Sample records for transmembrane helix structure

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  3. Consensus motif for integrin transmembrane helix association

    E-print Network

    Senes, Alessandro

    Consensus motif for integrin transmembrane helix association Bryan W. Bergera , Daniel W. Kulpa biological functions. For example, the TM helices of integrins are believed to interact heteromerically in the resting state; disruption of this interaction results in integrin activation and cellular adhesion

  4. Accurate prediction of the burial status of transmembrane residues of ?-helix membrane protein by incorporating the structural and physicochemical features

    Microsoft Academic Search

    Chengqi Wang; Shuyan Li; Lili Xi; Huanxiang Liu; Xiaojun Yao

    2011-01-01

    Predicting the burial status (the residue exposure to the lipid bilayer or buried within the protein core) of transmembrane\\u000a (TM) residues of ?-helix membrane protein (?HMP) is of great importance for genome-wide annotation and for experimental researchers\\u000a to elucidate diverse physiological processes. In this work, we developed a new computational model that can be used for predicting\\u000a the burial status

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

    PubMed

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

    2014-01-01

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

  6. Transmembrane helix-helix interactions involved in ErbB receptor signaling

    PubMed Central

    Cymer, Florian

    2010-01-01

    Among the many transmembrane receptor classes, the receptor tyrosine kinases represent an important superfamily, involved in many cellular processes like embryogenesis, development and cell division. Deregulation and dysfunctions of these receptors can lead to various forms of cancer and other diseases. Mostly, only fragmented knowledge exists about functioning of the entire receptors, and many studies have been performed on isolated receptor domains. In this review we focus on the function of the ErbB family of receptor tyrosine kinases with a special emphasis on the role of the transmembrane domain and on the mechanisms underlying regulated and deregulated signaling. Many general aspects of ErbB receptor structure and function have been analyzed and described. All human ErbBs appear to form homo- and heterodimers within cellular membranes and the single transmembrane domain of the receptors is involved in dimerization. Additionally, only defined structures of the transmembrane helix dimer allows signaling of ErbB receptors. PMID:20212358

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Goswami, Panchali; Paulino, Cristina; Hizlan, Dilem; Vonck, Janet; Yildiz, Özkan; Kühlbrandt, Werner

    2011-01-01

    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

  9. Helix Packing in Polytopic Membrane Proteins: Role of Glycine in Transmembrane Helix Association

    Microsoft Academic Search

    Maryam M. Javadpour; Markus Eilers; Michel Groesbeek; Steven O. Smith

    1999-01-01

    The nature and distribution of amino acids in the helix interfaces of four polytopic membrane proteins (cytochrome c oxidase, bacteriorhodopsin, the photosynthetic reaction center of Rhodobacter sphaeroides, and the potassium channel of Streptomyces lividans) are studied to address the role of glycine in transmembrane helix packing. In contrast to soluble proteins where glycine is a noted helix breaker, the backbone

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

    PubMed

    Ng, Derek P; Deber, Charles M

    2014-06-17

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

  11. Transmembrane helix uniformity examined by spectral mapping of torsion angles.

    PubMed

    Page, Richard C; Kim, Sanguk; Cross, Timothy A

    2008-05-01

    The environment and unique balance of molecular forces within lipid bilayers has a profound impact upon the structure, dynamics, and function of membrane proteins. We describe the biophysical foundations for the remarkable uniformity of many transmembrane helices that result from the molecular interactions within lipid bilayers. In fact, the characteristic uniformity of transmembrane helices leads to unique spectroscopic opportunities allowing for phi,psi torsion angles to be mapped directly onto solid state nuclear magnetic resonance (NMR) PISEMA spectra. Results from spectral simulations, the solid state NMR-derived structure of the influenza A M2 proton channel transmembrane domain, and high-resolution crystal structures of 27 integral membrane proteins demonstrate that transmembrane helices tend to be more uniform than previously thought. The results are discussed through the definition of a preferred range of backbone varphi,psi torsion angles for transmembrane alpha helices and are presented with respect to improving biophysical characterizations of integral membrane proteins. PMID:18462683

  12. Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs.

    PubMed

    Wang, C; Deber, C M

    2000-05-26

    Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis. PMID:10747951

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

    PubMed Central

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

    2014-01-01

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

  14. Integrin ?1 Has a Long Helix, Extending from the Transmembrane Region to the Cytoplasmic Tail in Detergent Micelles

    PubMed Central

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

    2013-01-01

    Integrin proteins are very important adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. They play essential roles in cell signaling and the regulation of cellular shape, motility, and the cell cycle. Here, the transmembrane and cytoplasmic (TMC) domains of integrin ?1 and ?1 were over-expressed and purified in detergent micelles. The structure and backbone relaxations of ?1-TMC in LDAO micelles were determined and analyzed using solution NMR. A long helix, extending from the transmembrane region to the cytoplasmic tail, was observed in ?1-TMC. Structural comparisons of ?1-TMC with reported ?IIb-TMC domains indicated different conformations in the transmembrane regions and cytoplasmic tails. An NMR titration experiment indicated weak interactions between ?1-TMC and ?1-TMC through several ?1-TMC residues located at its N-terminal juxta-transmembrane region and C-terminal extended helix region. PMID:23646163

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

    PubMed

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

    2015-05-01

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

  16. Direct Simulation of Transmembrane Helix Association: Role of Asparagines

    PubMed Central

    Stockner, Thomas; Ash, Walter L.; MacCallum, Justin L.; Tieleman, D. Peter

    2004-01-01

    The forces contributing to the association of transmembrane helices in folded membrane proteins have received considerable attention recently. In this study we investigate the importance of hydrogen bonding by studying the effect of a single Asn residue in the center of an otherwise hydrophobic transmembrane peptide using computer simulations. We use the model peptide MS1 which has been derived from the leucine zipper coiled-coil dimer of the transcription factor peptide GCN4-P1. We follow the trajectory of 36 initially monomeric MS1 transmembrane helical peptides in a membrane-mimicking octane layer as they associate into larger structures. These peptides predominately form dimers. The interaction between the polar asparagine residues, capable of simultaneously being a hydrogen-bond donor and acceptor, contributes strongly to the stability of associated helices. Only dimers with interhelical hydrogen bonds form stable structures, whereas aggregates without any hydrogen-bonding interactions form very transient structures. We examine the hydrogen-bonding patterns and find that there are two forms of dimer, one with symmetric hydrogen bonds and one with asymmetric hydrogen bonds. Based on the structures in our simulation we propose a model with a monomer ? symmetric dimer ? asymmetric dimer ? trimer equilibrium. PMID:15345544

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

    ERIC Educational Resources Information Center

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

    2008-01-01

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

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

    Microsoft Academic Search

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

    2007-01-01

    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

  19. Polar residues in membrane domains of proteins: molecular basis for helix-helix association in a mutant CFTR transmembrane segment.

    PubMed

    Partridge, Anthony W; Melnyk, Roman A; Deber, Charles M

    2002-03-19

    Polar side chains constitute over 20% of residues in the transmembrane (TM) helices of membrane proteins, where they may serve as hydrogen bond interaction sites for phenotypic polar mutations that arise in membrane protein-related diseases. To systematically explore the structural consequences of H-bonds between TM helices, we focused on TM4 of the cystic fibrosis conductance regulator (CFTR) and its cystic fibrosis- (CF-) phenotypic mutation, V232D, as a model system. Synthetic peptides corresponding to wild-type (TM4-wt) (residues 219-242: LQASAFCGLGFLIVLALFQAGLGR) and mutant (TM4-V232D) sequences both adopt helical structures in SDS micelles and display dimer bands on SDS-PAGE arising from disulfide bond formation via wild-type residue Cys-225. However, the TM4-V232D peptide additionally forms a ladder of noncovalent oligomers, including tetramers, hexamers, and octamers, mediated by a hydrogen bond network involving Asp-Gln side chain-side chain interactions. Ala-scanning mutagenesis of the TM4 sequence indicated that ladder formation minimally required the simultaneous presence of the Cys-225, Asp-232, and Gln-237 residues. As random hydrophobic sequences containing these three residues at TM4 equivalent positions did not oligomerize, specific van der Waals packing interactions between helix side chains were also shown to play a crucial role. Overall, the results suggest that polar mutations in membrane domains, in conjunction with critically positioned polar partner residues, potentially constitute a source of aberrant helix interactions that could contribute to loss of function when they arise in protein transmembrane domains. PMID:11888281

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

    PubMed

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

    2014-12-17

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

  1. The positive inside rule is stronger when followed by a transmembrane helix.

    PubMed

    Virkki, Minttu T; Peters, Christoph; Nilsson, Daniel; Sörensen, Therese; Cristobal, Susana; Wallner, Björn; Elofsson, Arne

    2014-08-12

    The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices. PMID:24927974

  2. Sequence-dependent backbone dynamics of a viral fusogen transmembrane helix.

    PubMed

    Stelzer, Walter; Langosch, Dieter

    2012-07-01

    The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing as indicated by mutational studies and functional reconstitution of peptide mimics. Here, we demonstrate that mutations of a GxxxG motif or of Ile residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics as determined by deuterium/hydrogen-exchange kinetics. Thus, the backbone dynamics of these helices may be linked to their fusogenicity which is consistent with the known over-representation of Gly and Ile in viral fusogen transmembrane helices. The transmembrane domains of membrane fusogenic proteins are known to contribute to lipid bilayer mixing. Our present results demonstrate that mutations of certain residues, that were previously shown to compromise the fusogenicity of the Vesicular Stomatitis virus G-protein transmembrane helix, reduce its backbone dynamics. Thus, the data suggest a relationship between sequence, backbone dynamics, and fusogenicity of transmembrane segments of viral fusogenic proteins. PMID:22593029

  3. Light-induced exposure of the cytoplasmic end of transmembrane helix seven in rhodopsin

    PubMed Central

    Abdulaev, Najmoutin G.; Ridge, Kevin D.

    1998-01-01

    A key step in signal transduction in the visual cell is the light-induced conformational change of rhodopsin that triggers the binding and activation of the guanine nucleotide-binding protein. Site-directed mAbs against bovine rhodopsin were produced and used to detect and characterize these conformational changes upon light activation. Among several antibodies that bound exclusively to the light-activated state, an antibody (IgG subclass) with the highest affinity (Ka ? 6 × 10?9 M) was further purified and characterized. The epitope of this antibody was mapped to the amino acid sequence 304–311. This epitope extends from the central region to the cytoplasmic end of the seventh transmembrane helix and incorporates a part of a highly conserved NPXXY motif, a critical region for signaling and agonist-induced internalization of several biogenic amine and peptide receptors. In the dark state, no binding of the antibody to rhodopsin was detected. Accessibility of the epitope to the antibody correlated with formation of the metarhodopsin II photointermediate and was reduced significantly at the metarhodopsin III intermediate. Further, incubation of the antigen–antibody complex with 11-cis-retinal failed to regenerate the native rhodopsin chromophore. These results suggest significant and reversible conformational changes in close proximity to the cytoplasmic end of the seventh transmembrane helix of rhodopsin that might be important for folding and signaling. PMID:9789004

  4. Structure of the Transmembrane Regions of a Bacterial Cyclic Nucleotide-Regulated Channel

    SciTech Connect

    Clayton,G.; Latieri, S.; Heginbotham, L.; Unger, V.; Morais-Cabral, J.

    2008-01-01

    The six-transmembrane helix (6 TM) tetrameric cation channels form the largest ion channel family, some members of which are voltage-gated and others are not. There are no reported channel structures to match the wealth of functional data on the non-voltage-gated members. We determined the structure of the transmembrane regions of the bacterial cyclic nucleotide-regulated channel MlotiK1, a non-voltage-gated 6 TM channel. The structure showed how the S1-S4 domain and its associated linker can serve as a clamp to constrain the gate of the pore and possibly function in concert with ligand-binding domains to regulate the opening of the pore. The structure also led us to hypothesize a new mechanism by which motions of the S6 inner helices can gate the ion conduction pathway at a position along the pore closer to the selectivity filter than the canonical helix bundle crossing.

  5. Transmembrane Protein Structure: Spin Labeling of Bacteriorhodopsin Mutants

    NASA Astrophysics Data System (ADS)

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

    1990-06-01

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

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

    PubMed Central

    Herzyk, P; Hubbard, R E

    1995-01-01

    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

  7. 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

    2010-01-01

    Background Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the ?-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

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

    PubMed Central

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

    2013-01-01

    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

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

    PubMed Central

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

    2005-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  11. 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

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

    PubMed Central

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

    2010-01-01

    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

  13. Structure of FGFR3 transmembrane domain dimer: implications for signaling and human pathologies

    PubMed Central

    Bocharov, Eduard V.; Lesovoy, Dmitry M.; Goncharuk, Sergey A.; Goncharuk, Marina V.; Hristova, Kalina; Arseniev, Alexander S.

    2013-01-01

    SUMMARY Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. At least 8 different pathogenic mutations, implicated in cancers and growth disorders, have been identified in FGFR3 transmembrane segment. Here we use heteronuclear NMR spectroscopy to determine the dimeric structure of FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, while others are located within a putative alternative interface. This implies that while the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the plasma membrane is complex. We propose a FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data. PMID:24120763

  14. Accurate analysis of helix slow-wave structures

    Microsoft Academic Search

    S. D'Agostino; F. Emma; C. Paoloni

    1998-01-01

    In this paper, the helix slow wave structure (SWS) of traveling wave tubes (TWT's) has been analyzed. Dielectric supporting rods of arbitrary cross section have been considered in this analysis. The inhomogeneous dielectric loading factor has been accounted for by modeling the discrete support with a number of continuous dielectric tubes of appropriate effective dielectric permittivity. The helix tape model

  15. 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

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

    2012-01-01

    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

  16. The C-Terminus of Transmembrane Helix 2 (TM2) of the Escherichia coli Tar Chemorecptor Determines Signal Output and Ligand Sensitivity

    E-print Network

    Adase, Christopher A. 1981-

    2012-11-20

    THE C-TERMINUS OF TRANSMEMBRANE HELIX 2 (TM2) OF THE ESCHERICHIA COLI TAR CHEMORECPTOR DETERMINES SIGNAL OUTPUT AND LIGAND SENSITIVITY A Dissertation by CHRISTOPHER A. ADASE Submitted to the Office of Graduate Studies... HAMP domain, although there are almost always some aromatic residues in this region. The question thus becomes what properties of this aromatic anchor are necessary for proper signal transduction. In this dissertation, I studied the effect on Tar...

  17. De novo design of a transmembrane Zn[superscript 2+]-transporting four-helix bundle

    E-print Network

    Wang, Tuo

    The design of functional membrane proteins from first principles represents a grand challenge in chemistry and structural biology. Here, we report the design of a membrane-spanning, four-helical bundle that transports ...

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

    PubMed Central

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

    2007-01-01

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

  19. Transmission line modelling of helix slow wave structures

    Microsoft Academic Search

    K. D. Ward; J. Wlodarczyk

    1993-01-01

    The numerical method Transmission Line Modelling has been used to determine the microwave characteristics of travelling wave tube helix slow wave structures. The capability to determine the ?-? diagram, and the Pierce interaction impedances have been demonstrated. The method has been used to determine the optimum tape to gap width for one structure, and to design a low dispersion structure

  20. Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  2. Dispersion Characteristics of a Rectangular Helix Slow-Wave Structure

    Microsoft Academic Search

    Chengfang Fu; Yanyu Wei; Wenxiang Wang; Yubin Gong

    2008-01-01

    A special type of helical slow-wave structure encompassing a rectangular geometry is investigated in this paper, and the slow-wave characteristics are studied taking into account the anisotropically conducting helix. By using the electromagnetic integral equations at the boundaries, the dispersion equation and the interaction impedance of transverse antisymmetric modes in this structure are derived. Moreover, the obtained complex dispersion equation

  3. Structural biology of human cannabinoid receptor-2 helix 6 in membrane-mimetic environments1

    PubMed Central

    Tiburu, Elvis K; Tyukhtenko, Sergiy; Deshmukh, Lalit; Vinogradova, Olga; Janero, David R.; Makriyannis, Alexandros

    2013-01-01

    We detail the structure and dynamics of a synthetic peptide corresponding to transmembrane helix 6 (TMH6) of human cannabinoid receptor 2 (hCB2) in biomembrane mimetic environments. The peptide’s NMR structural biology is characterized by two ?-helical domains bridged by a flexible, nonhelical hinge region containing a highly-conserved CWFP motif with an environmentally sensitive, Pro-based conformational switch. Buried within the peptide’s flexible region, W258 may hydrogen-bond with L255 to help stabilize the Pro-kinked hCB2 TMH6 structure and position C257 advantageously for interaction with agonist ligands. These characteristics of hCB2 TMH6 are potential structural features of ligand-induced hCB2 activation in vivo. PMID:19397896

  4. Transmembrane Helix 11 Is a Genuine Regulator of the Endoplasmic Reticulum Ca2+ Pump and Acts as a Functional Parallel of ?-Subunit on ?-Na+,K+-ATPase*

    PubMed Central

    Gorski, Przemek A.; Trieber, Catharine A.; Larivière, Els; Schuermans, Marleen; Wuytack, Frank; Young, Howard S.; Vangheluwe, Peter

    2012-01-01

    The housekeeping sarco(endo)plasmic reticulum Ca2+ ATPase SERCA2b transports Ca2+ across the endoplasmic reticulum membrane maintaining a vital Ca2+ gradient. Compared with the muscle-specific isoforms SERCA2a and SERCA1a, SERCA2b houses an 11th transmembrane segment (TM11) and a short luminal extension (LE) at its C terminus (2b-tail). The 2b-tail imposes a 2-fold higher apparent Ca2+ affinity and lower Vmax. Previously, we assumed that LE is the sole functional region of the 2b-tail and that TM11 is a passive element providing an additional membrane passage. However, here we show that peptides corresponding to the TM11 region specifically modulate the activity of the homologous SERCA1a in co-reconstituted proteoliposomes and mimic the 2b-tail effect (i.e. lower Vmax and higher Ca2+ affinity). Using truncated 2b-tail variants we document that TM11 regulates SERCA1a independently from LE, confirming that TM11 is a second, previously unrecognized functional region of the 2b-tail. A phylogenetic analysis further indicates that TM11 is the oldest and most conserved feature of the 2b-tail, found in the SERCA pump of all Bilateria, whereas LE is only present in Nematoda and vertebrates. Considering remarkable similarities with the Na+,K+-ATPase ?-? interaction, we now propose a model for interaction of TM11 with TM7 and TM10 in the anchoring subdomain of the Ca2+ pump. This model involves a TM11-induced helix bending of TM7. In conclusion, more than just a passive structural feature, TM11 acts as a genuine regulator of Ca2+ transport through interaction with the pump. PMID:22528494

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

    PubMed Central

    Somasekharan, Suma; Tanis, Jessica; Forbush, Biff

    2012-01-01

    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

  6. Modeling and mutagenesis of the human alpha 1a-adrenoceptor: orientation and function of transmembrane helix V sidechains.

