematically, some adaptations of the peptide-lipid interactions may be required to compensate the altered interfacial interactions. Recently we have developed a novel transmembrane peptide, termed GW5,19ALP23 (acetyl-GGALW5LALALALALALALW19LAGA-ethanolamide), which proves to be a well behaved sequence for an orderly investigation of protein-lipid interactions. Its roughly symmetric nature allows for shifting the anchoring Trp residues by one Leu-Ala pair inward (GW7,17ALP23) or outward (GW3,21ALP23), thus providing fine adjustments of the formal distance between the tryptophan residues. With no other obvious anchoring features present, we postulate that the inter-Trp distance may be crucial for aspects of the peptide-lipid interaction. Importantly, the amino acid composition is identical for each of the resulting related GWALP23 sequences, and the radial separation between the pairs of Trp residues on each side of the transmembrane ?-helix remains similar. Here we address the adaptation of the aforementioned peptides to the varying Trp locations by means of solid-state 2H NMR experiments in varying lipid bilayer membrane environments. All of the GWx,yALP23 sequence isomers adopt transmembrane orientations in DOPC, DMPC and DLPC environments, even when the Trp residues are quite closely spaced, in GW7,17ALP23. Furthermore, the dynamics for each peptide isomer are less extensive than for peptides possessing additional interfacial Trp residues. The helical secondary structure is maintained more strongly within the Trp-flanked core region than outside of the Trp boundaries. Deuterium labeled tryptophan indole rings in the GWx,yALP23 peptides provide additional insights into the behavior of the Trp side chains. A Trp side chain near the C-terminus adopts a different orientation and undergoes somewhat faster dynamics than a corresponding Trp side chain located an equivalent distance from the N-terminus. In contrast, as the inter-Trp distance changes, the variations among the average orientations of the Trp indole rings at either terminus are systematic yet fairly small. We conclude that subtle adjustments to the peptide tilt, and to the N- and C-terminal Trp side-chain torsion angles, permit the GWx,yALP23 peptides to maintain preferred transmembrane orientations while adapting to lipid bilayers of differing hydrophobic thickness. PMID:21800919

Vostrikov, Vitaly V.; Koeppe, Roger E.



Structure of the unique SEFIR domain from human interleukin 17 receptor A reveals a composite ligand-binding site containing a conserved ?-helix for Act1 binding and 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 ?-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 ?C and helix ?C in IL-17RA SEFIR is mostly well ordered, displaying a helix (?CC'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 ?CC'ins helix without forming any knots. Helix ?C 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 ?C 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 ?C could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling. PMID:24816115

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



A Helix Replacement Mechanism Directs Metavinculin Functions  

SciTech Connect

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.

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



Effects of side chains in helix nucleation differ from helix propagation  

PubMed Central

Helix–coil transition theory connects observable properties of the ?-helix to an ensemble of microstates and provides a foundation for analyzing secondary structure formation in proteins. Classical models account for cooperative helix formation in terms of an energetically demanding nucleation event (described by the ? constant) followed by a more facile propagation reaction, with corresponding s constants that are sequence dependent. Extensive studies of folding and unfolding in model peptides have led to the determination of the propagation constants for amino acids. However, the role of individual side chains in helix nucleation has not been separately accessible, so the ? constant is treated as independent of sequence. We describe here a synthetic model that allows the assessment of the role of individual amino acids in helix nucleation. Studies with this model lead to the surprising conclusion that widely accepted scales of helical propensity are not predictive of helix nucleation. Residues known to be helix stabilizers or breakers in propagation have only a tenuous relationship to residues that favor or disfavor helix nucleation. PMID:24753597

Miller, Stephen E.; Watkins, Andrew M.; Kallenbach, Neville R.; Arora, Paramjit S.



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

PubMed Central

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

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



Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins.  

PubMed Central

The interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X-ray or NMR data. Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins. The convex constraint analysis algorithm (Perczel, A., Hollósi, M., Tusnády, G., & Fasman, G. D., 1991, Protein Eng. 4, 669-679), on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived "pure" CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of alpha helices (the alpha helix in the soluble domain and the alpha T helix, for the transmembrane alpha helix), a beta-pleated sheet, a class C-like spectrum related to beta turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the alpha T helix was characterized by a positive red-shifted band in the range 195-200 nm (+95,000 deg cm2 dmol-1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222-nm band (-50,000 and -60,000 deg cm2 dmol-1, respectively) in comparison with the regular alpha helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, -30,000, and -30,000 deg cm2 dmol-1, respectively. PMID:1338977

Park, K.; Perczel, A.; Fasman, G. D.



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

PubMed Central

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 ?-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

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



Localized Influence of 2‘Hydroxyl Groups and Helix Geometry on Protein Recognition in the RNA Major Groove †  

Microsoft Academic Search

The local geometry of a DNA helix can influence protein recognition, but the sequence- specific features that contribute to helix structure are not fully understood, and even less is known about how RNA helix geometry may affect protein recognition. To begin to understand how local or global helix structure may influence binding in an RNA model system, we generated a

Stephen G. Landt; Alicia R. Tipton; Alan D. Frankel



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

PubMed Central

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 ?/? hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-Å 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 ?/? 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. PMID:20118260

Bahl, Christopher D.; Morisseau, Christophe; Bomberger, Jennifer M.; Stanton, Bruce A.; Hammock, Bruce D.; O'Toole, George A.; Madden, Dean R.



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

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.

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



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

PubMed Central

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

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



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

NASA Astrophysics Data System (ADS)

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.

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



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

SciTech Connect

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.

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



Double Helix Revisited.  

ERIC Educational Resources Information Center

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

Glickstein, Neil M.



Correlation of the Structure of the Transmembrane Domain of the neu Oncogene-Encoded p185 Protein with Its Function  

Microsoft Academic Search

The human homologue of the neu oncogene is frequently found in human tumors. Certain amino acid substitutions at position 664 in the transmembrance domain of the neu oncogene-encoded p185 protein product are known to cause malignant transformation of cells. Using conformational energy analysis based on ECEPP (empirical conformational energies for polypeptides program), we have previously determined the preferred three-dimensional structures

Paul W. Brandt-Rauf; Shalom Rackovsky; Matthew R. Pincus



NMR structures of the human ?7 nAChR transmembrane domain and associated anesthetic binding sites.  


The ?7 nicotinic acetylcholine receptor (nAChR), assembled as homomeric pentameric ligand-gated ion channels, is one of the most abundant nAChR subtypes in the brain. Despite its importance in memory, learning and cognition, no structure has been determined for the ?7 nAChR TM domain, a target for allosteric modulators. Using solution state NMR, we determined the structure of the human ?7 nAChR TM domain (PDB ID: 2MAW) and demonstrated that the ?7 TM domain formed functional channels in Xenopus oocytes. We identified the associated binding sites for the anesthetics halothane and ketamine; the former cannot sensitively inhibit ?7 function, but the latter can. The ?7 TM domain folds into the expected four-helical bundle motif, but the intra-subunit cavity at the extracellular end of the ?7 TM domain is smaller than the equivalent cavity in the ?4?2 nAChRs (PDB IDs: 2LLY; 2LM2). Neither drug binds to the extracellular end of the ?7 TM domain, but two halothane molecules or one ketamine molecule binds to the intracellular end of the ?7 TM domain. Halothane and ketamine binding sites are partially overlapped. Ketamine, but not halothane, perturbed the ?7 channel-gate residue L9'. Furthermore, halothane did not induce profound dynamics changes in the ?7 channel as observed in ?4?2. The study offers a novel high-resolution structure for the human ?7 nAChR TM domain that is invaluable for developing ?7-specific therapeutics. It also provides evidence to support the hypothesis: only when anesthetic binding perturbs the channel pore or alters the channel motion, can binding generate functional consequences. PMID:24384062

Bondarenko, Vasyl; Mowrey, David D; Tillman, Tommy S; Seyoum, Edom; Xu, Yan; Tang, Pei



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

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.

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 (SG); (Columbia); (JHU)



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

PubMed Central

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

Khadria, Ambalika S.; Senes, Alessandro



Structural constraints on the transmembrane and juxtamembrane regions of the phospholamban pentamer in membrane bilayers: Gln29 and Leu52.  


The Ca2+-ATPase of cardiac muscle cells transports Ca2+ ions against a concentration gradient into the sarcoplasmic reticulum and is regulated by phospholamban, a 52-residue integral membrane protein. It is known that phospholamban inhibits the Ca2+ pump during muscle contraction and that inhibition is removed by phosphorylation of the protein during muscle relaxation. Phospholamban forms a pentameric complex with a central pore. The solid-state magic angle spinning (MAS) NMR measurements presented here address the structure of the phospholamban pentamer in the region of Gln22-Gln29. Rotational echo double resonance (REDOR) NMR measurements show that the side chain amide groups of Gln29 are in close proximity, consistent with a hydrogen-bonded network within the central pore. 13C MAS NMR measurements are also presented on phospholamban that is 1-13C-labeled at Leu52, the last residue of the protein. pH titration of the C-terminal carboxyl group suggests that it forms a ring of negative charge on the lumenal side of the sarcoplasmic reticulum membrane. The structural constraints on the phospholamban pentamer described in this study are discussed in the context of a multifaceted mechanism for Ca2+ regulation that may involve phospholamban as both an inhibitor of the Ca2+ ATPase and as an ion channel. PMID:17996192

Liu, Wei; Fei, Jeffrey Z; Kawakami, Toru; Smith, Steven O



Structural constraints on the transmembrane and juxtamembrane regions of the phospholamban pentamer in membrane bilayers: Gln29 and Leu52  

PubMed Central

Summary The Ca2+-ATPase of cardiac muscle cells transports Ca2+ ions against a concentration gradient into the sarcoplasmic reticulum and is regulated by phospholamban, a 52-residue integral membrane protein. It is known that phospholamban inhibits the Ca2+ pump during muscle contraction and that inhibition is removed by phosphorylation of the protein during muscle relaxation. Phospholamban forms a pentameric complex with a central pore. The solid-state magic angle spinning (MAS) NMR measurements presented here address the structure of the phospholamban pentamer in the region of Gln22-Gln29. Rotational echo double resonance (REDOR) NMR measurements show that the side chain amide groups of Gln29 are in close proximity, consistent with a hydrogen-bonded network within the central pore. 13C MAS NMR measurements are also presented on phospholamban that is 1-13C-labeled at Leu52, the last residue of the protein. pH titration of the C-terminal carboxyl group suggests that it forms a ring of negative charge on the lumenal side of the sarcoplasmic reticulum membrane. The structural constraints on the phospholamban pentamer described in this study are discussed in the context of a multifaceted mechanism for Ca2+ regulation that may involve phospholamban as both an inhibitor of the Ca2+ ATPase and as an ion channel. PMID:17996192

Liu, Wei; Fei, Jeffrey Z.; Kawakami, Toru; Smith, Steven O.




E-print Network

on DNA double helix structure is very subtle. All double helices have comple- mentary base-pairs and look (1978) also predicted that the bases would have a propeller twist not seen in the refined fibre13 SIMULATING THE DYNAMICS OF THE DNA DOUBLE HELIX IN SOLUTION Miriam Hirshberg' and Michael Levitt

Levitt, Michael


Waves in tape helix loaded liquid crystal optical fiber  

NASA Astrophysics Data System (ADS)

This communication describes a rather new type of optical fiber composed of three layers with the outermost region being radially anisotropic liquid crystal, and the inner dielectric core-clad interface is loaded with conducting tape helix structure. Similarly to the fibers embedded with conducting sheath helix, the introduction of tape helix too would throw the impact of altering the dispersion features of the guide. However, the situation becomes more complex in the sense that, apart from the helix pitch angle (as generally considered in the case of sheath), the width of tape helix structure becomes the additional factor to affect the dispersion characteristics. We consider the core and the inner clad sections as made of linear, homogeneous and isotropic dielectrics, and the anisotropy remains in the outermost section due to the presence of nematic radially anisotropic liquid crystal material. Taking into account the zero-order guided modes in the fiber structure, effects on confinements due to the amalgamation of birefringence (optical property of liquid crystal) and tape helix pitch (geometrical/structural property of prefect conductor) are reported. Results reveal that such liquid crystal fibers with conducting tape helix loadings would be more useful than the sheath helix loaded fibers.

Ghasemi, Masih; Choudhury, P. K.



Transmembrane allosteric coupling of the gates in a potassium channel.  


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

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



Structure of the Retinoid X Receptor alpha DNA Binding Domain: A Helix Required for Homodimeric DNA Binding  

Microsoft Academic Search

The three-dimensional solution structure of the DNA binding domain (DBD) of the retinoid X receptor alpha (RXRalpha) was determined by nuclear magnetic resonance spectroscopy. The two zinc fingers of the RXR DBD fold to form a single structural domain that consists of two perpendicularly oriented helices and that resembles the corresponding regions of the glucocorticoid and estrogen receptors (GR and

Min S. Lee; Steven A. Kliewer; Joan Provencal; Peter E. Wright; Ronald M. Evans



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


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

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



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

NASA Astrophysics Data System (ADS)

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

Blinov, L. M.; Palto, S. P.



Malleable conformation of the elastic PEVK segment of titin: non-co-operative interconversion of polyproline II helix, beta-turn and unordered structures.  

PubMed Central

To understand the structural basis of molecular elasticity and protein interaction of the elastic PEVK (Pro-Glu-Val-Lys) segment of the giant muscle protein titin, we carried out a detailed analysis of a representative PEVK module and a 16-module PEVK protein under various environmental conditions. Three conformational states, polyproline II (PPII) helix, beta-turn and unordered coil were identified by CD and NMR. These motifs interconvert without long-range co-operativity. As a general trend, the relative content of PPII increases with lower temperature and higher polarity, beta-turn increases with lower temperature and lower polarity, and unordered coil increases with higher temperature and higher polarity. NMR studies demonstrate that trans -proline residues are the predominant form at room temperature (22 degrees C), with little trans -to- cis isomerization below 35 degrees C. Ionic strength affects salt bridges between charged side chains, but not the backbone conformation. We conclude that titin PEVK conformation is malleable and responds to subtle environmental changes without co-operativity. This gradual conformational transition may represent a regulatory mechanism for fine-tuning protein interactions and elasticity. PMID:12816538

Ma, Kan; Wang, Kuan



The role of transmembrane domain III in the lactose permease of Escherichia coli.  

PubMed Central

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to > 70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76-->Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96-->Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96-->Cys permease is enhanced at least 2-fold in the presence of beta-galactopyranosyl 1-thio-beta, D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport. PMID:7756986

Sahin-Toth, M.; Frillingos, S.; Bibi, E.; Gonzalez, A.; Kaback, H. R.



Dramatic structural changes resulting from the loss of a crucial hydrogen bond in the hinge region involved in C-terminal helix swapping in SurE: a survival protein from Salmonella typhimurium.  


Domain swapping is an interesting feature of some oligomeric proteins in which each protomer of the oligomer provides an identical surface for exclusive interaction with a segment or domain belonging to another protomer. Here we report results of mutagenesis experiments on the structure of C-terminal helix swapped dimer of a stationary phase survival protein from Salmonella typhimurium (StSurE). Wild type StSurE is a dimer in which a large helical segment at the C-terminus and a tetramerization loop comprising two ? strands are swapped between the protomers. Key residues in StSurE that might promote C-terminal helix swapping were identified by sequence and structural comparisons. Three mutants in which the helix swapping is likely to be avoided were constructed and expressed in E. coli. Three-dimensional X-ray crystal structures of the mutants H234A and D230A/H234A could be determined at 2.1 Å and 2.35 Å resolutions, respectively. Contrary to expectations, helix swapping was mostly retained in both the mutants. The loss of the crucial D230 OD2- H234 NE2 hydrogen bond (2.89 Å in the wild type structure) in the hinge region was compensated by new inter and intra-chain interactions. However, the two fold molecular symmetry was lost and there were large conformational changes throughout the polypeptide. In spite of these changes, the dimeric structure and an approximate tetrameric organization were retained, probably due to the interactions involving the tetramerization loop. Mutants were mostly functionally inactive, highlighting the importance of precise inter-subunit interactions for the symmetry and function of StSurE. PMID:23409101

Mathiharan, Yamuna Kalyani; Pappachan, Anju; Savithri, H S; Murthy, Mathur R N



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

NASA Astrophysics Data System (ADS)

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.

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



Structure of a Ca 2+ /CaM:Kv7.4 (KCNQ4) B-Helix  

E-print Network

.11.023 Edited by J. Bowie Abstract Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage 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

Lim, Wendell



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


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

PubMed Central

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

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



Structures of bacterial homologues of SWEET transporters in two distinct conformations.  


SWEETs and their prokaryotic homologues are monosaccharide and disaccharide transporters that are present from Archaea to plants and humans. SWEETs play crucial roles in cellular sugar efflux processes: that is, in phloem loading, pollen nutrition and nectar secretion. Their bacterial homologues, which are called SemiSWEETs, are among the smallest known transporters. Here we show that SemiSWEET molecules, which consist of a triple-helix bundle, form symmetrical, parallel dimers, thereby generating the translocation pathway. Two SemiSWEET isoforms were crystallized, one in an apparently open state and one in an occluded state, indicating that SemiSWEETs and SWEETs are transporters that undergo rocking-type movements during the transport cycle. The topology of the triple-helix bundle is similar yet distinct to that of the basic building block of animal and plant major facilitator superfamily (MFS) transporters (for example, GLUTs and SUTs). This finding indicates two possibilities: that SWEETs and MFS transporters evolved from an ancestral triple-helix bundle or that the triple-helix bundle represents convergent evolution. In SemiSWEETs and SWEETs, two triple-helix bundles are arranged in a parallel configuration to produce the 6- and 6 + 1-transmembrane-helix pores, respectively. In the 12-transmembrane-helix MFS transporters, four triple-helix bundles are arranged into an alternating antiparallel configuration, resulting in a much larger 2 × 2 triple-helix bundle forming the pore. Given the similarity of SemiSWEETs and SWEETs to PQ-loop amino acid transporters and to mitochondrial pyruvate carriers (MPCs), the structures characterized here may also be relevant to other transporters in the MtN3 clan. The insight gained from the structures of these transporters and from the analysis of mutations of conserved residues will improve the understanding of the transport mechanism, as well as allow comparative studies of the different superfamilies involved in sugar transport and the evolution of transporters in general. PMID:25186729

Xu, Yan; Tao, Yuyong; Cheung, Lily S; Fan, Chao; Chen, Li-Qing; Xu, Sophia; Perry, Kay; Frommer, Wolf B; Feng, Liang



PH-dependent Activities and Structural Stability of Loop-2-anchoring Helix of RadA Recombinase from Methanococcus voltae  

PubMed Central

RadA is an archaeal orthologue of human recombinase Rad51. This superfamily of recombinases, which also includes eukaryal meiosis-specific DMC1 and remotely related bacterial RecA, form filaments on single-stranded DNA in the presence of ATP and promote a strand exchange reaction between the single-stranded DNA and a homologous double-stranded DNA. Due to its feasibility of getting crystals and similarity (> 40% sequence identity) to eukaryal homologues, we have studied RadA from Methanococcus voltae (MvRadA) as a structural model for understanding the molecular mechanism of homologous strand exchange. Here we show this protein’s ATPase and strand exchange activities are minimal at pH 6.0. Interestingly, MvRadA’s pH dependence is similar to the properties of human Rad51 but dissimilar to that of the well-studied E. coli RecA. A structure subsequently determined at pH 6.0 reveals features indicative of an ATPase-inactive form with a disordered L2 loop. Comparison with a previously determined ATPase-active form at pH 7.5 implies that the stability of the ATPase-active conformation is reduced at the acidic pH. We interpret these results as further suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between single- and double-stranded DNA. His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent activities of MvRadA. PMID:24654848

D. E. C. S, Rao; Luo, Yu



Energetic Frustration of Apomyoglobin Folding: Role of the B Helix  

PubMed Central

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

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



Prototyping nanorod control: A DNA double helix sheathed within a DNA six-helix bundle.  


The control of the structure of matter is a key goal of nanoscience. DNA is an exciting molecule for control because it forms programmable intermolecular interactions. Stiff DNA structures, such as the double crossover motif, the tensegrity triangle, and the six-helix bundle (6HB) have been used to produce periodic arrays of DNA components. The 6HB motif consists of six DNA double helices flanking an inner cavity whose diameter is similar to that of a double helix. This motif appears to be an excellent candidate to sheathe and control nanorods by inserting them into the cavity, and then to control the placement and orientation of the rod by controlling the DNA sheath. Here, we prototype this kind of control by using a seventh DNA double helix as the nanorod and fixing it inside the 6HB motif. PMID:19716476

Wang, Risheng; Liu, Wenyan; Seeman, Nadrian C



Helix control in polymers  

PubMed Central

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

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



Lattice vibrational spectra of double-helix LiP  

NASA Astrophysics Data System (ADS)

The LiP infinite double-helix chain gives us the unique opportunity to investigate the double-helix structure at atomic level. Based on density-functional theory, we investigate the characteristic vibrational modes of this peculiar structure. Differently from single-wall carbon nanotubes, a dipole mode in LiP gives rise to high infrared intensity. Owing to the peculiar double-helix structure of LiP, the frequency of the out-of-phase radical breathing mode is lower than that of the in-phase one. It is similar to the condition of DNA. Furthermore, the out-of-phase breathing mode in LiP, instead of the in-phase one, has the highest Raman intensity. Most importantly, our finding is generally applicable to other double-helix structures.

Wang, Hui; Li, Tongwei; Lv, Zhenlong; Ju, Weiwei; Zhao, Xiaoyan; You, Jinghan; Li, Liben; Cai, Xiaowu; Han, Han



Alternative C-Terminal Helix Orientation Alters Chemokine Function  

PubMed Central

Chemokines, a subfamily of cytokines, are small, secreted proteins that mediate a variety of biological processes. Various chemokines adopt remarkable conserved tertiary structure comprising an anti-parallel ?-sheet core domain followed by a C-terminal helix that packs onto the ?-sheet. The conserved structural feature has been considered critical for chemokine function, including binding to cell surface receptor. The recently isolated variant, CXCL4L1, is a homologue of CXCL4 chemokine (or platelet factor 4) with potent anti-angiogenic activity and differed only in three amino acid residues of P58L, K66E, and L67H. In this study we show by x-ray structural determination that CXCL4L1 adopts a previously unrecognized structure at its C terminus. The orientation of the C-terminal helix protrudes into the aqueous space to expose the entire helix. The alternative helix orientation modifies the overall chemokine shape and surface properties. The L67H mutation is mainly responsible for the swing-out effect of the helix, whereas mutations of P58L and K66E only act secondarily. This is the first observation that reports an open conformation of the C-terminal helix in a chemokine. This change leads to a decrease of its glycosaminoglycan binding properties and to an enhancement of its anti-angiogenic and anti-tumor effects. This unique structure is recent in evolution and has allowed CXCL4L1 to gain novel functional properties. PMID:23536183

Kuo, Je-Hung; Chen, Ya-Ping; Liu, Jai-Shin; Dubrac, Alexandre; Quemener, Cathy; Prats, Herve; Bikfalvi, Andreas; Wu, Wen-guey; Sue, Shih-Che



Networks of helix-forming polymers  

Microsoft Academic Search

Biological molecules can form hydrogen bonds between nearby residues, leading to helical secondary structures. The associated reduction of configurational entropy leads to a temperature dependence of this effect: the helix-coil transition. Since the formation of helices implies a dramatic shortening of the polymer dimensions, an externally imposed end-to-end distance R affects the equilibrium helical fraction of the polymer and the

Samuel Kutter; Eugene M. Terentjev



DNA-like double helix formed by peptide nucleic acid  

Microsoft Academic Search

ALTHOUGH the importance of the nucleobases in the DNA double helix is well understood, the evolutionary significance of the deoxyribose phosphate backbone and the contribution of this chemical entity to the overall helical structure and stability of the double helix is not so clear. Peptide nucleic acid (PNA)1-7 is a DNA analogue with a backbone consisting of N-(2-aminoethyl)glycine units (Fig.

Pernilla Wittung; Peter E. Nielsen; Ole Buchardt; Michael Egholm; Bengt Nordén



Fluctuations of an Exposed ?-Helix Involved in Lipoxygenase Substrate Recognition  

PubMed Central

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 ?-helix, centrally located. A site-directed spin-label scan of 18 of the 21 helix 2 residues, and electron paramagnetic resonance, showed that the ?-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 ?-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 ?-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 ?-helix altered the intrinsic relaxation rates. Different symmetries of iron contributed to distance-dependent magnetic relaxation. We interpret these data to mean that a ?-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



Molecular Dynamics Simulations of Micelle Formation around Dimeric Glycophorin A Transmembrane Helices  

Microsoft Academic Search

Insertion and formation of membrane proteins involves the interaction of protein helices with one another in lipid environments. Researchers have studied glycophorin A (GpA) transmembrane helices embedded in sodium dodecyl sulfate (SDS) micelles to identify contacts significant for helix dimerization. However, a detailed picture of the conformation and dynamics of the GpA-SDS system cannot be obtained solely through experiment. Molecular

Rosemary Braun; Donald M. Engelman; Klaus Schulten



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

PubMed Central

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

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



Molecular Content of the Helix Nebula  

NASA Astrophysics Data System (ADS)

Multiple transitions of H_2CO, HCO^+, and CO were detected at nine positions across the planetary nebula NGC 7293, the Helix Nebula, using the 12m telescope and the Submillimeter Telescope (SMT) of the Arizona Radio Observatory (ARO). A complete map of the nebula has also been made in the J = 1 ? 0 transition of HCO^+ at 89 GHz. HCO^+ emission was found to be widespread across the Helix, and is coincident with the ionized gas as traced in optical images. A complex velocity structure is apparent in the HCO^+ spectra, as well. The CO and H_2CO data (J = 1 ? 0, 2 ? 1, and 3 ? 2) were modeled using a radiative transfer code at the nine positions observed in the Helix. Kinetic temperatures were typically found to be in the range Tkin ? 20 - 45 K and the gas density on the order of n(H_2) ? 105 cm-3 at these positions. The column densities for CO, H_2CO, and HCO^+ were determined to be 1015, 1012, and 1011 cm-2 respectively, corresponding to fractional abundances, relative to H_2, of f ? 10-4, 10-7, and 10-8. The extended distribution of HCO^+ suggests that dense clumps may exist throughout the nebula. Hence, the chemistry of evolved planetary nebulae may be more active than previously thought.