    PubMed

    Wetzel, J M; Salon, J A; Tamm, J A; Forray, C; Craig, D; Nakanishi, H; Cui, W; Vaysse, P J; Chiu, G; Weinshank, R L; Hartig, P R; Branchek, T A; Gluchowski, C

    1996-01-01

    A 3-dimensional model of the seven transmembrane helical segments (TMs) of the human alpha 1a-adrenoceptor was initially built by analogy to the known structure of bacteriorhodopsin. However, the rotational orientation of TM V about its helical axis, and the roles of several TM V residues in ligand binding and receptor activation remained in question. Accordingly, we determined the effects of six site-specific mutations in TM V on binding affinity and functional potency of a structurally diverse series of agonists and antagonists. Mutation of Ser 192 and Phe 193 disrupted the binding of many of the tested ligands, as measured by displacement of [3H]prazosin. In addition, mutation of Ser 188, Ser 192, and Phe 193 disrupted receptor activation, as measured by [3H]inositol phosphate formation. On the basis of these results, a specific rotational orientation of TM V is proposed as part of a revised receptor model, which also takes into account more recently reported information about the structure of rhodopsin. This revised alpha 1a-adrenoceptor model accounts for direct interactions which are proposed between Ser 188 and Ser 192 and the meta and para hydroxyl groups of norepinephrine, respectively, in the G-protein coupled receptor state. PMID:9014240

  7. Probabilistic grammatical model for helix?helix contact site classification

    PubMed Central

    2013-01-01

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

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

    PubMed

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

    2014-11-18

    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

  9. About the helix structure of the Lund string

    E-print Network

    Sarka Todorova

    2011-01-12

    The helix structure of the Lund string, first derived from studies devoted to the emission of soft gluons at the end of the parton cascade, may be at the origin of certain characteristic discrepancies observed in the low transverse momentum region at LEP and LHC. A study of the relation between different helix implementations and observable effects is presented. The model is extended to cover a multiparton string topology (result of parton shower), and compared with the experimental data. It is found that a helix-ordered string with a regular winding (proportional to the energy density stored in the string), is favoured by the inclusive single-particle spectra measured in the hadronic decay of Z$^0$.

  10. Tau could protect DNA double helix structure.

    PubMed

    Hua, Qian; He, Rong-qiao

    2003-02-21

    The hyperchromic effect has been used to detect the effect of tau on the transition of double-stranded DNA to single-stranded DNA. It was shown that tau increased the melting temperature of calf thymus DNA from 67 to 81 degrees C and that of plasmid from 75 to 85 degrees C. Kinetically, rates of increase in absorbance at 260 nm of DNA incubated with tau were markedly slower than those of DNA and DNA/bovine serum albumin used as controls during thermal denaturation. In contrast, rates of decrease in the DNA absorbance with tau were faster than those of controls when samples were immediately transferred from thermal conditions to room temperature. It revealed that tau prevented DNA from thermal denaturation, and improved renaturation of DNA. Circular dichroic spectra results indicated that there were little detectable conformational changes in DNA double helix when tau was added. Furthermore, tau showed its ability to protect DNA from hydroxyl radical (.OH) attacking in vitro, implying that tau functions as a DNA-protecting molecule to the radical. PMID:12573250

  11. TOPTMH: topology predictor for transmembrane alpha-helices.

    PubMed

    Ahmed, Rezwan; Rangwala, Huzefa; Karypis, George

    2010-02-01

    Alpha-helical transmembrane proteins mediate many key biological processes and represent 20%-30% of all genes in many organisms. Due to the difficulties in experimentally determining their high-resolution 3D structure, computational methods to predict the location and orientation of transmembrane helix segments using sequence information are essential. We present TOPTMH, a new transmembrane helix topology prediction method that combines support vector machines, hidden Markov models, and a widely used rule-based scheme. The contribution of this work is the development of a prediction approach that first uses a binary SVM classifier to predict the helix residues and then it employs a pair of HMM models that incorporate the SVM predictions and hydropathy-based features to identify the entire transmembrane helix segments by capturing the structural characteristics of these proteins. TOPTMH outperforms state-of-the-art prediction methods and achieves the best performance on an independent static benchmark. PMID:20183873

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

    PubMed

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

    2014-11-01

    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

  13. Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7) Structure

    PubMed Central

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

    2013-01-01

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

  14. Double-helix structure in multiwall boron nitride nanotubes.

    PubMed

    Celik-Aktas, Ayten; Zuo, Jian Min; Stubbins, James F; Tang, Chengchun; Bando, Yoshio

    2005-11-01

    A new nanotube structural form is reported that resembles a double helix in multiwall boron nitride nanotubes (MW-BNNT) grown by a carbon-free chemical-vapor-deposition process as documented by evidence obtained by transmission electron diffraction and microscopy. The double-helix structure is found in MW-BNNTs exhibiting the same chirality in its different walls. The MW-BNNTs deviate from the structure of ideal nested coaxial cylindrical tubes. Most significantly, bright- and dark-field electron imaging reveals regular zigzag dark and bright spots on the side walls of the nanotubes. The repeating distance between the bright, or dark, spots is related to the chiral angle of the nanotube. Electron diffraction patterns recorded from individual nanotubes show additional diffraction spots belonging to the 201 zone axes, which are not allowed in a perfectly cylindrical nanotube. These additional diffraction spots become asymmetrical as smaller sections of the nanotube are probed. A series of diffraction patterns recorded along the tube axis showed that the imperfections giving rise to these spots move in a regular fashion around the circumference of the tube. It is shown that all experimental evidence supports the structure model of two helices; one is polygonal in cross section and highly crystalline and the other is circular and less ordered. It is further suggested that the double-helix structure is a result of stronger wall-wall interactions associated with the ionic bonding in boron nitride. PMID:16244402

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

    E-print Network

    Bryan, Allen W.

    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 ...

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

    PubMed Central

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

    2013-01-01

    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

  17. Thermal Analysis of a Helix TWT Slow-Wave Structure

    Microsoft Academic Search

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

    2008-01-01

    A novel and effective analytical method using ANSYS has been developed for studying the heat-dissipation capability of a helix traveling-wave-tube slow-wave structure (SWS). This method, which is based on calibrating theoretical calculations with experimental data, is able to precisely predict the SWS heat dissipation, thereby reducing material costs and saving time. The consistency and feasibility of this method have been

  18. Study of planar-helix slow-wave structure for application to travelling-wave tubes

    Microsoft Academic Search

    D. Chadha; S. Aditya; R. K. Arora

    1984-01-01

    A planar helix, constituted of a pair of unidirectionally conducting screens conducting in different directions, is suggested as a slow-wave structure for application in a traveling-wave tube (TWT). Circuit parameters, such as interaction impedance and space-charge parameter, are derived for the suggested planar-helix TWT. Computed results for the planar helix indicate a performance comparable with that of its circular helix

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

    PubMed Central

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

    2006-01-01

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

  20. Helix-Destabilizing, ?-Branched, and Polar Residues in the Baboon Reovirus p15 Transmembrane Domain Influence the Modularity of FAST Proteins ?

    PubMed Central

    Clancy, Eileen K.; Duncan, Roy

    2011-01-01

    The fusogenic reoviruses induce syncytium formation using the fusion-associated small transmembrane (FAST) proteins. A recent study indicated the p14 FAST protein transmembrane domain (TMD) can be functionally replaced by the TMDs of the other FAST proteins but not by heterologous TMDs, suggesting that the FAST protein TMDs are modular fusion units. We now show that the p15 FAST protein is also a modular fusogen, as indicated by the functional replacement of the p15 ectodomain with the corresponding domain from the p14 FAST protein. Paradoxically, the p15 TMD is not interchangeable with the TMDs of the other FAST proteins, implying that unique attributes of the p15 TMD are required when this fusion module is functioning in the context of the p15 ecto- and/or endodomain. A series of point substitutions, truncations, and reextensions were created in the p15 TMD to define features that are specific to the functioning of the p15 TMD. Removal of only one or two residues from the N terminus or four residues from the C terminus of the p15 TMD eliminated membrane fusion activity, and there was a direct correlation between the fusion-promoting function of the p15 TMD and the presence of N-terminal, hydrophobic ?-branched residues. Substitution of the glycine residues and triserine motif present in the p15 TMD also impaired or eliminated the fusion-promoting activity of the p15 TMD. The ability of the p15 TMD to function in an ecto- and endodomain-specific context is therefore influenced by stringent sequence requirements that reflect the importance of TMD polar residues and helix-destabilizing residues. PMID:21367887

  1. Helix-destabilizing, beta-branched, and polar residues in the baboon reovirus p15 transmembrane domain influence the modularity of FAST proteins.

    PubMed

    Clancy, Eileen K; Duncan, Roy

    2011-05-01

    The fusogenic reoviruses induce syncytium formation using the fusion-associated small transmembrane (FAST) proteins. A recent study indicated the p14 FAST protein transmembrane domain (TMD) can be functionally replaced by the TMDs of the other FAST proteins but not by heterologous TMDs, suggesting that the FAST protein TMDs are modular fusion units. We now show that the p15 FAST protein is also a modular fusogen, as indicated by the functional replacement of the p15 ectodomain with the corresponding domain from the p14 FAST protein. Paradoxically, the p15 TMD is not interchangeable with the TMDs of the other FAST proteins, implying that unique attributes of the p15 TMD are required when this fusion module is functioning in the context of the p15 ecto- and/or endodomain. A series of point substitutions, truncations, and reextensions were created in the p15 TMD to define features that are specific to the functioning of the p15 TMD. Removal of only one or two residues from the N terminus or four residues from the C terminus of the p15 TMD eliminated membrane fusion activity, and there was a direct correlation between the fusion-promoting function of the p15 TMD and the presence of N-terminal, hydrophobic ?-branched residues. Substitution of the glycine residues and triserine motif present in the p15 TMD also impaired or eliminated the fusion-promoting activity of the p15 TMD. The ability of the p15 TMD to function in an ecto- and endodomain-specific context is therefore influenced by stringent sequence requirements that reflect the importance of TMD polar residues and helix-destabilizing residues. PMID:21367887

  2. Structure-based prediction reveals capping motifs that inhibit ?-helix aggregation

    PubMed Central

    Bryan, Allen W.; Starner-Kreinbrink, Jennifer L.; Hosur, Raghavendra; Clark, Patricia L.; Berger, Bonnie

    2011-01-01

    The parallel ?-helix is a geometrically regular fold commonly found in the proteomes of bacteria, viruses, fungi, archaea, and some vertebrates. ?-helix structure has been observed in monomeric units of some aggregated amyloid fibers. In contrast, soluble ?-helices, both right- and left-handed, are usually “capped” on each end by one or more secondary structures. Here, an in-depth classification of the diverse range of ?-helix cap structures reveals subtle commonalities in structural components and in interactions with the ?-helix core. Based on these uncovered commonalities, a toolkit of automated predictors was developed for the two distinct types of cap structures. In vitro deletion of the toolkit-predicted C-terminal cap from the pertactin ?-helix resulted in increased aggregation and the formation of soluble oligomeric species. These results suggest that ?-helix cap motifs can prevent specific, ?-sheet-mediated oligomeric interactions, similar to those observed in amyloid formation. PMID:21685332

  3. Study of the helix structure of QCD string

    NASA Astrophysics Data System (ADS)

    Todorova-Nová, Šárka

    2012-08-01

    The properties of a helixlike shaped QCD string are studied in the context of the Lund fragmentation model, where the concept of a three-dimensional structure of the gluon field offers an alternative approach to the modeling of the transverse momentum of hadrons. The paper is focused on the phenomenology of the model, which introduces correlations between transverse and longitudinal components of hadrons, as well as azimuthal ordering of hadrons along the string. The similarities between 2-particle correlations stemming from the helix-string structure and those commonly attributed to the Bose-Einstein interference are pointed out. It is shown that the charged asymmetry observed in hadron production of close hadron pairs can be associated with fluctuations in the space-time history of the string breakup and with the presence of resonant hadronic states.

  4. Analysis of Helix Slow Wave Structure for High Efficiency Space TWT

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

    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.

  5. Determination of the interaction impedance of helix slow-wave structures

    Microsoft Academic Search

    Richard G. Carter

    2000-01-01

    The computer simulation of helix travelling-wave tubes requires accurate information about the phase velocity, interaction impedance and attenuation of the helix slow wave structure at each frequency. This has received considerable attention, from the pioneering work of Pierce onwards, and it is possible to compute or measure the phase velocity with sufficient accuracy. The methods available for determining the interaction

  6. Design of helix slow-wave structures for high efficiency TWTs

    Microsoft Academic Search

    Vishnu Srivastava; Richard G. Carter; B. Ravinder; A. K. Sinha; S. N. Joshi

    2000-01-01

    TWTs for space applications commonly have a helix pitch profile which incorporates a section with increased phase velocity followed by a negative phase velocity taper. A simple method is described for the initial design of a helix slow-wave structure of this kind to achieve high overall efficiency. It is shown that the use of a section with increased phase velocity

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

    SciTech Connect

    Xiang, Ye; Rossmann, Michael G. (Purdue)

    2011-12-22

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

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

    PubMed

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

    2005-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  10. Analysis of Helix Slow Wave Structure for High Efficiency Space TWT

    Microsoft Academic Search

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

    2009-01-01

    This paper describes the analysis of helix slow-wave structure (SWS) for a high efficiency space traveling wave tube that\\u000a is carried out using Ansoft HFSS and CST microwave studio, which is a 3D electromagnetic field simulators. Two approaches\\u000a of simulating the dispersion and impedance characteristics of the helix slow wave structure have been discussed and compared\\u000a with measured results. The

  11. Analysis of the Dispersion Characteristic and Interaction Impedance of a Tape Helix Slow Wave Structure with Novel Supporting Mode

    Microsoft Academic Search

    Zhaoyun Duan; Yubin Gong; Yanyu Wei; Wenxiang Wang; Minzhi Huang

    2004-01-01

    In order to enhance the power capability of the helix travelling wave tube, a novel tape helix slow wave structure (SWS), which is supported by helically arrayed radial dielectric-support posts, is developed. Each dielectric post can be easily brazed with the tape helix and the metal envelope by means of a special soldering. This kind of supporting mode can protect

  12. Structural Basis of Amino Acid alpha Helix Propensity

    Microsoft Academic Search

    Michael Blaber; Xue-Jun Zhang; Brian W. Matthews

    1993-01-01

    The propensity of an amino acid to form an alpha helix in a protein was determined by multiple amino substitutions at positions 44 and 131 in T4 lysozyme. These positions are solvent-exposed sites within the alpha helices that comprise, respectively, residues 39 to 50 and 126 to 134. Except for two acidic substitutions that may be involved in salt bridges,

  13. Cell-Cell Membrane Fusion Induced by p15 Fusion-associated Small Transmembrane (FAST) Protein Requires a Novel Fusion Peptide Motif Containing a Myristoylated Polyproline Type II Helix*

    PubMed Central

    Top, Deniz; Read, Jolene A.; Dawe, Sandra J.; Syvitski, Raymond T.; Duncan, Roy

    2012-01-01

    The p15 fusion-associated small transmembrane (FAST) protein is a nonstructural viral protein that induces cell-cell fusion and syncytium formation. The exceptionally small, myristoylated N-terminal ectodomain of p15 lacks any of the defining features of a typical viral fusion protein. NMR and CD spectroscopy indicate this small fusion module comprises a left-handed polyproline type II (PPII) helix flanked by small, unstructured N and C termini. Individual prolines in the 6-residue proline-rich motif are highly tolerant of alanine substitutions, but multiple substitutions that disrupt the PPII helix eliminate cell-cell fusion activity. A synthetic p15 ectodomain peptide induces lipid mixing between liposomes, but with unusual kinetics that involve a long lag phase before the onset of rapid lipid mixing, and the length of the lag phase correlates with the kinetics of peptide-induced liposome aggregation. Lipid mixing, liposome aggregation, and stable peptide-membrane interactions are all dependent on both the N-terminal myristate and the presence of the PPII helix. We present a model for the mechanism of action of this novel viral fusion peptide, whereby the N-terminal myristate mediates initial, reversible peptide-membrane binding that is stabilized by subsequent amino acid-membrane interactions. These interactions induce a biphasic membrane fusion reaction, with peptide-induced liposome aggregation representing a distinct, rate-limiting event that precedes membrane merger. Although the prolines in the proline-rich motif do not directly interact with membranes, the PPII helix may function to force solvent exposure of hydrophobic amino acid side chains in the regions flanking the helix to promote membrane binding, apposition, and fusion. PMID:22170056

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

    SciTech Connect

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

    2011-12-31

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

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

    PubMed Central

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

    2015-01-01

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

  16. Analysis of a Chiral Dielectric Supported Broadband Helix Slow-Wave Structure for Millimeter-Wave TWTs

    Microsoft Academic Search

    S. K. Datta; E. V. Jayashree; S. D. Veena; Lalit Kumar

    2007-01-01

    A novel technique of broadbanding a helical slow-wave structure through negative dispersion shaping is proposed. The model\\u000a considers a simple continuous chiral dielectric support for the helix inside a metallic barrel, unlike conventional helix\\u000a slow-wave structures with three discrete dielectric supports at 1200 apart. The dispersion relation of the slow-wave structure was derived following sheath-helix abstraction, suitably benchmarked\\u000a for special

  17. Role of Putative Second Transmembrane Region of Nox2 Protein in the Structural Stability and Electron Transfer of the Phagocytic NADPH Oxidase*

    PubMed Central

    Picciocchi, Antoine; Debeurme, Franck; Beaumel, Sylvain; Dagher, Marie-Claire; Grunwald, Didier; Jesaitis, Algirdas J.; Stasia, Marie-José

    2011-01-01

    Flavocytochrome b558 (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22phox and gp91phox. The latter subunit, also known as Nox2, has a cytosolic C-terminal “dehydrogenase domain” containing FAD/NADPH-binding sites. The N-terminal half of Nox2 contains six predicted transmembrane ?-helices coordinating two hemes. We studied the role of the second transmembrane ?-helix, which contains a “hot spot” for mutations found in rare X+ and X? chronic granulomatous disease. By site-directed mutagenesis and transfection in X-CGD PLB-985 cells, we examined the functional and structural impact of seven missense mutations affecting five residues. P56L and C59F mutations drastically influence the level of Nox2 expression indicating that these residues are important for the structural stability of Nox2. A53D, R54G, R54M, and R54S mutations do not affect spectral properties of oxidized/reduced cytb, oxidase complex assembly, FAD binding, nor iodonitrotetrazolium (INT) reductase (diaphorase) activity but inhibit superoxide production. This suggests that Ala-53 and Arg-54 are essential in control of electron transfer from FAD. Surprisingly, the A57E mutation partially inhibits FAD binding, diaphorase activity, and oxidase assembly and affects the affinity of immunopurified A57E cytochrome b558 for p67phox. By competition experiments, we demonstrated that the second transmembrane helix impacts on the function of the first intracytosolic B-loop in the control of diaphorase activity of Nox2. Finally, by comparing INT reductase activity of immunopurified mutated and wild type cytb under aerobiosis versus anaerobiosis, we showed that INT reduction reflects the electron transfer from NADPH to FAD only in the absence of superoxide production. PMID:21659519

  18. Electromagnetic Wave Propagation through an Azimuthally Asymmetric Helix Slow Wave Structure

    Microsoft Academic Search

    Young-Do Joo; Ashok Kumar Sinha; Gun-Sik Park

    2003-01-01

    In the fabrication of the practical helix slow wave structure (SWS), the misalignment or the dielectric constant variation in its support is not avoidable and, thus, causing azimuthal asymmetry in the structure. Therefore, the analysis of the asymmetric structure becomes an important issue. In this study, rigorous field analysis of a general asymmetric helical SWS was done by considering the

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

    E-print Network

    Jakob Bohr; Kasper Olsen

    2010-04-11

    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.

  20. 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

    Rhode Island, University of

    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

  1. Structural basis of the role of the NikA ribbon-helix-helix domain in initiating bacterial conjugation.

    PubMed

    Yoshida, Hitoshi; Furuya, Nobuhisa; Lin, Yi-Jan; Güntert, Peter; Komano, Teruya; Kainosho, Masatsune

    2008-12-19

    Conjugation is a fundamental process for the rapid evolution of bacteria, enabling them, for example, to adapt to various environmental conditions or to acquire multi-drug resistance. NikA is one of the relaxosomal proteins that initiate the intercellular transfer of the R64 conjugative plasmid with the P-type origin of transfer, oriT. The three-dimensional structure of the N-terminal 51 residue fragment of NikA, NikA(1-51), which binds to the 17-bp repeat A sequence in R64 oriT, was determined by NMR to be a homodimer composed of two identical ribbon-helix-helix (RHH) domains, which are commonly found in transcriptional repressors. The structure determination of NikA(1-51) was achieved using automated NOE assignment with CYANA, without measuring filtered NOESY experiments to distinguish between the intra- and intermolecular NOEs, and without any a priori assumption on the tertiary or quaternary structure of the protein. Mutational experiments revealed that the DNA-binding region of the NikA(1-51) dimer is an anti-parallel beta-sheet composed of one beta-strand from each of the N-terminal ends of the two domains. Various biochemical experiments have indicated that the full length NikA(1-109) exists as a homotetramer formed through an alpha-helical domain at the C-terminus, and that the anti-parallel beta-sheets of both dimeric domains bind to two homologous 5 bp internal repeats within repeat A. As a tetramer, the full length NikA(1-109) showed higher affinity to repeat A and bent the oriT duplex more strongly than NikA(1-51) did. Many RHH proteins are involved in specific DNA recognition and in protein-protein interactions. The discovery of the RHH fold in NikA suggests that NikA binds to oriT and interacts with the relaxase, NikB, which is unable to bind to the nick region in oriT without NikA. PMID:18929573

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

    NASA Astrophysics Data System (ADS)

    Eisenberg, David

    2003-09-01

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

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

    E-print Network

    Bertram, Richard

    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

  4. Structural Insights into Triglyceride Storage Mediated by Fat Storage-Inducing Transmembrane (FIT) Protein 2

    E-print Network

    Snapp, Erik Lee

    Structural Insights into Triglyceride Storage Mediated by Fat Storage-Inducing Transmembrane (FIT in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence

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

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  6. Structural Transitions in the Polyalanine r-Helix under Uniaxial Strain

    E-print Network

    polyalanine chain in the R-helical conformation using density functional theory. Under compressive strainStructural Transitions in the Polyalanine r-Helix under Uniaxial Strain Joel Ireta,*, Jo 31, 2005; E-mail: ireta@fhi-berlin.mpg.de Abstract: We analyzed the response to strain of an infinite

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

    PubMed Central

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

    2011-01-01

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

  8. Structural and Functional Analysis of Transmembrane XI of the NHE1 Isoform of the Na+/H+ Exchanger*S?

    PubMed Central

    Lee, Brian L.; Li, Xiuju; Liu, Yongsheng; Sykes, Brian D.; Fliegel, Larry

    2009-01-01

    The Na+/H+ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals by extruding an intracellular H+ in exchange for one extracellular Na+. We characterized structural and functional aspects of the critical transmembrane (TM) segment XI (residues 449-470) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM XI was mutated to cysteine in the background of the cysteine-less protein and the sensitivity to water-soluble sulfhydryl reactive compounds MTSET ((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES ((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues with at least moderate activity remaining. Of the residues tested, only proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The activity of the L465C mutant was almost completely eliminated, whereas that of the L457C and I461C mutants was partially affected. The structure of a peptide representing TM XI (residues Lys447-Lys472) was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. The structure consisted of helical regions between Asp447-Tyr454 and Phe460-Lys471 at the N and C termini of the peptide, respectively, connected by a region with poorly defined, irregular structure consisting of residues Gly455-Gly459. TM XI of NHE1 had a structural similarity to TM XI of the Escherichia coli Na+/H+ exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with residue Leu465 contributing to the pore. PMID:19176522

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

    E-print Network

    Granada, Universidad de

    Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein the conserva- tion patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of a-helical transmembrane (TM) segments. We find that Ser/ Thr motifs are often

  10. Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix

    PubMed Central

    Brown, Jessica A.; Bulkley, David; Wang, Jimin; Valenstein, Max L.; Yario, Therese A.; Steitz, Thomas A.; Steitz, Joan A.

    2014-01-01

    Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly-abundant nuclear long noncoding RNA that promotes malignancy. A 3?-stem-loop structure is predicted to confer stability by engaging a downstream A-rich tract in a triple helix, similar to the expression and nuclear retention element (ENE) from the KSHV polyadenylated nuclear RNA. The 3.1-Å resolution crystal structure of the human MALAT1 ENE and A-rich tract reveals a bipartite triple helix containing stacks of five and four U•A-U triples separated by a C+•G-C triplet and C-G doublet, extended by two A-minor interactions. In vivo decay assays indicate that this blunt-ended triple helix, with the 3? nucleotide in a U•A-U triple, inhibits rapid nuclear RNA decay. Interruption of the triple helix by the C-G doublet induces a “helical reset” that explains why triple-helical stacks longer than six do not occur in nature. PMID:24952594

  11. Estimation of conductivity losses in a helix slow-wave structure using eigen-mode solutions

    Microsoft Academic Search

    P. Raja Ramana Rao; Subrata Kumar Datta

    2008-01-01

    This paper presents a simple method of estimating the attenuation constant due to conductivity losses and surface finish in a helix slow-wave structure, using the quality factor and dispersion characteristics obtainable from the eigen-mode solutions through 3D HFSS modeling. The method has been benchmarked against two practical structures published in the literature, and effect of surface finish on the attenuation

  12. Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator

    PubMed Central

    Lewis, Hal A; Buchanan, Sean G; Burley, Stephen K; Conners, Kris; Dickey, Mark; Dorwart, Michael; Fowler, Richard; Gao, Xia; Guggino, William B; Hendrickson, Wayne A; Hunt, John F; Kearins, Margaret C; Lorimer, Don; Maloney, Peter C; Post, Kai W; Rajashankar, Kanagalaghatta R; Rutter, Marc E; Sauder, J Michael; Shriver, Stephanie; Thibodeau, Patrick H; Thomas, Philip J; Zhang, Marie; Zhao, Xun; Emtage, Spencer

    2004-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1–NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments. PMID:14685259

  13. Defining the structural requirements for a helix in 23 S ribosomal RNA that confers erythromycin resistance.

    PubMed

    Douthwaite, S; Powers, T; Lee, J Y; Noller, H F

    1989-10-20

    The helix spanning nucleotides 1198 to 1247 (helix 1200-1250) in Escherichia coli 23 S ribosomal RNA (rRNA) is functionally important in protein synthesis, and deletions in this region confer erythromycin resistance. In order to define the structural requirements for resistance, we have dissected this region using in vitro mutagenesis. Erythromycin resistance is established after a minimal deletion of three bases, CAU1231 or AUG1232. The maximum deletion observed to confer resistance is 25 bases. The level of erythromycin resistance conferred by intermediate sized deletions is variable and some deletion mutants show a sensitive phenotype. Deletions that extend into the base-pairing between GCC1208 and GGU1240 result in non-functional 23 S RNAs, which consequently do not confer resistance. A number of phylogenetically conserved nucleotides have been shown to be non-essential for 23 S RNA function. However, removal of either these or non-conserved nucleotides from helix 1200-1250 measurably reduces the efficiency of 23 S RNA in forming functional ribosomes. We have used chemical probing and a modified primer extension method to investigate erythromycin binding to wild-type and resistant ribosomes with a 12-base deletion in 23 S RNA. Erythromycin interacts as strongly with mutant 23 S RNA as with wild-type 23 S RNA. Deletions in the 1200-1250 helix do not therefore confer resistance by reducing erythromycin binding, but by suppressing the effects of the drug at the level of its mechanism of action. PMID:2685326

  14. Structure based aggregation studies reveal the presence of helix-rich intermediate during ?-Synuclein aggregation

    PubMed Central

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

    2015-01-01

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

  15. Structural parameters of the myelin transmembrane proteolipid in reverse micelles.

    PubMed Central

    Binks, B P; Chatenay, D; Nicot, C; Urbach, W; Waks, M

    1989-01-01

    The Folch-Pi proteolipid is the most abundant structural protein from the central nervous system myelin. This protein-lipid complex, normally insoluble in water, requires only a small amount of water for solubilization in reverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The characterization of the proteolipid-free and proteolipid-containing micelles was undertaken by light scattering and fluorescence recovery after fringe pattern photobleaching (FRAPP) experiments. Quasi elastic light scattering (QELS) was carried out at a high (200 mM) AOT concentration, at low water-to-surfactant mole ratio (Wo = 7) and at increasing protein occupancy. Two apparent hydrodynamic radii, differing tenfold in size, were obtained from correlation functions. The smaller one (RaH = 5.2 nm) remains constant and corresponds to that measured for protein-free micelles. The larger one increases linearly with protein concentration. In contrast, FRAPP measurements of self-diffusion coefficients were found unaffected by the proteolipid concentration. Accordingly, they have been performed at constant protein/surfactant mole ratios. The equivalent RH, extrapolated to zero AOT concentration for protein-free reverse micelles (2.9 nm) and in the presence of the proteolipid (4.6 nm), do not reveal the mode of organization previously suggested by QELS measurements. The complex picture emerging from this work represents a first step in the characterization of an integral membrane protein in reverse micelles. PMID:2470431

  16. Structural parameters of the myelin transmembrane proteolipid in reverse micelles.

    PubMed

    Binks, B P; Chatenay, D; Nicot, C; Urbach, W; Waks, M

    1989-05-01

    The Folch-Pi proteolipid is the most abundant structural protein from the central nervous system myelin. This protein-lipid complex, normally insoluble in water, requires only a small amount of water for solubilization in reverse micelles of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The characterization of the proteolipid-free and proteolipid-containing micelles was undertaken by light scattering and fluorescence recovery after fringe pattern photobleaching (FRAPP) experiments. Quasi elastic light scattering (QELS) was carried out at a high (200 mM) AOT concentration, at low water-to-surfactant mole ratio (Wo = 7) and at increasing protein occupancy. Two apparent hydrodynamic radii, differing tenfold in size, were obtained from correlation functions. The smaller one (RaH = 5.2 nm) remains constant and corresponds to that measured for protein-free micelles. The larger one increases linearly with protein concentration. In contrast, FRAPP measurements of self-diffusion coefficients were found unaffected by the proteolipid concentration. Accordingly, they have been performed at constant protein/surfactant mole ratios. The equivalent RH, extrapolated to zero AOT concentration for protein-free reverse micelles (2.9 nm) and in the presence of the proteolipid (4.6 nm), do not reveal the mode of organization previously suggested by QELS measurements. The complex picture emerging from this work represents a first step in the characterization of an integral membrane protein in reverse micelles. PMID:2470431

  17. A novel seven-helix transmembrane protein BTP1 of Botrytis cinerea controls the expression of GST-encoding genes, but is not essential for pathogenicity.

    PubMed

    Gronover, Christian Schulze; Schumacher, Julia; Hantsch, Phillip; Tudzynski, Bettina

    2005-05-01

    SUMMARY To gain new insights into the signalling mechanisms of the grey mould Botrytis cinerea, which causes several pre- and post-harvest diseases on a variety of host plants, we cloned, sequenced and functionally characterized a gene, btp1, encoding a novel 391-amino acid transmembrane protein. The protein BTP1 shows similarity to the transmembrane protein pth11, which is essential for appressorium formation and successful colonization of plant tissue in the rice blast fungus Magnaporthe grisea. Analyses of the deduced amino acid sequence of btp1 predicted a seven alpha-helical transmembrane topology, which is known to be typical for G protein-coupled receptors (GPCRs) and therefore the protein is thought to play a role in mediation of extracellular signals to intracellular effectors. The gene is located next to the gene bcgstII encoding a new putative glutathione S-transferase, and both genes are transcribed in opposite directions from the same promoter. BcGSTII shows similarity to the glutathione S-transferase GSTII of Schizosaccharomyces pombe, a protein thought to be involved in detoxification of several antifungal drugs. From the sequence similarity of BTP1 to GPCRs, and its expression in planta, we suggested that it might play a role in mediation of plant signals and therefore in pathogenicity. However, targeted gene replacement of btp1 did not result in a phenotype markedly affecting either pathogenicity or sensitivity to chemical stress when compared with the wild-type strain; however, the ten-fold dilution of conidial suspension used for the pathogenicity assay resulted in slight reduction of virulence. Visible symptom development of the mutants on bean plants was also different from the wild-type. The brownish ring, which appears at the margin of secondary lesions in wild-type infections, was brighter and almost absent in Deltabtp1 mutants. Interestingly, deletion of btp1 not only affected the expression of the physically linked bcgstII gene, but in addition the expression of the other two GST-encoding genes in B. cinerea for bcgstI was down-regulated, bcgstII was slightly up-regulated and bcgstIII was strongly up-regulated in the mutant. PMID:20565654

  18. An Improved Technique for Measuring the Pierce Impedance of Helix Slow-Wave Structures

    Microsoft Academic Search

    P. Wang; R. G. Carter; B. N. Basu

    1994-01-01

    The Pierce impedance of helix slow-wave structures has been measured using nonresonant perturbation techniques. Effects of space harmonics and TE fields were demonstrated using experimental results for the first time. It was found that there was an error in the derivation of the correction factor commonly used for the uniform-axial-field assumption. It was shown that this error should be corrected

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

    PubMed Central

    Puljung, Michael C.; Zagotta, William N.

    2013-01-01

    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

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

    PubMed Central

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

    2014-01-01

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

  1. Shielded helix traveling wave cathode ray tube deflection structure

    DOEpatents

    Norris, Neil J. (Santa Barbara, CA); Hudson, Charles L. (Santa Barbara, CA)

    1992-01-01

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

  2. Shielded helix traveling wave cathode ray tube deflection structure

    DOEpatents

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

    1992-12-15

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

  3. A novel slotted helix slow-wave structure for high power Ka-band traveling-wave tubes

    NASA Astrophysics Data System (ADS)

    Liu, Lu-Wei; Wei, Yan-Yu; Wang, Shao-Meng; Hou, Yan; Yin, Hai-Rong; Zhao, Guo-Qing; Duan, Zhao-Yun; Xu, Jin; Gong, Yu-Bin; Wang, Wen-Xiang; Yang, Ming-Hua

    2013-10-01

    A novel slotted helix slow-wave structure (SWS) is proposed to develop a high power, wide-bandwidth, and high reliability millimeter-wave traveling-wave tube (TWT). This novel structure, which has higher heat capacity than a conventional helix SWS, evolves from conventional helix SWS with three parallel rows of rectangular slots made in the outside of the helix tape. In this paper, the electromagnetic characteristics and the beam-wave interaction of this novel structure operating in the Ka-band are investigated. From our calculations, when the designed beam voltage and beam current are set to be 18.45 kV and 0.2 A, respectively, this novel circuit can produce over 700-W average output power in a frequency range from 27.5 GHz to 32.5 GHz, and the corresponding conversion efficiency values vary from 19% to 21.3%, and the maximum output power is 787 W at 30 GHz.

  4. Structural organization of FtsB, a transmembrane protein of the bacterial divisome

    PubMed Central

    LaPointe, Loren M.; Taylor, Keenan C.; Subramaniam, Sabareesh; Khadria, Ambalika; Rayment, Ivan; Senes, Alessandro

    2013-01-01

    We report the first structural analysis of an integral membrane protein of the bacterial divisome. FtsB is a single-pass membrane protein with a periplasmic coiled coil. Its heterologous association with its partner FtsL represents an essential event for the recruitment of the late components to the division site. Using a combination of mutagenesis, computational modeling and X-ray crystallography, we determined that FtsB self-associates and we investigated its structural organization. We found that the transmembrane domain of FtsB homo-oligomerizes through an evolutionarily conserved interaction interface where a polar residue (Gln 16) plays a critical role through the formation of an inter-helical hydrogen bond. The crystal structure of the periplasmic domain, solved as a fusion with Gp7, shows that 30 juxta-membrane amino acids of FtsB form a canonical coiled coil. The presence of conserved Gly residue in the linker region suggests that flexibility between the transmembrane and coiled coil domains is functionally important. We hypothesize that the transmembrane helices of FtsB form a stable dimeric core for its association with FtsL into a higher-order oligomer, and that FtsL is required to stabilize the periplasmic domain of FtsB, leading to the formation of a complex that is competent for binding to FtsQ, and to their consequent recruitment to the divisome. The study provides an experimentally validated structural model and identifies point mutations that disrupt association, thereby establishing important groundwork for the functional characterization of FtsB in vivo. PMID:23520975

  5. CLASSICAL AREAS OF PHENOMENOLOGY: Small-signal analysis of a rectangular helix structure traveling-wave-tube

    NASA Astrophysics Data System (ADS)

    Fu, Cheng-Fang; Wei, Yan-Yu; Duan, Zhao-Yun; Wang, Wen-Xiang; Gong, Yu-Bin

    2009-07-01

    This paper investigates the properties of traveling wave-beam interaction in a rectangular helix traveling-wave-tube (TWT) for a solid sheet electron beam. The 'hot' dispersion equation is obtained by means of the self-consistent field theory. The small signal analysis, which includes the effects of the beam parameters and slow-wave structure (SWS) parameters, is carried out by theoretical computation. The numerical results show that the bandwidth and the small-signal gain of the rectangular helix TWT increase as the beam current increases; and the beam voltage not obviously influences the small signal gain. Among different rectangular helix structures, the small-signal gain increases as the width of the rectangular helix SWS increases, however, the bandwidth decreases whether structure parameters a and L or ? and L are fixed or not. In addition, a comparison of the small-signal gain of this structure with a conventional round helix is made. The presented analysis will be useful for the design of the TWT with a rectangular helix circuit.