Zack, L. N.; Zeigler, N. R.; Ziurys, L. M.



Polyproline and Triple Helix Motifs in Host-Pathogen Recognition  

PubMed Central

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

Berisio, Rita; Vitagliano, Luigi



Genetic systems for monitoring interactions of transmembrane domains in bacterial membranes.  


In recent years several systems have been developed to study interactions of TM domains within the inner membrane of the Gram-negative bacterium Escherichia coli. Mostly, a transmembrane domain of interest is fused to a soluble DNA-binding domain, which dimerizes in E. coli cytoplasm after interactions of the transmembrane domains. The dimeric DNA-binding domain subsequently binds to a promoter/operator region and thereby activates or represses a reporter gene. In 1996 the first bacterial system has been introduced to measure interactions of TM helices within a bacterial membrane, which is based on fusion of a transmembrane helix of interest to the DNA-binding domain of the Vibrio cholerae ToxR protein. Interaction of a transmembrane helix of interest within the membrane environment results in dimerization of the DNA-binding domain in the bacterial cytoplasm, and the dimeric DNA-binding domain then binds to the DNA and activates a reporter gene. Subsequently, systems with improved features, such as the TOXCAT- or POSSYCCAT system, which allow screening of TM domain libraries, or the GALLEX system, which allows measuring heterotypic interactions of TM helices, have been developed and successfully applied. Here we briefly introduce the currently most applied systems and discuss their advantages together with their limitations. PMID:23975772

Tome, Lydia; Steindorf, Dominik; Schneider, Dirk



Folding of Aquaporin 1: multiple evidence that helix 3 can shift out of the membrane core.  


The folding of most integral membrane proteins follows a two-step process: initially, individual transmembrane helices are inserted into the membrane by the Sec translocon. Thereafter, these helices fold to shape the final conformation of the protein. However, for some proteins, including Aquaporin 1 (AQP1), the folding appears to follow a more complicated path. AQP1 has been reported to first insert as a four-helical intermediate, where helix 2 and 4 are not inserted into the membrane. In a second step, this intermediate is folded into a six-helical topology. During this process, the orientation of the third helix is inverted. Here, we propose a mechanism for how this reorientation could be initiated: first, helix 3 slides out from the membrane core resulting in that the preceding loop enters the membrane. The final conformation could then be formed as helix 2, 3, and 4 are inserted into the membrane and the reentrant regions come together. We find support for the first step in this process by showing that the loop preceding helix 3 can insert into the membrane. Further, hydrophobicity curves, experimentally measured insertion efficiencies and MD-simulations suggest that the barrier between these two hydrophobic regions is relatively low, supporting the idea that helix 3 can slide out of the membrane core, initiating the rearrangement process. PMID:24777974

Virkki, Minttu T; Agrawal, Nitin; Edsbäcker, Elin; Cristobal, Susana; Elofsson, Arne; Kauko, Anni



The role of ?-, 310-, and ?-helix in helix->coil transitions  

PubMed Central

The conformational equilibrium between 310- and ?-helical structure has been studied via high-resolution NMR spectroscopy by Millhauser and coworkers using the MW peptide Ac-AMAAKAWAAKA AAARA-NH2. Their 750-MHz nuclear Overhauser effect spectroscopy (NOESY) spectra were interpreted to reflect appreciable populations of 310-helix throughout the peptide, with the greatest contribution at the N and C termini. The presence of simultaneous ?N(i,i + 2) and ?N(i,i + 4) NOE cross-peaks was proposed to represent conformational averaging between 310- and ?-helical structures. In this study, we describe 25-nsec molecular dynamics simulations of the MW peptide at 298 K, using both an 8 ? and a 10 ? force-shifted nonbonded cutoff. The ensemble averages of both simulations are in reasonable agreement with the experimental helical content from circular dichroism (CD), the 3JHN? coupling constants, and the 57 observed NOEs. Analysis of the structures from both simulations revealed very little formation of contiguous i ? i + 3 hydrogen bonds (310-helix); however, there was a large population of bifurcated i ? i + 3 and i ? i + 4 ?-helical hydrogen bonds. In addition, both simulations contained considerable populations of ?-helix (i ? i + 5 hydrogen bonds). Individual turns formed over residues 1–9, which we predict contribute to the intensities of the experimentally observed ?N(i,i + 2) NOEs. Here we show how sampling of both folded and unfolded structures can provide a structural framework for deconvolution of the conformational contributions to experimental ensemble averages. PMID:12761385

Armen, Roger; Alonso, Darwin O.V.; Daggett, Valerie



Predicted alpha-helix/beta-sheet secondary structures for the zinc-binding motifs of human papillomavirus E7 and E6 proteins by consensus prediction averaging and spectroscopic studies of E7.  

PubMed Central

The E7 and E6 proteins are the main oncoproteins of human papillomavirus types 16 and 18 (HPV-16 and HPV-18), and possess unknown protein structures. E7 interacts with the cellular tumour-suppressor protein pRB and contains a zinc-binding site with two Cys-Xaa2-Cys motifs spaced 29 or 30 residues apart. E6 interacts with another cellular tumour-suppressor protein p53 and contains two zinc-binding sites, each with two Cys-Xaa2-Cys motifs at a similar spacing of 29 or 30 residues. By using the GOR I/III, Chou-Fasman, SAPIENS and PHD methods, the effectiveness of consensus secondary structure predictions on zinc-finger proteins was first tested with sequences for 160 transcription factors and 72 nuclear hormone receptors. These contain Cys2His2 and Cys2Cys2 zinc-binding regions respectively, and possess known atomic structures. Despite the zinc- and DNA-binding properties of these protein folds, the major alpha-helix structures in both zinc-binding regions were correctly identified. Thus validated, the use of these prediction methods with 47 E7 sequences indicated four well-defined alpha-helix (alpha) and beta-sheet (beta) secondary structure elements in the order beta beta alpha beta in the zinc-binding region of E7 at its C-terminus. The prediction was tested by Fourier transform infrared spectroscopy of recombinant HPV-16 E7 in H2O and 2H2O buffers. Quantitative integration showed that E7 contained similar amounts of alpha-helix and beta-sheet structures, in good agreement with the averaged prediction of alpha-helix and beta-sheet structures in E7 and also with previous circular dichroism studies. Protein fold recognition analyses predicted that the structure of the zinc-binding region in E7 was similar to a beta beta alpha beta motif found in the structure of Protein G. This is consistent with the E7 structure predictions, despite the low sequence similarities with E7. This predicted motif is able to position four Cys residues in proximity to a zinc atom. A model for the zinc-binding motif of E7 was constructed by combining the Protein G coordinates with those for the zinc-binding site in transcription factor TFIIS. Similar analyses for the two zinc-binding motifs in E6 showed that they have different alpha/beta secondary structures from that in E7. When compared with 12 other zinc-binding proteins, these results show that E7 and E6 are predicted to possess novel types of zinc-binding structure. PMID:8870673

Ullman, C G; Haris, P I; Galloway, D A; Emery, V C; Perkins, S J



Misplaced helix slows down ultrafast pressure-jump protein folding  

PubMed Central

Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein ?*YA, the Y22W/Q33Y/G46,48A mutant of ?-repressor fragment 6–85, from 3 ?s 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 ?s 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 ?-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of ?*YA contains mainly residual helix and little ?-sheet; following a fast P-drop, at least some ?*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

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



Statistical mechanics of helix bundles using a dynamic programming approach.  


Despite much study, biomolecule folding cooperativity is not well understood. There are quantitative models for helix-coil transitions and for coil-to-globule transitions, but no accurate models yet treat both chain collapse and secondary structure formation together. We develop here a dynamic programming approach to statistical mechanical partition functions of foldamer chain molecules. We call it the ascending levels model. We apply it to helix-coil and helix-bundle folding and cooperativity. For 14- to 50-mer Baldwin peptides, the model gives good predictions for the heat capacity and helicity versus temperature and urea. The model also gives good fits for the denaturation of Oas's three-helix bundle B domain of protein A (F13W*) and synthetic protein alpha3C by temperature and guanidine. The model predicts the conformational distributions. It shows that these proteins fold with transitions that are two-state, although the transitions in the Baldwin helices are nearly higher order. The model shows that the recently developed three-helix bundle polypeptoids of Lee et al. fold anti-cooperatively, with a predicted value of DeltaHvH/DeltaHcal = 0.72. The model also predicts that two-helix bundles are unstable in proteins but stable in peptoids. Our dynamic programming approach provides a general way to explore cooperativity in complex foldable polymers. PMID:17362002

Lucas, Adam; Huang, Liang; Joshi, Aravind; Dill, Ken A



A Synthetic Peptide Inhibitor of Human Immunodeficiency Virus Replication: Correlation between Solution Structure and Viral Inhibition  

Microsoft Academic Search

A peptide designated DP-107 was synthesized containing amino acid residues 558-595 of the envelope glycoprotein gp160 of human immunodeficiency virus type 1 strain LAI (HIV-1LAI). Algorithms for secondary structure have predicted that this region of the envelope transmembrane protein should form an extended alpha-helix. Consistent with this prediction, analysis by circular dichroism (CD) indicated that, under physiological conditions, DP-107 is

Carl Wild; Terrence Oas; Charlene McDanal; Dani Bolognesi; Thomas Matthews



Self-recognition behavior of a helix-loop-helix domain by a fragment scan.  


The inhibitors of DNA binding Id1-4 are helix-loop-helix (HLH) proteins that exert their biological function by interacting with members of the basic-HLH (bHLH) transcription-factor family. The HLH domains of the Id and bHLH proteins allow both self- and hetero-association. Due to their abnormal expression in cancer cells, the Id proteins are potential protein targets for cancer treatment. Suitable Id-protein inactivators should promote self-association and/or prevent hetero-association. In this work we evaluated the ability of the Id-protein HLH domain to recognize itself in form of short sequences extracted from the helical and loop regions. We performed a peptide scan of the Id1 HLH domain 64-106 based on three-residue overlapping octapeptides. Interaction of each octapeptide with the natively folded Id1 HLH domain was investigated by CD and fluorescence spectroscopy. The results from both techniques showed that the helix-based but not the loop-based octapeptides interacted with the Id1 HLH domain in the low-micromolar range. In contrast, a nitrotyrosine-containing analog of the Id1 HLH region, which was unable to reproduce the native-like conformation, quenched only the 2-amino-benzoyl-(Abz)-labeled loop-based octapeptides. This opposite self-recognition pattern suggests that the short helix-based and loop-based sequences should be able to distinguish different folding states of the Id1 HLH domain. This feature may be biologically relevant, as the Id proteins are predicted to behave as intrinsically disordered proteins, being in equilibrium between rapidly exchanging monomeric conformations and structurally better-defined homo-/heterodimers displaying the parallel four-helix bundle. PMID:24981796

Beisswenger, Michael; Cabrele, Chiara



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

PubMed Central

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

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.



Structure-Activity Relationships of Cyanoquinolines with Corrector-Potentiator Activity in delta-F508-Cystic Fibrosis Transmembrane Conductance Regulator Protein  

PubMed Central

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The most common CF-causing mutation, ?F508-CFTR, produces CFTR loss-of-function by impairing its cellular targeting to the plasma membrane and its chloride channel gating. We recently identified, cyanoquinolines with both corrector (“Co”; normalizing ?F508-CFTR targeting) and potentiator (“Po”; normalizing ?F508-CFTR channel gating) activities. Here, we synthesized and characterized twenty-four targeted cyanoquinoline analogues to elucidate the conformational requirements for corrector and potentiator activities. Compounds with potentiator-only, corrector-only and dual potentiator-corrector activities were found. Molecular modeling studies (conformational search ? force-field lowest energy assessment ? geometry optimization] suggest that (1) a flexible tether and (2) a relatively short bridge between the cyanoquinoline and aryl amide moieties are important cyanoquinoline-based CoPo features. Further, these CoPo’s should adopt two distinct ?-stacking conformations to elicit corrector and potentiator activities. PMID:22214395

Knapp, John M.; Wood, Alex B.; Phuan, Puay-Wah; Lodewyk, Michael W.; Tantillo, Dean J.; Verkman, A. S.; Kurth, Mark J.



A GxxxG-like Motif within HIV-1 Fusion Peptide Is Critical to Its Immunosuppressant Activity, Structure, and Interaction with the Transmembrane Domain of the T-cell Receptor*  

PubMed Central

To thrive in the human body, HIV fuses to its target cell and evades the immune response via several mechanisms. The fusion cascade is initiated by the fusion peptide (FP), which is located at the N-terminal of gp41, the transmembrane protein of HIV. Recently, it has been shown that the HIV-1 FP, particularly its 5–13 amino acid region (FP5–13), suppresses T-cell activation and interacts with the transmembrane domain (TMD) of the T-cell receptor (TCR) complex. Specific amino acid motifs often contribute to such interactions in TMDs of membrane proteins. Using bioinformatics and experimental studies, we report on a GxxxG-like motif (AxxxG), which is conserved in the FP throughout different clades and strains of HIV-1. Biological activity studies and FTIR spectroscopy revealed that HIV FP5–13-derived peptides, in which the motif was altered either by randomization or by a single amino acid shift, lost their immunosuppressive activity concomitant with a loss of the ?-sheet structure in a membranous environment. Furthermore, fluorescence studies revealed that the inactive mutants lost their ability to interact with their target site, namely, the TMD of TCR?, designated CP. Importantly, lipotechoic acid activated macrophages (lacking TCR) were not affected by FP, further demonstrating the specificity of the immunosuppressant activity of CP. Finally, although the AxxxG WT and the GxxxG analog both associated with the CP and immunosuppressed T-cells, the AxxxG WT but not the GxxxG analog induced lipid mixing. Overall, the data support an important role for the AxxxG motif in the function of FP and might explain the natural selection of the AxxxG motif rather than the classical GxxxG motif in FP. PMID:22872636

Faingold, Omri; Cohen, Tomer; Shai, Yechiel



Helix XI contributes to the entrance of the serotonin transporter permeation pathway  

PubMed Central

The sodium-dependent transporters for dopamine, norepinephrine, and serotonin that regulate neurotransmission, also translocate the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Previous studies implicated residues in transmembrane helix (TMH) XI of DAT as important sites for MPP+ transport. We examined the importance of TMH XI residues F551 and F556 for MPP+ translocation by human SERT. Mutations at hSERT F556, but not F551, reduced both 5-HT and MPP+ transport compared to wild type. However, F556S/hSERT showed a reduction in surface expression explaining the decrease of transport activity for 5-HT, but did not account for the decrease in MPP+ transport observed. Cysteine mutants at those positions confirmed the accessibility of hSERT/F556 to different methanethiosulfonate (MTS) reagents, suggesting its presence in a hydrophilic environment of the protein. In the presence of MTSET, current induced by 5-HT and MPP+ was inhibited at the F556C mutant. In agreement with our homology model of SERT, based on the leucine transporter (LeuTAa) from Aquifex aeolicus structure, these results are consistent with the hypothesis that a portion of TMH XI lines the entrance into the substrate permeation pathway. PMID:18628241

Torres-Altoro, Melissa I.; White, Kellie J.; Rodriguez, Gustavo J.; Nichols, David E.; Barker, Eric L.



Morphology and Composition of the Helix Nebula  

E-print Network

We present new narrow-band filter imagery in H-alpha and [N II] 6584 along with UV and optical spectrophotometry measurements from 1200 to 9600 Angstroms of NGC 7293, the Helix Nebula, a nearby, photogenic planetary nebula of large diameter and low surface brightness. Detailed models of the observable ionized nebula support the recent claim that the Helix is actually a flattened disk whose thickness is roughly one-third its diameter with an inner region containing hot, highly ionized gas which is generally invisible in narrow-band images. The outer visible ring structure is of lower ionization and temperature and is brighter because of a thickening in the disk. We also confirm a central star effective temperature and luminosity of 120,000K and 100L_sun, and we estimate a lower limit to the nebular mass to be 0.30M_sun. Abundance measurements indicate the following values: He/H=0.12 (+/-0.017), O/H=4.60x10^-4 (+/-0.18), C/O=0.87 (+/-0.12), N/O=0.54 (+/-0.14), Ne/O=0.33 (+/-0.04), S/O=3.22x10^-3 (+/-0.26), and Ar/O=6.74x10^-3 (+/-0.76). Our carbon abundance measurements represent the first of their kind for the Helix Nebula. The S/O ratio which we derive is anomalously low; such values are found in only a few other planetary nebulae. The central star properties, the super-solar values of He/H and N/O, and a solar level of C/O are consistent with a 6.5M_sun progenitor which underwent three phases of dredge-up and hot bottom burning before forming the planetary nebula.

R. B. C. Henry; K. B. Kwitter; R. J Dufour




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



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



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



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


An inverse correlation between loop length and stability in a four-helix-bundle protein  

E-print Network

An inverse correlation between loop length and stability in a four-helix-bundle protein Athena D Nagi and Lynne Regan Background: The loops in proteins are less well characterized than the secondary structural elements that they connect. We have used the four-helix- bundle protein Rop as a model system

Mochrie, Simon


A critical role of helix 3-helix 5 interaction in steroid hormone receptor function  

PubMed Central

The ligand-binding domains of steroid hormone receptors possess a conserved structure with 12 ?-helices surrounding a central hydrophobic core. On agonist binding, a repositioned helix 12 forms a pocket with helix 3 (H3) and helix 5 (H5), where transcriptional coactivators bind. The precise molecular interactions responsible for activation of these receptors remain to be elucidated. We previously identified a H3–H5 interaction that permits progesterone-mediated activation of a mutant mineralocorticoid receptor. We were intrigued to note that the potential for such interaction is widely conserved in the nuclear receptor family, indicating a possible functional significance. Here, we demonstrate via transcriptional activation studies in cell culture that alteration of residues involved in H3–H5 interaction consistently produces a gain of function in steroid hormone receptors. These data suggest that H3–H5 interaction may function as a molecular switch regulating the activity of nuclear receptors and suggest this site as a general target for pharmacologic intervention. Furthermore, they reveal a general mechanism for the creation of nuclear receptors bearing increased activity, providing a potentially powerful tool for the study of physiologic pathways in vivo. PMID:15710879

Zhang, Junhui; Simisky, Jessica; Tsai, Francis T. F.; Geller, David S.



Design of Coaxial Couplers for High Efficiency Helix TWT  

NASA Astrophysics Data System (ADS)

The main objective of the paper is to make an efficient design of the input and output coaxial coupler for a helix TWTs. An approach has been developed for the efficient design and analysis of the coaxial couplers in the practical situation. Normally multi-section impedance transformer approach is used for any wide band coupler. For a space helix TWT, coupler should be wide bandwidth and small size. In this case coupler is matched with helix slow wave structure and the standard 50-ohm connectors. The simulated return loss (dB) profile for different type of couplers is obtained by using Ansoft HFSS, CST microwave studio and compares those with experimental results. The tip loss design at sever ends for the input and the output section has been also optimized.

Alaria, Mukesh Kumar; Sinha, A. K.; Bera, A.; Srivastava, V.



HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Requirement of and subunit transmembrane helix separation for integrin  

E-print Network

in adhesion-induced outside-in signaling as measured by cell spreading, actin stress-fiber and focal adhesion formation, and focal adhesion kinase activation. This defect was res- cued by reduction of the disulfide organization that critically influence cell shape, migration, growth, and survival.1 The mechanism by which

Springer, Timothy A.


X-ray structure of dopamine transporter elucidates antidepressant mechanism.  


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

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



Palmitoylated transmembrane adaptor proteins in leukocyte signaling.  


Transmembrane adaptor proteins (TRAPs) are structurally related proteins that have no enzymatic function, but enable inducible recruitment of effector molecules to the plasma membrane, usually in a phosphorylation dependent manner. Numerous surface receptors employ TRAPs for either propagation or negative regulation of the signal transduction. Several TRAPs (LAT, NTAL, PAG, LIME, PRR7, SCIMP, LST1/A, and putatively GAPT) are known to be palmitoylated that could facilitate their localization in lipid rafts or tetraspanin enriched microdomains. This review summarizes expression patterns, binding partners, signaling pathways, and biological functions of particular palmitoylated TRAPs with an emphasis on the three most recently discovered members, PRR7, SCIMP, and LST1/A. Moreover, we discuss in silico methodology used for discovery of new family members, nature of their binding partners, and microdomain localization. PMID:24440308

Stepanek, Ondrej; Draber, Peter; Horejsi, Vaclav



Accommodation of a Central Arginine in a Transmembrane Peptide by Changing the Placement of Anchor Residues†  

PubMed Central

Both Trp and Arg in transmembrane protein domains make important interactions with lipids at the membrane/water interface, but at different depths. Derivatives of the designed peptide GWALP23, acetyl-GGALW5LALALALALALALW19LAGA-amide, with single Trp anchors, have proven useful for characterizing such interactions. Indeed previous work revealed quite different effects emanating from Arg substitutions at positions 12 and 14 within GWALP23, with the R12 peptide exhibiting multiple positions and orientations with respect to DOPC bilayer membranes (Vostrikov et al. [2010] J. Am. Chem. Soc. 132, 5803–5811). To gain further understanding of the multi-state behavior, we moved the Trp “anchor” residues to more outer positions 3 and 21 in GWALP23 itself, and in the R12 and R14 derivatives. The locations and orientations of the peptides with respect to lipid bilayer membranes of differing thickness were investigated by means of solid-state 2H NMR spectroscopy, using labeled alanines, and coarse-grained molecular dynamics simulations. Interestingly, relatively intense and narrow 2H resonances from selected backbone C? deuterons were observed over quite narrow ranges of frequency and sample orientation. The backbone resonances reflect dynamic complexities and at the same time provide important contributions for the analysis of peptide transmembrane orientation. With the Trp3,21 anchors relatively far from the peptide and bilayer center, the results indicate significantly large apparent tilt angles, for example close to 30° for the new R12 and R14 peptides with respect to the bilayer normal of DLPC membranes. The R12 side chain indeed is “rescued” to a stable position, where it is accommodated within the transmembrane helix, when the Trp anchors are moved outward and to another face of the helix. At the same time, the R14 side chain of transmembrane GW3,21ALP23 also retains a stable favored position. PMID:23030363

Vostrikov, Vitaly V.; Hall, Benjamin A.; Sansom, Mark S. P.; II, Roger E. Koeppe



The Helix-Loop-Helix Factors Id3 and E47 Are Novel Regulators of Adiponectin  

Microsoft Academic Search

Adiponectin is an adipocyte-derived cytokine with beneficial effects on insulin sensitivity and the development of atherosclerosis. Id3 is a helix-loop-helix factor that binds to E-proteins such as E47 and inhibits their binding to DNA. Although the helix-loop-helix factor sterol regulatory element binding protein (SREBP)-1c is a known activator of adiponectin transcription, this study provides the first evidence of a role

Amanda C. Doran; Nahum Meller; Alexis Cutchins; Hamid Deliri; R. Parker Slayton; Stephanie N. Oldham; Jae B. Kim; Susanna R. Keller; Coleen A. McNamara



Predicting helix-helix interactions from residue contacts in membrane proteins  

Microsoft Academic Search

Motivation: Helix-helix interactions play a critical role in the struc- ture assembly, stability, and function of membrane proteins. On the molecular level, the interactions are mediated by one or more resi- due contacts. Although previous studies focused on helix-packing patterns and sequence motifs, few of them developed methods spe- cifically for contact prediction. Results: We present a new hierarchical framework

Allan Lo; Yi-yuan Chiu; Einar Andreas Rødland; Ping-chiang Lyu; Ting-yi Sung; Wen-lian Hsu



Design and Synthesis of Peptides with Hybrid Helix-Turn-Helix (HTH) Motif and Their Conformational Study.  


The present study is aimed at the design and synthesis of peptides with hybrid helix-turn-helix (HTH) motif and their conformational analysis (NMR, MD, and CD studies). The requisite peptides with heterogeneous backbones were prepared from ?-, ?-, and ?-amino acids with carbohydrate side chains and ?-amino acid, l-Ala. The ?/?-peptides were prepared from (S)-?-Caa(l) (C-linked carbo-?-amino acid with D-lyxo furanoside side chain) and l-Ala with a 1:1 alternation. The ?/?-peptides with "helix-turn" motif displayed a 11/9-helix nucleating a 13-atom H-bonding turn. The ?/?-octapeptides showed the presence of HTH structures with bifurcated 11/15-H-bonded turn. Further, the ?/?-hexapeptide with HT motif, independently on coupling with ?/?/?/?- and ?/?/?/?-tetrapeptides at the C-terminus provided access to the decapeptides with "hybrid HTH" motifs. The decapeptide ("?-?-?-?-?-?-?-?-?-?") showed a hybrid HTH with "11/9/11/9/11/16/9/12/10" H-bonding, while the decapeptide ("?-?-?-?-?-?-?-?-?-?") revealed the presence of a "11/9/11/9/11/17/9/13/11" helical pattern. The above peptides thus have shown compatibility between different types of helices and serendipitous bifurcated 11/16- and 11/17-turns. The present study thus provided the first opportunity for the design and study of "hybrid HTH" motifs with more than one kind of helical structures in them. PMID:25180942

Sharma, Gangavaram V M; Thodupunuri, Prashanth; Sirisha, Katukuri; Basha, Shaik Jeelani; Gurava Reddy, Pottireddygari; Sarma, Akella V S



Functional Isoforms of IkB Kinase a (IKKa) Lacking Leucine Zipper and Helix-Loop-Helix Domains Reveal that IKKa and IKKb Have Different Activation Requirements  

Microsoft Academic Search

The activity of the NF-kB family of transcription factors is regulated principally by phosphorylation and subsequent degradation of their inhibitory IkB subunits. Site-specific serine phosphorylation of IkBs by two IkB kinases (IKKa (also known as CHUK) and IKKb) targets them for proteolysis. IKKa and -b have a unique structure, with an amino-terminal serine-threonine kinase catalytic domain and carboxy-proximal helix-loop-helix (HLH)




The Origins of Transmembrane Ion Channels  

NASA Technical Reports Server (NTRS)

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.

Pohorille, Andrew; Wilson, Michael A.



Modeling the flexibility of alpha helices in protein interfaces : structure based design and prediction of helix-mediated protein-protein interactions  

E-print Network

Protein-protein interactions play an essential role in many biological functions. Prediction and design of these interactions using computational methods requires models that can be used to efficiently sample structural ...

Apgar, James R. (James Reasoner)



Discovering the double helix structure of DNA, James Watson, video with 3D animation and narrationSite: DNA Interactive (  

NSDL National Science Digital Library

DNAi Location:Code>Finding the structure>pieces of the puzzle>Watson's base pairing Watson's base pairing James Watson used cardboard cutouts representing the shapes of the DNA bases to figure out how bases pair. He realized that the adenine-thymine and cytosine-guanine pairings fit all the dimensions.



Tryptophan at the transmembrane-cytosolic junction modulates thrombopoietin receptor dimerization and activation  

PubMed Central

Dimerization of single-pass membrane receptors is essential for activation. In the human thrombopoietin receptor (TpoR), a unique amphipathic RWQFP motif separates the transmembrane (TM) and intracellular domains. Using a combination of mutagenesis, spectroscopy, and biochemical assays, we show that W515 of this motif impairs dimerization of the upstream TpoR TM helix. TpoR is unusual in that a specific residue is required for this inhibitory function, which prevents receptor self-activation. Mutations as diverse as W515K and W515L cause oncogenic activation of TpoR and lead to human myeloproliferative neoplasms. Two lines of evidence support a general mechanism in which W515 at the intracellular juxtamembrane boundary inhibits dimerization of the TpoR TM helix by increasing the helix tilt angle relative to the membrane bilayer normal, which prevents the formation of stabilizing TM dimer contacts. First, measurements using polarized infrared spectroscopy show that the isolated TM domain of the active W515K mutant has a helix tilt angle closer to the bilayer normal than that of the wild-type receptor. Second, we identify second-site R514W and Q516W mutations that reverse dimerization and tilt angle changes induced by the W515K and W515L mutations. The second-site mutations prevent constitutive activation of TpoR W515K/L, while preserving ligand-induced signaling. The ability of tryptophan to influence the angle and dimerization of the TM helix in wild-type TpoR and in the second-site revertants is likely associated with its strong preference to be buried in the headgroup region of membrane bilayers. PMID:23359689

Defour, Jean-Philippe; Itaya, Miki; Gryshkova, Vitalina; Brett, Ian C.; Pecquet, Christian; Sato, Takeshi; Smith, Steven O.; Constantinescu, Stefan N.



A left-handed ?-helix containing bothL- andD-amino acids: The solution structure of the antimicrobial lipodepsipeptide tolaasin  

Microsoft Academic Search

The 18-amino acid cytolytic li- podepsipeptide tolaasin, produced in culture by virulent strains of Pseudomonas tolaasii ,i s the causal agent of the brown blotch disease of the cultivated mushroom. Tolaasin has a sequence of D-amino acids in its N-terminal region, then alter- nates L- and D-amino acids, and bears a C-terminal lactone macrocycle composed of 5-residues. The solution structure

Fabrice Jourdan; Silvia Lazzaroni; Pietro Lo Cantore; Marianna de Julio; Pietro Amodeo; Nicola S. Iacobellis; Antonio Evidente; Andrea Motta



Peptide probes for protein transmembrane domains.  


Much current interest in chemical biology focuses on the transmembrane domains of proteins, which have emerged as targets for the development of novel diagnostics and therapeutics. Integral membrane proteins are a group of important biomolecules that play pivotal roles in many cellular activities. Previous studies primarily focused on the extra- and/or intracellular domains of membrane proteins. However, the importance of transmembrane regions in the regulation of protein complexes is beginning to emerge. As such, a number of methods for designing and testing novel exogenous peptides that recognize transmembrane targets and modulate cellular functions have been developed. This Review outlines current methodologies for developing these transmembrane probes that may provide useful tools to study a variety of biological phenomena in the membrane. PMID:18533658

Slivka, Peter F; Wong, Johnny; Caputo, Gregory A; Yin, Hang



BuD, a helix-loop-helix DNA-binding domain for genome modification  

PubMed Central

DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19?bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin ? (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing. PMID:25004980

Stella, Stefano; Molina, Rafael; Lopez-Mendez, Blanca; Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza; Campos-Olivas, Ramon; Duchateau, Phillippe; Montoya, Guillermo



A Genome-Wide Survey on Basic Helix-Loop-Helix Transcription Factors in Giant Panda  

PubMed Central

The giant panda (Ailuropoda melanoleuca) is a critically endangered mammalian species. Studies on functions of regulatory proteins involved in developmental processes would facilitate understanding of specific behavior in giant panda. The basic helix-loop-helix (bHLH) proteins play essential roles in a wide range of developmental processes in higher organisms. bHLH family members have been identified in over 20 organisms, including fruit fly, zebrafish, mouse and human. Our present study identified 107 bHLH family members being encoded in giant panda genome. Phylogenetic analyses revealed that they belong to 44 bHLH families with 46, 25, 15, 4, 11 and 3 members in group A, B, C, D, E and F, respectively, while the remaining 3 members were assigned into “orphan”. Compared to mouse, the giant panda does not encode seven bHLH proteins namely Beta3a, Mesp2, Sclerax, S-Myc, Hes5 (or Hes6), EBF4 and Orphan 1. These results provide useful background information for future studies on structure and function of bHLH proteins in the regulation of giant panda development. PMID:22096504

Dang, Chunwang; Wang, Yong; Zhang, Debao; Yao, Qin; Chen, Keping



BuD, a helix-loop-helix DNA-binding domain for genome modification.  


DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein-DNA interactions in protein scaffolds is key to providing `toolkits' for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19?bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix-loop-helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin ? (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing. PMID:25004980

Stella, Stefano; Molina, Rafael; López-Méndez, Blanca; Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza; Campos-Olivas, Ramon; Duchateau, Phillippe; Montoya, Guillermo



Playing with peptides: how to build a supramolecular peptide nanostructure by exploiting helix···helix macrodipole interactions.  


A novel method to build bicomponent peptide self-assembled monolayers (SAMs) has been developed, by exploiting helix···helix macrodipole interactions. In this work, a peptide-based self-assembled monolayer composed of two helical peptides was immobilized on a gold surface. Specifically, a pyrene-containing octapeptide, devoid of any sulfur atom (A8Pyr), and a hexapeptide, functionalized at the N-terminus with (S,R) lipoic acid, for binding to gold substrates (SSA4WA) via a Au-S linkage, have been employed. Both peptides investigated attain a helical structure, because they are almost exclusively formed by strongly folding inducer C(?)-tetrasubstituted ?-amino acids. We demonstrate that the two peptides generate a stable supramolecular nanostructure (a densely packed bicomponent peptide monolayer), where A8Pyr is incorporated into the SSA4WA palisade by exploiting helix···helix macrodipole interactions. The presence of both peptides on the gold surface was investigated by spectroscopic and electrochemical techniques, while the morphology of the monolayer was analyzed by ultra high-vacuum scanning tunnelling microscopy. The composition of the bicomponent SAM on the surface was studied by a combination of electrochemical and spectroscopic techniques. In particular, the amount of Au-S linkages from the sulfur-containing peptides was quantified from reductive desorption of the peptide-based SAM, while the amount of A8Pyr was estimated by fluorescence spectroscopy. The antiparallel orientation of the A8Pyr and SSA4WA peptide chains minimizes the interaction energy between the helix dipoles, suggesting that this kind of electrostatic phenomenon is the driving force that stabilizes the bicomponent SAM. PMID:22214420

Gatto, E; Porchetta, A; Scarselli, M; De Crescenzi, M; Formaggio, F; Toniolo, C; Venanzi, M



?-Synuclein Populates Both Elongated and Broken Helix States on Small Unilamellar Vesicles  

PubMed Central

The misfolding of the protein ?-synuclein (?S) has been implicated in the molecular chain of events leading to Parkinson disease. Physiologically, ?S undergoes a transition from a random coil to helical conformation upon encountering synaptic vesicle membranes. On analogous small unilamellar vesicles (SUVs), the conformation of ?S is dominated by a single elongated ?S helix. However, alternative broken helix states have been postulated, mandating experimental clarification. Here, the upper limit for the free energy difference between elongated and broken helix conformations on SUVs resembling synaptic vesicles was determined to be 1.2 ± 0.4 kcal/mol, which amounts to a population ratio of 7.6:1 between both states (12% broken helices). In response to helix breaks at different positions, ?S rearranged in an opportunistic manner, thereby minimizing helix abrogations to as little as one to two turns. Enthalpy and entropy measurements of gel state SUV-?S interactions indicated that broken helix states retain the ability to relieve membrane-packing stress. Thus, broken helix states are a distinct physiological feature of the vesicle-bound ?S state, making it a “checkered” protein of multiple parallel conformations. A continuous interconversion between structural states may contribute to pathological ?S misfolding. PMID:21524999

Lokappa, Sowmya Bekshe; Ulmer, Tobias S.



Nonequilibrium dynamics of helix reorganization observed by transient 2D IR spectroscopy  

PubMed Central

The relaxation of helical structures very close to equilibrium is observed via transient 2D IR spectroscopy. An initial distribution of synthetically distorted helices having an unnatural bridge linking the 10th and 12th residues of an alanine-rich ?-helix is released to evolve into the equilibrium distribution of ?-helix conformations. The bridge constrains the structure to be slightly displaced from the full ?-helix equilibrium near these residues, yet the peptide is not unfolded completely. The release is accomplished by a subpicosecond pulse of UV irradiation. The resulting 2D IR signals are used to obtain snapshots of the ?100-ps helical conformational reorganization of the distorted dihedral angle and distance between amide units at chemical bond length-scale resolution. The decay rates of the angle between the dipoles, dihedral angles, and distance autocorrelations obtained from molecular dynamics simulations support the experiments, providing evidence that the final helix collapse conforms to linear response theory. PMID:24106309

Tucker, Matthew J.; Abdo, Mohannad; Courter, Joel R.; Chen, Jianxin; Brown, Stephen P.; Smith, Amos B.; Hochstrasser, Robin M.



Linus Pauling' s manuscript describing incorrect triple helix, Peter PaulingSite: DNA Interactive (  

NSDL National Science Digital Library

Interviewee: Peter Pauling DNAi Location:Code>Finding the Structure>players The triple helix In 1952, Peter Pauling was a student at Cambridge when his father, Linus, sent him a paper proposing that DNA was a triple helix. James (Jim) Watson eagerly read the paper and realized that Pauling got it wrong.



Equilibrium crossing exhibited by an ethynylhelicene (M)-nonamer during random-coil-to-double-helix thermal transition in solution.  


The structural change between the random-coil and the double-helix of an ethynylhelicene (M)-nonamer during heating crosses equilibrium. This is a phenomenon where a chemical reaction crosses equilibrium and returns to equilibrium. It is due to an accelerated rate of formation of the double-helix by self-catalysis and an equilibrium shift. PMID:25303723

Miyagawa, Masamichi; Yagi, Atsushi; Shigeno, Masanori; Yamaguchi, Masahiko



Structural Analysis of Cloned Plasma Membrane Proteins by Freeze-Fracture Electron Microscopy  

NASA Astrophysics Data System (ADS)

We have used freeze-fracture electron microscopy to examine the oligomeric structure and molecular asymmetry of integral plasma membrane proteins. Recombinant plasma membrane proteins were functionally expressed in Xenopus laevis oocytes, and the dimensions of their freeze-fracture particles were analyzed. To characterize the freeze-fracture particles, we compared the particle cross-sectional area of proteins with ? -helical transmembrane domains (opsin, aquaporin 1, and a connexin) with their area obtained from existing maps calculated from two-dimensional crystals. We show that the cross-sectional area of the freeze-fracture particles corresponds to the area of the transmembrane domain of the protein, and that the protein cross-sectional area varies linearly with the number membrane-spanning helices. On average, each helix occupies 1.40 ± 0.03 nm2. By using this information, we examined members from three classes of plasma membrane proteins: two ion channels, the cystic fibrosis transmembrane conductance regulator and connexin 50 hemi-channel; a water channel, the major intrinsic protein (the aquaporin 0); and a cotransporter, the Na+/glucose cotransporter. Our results suggest that the cystic fibrosis transmembrane conductance regulator is a dimer containing 25 ± 2 transmembrane helices, connexin 50 is a hexamer containing 24 ± 3 helices, the major intrinsic protein is a tetramer containing 24 ± 3 helices, and the Na+/glucose cotransporter is an asymmetrical monomer containing 15 ± 2 helices.

Eskandari, Sepehr; Wright, Ernest M.; Kreman, Mike; Starace, Dorine M.; Zampighi, Guido A.



Molecular dynamics investigation of membrane-bound bundles of the channel-forming transmembrane domain of viral protein U from the human immunodeficiency virus HIV-1.  


Molecular dynamics (MD) simulations have been carried out on bundles of the channel-forming transmembrane (TM) domain of the viral protein U (VPU(1-27) and VPU(6-27)) from the human immunodeficiency virus (HIV-1). Simulations of hexameric and pentameric bundles of VPU(6-27) in an octane/water membrane mimetic system suggested that the pentamer is the preferred oligomer. Accordingly, an unconstrained pentameric helix bundle of VPU(1-27) was then placed in a hydrated palmitoyl-oleyl-3-n-glycero-phosphatidylethanolamine (POPE) lipid bilayer and its structural properties calculated from a 3-ns MD run. Some water molecules, initially inside the channel lumen, were expelled halfway through the simulation and the bundle adopted a conical structure reminiscent of previous MD results obtained for VPU(6-27) in an octane/water system. The pore constriction generated may correspond to a closed state of the channel and underlies the relocation of the W residue toward the pore lumen. The relative positions of the helices with respect to the bilayer and their interactions with the lipids are discussed. The observed structure is stabilized via specific interactions between the VPU helices and the carbonyl oxygen atoms of the lipid molecules, particularly at the Q and S residues. PMID:12202353

Lopez, Carlos F; Montal, Mauricio; Blasie, J Kent; Klein, Michael L; Moore, Preston B



An analysis of oligomerization interfaces in transmembrane proteins  

PubMed Central

Background The amount of transmembrane protein (TM) structures solved to date is now large enough to attempt large scale analyses. In particular, extensive studies of oligomeric interfaces in the transmembrane region are now possible. Results We have compiled the first fully comprehensive set of validated transmembrane protein interfaces in order to study their features and assess what differentiates them from their soluble counterparts. Conclusions The general features of TM interfaces do not differ much from those of soluble proteins: they are large, tightly packed and possess many interface core residues. In our set, membrane lipids were not found to significantly mediate protein-protein interfaces. Although no G protein-coupled receptor (GPCR) was included in the validated set, we analyzed the crystallographic dimerization interfaces proposed in the literature. We found that the putative dimer interfaces proposed for class A GPCRs do not show the usual patterns of stable biological interfaces, neither in terms of evolution nor of packing, thus they likely correspond to crystal interfaces. We cannot however rule out the possibility that they constitute transient or weak interfaces. In contrast we do observe a clear signature of biological interface for the proposed dimer of the class F human Smoothened receptor. PMID:24134166



Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry  

NASA Technical Reports Server (NTRS)

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.

Wang, N.; Ingber, D. E.



Contribution of dipole-dipole interactions to the stability of the collagen triple helix  

PubMed Central

Unveiling sequence–stability and structure–stability relationships is a major goal of protein chemistry and structural biology. Despite the enormous efforts devoted, answers to these issues remain elusive. In principle, collagen represents an ideal system for such investigations due to its simplified sequence and regular structure. However, the definition of the molecular basis of collagen triple helix stability has hitherto proved to be a difficult task. Particularly puzzling is the decoding of the mechanism of triple helix stabilization/destabilization induced by imino acids. Although the propensity-based model, which correlates the propensities of the individual imino acids with the structural requirements of the triple helix, is able to explicate most of the experimental data, it is unable to predict the rather high stability of peptides embedding Gly–Hyp–Hyp triplets. Starting from the available X-ray structures of this polypeptide, we carried out an extensive quantum chemistry analysis of the mutual interactions established by hydroxyproline residues located at the X and Y positions of the Gly–X–Y motif. Our data clearly indicate that the opposing rings of these residues establish significant van der Waals and dipole–dipole interactions that play an important role in triple helix stabilization. These findings suggest that triple helix stabilization can be achieved by distinct structural mechanisms. The interplay of these subtle but recurrent effects dictates the overall stability of this widespread structural motif. PMID:18369197

Improta, Roberto; Berisio, Rita; Vitagliano, Luigi



A role for U2/U6 helix Ib in 5' splice site selection.  

PubMed Central

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

Luukkonen, B G; Seraphin, B



N-Terminal Helix-Cap in ?-Helix 2 Modulates ?-State Misfolding in Rabbit and Hamster Prion Proteins  

PubMed Central

Susceptibility of a particular species to prion disease is affected by small differences in the sequence of PrP and correlates with the propensity of its PrP to assume the ?-state. A helix-cap motif in the ?2??2-loop of native ?-helical rabbit PrP, a resistant species, contains sequence differences that influence intra- and interspecies transmission. To determine the effect the helix-cap motif on ?-state refolding propensity, we mutated S170N, S174N, and S170N/S174N of the rabbit PrP helix-cap to resemble that of hamster PrP and conversely, N170S, N174S, and N170S/N174S of hamster PrP to resemble the helix-cap of rabbit PrP. High-resolution crystal structures (1.45–1.6 Å) revealed that these mutations ablate hydrogen-bonding interactions within the helix-cap motif in rabbit PrPC. They also alter the ?-state-misfolding propensity of PrP; the serine mutations in hamster PrP decrease the propensity up to 35%, whereas the asparagine mutations in rabbit PrP increase it up to 42%. Rapid dilution of rabbit and hamster into ?-state buffer conditions causes quick conversion to ?-state monomers. Kinetic monitoring using size-exclusion chromatography showed that the monomer population decreases exponentially mirrored by an increase in an octameric species. The monomer-octamer transition rates are faster for hamster than for rabbit PrP. The N170S/N174S mutant of hamster PrP has a smaller octamer component at the endpoint compared to the wild-type, whereas the kinetics of octamer formation in mutant and wild-type rabbit PrP are comparable. These findings demonstrate that the sequence of the ?2??2 helix-cap affects refolding to the ?-state and subsequently, may influence susceptibility to prion disease. PMID:23675452

Sweeting, Braden; Brown, Eric; Khan, M. Qasim; Chakrabartty, Avijit; Pai, Emil F.



Enantioselective assembly of a ruthenium(II) polypyridyl complex into a double helix.  


Evolution can increase the complexity of matter by self-organization into helical architectures, the best example being the DNA double helix. One common aspect, apparently shared by most of these architectures, is the presence of covalent bonds within the helix backbone. Here, we report the unprecedented crystal structures of a metal complex that self-organizes into a continuous double helical structure, assembled by non-covalent building blocks. Built up solely by weak stacking interactions, this alternating tread stairs-like double helical assembly mimics the DNA double helix structure. Starting from a racemic mixture in aqueous solution, the ruthenium(II) polypyridyl complex forms two polymorphic structures of a left-handed double helical assembly of only the ?-enantiomer. The stacking of the helices is different in both polymorphs: a crossed woodpile structure versus a parallel columnar stacking. PMID:25044471

Van Hecke, Kristof; Cardinaels, Thomas; Nockemann, Peter; Jacobs, Jeroen; Vanpraet, Louis; Parac-Vogt, Tatjana N; Van Deun, Rik; Binnemans, Koen; Van Meervelt, Luc



A helix-loop-helix protein related to the immunoglobulin E box-binding proteins.  

PubMed Central

A human cDNA encoding a novel protein in the helix-loop-helix family has been isolated by screening a bacteriophage expression library with a probe containing the binding site for major late transcription factor. The protein encoded by this cDNA, TFEB, probably recognizes E-box sequences in the heavy-chain immunoglobulin enhancer. Images PMID:2115126

Carr, C S; Sharp, P A



Homologue Structure of the SLAC1 Anion Channel for Closing Stomata in Leaves  

SciTech Connect

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.

Y Chen; L Hu; M Punta; R Bruni; B Hillerich; B Kloss; B Rost; J Love; S Siegelbaum; W Hendrickson



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


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

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



Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases  

PubMed Central

Helix–hairpin–helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH)2 domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo V's HhH motifs for modulating DNA-binding properties of the Stoffel fragment of TaqDNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH2 terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the processivity of chimeric polymerases to salts depend on the structure of added HhH motifs. Regardless of the type of the construct, the thermal stability of chimeric Taq polymerases increases under the optimal ionic conditions, as compared with that of TaqDNA polymerase or its Stoffel fragment. Our approach to raise the salt tolerance, processivity, and thermostability of Taq and Pfu DNA polymerases may be applied to all pol1- and polB-type polymerases, as well as to other DNA processing enzymes. PMID:12368475

Pavlov, Andrey R.; Belova, Galina I.; Kozyavkin, Sergei A.; Slesarev, Alexei I.



Linus Pauling's triple DNA helix model, 3D animation with basic narrationSite: DNA Interactive (  

NSDL National Science Digital Library

DNAi Location:Code>Finding the Structure>pieces of the puzzle>Pauling's triple helix This is Linus Pauling's failed attempt to predict the structure of DNA. The problem with his triple helix model is that the phosphates form the helical core, with the bases pointing outwards. This would be impossible under normal cellular conditions. Each phosphate group is negatively charged, and so many negative charges forced together would repel each other, literally driving the structure apart.



Structural Basis for the Trembler-J Phenotype of Charcot-Marie-Tooth Disease  

PubMed Central

Summary Mutations in peripheral myelin protein 22 (PMP22) can result in the common peripheral neuropathy, Charcot-Marie-Tooth disease (CMTD). The Leu16Pro mutation in PMP22 results in misassembly of the protein, which causes the Trembler-J (TrJ) disease phenotype. Here we elucidate the structural defects present in a partially folded state of TrJ PMP22 that are decisive in promoting CMTD-causing misfolding. In this state transmembrane helices 2-4 (TM2-4) form a molten-globular bundle while transmembrane helix 1 (TM1) is dissociated from this bundle. The TrJ mutation was seen to profoundly disrupt the TM1 helix, resulting in increased backbone dynamics and changes in the tertiary interactions of TM1 with the PMP22 TM2-4 core in the folded state. Consequently, TM1 undergoes enhanced dissociation from the other transmembrane segments in TrJ PMP22, becoming available for recognition and sequestration by protein folding quality control, leading to loss of function and toxic accumulation of aggregates that results in CMTD. PMID:21827951

Sakakura, Masayoshi; Hadziselimovic, Arina; Wang, Zhen; Schey, Kevin L.; Sanders, Charles R.



Interface connections of a transmembrane voltage sensor  

E-print Network

Interface connections of a transmembrane voltage sensor J. Alfredo Freites*, Douglas J. Tobias by the motion of the S4 voltage sensors. These sensors are ­helices that include four or more positively charged of the KvAP K channel [Jiang, et al. (2003) Nature 423, 33­41], posits that the S4 sensors move within

White, Stephen


Seven transmembrane receptors: something old, something new  

Microsoft Academic Search

Receptors for hormones, neurotransmitters, drugs, sensory stimuli and many other agents represent the gateway to cellular metabolism and activity. They regulate virtually all physiological processes in mammals. Yet as recently as 40 years ago their very existence was still in question. One class of receptors, those coupled to G proteins (also known as GPCRs or seven transmembrane receptors) comprise by

R. J. Lefkowitz



Introduction The cystic fibrosis transmembrane conductance regu-  

E-print Network

Introduction The cystic fibrosis transmembrane conductance regu- lator (CFTR) is a c, and exocrine glands (1). CFTR is causal in 2 major human diseases: cystic fibrosis (CF) and secretory diarrhea Ulrich Thome,6 John F. Engelhardt,4 Deborah J. Nelson,2 and Kevin L. Kirk1 1Gregory Fleming James Cystic

Engelhardt, John F.


Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation.  


Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 degrees C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes. PMID:19922971

Yunus, Abdul S; Jackson, Trent P; Crisafi, Katherine; Burimski, Irina; Kilgore, Nicole R; Zoumplis, Dorian; Allaway, Graham P; Wild, Carl T; Salzwedel, Karl



Elevated Temperature Triggers Human Respiratory Syncytial Virus F Protein Six-Helix Bundle Formation  

PubMed Central

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°C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes. PMID:19922971

Yunus, Abdul S.; Jackson, Trent P.; Crisafi, Katherine; Burimski, Irina; Kilgore, Nicole R.; Zoumplis, Dorian; Allaway, Graham P.; Wild, Carl T.; Salzwedel, Karl



Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation  

SciTech Connect

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 deg. C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes.

Yunus, Abdul S.; Jackson, Trent P.; Crisafi, Katherine; Burimski, Irina; Kilgore, Nicole R.; Zoumplis, Dorian; Allaway, Graham P.; Wild, Carl T. [Panacos Pharmaceuticals, Inc., 209 Perry Parkway, Suite 7, Gaithersburg, MD 20877 (United States); Salzwedel, Karl, E-mail: salzwedelkd@niaid.nih.go [Panacos Pharmaceuticals, Inc., 209 Perry Parkway, Suite 7, Gaithersburg, MD 20877 (United States)



Evolutionary analysis of the segment from helix 3 through helix 5 in vertebrate progesterone receptors.  