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

    PubMed

    Páli, Tibor; Bashtovyy, Denys; Marsh, Derek

    2006-05-01

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

  7. Analysis of dielectric rods with arbitrary shape for low-dispersion slow-wave structures in helix TWTs

    Microsoft Academic Search

    Claudio Paoloni

    2006-01-01

    The introduction of rods with optimized cross-sectional shape in slow-wave structures (SWSs) for wideband helix traveling-wave tubes can sensibly improve the dispersion maintaining an adequate interaction-impedance level. In this brief, a procedure for analyzing helix SWSs loaded with rods of arbitrary shape, by using an analysis method adopting the inhomogeneous dielectric loading approach, is proposed. The advantages, in terms of

  8. Correction of the dispersion characteristic of a helix slow-wave structure in traveling-wave tubes

    Microsoft Academic Search

    Yu. N. Pchelnikov

    2011-01-01

    On the basis of a physical notions of the specific features of the slow-wave field distribution in the helix structure of\\u000a a traveling-wave tube (TWT), various methods for controlling the amount of wave slowing and the slope of the dispersion characteristic\\u000a are analyzed. The effect of the dielectric supports and correcting elements placed outside the helix is considered. In addition

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

    PubMed

    Samoylovich, Mikhail; Talis, Alexander

    2014-03-01

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

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

    SciTech Connect

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

    2009-05-26

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

  11. Interfacial water screens the protein-induced transmembrane voltage.

    PubMed

    Zarzycki, Piotr

    2015-01-29

    Transmembrane proteins are crucial in cellular traffic, signal transduction, and energy storage in a form of transmembrane voltage. These proteins are stabilized by hydrophobic and hydrophilic interactions, in which cytoplasmic and exoplasmic water plays a special role. Water structural ordering generates the dipole potential that typically overcompensates for an intrinsic membrane-protein potential gradient, and thus it modifies and sustains an overall cellular electrostatics. Although the transmembrane voltage has been extensively studied, the dipole potential has attracted very little attention. Here, by using molecular dynamics, we examined water electrostatic response to the transmembrane charge, field, and potential asymmetry introduced by the presence of four integral membrane proteins: typical of inner (?-helix) and outer membrane (?-barrel). In all cases, the protein presence introduces electrostatic directionality in the transmembrane dipole field and voltage. In particular, water generates a deep potential sink if strongly polar residues are densely packed on one side of bilayer, as frequently occurs in a selectivity filter of the K(+) channel. We also found that protein secondary structure is less important than the polar residue distribution along the protein channel. Our findings are relevant for understanding the driving force behind biomembrane conductivity: the ability of biological water to electrostatically screen the transmembrane voltage. PMID:25563965

  12. Crystal structure of phenylalanine ammonia lyase: multiple helix dipoles implicated in catalysis.

    PubMed

    Calabrese, Joseph C; Jordan, Douglas B; Boodhoo, Amechand; Sariaslani, Sima; Vannelli, Todd

    2004-09-14

    The first three-dimensional structure of phenylalanine ammonia lyase (PAL) has been determined at 2.1 A resolution for PAL from Rhodosporidium toruloides. The enzyme is structurally similar to the mechanistically related histidine ammonia lyase (HAL), with PAL having an additional approximately 160 residues extending from the common fold. We propose that catalysis (including lowering the pK(a) of nonacidic C3 of l-phenylalanine for an E1cb mechanism) is potentially governed by dipole moments of seven alpha helices associated with the PAL active site (six positive poles and one negative pole). Cofactor 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) resides atop the positive poles of three helices, for increasing its electrophilicity. The helix dipoles appear fully compatible with a model of phenylalanine docked in the active site of PAL having the first covalent bond formed between the amino group of substrate and the methylidene group of MIO: 12 highly conserved residues (near the N termini of helices for enhancing function) are poised to serve roles in substrate recognition, MIO activation, product separation, proton donation, or polarizing electrons from the phenyl ring of substrate for activation of C3; and a highly conserved His residue (near the C terminus of the one helix that directs its negative pole toward the active site to increase the residue's basicity) is positioned to act as a general base, abstracting the pro-S hydrogen from C3 of substrate. A similar mechanism is proposed for HAL, which has a similar disposition of seven alpha helices and similar active-site residues. The helix dipoles appear incompatible with a proposed mechanism that invokes a carbocation intermediate. PMID:15350127

  13. NMR structures and localization of the potential fusion peptides and the pre-transmembrane region of SARS-CoV: Implications in membrane fusion.

    PubMed

    Mahajan, Mukesh; Bhattacharjya, Surajit

    2015-02-01

    Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) poses a serious public health hazard. The S2 subunit of the S glycoprotein of SARS-CoV carries out fusion between the virus and the host cells. However, the exact mechanism of the cell fusion process is not well understood. Current model suggests that a conformational transition, upon receptor recognition, of the two heptad core regions of S2 may expose the hydrophobic fusogenic peptide or fusion peptide for membrane insertion. Three regions of the S2 subunit have been proposed to be involved in cell-cell fusion. The N-terminal fusion peptide (FP, residues 770-788), an internal fusion peptide (IFP, residues 873-888) and the pre-transmembrane region (PTM, residues 1185-1202) demonstrated interactions with model lipid membranes and potentially involved in the fusion process. Here, we have determined atomic resolution structures of these three peptides in DPC detergent micelles by solution NMR. FP assumes ?-helical conformation with significant distortion at the central Gly residues; enabling a close packing among sidechains of aromatic residues including W, Y and F. The 3-D structure of PMT is characterized by a helix-loop-helix with extensive aromatic interactions within the helices. IFP adopts a rather straight ?-helical conformation defined by packing among sidechains of aromatic and aliphatic residues. Paramagnetic spin labeled NMR has demonstrated surface localization of PMT whereas FP and IFP inserted into the micelles. Collectively, data presented in this study will aid in understanding fusion mechanism of SARS-CoV. PMID:25475644

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

    PubMed Central

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

    2014-01-01

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

  15. Analysis of a Chiral Dielectric Supported Broadband Helix Slow-Wave Structure for Millimeter-Wave TWTs

    NASA Astrophysics Data System (ADS)

    Datta, S. K.; Jayashree, E. V.; Veena, S. D.; Kumar, Lalit

    2007-09-01

    A novel technique of broadbanding a helical slow-wave structure through negative dispersion shaping is proposed. The model considers a simple continuous chiral dielectric support for the helix inside a metallic barrel, unlike conventional helix slow-wave structures with three discrete dielectric supports at 1200 apart. The dispersion relation of the slow-wave structure was derived following sheath-helix abstraction, suitably benchmarked for special cases, and was used for analyzing the dispersion behavior of a typical slow-wave structure. Chiral dielectric loading could easily provide negative dispersion characteristics (required for broadband operation) by merely controlling the chirality parameter alone. The scheme with its simple geometric configuration is expected to be useful for millimeter-wave devices providing better thermal management.

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

    PubMed Central

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

    2014-01-01

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

  17. High-resolution modeling of transmembrane helical protein structures from distant homologues.

    PubMed

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

    2014-05-01

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

  18. Helix coupling

    DOEpatents

    Ginell, W.S.

    1982-03-17

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

  19. Helix coupling

    DOEpatents

    Ginell, W.S.

    1989-04-25

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

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

    PubMed

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

    2012-08-28

    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. PMID:22938259

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

    PubMed Central

    Palasingam, Paaventhan; Kolatkar, Prasanna R.

    2012-01-01

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

  2. A novel alpha-helix-liked metallohelicate series and their structural adjustments for the isomorphous substitution.

    PubMed

    Zhang, Y; Li, J; Chen, J; Su, Q; Deng, W; Nishiura, M; Imamoto, T; Wu, X; Wang, Q

    2000-05-29

    A novel metallohelical motif is well designed and synthesized by mimicking the alpha-helical fold structure of protein. The 1D helical structures of [Cd(CH2(COO)2)(SC(NH2)2)2]n (I) and [Zn(CH2(COO)2) (SC(NH2)2)2]n (II) are primarily induced and stabilized by the multiple long-range intrahelix hydrogen bonds. Malonate dianion acts as a bidentate ligand coordinated with metal ions to form the backbone of the helix, and thiourea molecules that bend into the helical turn are involved in the intrahelix hydrogen-bond system. The metal ion occupations in the helix of I and II can be freely substituted by simply controlling the initial ratio of those two metal ions. Single crystals of three mixed metal ion complexes of [Cd0.77Zn0.23(CH2(COO)2)(SC(NH2)2)2]n (III), [Cd0.50Zn0.50(CH2(COO)2)(SC(NH2)2)2]n (IV), and [Cd0.21Zn0.79(CH2(COO)2)(SC(NH2)2)2]n (V) were synthesized from systems with an initial Cd/Zn mole ratio of 1:1 for III, 1:2 for IV, and 1:8 for V. They are isomorphous as confirmed by X-ray characterization. When the metal ion is substituted, the multiple intrahelix hydrogen interaction motifs of the coordination polymer structure are self-adjusted to sustain their 1D helical motifs. PMID:12526493

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

    PubMed Central

    Studer, Gabriel; Biasini, Marco; Schwede, Torsten

    2014-01-01

    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: torsten.schwede@unibas.ch Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:25161240

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

    PubMed Central

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

    2012-01-01

    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

  5. Allosteric and hyperekplexic mutant phenotypes investigated on an ?1 glycine receptor transmembrane structure.

    PubMed

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

    2015-03-01

    The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human ?1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the ?1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the ?1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the ?1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the ?1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor. PMID:25730860

  6. The 2 Å structure of helix 6 of the human signal recognition particle RNA

    Microsoft Academic Search

    Klemens Wild; Oliver Weichenrieder; Gordon A Leonard; Stephen Cusack

    1999-01-01

    Background: The mammalian signal recognition particle (SRP) is an essential cytoplasmic ribonucleoprotein complex involved in targeting signal-peptide-containing proteins to the endoplasmic reticulum. Assembly of the SRP requires protein SRP19 to bind first to helix 6 of the SRP RNA before the signal-peptide-recognizing protein, SRP54, can bind to helix 8 of the RNA. Helix 6 is closed by a GGAG tetraloop,

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

    PubMed

    Lei, Hsiang-Ting; Bolla, Jani Reddy; Bishop, Nicholas R; Su, Chih-Chia; Yu, Edward W

    2014-01-23

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

  8. Simplified Approach to the Nonlinear Analysis in Helix Slow-Wave-Structure for a Traveling Wave Tube

    Microsoft Academic Search

    Young-Do Joo; Ashok Kumar Sinha; Yanyu Wei; Gun-Sik Park

    2003-01-01

    A stationary 1-D nonlinear code based on Lagrangian disk model is developed on the basis of a simple set of analytical expressions to study nonlinear dynamics in the helix slow-wave structure used in a traveling wave tube. The loss profiles such as triangular and Gaussian types are modeled as stairsteps and a simple formula is developed to found the loss

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

    PubMed

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

    2011-08-16

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

  10. Structure-based prediction reveals capping motifs that inhibit -helix aggregation

    E-print Network

    Clark, Patricia L.

    amyloid fibers. In contrast, soluble - helices, both right- and left-handed, are usually "capped" on each associate to form multimeric fibers similar to amyloid. Thus, disruption of -helix caps could sequester, similar to those observed in amyloid formation. beta-helix hidden Markov model threading aggregation

  11. Production of disulfide-stabilized transmembrane peptide complexes for structural studies.

    PubMed

    Sharma, Pooja; Kaywan-Lutfi, Mariam; Krshnan, Logesvaran; Byrne, Eamon F X; Call, Melissa Joy; Call, Matthew Edwin

    2013-01-01

    Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences. PMID:23486227

  12. Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

    PubMed Central

    Sharma, Pooja; Kaywan-Lutfi, Mariam; Krshnan, Logesvaran; Byrne, Eamon F. X.; Call, Melissa Joy; Call, Matthew Edwin

    2013-01-01

    Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences. PMID:23486227

  13. Study on Effects of Different Metallic Vane-Loaded Helix Slow-Wave Structures in Traveling-Wave Tubes

    NASA Astrophysics Data System (ADS)

    Yang, Jianhong; Zhang, Yong; Cai, Xueyuan; Li, Lin

    2009-06-01

    The effects of different metallic vane-loaded helix slow-wave structures of a traveling-wave tube are proposed based on the analysis of the Fourier expansions of the exterior region with metallic vanes. The influences of the metallic vanes dimensions on the phase velocity and interaction impedance are considered in detail. The computed data is compared with the reference data in the 0-16 GHz frequency range with a good consistency. The analytical results reveal that the method of using Fourier expansions can contribute effectively to the reducing of the error between the theoretical and experimented data (around 1.2%). By analyzing the computed results, the performances of the helix slow-wave structure, with T-shaped metallic vanes are superior to the sector-shaped with the same designed parameters. Adjustments can be made to the outer radius of T-shaped metallic vanes which then control the dispersion relation showing either negative or positive, and it is similar to sector-shaped vanes by adjusting its inner radius. And with increasing the distance between the helix and metallic vanes, the dispersion characteristics and interaction impedance of the helix slow-wave structure with T-shaped/sector-shaped metallic vane are all improved.

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

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

    E-print Network

    Kim, Taehoon

    2011-12-31

    Membrane proteins with one or a few transmembrane (TM) helices are abundant and often involved in important TM-included signaling and regulation through formation of hetero- and homo-oligomers. Especially, solid-state NMR ...

  16. Predicting residue and helix contacts in membrane proteins

    NASA Astrophysics Data System (ADS)

    Fuchs, Angelika; Kirschner, Andreas; Frishman, Dmitrij

    Helix-helix contacts are an important feature of alpha-helical membrane proteins as they define their characteristic helix bundle structure. No bioinformatics approaches for the prediction of pairwise residue contacts in membrane proteins have existed until recently. In this chapter we describe novel contact prediction methods based on residue coevolution and machine learning techniques specifically geared towards membrane proteins. While contact prediction accuracies are limited to ~10% using co-evolving residues alone, machine learning methods are able to improve these accuracies significantly to more than 25% by using available membrane protein structures as a training dataset and incorporating membrane protein specific sequence features into the prediction process. Importantly, predicted residue contacts allow for identification of interacting transmembrane helices with high accuracy. As different membrane protein structures can be distinguished by their specific pattern of helix interactions, predicted residue contacts may not only serve as structural constraints in modeling experiments, but also constitute valuable information for structural classification of membrane proteins with unknown structure.

  17. The type II transmembrane serine protease matriptase-2--identification, structural features, enzymology, expression pattern and potential roles.

    PubMed

    Ramsay, Andrew J; Reid, Janet C; Velasco, Gloria; Quigley, James P; Hooper, John D

    2008-01-01

    Matriptase-2 (also known as TMPRSS6) is a recently identified member of the type II transmembrane serine protease (TTSP) family. Structurally this enzyme contains a short cytoplasmic amino terminal tail, a transmembrane region, a stem region containing two CUB domains and three LDL receptor class A domains, and at the carboxy terminal a trypsin-like serine protease domain. The matriptase-2 gene and encoded protein are highly conserved in mammals. Biochemically matriptase-2 has substrate specificity similar to the structurally related protein matriptase (also known as MT-SP1). Although the patho-physiological functions of matriptase-2 are not known, its high mRNA expression in liver and several cancers indicate that this enzyme, similar to other TTSPs, will likely have important cell surface associated roles in normal and disease states. Here we overview the identification of matriptase-2, summarise its structural features, biochemistry, expression pattern and disease associations and discuss its potential functions. PMID:17981570

  18. Intrinsic instability of the helix spin structure in MnGe and order-disorder phase transition

    NASA Astrophysics Data System (ADS)

    Altynbaev, E.; Siegfried, S.-A.; Dyadkin, V.; Moskvin, E.; Menzel, D.; Heinemann, A.; Dewhurst, C.; Fomicheva, L.; Tsvyashchenko, A.; Grigoriev, S.

    2014-11-01

    The magnetic structure of the cubic helimagnet MnGe was studied by small-angle neutron scattering in a wide temperature range from 10 to 300 K. We show that MnGe undergoes a complex order-disorder phase transition covering more than 100 K above the critical temperature TN=(130 ±2 ) K. Moreover, the helical structure is intrinsically unstable below TN, since the profile of the reflection at kh˜2 nm-1 associated with the spin helix can be described by the sum of Gaussian and Lorentzian contributions, indicating a stable helix and spin-helix fluctuations, respectively. The Gaussian behavior determines the system at low temperature, whereas the Lorentzian is negligible. With increasing temperature, however, the Lorentzian starts to contribute and dominates at TN. The spin-helix fluctuations are accompanied by intensive spin excitations observed in small-angle neutron scattering as Q -independent scattering at Q

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-03-01

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

  1. Biochemical and structural analysis of Helix pomatia agglutinin. A hexameric lectin with a novel fold.

    PubMed

    Sanchez, Jean-Frederic; Lescar, Julien; Chazalet, Valérie; Audfray, Aymeric; Gagnon, Jean; Alvarez, Richard; Breton, Christelle; Imberty, Anne; Mitchell, Edward P

    2006-07-21

    Helix pomatia agglutinin (HPA) is a N-acetylgalactosamine (GalNAc) binding lectin found in the albumen gland of the roman snail. As a constituent of perivitelline fluid, HPA protects fertilized eggs from bacteria and is part of the innate immunity system of the snail. The peptide sequence deduced from gene cloning demonstrates that HPA belongs to a family of carbohydrate-binding proteins recently identified in several invertebrates. This domain is also present in discoidin from the slime mold Dictyostelium discoideum. Investigation of the lectin specificity was performed with the use of glycan arrays, demonstrating that several GalNAc-containing oligosaccharides are bound and rationalizing the use of this lectin as a cancer marker. Titration microcalorimetry performed on the interaction between HPA and GalNAc indicates an affinity in the 10(-4) M range with an enthalpy-driven binding mechanism. The crystal structure of HPA demonstrates the occurrence of a new beta-sandwich lectin fold. The hexameric quaternary state was never observed previously for a lectin. The high resolution structure complex of HPA with GalNAc characterizes a new carbohydrate binding site and rationalizes the observed preference for alphaGalNAc-containing oligosaccharides. PMID:16704980

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

    SciTech Connect

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

    2009-05-26

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

  3. Structure, mechanism, and regulation of soluble adenylyl cyclases - similarities and differences to transmembrane adenylyl cyclases.

    PubMed

    Steegborn, Clemens

    2014-12-01

    The second messenger cyclic adenosine 3',5'-monophosphate (cAMP) regulates a wide range of physiological processes in almost all organisms. cAMP synthesis is catalyzed by adenylyl cyclases (ACs). All ten mammalian AC isoenzymes (AC1-10) belong to AC Class III, which is defined by sequence homologies in the catalytic domains. Nevertheless, the mammalian AC can be separated into two distinct types, nine transmembrane enzymes (tmAC; AC1-9) and one soluble AC (sAC; AC10). tmACs are mainly regulated by heterotrimeric G-proteins as part of the G-protein coupled receptor pathways, while sAC is directly activated by bicarbonate and Ca²? and acts as a sensor for ATP, Ca²?, and bicarbonate/CO?/pH at various intracellular locations. Mammalian sAC has been implicated in processes such as sperm activation, glucose metabolism, and prostate and skin cancer, making it a potential therapeutic target, and first sAC-specific inhibitors have been developed. Mammalian sAC appears evolutionarily closer related to microbial Class III ACs than to tmACs, and sAC-like bicarbonate activated ACs are indeed found in lower organisms and can contribute, e.g., to virulence regulation in microbial pathogens. Here, we review work on the architecture, catalysis, and physiological and pharmacological regulation of sAC-like enzymes, with a main focus on the mammalian enzyme. We further compare the biochemical, regulatory, and structural characteristics of sAC-like enzymes to the evolutionarily and structurally related mammalian tmACs, pointing out common features as well as sAC-specific properties and modulators. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease. PMID:25193033

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

    SciTech Connect

    Darnell, Gregory; Orgel, Joseph P.R.O.; Pahl, Reinhard; Meredith, Stephen C. (IIT); (UC)

    2008-06-24

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

  5. Crystal structure of the catalytic domain of DESC1, a new member of the type II transmembrane serine proteinase family.

    PubMed

    Kyrieleis, Otto J P; Huber, Robert; Ong, Edgar; Oehler, Ryan; Hunter, Mike; Madison, Edwin L; Jacob, Uwe

    2007-04-01

    DESC1 was identified using gene-expression analysis between squamous cell carcinoma of the head and neck and normal tissue. It belongs to the type II transmembrane multidomain serine proteinases (TTSPs), an expanding family of serine proteinases, whose members are differentially expressed in several tissues. The biological role of these proteins is currently under investigation, although in some cases their participation in specific functions has been reported. This is the case for enteropeptidase, hepsin, matriptase and corin. Some members, including DESC1, are associated with cell differentiation and have been described as tumor markers. TTSPs belong to the type II transmembrane proteins that display, in addition to a C-terminal trypsin-like serine proteinase domain, a differing set of stem domains, a transmembrane segment and a short N-terminal cytoplasmic region. Based on sequence analysis, the TTSP family is subdivided into four subfamilies: hepsin/transmembrane proteinase, serine (TMPRSS); matriptase; corin; and the human airway trypsin (HAT)/HAT-like/DESC subfamily. Members of the hepsin and matriptase subfamilies are known structurally and here we present the crystal structure of DESC1 as a first member of the HAT/HAT-like/DESC subfamily in complex with benzamidine. The proteinase domain of DESC1 exhibits a trypsin-like serine proteinase fold with a thrombin-like S1 pocket, a urokinase-type plasminogen activator-type S2 pocket, to accept small residues, and an open hydrophobic S3/S4 cavity to accept large hydrophobic residues. The deduced substrate specificity for DESC1 differs markedly from that of other structurally known TTSPs. Based on surface analysis, we propose a rigid domain association for the N-terminal SEA domain with the back site of the proteinase domain. PMID:17388811

  6. A structural role for the synaptobrevin 2 transmembrane domain in dense-core vesicle fusion pores.

    PubMed

    Chang, Che-Wei; Hui, Enfu; Bai, Jihong; Bruns, Dieter; Chapman, Edwin R; Jackson, Meyer B

    2015-04-01

    Ca(2+)-triggered release of neurotransmitters and hormones depends on soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) to drive the fusion of the vesicle and plasma membranes. The formation of the SNARE complex by the vesicle SNARE synaptobrevin 2 (syb2) and the two plasma membrane SNAREs syntaxin (syx) and SNAP-25 draws the two membranes together, but the events that follow membrane juxtaposition, and the ways that SNAREs remodel lipid membranes remain poorly understood. The SNAREs syx and syb2 have transmembrane domains (TMDs) that can exert force directly on the lipid bilayers. The TMD of syx influences fusion pore flux in a manner that suggests it lines the nascent fusion pore through the plasma membrane. The TMD of syb2 traverses the vesicle membrane and is the most likely partner to syx in completing a proteinaceous fusion pore through the vesicle membrane, but the role of this vesicle SNARE in fusion pores has yet to be tested. Here amperometry and conductance measurements were performed to probe the function of the syb2 TMD in fusion pores formed during catecholamine exocytosis in mouse chromaffin cells. Fusion pore flux was sensitive to the size and charge of TMD residues near the N terminus; fusion pore conductance was altered by substitutions at these sites. Unlike syx, the syb2 residues that influence fusion pore permeation fell along two ?-helical faces of its TMD, rather than one. These results indicate a role for the syb2 TMD in nascent fusion pores, but in a very different structural arrangement from that of the syx TMD. PMID:25855187

  7. Computational design of a ?–peptide that targets transmembrane helices

    PubMed Central

    Shandler, Scott J.; Korendovych, Ivan V.; Moore, David T.; Smith-Dupont, Kathryn B.; Streu, Craig N.; Litvinov, Rustem I.; Billings, Paul C.; Gai, Feng; Bennett, Joel S.; DeGrado, William F.

    2011-01-01

    The design of ?-peptide foldamers targeting the transmembrane (TM) domains of complex natural membrane proteins has been a formidable challenge. A series of ?-peptides was designed to stably insert in TM orientations in phospholipid bilayers. Their secondary structures and orientation in the phospholipid bilayer was characterized using biophysical methods. Computational methods were then devised to design a ?-peptide that targeted a TM helix of the integrin ?IIb?3. The designed peptide (?-CHAMP) interacts with the isolated target TM domain of the protein, and activates the intact integrin in vitro. PMID:21780757

  8. Improvement of the Performance of a Helix Slow-Wave Structure With a Copper-Embedded Barrel

    Microsoft Academic Search

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

    2010-01-01

    The effect of a copper-embedded barrel on the performance of a helix slow-wave structure (SWS) has been studied theoretically and experimentally. The heat dissipation capability and cold-test characteristics of the SWSs with a monel barrel, a normal copper barrel, and a copper-embedded monel barrel have been analyzed and compared. With its combination of high mechanical strength and good thermal conductivity,

  9. Global Topology & Stability and Local Structure & Dynamics in a Synthetic Spin-Labeled Four-Helix Bundle Protein

    E-print Network

    Gibney, Brian R.

    Global Topology & Stability and Local Structure & Dynamics in a Synthetic Spin-Labeled Four to examine the designed self-association of a four-helix bundle ([R2]2), focusing on the bundle topology an antiparallel topology of the monomers in [MAL- 6-R2]2. The parent [R2]2 and the modified [MAL-6-R2]2 and [CP-R2

  10. Study on Effects of Different Metallic Vane-Loaded Helix Slow-Wave Structures in Traveling-Wave Tubes

    Microsoft Academic Search

    Jianhong Yang; Yong Zhang; Xueyuan Cai; Lin Li

    2009-01-01

    The effects of different metallic vane-loaded helix slow-wave structures of a traveling-wave tube are proposed based on the\\u000a analysis of the Fourier expansions of the exterior region with metallic vanes. The influences of the metallic vanes dimensions\\u000a on the phase velocity and interaction impedance are considered in detail. The computed data is compared with the reference\\u000a data in the 0?16 GHz

  11. Microfabrication and Characterization of W-Band Planar Helix Slow-Wave Structure With StraightEdge Connections

    Microsoft Academic Search

    Ciersiang Chua; Julius M. Tsai; Sheel Aditya; Min Tang; Soon Wee Ho; Zhongxiang Shen; Lei Wang

    2011-01-01

    A slow-wave structure (SWS) consisting of a planar helix with straight-edge connections and incorporating a coplanar waveguide feed has been designed for operation at W-band and has been fabricated using microfabrication technique. On-wafer cold measurements have been carried out on a number of fab- ricated SWSs, and the results are reported here for the first time. The parameters measured are

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

    SciTech Connect

    Samoylovich, M. I., E-mail: samoylovich@technomash.ru [Central Research Technological Institute 'Technomash' (Russian Federation); Talis, A. L. [Russian Academy of Sciences, Nesmeyanov Institute of Organoelement Compounds (Russian Federation)] [Russian Academy of Sciences, Nesmeyanov Institute of Organoelement Compounds (Russian Federation)

    2013-09-15

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

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

    SciTech Connect

    Pilch, D.S.; Shafer, R.H. (Univ. of California, San Francisco (United States)); Levenson, C. (Cetus Corp., Emeryville, CA (United States))

    1991-06-25

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

  14. Three complete turns of a 310-helix at atomic resolution: the crystal structure of Z-(Aib)11-OtBu

    Microsoft Academic Search

    Renate Gessmann; Kyriacos Petratos

    2003-01-01

    The crystal structure of the synthetic protected oligopeptide Z-(Aib)11-OtBu was determined by x-ray crystallography. The undecapeptide folds in a regular 310-helix with nine consecutive 4 ? 1 hydrogen bonds. At present, this is the largest available structure of a homopeptide (including homopeptides consisting of standard amino acids) and also the longest observed regular 310-helix at atomic resolution. Z-(Aib)11-OtBu crystallizes readily

  15. The inhomogeneous dielectric loading effects of practical helix supports on the interaction impedance of the slow-wave structure of a TWT

    Microsoft Academic Search

    Pradip K. Jain; B. N. Basu

    1992-01-01

    The helical slow-wave structure of a traveling-wave tube is usually supported by wedge-shaped bars or rods or circular or rectangular cross section. Such supports present an inhomogeneous loading of the helix caused by the variation of the effective permittivity of the helix surrounding with the radial coordinate. In one of the approaches to the analysis of such structures for dispersion

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

    PubMed Central

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

    2012-01-01

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

  17. GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity

    Microsoft Academic Search

    George W. Kemble; Yoav I. Henis; Judith M. White

    1993-01-01

    We investigated the influence of a glycosyl- phosphatidylinositol (GPI) anchor on the ectodomain of the influenza hemagglutinin (HA) by replacing the wild type (wt) transmembrane and cytoplasmic do- mains with a GPI lipid anchor. GPI-anchored HA (GPI-HA) was transported to the cell surface with equal efficiency and at the same rate as wt-HA. Like wt-HA, cell surface GPI-HA, and its

  18. Linear theory of rectangular helix slow-wave structure for traveling wave tube

    Microsoft Academic Search

    Cheng-fang Fu; Zhao Bo

    2011-01-01

    Linear gain properties of a rectangular helix traveling wave tubes (TWT) loaded a solid sheet electron beam are investigated. The working dispersion equation was obtained by the self-consistent field theory, and then small signal gain of it was computed. The numerical results show that, the small signal gain and the bandwidth increase as the beam current increasing; while the beam

  19. The Arabidopsis Histone Methyltransferase SUVR4 Binds Ubiquitin via a Domain with a Four-Helix Bundle Structure

    PubMed Central

    Rahman, Mohummad Aminur; Kristiansen, Per E.; Veiseth, Silje V.; Andersen, Jan Terje; Yap, Kyoko L.; Zhou, Ming-Ming; Sandlie, Inger; Thorstensen, Tage; Aalen, Reidunn B.

    2014-01-01

    In eukaryotes, different chromatin states facilitate or repress gene expression and restrict the activity of transposable elements. Post-translational modifications (PTMs) of amino acid residues on the N-terminal tails of histones are suggested to define such states. The histone lysine methyltransferase (HKMTase) SU(VAR)3-9 RELATED4 (SUVR4) of Arabidopsis thaliana functions as a repressor of transposon activity. Binding of ubiquitin by the WIYLD domain facilitates the addition of two methyl groups to monomethylated lysine 9 of histone H3. By using nuclear magnetic resonance (NMR) spectroscopy, we identified SUVR4 WIYLD (S4WIYLD) as a domain with a four-helix bundle structure, in contrast to three-helix bundles of other ubiquitin binding domains. NMR titration analyses showed that residues of helix ?1 (Q38, L39, and D40) and helix ?4 (N68, T70, A71, V73, D74, I76, S78, and E82) of S4WIYLD and residues between the first and second ?-strands (T9 and G10) and on ?-strands 3 (R42, G47, K48, and Q49) and 4 (H68, R72, and L73) undergo significant chemical shift changes when the two proteins interact. A model of the complex, generated using HADDOCK, suggests that the N-terminal and C-terminal parts of S4WIYLD constitute a surface that interacts with charged residues close to the hydrophobic patch of ubiquitin. The WIYLD domains of the closely related SUVR1 and SUVR2 Arabidopsis proteins also bind ubiquitin, indicating that this is a general feature of this domain. The question of whether SUVR proteins act as both readers of monoubiquitinated H2B and writers of histone PTMs is discussed. PMID:24625295

  20. Solution Nuclear Magnetic Resonance Structure of Membrane-Integral Diacylglycerol Kinase

    Microsoft Academic Search

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

    2009-01-01

    Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer with the use of solution nuclear magnetic resonance. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK's

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

    SciTech Connect

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

    2011-09-28

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

  2. Membrane Protein Crystallization in Lipidic Mesophases. Hosting lipid affects on the crystallization and structure of a transmembrane peptide.

    PubMed

    Höfer, Nicole; Aragão, David; Lyons, Joseph A; Caffrey, Martin

    2011-04-01

    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 synthesised 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. PMID:22933857

  3. Confinement in nanopores can destabilize ?-helix folding proteins and stabilize the ? structures

    NASA Astrophysics Data System (ADS)

    Javidpour, Leili; Sahimi, Muhammad

    2011-09-01

    Protein folding in confined media has attracted wide attention over the past decade due to its importance in both in vivo and in vitro applications. Currently, it is generally believed that protein stability increases by decreasing the size of the confining medium, if its interaction with the confining walls is repulsive, and that the maximum folding temperature in confinement occurs for a pore size only slightly larger than the smallest dimension of the folded state of a protein. Protein stability in pore sizes, very close to the size of the folded state, has not however received the attention that it deserves. Using detailed, 0.3-ms-long molecular dynamics simulations, we show that proteins with an ?-helix native state can have an optimal folding temperature in pore sizes that do not affect the folded-state structure. In contradiction to the current theoretical explanations, we find that the maximum folding temperature occurs in larger pores for smaller ?-helices. In highly confined pores the free energy surface becomes rough, and a new barrier for protein folding may appear close to the unfolded state. In addition, in small nanopores the protein states that contain the ? structures are entropically stabilized, in contrast to the bulk. As a consequence, folding rates decrease notably and the free energy surface becomes rougher. The results shed light on many recent experimental observations that cannot be explained by the current theories, and demonstrate the importance of entropic effects on proteins' misfolded states in highly confined environments. They also support the concept of passive effect of chaperonin GroEL on protein folding by preventing it from aggregation in crowded environment of biological cells, and provide deeper clues to the ? ? ? conformational transition, believed to contribute to Alzheimer's and Parkinson's diseases. The strategy of protein and enzyme stabilization in confined media may also have to be revisited in the case of tight confinement. For in silico studies of protein folding in confined media, use of non-Go potentials may be more appropriate.

  4. Simplified Approach to the Nonlinear Analysis in Helix Slow-Wave-Structure for a Traveling Wave Tube

    NASA Astrophysics Data System (ADS)

    Joo, Young-Do; Sinha, Ashok Kumar; Wei, Yanyu; Park, Gun-Sik

    2003-11-01

    A stationary 1-D nonlinear code based on Lagrangian disk model is developed on the basis of a simple set of analytical expressions to study nonlinear dynamics in the helix slow-wave structure used in a traveling wave tube. The loss profiles such as triangular and Gaussian types are modeled as stairsteps and a simple formula is developed to found the loss at a plane for such loss profile. In contrast to the earlier works in nonlinear theory, at present, no numerical method is used at any stage. The method, introduced in this work, is general in nature because it can handle (a) multi-section structure with sever, (b) different loss profiles, namely, center (Gaussian) and tip (triangular: increasing or decreasing), (c) space charge effect on the electrons, (d) backward waves arising due to reflections, etc. Accuracy of the theory and code is verified with comparison of the computed present results with the results from simulation code MAGIC and published elsewhere and found to be in good agreement. The generation and suppression of the harmonic power are studied for a typical structure. It is found that the introduction of resynchronization section of the reduced pitch enhances the fundamental power with the reduction of the second harmonic power. In addition, the method can be used for any helix slow-wave-structure consisting of homogeneous/inhomogeneous dielectric support rods in isotropic/anisotropic overall metallic enclosure, because the axial propagation constant and interaction impedance obtained for any structure and model such as sheath and tape helix approximations or from any simulation codes can be used as the input in the program to make the code more general.

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

    Microsoft Academic Search

    Jason A. Holland; David W. Hoffman

    1996-01-01

    A 30 nt RNA with a sequence designed to form an intramolecular triple helix was analyzed by one- and two-dimensional NMR spectroscopy and UV absorption measurements. NMR data show that the RNA contains seven pyrimidine-purine-pyrimidine base triples stabilized by Watson-Crick and Hoog- steen interactions. The temperature dependence of the imino proton resonances, as well as UV absorption data, indicate that

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

    PubMed Central

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

    2013-01-01

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

  7. Yummy Gummy Double Helix

    NSDL National Science Digital Library

    Wellcome Trust Sange Institute

    2013-10-10

    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.

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

    PubMed

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

    2015-03-11

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

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

    PubMed Central

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

    2015-01-01

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

  10. Crystal Structure of Human T Cell Leukemia Virus Type 1 gp21 Ectodomain Crystallized as a Maltose-Binding Protein Chimera Reveals Structural Evolution of Retroviral Transmembrane Proteins

    Microsoft Academic Search

    Bostjan Kobe; Bruce E. Kemp; Pantelis Poumbourios

    1999-01-01

    Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5- angstrom resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the

  11. Explicit Water Near the Catalytic I Helix Thr in the Predicted Solution Structure of CYP2A4

    PubMed Central

    Gorokhov, Anna; Negishi, Masahiko; Johnson, Eric F.; Pedersen, Lars C.; Perera, Lalith; Darden, Tom A.; Pedersen, Lee G.

    2003-01-01

    The solution structure of mouse cytochrome P450 2A4 (CYP2A4), a monooxygenase of deoxysteroids, was obtained using homology modeling and molecular dynamics. The solvent-equilibrated CYP2A4 preserves the essential features of CYP450s. A comparison of the models CYP2A4 and CYP2A4 with testosterone bound CYP2A4/T illustrates the changes induced by the binding of the substrate. Experimental evidence links four amino acid residues to the catalytic activity, substrate specificity, and regioselectivity of this enzyme. Three of the four amino acids are found within contact distance of the testosterone substrate, and therefore may control the binding of the substrate through direct interaction. Remarkably, a water complex previously observed in x-ray crystal structure forms near the bulge in the central I helix that contains a conserved Thr. The properties of the I helix are computed in the context of the presence or absence of ligand. PMID:12524265

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

    PubMed Central

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

    2014-01-01

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

  13. Structure and dynamics of the lipid modifications of a transmembrane ?-helical peptide determined by ²H solid-state NMR spectroscopy.

    PubMed

    Penk, Anja; Müller, Matthias; Scheidt, Holger A; Langosch, Dieter; Huster, Daniel

    2011-03-01

    The fusion of biological membranes is mediated by integral membrane proteins with ?-helical transmembrane segments. Additionally, those proteins are often modified by the covalent attachment of hydrocarbon chains. Previously, a series of de novo designed ?-helical peptides with mixed Leu/Val sequences was presented, mimicking fusiogenically active transmembrane segments in model membranes (Hofmann et al., Proc. Natl. Acad. Sci. USA 101 (2004) 14776-14781). From this series, we have investigated the peptide LV16 (KKKW LVLV LVLV LVLV LVLV KKK), which was synthesized featuring either a free N-terminus or a saturated N-acylation of 2, 8, 12, or 16 carbons. We used ²H and ³¹P NMR spectroscopy to investigate the structure and dynamics of those peptide lipid modifications in POPC and DLPC bilayers and compared them to the hydrocarbon chains of the surrounding membrane. Except for the C2 chain, all peptide acyl chains were found to insert well into the membrane. This can be explained by the high local lipid concentrations the N-terminal lipid chains experience. Further, the insertion of these peptides did not influence the membrane structure and dynamics as seen from the ²H and ³¹P NMR data. In spite of the fact that the longer acyl chains insert into the membrane, they do not adapt their lengths to the thickness of the bilayer. Even the C16 lipid chain on the peptide, which could match the length of the POPC palmitoyl chain, exhibited lower order parameters in the upper chain, which get closer and finally reach similar values in the lower chain region. ²H NMR square law plots reveal motions of slightly larger amplitudes for the peptide lipid chains compared to the surrounding phospholipids. In spite of the significantly different chain lengths of the acylations, the fraction of gauche defects in the inserted chains is constant. PMID:21192915

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

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

  16. Predicting the topology of transmembrane helical proteins using mean burial propensity and a hidden-Markov-model-based method.