The interaction between helix 3 and helix 5 in the human mineralocorticoid receptor [MR], progesterone receptor [PR] and glucocorticoid receptor [GR] influences their response to steroids. For the human PR, mutations at Gly-722 on helix 3 and Met-759 on helix 5 alter responses to progesterone. We analyzed the evolution of these two sites and the rest of a 59 residue segment containing helices 3, 4 and 5 in vertebrate PRs and found that a glycine corresponding to Gly-722 on helix 3 in human PR first appears in platypus, a monotreme. In lamprey, skates, fish, amphibians and birds, cysteine is found at this position in helix 3. This suggests that the cysteine to glycine replacement in helix 3 in the PR was important in the evolution of mammals. Interestingly, our analysis of the rest of the 59 residue segment finds 100% sequence conservation in almost all mammal PRs, substantial conservation in reptile and amphibian PRs and divergence of land vertebrate PR sequences from the fish PR sequences. The differences between fish and land vertebrate PRs may be important in the evolution of different biological progestins in fish and mammalian PR, as well as differences in susceptibility to environmental chemicals that disrupt PR-mediated physiology. PMID:22575083

Baker, Michael E; Uh, Kayla Y



The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae  

PubMed Central

The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before. The long C-terminal helix ?6 protrudes from the body of the domain and most likely continues as the first transmembrane helix of EpsF. Two N-terminal EpsF domains form a tight dimer with a conserved interface, suggesting that the observed dimer occurs in the T2SS of many bacteria. Two calcium binding sites are present in the dimer interface with ligands provided for each site by both subunits. Based on this new structure, sequence comparisons of EpsF homologs and localization studies of GFP fused with EpsF, we propose that the second cytoplasmic domain of EpsF adopts a similar fold as the first cytoplasmic domain and that full-length EpsF, and its T2SS homologs, have a three-transmembrane helix topology. PMID:19324092

Abendroth, Jan; Mitchell, Daniel D.; Korotkov, Konstantin V.; Johnson, Tanya L.; Kreger, Allison; Sandkvist, Maria; Hol, Wim G. J.



Helix A Stabilization Precedes Amino-terminal Lobe Activation upon Calcium Binding to Calmodulin  

SciTech Connect

The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)-resorufin (ReAsH), which upon binding to an engineered tetracysteine binding motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the 1H-15N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe (Kd = 0.36 ? 0.04 ?M), which results in a reduction in the rate of ReAsH binding from 4900 M-1 sec-1 to 370 M-1 sec-1. In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe requires calcium-occupancy of amino-terminal sites (Kd = 18 ? 3 ?M). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain linker that release helix A, thereby facilitating the formation of calcium binding sites in the amino-terminal lobe and linked tertiary structural rearrangements to form a high-affinity binding cleft that can associate with target proteins.

Chen, Baowei; Lowry, David; Mayer, M. Uljana; Squier, Thomas C.



[Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].  


A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy. PMID:22393787

Goncharuk, M V; Shul'ga, A A; Ermoliuk, Ia S; Tkach, E N; Goncharuk, S A; Pustovalova, Iu E; Mineev, K S; Bocharov, É V; Maslennikov, I V; Arsen'ev, A S; Kirpichnikov, M P



Essential Molecular Determinants for Thyroid Hormone Transport and First Structural Implications for Monocarboxylate Transporter 8*  

PubMed Central

Monocarboxylate transporter 8 (MCT8, SLC16A2) is a thyroid hormone (TH) transmembrane transport protein mutated in Allan-Herndon-Dudley syndrome, a severe X-linked psychomotor retardation. The neurological and endocrine phenotypes of patients deficient in MCT8 function underscore the physiological significance of carrier-mediated TH transmembrane transport. MCT8 belongs to the major facilitator superfamily of 12 transmembrane-spanning proteins and mediates energy-independent bidirectional transport of iodothyronines across the plasma membrane. Structural information is lacking for all TH transmembrane transporters. To gain insight into structure-function relations in TH transport, we chose human MCT8 as a paradigm. We systematically performed conventional and liquid chromatography-tandem mass spectrometry-based uptake measurements into MCT8-transfected cells using a large number of compounds structurally related to iodothyronines. We found that human MCT8 is specific for l-iodothyronines and requires at least one iodine atom per aromatic ring. Neither thyronamines, decarboxylated metabolites of iodothyronines, nor triiodothyroacetic acid and tetraiodothyroacetic acid, TH derivatives lacking both chiral center and amino group, are substrates for MCT8. The polyphenolic flavonoids naringenin and F21388, potent competitors for TH binding at transthyretin, did not inhibit T3 transport, suggesting that MCT8 can discriminate its ligand better than transthyretin. Bioinformatic studies and a first molecular homology model of MCT8 suggested amino acids potentially involved in substrate interaction. Indeed, alanine mutation of either Arg445 (helix 8) or Asp498 (helix 10) abrogated T3 transport activity of MCT8, supporting their predicted role in substrate recognition. The MCT8 model allows us to rationalize potential interactions of amino acids including those mutated in patients with Allan-Herndon-Dudley syndrome. PMID:20628049

Kinne, Anita; Kleinau, Gunnar; Hoefig, Carolin S.; Gruters, Annette; Kohrle, Josef; Krause, Gerd; Schweizer, Ulrich



Solid-state NMR investigations of peptide-lipid interactions of the transmembrane domain of a plant-derived protein, Hcf106.  


The chloroplast twin arginine translocation system transports highly folded precursor proteins across the thylakoid using the protonmotive force as its only energy source. Hcf106 and another thylakoid protein, cpTatC compose the precursor receptor complex. Hcf106 is predicted to contain a single amino terminal transmembrane domain (TMD) followed by a Pro-Gly hinge, an amphipathic ?-helix, and a loosely structured carboxyl terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes; however, how this occurs is not understood. To investigate how Hcf106 inserts itself into the membrane unassisted, solid-state NMR spectroscopy was used to investigate the membrane activity of the TMD. A synthetic peptide of the Hcf106 TMD was incorporated into multilamellar vesicles made of 100% 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) or 85%:15% ratio with monogalactosyl diacylglycerol (POPC/MGDG) to probe peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic techniques were used to reveal peptide perturbations of the phospholipid membranes. Changes in spectral lineshape, chemical shift anisotropy width, (31)P T1 relaxation time and SCD order parameters demonstrated that the Hcf106 TMD peptide interacted with the phospholipids. Furthermore, the comparison between POPC and POPC/MGDG multilamellar vesicles indicated that lipid bilayer composition affected the peptide-lipid interaction with the peptide interacting preferentially with vesicles that more closely mimic the thylakoid. PMID:24075840

Zhang, Lei; Liu, Lishan; Maltsev, Sergey; Lorigan, Gary A; Dabney-Smith, Carole



310-Helix Conformation Facilitates the Transition of a Voltage Sensor S4 Segment toward the Down State  

PubMed Central

The activation of voltage-gated ion channels is controlled by the S4 helix, with arginines every third residue. The x-ray structures are believed to reflect an open-inactivated state, and models propose combinations of translation, rotation, and tilt to reach the resting state. Recently, experiments and simulations have independently observed occurrence of 310-helix in S4. This suggests S4 might make a transition from ?- to 310-helix in the gating process. Here, we show 310-helix structure between Q1 and R3 in the S4 segment of a voltage sensor appears to facilitate the early stage of the motion toward a down state. We use multiple microsecond-steered molecular simulations to calculate the work required for translating S4 both as ?-helix and transformed to 310-helix. The barrier appears to be caused by salt-bridge reformation simultaneous to R4 passing the F233 hydrophobic lock, and it is almost a factor-two lower with 310-helix. The latter facilitates translation because R2/R3 line up to face E183/E226, which reduces the requirement to rotate S4. This is also reflected in a lower root mean-square deviation distortion of the rest of the voltage sensor. This supports the 310 hypothesis, and could explain some of the differences between the open-inactivated- versus activated-states. PMID:21402026

Schwaiger, Christine S.; Bjelkmar, Par; Hess, Berk; Lindahl, Erik



Crystal Structure of the RC-LH1 Core Complex from Rhodopseudomonas palustris  

NASA Astrophysics Data System (ADS)

The crystal structure at 4.8 angstrom resolution of the reaction center-light harvesting 1 (RC-LH1) core complex fromRhodopseudomonas palustris shows the reaction center surrounded by an oval LH1 complex that consists of 15 pairs of transmembrane helical ?- and ?-apoproteins and their coordinated bacteriochlorophylls. Complete closure of the RC by the LH1 is prevented by a single transmembrane helix, out of register with the array of inner LH1 ?-apoproteins. This break, located next to the binding site in the reaction center for the secondary electron acceptor ubiquinone (UQB), may provide a portal through which UQB can transfer electrons to cytochrome b/c1.

Roszak, Aleksander W.; Howard, Tina D.; Southall, June; Gardiner, Alastair T.; Law, Christopher J.; Isaacs, Neil W.; Cogdell, Richard J.



Voltage Distribution Over Emg Helix Wires  

Microsoft Academic Search

The efficiency of EMG operation depends on the magnetic flux losses produced under circuit deformation. Voltage overloads in the circuit generator resulting in electrical breakdowns are among the potential reasons for these losses. A sectionalized EMG with a 100 mm inner diameter of its helix turns is considered as an example. The initial inductance of this generator is 17 muH

V. R. Chernyshev; E. I. Zharinov; V. N. Buzin; B. E. Grinevich; K. N. Klimushkin; I. D. Kudelkin; A. A. Bazanov; O. V. Sokolova; M. M. Gubin; S. V. Pak; A. N. Skobelev



The Triple Helix Perspective of Innovation Systems  

Microsoft Academic Search

Alongside the neo-institutional model of networked relations among universities, industries, and governments, the Triple Helix can be provided with a neo-evolutionary interpretation as three selection environments operating upon one another: markets, organizations, and technological opportunities. How are technological innovation systems different from national ones? The three selection environments fulfill social functions: wealth creation, organization control, and organized knowledge production. The

Loet Leydesdorff; Girma Zawdie



Exploring transmembrane diffusion pathways with molecular dynamics.  


Transmembrane exchange of materials is a fundamental process in biology. Molecular dynamics provides a powerful method to investigate in great detail various aspects of the phenomenon, particularly the permeation of small uncharged molecules, which continues to pose a challenge to experimental studies. We will discuss some of the recent simulation studies investigating the role of lipid-mediated and protein-mediated mechanisms in permeation of water and gas molecules across the membrane. PMID:20551228

Wang, Yi; Shaikh, Saher A; Tajkhorshid, Emad



Exploring Transmembrane Diffusion Pathways With Molecular Dynamics  

NSDL National Science Digital Library

Transmembrane exchange of materials is a fundamental process in biology. Molecular dynamics provides a powerful method to investigate in great detail various aspects of the phenomenon, particularly the permeation of small uncharged molecules, which continues to pose a challenge to experimental studies. We will discuss some of the recent simulation studies investigating the role of lipid-mediated and protein-mediated mechanisms in permeation of water and gas molecules across the membrane.

Yi Wang (University of Illinois at Urbana-Champaign); Saher Shaikh (University of Illinois at Urbana-Champaign); Emad Tajkhorshid (University of Illinois at Urbana-Champaign)



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

PubMed Central

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 ? 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 ?-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in ? 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 ?’ H1242 and highly conserved active site residues ? 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

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



Regulatory module network of basic\\/helix-loop-helix transcription factors in mouse brain  

Microsoft Academic Search

BACKGROUND: The basic\\/helix-loop-helix (bHLH) proteins are important components of the transcriptional regulatory network, controlling a variety of biological processes, especially the development of the central nervous system. Until now, reports describing the regulatory network of the bHLH transcription factor (TF) family have been scarce. In order to understand the regulatory mechanisms of bHLH TFs in mouse brain, we inferred their

Jing Li; Zijing J Liu; Yuchun C Pan; Qi Liu; Xing Fu; Nigel GF Cooper; Yixue X Li; Mengsheng S Qiu; Tieliu L Shi



Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs  

Microsoft Academic Search

DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecu- lar interactions. The helix-loop-helix (HLH)-contain- ing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2\\/Net\\/ ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also con- tains an

Julie Stinson; Toshiaki Inoue; Paula Yates; Anne Clancy; John D. Norton; Andrew D. Sharrocks




E-print Network

MODELS OF THE ACTIN-LIKE MREB HELIX IN PROKARYOTES by Jun Allard SUBMITTED IN PARTIAL FULFILLMENT a thesis entitled "Models of the actin-like MreB helix in prokaryotes" by Jun Allard in partial fulfillment Allard Title: Models of the actin-like MreB helix in prokaryotes Department: Physics and Atmospheric

Allard, Jun


Myocardial activation of the human cardiac alpha-actin promoter by helix-loop-helix proteins.  

PubMed Central

The cardiac alpha-actin gene is expressed in both heart and skeletal muscle. In skeletal myogenic cells, the 177-base-pair promoter of the human cardiac alpha-actin (HCA) gene requires three transcription factors for activation: Sp1, serum response factor (SRF), and MyoD. However, MyoD is undetectable in heart. To search for a functional equivalent of MyoD, we analyzed the transcriptional regulation of the HCA promoter in primary cultures of rat cardiac myocytes. The same DNA sequence elements recognized by SRF, Sp1, and MyoD and required for HCA transcription in skeletal muscle cells were also found to be necessary for expression in cardiomyocytes. Overexpression of Id, a negative regulator of basic helix-loop-helix proteins, selectively attenuated expression of the HCA promoter. Cardiomyocyte nuclei contain a protein complex that specifically interacts with the same required sequence (E box) in the HCA promoter that is bound by MyoD in skeletal myogenic cells. Furthermore, these complexes contain a peptide that is a member of the E2A family of basic helix-loop-helix proteins. Cardiomyocyte nuclei appear to be enriched for a protein that can bind to the E-box site as dimers with the E12 protein. These results suggest that a member of the basic helix-loop-helix family, together with SRF and Sp1, activates the HCA promoter in heart. Alternative strategies for myocardial transcription of HCA are discussed. Images PMID:1570331

Sartorelli, V; Hong, N A; Bishopric, N H; Kedes, L



A novel type of PTD, common helix–loop–helix motif, could efficiently mediate protein transduction into mammalian cells  

Microsoft Academic Search

Protein transduction domains (PTDs), such as HIV TAT PTD, have been widely used as delivery tools into living cells. Here we reported for the first time that the helix–loop–helix (HLH) domain of basic helix–loop–helix (bHLH) family was a novel type of PTD. Efficient internalization has been obtained with HLH domains derived from bHLH proteins, NeuroD\\/BETA2, Neurogenin3, and Mitf, in various

Jing Chen; Ge Li; Jun Lu; Lei Chen; Yin Huang; Huiling Wu; Jiaxin Zhang; Daru Lu



The double helix and immunology  

NASA Astrophysics Data System (ADS)

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.

Nossal, Gustav J. V.



Amide vibrations are delocalized across the hydrophobic interface of a transmembrane  

E-print Network

° from 2D IR spectroscopy, combined with the coupling led to a structural picture of the hydrophobic param- eters for the helix dimers from an analysis of the coupling constant distribution. tertiary spectral maps in the IR, analogous to 2D NMR spectra. The amide-I vibrational bands of polypeptides

Senes, Alessandro


Transmembrane Organization of the Bacillus subtilis Chemoreceptor McpB Deduced by Cysteine  

E-print Network

differences exist in the signaling mechanism between E. coli and Bacillus subtilis chemoreceptors. Although chemo- taxis receptors in both B. subtilis and E. coli are subject to methylation by Che Elsevier Ltd. All rights reserved. Keywords: chemotaxis; chemoreceptor; crosslinking; structure; helix

Ordal, George W.


Dominance of misfolded intermediates in the dynamics of ?-helix folding.  


Helices are the "hydrogen atoms" of biomolecular complexity; the DNA/RNA double hairpin and protein ?-helix ubiquitously form the building blocks of life's constituents at the nanometer scale. Nevertheless, the formation processes of these structures, especially the dynamical pathways and rates, remain challenging to predict and control. Here, we present a general analytical method for constructing dynamical free-energy landscapes of helices. Such landscapes contain information about the thermodynamic stabilities of the possible macromolecular conformations, as well as about the dynamic connectivity, thus enabling the visualization and computation of folding pathways and timescales. We elucidate the methodology using the folding of polyalanine, and demonstrate that its ?-helix folding kinetics is dominated by misfolded intermediates. At the physiological temperature of T = 298 K and midfolding time t = 250 ns, the fraction of structures in the native-state (?-helical) basin equals 22%, which is in good agreement with time-resolved experiments and massively distributed, ensemble-convergent molecular-dynamics simulations. We discuss the prominent role of ?-strand-like intermediates in flight toward the native fold, and in relation to the primary conformational change precipitating aggregation in some neurodegenerative diseases. PMID:25246551

Lin, Milo M; Shorokhov, Dmitry; Zewail, Ahmed H



Porous silicon membrane for investigation of transmembrane proteins  

NASA Astrophysics Data System (ADS)

This article presents the assembly and signal transduction of an artificial biological membrane suspended on a thin porous silicon template. The electrochemically-fabricated porous silicon membrane has average pore diameters in the range 0.50-2 ?m and dimensions of about 200 × 200 × 3 ?m3 and may be batch fabricated in large arrays for combinatorial testing. Biological membranes may be deposited on one or both sides of this template are fully accessible for studies using electrochemical impedance spectroscopy. Initial results using a two probe impedance measurement clearly show a significant impedance change between the porous silicon structure and the lipid bilayer. Furthermore, there is a clear reduction in the impedance of lipid bilayer when fused with a transmembrane ion channel protein. The photoluminescence and biodegradability properties of porous silicon in addition to lower cost and ease of fabrication make it superior over e-beam patterned silicon structures used in previous works, and thus suitable for in vivo monitoring.

Tantawi, Khalid Hasan; Berdiev, Bakhrom; Cerro, Ramon; Williams, John D.



Triple helix formation inhibits DNA gyrase activity.  


The goal of this work was to examine the effect of triple helix-forming oligonucleotides on a gyrase target region and on the activity of the enzyme. Using melting temperature measurements and gel mobility shift analysis, it was found that modified oligonucleotides can form a triple helix along the 29-nucleotide region of a 32-bp duplex representing part of the gyrase DNA-target sequence of the 162-bp fragment from pBR322. Triplex formation with this target region has been achieved at pH 7.5 by using a synthetic oligonucleotide in which cytosine was replaced by the C-nucleoside of 2-aminopyridine. The results of the enzymic experiments in vitro with the 162-bp fragment demonstrated that the cleavage reaction mediated by gyrase can be efficiently inhibited by the triplex-forming oligonucleotide modified with 2-aminopyridine. A possible inhibitory mechanism is discussed. PMID:10645778

Simon, H; Förtsch, I; Burckhardt, G; Gabrielyan, A; Birch-Hirschfeld, E; Stelzner, A; Prévot-Halter, I; Leumann, C; Zimmer, C



DNA helix, still imageSite: DNA Interactive (  

NSDL National Science Digital Library

Image depicting DNA helix model and table. In 1953, Watson and Crick published a paper: A Structure for Deoxyribose Nucleic Acid. Based on available data from X-ray diffraction patterns and 3D conceptualization through model building, Watson and Crick proposed that DNA is a double helix ÃÂÃÂì a twisted ladder ÃÂÃÂì with two phosphate-based backbones and "runged" nucleotides that pair. Their paper ended with this comment: It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.



The Structure of a Soluble Chemoreceptor Suggests a Mechanism for Propagating Conformational Signals  

SciTech Connect

Transmembrane chemoreceptors, also known as methyl-accepting chemotaxis proteins (MCPs), translate extracellular signals into intracellular responses in the bacterial chemotaxis system. MCP ligand binding domains control the activity of the CheA kinase, situated {approx}200 {angstrom} away, across the cytoplasmic membrane. The 2.17 {angstrom} resolution crystal structure of a Thermotoga maritima soluble receptor (Tm14) reveals distortions in its dimeric four-helix bundle that provide insight into the conformational states available to MCPs for propagating signals. A bulge in one helix generates asymmetry between subunits that displaces the kinase-interacting tip, which resides more than 100 {angstrom} away. The maximum bundle distortion maps to the adaptation region of transmembrane MCPs where reversible methylation of acidic residues tunes receptor activity. Minor alterations in coiled-coil packing geometry translate the bulge distortion to a >25 {angstrom} movement of the tip relative to the bundle stalks. The Tm14 structure discloses how alterations in local helical structure, which could be induced by changes in methylation state and/or by conformational signals from membrane proximal regions, can reposition a remote domain that interacts with the CheA kinase.

Pollard, Abiola M.; Bilwes, Alexandrine M.; Crane, Brian R.; Cornell



Novel Antimicrobial Peptides Derived from Human Immunodeficiency Virus Type 1 and Other Lentivirus Transmembrane Proteins  

Microsoft Academic Search

We have previously described a conserved set of peptides derived from lentiviral envelope transmembrane proteins that are similar to the natural antimicrobial peptides cecropins and magainins in overall structure but bear no sequence homology to them or other members of their class. We describe here an evaluation of the antimicrobial properties of these virally derived peptides, designated lentivirus lytic peptides




"Frozen" dynamic dimer model for transmembrane signaling in bacterial chemotaxis receptors.  

PubMed Central

The crystal structures of the ligand binding domain of a bacterial aspartate receptor suggest a simple mechanism for transmembrane signaling by the dimer of the receptor. On ligand binding, one domain rotates with respect to the other, and this rotational motion is proposed to be transmitted through the membrane to the cytoplasmic domains of the receptor. PMID:8003953

Kim, S. H.



The Multitude of Molecular Hydrogen Knots in the Helix Nebula  

NASA Astrophysics Data System (ADS)

We present Hubble Space Telescope NICMOS imaging of the H2 2.12 ?m emission in five fields in the Helix Nebula ranging in radial distance from 250" to 450" from the central star. The images reveal arcuate structures with their apexes pointing toward the central star. These molecular hydrogen knots are most highly structured in the fields closest to the central star and become increasingly less structured with increasing radius. Comparison of these images with ground-based images of comparable resolution reveals that the molecular gas is more highly clumped than the ionized gas line tracers. From our images, we determine an average number density of knots in the molecular gas ranging from 162 knots arcmin-2 in the denser regions to 18 knots arcmin-2 in the lower density outer regions. The decreasing number density of H2 knots in the outer regions creates a lower filling factor of neutral and molecular gas emission in the radio observations of CO and H I and may explain why these outer regions, where we clearly detect H2 2.12 ?m, fall below the detection limit of the radio observations. Using this new number density, we estimate the total number of knots in the Helix to be ~23,000, which is a factor of 6.5 larger than previous estimates. The total neutral gas mass in the Helix is 0.35 Msolar assuming a mass of ~1.5×10-5 Msolar for the individual knots. The H2 emission structure of the entire Helix Nebula supports the recent interpretation of the Helix as a nearly pole-on polypolar planetary nebula (PN). The H2 intensity, (5-9)×10-5 ergs s-1 cm-2 sr-1, remains relatively constant with projected distance from the central star, suggesting a heating mechanism for the molecular gas that is distributed almost uniformly in the knots throughout the nebula. The temperature and H2 2.12 ?m intensity of the knots can be approximately explained by photodissociation regions (PDRs) in the individual knots; however, theoretical PDR models of PNs underpredict the intensities of some knots by a factor of 10. The brightest H2 emission (~3×10-4 ergs s-1 cm-2 sr-1) may be enhanced by a larger than unity area filling factor of H2 knots or may be an individual H2 knot exposed to direct starlight, causing rapid photoevaporation compared with the more embedded knots of the disk. Based in part on observations with the NASA/ESA Hubble Space Telescope obtained at the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS5-26555.

Meixner, Margaret; McCullough, Peter; Hartman, Joel; Son, Minho; Speck, Angela



3-Helix Micelles Stabilized by Polymer Springs  

PubMed Central

Despite increasing demands to employ amphiphilic micelles as nanocarriers and nanoreactors, it remains a significant challenge to simultaneously reduce the particle size and enhance the particle stability. Complementary to covalent chemical bonding and attractive intermolecular interactions, entropic repulsion can be incorporated by rational design in the headgroup of an amphiphile to generate small micelles with enhanced stability. A new family of amphiphilic peptide-polymer conjugates is presented where the hydrophilic headgroup is composed of a 3-helix coiled-coil with poly(ethylene glycol) attached to the exterior of the helix bundle. When micelles form, the PEG chains are confined in close proximity and are compressed to act as a spring to general lateral pressure. The formation of 3-helix bundles determines the location and the directionalities of the force vector of each PEG elastic spring so as to slow down amphiphile desorption. Since each component of the amphiphile can be readily tailored, these micelles provide numerous opportunities to meet current demands for organic nanocarriers with tunable stability in life science and energy science. Furthermore, present studies open new avenues to use energy arising from entropic polymer chain deformation to self-assemble energetically stable single nanoscopic objects, much like repulsion that stabilizes bulk assemblies of colloidal particles. PMID:22731391

Dong, He; Shu, Jessica Y.; Dube, Nikhil; Ma, Yufei; Tirrell, Matthew V.; Downing, Kenneth H.; Xu, Ting



The Auto-Inhibitory Role of the EPAC Hinge Helix as Mapped by NMR  

PubMed Central

The cyclic-AMP binding domain (CBD) is the central regulatory unit of exchange proteins activated by cAMP (EPAC). The CBD maintains EPAC in a state of auto-inhibition in the absence of the allosteric effector, cAMP. When cAMP binds to the CBD such auto-inhibition is released, leading to EPAC activation. It has been shown that a key feature of such cAMP-dependent activation process is the partial destabilization of a structurally conserved hinge helix at the C-terminus of the CBD. However, the role of this helix in auto-inhibition is currently not fully understood. Here we utilize a series of progressive deletion mutants that mimic the hinge helix destabilization caused by cAMP to show that such helix is also a pivotal auto-inhibitory element of apo-EPAC. The effect of the deletion mutations on the auto-inhibitory apo/inactive vs. apo/active equilibrium was evaluated using recently developed NMR chemical shift projection and covariance analysis methods. Our results show that, even in the absence of cAMP, the C-terminal region of the hinge helix is tightly coupled to other conserved allosteric structural elements of the CBD and perturbations that destabilize the hinge helix shift the auto-inhibitory equilibrium toward the apo/active conformations. These findings explain the apparently counterintuitive observation that cAMP binds more tightly to shorter than longer EPAC constructs. These results are relevant for CBDs in general and rationalize why substrates sensitize CBD-containing systems to cAMP. Furthermore, the NMR analyses presented here are expected to be generally useful to quantitatively evaluate how mutations affect conformational equilibria. PMID:23185272

Selvaratnam, Rajeevan; Mazhab-Jafari, Mohammad T.; Das, Rahul; Melacini, Giuseppe



Calculations of the Mueller scattering matrix for a DNA plectonemic helix  

SciTech Connect

The polarization state of a beam of light is fully described by the four elements of the Stokes vector. How the Stokes vector is transformed upon scattering is described by the sixteen element Mueller scattering matrix. Polarized light scattering provides a useful tool to study macromolecular structure. In particular, measurements of Mueller matrix elements have been used to study chromosome structure; changes in these measured quantities can be used to monitor structure as a function of cell cycle. Such measurements done on helical structures can be compared to theoretical computations to determine the geometrical parameters describing the helix. The matrix elements that are most sensitive to the chiral parameters of the helix are largest when the wavelength of light is of the order of the pitch of the helix. Therefore, polarized light scattering measurements made on DNA plectonemic helices would provide the most information in the far ultraviolet (UV) and x-ray region. The Mueller matrix elements are calculated using the coupled dipole approximation in the orientation average at wavelengths in the visible, ultraviolet, and x-ray regions. Each base-pair of the idealized plectonemic helix is represented by a single dipole. A complex polarizability tensor is assigned to each dipole. Calculations are sensitive to the writhe and polarizability of the DNA molecule.

Shapiro, D.B. (Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720 (United States) Graduate Group in Biophysics, University of California, Berkeley, California 94720 (United States)); Hull, P.G. (Department of Physics, Tennessee State University, Nashville, Tennessee 37209-1561 (United States)); Hunt, A.J. (Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720 (United States)); Hearst, J.E. (Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720 (United States) Graduate Group in Biophysics, University of California, Berkeley, California 94720 (United States))



Effect of helix inversion of poly(?-phenethyl l-aspartate) on macroscopic piezoelectricity  

NASA Astrophysics Data System (ADS)

It has been reported that poly(?-phenethyl l-aspartate) (PPLA) exhibits irreversible inversion from right-handed to left-handed helixes in a solid state at 130-140 °C from the results of circular dichroism measurement and X-ray diffraction. On the other hand, it is well known that semicrystalline chiral polymers show shear piezoelectricity due to their asymmetric crystal structure. The results of macroscopic shear piezoelectric measurement include information on macroscopic and microscopic properties due to the helix structure of the chain molecules. However, in general, no direct correspondence has been reported between the macroscopic piezoelectric properties and crystal characteristics because of the existence of a unique complex high-order structure in polymer films. We measured the temperature dependence of the piezoelectricity of a PPLA film in order to obtain more information concerning the helix inversion process. As a result, the sign inversion of the piezoelectric constant was observed at the temperature at which the helix inversion took place. It was also shown that the macroscopic orientation of the crystalline structure was maintained in the PPLA film after the sign inversion, even though molecular motion such as the segmental motion and side-chain motion of PPLA molecules was activated.

Tanimoto, Kazuhiro; Nishizaki, Hisayoshi; Tada, Teruyuki; Shiomi, Yuki; Ito, Nariaki; Shibata, Kouki; Furuya, Hidemine; Abe, Akihiro; Imoto, Kenji; Date, Munehiro; Fukada, Eiichi; Tajitsu, Yoshiro



An inverse correlation between loop length and stability in a four-helix-bundle protein  

Microsoft Academic Search

Background: The loops in proteins are less well characterized than the secondary structural elements that they connect. We have used the four-helix- bundle protein Rop as a model system in which to explore the role of loop length in protein folding and stability. Results: A natural two-residue loop was replaced with a series of glycine linkers up to 10 residues

Athena D. Nagi; Lynne Regan



Arginines in the First Transmembrane Segment Promote Maturation of a P-glycoprotein Processing Mutant by Hydrogen Bond Interactions with Tyrosines in Transmembrane Segment 11*  

PubMed Central

A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions. PMID:18596043

Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.



Interferon-induced transmembrane protein 3 is a type II transmembrane protein.  


The interferon-induced transmembrane (IFITM) proteins are a family of small membrane proteins that inhibit the cellular entry of several genera of viruses. These proteins had been predicted to adopt a two-pass, type III transmembrane topology with an intracellular loop, two transmembrane helices (TM1 and TM2), and extracellular N and C termini. Recent work, however, supports an intramembrane topology for the helices with cytosolic orientation of both termini. Here we determined the topology of murine Ifitm3. We found that the N terminus of Ifitm3 could be stained by antibodies at the cell surface but that this conformation was cell type-dependent and represented a minority of the total plasma membrane pool. In contrast, the C terminus was readily accessible to antibodies at the cell surface and extracellular C termini comprised most or all of those present at the plasma membrane. The addition of a C-terminal KDEL endoplasmic reticulum retention motif to Ifitm3 resulted in sequestration of Ifitm3 in the ER, demonstrating an ER-luminal orientation of the C terminus. C-terminal, but not N-terminal, epitope tags were also degraded within lysosomes, consistent with their luminal orientation. Furthermore, epitope-tagged Ifitm3 TM2 functioned as a signal anchor sequence when expressed in isolation. Collectively, our results demonstrate a type II transmembrane topology for Ifitm3 and will provide insight into its interaction with potential targets and cofactors. PMID:24067232

Bailey, Charles C; Kondur, Hema R; Huang, I-Chueh; Farzan, Michael



Membrane shape modulates transmembrane protein distribution.  


Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins. PMID:24480645

Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E S; Bassereau, Patricia



Metal concentrations in Helix pomatia, Helix aspersa and Arion rufus: a comparative study.  


In this study we evaluated the differences between concentrations of copper, iron, zinc, manganese, lead and cadmium in three terrestrial gastropods: Helix pomatia, Helix aspersa and Arion rufus, collected in a semi-rural location in Northern Italy. Metal concentrations in the foot and in the digestive gland were measured. In the hepatopancreas, copper and zinc did not differ significantly in the three species; the levels of copper were also similar in the foot. In comparison to Helix sps., A. rufus demonstrated lower concentrations of manganese and cadmium in the hepatopancreas and higher concentrations in the foot. In the slug the mucus produced in the foot could represent an efficient elimination mechanism of some elements. We also investigated correlations between the trace element content in the soft tissues and the mollusk size (weight of the body and height of the shell). PMID:11706793

Menta, C; Parisi, V



Involvement of disulfide bonds and histidine 172 in a unique beta-sheet to alpha-helix transition of alpha 1-acid glycoprotein at the biomembrane interface.  


Human alpha(1)-acid glycoprotein (AGP), which is comprised of 183 amino acid residues and 5 carbohydrate chains, is a major plasma protein that binds to basic and neutral drugs as well as to steroid hormones. It has a beta-sheet-rich structure in aqueous solution. Our previous findings suggest that AGP forms an alpha-helix structure through an interaction with biomembranes. We report herein on a study of the mechanism of alpha-helix formation in AGP using various modified AGPs. The disulfide reduced AGP (R-AGP) was extensively unfolded, whereas asialylated AGP (A-AGP) maintained the native structure. Intriguingly, reduced and asialylated AGP (RA-AGP) increased the alpha-helix content as observed in the presence of biomembrane models, and showed a significant decrease in ligand binding capacity. This suggests that AGP has an innate tendency to form an alpha-helix structure, and disulfide bonds are a key factor in the conformational transition between the beta-sheet and alpha-helix structures. However, RA-AGP with all histidine residues chemically modified (HRA-AGP) was found to lose the intrinsic ability to form an alpha-helix structure. Furthermore, disulfide reduction of the H172A mutant expressed in Pichia pastoris also caused a similar loss of folding ability. The present results indicate that disulfide bonds and the C-terminal region, including H172 of AGP, play important roles in alpha-helix formation in the interaction of the protein with biomembranes. PMID:16470806

Nishi, Koji; Komine, Yoshio; Fukunaga, Naoko; Maruyama, Toru; Suenaga, Ayaka; Otagiri, Masaki



Anchors aweigh: protein localization and transport mediated by transmembrane domains.  


The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in cytosolic domains, TMD sorting determinants are not conserved amino acid sequences but physical properties such as the length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized, but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport. PMID:23806646

Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S



Anchors Aweigh: Protein Traffic Mediated by Transmembrane Domains  

PubMed Central

The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in the cytosolic domains, TMD sorting determinants are not conserved amino-acid sequences but physical properties such as length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport. PMID:23806646

Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S.



The helix-turn-helix motif of bacterial insertion sequence IS911 transposase is required for DNA binding  

PubMed Central

The transposase of IS911, a member of the IS3 family of bacterial insertion sequences, is composed of a catalytic domain located at its C-terminal end and a DNA binding domain located at its N-terminal end. Analysis of the transposases of over 60 members of the IS3 family revealed the presence of a helix–turn–helix (HTH) motif within the N-terminal region. Alignment of these potential secondary structures further revealed a completely conserved tryptophan residue similar to that found in the HTH motifs of certain homeodomain proteins. The analysis also uncovered a similarity between the IS3 family HTH and that of members of the LysR family of bacterial transcription factors. This information was used to design site-directed mutations permitting an assessment of its role in transposase function. A series of in vivo and in vitro tests demonstrated that the HTH domain is important in directing the transposase to bind the terminal inverted repeats of IS911. PMID:14981152

Rousseau, Philippe; Gueguen, Erwan; Duval-Valentin, Guy; Chandler, Mick



The basic helix-loop-helix leucine zipper transcription factor Mitf is conserved in Drosophila and functions in eye development.  

PubMed Central

The MITF protein is a member of the MYC family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors and is most closely related to the TFE3, TFEC, and TFEB proteins. In the mouse, MITF is required for the development of several different cell types, including the retinal pigment epithelial (RPE) cells of the eye. In Mitf mutant mice, the presumptive RPE cells hyperproliferate, abnormally express the retinal transcriptional regulator Pax6, and form an ectopic neural retina. Here we report the structure of the Mitf gene in Drosophila and demonstrate expression during embryonic development and in the eye-antennal imaginal disc. In vitro, transcriptional regulation by Drosophila Mitf, like its mouse counterpart, is modified by the Eyeless (Drosophila Pax6) transcription factor. In vivo, targeted expression of wild-type or dominant-negative Drosophila Mitf results in developmental abnormalities reminiscent of Mitf function in mouse eye development. Our results suggest that the Mitf gene is the original member of the Mitf-Tfe subfamily of bHLH-Zip proteins and that its developmental function is at least partially conserved between vertebrates and invertebrates. These findings further support the common origin of the vertebrate and invertebrate eyes. PMID:15166150

Hallsson, Jon H; Haflidadottir, Benedikta S; Stivers, Chad; Odenwald, Ward; Arnheiter, Heinz; Pignoni, Francesca; Steingrimsson, Eirikur



Structure and selectivity in bestrophin ion channels.  


Human bestrophin-1 (hBest1) is a calcium-activated chloride channel from the retinal pigment epithelium, where mutations are associated with vitelliform macular degeneration, or Best disease. We describe the structure of a bacterial homolog (KpBest) of hBest1 and functional characterizations of both channels. KpBest is a pentamer that forms a five-helix transmembrane pore, closed by three rings of conserved hydrophobic residues, and has a cytoplasmic cavern with a restricted exit. From electrophysiological analysis of structure-inspired mutations in KpBest and hBest1, we find a sensitive control of ion selectivity in the bestrophins, including reversal of anion/cation selectivity, and dramatic activation by mutations at the cytoplasmic exit. A homology model of hBest1 shows the locations of disease-causing mutations and suggests possible roles in regulation. PMID:25324390

Yang, Tingting; Liu, Qun; Kloss, Brian; Bruni, Renato; Kalathur, Ravi C; Guo, Youzhong; Kloppmann, Edda; Rost, Burkhard; Colecraft, Henry M; Hendrickson, Wayne A



Surfactant protein C peptides with salt-bridges ("ion-locks") promote high surfactant activities by mimicking the ?-helix and membrane topography of the native protein  

PubMed Central

Background. Surfactant protein C (SP-C; 35 residues) in lungs has a cationic N-terminal domain with two cysteines covalently linked to palmitoyls and a C-terminal region enriched in Val, Leu and Ile. Native SP-C shows high surface activity, due to SP-C inserting in the bilayer with its cationic N-terminus binding to the polar headgroup and its hydrophobic C-terminus embedded as a tilted, transmembrane ?-helix. The palmitoylcysteines in SP-C act as ‘helical adjuvants’ to maintain activity by overriding the ?-sheet propensities of the native sequences. Objective. We studied SP-C peptides lacking palmitoyls, but containing glutamate and lysine at 4-residue intervals, to assess whether SP-C peptides with salt-bridges (“ion-locks”) promote surface activity by mimicking the ?-helix and membrane topography of native SP-C. Methods. SP-C mimics were synthesized that reproduce native sequences, but without palmitoyls (i.e., SP-Css or SP-Cff, with serines or phenylalanines replacing the two cysteines). Ion-lock SP-C molecules were prepared by incorporating single or double Glu?–Lys+ into the parent SP-C’s. The secondary structures of SP-C mimics were studied with Fourier transform infrared (FTIR) spectroscopy and PASTA, an algorithm that predicts ?-sheet propensities based on the energies of the various ?-sheet pairings. The membrane topography of SP-C mimics was investigated with orientated and hydrogen/deuterium (H/D) exchange FTIR, and also Membrane Protein Explorer (MPEx) hydropathy analysis. In vitro surface activity was determined using adsorption surface pressure isotherms and captive bubble surfactometry, and in vivo surface activity from lung function measures in a rabbit model of surfactant deficiency. Results. PASTA calculations predicted that the SP-Css and SP-Cff peptides should each form parallel ?-sheet aggregates, with FTIR spectroscopy confirming high parallel ?-sheet with ‘amyloid-like’ properties. The enhanced ?-sheet properties for SP-Css and SP-Cff are likely responsible for their low surfactant activities in the in vitro and in vivo assays. Although standard 12C-FTIR study showed that the ?-helicity of these SP-C sequences in lipids was uniformly increased with Glu?–Lys+ insertions, elevated surfactant activity was only selectively observed. Additional results from oriented and H/D exchange FTIR experiments indicated that the high surfactant activities depend on the SP-C ion-locks recapitulating both the ?-helicity and the membrane topography of native SP-C. SP-Css ion-lock 1, an SP-Css with a salt-bridge for a Glu?–Lys+ ion-pair predicted from MPEx hydropathy calculations, demonstrated enhanced surfactant activity and a transmembrane helix simulating those of native SP-C. Conclusion. Highly active SP-C mimics were developed that replace the palmitoyls of SP-C with intrapeptide salt-bridges and represent a new class of synthetic surfactants with therapeutic interest. PMID:25083348

Waring, Alan J.; Hernandez-Juviel, Jose M.; Ruchala, Piotr; Wang, Zhengdong; Notter, Robert H.; Gordon, Larry M.



Crystal structure of aura virus capsid protease and its complex with dioxane: new insights into capsid-glycoprotein molecular contacts.  


The nucleocapsid core interaction with endodomains of glycoproteins plays a critical role in the alphavirus life cycle that is essential to virus budding. Recent cryo-electron microscopy (cryo-EM) studies provide structural insights into key interactions between capsid protein (CP) and trans-membrane glycoproteins E1 and E2. CP possesses a chymotrypsin-like fold with a hydrophobic pocket at the surface responsible for interaction with glycoproteins. In the present study, crystal structures of the protease domain of CP from Aura virus and its complex with dioxane were determined at 1.81 and 1.98 Å resolution respectively. Due to the absence of crystal structures, homology models of E1 and E2 from Aura virus were generated. The crystal structure of CP and structural models of E1 and E2 were fitted into the cryo-EM density map of Venezuelan equine encephalitis virus (VEEV) for detailed analysis of CP-glycoprotein interactions. Structural analysis revealed that the E2 endodomain consists of a helix-loop-helix motif where the loop region fits into the hydrophobic pocket of CP. Our studies suggest that Cys397, Cys418 and Tyr401 residues of E2 are involved in stabilizing the structure of E2 endodomain. Density map fitting analysis revealed that Pro405, a conserved E2 residue is present in the loop region of the E2 endodomain helix-loop-helix structure and makes intermolecular hydrophobic contacts with the capsid. In the Aura virus capsid protease (AVCP)-dioxane complex structure, dioxane occupies the hydrophobic pocket on CP and structurally mimics the hydrophobic pyrollidine ring of Pro405 in the loop region of E2. PMID:23251484

Aggarwal, Megha; Tapas, Satya; Preeti; Siwach, Anjul; Kumar, Pravindra; Kuhn, Richard J; Tomar, Shailly



Serine Cluster Phosphorylation Liberates the C-Terminal Helix of IFN Regulatory Factor 7 To Bind Histone Acetyltransferase p300.  


IFN regulatory factor 7 (IRF7) is a major regulator of type I (??) IFN secretion. A growing body of evidence shows that IRF7 is involved in a wide variety of pathologic conditions in addition to infections; however, the detailed mechanism of IRF7 transactivation remains elusive. Our current knowledge of IRF7 transactivation is based on studies of IRF3, another major regulator of IFN-? secretion. IRF3 and IRF7 are closely related homologs with high sequence similarity in their C-terminal regions, and both proteins are activated by phosphorylation of a specific serine cluster (SC). Nevertheless, the functional domains of the two proteins are arranged in an inverted manner. We generated a model structure of the IRF7 C-terminal region using homology modeling and used it to guide subsequent functional domain studies. The model structure led to the identification of a tripod-helix structure containing the SC. Based on the model and experimental data, we hypothesized that phosphorylation-mediated IRF7 transactivation is controlled by a tripod-helix structure. Inducible I?B kinase binds a tripod-helix structure. Serial phosphorylation of the SC by the kinase liberates C-terminal helix from an inhibitory hydrophobic pocket. A histone acetyltransferase P300 binds the liberated helix. The difference in the P300 binding sites explains why the domain arrangement of IRF7 is inverted relative to that of IRF3. PMID:25225665

Lee, Kyoung Jin; Ye, Jung Sook; Choe, Han; Nam, Young Ran; Kim, Nari; Lee, Uk; Joo, Chul Hyun



Evaluation of Diverse ?/? Backbone Patterns for Functional ?-Helix Mimicry: Analogues of the Bim BH3 Domain  

PubMed Central

Peptidic oligomers that contain both ?- and ?-amino acid residues, in regular patterns throughout the backbone, are emerging as structural mimics of ?-helix-forming conventional peptides (composed exclusively of ?-amino acid residues). Here we describe a comprehensive evaluation of diverse ?/?-peptide homologues of the Bim BH3 domain in terms of their ability to bind to the BH3-recognition sites on two partner proteins, Bcl-xL and Mcl-1. These proteins are members of the anti-apoptotic Bcl-2 family, and both bind tightly to the Bim BH3 domain itself. All ?/?-peptide homologues retain the side chain sequence of the Bim BH3 domain, but each homologue contains periodic ?-residue ? ?3-residue substitutions. Previous work has shown that the ??????? pattern, which aligns the ?3-residues in a 'stripe' along one side of the helix, can support functional ?-helix mimicry, and the results reported here support this conclusion. The present study provides the first evaluation of functional mimicry by ??? and ???? patterns, which cause the ?3-residues to spiral around the helix periphery. We find that the ???? pattern can support effective mimicry of the Bim BH3 domain, as manifested by the crystal structure of an ?/?-peptide bound to Bcl-xL, affinity for a variety of Bcl-2 family proteins, and induction of apoptotic signaling in mouse embryonic fibroblast extracts. The best ???? homologue shows substantial protection from proteolytic degradation relative to the Bim BH3 ?-peptide. PMID:22040025

Boersma, Melissa D.; Haase, Holly S.; Peterson-Kaufman, Kimberly J.; Lee, Erinna F.; Clarke, Oliver B.; Colman, Peter M.; Smith, Brian J.; Horne, W. Seth; Fairlie, W. Douglas; Gellman, Samuel H.



Phased psoralen cross-links do not bend the DNA double helix  

SciTech Connect

Although the chemical reaction of psoralens with nucleic acids is well understood, the structure of psoralen-DNA cross-linked products is still not clear. Model building studies based on the crystal structure of the psoralen-thymine monoadduct suggest that each cross-link bends the DNA double helix by 46.5/sup 0/. Here the authors use gel electrophoresis to test the validity of the current models. They have synthesized a series of DNA fragments (21-24 base pairs in length), each containing one unique T-A site for 4'-(hydroxymethyl)-4,5'8-trimethylpsoralen (HMT) cross-linking. Because of an estimated 28/sup 0/ unwinding of the helix by HMT, one expects that the 22-bp cross-linked fragment will be repeated nearly in phase with the average helical screw when multimerized. In that sequence ligation will maximally amplify any deformation to the double helix. They find that the ligated multimers of cross-linked DNA migrate close to the multimers of non-cross-linked DNA on polyacrylamide gels. These observations place an upper limit of 10/sup 0/ on DNA bending induced by psoralen cross-linking and indicate unwinding by about 1 bp, as well as stiffening of the double helix. These properties are not unexpected for classical intercalators.

Haran, T.E.; Crothers, D.M.



Role of potentially charged transmembrane residues in targeting proteins for retention and degradation within the endoplasmic reticulum.  

PubMed Central

The selective breakdown of newly synthesized proteins retained within the endoplasmic reticulum (ER) is probably mediated by the specific recognition of structural features of protein substrates by components of a degradative system. Within the alpha chain of the multisubunit T-cell antigen receptor (TCR) complex, a transmembrane sequence containing two basic amino acid residues has been shown to act as a determinant for retention and rapid degradation in the ER. We now demonstrate that single basic or acidic amino acid residues can cause targeting for retention and degradation in the ER when placed within the transmembrane domain of an integral membrane protein normally destined for the cell surface. The effect of such potentially charged residues is dependent on their relative position within the transmembrane sequence and on the nature of the amino acid side chains. The phenotypic changes induced by potentially charged transmembrane residues occur without apparent alterations of the global folding or transmembrane topology of the mutant proteins. These observations test the hypothesis that potentially charged residues within transmembrane domains can provide the basis for a motif for ER degradation and explain the selective breakdown of some proteins retained within the ER. Images PMID:1915263

Bonifacino, J S; Cosson, P; Shah, N; Klausner, R D



Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix.  


The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin was solved. Recombinant hemoglobin was expressed in Escherichia coli and was purified by ion-exchange chromatography. Recombinant hemoglobin was crystallized by the hanging-drop vapor-diffusion method using polyethylene glycol as a precipitant. The obtained orthorhombic crystal contained two subunits in the asymmetric unit. The refined structure was interpreted as the aquo-met form. Structural comparisons were performed among hemoglobins from deer mouse, house mouse and human. In contrast to human hemoglobin, deer mouse hemoglobin lacks the hydrogen bond between ?1Trp14 in the A helix and ?1Thr67 in the E helix owing to the Thr67Ala substitution. In addition, deer mouse hemoglobin has a unique hydrogen bond at the ?1?1 interface between residues ?1Cys34 and ?1Ser128. PMID:23545644

Inoguchi, Noriko; Oshlo, Jake R; Natarajan, Chandrasekhar; Weber, Roy E; Fago, Angela; Storz, Jay F; Moriyama, Hideaki



Orbital dynamics in the Tevatron double helix  

SciTech Connect

A key feature of the Tevatron upgrade is the placement of proton and anti-proton bunches on the branches of a double helix which winds around the current closed orbit. Electrostatic separators will transfer the bunches on and off the double helix so that they experience head-on collisions only at the experimental areas, BO and DO, all other encounters occurring at large transverse separation. In this way the number of bunches, and the luminosity, can be increased without a proportional growth in the beam-beam tune shift. The scenario raises a number of beam dynamics issues, especially the consequences of sampling magnetic fields far from the magnets' center lines, and the effects of the long-range beam-beam interaction. This report presents the results of (admittedly incomplete) calculations and simulations done to date to explore: a Fermilab team have been studying, both experimentally and theoretically, but we shall not review those efforts here. The constraint of a page limit has forced us to bound this discussion rather stringently, but a more complete paper will be available as a Fermilab Technical Memo. 7 refs., 7 figs.

Michelotti, L.; Saritepe, S.