    PubMed

    Zhou, Hongyi; Zhou, Yaoqi

    2003-07-01

    Helices in membrane spanning regions are more tightly packed than the helices in soluble proteins. Thus, we introduce a method that uses a simple scale of burial propensity and a new algorithm to predict transmembrane helical (TMH) segments and a positive-inside rule to predict amino-terminal orientation. The method (the topology predictor of transmembrane helical proteins using mean burial propensity [THUMBUP]) correctly predicted the topology of 55 of 73 proteins (or 75%) with known three-dimensional structures (the 3D helix database). This level of accuracy can be reached by MEMSAT 1.8 (a 200-parameter model-recognition method) and a new HMM-based method (a 111-parameter hidden Markov model, UMDHMM(TMHP)) if they were retrained with the 73-protein database. Thus, a method based on a physiochemical property can provide topology prediction as accurate as those methods based on more complicated statistical models and learning algorithms for the proteins with accurately known structures. Commonly used HMM-based methods and MEMSAT 1.8 were trained with a combination of the partial 3D helix database and a 1D helix database of TMH proteins in which topology information were obtained by gene fusion and other experimental techniques. These methods provide a significantly poorer prediction for the topology of TMH proteins in the 3D helix database. This suggests that the 1D helix database, because of its inaccuracy, should be avoided as either a training or testing database. A Web server of THUMBUP and UMDHMM(TMHP) is established for academic users at http://www.smbs.buffalo.edu/phys_bio/service.htm. The 3D helix database is also available from the same Web site. PMID:12824500

  17. The structure of the CD3 ?? transmembrane dimer in POPC and raft-like lipid bilayer: a molecular dynamics study.

    PubMed

    Petruk, Ariel Alcides; Varriale, Sonia; Coscia, Maria Rosaria; Mazzarella, Lelio; Merlino, Antonello; Oreste, Umberto

    2013-11-01

    Plasma membrane lipids significantly affect assembly and activity of many signaling networks. The present work is aimed at analyzing, by molecular dynamics simulations, the structure and dynamics of the CD3 ?? dimer in palmitoyl-oleoyl-phosphatidylcholine bilayer (POPC) and in POPC/cholesterol/sphingomyelin bilayer, which resembles the raft membrane microdomain supposed to be the site of the signal transducing machinery. Both POPC and raft-like environment produce significant alterations in structure and flexibility of the CD3 ?? with respect to nuclear magnetic resonance (NMR) model: the dimer is more compact, its secondary structure is slightly less ordered, the arrangement of the Asp6 pair, which is important for binding to the Arg residue in the alpha chain of the T cell receptor (TCR), is stabilized by water molecules. Different interactions of charged residues with lipids at the lipid-cytoplasm boundary occur when the two environments are compared. Furthermore, in contrast to what is observed in POPC, in the raft-like environment correlated motions between transmembrane and cytoplasmic regions are observed. Altogether the data suggest that when the TCR complex resides in the raft domains, the CD3 ?? dimer assumes a specific conformation probably necessary to the correct signal transduction. PMID:23896554

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

    SciTech Connect

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

    2009-01-01

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

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

    SciTech Connect

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

    2009-11-01

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

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

    SciTech Connect

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

    2009-01-01

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

  1. Perturbations of the Straight Transmembrane ?-Helical Structure of the Amyloid Precursor Protein Affect Its Processing by ?-Secretase*

    PubMed Central

    Lemmin, Thomas; Dimitrov, Mitko; Fraering, Patrick C.; Dal Peraro, Matteo

    2014-01-01

    The amyloid precursor protein (APP) is a widely expressed type I transmembrane (TM) glycoprotein present at the neuronal synapse. The proteolytic cleavage by ?-secretase of its C-terminal fragment produces amyloid-? (A?) peptides of different lengths, the deposition of which is an early indicator of Alzheimer disease. At present, there is no consensus on the conformation of the APP-TM domain at the biological membrane. Although structures have been determined by NMR in detergent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight ?-helical conformation once embedded in a membrane bilayer. However, APP-TM is highly flexible, and its secondary structure is strongly influenced by the surrounding lipid environment, as when enclosed in detergent micelles. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance spectroscopy. These structural and dynamic features are critical in the proteolytic processing of APP by the ?-secretase enzyme, as suggested by a series of Gly700 mutants. Affecting the hydration and flexibility of APP-TM, these mutants invariantly show an increase in the production of A?38 compared with A?40 peptides, which is reminiscent of the effect of ?-secretase modulators inhibitors. PMID:24469457

  2. The discovery of the double helix structure of DNA, James Watson, 3D animation with basic narrationSite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-10-06

    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."

  3. Role of transmembrane segment 10 in efflux mediated by the staphylococcal multidrug transport protein QacA.

    PubMed

    Xu, Zhiqiang; O'Rourke, Brendon A; Skurray, Ronald A; Brown, Melissa H

    2006-01-13

    The staphylococcal multidrug exporter QacA confers resistance to a wide range of structurally dissimilar monovalent and bivalent cationic antimicrobial compounds. To understand the functional importance of transmembrane segment 10, which is thought to be involved in substrate binding, cysteine-scanning mutagenesis was performed in which 35 amino acid residues in the putative transmembrane helix and its flanking regions were replaced in turn with cysteine. Solvent accessibility analysis of the introduced cysteine residues using fluorescein maleimide indicated that transmembrane segment 10 of QacA contains a 20-amino-acid hydrophobic core and may extend from Pro-309 to Ala-334. Phenotypic analysis and fluorimetric transport assays of these mutants showed that Gly-313 is important for the efflux of both monovalent and bivalent cationic substrates, whereas Asp-323 is only important for the efflux of bivalent substrates and probably forms part of the bivalent substrate-binding site(s) together with Met-319. Furthermore, the effects of N-ethyl-maleimide treatment on ethidium and 4',6-diamidino-2-phenylindole export mediated by the QacA mutants suggest that the face of transmembrane segment 10 that contains Asp-323 may also be close to the monovalent substrate-binding site(s), making this helix an integral component of the QacA multidrug-binding pocket. PMID:16282328

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

    PubMed Central

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

    2014-01-01

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

  5. Crystal structure of the complete integrin [alpha]V[beta]3 ectodomain plus an [alpah/beta] transmembrane fragment

    SciTech Connect

    Xiong, Jian-Ping; Mahalingham, Bhuvaneshwari; Alonso, Jose Luis; Borrelli, Laura Ann; Rui, Xianliang; Anand, Saurabh; Hyman, Bradley T.; Rysiok, Thomas; Müller-Pompalla, Dirk; Goodman, Simon L.; Arnaout, M. Amin; (Harvard-Med); (Merck-Serono)

    2010-02-22

    We determined the crystal structure of 1TM-{alpha}V{beta}3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the {alpha}V and {beta}3 subunits. 1TM-{alpha}V{beta}3 is more compact and less active in solution when compared with {Delta}TM-{alpha}V{beta}3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the {alpha}-{beta} interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length {alpha}V{beta}3 on live cells yields a donor-membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2-thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state.

  6. The conserved transmembrane proteoglycan Perdido/Kon-tiki is essential for myofibrillogenesis and sarcomeric structure in Drosophila

    PubMed Central

    Pérez-Moreno, Juan J.; Bischoff, Marcus; Martín-Bermudo, Maria D.; Estrada, Beatriz

    2014-01-01

    ABSTRACT Muscle differentiation requires the assembly of high-order structures called myofibrils, composed of sarcomeres. Even though the molecular organization of sarcomeres is well known, the mechanisms underlying myofibrillogenesis are poorly understood. It has been proposed that integrin-dependent adhesion nucleates myofibrils at the periphery of the muscle cell to sustain sarcomere assembly. Here, we report a role for the gene perdido (perd, also known as kon-tiki, a transmembrane chondroitin proteoglycan) in myofibrillogenesis. Expression of perd RNAi in muscles, prior to adult myogenesis, can induce misorientation and detachment of Drosophila adult abdominal muscles. In comparison to controls, perd-depleted muscles contain fewer myofibrils, which are localized at the cell periphery. These myofibrils are detached from each other and display a defective sarcomeric structure. Our results demonstrate that the extracellular matrix receptor Perd has a specific role in the assembly of myofibrils and in sarcomeric organization. We suggest that Perd acts downstream or in parallel to integrins to enable the connection of nascent myofibrils to the Z-bands. Our work identifies the Drosophila adult abdominal muscles as a model to investigate in vivo the mechanisms behind myofibrillogenesis. PMID:24794494

  7. Solution structure of the transmembrane 2 domain of the human melanocortin-4 receptor in sodium dodecyl sulfate (SDS) micelles and the functional implication of the D90N mutant.

    PubMed

    Yun, Ji-Hye; Kim, Minsup; Kim, Kuglae; Lee, Dongju; Jung, Youngjin; Oh, Daeseok; Ko, Yoon-Joo; Cho, Art E; Cho, Hyun-Soo; Lee, Weontae

    2015-06-01

    The melanocortin receptors (MCRs) are members of the G protein-coupled receptor (GPCR) 1 superfamily with seven transmembrane (TM) domains. Among them, the melanocortin-4 receptor (MC4R) subtype has been highlighted recently by genetic studies in obese humans. In particular, in a patient with severe early-onset obesity, a novel heterozygous mutation in the MC4R gene was found in an exchange of Asp to Asn in the 90th amino acid residue located in the TM 2 domain (MC4R(D90N)). Mutations in the MC4R gene are the most frequent monogenic causes of severe obesity and are described as heterozygous with loss of function. We determine solution structures of the TM 2 domain of MC4R (MC4R(TM2)) and compared secondary structure of Asp90 mutant (MC4R(TM2-D90N)) in a micelle environment by nuclear magnetic resonance (NMR) spectroscopy. NMR structure shows that MC4R(TM2) forms a long ?-helix with a kink at Gly98. Interestingly, the structure of MC4R(TM2-D90N) is similar to that of MC4R(TM2) based on data from CD and NMR spectrum. However, the thermal stability and homogeneity of MC4R(D90N) is quite different from those of MC4R. The structure from molecular modeling suggests that Asp90(2.50) plays a key role in allosteric sodium ion binding. Our data suggest that the sodium ion interaction of Asp90(2.50) in the allosteric pocket of MC4R is essential to its function, explaining the loss of function of the MC4R(D90N) mutant. PMID:25753114

  8. Three Dimensional Structure Prediction of Fatty Acid Binding Site on Human Transmembrane Receptor CD36.

    PubMed

    Tarhda, Zineb; Semlali, Oussama; Kettani, Anas; Moussa, Ahmed; Abumrad, Nada A; Ibrahimi, Azeddine

    2013-01-01

    CD36 is an integral membrane protein which is thought to have a hairpin-like structure with alpha-helices at the C and N terminals projecting through the membrane as well as a larger extracellular loop. This receptor interacts with a number of ligands including oxidized low density lipoprotein and long chain fatty acids (LCFAs). It is also implicated in lipid metabolism and heart diseases. It is therefore important to determine the 3D structure of the CD36 site involved in lipid binding. In this study, we predict the 3D structure of the fatty acid (FA) binding site [127-279 aa] of the CD36 receptor based on homology modeling with X-ray structure of Human Muscle Fatty Acid Binding Protein (PDB code: 1HMT). Qualitative and quantitative analysis of the resulting model suggests that this model was reliable and stable, taking in consideration over 97.8% of the residues in the most favored regions as well as the significant overall quality factor. Protein analysis, which relied on the secondary structure prediction of the target sequence and the comparison of 1HMT and CD36 [127-279 aa] secondary structures, led to the determination of the amino acid sequence consensus. These results also led to the identification of the functional sites on CD36 and revealed the presence of residues which may play a major role during ligand-protein interactions. PMID:24348024

  9. Three Dimensional Structure Prediction of Fatty Acid Binding Site on Human Transmembrane Receptor CD36

    PubMed Central

    Tarhda, Zineb; Semlali, Oussama; Kettani, Anas; Moussa, Ahmed; Abumrad, Nada A.; Ibrahimi, Azeddine

    2013-01-01

    CD36 is an integral membrane protein which is thought to have a hairpin-like structure with alpha-helices at the C and N terminals projecting through the membrane as well as a larger extracellular loop. This receptor interacts with a number of ligands including oxidized low density lipoprotein and long chain fatty acids (LCFAs). It is also implicated in lipid metabolism and heart diseases. It is therefore important to determine the 3D structure of the CD36 site involved in lipid binding. In this study, we predict the 3D structure of the fatty acid (FA) binding site [127–279 aa] of the CD36 receptor based on homology modeling with X-ray structure of Human Muscle Fatty Acid Binding Protein (PDB code: 1HMT). Qualitative and quantitative analysis of the resulting model suggests that this model was reliable and stable, taking in consideration over 97.8% of the residues in the most favored regions as well as the significant overall quality factor. Protein analysis, which relied on the secondary structure prediction of the target sequence and the comparison of 1HMT and CD36 [127–279 aa] secondary structures, led to the determination of the amino acid sequence consensus. These results also led to the identification of the functional sites on CD36 and revealed the presence of residues which may play a major role during ligand-protein interactions. PMID:24348024

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

    PubMed Central

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

    2013-01-01

    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

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

    PubMed

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

    2013-01-01

    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 (ZNRF3(ecto)), a signalling-competent Furin1-Furin2 (Fu1-Fu2) fragment of Rspo2 (Rspo2(Fu1-Fu2)), and Rspo2(Fu1-Fu2) in complex with ZNRF3(ecto), or RNF43(ecto). A prominent loop in Fu1 clamps into equivalent grooves in the ZNRF3(ecto) and RNF43(ecto) surface. Rspo binding enhances dimerization of ZNRF3(ecto) but not of RNF43(ecto). 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

  12. Role of sequence and membrane composition in structure of transmembrane domain of Amyloid Precursor Protein

    NASA Astrophysics Data System (ADS)

    Straub, John

    2013-03-01

    Aggregation of proteins of known sequence is linked to a variety of neurodegenerative disorders. The amyloid ? (A?) protein associated with Alzheimer's Disease (AD) is derived from cleavage of the 99 amino acid C-terminal fragment of Amyloid Precursor Protein (APP-C99) by ?-secretase. Certain familial mutations of APP-C99 have been shown to lead to altered production of A? protein and the early onset of AD. We describe simulation studies exploring the structure of APP-C99 in micelle and membrane environments. Our studies explore how changes in sequence and membrane composition influence (1) the structure of monomeric APP-C99 and (2) APP-C99 homodimer structure and stability. Comparison of simulation results with recent NMR studies of APP-C99 monomers and dimers in micelle and bicelle environments provide insight into how critical aspects of APP-C99 structure and dimerization correlate with secretase processing, an essential component of the A? protein aggregation pathway and AD.

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

    PubMed Central

    2014-01-01

    EphA1 is a receptor tyrosine kinase (RTK) that plays a key role in developmental processes, including guidance of the migration of axons and cells in the nervous system. EphA1, in common with other RTKs, contains an N-terminal extracellular domain, a single transmembrane (TM) ?-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

  14. CROSS-DISCIPLINARY PHYSICS AND RELATED AREAS OF SCIENCE AND TECHNOLOGY: Radio-Frequency Characteristics of a Printed Rectangular Helix Slow-Wave Structure

    Microsoft Academic Search

    Cheng-Fang Fu; Yan-Yu Wei; Wen-Xiang Wang; Yu-Bin Gong

    2008-01-01

    A new type of printed rectangular helix slow-wave structure (SWS) is investigated using the field-matching method and the electromagnetic integral equations at the boundaries. The radio-frequency characteristics including the dispersion equation and the coupling impedance for transverse antisymmetric (odd) modes of this structure are analysed. The numerical results agree well with the results obtained by the EM simulation software HFSS.

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

    PubMed Central

    Marko, John F.; Neukirch, Sébastien

    2014-01-01

    We present a free energy model for structural transitions of the DNA double helix driven by tensile and torsional stress. Our model is coarse grained, and is based on semiflexible polymer descriptions of B-DNA, underwound L-DNA, and highly overwound P-DNA. The statistical-mechanical model of plectonemic supercoiling previously developed for B-DNA is applied to semiflexible polymer models of P and L-DNA, to obtain a model of DNA structural transitions in quantitative accord with experiment. We identify two distinct plectonemic states, one “inflated” by electrostatic repulsion and thermal fluctuations, and the other “collapsed”, with the two double helices inside the supercoils driven to close contact. We find that supercoiled B and L are stable only in inflated form, while supercoiled P is always collapsed. We also predict the behavior and experimental signatures of highly underwound “Q”-DNA, the left-handed analog of P-DNA; as for P, supercoiled Q is always collapsed. Overstretched “S”-DNA and strand-separated “stress-melted” DNA are also included in our model, allowing prediction of a global phase diagram for forces up to 1000 pN and torques between ±60 pN nm, or in terms of linking number density, from ? = ?5 to +3. PMID:24483501

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

    PubMed

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

    2014-01-01

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

  17. Structural Basis for Autoinhibition of CTP:Phosphocholine Cytidylyltransferase (CCT), the Regulatory Enzyme in Phosphatidylcholine Synthesis, by Its Membrane-binding Amphipathic Helix*

    PubMed Central

    Lee, Jaeyong; Taneva, Svetla G.; Holland, Bryan W.; Tieleman, D. Peter; Cornell, Rosemary B.