Wide-band semivane and heavily dielectric loaded helix traveling-wave tubes  

Microsoft Academic Search

Theoretical simulation of the dispersion and interaction impedance characteristics of a semivane loaded negative dispersion helical slow-wave structure, which is a slight variant of a conventional vane-loaded helix of a wide-band traveling-wave tube (TWT), was validated for the nonresonant perturbation measurement. While the experimental and theoretical results agreed well with respect to interaction impedance for both the semivane structure and

Sun-Shin Jung; Chan-Wook Baik; Seong-Tae Han; Seok-Gy Jeon; Hyun-Jun Ha; A. V. Soukhov; Baofu Jia; Gun-Sik Park; H. S. Kim; H. S. Uhm; B. N. Basu



Transmembrane and Coiled-Coil Domain Family 1 Is a Novel Protein of the Endoplasmic Reticulum  

PubMed Central

The endoplasmic reticulum (ER) is a continuous membrane network in eukaryotic cells comprising the nuclear envelope, the rough ER, and the smooth ER. The ER has multiple critical functions and a characteristic structure. In this study, we identified a new protein of the ER, TMCC1 (transmembrane and coiled-coil domain family 1). The TMCC family consists of at least 3 putative proteins (TMCC1–3) that are conserved from nematode to human. We show that TMCC1 is an ER protein that is expressed in diverse human cell lines. TMCC1 contains 2 adjacent transmembrane domains near the C-terminus, in addition to coiled-coil domains. TMCC1 was targeted to the rough ER through the transmembrane domains, whereas the N-terminal region and C-terminal tail of TMCC1 were found to reside in the cytoplasm. Moreover, the cytosolic region of TMCC1 formed homo- or hetero-dimers or oligomers with other TMCC proteins and interacted with ribosomal proteins. Notably, overexpression of TMCC1 or its transmembrane domains caused defects in ER morphology. Our results suggest roles of TMCC1 in ER organization. PMID:24454821

Wang, Zhe; Chiang, Yan Ting; Ng, Gary K. H.; Shaw, Pang-Chui; Wang, Yuzhuo; Qi, Robert Z.



Lipid functions in cytochrome bc complexes: an odd evolutionary transition in a membrane protein structure  

PubMed Central

Lipid-binding sites and properties were compared in the hetero-oligomeric cytochrome (cyt) b6f and the yeast bc1 complexes that function, respectively, in photosynthetic and respiratory electron transport. Seven lipid-binding sites in the monomeric unit of the dimeric cyanobacterial b6f complex overlap four sites in the Chlamydomonas reinhardtii algal b6f complex and four in the yeast bc1 complex. The proposed lipid functions include: (i) interfacial–interhelix mediation between (a) the two 8-subunit monomers of the dimeric complex, (b) between the core domain (cyt b, subunit IV) and the six trans membrane helices of the peripheral domain (cyt f, iron–sulphur protein (ISP), and four small subunits in the boundary ‘picket fence’); (ii) stabilization of the ISP domain-swapped trans-membrane helix; (iii) neutralization of basic residues in the single helix of cyt f and of the ISP; (iv) a ‘latch’ to photosystem I provided by the ?-carotene chain protruding through the ‘picket fence’; (v) presence of a lipid and chlorophyll a chlorin ring in b6f in place of the eighth helix in the bc1 cyt b polypeptide. The question is posed of the function of the lipid substitution in relation to the evolutionary change between the eight and seven helix structures of the cyt b polypeptide. On the basis of the known n-side activation of light harvesting complex II (LHCII) kinase by the p-side level of plastoquinol, one possibility is that the change was directed by the selective advantage of p- to n-side trans membrane signalling functions in b6f, with the lipid either mediating this function or substituting for the trans membrane helix of a signalling protein lost in crystallization. PMID:23148267

Hasan, S. Saif; Cramer, William A.



Structure of a beta1-adrenergic G-protein-coupled receptor.  


G-protein-coupled receptors have a major role in transmembrane signalling in most eukaryotes and many are important drug targets. Here we report the 2.7 A resolution crystal structure of a beta(1)-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey (Meleagris gallopavo) receptor was selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane alpha-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilized by two disulphide bonds and a sodium ion. Binding of cyanopindolol to the beta(1)-adrenergic receptor and binding of carazolol to the beta(2)-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the beta(2)-adrenergic receptor, directly interacts by means of a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation. PMID:18594507

Warne, Tony; Serrano-Vega, Maria J; Baker, Jillian G; Moukhametzianov, Rouslan; Edwards, Patricia C; Henderson, Richard; Leslie, Andrew G W; Tate, Christopher G; Schertler, Gebhard F X



Empirical parameterization of a model for predicting peptide helix/coil equilibrium populations.  

PubMed Central

A modification of the Lifson-Roig formulation of helix/coil transitions is presented; it (1) incorporates end-capping and coulombic (salt bridges, hydrogen bonding, and side-chain interactions with charged termini and the helix dipole) effects, (2) helix-stabilizing hydrophobic clustering, (3) allows for different inherent termination probabilities of individual residues, and (4) differentiates helix elongation in the first versus subsequent turns of a helix. Each residue is characterized by six parameters governing helix formation. The formulation of the conditional probability of helix initiation and termination that we developed is essentially the same as one presented previously (Shalongo W, Stellwagen, E. 1995. Protein Sci 4:1161-1166) and nearly the mathematical equivalent of the new capping formulation incorporated in the model presented by Rohl et al. (1996. Protein Sci 5:2623-2637). Side-chain/side-chain interactions are, in most cases, incorporated as context dependent modifications of propagation rather than nucleation parameters. An alternative procedure for converting [theta]221 values to experimental fractional helicities () is presented. Tests of the program predictions suggest this method may have some advantages both for designed peptides and for the analysis of secondary structure preferences that could drive the formation of molten-globule intermediates on protein folding pathways. The model predicts the fractional helicity of 385 peptides with a root-mean-square deviation (RMSD) of 0.050 and locates (with precise definition of the termini in many cases) helices in proteins as well as competing methods. The propagation and nucleation parameters were derived from NMR data and from the CD data for a 79 peptide "learning set" for which an excellent fit resulted (RMSD = 0.0295). The current set of parameter corrections for capping boxes, helix dipole interactions, and side-chain/side-chain interactions (coulombic, hydrogen bonding and hydrophobic clustering), although still under development provide a significant improvement in both helix/coil equilibrium prediction for peptides and helix location in protein sequences. This is clearly evident in the rms deviations between CD measures and calculated values of fractional helicity for different classes of peptides before and after applying the corrections: for peptides lacking capping boxes and i/i + 3 and i/i + 4 side-chain/side-chain interactions RMSD = 0.044 (n = 164) versus RMSD = 0.054 (0.172 without the corrections, n = 221) for peptides that required context-dependent corrections of the parameters. If we restrict the analysis to N-acylated peptides with helix stabilizing side-chain/side-chain interactions (including N-capping boxes), the degree to which our corrections account for the stabilizing interaction can be judged from the change in helicity underestimation, (calc-CD): -0.15 +/- 0.10, which is reduced to -0.018 +/- 0.048 (n = 191) upon applying the corrections. PMID:9300492

Andersen, N. H.; Tong, H.



Large Deformation of Helix F during the Photoreaction Cycle of Pharaonis Halorhodopsin in Complex with Azide  

PubMed Central

Halorhodopsin from Natronomonas pharaonis (pHR), a retinylidene protein that functions as a light-driven chloride ion pump, is converted into a proton pump in the presence of azide ion. To clarify this conversion, we investigated light-induced structural changes in pHR using a C2 crystal that was prepared in the presence of Cl? and subsequently soaked in a solution containing azide ion. When the pHR-azide complex was illuminated at pH 9, a profound outward movement (?4 Å) of the cytoplasmic half of helix F was observed in a subunit with the EF loop facing an open space. This movement created a long water channel between the retinal Schiff base and the cytoplasmic surface, along which a proton could be transported. Meanwhile, the middle moiety of helix C moved inward, leading to shrinkage of the primary anion-binding site (site I), and the azide molecule in site I was expelled out to the extracellular medium. The results suggest that the cytoplasmic half of helix F and the middle moiety of helix C act as different types of valves for active proton transport. PMID:23442859

Nakanishi, Taichi; Kanada, Soun; Murakami, Midori; Ihara, Kunio; Kouyama, Tsutomu



A peptide study of the relationship between the collagen triple-helix and amyloid  

PubMed Central

Type XXV collagen, or Collagen-Like Amyloidogenic Component (CLAC), is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer’s disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro-Hyp-Gly)10; an amyloidogenic peptide GNNQQNY; and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)n domains. CD and NMR spectroscopy showed the GNNQQNY peptide formed a random coil structure, while the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple-helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, while amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple-helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple-helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly-Xaa-Yaa sequence and required the triple-helix conformation. The inhibitory effect of the collagen triple-helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation. PMID:22806499

Parmar, Avanish S.; Nunes, Ana Monica; Baum, Jean; Brodsky, Barbara



Comets Kick up Dust in Helix Nebula  

NASA Technical Reports Server (NTRS)

This infrared image from NASA's Spitzer Space Telescope shows the Helix nebula, a cosmic starlet often photographed by amateur astronomers for its vivid colors and eerie resemblance to a giant eye.

The nebula, located about 700 light-years away in the constellation Aquarius, belongs to a class of objects called planetary nebulae. Discovered in the 18th century, these colorful beauties were named for their resemblance to gas-giant planets like Jupiter.

Planetary nebulae are the remains of stars that once looked a lot like our sun. When sun-like stars die, they puff out their outer gaseous layers. These layers are heated by the hot core of the dead star, called a white dwarf, and shine with infrared and visible colors. Our own sun will blossom into a planetary nebula when it dies in about five billion years.

In Spitzer's infrared view of the Helix nebula, the eye looks more like that of a green monster's. Infrared light from the outer gaseous layers is represented in blues and greens. The white dwarf is visible as a tiny white dot in the center of the picture. The red color in the middle of the eye denotes the final layers of gas blown out when the star died.

The brighter red circle in the very center is the glow of a dusty disk circling the white dwarf (the disk itself is too small to be resolved). This dust, discovered by Spitzer's infrared heat-seeking vision, was most likely kicked up by comets that survived the death of their star. Before the star died, its comets and possibly planets would have orbited the star in an orderly fashion. But when the star blew off its outer layers, the icy bodies and outer planets would have been tossed about and into each other, resulting in an ongoing cosmic dust storm. Any inner planets in the system would have burned up or been swallowed as their dying star expanded.

So far, the Helix nebula is one of only a few dead-star systems in which evidence for comet survivors has been found.

This image is made up of data from Spitzer's infrared array camera and multiband imaging photometer. Blue shows infrared light of 3.6 to 4.5 microns; green shows infrared light of 5.8 to 8 microns; and red shows infrared light of 24 microns.



A new family of ?-helix proteins with similarities to the polysaccharide lyases.  


Microorganisms that degrade biomass produce diverse assortments of carbohydrate-active enzymes and binding modules. Despite tremendous advances in the genomic sequencing of these organisms, many genes do not have an ascribed function owing to low sequence identity to genes that have been annotated. Consequently, biochemical and structural characterization of genes with unknown function is required to complement the rapidly growing pool of genomic sequencing data. A protein with previously unknown function (Cthe_2159) was recently isolated in a genome-wide screen using phage display to identify cellulose-binding protein domains from the biomass-degrading bacterium Clostridium thermocellum. Here, the crystal structure of Cthe_2159 is presented and it is shown that it is a unique right-handed parallel ?-helix protein. Despite very low sequence identity to known ?-helix or carbohydrate-active proteins, Cthe_2159 displays structural features that are very similar to those of polysaccharide lyase (PL) families 1, 3, 6 and 9. Cthe_2159 is conserved across bacteria and some archaea and is a member of the domain of unknown function family DUF4353. This suggests that Cthe_2159 is the first representative of a previously unknown family of cellulose and/or acid-sugar binding ?-helix proteins that share structural similarities with PLs. Importantly, these results demonstrate how functional annotation by biochemical and structural analysis remains a critical tool in the characterization of new gene products. PMID:25286843

Close, Devin W; D'Angelo, Sara; Bradbury, Andrew R M



Guided Transport of a Transmembrane Nanochannel  

NASA Astrophysics Data System (ADS)

Via the Dissipative Particle Dynamics approach, we design a system that allows transport of a nanochannel to a desired location by applying an external force. Each nanochannel encompasses an ABA architecture, with a hydrophobic shaft (B) with two hydrophilic ends (A). One of the hydrophilic ends of the nanochannel is functionalized with hydrophilic functional groups, or hairs. The hydrophilic hairs serve a dual role: (1) control transport across the membrane barrier when the channel diffuses freely in the membrane, and (2) enable the channel relocation to a specific membrane site. Our system comprises a transmembrane hairy nanochannel with the hairs extending into solution. In our earlier work, we demonstrated the spontaneous insertion of such a hairy nanochannel into a lipid bilayer (Nanoscale DOI: 10.1039/C0NR00578A). First, we hold a suitably functionalized pipette stationary above the membrane while the nanochannel freely diffuses within the membrane. For an optimal range of parameters, we demonstrate that the hairs find the pipette and spontaneously anchor onto it. We then show that by moving the pipette for a range of velocities, we can effectively transport the channel to any location within the membrane. This prototype system can provide guidelines for designing a number of biomimetic applications.

Dutt, Meenakshi; Kuksenok, Olga; Balazs, Anna



A natural classification of the basic helix-loop-helix class of transcription factors  

PubMed Central

A natural (evolutionary) classification is provided for 242 basic helix–loop–helix (bHLH) motif-containing proteins. Phylogenetic analyses of amino acid sequences describe the patterns of evolutionary change within the motif and delimit evolutionary lineages. These evolutionary lineages represent well known functional groups of proteins and can be further arranged into five groups based on binding to DNA at the hexanucleotide E-box, the amino acid patterns in other components of the motif, and the presence/absence of a leucine zipper. The hypothesized ancestral amino acid sequence for the bHLH transcription factor family is given together with the ancestral sequences of the subgroups. It is suggested that bHLH proteins containing a leucine zipper are not a natural, monophyletic group. PMID:9144210

Atchley, William R.; Fitch, Walter M.



Helical Structure and Packing Orientation of the S2 Segment in the Shaker K+ Channel  

PubMed Central

Six transmembrane segments, S1–S6, cluster around the central pore-forming region in voltage-gated K+ channels. To investigate the structural characteristics of the S2 segment in the Shaker K+ channel, we replaced each residue in S2 singly with tryptophan (or with alanine for the native tryptophan). All but one of the 23 Trp mutants expressed voltage-dependent K+ currents in Xenopus oocytes. The effects of the mutations were classified as being of low or high impact on channel gating properties. The periodicity evident in the effects of these mutations supports an ?-helical structure for the S2 segment. The high- and low-impact residues cluster onto opposite faces of a helical wheel projection of the S2 segment. The low-impact face is also tolerant of single mutations to asparagine. All results are consistent with the idea that the low-impact face projects toward membrane lipids and that changes in S2 packing occur upon channel opening. We conclude that the S2 segment is a transmembrane ? helix and that the high-impact face packs against other transmembrane segments in the functional channel. PMID:10051517

Monks, Stephen A.; Needleman, Daniel J.; Miller, Christopher



Transmembrane potential induced in a spherical cell model under low-frequency magnetic stimulation  

NASA Astrophysics Data System (ADS)

Time-varying magnetic fields can induce electric fields in the neuronal tissue, a phenomenon that has been recently explored in clinical applications such as peripheral nerve stimulation and transcranial magnetic stimulation. Although the transmembrane potential induced during direct electric stimulation has already been the subject of a number of theoretical studies, an analytical solution for the magnetically induced transmembrane potential change is still unavailable. In addition, although several studies have analyzed the impact of stimulation parameters, including stimulation intensity and frequency, as well as coil design and position, on the amount of tissue polarization, the effects of tissue non-homogeneity on cell polarization have not been fully elucidated. In this study, we have derived an analytical expression for the transmembrane potential induced by a low-frequency magnetic field in a spherical neuronal structure. This model is representative of a spherical cell body or any neuronal structure of a similar shape. The model cell is located in an extracellular medium and possesses a low-conductive membrane and an internal cytoplasm. These three regions represent the basic tissue non-homogeneity of a neuron at a microscopic level. The sensitivity of the induced transmembrane potential to the coil position and to the geometrical and electrical parameters of the model structure was studied in a broad physiologically relevant range. Our results demonstrate that the structure is regionally polarized, with the pattern of polarization depending on the relative positioning between the model cell and the stimulation coil. In addition, both the geometrical and electrical parameters of the structure affect the amount of polarization. These results may be generalized to other neuronal tissues that possess similar non-homogenous properties, but different shapes, such as an axon. Our results support the idea that aside from coil design and position, tissue non-homogeneity could play an important role in determining the effects of magnetic stimulation.

Ye, Hui; Cotic, Marija; Carlen, Peter L.



Relaxed Constraint and Evolutionary Rate Variation between Basic Helix-Loop-Helix Floral Anthocyanin Regulators in Ipomoea  

E-print Network

of anthocyanin pigmentation genes in flowers. We cloned the full-length coding region from 2 basic helix-loop-helix transcription factors from several species of Ipomoea with diverse flower colors and determined the selective previously characterized plant transcription factors. Moreover, codon models of substitution rates and models

Oregon, University of


Target genes for OBP3, a Dof transcription factor, include novel basic helix-loop-helix domain proteins inducible by  

E-print Network

proteins inducible by salicylic acid Hong-Gu Kang1,y,z , Rhonda C. Foley2,z , Luis On� ate-Sa�nchez2 contributed equally to the paper. Summary Overexpression of a salicylic-acid (SA)-inducible Arabidopsis DNA helix-loop-helix, salicylic acid, jasmonic acid, AtDof3.6. Introduction Although there is considerable

Lin, Chentao


Structure of the uncleaved ectodomain of the paramyxovirus (hPIV3) fusion protein  

SciTech Connect

Class I viral fusion proteins share common mechanistic and structural features but little sequence similarity. Structural insights into the protein conformational changes associated with membrane fusion are based largely on studies of the influenza virus hemagglutinin in pre- and postfusion conformations. Here, we present the crystal structure of the secreted, uncleaved ectodomain of the paramyxovirus, human parainfluenza virus 3 fusion (F) protein, a member of the class I viral fusion protein group. The secreted human parainfluenza virus 3 F forms a trimer with distinct head, neck, and stalk regions. Unexpectedly, the structure reveals a six-helix bundle associated with the postfusion form of F, suggesting that the anchor-minus ectodomain adopts a conformation largely similar to the postfusion state. The transmembrane anchor domains of F may therefore profoundly influence the folding energetics that establish and maintain a metastable, prefusion state.

Yin, Hsien-Sheng; Paterson, Reay G.; Wen, Xiaolin; Lamb, Robert A.; Jardetzky, Theodore S. (NWU)



The Structure of a Conserved Piezo Channel Domain Reveals a Topologically Distinct ? Sandwich Fold.  


Piezo has recently been identified as a family of eukaryotic mechanosensitive channels composed of subunits containing over 2,000 amino acids, without recognizable sequence similarity to other channels. Here, we present the crystal structure of a large, conserved extramembrane domain located just before the last predicted transmembrane helix of C. elegans PIEZO, which adopts a topologically distinct ? sandwich fold. The structure was also determined of a point mutation located on a conserved surface at the position equivalent to the human PIEZO1 mutation found in dehydrated hereditary stomatocytosis patients (M2225R). While the point mutation does not change the overall domain structure, it does alter the surface electrostatic potential that may perturb interactions with a yet-to-be-identified ligand or protein. The lack of structural similarity between this domain and any previously characterized fold, including those of eukaryotic and bacterial channels, highlights the distinctive nature of the Piezo family of eukaryotic mechanosensitive channels. PMID:25242456

Kamajaya, Aron; Kaiser, Jens T; Lee, Jonas; Reid, Michelle; Rees, Douglas C



Crystal structure of the Marburg virus GP2 core domain in its postfusion conformation.  


Marburg virus (MARV) and Ebola virus (EBOV) are members of the family Filoviridae ("filoviruses") and cause severe hemorrhagic fever with human case fatality rates of up to 90%. Filovirus infection requires fusion of the host cell and virus membranes, a process that is mediated by the envelope glycoprotein (GP). GP contains two subunits, the surface subunit (GP1), which is responsible for cell attachment, and the transmembrane subunit (GP2), which catalyzes membrane fusion. The GP2 ectodomain contains two heptad repeat regions, N-terminal and C-terminal (NHR and CHR, respectively), that adopt a six-helix bundle during the fusion process. The refolding of this six-helix bundle provides the thermodynamic driving force to overcome barriers associated with membrane fusion. Here we report the crystal structure of the MARV GP2 core domain in its postfusion (six-helix bundle) conformation at 1.9 Å resolution. The MARV GP2 core domain backbone conformation is virtually identical to that of EBOV GP2 (reported previously), and consists of a central NHR core trimeric coiled coil packed against peripheral CHR ?-helices and an intervening loop and helix-turn-helix segments. We previously reported that the stability of the MARV GP2 postfusion structure is highly pH-dependent, with increasing stability at lower pH [Harrison, J. S., Koellhoffer, J. K., Chandran, K., and Lai, J. R. (2012) Biochemistry51, 2515-2525]. We hypothesized that this pH-dependent stability provides a mechanism for conformational control such that the postfusion six-helix bundle is promoted in the environments of appropriately mature endosomes. In this report, a structural rationale for this pH-dependent stability is described and involves a high-density array of core and surface acidic side chains at the midsection of the structure, termed the "anion stripe". In addition, many surface-exposed salt bridges likely contribute to the stabilization of the postfusion structure at low pH. These results provide structural insights into the mechanism of MARV GP2-mediated membrane fusion. PMID:22935026

Koellhoffer, Jayne F; Malashkevich, Vladimir N; Harrison, Joseph S; Toro, Rafael; Bhosle, Rahul C; Chandran, Kartik; Almo, Steven C; Lai, Jonathan R



Expression and initial structural insights from solid-state NMR of the M2 proton channel from influenza A virus.  


The M2 protein from influenza A virus has been expressed, purified, and reconstituted into DMPC/DMPG liposomes. SDS-PAGE analysis of reconstituted M2 protein in DMPC/DMPG liposomes demonstrates a stable tetrameric preparation. Circular dichroism spectra of the intact M2 protein in detergent indicate 67% alpha-helix. The uniformly (15)N-labeled M2 protein and both (15)N-Val- and (15)N-Leu-labeled M2 protein have been expressed from defined M9 media. The (1)H-(15)N HSQC (heteronuclear single quantum correlation) solution NMR experiments have been performed on the amino acid specific labeled protein in 300 mM SDS-d(25) micelles, and the data indicate a homogeneous preparation. The reconstituted M2/DMPC/DMPG proteoliposomes were used for preparing uniformly aligned solid-state NMR samples for (15)N-(1)H dipolar/(15)N chemical shift correlation experiments. The spectra support a transmembrane helix in M2 protein having a tilt angle of approximate 25 degrees, quantitatively similar to results obtained on the isolated M2 transmembrane peptide reconstituted in DMPC bilayers (38 degrees ). In addition, the spectra suggest that the tetrameric protein forms a symmetric or at least pseudosymmetric bundle consistent with data obtained by other research groups based on electrophysiological measurements and substituted cysteine scanning mutagenesis experiments that characterize a tetrameric structure. PMID:12220196

Tian, Changlin; Tobler, Kurt; Lamb, Robert A; Pinto, Lawrence H; Cross, T A



Molecular mechanisms of intercellular communication: transmembrane signaling  

SciTech Connect

This short discussion of transmembrane signaling depicts a particular class of signaling devices whose functional characteristics may well be representative of broader classes of membrane switches. These multicomponent aggregates are characterized by tight organization of interacting components which function by conformational interactions to provide sensitive, amplified, rapid, and modulated responses. It is clear that the essential role of such switches in cell-cell interactions necessitated their appearance early in the history of the development of multicellular organisms. It also seems clear that once such devices made their appearance, the conformationally interactive moieties were firmly locked into a regulatory relationship. Since modification of interacting components could perturb or interfere with the functional integrity of the whole switch, genetic drift was only permitted at the input and outflow extremes. However, the GTP binding moiety and its interacting protein domains on contiguous portions of the receptor and readout components were highly conserved. The observed stringent evolutionary conservation of the molecular features of these membrane switches thus applies primarily to the central (GTP binding) elements. An extraordinary degree of variation was permitted within the domains of signal recognition and enzymatic output. Thus, time and evolution have adapted the central logic of the regulatory algorithm to serve a great variety of cellular purposes and to recognize a great variety of chemical and physical signals. This is exemplified by the richness of the hormonal and cellular dialogues found in primates such as man. Here the wealth of intercellular communiation can support the composition and performance of symphonies and the study of cellular immunology.

Bitensky, M.W.; George, J.S.; Siegel, H.N.; McGregor, D.M.



Lipid bilayer microarray for parallel recording of transmembrane ion currents.  


This paper describes a multiwell biochip for simultaneous parallel recording of ion current through transmembrane pores reconstituted in planar lipid bilayer arrays. Use of a thin poly(p-xylylene) (parylene) film having micrometer-sized apertures (phi=15-50 microm, t=20 microm) led to formation of highly stable bilayer lipid membranes (BLMs) for incorporation of transmembrane pores; thus, a large number of BLMs could be arrayed without any skillful technique. We optically confirmed the simultaneous formation of BLMs in a 5x5 matrix, and in our durability test, the BLM lasted more than 15 h. Simultaneous parallel recording of alamethicin and gramicidin transmembrane pores in multiple contiguous recording sites demonstrated the feasibility of high-throughput screening of transmembrane ion currents in artificial lipid bilayers. PMID:18001126

Le Pioufle, Bruno; Suzuki, Hiroaki; Tabata, Kazuhito V; Noji, Hiroyuki; Takeuchi, Shoji



DNA helix model closeup, still imageSite: DNA Interactive (  

NSDL National Science Digital Library

Image of DNA helix model (closeup). By this point, most scientists believed that DNA carried hereditary information. So having solved the structure, Watson and Crick's comment was a teaser, in reference to two fairly obvious questions. If DNA is in the shape of a twisted ladder, then: how is the information stored in the DNA molecule passed on to the next generation? how is the information stored in the DNA molecule used to make protein?



Enhanced orientational Kerr effect in vertically aligned deformed helix ferroelectric liquid crystals.  