    2014-01-01

    CTP:phosphocholine cytidylyltransferase (CCT) interconverts between an inactive soluble and active membrane-bound form in response to changes in membrane lipid composition. Activation involves disruption of an inhibitory interaction between the ?E helices at the base of the active site and an autoinhibitory (AI) segment in the regulatory M domain and membrane insertion of the M domain as an amphipathic helix. We show that in the CCT soluble form the AI segment functions to suppress kcat and elevate the Km for CTP. The crystal structure of a CCT dimer composed of the catalytic and AI segments reveals an AI-?E interaction as a cluster of four amphipathic helices (two ?E and two AI helices) at the base of the active sites. This interaction corroborates mutagenesis implicating multiple hydrophobic residues within the AI segment that contribute to its silencing function. The AI-?E interaction directs the turn at the C-terminal end of the AI helix into backbone-to-backbone contact with a loop (L2) at the opening to the active site, which houses the key catalytic residue, lysine 122. Molecular dynamics simulations suggest that lysine 122 side-chain orientations are constrained by contacts with the AI helix-turn, which could obstruct its engagement with substrates. This work deciphers how the CCT regulatory amphipathic helix functions as a silencing device. PMID:24275660

  18. Helix Movement is Coupled to Displacement of the Second Extracellular Loop in Rhodopsin Activation

    PubMed Central

    Ahuja, Shivani; Hornak, Viktor; Yan, Elsa C. Y.; Syrett, Natalie; Goncalves, Joseph A.; Hirshfeld, Amiram; Ziliox, Martine; Sakmar, Thomas P.; Sheves, Mordechai; Reeves, Philip J.; Smith, Steven O.; Eilers, Markus

    2009-01-01

    The second extracellular loop (EL2) of rhodopsin forms a cap over the binding site of its photoreactive 11-cis retinylidene chromophore. A critical question has been whether EL2 forms a reversible gate that opens upon activation or acts as a rigid barrier. Distance measurements using solid-state 13C NMR spectroscopy between the retinal chromophore and the ?4 strand of EL2 show the loop is displaced from the retinal binding site upon activation, and there is a rearrangement in the hydrogen-bonding networks connecting EL2 with the extracellular ends of transmembrane helices H4, H5 and H6. NMR measurements further reveal that structural changes in EL2 are coupled to the motion of helix H5 and breaking of the ionic lock that regulates activation. These results provide a comprehensive view of how retinal isomerization triggers helix motion and activation in this prototypical G protein-coupled receptor. PMID:19182802

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

    PubMed

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

    2010-09-01

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

  20. Detergents Modulate Dimerization, but not Helicity, of the Glycophorin A Transmembrane Domain

    E-print Network

    Detergents Modulate Dimerization, but not Helicity, of the Glycophorin A Transmembrane Domain to measure dimerization of the glyco- phorin A transmembrane helix in detergent micelles. The observed Kd is at least two orders of magnitude weaker in sodium dodecyl sulfate than it is in zwitterionic detergents

  1. Structure-function analysis of a double-mutant cystic fibrosis transmembrane conductance regulator protein occurring in disorders related to cystic fibrosis

    Microsoft Academic Search

    Pascale Fanen; Jérôme Clain; Régis Labarthe; Philippe Hulin; Emmanuelle Girodon; Patrick Pagesy; Michel Goossens; Aleksander Edelman

    1999-01-01

    A number of disorders related to cystic fibrosis have been described since the cloning of the cystic fibrosis gene, including infertility due to the congenital bilateral absence of the vas deferens. We have identified, in several patients, complex cystic fibrosis transmembrane conductance regulator genotypes like double-mutant alleles. We have now analyzed the structure-function relationships of one of these mutants, R74W-D1270N

  2. New simulated annealing approach considering helix bending applied to determine the 8.8Å structure of 15-protofilament microtubules.

    PubMed

    Ogura, Toshihiko; Yajima, Hiroaki; Nitta, Ryo; Hirokawa, Nobutaka; Sato, Chikara

    2014-11-01

    The helix is an important motif in biological architectures. The helical structures of nanoscale proteins are principally determined by three-dimensional (3D) reconstruction from electron micrographs. However, bending or distortion of flexible helices and the low contrast of the images recorded by cryo-electron microscopy, prevent the analysis from reaching high resolution. We have developed a novel helical reconstruction method that overcomes these issues, and present the processing of microtubule images to demonstrate its application. Cropping long helical structures into small square pieces allows bending or distortion of the helices to be accounted for. The initial image-frames are automatically positioned assuming perfect helical symmetry. A simulated annealing (SA)-based algorithm is then used to adjust the framing. This is guided by the contrast of 2D averages, which serve as an accuracy index. After the initial 3D reconstruction, the position and orientation of each average image is iteratively adjusted to give the best match between the input average and the reprojection from the reconstruction. Finally, reconstructions from images recorded at different defocus values, are aligned and averaged to compensate the contrast transfer modulation and improve the resolution. The method successfully determined the structure of a 15-protofilament microtubule. The 8.8Å resolution (7.8Å using the 0.143 FSC criterion) attained allows differences between the ?- and ?- tubulins to be discerned in the absence of a molecular landmark such as microtubule-associated proteins, for the first time by electron microscopy. The SA-based method is applicable to other helical protein complexes and in general to helical structures. PMID:25193738

  3. Conformational variability of the N-terminal helix in the structure of ribosomal protein S15

    E-print Network

    Ramakrishnan, Venki

    basic protein. The protein from Bacillus stearothermophilus has a molecular weight of 10 for the study of protein­RNA interactions. Results: The crystal structure of S15 from Bacillus stearothermophilus has been solved to 2.1 � resolution. The structure consists of four helices. Three

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

    SciTech Connect

    Yuan, Ping; Swanson, Kurt A.; Leser, George P.; Paterson, Reay G.; Lamb, Robert A.; Jardetzky, Theodore S. (Stanford-MED); (NWU)

    2014-10-02

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

  5. Partitivirus Structure Reveals a 120-Subunit, Helix-Rich Capsid with Distinctive Surface Arches

    E-print Network

    Baker, Timothy S.

    partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3 A° resolution

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

    PubMed

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

    2013-09-13

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

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

    E-print Network

    Schubert, Christian Reinhold

    2009-01-01

    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 ...

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

    PubMed

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

    2015-04-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to the ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. To investigate the conformation of the CFTR in the membrane, we applied the small-angle x-ray scattering (SAXS) technique on microsomal membranes extracted from NIH/3T3 cells permanentely transfected with wild-type (WT) CFTR and with CFTR carrying the ?F508 mutation. The electronic density profile of the membranes was calculated from the SAXS data, assuming the lipid bilayer electronic density to be composed by a series of Gaussian shells. The data indicate that membranes in the microsome vesicles, that contain mostly endoplasmic reticulum membranes, are oriented in the outside-out conformation. Phosphorylation does not change significantly the electronic density profile, while dephosphorylation produces a significant modification in the inner side of the profile. Thus, we conclude that the CFTR and its associated protein complex in microsomes are mostly phosphorylated. The electronic density profile of the ?F508-CFTR microsomes is completely different from WT, suggesting a different assemblage of the proteins in the membranes. Low-temperature treatment of cells rescues the ?F508-CFTR protein, resulting in a conformation that resembles the WT. Differently, treatment with the corrector VX-809 modifies the electronic profile of ?F508-CFTR membrane, but does not recover completely the WT conformation. To our knowledge, this is the first report of a direct physical measurement of the structure of membranes containing CFTR in its native environment and in different functional and pharmacological conditions. PMID:25274064

  9. Triplex Crosslinking through Furan Oxidation Requires Perturbation of the Structured Triple-Helix.

    PubMed

    Gyssels, Ellen; Carrette, Lieselot L G; Vercruysse, Emma; Stevens, Kristof; Madder, Annemieke

    2015-03-01

    Short oligonucleotides can selectively recognize duplexes by binding in the major groove thereby forming triplexes. Based on the success of our recently developed strategy for furan-based crosslinking in DNA duplexes, we here investigated for the first time the use of the furan-oxidation crosslink methodology for the covalent locking of triplex structures by an interstrand crosslink. It was shown that in a triplex context, although crosslinking yields are surprisingly low (to nonexistent) when targeting fully complementary duplexes, selective crosslinking can be achieved towards mismatched duplex sites at the interface of triplex to duplex structures. We show the promising potential of furan-containing probes for the selective detection of single-stranded regions within nucleic acids containing a variety of structural motifs. PMID:25630588

  10. A Specific Interface between Integrin Transmembrane Helices

    E-print Network

    Springer, Timothy A.

    A Specific Interface between Integrin Transmembrane Helices and Affinity for Ligand Bing-Hao Luo of integrins is beginning to be defined by structural work on both domains. However, the role of the a and b elusive. Disulfide bond scanning of the exofacial portions of the integrin aIIb and b3 transmembrane

  11. Molecular Architecture of a Sodium Channel S6 Helix

    PubMed Central

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

    2013-01-01

    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

  12. Solution structure of a DNA double helix with consecutive metal-mediated base pairs

    NASA Astrophysics Data System (ADS)

    Johannsen, Silke; Megger, Nicole; Böhme, Dominik; Sigel, Roland K. O.; Müller, Jens

    2010-03-01

    Metal-mediated base pairs represent a powerful tool for the site-specific functionalization of nucleic acids with metal ions. The development of applications of the metal-modified nucleic acids will depend on the availability of structural information on these double helices. We present here the NMR solution structure of a self-complementary DNA oligonucleotide with three consecutive imidazole nucleotides in its centre. In the absence of transition-metal ions, a hairpin structure is adopted with the artificial nucleotides forming the loop. In the presence of Ag(I) ions, a duplex comprising three imidazole-Ag+-imidazole base pairs is formed. Direct proof for the formation of metal-mediated base pairs was obtained from 1J(15N,107/109Ag) couplings upon incorporation of 15N-labelled imidazole. The duplex adopts a B-type conformation with only minor deviations in the region of the artificial bases. This work represents the first structural characterization of a metal-modified nucleic acid with a continuous stretch of metal-mediated base pairs.

  13. Solution structure of a DNA double helix with consecutive metal-mediated base pairs.

    PubMed

    Johannsen, Silke; Megger, Nicole; Böhme, Dominik; Sigel, Roland K O; Müller, Jens

    2010-03-01

    Metal-mediated base pairs represent a powerful tool for the site-specific functionalization of nucleic acids with metal ions. The development of applications of the metal-modified nucleic acids will depend on the availability of structural information on these double helices. We present here the NMR solution structure of a self-complementary DNA oligonucleotide with three consecutive imidazole nucleotides in its centre. In the absence of transition-metal ions, a hairpin structure is adopted with the artificial nucleotides forming the loop. In the presence of Ag(i) ions, a duplex comprising three imidazole-Ag(+)-imidazole base pairs is formed. Direct proof for the formation of metal-mediated base pairs was obtained from ¹J(¹?N,¹??/¹??Ag) couplings upon incorporation of ¹?N-labelled imidazole. The duplex adopts a B-type conformation with only minor deviations in the region of the artificial bases. This work represents the first structural characterization of a metal-modified nucleic acid with a continuous stretch of metal-mediated base pairs. PMID:21124482

  14. Crystal structure of an intramolecular chaperone mediating triple–?-helix folding

    Microsoft Academic Search

    Eike C Schulz; Achim Dickmanns; Henning Urlaub; Andreas Schmitt; Martina Mühlenhoff; Katharina Stummeyer; David Schwarzer; Rita Gerardy-Schahn; Ralf Ficner

    2010-01-01

    Protein folding is often mediated by molecular chaperones. Recently, a novel class of intramolecular chaperones has been identified in tailspike proteins of evolutionarily distant viruses, which require a C-terminal chaperone for correct folding. The highly homologous chaperone domains are interchangeable between pre-proteins and release themselves after protein folding. Here we report the crystal structures of two intramolecular chaperone domains in

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

    E-print Network

    with -sheet structures is related to some important neurodegenerative diseases such as Huntington's disease #12;INTRODUCTION Neurodegenerative diseases such as Huntington's disease (HD)1 , Alzheimer's disease2 by expanded CAG repeats in genomic sequence16­20 . Huntington's disease is the most common among

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

    SciTech Connect

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

    2009-06-02

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

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

    PubMed

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

    2007-05-15

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

  18. Annular Anionic Lipids Stabilize the Integrin ?IIb?3 Transmembrane Complex.

    PubMed

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

    2015-03-27

    Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin ?IIb?3 transmembrane (TM) complex is stabilized by a membrane-proximal ?IIb(Arg(995))-?3(Asp(723)) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for ?IIb(Arg(995)) contacts with ?3(Asp(723)) but paradoxically do not diminish the contribution of ?IIb(Arg(995))-?3(Asp(723)) to TM complex stability. Overall, anionic lipids in annular positions stabilize the ?IIb?3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact. PMID:25632962

  19. Structural Model for 12Helix Transporters Belonging to the Major Facilitator Superfamily

    Microsoft Academic Search

    Teruhisa Hirai; Jurgen A. W. Heymann; Peter C. Maloney; Sriram Subramaniam

    2003-01-01

    oxalate transporter, OxlT, a representative member of this superfamily. In the oxalate-bound state, 12 helices surround a central cavity to form a remarkably symmetrical structure that displays a well-defined pseudo twofold axis perpendicular to the plane of the membrane as well as two less pronounced, mutually perpen- dicular pseudo twofold axes in the plane of the membrane. Here, we combined

  20. ?-Helix is a likely core structure of yeast prion Sup35 amyloid fibers

    Microsoft Academic Search

    Aiko Kishimoto; Kazuya Hasegawa; Hirofumi Suzuki; Hideki Taguchi; Keiichi Namba; Masasuke Yoshida

    2004-01-01

    We have studied the core structure of amyloid fibers of yeast prion protein Sup35. We developed procedures to prepare straight fibers of relatively uniform diameters from three kinds of fragments; N (1–123), NMp (1–189), and NM (1–253). X-ray fiber diffraction patterns from dried oriented fibers gave common reflections in all three cases; a sharp meridional reflection at 4.7Å, and a

  1. Double helix

    NSDL National Science Digital Library

    Dennis Myts (None; )

    2007-02-19

    Watson and Crick used previous background research to form a hypothesis about the structure of DNA. They then performed experiments to test their hypothesis. They analyzed their results and finally found their predictions to be true after several attempts.

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

    PubMed

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

    2014-01-01

    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

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

    SciTech Connect

    Storrs, R.W.

    1992-08-01

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

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

    SciTech Connect

    Storrs, R.W.

    1992-08-01

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

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

    SciTech Connect

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

    2001-10-30

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

  6. Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H +-V-ATPase

    Microsoft Academic Search

    Afonso M. S. Duarte; Cor J. A. M. Wolfs; Nico A. J. van Nuland; Michael A. Harrison; John B. C. Findlay; Carlo P. M. van Mierlo; Marcus A. Hemminga

    2007-01-01

    Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more

  7. CHUK, a Conserved Helix-Loop-Helix Ubiquitous Kinase, Maps to Human Chromosome 10 and Mouse Chromosome 19

    Microsoft Academic Search

    Beverly A. Mock; Margery A. Connelly; O. Wesley McBride; Christine A. Kozak; Kenneth B. Marcu

    1995-01-01

    Helix-loop-helix proteins contain stretches of DNA that encode two amphipathic ?-helices joined by a loop structure and are involved in protein dimerization and transcriptional regulation essential to a variety of cellular processes. CHUK, a newly described conserved helix-loop-helix ubiquitos kinase, was mapped by somatic cell hybrid analyses to human Chr 10q24-q25. Chuk and a related sequence, Chuk-rs1, were mapped to

  8. The structure of the XPF-ssDNA complex underscores the distinct roles of the XPF and ERCC1 helix- hairpin-helix domains in ss/ds DNA recognition.

    PubMed

    Das, Devashish; Folkers, Gert E; van Dijk, Marc; Jaspers, Nicolaas G J; Hoeijmakers, Jan H J; Kaptein, Robert; Boelens, Rolf

    2012-04-01

    Human XPF/ERCC1 is a structure-specific DNA endonuclease that nicks the damaged DNA strand at the 5' end during nucleotide excision repair. We determined the structure of the complex of the C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the second helix of the first HhH motif contacts the ssDNA phosphate backbone. One guanine base is flipped out of register and positioned in a pocket contacting residues from both HhH motifs of XPF. Comparison to other HhH-containing proteins indicates a one-residue deletion in the second HhH motif of XPF that has altered the hairpin conformation, thereby permitting ssDNA interactions. Previous nuclear magnetic resonance studies showed that ERCC1 in the XPF-ERCC1 heterodimer can bind dsDNA. Combining the two observations gives a model that underscores the asymmetry of the human XPF/ERCC1 heterodimer in binding at an ss/ds DNA junction. PMID:22483113

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

    PubMed Central

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

    2015-01-01

    The sigma-1 receptor (S1R) is a ligand-regulated membrane chaperone protein associated with endoplasmic reticulum stress response, and modulation of ion channel activities at the plasma membrane. We report here a solution NMR study of a S1R construct (S1R(?35)) in which only the first transmembrane domain and the eight-residue N-terminus have been removed. The second transmembrane helix is found to be composed of residues 91–107, which corresponds to the first steroid binding domain-like region. The cytosolic domain is found to contain three helices, and the secondary structure and backbone dynamics of the chaperone domain are consistent with that determined previously for the chaperone domain alone. The position of TM2 provides a framework for ongoing studies of S1R ligand binding and oligomerisation. PMID:25647032

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

    PubMed

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

    2015-02-27

    The sigma-1 receptor (S1R) is a ligand-regulated membrane chaperone protein associated with endoplasmic reticulum stress response, and modulation of ion channel activities at the plasma membrane. We report here a solution NMR study of a S1R construct (S1R(?35)) in which only the first transmembrane domain and the eight-residue N-terminus have been removed. The second transmembrane helix is found to be composed of residues 91-107, which corresponds to the first steroid binding domain-like region. The cytosolic domain is found to contain three helices, and the secondary structure and backbone dynamics of the chaperone domain are consistent with that determined previously for the chaperone domain alone. The position of TM2 provides a framework for ongoing studies of S1R ligand binding and oligomerisation. PMID:25647032

  11. Analysis of pi-point instability in an asymmetric helical slow-wave structure in helix traveling wave tubes

    Microsoft Academic Search

    Y. D. Joo; A. K. Sinha; B. N. Basu; G. S. Park

    2004-01-01

    The energy exchange takes place between the linear electron beam and the electromagnetic wave propagating in the helix (E.I. Lien, IEDM Tech. Digest, pp. 412-415, 1979; T. Onodera, IEEE Trans. ED, vol. 35, no 10, 1988; B. Levush, Int. Vacuum Electron. Conf., pp. 71-72, 2002; B. N. Basu, Electromagnetic Theory and Applications in Beam-Wave Electronics, World Publ. Co. Inc. 1996;

  12. Transmembrane Signaling and Assembly of the Cytochrome b6f-Lipidic Charge Transfer Complex

    PubMed Central

    Hasan, S. Saif; Yamashita, E.; Cramer, William A.