We disclose the vertically aligned deformed helix ferroelectric liquid crystal whose Kerr constant (Kkerr?130??nm/V2 at ?=543??nm) is around one order of magnitude higher than any other value previously reported for liquid crystalline structures. Under certain conditions, the phase modulation with ellipticity less than 0.05 over the range of continuous and hysteresis-free electric adjustment of the phase shift from zero to 2? has been obtained at subkilohertz frequency. PMID:24978232

Pozhidaev, Evgeny P; Srivastava, Abhishek Kumar; Kiselev, Alexei D; Chigrinov, Vladimir G; Vashchenko, Valery V; Krivoshey, Alexander I; Minchenko, Maxim V; Kwok, Hoi-Sing



Exploring novel strategies for AIDS protozoal pathogens: ?-helix mimetics targeting a key allosteric protein-protein interaction in C. hominis TS-DHFR  

PubMed Central

The bifunctional enzyme thymidylate synthase–dihydrofolate reductase (TS–DHFR) from the protozoal parasite Cryptosporidium hominis is a potential molecular target for the design of antiparasitic therapies for AIDS-related opportunistic infections. The enzyme exists as a homodimer with each monomer containing a unique swap domain known as a “crossover helix” that binds in a cleft on the adjacent DHFR active site. This crossover helix is absent in species containing monofunctional forms of DHFR such as human. An in-depth understanding of protein–protein interactions between the crossover helix and adjacent DHFR active site that might modulate enzyme integrity or function would allow for insights into rational design of species-specific allosteric inhibitors. Mutational analysis coupled with structural studies and biophysical and kinetic characterization of crossover helix mutants identifies this domain as essential for full enzyme stability and catalytic activity, and pinpoints these effects to distinct faces of the crossover helix important in protein–protein interactions. Moreover, targeting this helical protein interaction with ?-helix mimetics of the crossover helix leads to selective inhibition and destabilization of the C. hominis TS–DHFR enzyme, thus validating this region as a new avenue to explore for species-specific inhibitor design. PMID:24324854

Martucci, W. Edward; Rodriguez, Johanna M.; Vargo, Melissa A.; Marr, Matthew; Hamilton, Andrew D.



Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein  

PubMed Central

The coronavirus responsible for the severe acute respiratory syndrome (SARS-CoV) contains a small envelope protein, E, with putative involvement in host cell apoptosis and virus morphogenesis. It has been suggested that E protein can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a regular TM ?-helical bundle. We have shown previously that the topology of the ?-helical putative TM domain of E protein (ETM), flanked by two lysine residues at C and N termini to improve solubility, is consistent with a regular TM ?-helix, with orientational parameters in lipid bilayers that are consistent with a homopentameric model. Herein, we show that this peptide, reconstituted in lipid bilayers, shows sodium conductance. Channel activity is inhibited by the anti-influenza drug amantadine, which was found to bind our preparation with moderate affinity. Results obtained from single or double mutants indicate that the organization of the transmembrane pore is consistent with our previously reported pentameric ?-helical bundle model. PMID:17766393

Torres, Jaume; Maheswari, Uma; Parthasarathy, Krupakar; Ng, Lifang; Liu, Ding Xiang; Gong, Xiandi



Investigating the interaction between peptides of the amphipathic helix of Hcf106 and the phospholipid bilayer by solid-state NMR spectroscopy.  


The chloroplast twin arginine translocation (cpTat) system transports highly folded precursor proteins into the thylakoid lumen using the protonmotive force as its only energy source. Hcf106, as one of the core components of the cpTat system, is part of the precursor receptor complex and functions in the initial precursor-binding step. Hcf106 is predicted to contain a single amino terminal transmembrane domain followed by a Pro-Gly hinge, a predicted amphipathic ?-helix (APH), and a loosely structured carboxy terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes. To better understand the membrane active capabilities of Hcf106, we used solid-state NMR spectroscopy to investigate those properties of the APH. In this study, synthesized peptides of the predicted Hcf106 APH (amino acids 28-65) were incorporated at increasing mol.% into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and POPC/MGDG (monogalactosyldiacylglycerol; mole ratio 85:15) multilamellar vesicles (MLVs) to probe the peptide-lipid interaction. Solid-state (31)P NMR and (2)H NMR spectroscopic experiments revealed that the peptide perturbs the headgroup and the acyl chain regions of phospholipids as indicated by changes in spectral lineshape, chemical shift anisotropy (CSA) line width, and (2)H order SCD parameters. In addition, the comparison between POPC MLVs and POPC/MGDG MLVs indicated that the lipid bilayer composition affected peptide perturbation of the lipids, and such perturbation appeared to be more intense in a system more closely mimicking a thylakoid membrane. PMID:24144541

Zhang, Lei; Liu, Lishan; Maltsev, Sergey; Lorigan, Gary A; Dabney-Smith, Carole



Polar residues and their positional context dictate the transmembrane domain interactions of influenza A neuraminidases.  


Interactions that facilitate transmembrane domain (TMD) dimerization have been identified mainly using synthetic TMDs. Here, we investigated how inherent properties within natural TMDs modulate their interaction strength by exploiting the sequence variation in the nine neuraminidase subtypes (N1-N9) and the prior knowledge that a N1 TMD oligomerizes. Initially, consensus TMDs were created from the influenza A virus database, and their interaction strengths were measured in a biological membrane system. The TMD interactions increased with respect to decreasing hydrophobicity across the subtypes (N1-N9) and within the human N1 subtype where the N1 TMDs from the pandemic H1N1 strain of swine origin were found to be significantly less hydrophobic. The hydrophobicity correlation was attributed to the conserved amphipathicity within the TMDs as the interactions were abolished by mutating residues on the polar faces that are unfavorably positioned in the membrane. Similarly, local changes enhanced the interactions only when a larger polar residue existed on the appropriate face in an unfavorable membrane position. Together, the analysis of this unique natural TMD data set demonstrates how polar-mediated TMD interactions from bitopic proteins depend on which polar residues are involved and their positioning with respect to the helix and the membrane bilayer. PMID:23447533

Nordholm, Johan; da Silva, Diogo V; Damjanovic, Justina; Dou, Dan; Daniels, Robert



GTF2IRD2 is located in the Williams-Beuren syndrome critical region 7q11.23 and encodes a protein with two TFII-I-like helix-loop-helix repeats  

Microsoft Academic Search

Williams-Beuren syndrome (also known as Williams syndrome) is caused by a deletion of a 1.55- to 1.84-megabase region from chromosome band 7q11.23. GTF2IRD1 and GTF2I, located within this critical region, encode proteins of the TFII-I family with multiple helix-loop-helix domains known as I repeats. In the present work, we characterize a third member, GTF2IRD2, which has sequence and structural similarity

Aleksandr V. Makeyev; Lkhamsuren Erdenechimeg; Ognoon Mungunsukh; Jutta J. Roth; Badam Enkhmandakh; Frank H. Ruddle; Dashzeveg Bayarsaihan



Transition state and ground state properties of the helix-coil transition in peptides deduced from high-pressure studies  

PubMed Central

Volume changes associated with protein folding reactions contain valuable information about the folding mechanism and the nature of the transition state. However, meaningful interpretation of such data requires that overall volume changes be deconvoluted into individual contributions from different structural components. Here we focus on one type of structural element, the ?-helix, and measure triplet–triplet energy transfer at high pressure to determine volume changes associated with the helix–coil transition. Our results reveal that the volume of a 21-amino-acid alanine-based peptide shrinks upon helix formation. Thus, helices, in contrast with native proteins, become more stable with increasing pressure, explaining the frequently observed helical structures in pressure-unfolded proteins. Both helix folding and unfolding become slower with increasing pressure. The volume changes associated with the addition of a single helical residue to a preexisting helix were obtained by comparing the experimental results with Monte Carlo simulations based on a kinetic linear Ising model. The reaction volume for adding a single residue to a helix is small and negative (?0.23 cm3 per mol = ?0.38 Å3 per molecule) implying that intrahelical hydrogen bonds have a smaller volume than peptide-water hydrogen bonds. In contrast, the transition state has a larger volume than either the helical or the coil state, with activation volumes of 2.2 cm3/mol (3.7 Å3 per molecule) for adding and 2.4 cm3/mol (4.0 Å3 per molecule) for removing one residue. Thus, addition or removal of a helical residue proceeds through a transitory high-energy state with a large volume, possibly due to the presence of unsatisfied hydrogen bonds, although steric effects may also contribute. PMID:24324160

Neumaier, Sabine; Buttner, Maren; Bachmann, Annett; Kiefhaber, Thomas



Atomic resolution structure of the E. coli YajR transporter YAM domain.  


YajR is an Escherichia coli transporter that belongs to the major facilitator superfamily. Unlike most MFS transporters, YajR contains a carboxyl terminal, cytosolic domain of 67 amino acid residues termed YAM domain. Although it is speculated that the function of this small soluble domain is to regulate the conformational change of the 12-helix transmembrane domain, its precise regulatory role remains unclear. Here, we report the crystal structure of the YAM domain at 1.07-Å resolution, along with its structure determined using nuclear magnetic resonance. Detailed analysis of the high resolution structure revealed a symmetrical dimer in which a belt of well-ordered poly-pentagonal water molecules is embedded. A mutagenesis experiment and a thermal stability assay were used to analyze the putative role of this dimerization in response to changes in halogen concentration. PMID:24952155

Jiang, Daohua; Zhao, Yan; Fan, Junping; Liu, Xuehui; Wu, Yan; Feng, Wei; Zhang, Xuejun C



Conformational flexibility and structural dynamics in GPCR-mediated G protein activation: a perspective.  


Structure and dynamics of G proteins and their cognate receptors, both alone and in complex, are becoming increasingly accessible to experimental techniques. Understanding the conformational changes and timelines that govern these changes can lead to new insights into the processes of ligand binding and associated G protein activation. Experimental systems may involve the use of, or otherwise stabilize, non-native environments. This can complicate our understanding of structural and dynamic features of processes such as the ionic lock, tryptophan toggle, and G protein flexibility. While elements in the receptor's transmembrane helices and the C-terminal ?5 helix of G? undergo well-defined structural changes, regions subject to conformational flexibility may be important in fine-tuning the interactions between activated receptors and G proteins. The pairing of computational and experimental approaches will continue to provide powerful tools to probe the conformation and dynamics of receptor-mediated G protein activation. PMID:23602809

Preininger, Anita M; Meiler, Jens; Hamm, Heidi E



Transmembrane domain length of viral K+ channels is a signal for mitochondria targeting  

PubMed Central

K+ channels operate in the plasma membrane and in membranes of organelles including mitochondria. The mechanisms and topogenic information for their differential synthesis and targeting is unknown. This article describes 2 similar viral K+ channels that are differentially sorted; one protein (Kesv) is imported by the Tom complex into the mitochondria, the other (Kcv) to the plasma membrane. By creating chimeras we discovered that mitochondrial sorting of Kesv depends on a hierarchical combination of N- and C-terminal signals. Crucial is the length of the second transmembrane domain; extending its C terminus by ?2 hydrophobic amino acids redirects Kesv from the mitochondrial to the plasma membrane. Activity of Kesv in the plasma membrane is detected electrically or by yeast rescue assays only after this shift in sorting. Hence only minor structural alterations in a transmembrane domain are sufficient to switch sorting of a K+ channel between the plasma membrane and mitochondria. PMID:18719119

Balss, Jorg; Papatheodorou, Panagiotis; Mehmel, Mario; Baumeister, Dirk; Hertel, Brigitte; Delaroque, Nicolas; Chatelain, Franck C.; Minor, Daniel L.; Van Etten, James L.; Rassow, Joachim; Moroni, Anna; Thiel, Gerhard



High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells  

PubMed Central

Membrane halves of boar sperm flagella were produced by freeze-fracture and labeled in situ with concanavalin A and wheat germ agglutinin; the lectins were visualized with protein-gold complexes. Concanavalin A and wheat germ agglutinin binding sites partition with both protoplasmic and exoplasmic halves of the membrane. A high density of lectin marking was found on protoplasmic membrane halves; we conclude that the label corresponds to transmembrane glycoproteins that, on freeze-fracture, are dragged across the outer (exoplasmic) half of the phospholipid bilayer. Our demonstration of numerous transmembrane proteins in sperm flagella offers the structural setting for previous models on flagellar surface motility that postulate accessibility of motile membrane components to the submembranous cytoskeleton. PMID:6746741



Failure of Prion Protein Oxidative Folding Guides the Formation of Toxic Transmembrane Forms*  

PubMed Central

The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrPC) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations. PMID:22955286

Lisa, Silvia; Domingo, Beatriz; Martinez, Javier; Gilch, Sabine; Llopis, Juan F.; Schatzl, Hermann M.; Gasset, Maria



The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix  

PubMed Central

Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD. The WAT chains are related by an ?4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT–WAT and WAT–PRAD interactions. A model is proposed for the synaptic AChET tetramer. PMID:15526038

Dvir, Hay; Harel, Michal; Bon, Suzanne; Liu, Wang-Qing; Vidal, Michel; Garbay, Christiane; Sussman, Joel L; Massoulie, Jean; Silman, Israel



Helix-Coil Transitions in Nonpolar Peptides  

NASA Astrophysics Data System (ADS)

A Monte Carlo simulation of the folding and unfolding of a two-dimensional model polypeptide, which has ?-helical motifs, in nonpolar side chains were carried out. The terms that approximate the effect of the hydrophobic interaction in a solvent are phenomenologically included. Only three states were considered for each unit: extended with hydrogen bonds, turns and coils. We investigated the nature of hydration in the assembly process of ?-helical stretches. The conversion exhibited transitions between three states: random coils (C) at a high temperature, helix bundles (B) at a medium temperature, and ?-helices (H) without lateral hydrophobic bonds at a low temperature. Small hydrophobic interactions result in only the H-C transition lacking the B state. On the other hand, as the hydrophobic effect strengthens, the B state appears at a medium temperature. The B-C and B-H transitions appear as an all-or-none-type transition and a gradually diffused type transition, respectively. This problem is related to protein folding. In the simulation performed in this study, we discuss the cold denaturation of proteins from the viewpoint of B-H transitions.

Kanô, Fumiaki



Effects of radiation on DNA's double helix  

NASA Technical Reports Server (NTRS)

The blueprint of life, DNA's double helix is found in the cells of everything from bacteria to astronauts. Exposure to radiation(depicted at right) such as X-rays (upper) or heavy ion particles (lower), can damage DNA and cause dire consequences both to the organism itself and to future generations. One of NASA's main goals is to develop better radiation shielding materials to protect astronauts from destructive radiation in space. This is particularly important for long space missions. NASA has selected researchers to study materials that provide better shielding. This research is managed by NASA's Office of Biological and Physical Research and is supported by the Microgravity Science and Applications Department at NASA's Marshall Center. During International Space Station Expedition Six, the Extravehicular Activity Radiation Monitoring (EVARM) will continue to measure radiation dosage encountered by the eyes, internal organs and skin during specific spacewalks, and relate it to the type of activity, location and other factors. An analysis of this information may be useful in mitigating potential exposure to space walkers in the future. (Illustration by Dr. Frank Cucinotta, NASA/Johnson Space Center, and Prem Saganti, Lockheed Martin)



The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix  

E-print Network

The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix Hay Dvir1 localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction; neuroscience Keywords: acetylcholinesterase; polyproline II; tetrameriza- tion domain; WWW motif; X

Sussman, Joel L.


Mapping the Homodimer Interface of an Optimized, Artificial, Transmembrane Protein Activator of the Human Erythropoietin Receptor  

PubMed Central

Transmembrane proteins constitute a large fraction of cellular proteins, and specific interactions involving membrane-spanning protein segments play an important role in protein oligomerization, folding, and function. We previously isolated an artificial, dimeric, 44-amino acid transmembrane protein that activates the human erythropoietin receptor (hEPOR) in trans. This artificial protein supports limited erythroid differentiation of primary human hematopoietic progenitor cells in vitro, even though it does not resemble erythropoietin, the natural ligand of this receptor. Here, we used a directed-evolution approach to explore the structural basis for the ability of transmembrane proteins to activate the hEPOR. A library that expresses thousands of mutants of the transmembrane activator was screened for variants that were more active than the original isolate at inducing growth factor independence in mouse cells expressing the hEPOR. The most active mutant, EBC5-16, supports erythroid differentiation in human cells with activity approaching that of EPO, as assessed by cell-surface expression of glycophorin A, a late-stage marker of erythroid differentiation. EBC5-16 contains a single isoleucine to serine substitution at position 25, which increases its ability to form dimers. Genetic studies confirmed the importance of dimerization for activity and identified the residues constituting the homodimer interface of EBC5-16. The interface requires a GxxxG dimer packing motif and a small amino acid at position 25 for maximal activity, implying that tight packing of the EBC5-16 dimer is a crucial determinant of activity. These experiments identified an artificial protein that causes robust activation of its target in a natural host cell, demonstrated the importance of dimerization of this protein for engagement of the hEPOR, and provided the framework for future structure-function studies of this novel mechanism of receptor activation. PMID:24788775

Bears, Zachary; Barrera, Francisco N.; Alonso, Miriam; Engelman, Donald M.; DiMaio, Daniel



Induction of peptide bond dipoles drives cooperative helix formation in the (AAQAA)3 peptide.  


Cooperativity is a central feature in the formation of secondary structures in proteins. However, the driving forces behind this cooperativity are poorly understood. The present work shows that the cooperativity of helix formation in the acetyl-(AAQAA)3-NH2 peptide is significantly enhanced using an empirical force field that explicitly includes the treatment of electronic polarizability. Polarizable simulations yield helical content consistent with experimental measurements and indicate that the dependence of helical content on temperature is improved over additive models, though further sampling is required to fully validate this conclusion. Cooperativity is indicated by the peptide sampling either the coiled state or long helices with relatively low populations of short helices. The cooperativity is shown to be associated with enhanced dipole moments of the peptide backbone upon helix formation. These results indicate the polarizable force field to more accurately model peptide-folding cooperativity based on its physically realistic treatment of electronic polarizability. PMID:25140435

Huang, Jing; MacKerell, Alexander D



On the surface lattice of microtubules: helix starts, protofilament number, seam, and handedness  

PubMed Central

The tubulin monomers of brain microtubules reassembled in vitro are arranged on a 3-start helix, irrespective of whether the number of protofilaments is 13 or 14. The dimer packing is that of the B-lattice described for flagellar microtubules. This implies that the tubulin core of microtubules contains at least one helical discontinuity. Neither 5-start nor 8-start helices have a physical significance and thus cannot be implicated in models of microtubule elongation, but the structure is compatible with elongation of protofilaments by dimers or protofilamentous oligomers. The inner and outer surfaces of the microtubule wall can be visualized by propane jet freezing, freeze fracturing, and metal replication, at a resolution of at least 4 nm. The 3-start helix is left-handed, in contrast to a previous study based on negative staining and shadowing. The reasons for this discrepancy are discussed. PMID:3949873



HELIX-Atlanta Birth Defects Team: Air Pollution and Congenital Heart Defects in Atlanta  

E-print Network

HELIX-Atlanta Birth Defects Team: Air Pollution and Congenital Heart Defects in Atlanta Matt StricklandMatt Strickland On behalf of the HELIXOn behalf of the HELIX--Atlanta Birth Defects teamAtlantaPurpose HELIX (HELIX (HHealth andealth and EEnvironmentnvironment LLinked forinked for IInformationnformation eeXXchangechange))--Atlanta:Atlanta


Solvation model dependency of helix-coil transition in polyalanine.  


Helix-coil transitions in polyalanine molecules of length 10 are studied by multi-canonical Monte Carlo simulations. The solvation effects are included by either a distance-dependent dielectric permittivity or by a term that is proportional to the solvent-accessible surface area of the peptide. We found a strong dependence of the characteristics of the helix-coil transition from the details of the solvation model. PMID:12023250

Peng, Yong; Hansmann, Ulrich H E



Synthesis of Triple Helix Forming Oligonucleotides Containing 2?-Deoxyformycin A  

Microsoft Academic Search

N-Benzoyl-2?-deoxyformycin A (5) was prepared from formycin A and incorporated into the triple helix forming oligonucleotide PRE2ap at CG inversion sites. The modified oligonucleotide containing three substitutions of 2?-deoxyformycin A displayed a 10-fold increase in binding affinity as compared to its unmodified counterpart. This provided a method to accommodate CG inversion sites within target sites for antiparallel triple helix formation.

T. Sudhakar Rao; Michael E. Hogan; Ganapathi R. Revankar




E-print Network

: COMPUTATIONAL MOLECULAR BIOLOGY FINAL PROJECT DECEMBER 6TH , 2002 #12;Introduction: Protein crystal structures. Their importance is illustrated by the high occurrence of journal articles publishing protein structures in highly respected journals such as Science and Nature. While the number of known protein crystal structures has


X-ray structure of a protein-conducting channel.  


A conserved heterotrimeric membrane protein complex, the Sec61 or SecY complex, forms a protein-conducting channel, allowing polypeptides to be transferred across or integrated into membranes. We report the crystal structure of the complex from Methanococcus jannaschii at a resolution of 3.2 A. The structure suggests that one copy of the heterotrimer serves as a functional translocation channel. The alpha-subunit has two linked halves, transmembrane segments 1-5 and 6-10, clamped together by the gamma-subunit. A cytoplasmic funnel leading into the channel is plugged by a short helix. Plug displacement can open the channel into an 'hourglass' with a ring of hydrophobic residues at its constriction. This ring may form a seal around the translocating polypeptide, hindering the permeation of other molecules. The structure also suggests mechanisms for signal-sequence recognition and for the lateral exit of transmembrane segments of nascent membrane proteins into lipid, and indicates binding sites for partners that provide the driving force for translocation. PMID:14661030

Van den Berg, Bert; Clemons, William M; Collinson, Ian; Modis, Yorgo; Hartmann, Enno; Harrison, Stephen C; Rapoport, Tom A



Structural models of the MscL gating mechanism  

NASA Technical Reports Server (NTRS)

Three-dimensional structural models of the mechanosensitive channel of large conductance, MscL, from the bacteria Mycobacterium tuberculosis and Escherichia coli were developed for closed, intermediate, and open conformations. The modeling began with the crystal structure of M. tuberculosis MscL, a homopentamer with two transmembrane alpha-helices, M1 and M2, per subunit. The first 12 N-terminal residues, not resolved in the crystal structure, were modeled as an amphipathic alpha-helix, called S1. A bundle of five parallel S1 helices are postulated to form a cytoplasmic gate. As membrane tension induces expansion, the tilts of M1 and M2 are postulated to increase as they move away from the axis of the pore. Substantial expansion is postulated to occur before the increased stress in the S1 to M1 linkers pulls the S1 bundle apart. During the opening transition, the S1 helices and C-terminus amphipathic alpha-helices, S3, are postulated to dock parallel to the membrane surface on the perimeter of the complex. The proposed gating mechanism reveals critical spatial relationships between the expandable transmembrane barrel formed by M1 and M2, the gate formed by S1 helices, and "strings" that link S1s to M1s. These models are consistent with numerous experimental results and modeling criteria.

Sukharev, S.; Durell, S. R.; Guy, H. R.



Possible roles of basic helix-loop-helix transcription factors in adaptation to drought.  


Water deficiency decreases plant growth and productivity. Several mechanisms are activated in response to dehydration that allows plants to cope with stress, including factors controlling stomatal aperture and ramified root system development. In addition, ABA metabolism is also implicated in the regulation of drought responses. The basic helix-loop-helix (bHLH) proteins, a large family of conserved transcription factors that regulates many cellular processes in eukaryotic organisms, are also involved in several responses that are important for plants to cope with drought stress. This review discusses distinct mechanisms related to drought-adaptive responses, especially the possible involvement of the bHLH transcription factors such as MUTE, implicated in stomatal development; RD29, an ABA-responsive gene; EGL3 and GL3, involved in thichome and root hair development; and SPT, which play roles in repressing leaf expansion. Transcription factors are potential targets for new strategies to increase the tolerance of cultivars to drought stress. Recognition of gene regulatory networks in crops is challenging, and the manipulation of bHLH genes as well as components that mediate bHLH transcription factor responses in different pathways could be essential to achieve abiotic stress tolerance in plants through genetic manipulation. PMID:24767109

Castilhos, Graciela; Lazzarotto, Fernanda; Spagnolo-Fonini, Leila; Bodanese-Zanettini, Maria Helena; Margis-Pinheiro, Márcia



ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation.  

PubMed Central

DNA-binding proteins containing the basic helix-loop-helix (bHLH) domain have been implicated in lineage determination and the regulation of specific gene expression in a number of cell types. By oligonucleotide screening of an adipocyte cDNA expression library, we have identified a novel member of the bHLH-leucine zipper transcription factor family designated ADD1. ADD1 mRNA is expressed predominantly in brown adipose tissue in vivo and is regulated during both determination and differentiation of cultured adipocyte cell lines. ADD1 can function as a sequence-specific transcriptional activator in that it stimulates expression of a chloramphenicol acetyltransferase vector containing multiple ADD1 binding sequences but is unable to activate the myosin light-chain enhancer, which contains multiple binding sites for another bHLH factor, MyoD. ADD1 can also activate transcription through a binding site present in the 5'-flanking region of the fatty acid synthetase gene which is expressed in a differentiation-dependent manner in adipose cells. These data suggest that ADD1 plays a role in the regulation of determination- and differentiation-specific gene expression in adipocytes. Images PMID:8336713

Tontonoz, P; Kim, J B; Graves, R A; Spiegelman, B M



Phylogenetic analysis of the human basic helix-loop-helix proteins  

PubMed Central

Background The basic helix-loop-helix (bHLH) proteins are a large and complex multigene family of transcription factors with important roles in animal development, including that of fruitflies, nematodes and vertebrates. The identification of orthologous relationships among the bHLH genes from these widely divergent taxa allows reconstruction of the putative complement of bHLH genes present in the genome of their last common ancestor. Results We identified 39 different bHLH genes in the worm Caenorhabditis elegans, 58 in the fly Drosophila melanogaster and 125 in human (Homo sapiens). We defined 44 orthologous families that include most of these bHLH genes. Of these, 43 include both human and fly and/or worm genes, indicating that genes from these families were already present in the last common ancestor of worm, fly and human. Only two families contain both yeast and animal genes, and no family contains both plant and animal bHLH genes. We suggest that the diversification of bHLH genes is directly linked to the acquisition of multicellularity, and that important diversification of the bHLH repertoire occurred independently in animals and plants. Conclusions As the last common ancestor of worm, fly and human is also that of all bilaterian animals, our analysis indicates that this ancient ancestor must have possessed at least 43 different types of bHLH, highlighting its genomic complexity. PMID:12093377



Gene replacement strategies to test the functional redundancy of basic helix-loop-helix transcription factor.  


Basic helix-loop-helix (bHLH) transcription factors control developmental decisions for a wide range of embryonic cell types. Hand1 and Hand2 are closely related bHLH proteins that control cardiac, craniofacial, and limb development. Within the developing heart, Hand1 expression becomes restricted predominantly to the left ventricle, whereas Hand2 becomes restricted predominantly to the left ventricle, for which findings have shown each Hand factor to be necessary for normal chamber formation. Forced overexpression of Hand1 throughout the early developing heart induces abnormal interventricular septal development, with resulting pathogenesis of congenital heart defects. To investigate the potential transcriptional mechanisms involved in heart morphogenesis by Hand2, this study used a replacement targeting approach to knock Hand2 into the Hand1 locus and ectopically express one copy of Hand2 within the endogenous Hand1 expression domain in the developing hearts of transgenic mice. The findings show that high-percentage Hand1 ( Hand2 ) chimeras die at birth and exhibit a range of congenital heart defects. These findings suggest that Hand factors may act via unique transcriptional mechanisms mediated by bHLH factor partner choice, supporting the notion that alterations of Hand factor stoichiometry may be as deleterious to normal heart morphogenesis as Hand factor loss of function. PMID:20155416

Firulli, Anthony B; Firulli, Beth A; Wang, Jian; Rogers, Rhonda H; Conway, Simon J



Combinatorial targeting of ribbon-helix-helix artificial transcription factors to chimeric recognition sites  

PubMed Central

Artificial transcription factors (ATFs) are potent synthetic biology tools for modulating endogenous gene expression and precision genome editing. The ribbon–helix–helix (RHH) superfamily of transcription factors are widespread in bacteria and archaea. The principal DNA binding determinant in this family comprises a two-stranded antiparallel ?-sheet (ribbons) in which a pair of eight-residue motifs insert into the major groove. Here, we demonstrate that ribbons of divergent RHH proteins are compact and portable elements that can be grafted into a common ?-helical scaffold producing active ATFs. Hybrid proteins cooperatively recognize DNA sites possessing core tetramer boxes whose functional spacing is dictated by interactions between the ?-helical backbones. These interactions also promote combinatorial binding of chimeras with different transplanted ribbons, but identical backbones, to synthetic sites bearing cognate boxes for each protein either in vitro or in vivo. The composite assembly of interacting hybrid proteins offers potential advantages associated with combinatorial approaches to DNA recognition compared with ATFs that involve binding of a single protein. Moreover, the new class of RHH ATFs may be utilized to re-engineer transcriptional circuits, or may be enhanced with affinity tags, fluorescent moieties or other elements for targeted genome marking and manipulation in bacteria and archaea. PMID:22492712

Zampini, Massimiliano; Hayes, Finbarr



Identification of a Novel Family of Oligodendrocyte Lineage-Specific Basic Helix–Loop–Helix Transcription Factors  

Microsoft Academic Search

Basic helix–loop–helix (bHLH) transcription factors have been identified for neurons and their precursors but not for glial cells. We have identified two bHLH factors, Oligo1 and Oligo2, that are specifically expressed in zones of neuroepithelium from which oligodendrocyte precursors emerge, as well as in the precursors themselves. Expression of Oligo2 in the spinal cord precedes that of platelet-derived growth factor

Qiao Zhou; Songli Wang; David J. Anderson



Nanoparticle biofabrication using English ivy (Hedera helix)  

PubMed Central

Background English ivy (Hedera helix) is well known for its adhesive properties and climbing ability. Essential to its ability to adhere to vertical surfaces is the secretion of a nanocomposite adhesive containing spherical nanoparticles, 60–85 nm in diameter, produced exclusively by root hairs present on adventitious roots. These organic nanoparticles have shown promise in biomedical and cosmetic applications, and represent a safer alternative to metal oxide nanoparticles currently available. Results It was discovered that the maximum adventitious root production was achieved by a 4 h application of 1 mg/ml indole-3 butyric acid (IBA) to juvenile English ivy shoot segments cultured in custom vessels. After incubation of the shoots under continuous light at 83 ?mol/m2 s at 20°C for 2 weeks, the adventitious roots were harvested from the culture system and it was possible to isolate 90 mg of dry weight nanoparticles per 12 g of roots. The nanoparticle morphology was characterized by atomic force microscopy, and found to be similar to previous studies. Conclusions An enhanced system for the production of English ivy adventitious roots and their nanoparticles by modifying GA7 Magenta boxes and identifying the optimal concentration of IBA for adventitious root growth was developed. This system is the first such platform for growing and harvesting organic nanoparticles from plants, and represents an important step in the development of plant-based nanomanufacturing. It is a significant improvement on the exploitation of plant systems for the formation of metallic nanoparticles, and represents a pathway for the generation of bulk ivy nanoparticles for translation into biomedical applications. PMID:23095780



Influence of substituents on conformational preferences of helix foldamers of ?-dipeptides.  


Conformational preferences of 9- and 14-helix foldamers have been studied for ?-dipeptides of 2-aminocyclohexylacetic acid (?Ac6 a) residues such as Ac-(?Ac6 a)2 -NHMe (1), Ac-(C(?) -Et-?Ac6 a)2 -NHMe (2), Ac-(?Ac6 a)2 -NHBn (3), and Ac-(C(?) -Et-?Ac6 a)2 -NHBn (4) at the M06-2X/cc-pVTZ//M06-2X/6-31?+?G(d) level of theory to explore the influence of substituents on their conformational preferences. In the gas phase, the 9-helix foldamer H9 and 14-helix foldamer H14-z are found to be most preferred for dipeptides 2 and 4, respectively, as for dipeptides 1 and 3, which indicates no remarkable influence of the C(?) -ethyl substitution on conformational preferences. The benzyl substitution at the C-terminal end lead H14-z to be the most preferred conformer for dipeptides 3 and 4, whereas it is H9 for dipeptides 1 and 2, which can be ascribed to the favored C?H···? interactions between the cyclohexyl group of the first residue and the C-terminal benzyl group. There are only marginal changes in backbone structures and the distances and angles of H-bonds for all local minima by C(?) -ethyl and/or benzyl substitutions. Although vibrational frequencies and intensities of the dipeptide 4 calculated at both M06-2X/6-31?+?G(d) and M05-2X/6-31?+?G(d) levels of theory are consistent with observed results in the gas phase, H14-z is predicted to be most preferred by ?G only at the former level of theory. Hydration did not bring the significant changes in backbone structures of helix foldamers for both dipeptide 1 and 4. It is expected that the different substitutions at the C-terminal end lead to the different helix foldamers, which may increase the resistance of helical structures to proteolysis and provide the more surface to the helical structures suitable for molecular recognition. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1077-1087, 2014. PMID:24838173

Kee Kang, Young; Yoo, In Kee



A Functional-Phylogenetic Classification System for Transmembrane Solute Transporters  

PubMed Central

A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional assignments of newly sequenced transport proteins that will result from future genome sequencing projects. PMID:10839820

Saier, Milton H.



Length Requirements for Membrane Fusion of Influenza Virus Hemagglutinin Peptide Linkers to Transmembrane or Fusion Peptide Domains  

Microsoft Academic Search

During membrane fusion, the influenza A virus hemagglutinin (HA) adopts an extended helical structure that contains the viral transmembrane and fusion peptide domains at the same end of the molecule. The peptide segments that link the end of this rod-like structure to the membrane-associating domains are approximately 10 amino acids in each case, and their structure at the pH of

Zhu-Nan Li; Byeong-Jae Lee; William A. Langley; Konrad C. Bradley; Rupert J. Russell; David A. Steinhauer



Osmotic and pH transmembrane gradients control the lytic power of melittin.  


Transmembrane osmotic gradients applied on large unilamellar 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles were used to modulate the potency of melittin to induce leakage. Melittin, an amphipathic peptide, changes the permeability of vesicles, as studied using the release of entrapped calcein, a fluorescent marker. A promotion of the ability of melittin to induce leakage was observed when a hyposomotic gradient (i.e., internal salt concentration higher than the external one) was imposed on the vesicles. It is proposed that structural perturbations caused by the osmotic pressure loosen the compactness of the outer leaflet, which facilitates the melittin-induced change in membrane permeability. Additionally, we have shown that this phenomenon is not due to enhanced binding of melittin to the vesicles using intrinsic fluorescence of the melittin tryptophan. Furthermore, we investigated the possibility of using a transmembrane pH gradient to control the lytic activity of melittin. The potency of melittin in inducing release is known to be inhibited by increased negative surface charge density. A transmembrane pH gradient causing an asymmetric distribution of unprotonated palmitic acid in the bilayer is shown to be an efficient way to modulate the lytic activity of melittin, without changing the overall lipid composition of the membrane. We demonstrate that the protective effect of negatively charged lipids is preserved for asymmetric membranes. PMID:8789100

Benachir, T; Lafleur, M



Structure of Concatenated HAMP Domains Provides a Mechanism for Signal Transduction  

SciTech Connect

HAMP domains are widespread prokaryotic signaling modules found as single domains or poly-HAMP chains in both transmembrane and soluble proteins. The crystal structure of a three-unit poly-HAMP chain from the Pseudomonas aeruginosa soluble receptor Aer2 defines a universal parallel four-helix bundle architecture for diverse HAMP domains. Two contiguous domains integrate to form a concatenated di-HAMP structure. The three HAMP domains display two distinct conformations that differ by changes in helical register, crossing angle, and rotation. These conformations are stabilized by different subsets of conserved residues. Known signals delivered to HAMP would be expected to switch the relative stability of the two conformations and the position of a coiled-coil phase stutter at the junction with downstream helices. We propose that the two conformations represent opposing HAMP signaling states and suggest a signaling mechanism whereby HAMP domains interconvert between the two states, which alternate down a poly-HAMP chain.

Airola, Michael V.; Watts, Kylie J.; Bilwes, Alexandrine M.; Crane, Brian R. (Cornell); (Lorma Linda U)



Ligatoxin B, a new cytotoxic protein with a novel helix-turn-helix DNA-binding domain from the mistletoe Phoradendron liga.  

PubMed Central

A new basic protein, designated ligatoxin B, containing 46 amino acid residues has been isolated from the mistletoe Phoradendron liga (Gill.) Eichl. (Viscaceae). The protein's primary structure, determined unambiguously using a combination of automated Edman degradation, trypsin enzymic digestion, and tandem MS analysis, was 1-KSCCPSTTAR-NIYNTCRLTG-ASRSVCASLS-GCKIISGSTC-DSGWNH-46. Ligatoxin B exhibited in vitro cytotoxic activities on the human lymphoma cell line U-937-GTB and the primary multidrug-resistant renal adenocarcinoma cell line ACHN, with IC50 values of 1.8 microM and 3.2 microM respectively. Sequence alignment with other thionins identified a new member of the class 3 thionins, ligatoxin B, which is similar to the earlier described ligatoxin A. As predicted by the method of homology modelling, ligatoxin B shares a three-dimensional structure with the viscotoxins and purothionins and so may have the same mode of cytotoxic action. The novel similarities observed by structural comparison of the helix-turn-helix (HTH) motifs of the thionins, including ligatoxin B, and the HTH DNA-binding proteins, led us to propose the working hypothesis that thionins represent a new group of DNA-binding proteins. This working hypothesis could be useful in further dissecting the molecular mechanisms of thionin cytotoxicity and of thionin opposition to multidrug resistance, and useful in clarifying the physiological function of thionins in plants. PMID:12049612

Li, Shi-Sheng; Gullbo, Joachim; Lindholm, Petra; Larsson, Rolf; Thunberg, Eva; Samuelsson, Gunnar; Bohlin, Lars; Claeson, Per



CD spectra of indolicidin antimicrobial peptides suggest turns, not polyproline helix.  


Indolicidin is a 13-residue antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils that contains five Trp and three Pro residues. Falla et al. [(1996) J. Biol. Chem. 271, 19298] suggested that indolicidin forms a poly-L-proline II helix based upon the circular dichroism (CD) spectra of a closely related peptide (indolicidin methyl ester). In contrast, we found no evidence of poly-L-proline II helix formation in the CD spectra of native indolicidin in various solvents or when bound to micelles and membranes [Ladokhin et al. (1997) Biophys. J. 72, 794]. We interpreted the spectra as arising from unordered and/or beta-turn structures, but noted a sharp negative band at 227 nm arising from the tryptophan residues that would mask spectral features characteristic of poly-L-proline II helix. We have reexamined this issue by means of CD measurements of native indolicidin and several of its analogues. None of the features characteristic of a poly-L-proline helix (or alpha- or 3(10)-helix) were observed for any of the peptides studied. To eliminate artifacts associated with tryptophan, we synthesized indolicidin-L and indolicidin-F in which all five tryptophans were replaced with leucines or phenylalanines, respectively. The changes in CD spectra of these Trp-free peptides upon transfer into membrane-like environments were found to be consistent with the formation of beta-turns. For the native indolicidin in SDS micelles, temperature increases resulted in a coupled diminution of two sharp bands, a negative one at 227 nm and a positive one at 217 nm. This phenomenon, which is absent in indolicidin-L variants with single Leu-->Trp substitutions, is consistent with exciton splitting produced by the stacking of indole rings. Type VI turns in model peptides in aqueous solution are known to be promoted by stacking interactions between cis-proline and neighboring aromatic residues [Yao et al. (1994) J. Mol. Biol. 243, 754]. Molecular modeling of indolicidin with a -Trp(6)-cis-Pro(7)-Trp(8)- type VIa turn demonstrated the feasibility of this turn conformation and revealed the possibility of an accompanying amphipathic structure. We therefore suggest that turn conformations are the principal structural motif of indolicidin and that these turns greatly enhance membrane activity. PMID:10493799

Ladokhin, A S; Selsted, M E; White, S H



Herschel spectral-mapping of the Helix Nebula (NGC 7293): Extended CO photodissociation and OH+ emission  

E-print Network

The Helix Nebula (NGC 7293) is the closest planetary nebulae. Therefore, it is an ideal template for photochemical studies at small spatial scales in planetary nebulae. We aim to study the spatial distribution of the atomic and the molecular gas, and the structure of the photodissociation region along the western rims of the Helix Nebula as seen in the submillimeter range with Herschel. We use 5 SPIRE FTS pointing observations to make atomic and molecular spectral maps. We analyze the molecular gas by modeling the CO rotational lines using a non-local thermodynamic equilibrium (non-LTE) radiative transfer model. For the first time, we have detected extended OH+ emission in a planetary nebula. The spectra towards the Helix Nebula also show CO emission lines (from J= 4 to 8), [NII] at 1461 GHz from ionized gas, and [CI] (2-1), which together with the OH+ lines, trace extended CO photodissociation regions along the rims. The estimated OH+ column density is (1-10)x1e12 cm-2. The CH+ (1-0) line was not detected at...

Etxaluze, M; Goicoechea, J R; van Hoof, P A M; Swinyard, B M; Barlow, M J; van de Steene, G C; Groenewegen, M A T; Kerschbaum, F; Lim, T L; Lique, F; Matsuura, M; Pearson, C; Polehampton, E T; Royer, P; Ueta, T



Positional editing of transmembrane domains during ion channel assembly.  


The integration of transmembrane (TM)-spanning regions of many channels and ion transporters is potentially compromised by the presence of polar and charged residues required for biological function. Although the two TMs of the ATP-gated ion channel subunit P2X2 each contain charged/polar amino acids, we found that each TM is efficiently membrane inserted when it is analysed in isolation, and uncovered no evidence for cooperativity between these two TMs during P2X2 integration. However, using minimal N-glycosylation distance mapping, we find that the positioning of TM2 in newly synthesized P2X2 monomers is distinct from that seen in subunits of the high-resolution structures of assembled homologous trimers. We conclude that P2X2 monomers are initially synthesised at the endoplasmic reticulum in a distinct conformation, where the extent of the TM-spanning regions is primarily defined by the thermodynamic cost of their membrane integration at the Sec61 translocon. In this model, TM2 of P2X2 subsequently undergoes a process of positional editing within the membrane that correlates with trimerisation of the monomer, a process requiring specific polar/charged residues in both TM1 and TM2. We postulate that the assembly process offsets any energetic cost of relocating TM2, and find evidence that positional editing of TM2 in the acid-sensing ion channel (ASIC1a) is even more pronounced than that observed for P2X2. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains. We propose that the orchestrated repositioning of TM segments during subunit oligomerisation plays an important role in generating the functional architecture of active ion channels, and suggest that the regulation of this underappreciated biosynthetic step may provide an elegant mechanism for maintaining ER homeostasis. PMID:23230148

Öjemalm, Karin; Watson, Helen R; Roboti, Peristera; Cross, Benedict C S; Warwicker, Jim; von Heijne, Gunnar; High, Stephen



Two new zinc(ii) coordination complexes with helix characteristics showing both interpenetration and self-catenation features: a platform for the synthesis of chiral and catenated structures assembled by length-modulated dicarboxylates.  


Two new zinc coordination complexes, namely [Zn2(tib)4/3(L(1))2]·DMA (1) and [Zn2(tib)4/3(L(2))2]·H2O (2) (tib = 1,3,5-tris(1-imidazolyl)benzene, H2L(1) = biphenyl-4,4'-dicarboxylic acid, H2L(2) = 4,4'-(2,2'-oxybis(ethane-2,1-diyl)bis(oxy))dibenzoic acid and DMA = N,N-dimethylacetamide), are obtained using achiral mixed ligands and characterized using elemental analysis, IR and X-ray crystallography. Compounds 1 and 2 both display intriguing structural features of both interpenetration and self-catenation. By careful inspection of the two structures, we found that the Zn(ii) cations, the tib ligands, and the dicarboxylic anions show the same coordination modes or analogous configurations. Compound 1 is chiral, which was confirmed by measuring the optical rotation of the bulk samples using solid-state circular dichroism (CD). It is comprised of two crystallographically independent interpenetrated 3D motifs, each containing interlaced triple-stranded right- and left-handed Zn-L(1)-Zn helical chains and chiral 2D [Zn(tib)2/3] layers. Both motifs display binodal (3,4)-coordinated 3D self-catenated networks with the point symbol (10(3))2(10(6))3 and the vertex symbols [1013·1013·1013] and [107·107·108·1010·1011·1011]. However, the two types of helical chains are not racemic due to the differences between the two kinds of L(1) anions, and the two types of chiral 2D [Zn(tib)2/3](2+) layers are not enantiomeric either due to the different configurations of the tib ligands. Therefore, the two motifs are not enantiomers. Compound 2 is achiral, containing Zn-L(2)-Zn zigzag chains that span into three directions and chiral 2D [Zn(tib)2/3] layers. The overall 3D network is a new binodal (3,4)-coordinated self-catenated network with the point symbol (10(3))2(10(6))3 and the vertex symbols [107·107·107] and [102·104·105·105·105·105]. Two of these networks interpenetrate. Their chiral and achiral structures are mainly modulated by the length of the dicarboxylates. As expected, compounds 1 and 2 show photoluminescence behaviors and compound 1 shows a ferroelectric behavior. Thermogravimetric studies of 1 and 2 have also been performed. We examined all 48 known structures containing the tib ligand and drew the conclusion that the metal + tib combination, modulated by the prolonged L ligands, can be a good basis for new chiral and catenated structures. PMID:25182167

Wang, Yue; Qi, Yan; Blatov, Vladislav A; Zheng, Jimin; Li, Qun; Zhang, Chao



Expression and characterization of a four-alpha-helix bundle protein that binds the volatile general anesthetic halothane.  


The structural features of volatile anesthetic binding sites on proteins are being investigated with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. The current study describes the bacterial expression, purification, and initial characterization of the four-alpha-helix bundle (Aalpha(2)-L1M/L38M)(2). The alpha-helical content and stability of the expressed protein are comparable to that of the chemically synthesized four-alpha-helix bundle (Aalpha(2)-L38M)(2) reported earlier. The affinity for binding halothane is somewhat improved with a K(d) = 120 +/- 20 microM as determined by W15 fluorescence quenching, attributed to the L1M substitution. Near-UV circular dichroism spectroscopy demonstrated that halothane binding changes the orientation of the aromatic residues in the four-alpha-helix bundle. Nuclear magnetic resonance experiments reveal that halothane binding results in narrowing of the peaks in the amide region of the one-dimensional proton spectrum, indicating that bound anesthetic limits protein dynamics. This expressed protein should prove to be amenable to nuclear magnetic resonance structural studies on the anesthetic complexes, because of its relatively small size (124 residues) and the high affinities for binding volatile anesthetics. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules and will provide guidelines regarding the general architecture of binding sites on central nervous system proteins. PMID:15877373

Pidikiti, Ravindernath; Shamim, Mohammad; Mallela, Krishna M G; Reddy, Konda S; Johansson, Jonas S



Substrate-induced changes in the structural properties of LacY  

PubMed Central

The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H+ across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H+-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H+- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H+ symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154?Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154?Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties. PMID:24711390

Serdiuk, Tetiana; Madej, M. Gregor; Sugihara, Junichi; Kawamura, Shiho; Mari, Stefania A.; Kaback, H. Ronald; Muller, Daniel J.



Preferred sequences for DNA recognition by the TAL1 helix-loop-helix proteins  

SciTech Connect

Tumor-specific activation of the TAL1 gene is the most common genetic alteration seen in patients with T-cell acute lymphoblastic leukemia. The TAL1 gene products contain the basic helix-loop-helix (bHLH) domain, a protein dimerization and DNA-binding motif common to several known transcription factors. A binding-site selection procedure has now been used to evaluate the DNA recognition properties of TAL1. These studies demonstrate that TAL1 polypeptides do not have intrinsic DNA-binding activity, presumably because of their inability to form bHLH homodimers. However, TAL1 readily interacts with any of the known class A bHLH proteins (E12, E47, E2-2, and HEB) to form heterodimers that bind DNA in a sequence-specific manner. The TAL1 heterodimers preferentially recognize a subset of E-box elements (CANNTG) that can be represented by the consensus sequence AACAGATGGT. This consensus is composed of half-sites for recognition by the participating class A bHLH polypeptide (AACAG) and the TAL1 polypeptide (ATGGT). TAL1 heterodimers with DNA-binding activity are readily detected in nuclear extracts of Jurkat, a leukemic cell line derived from a patient with T-cell acute lymphoblastic leukemia. Hence, TAL1 is likely to bind and regulate the transcription of a unique subset of subordinate target genes, some of which may mediate the malignant function of TAL1 during T-cell leukemogenesis. 48 refs., 10 figs.

Hai-Ling Hsu; Lan Huang; Julia Tsou Tsan [Univ. of Texas Southwestern Medical Center, Dallas, TX (United States)] [and others



Common Regulation of Growth Arrest and Differentiation of Osteoblasts by Helix-Loop-Helix Factors  

PubMed Central

Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21WAF1/Cip1. Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWIST and FGFR3 genes. PMID:11585922

Funato, Noriko; Ohtani, Kiyoshi; Ohyama, Kimie; Kuroda, Takayuki; Nakamura, Masataka



Evaluation of Doxorubicin-loaded 3-Helix Micelles as Nanocarriers  

PubMed Central

Designing stable drug nanocarriers, 10-30 nm in size, would have significant impact on their transport in circulation, tumor penetration and therapeutic efficacy. In the present study, biological properties of 3-helix micelles loaded with 8 wt% doxorubicin (DOX), ~15 nm in size, were characterized to validate their potential as a nanocarrier platform. DOX-loaded micelles exhibited high stability in terms of size and drug retention in concentrated protein environments similar to conditions after intravenous injections. DOX-loaded micelles were cytotoxic to PPC-1 and 4T1 cancer cells at levels comparable to free DOX. 3-helix micelles can be disassembled by proteolytic degradation of peptide shell to enable drug release and clearance to minimize long-term accumulation. Local administration to normal rat striatum by convection enhanced delivery (CED) showed greater extent of drug distribution and reduced toxicity relative to free drug. Intravenous administration of DOX-loaded 3-helix micelles demonstrated improved tumor half-life and reduced toxicity to healthy tissues in comparison to free DOX. In vivo delivery of DOX-loaded 3-helix micelles through two different routes clearly indicates the potential of 3-helix micelles as safe and effective nanocarriers for cancer therapeutics. PMID:24050265

Dube, Nikhil; Shu, Jessica Y.; Dong, He; Seo, Jai W.; Ingham, Elizabeth; Kheirolomoom, Azadeh; Chen, Pin-Yuan; Forsayeth, John; Bankiewicz, Krystof; Ferrara, Katherine W.; Xu, Ting



Gold helix photonic metamaterials: a numerical parameter study.  


We have recently shown that metamaterials composed of three-dimensional gold helices periodically arranged on a square lattice can be used as compact "thin-film" circular polarizers with one octave bandwidth. The physics of the motif of these artificial crystals is closely related to that of microwave sub-wavelength helical antennas in end-fire geometry. Here, we systematically study the dependence of the metamaterial's chiral optical properties on helix pitch, helix radius, two-dimensional lattice constant, wire radius, number of helix pitches, and angle of incidence. Our numerical calculations show that the optical properties are governed by resonances of the individual helices, yet modified by interaction effects. Furthermore, our study shows possibilities and limitations regarding performance optimization. PMID:20173927

Gansel, Justyna K; Wegener, Martin; Burger, Sven; Linden, Stefan