    2014-01-01

    Structure-function properties of the cytochrome b6f complex are sufficiently unique compared to those of the cytochrome bc1 complex that b6f should not be considered a trivially modified bc1 complex. A unique property of the dimeric b6f complex is its involvement in trans-membrane signaling associated with the p-side oxidation of plastoquinol. Structure analysis of lipid binding sites in the cyanobacterial b6f complex prepared by hydrophobic chromatography shows that the space occupied by the H transmembrane helix in the cytochrome b subunit of the bc1 complex is mostly filled by a lipid in the b6f crystal structure. It is suggested that this space can be filled by the domain of a transmembrane signaling protein. The identification of lipid sites and likely function define the intra-membrane conserved central core of the b6f complex, consisting of the seven trans-membrane helices of the cytochrome b and subunit IV polypeptides. The other six TM helices, contributed by cytochrome f, the iron-sulfur protein, and the four peripheral single span subunits, define a peripheral less conserved domain of the complex. The distribution of conserved and non-conserved domains of each monomer of the complex, and the position and inferred function of a number of the lipids, suggests a model for the sequential assembly in the membrane of the eight subunits of the b6f complex, in which the assembly is initiated by formation of the cytochrome b6-subunit IV core sub-complex in a monomer unit. Two conformations of the unique lipidic chlorophyll a, defined in crystal structures, are described, and functions of the outlying ?-carotene, a possible ’latch’ in supercomplex formation, are discussed. PMID:23507619

  13. The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision.

    PubMed

    Lee, Hsiao-Lu D; Sahl, Steffen J; Lew, Matthew D; Moerner, W E

    2012-04-01

    The double-helix point spread function microscope encodes the axial (z) position information of single emitters in wide-field (x,y) images, thus enabling localization in three dimensions (3D) inside extended volumes. We experimentally determine the statistical localization precision ? of this approach using single emitters in a cell under typical background conditions, demonstrating ??structures in fixed mammalian cells over a large axial range in three dimensions. PMID:22550359

  14. Structure and stability of a DNA triple helix in solution: NMR studies on d(T) sub 6 ter dot d(T) sub 6 and its complex with a minor groove binding drug

    SciTech Connect

    Umemoto, Kimiko; Sarma, Mukti H.; Gupta, Goutam; Luo, Jia; Sarma, Ramaswamy H. (State Univ. of New York, Albany (USA))

    1990-05-23

    The possibility of both Watson-Crick and Hoogsteen A{center dot}T pairs can result in a triple helical structure for d(T){sub 6}{center dot}d(A){sub 6}{center dot}d(T){sub 6} in solution. In the triple helix the Watson-Crick paired T strand can run antiparallel, while the Hoogsteen paired T strand can run parallel to the A strand. On the basis of 1D/2D NMR studies, we have characterized the structural properties of the triple helix in terms of (a) nature of H-bonding, (b) chain conformations and relative chain orientations, (c) location of triplets T{center dot}A{center dot}T with respect to the helix axis, and (d) effects of NaCl and MgCl{sub 2}. In addition, we experimentally demonstrate that a minor groove specific drug Dst2 (a distamycin analogue) can bind to the triple helix. We show that the nature of thermal transition is altered by Dst2 binding; i.e., the host triple helix shows triple {yields} coil (monophasic) transition in the absence of Dst2, while in its presence the helix shows a triplex {yields} duplex {yields} coil (biphasic) transition.

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

    PubMed Central

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

    1987-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

  17. Contributions of the C-Terminal Helix to the Structural Stability of a Hyperthermophilic Fe-Superoxide Dismutase (TcSOD)

    PubMed Central

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

    2009-01-01

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

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

    SciTech Connect

    Yu,P.

    2007-01-01

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

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

    SciTech Connect

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

    2011-09-06

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

  20. A Helix Replacement Mechanism Directs Metavinculin Functions

    SciTech Connect

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

    2010-10-11

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

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

    PubMed Central

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

    1992-01-01

    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

  2. Numerical simulation of coil–helix transition processes of gelatin

    Microsoft Academic Search

    Xiliang Chen; Yuxi Jia; Ligang Feng; Sheng Sun; Lijia An

    2009-01-01

    Gelatin is widely used in food, pharmaceutical, and photographic industries due to the coil–helix transition, whereas the structural inhomogeneity considerably affects its essential properties closely connecting with the industrial applications. The spatially structural inhomogeneity of the gelatin caused by the uneven and unstable temperature field is analyzed by the finite element method during the cooling-induced coil–helix transition process. The helix

  3. SIMULATING THE DYNAMICS OF THE DNA DOUBLE HELIX IN SOLUTION

    E-print Network

    Levitt, Michael

    . Although Watson and Crick's model building study predicted the double-helix structure over 40 vears ago (Watson and Crick. 1953) and the structure was soon confirmed by fibre diffraction (Langridge et al.. 1960 structures. Watson and Crick's ( 1953) famous modeling of the double helix was theoretical in nature

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

    PubMed Central

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

    2014-01-01

    ABSTRACT Agnoprotein is a small multifunctional regulatory protein required for sustaining the productive replication of JC virus (JCV). It is a mostly cytoplasmic protein localizing in the perinuclear area and forms highly stable dimers/oligomers through a Leu/Ile/Phe-rich domain. There have been no three-dimensional structural data available for agnoprotein due to difficulties associated with the dynamic conversion from monomers to oligomers. Here, we report the first nuclear magnetic resonance (NMR) structure of a synthetic agnoprotein peptide spanning amino acids Thr17 to Glu55 where Lys23 to Phe39 encompassing the Leu/Ile/Phe-rich domain forms an amphipathic ?-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

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

    PubMed Central

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

    2012-01-01

    Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage-gated potassium channels. Channels from this family produce neuronal M-currents and cardiac and auditory IKS currents, and harbor mutations that cause arrhythmias, epilepsy, and deafness. Despite extensive functional characterization, biochemical and structural details of the interaction between CaM and the channel have remained elusive. Here, we show that both apo-CaM and Ca2+/CaM bind to the C-terminal tail of the neuronal channel Kv7.4 (KCNQ4), which is involved both hearing and mechanosensation. Interactions between apo-CaM and the Kv7.4 tail involve two C-terminal tail segments, known as the A and B segments, whereas the interaction between Ca2+/CaM and the Kv7.4 C-terminal tail requires only the B segment. Biochemical studies show that the calcium dependence of the CaM:B segment interaction is conserved in all Kv7 subtypes. X-ray crystallographic determination of the structure of the Ca2+/CaM:Kv7.4 B segment complex shows that Ca2+/CaM wraps around the Kv7.4 B segment, which forms an ?-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

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

    PubMed Central

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

    2013-01-01

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

  7. Hydrophobic Matching Controls the Tilt and Stability of the Dimeric Platelet-derived Growth Factor Receptor (PDGFR) ? Transmembrane Segment*

    PubMed Central

    Muhle-Goll, Claudia; Hoffmann, Silke; Afonin, Sergii; Grage, Stephan L.; Polyansky, Anton A.; Windisch, Dirk; Zeitler, Marcel; Bürck, Jochen; Ulrich, Anne S.

    2012-01-01

    The platelet-derived growth factor receptor ? is a member of the cell surface receptor tyrosine kinase family and dimerizes upon activation. We determined the structure of the transmembrane segment in dodecylphosphocholine micelles by liquid-state NMR and found that it forms a stable left-handed helical dimer. Solid-state NMR and oriented circular dichroism were used to measure the tilt angle of the helical segments in macroscopically aligned model membranes with different acyl chain lengths. Both methods showed that decreasing bilayer thickness (DEPC-POPC-DMPC) led to an increase in the helix tilt angle from 10° to 30° with respect to the bilayer normal. At the same time, reconstitution of the comparatively long hydrophobic segment became less effective, eventually resulting in complete protein aggregation in the short-chain lipid DLPC. Unrestrained molecular dynamics simulations of the dimer were carried out in explicit lipid bilayers (DEPC, POPC, DMPC, sphingomyelin), confirming the observed dependence of the helix tilt angle on bilayer thickness. Notably, molecular dynamics revealed that the left-handed dimer gets tilted en bloc, whereas conformational transitions to alternative (e.g. right-handed dimeric) states were not supported. The experimental data along with the simulation results demonstrate a pronounced interplay between the platelet-directed growth factor receptor ? transmembrane segment and the bilayer thickness. The effect of hydrophobic mismatch might play a key role in the redistribution and activation of the receptor within different lipid microdomains of the plasma membrane in vivo. PMID:22619173

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-06-01

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

  10. Primary structure of a copper-binding metallothionein from mantle tissue of the terrestrial gastropod Helix pomatia L.

    PubMed Central

    Berger, B; Dallinger, R; Gehrig, P; Hunziker, P E

    1997-01-01

    A novel copper-binding metallothionein (MT) has been purified from mantle tissue of the terrestrial snail Helix pomatia using gel-permeation chromatography, ion-exchange chromatography and reverse-phase HPLC. Copper was removed from the thionein by addition of ammonium tetrathiomolybdate. The resulting apothionein (molecular mass 6247 Da) was S-methylated and digested with trypsin, endoproteinase Arg-C and endoproteinase Lys-C. Amino acid sequences of the resulting peptides were determined by collision-induced dissociation tandem MS. The protein is acetylated at its N-terminus, and consists of 64 amino acids, 18 of which are cysteine residues. A comparison with the cadmium-binding MT isolated from the midgut gland of the same species shows an identical arrangement of the cysteines, but an unexpectedly high variability in the other amino acids. The two MT isoforms differ in total length and at 26 positions of their peptide chains. We suggest that the copper-binding MT isoform from the mantle of H. pomatia is responsible for regulatory functions in favour of copper, probably in connection with the metabolism of the copper-bearing protein, haemocyanin. PMID:9359856

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

    PubMed

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

    2015-02-20

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

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

    PubMed Central

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

    2013-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-08-01

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

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

    SciTech Connect

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

    2010-01-01

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

  15. 1H, 15N and 13C resonance assignments and secondary structure of group II phospholipase A2 from Agkistrodon piscivorus piscivorus: presence of an amino-terminal helix in solution.

    PubMed

    Jerala, R; Almeida, P F; Ye, Q; Biltonen, R L; Rule, G S

    1996-03-01

    1H, 15N and 13C resonance assignments are presented for the group II phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus. The secondary structure of the enzyme has been inferred from an analysis of coupling constants, interproton distances, chemical shifts, and kinetics of amide exchange. Overall, the secondary structure of this PLA2 is similar to the crystal structure of the homologous group II human nonpancreatic secretory phospholipase [Scott, D.L., White, S.P., Browning, J.L., Rosa, J.J., Gelb, M.H. and Sigler, P.B. (1991) Science, 254, 1007-1010]. In the group I enzyme from porcine pancreas, the amino-terminal helix becomes fully ordered in the ternary complex of enzyme, lipid micelles and inhibitor. The formation of this helix is thought to be important for the increase in activity of phospholipases on aggregated substrates [Van den Berg, B., Tessari, M., Boelens, R., Dijkman, R., De Haas, G. H., Kaptein, R. and Verheij, H.M. (1995) Nature Strct. Biol., 2, 402-406]. However, the group II enzyme from Agkistrodon piscivorus piscivorus possesses a defined and well-positioned amino-terminal helix in the absence of substrate. Therefore, there is a clear difference between the conformation group I and group II enzymes in solution. These conformational differences suggest that formation of the amino-terminal helix is a necessary, but not sufficient, step in interfacial activation of phospholipases. PMID:8616268

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

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

    PubMed Central

    Khadria, Ambalika S.; Senes, Alessandro

    2014-01-01

    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

  18. Propofol Binding to the Resting State of the Gloeobacter violaceus Ligand-gated Ion Channel (GLIC) Induces Structural Changes in the Inter- and Intrasubunit Transmembrane Domain (TMD) Cavities*

    PubMed Central

    Ghosh, Borna; Satyshur, Kenneth A.; Czajkowski, Cynthia

    2013-01-01

    General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428–431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state. PMID:23640880

  19. Development of 200 watt CW helix traveling wave tube

    Microsoft Academic Search

    P. V. Bhaskar; K. S. Prasad; R. R. Singh; S. Ghosh; V. Kiran

    2003-01-01

    In this paper, authors have presented design and development of a S, C band 200 W, 2-6 GHz CW helix travelling wave tube (TWT) with saturated gain of 40 dB. In the TWT, wide bandwidth is achieved when helix is used as a slow wave structure. The electron gun of this tube is a Peirce convergent type tube. The electron

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

    PubMed Central

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

    2014-01-01

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

  1. Role of Amphipathic Helix of a Herpesviral Protein in Membrane Deformation and T Cell Receptor Downregulation

    Microsoft Academic Search

    Chan-Ki Min; Sun-Young Bang; Bon-A Cho; Yun-Hui Choi; Jae-Seong Yang; Sun-Hwa Lee; Seung-Yong Seong; Ki Woo Kim; Sanguk Kim; Jae Ung Jung; Myung-Sik Choi; Ik-Sang Kim; Nam-Hyuk Cho

    2008-01-01

    Lipid rafts are membrane microdomains that function as platforms for signal transduction and membrane trafficking. Tyrosine kinase interacting protein (Tip) of T lymphotropic Herpesvirus saimiri (HVS) is targeted to lipid rafts in T cells and downregulates TCR and CD4 surface expression. Here, we report that the membrane-proximal amphipathic helix preceding Tip's transmembrane (TM) domain mediates lipid raft localization and membrane

  2. The structure of a receptor with two associating transmembrane domains on the cell surface: integrin IIb3

    E-print Network

    Baker, David

    : integrin IIb3 Jieqing Zhu1,2, Bing-Hao Luo2,3, Patrick Barth2,4, Jack Schonbrun4,5, David Baker4. An integrin heterodimer structure reveals association over most of the lengths of the and TM domains, shows how TM association/dissociation regulates integrin signaling. A joint ectodomain and membrane

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

    SciTech Connect

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

    2010-07-19

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

  4. Solid-state NMR spectroscopy of the HIV gp41 membrane fusion protein supports intermolecular antiparallel ? sheet fusion peptide structure in the final six-helix bundle state

    PubMed Central

    Sackett, Kelly; Nethercott, Matthew J.; Zheng, Zhaoxiong; Weliky, David P.

    2013-01-01

    The HIV gp41 protein catalyzes fusion between viral and target cell membranes. Although the ~20-residue N-terminal fusion peptide (FP) region is critical for fusion, the structure of this region is not well-characterized in large gp41 constructs that model the gp41 state at different times during fusion. This paper describes solid-state NMR (SSNMR) studies of FP structure in a membrane-associated construct (FP-Hairpin) which likely models the final fusion state thought to be thermostable trimers with six-helix bundle structure in the region C-terminal of the FP. The SSNMR data show that there are populations of FP-Hairpin with either ? helical or ? sheet FP conformation. For the ? sheet population, measurements of intermolecular 13C-13C proximities in the FP are consistent with a significant fraction of intermolecular antiparallel ? sheet FP structure with adjacent strand crossing near L7 and F8. There appears to be negligible in-register parallel structure. These findings support assembly of membrane-associated gp41 trimers through inter-leaving of N-terminal FPs from different trimers. Similar SSNMR data are obtained for FP-Hairpin and a construct containing the 70 N-terminal residues of gp41 (N70) which is a model for part of the putative pre-hairpin intermediate state of gp41. FP assembly may therefore occur at an early fusion stage. On a more fundamental level, similar SSNMR data are obtained for FP-Hairpin and a construct containing the 34 N-terminal gp41 residues (FP34) and support the hypothesis that the FP is an autonomous folding domain. PMID:24246500

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

    PubMed

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

    2014-11-20

    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

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

    PubMed

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

    2015-01-16

    Bacterial cell membranes contain several protein pumps that resist the toxic effects of drugs by efficiently extruding them. One family of these pumps, the small multidrug resistance proteins (SMRs), consists of proteins of about 110 residues that need to oligomerize to form a structural pathway for substrate extrusion. As such, SMR oligomerization sites should constitute viable targets for efflux inhibition, by disrupting protein-protein interactions between helical segments. To explore this proposition, we are using Hsmr, an SMR from Halobacter salinarum that dimerizes to extrude toxicants. Our previous work established that (i) Hsmr dimerization is mediated by a helix-helix interface in Hsmr transmembrane (TM) helix 4 (residues (90)GLALIVAGV(98)); and (ii) a peptide comprised of the full TM4(85-105) sequence inhibits Hsmr-mediated ethidium bromide efflux from bacterial cells. Here we define the minimal linear sequence for inhibitor activity (determined as TM4(88-100), and then "staple" this sequence via Grubbs metathesis to produce peptides typified by acetyl-A-(Sar)3-(88)VVGLXLIZXGVVV(100)-KKK-NH2 (X = 2-(4'-pentenyl)alanine at positions 92 and 96; Z = Val, Gly, or Asn at position 95)). The Asn(95) peptide displayed specific efflux inhibition and resensitization of Hsmr-expressing cells to ethidium bromide; and was non-hemolytic to human red blood cells. Stapling essentially prevented peptide degradation in blood plasma and liver homogenates versus an unstapled counterpart. The overall results confirm that the stapled analog of TM4(88-100) retains the structural complementarity required to disrupt the Hsmr TM4-TM4 locus in Hsmr, and portend the general validity of stapled peptides as therapeutics for the disruption of functional protein-protein interactions in membranes. PMID:25425644

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

    PubMed

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

    2014-05-01

    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

  8. Model for an RNA tertiary interaction from the structure of an intermolecular complex between a GAAA tetraloop and an RNA helix.

    PubMed

    Pley, H W; Flaherty, K M; McKay, D B

    1994-11-01

    In large structured RNAs, RNA hairpins in which the strands of the duplex stem are connected by a tetraloop of the consensus sequence 5'-GNRA (where N is any nucleotide, and R is either G or A) are unusually frequent. In group I introns there is a covariation in sequence between nucleotides in the third and fourth positions of the loop with specific distant base pairs in putative RNA duplex stems: GNAA loops correlate with successive 5'-C-C.G-C base pairs in stems, whereas GNGA loops correlate with 5'-C-U.G-A. This has led to the suggestion that GNRA tetraloops may be involved in specific long-range tertiary interactions, with each A in position 3 or 4 of the loop interacting with a C-G base pair in the duplex, and G in position 3 interacting with a U-A base pair. This idea is supported experimentally for the GAAA loop of the P5b extension of the group I intron of Tetrahymena thermophila and the L9 GUGA terminal loop of the td intron of bacteriophage T4 (ref. 4). NMR has revealed the overall structure of the tetraloop for 12-nucleotide hairpins with GCAA and GAAA loops and models have been proposed for the interaction of GNRA tetraloops with base pairs in the minor groove of A-form RNA. Here we describe the crystal structure of an intermolecular complex between a GAAA tetraloop and an RNA helix. The interactions we observe correlate with the specificity of GNRA tetraloops inferred from phylogenetic studies, suggesting that this complex is a legitimate model for intramolecular tertiary interactions mediated by GNRA tetraloops in large structured RNAs. PMID:7526219

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

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

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

  10. 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

    PubMed Central

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

    2013-01-01

    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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2013-01-15

    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

  13. A Numerical Study on Helix Nanowire Metamaterials as Optical Circular Polarizers in the Visible Region

    Microsoft Academic Search

    Z. Y. Yang; M. Zhao; P. X. Lu

    2010-01-01

    Broadband optical circular polarizers functioning in the visible region were developed based on structural design and numerical simulation using the finite-difference time-domain method. By properly selecting the parameters of helix nanowire metamaterials including metal materials, diameters of wires, numbers of helix-period, spacing of grids, lengths of helix-period, and diameters of helixes, we proposed a broadband optical circular polarizer using aluminium

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

    SciTech Connect

    Bose, Sayantan; Welch, Brett D.; Kors, Christopher A.; Yuan, Ping; Jardetzky, Theodore S.; Lamb, Robert A. (NWU); (Stanford-MED)

    2014-10-02

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-07-01

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

  16. Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs

    Microsoft Academic Search

    Stephen R. Holbrook; Chaejoon Cheong; Ignacio Tinoco; Sung-Hou Kim

    1991-01-01

    THE crystal structure of the RNA dodecamer duplex (r-GGACUUCGGUCC)2 has been determined. The dodecamers stack end-to-end in the crystal, simulating infinite A-form helices with only a break in the phosphodiester chain. These infinite helices are held together in the crystal by hydrogen bonding between ribose hydroxyl groups and a variety of donors and acceptors. The four noncomplementary nucleotides in the

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

    PubMed Central

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

    2002-01-01

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

  18. Human immunodeficiency virus-1 BF intersubtype recombinant viral protein U second ? helix plays an important role in viral release and BST-2 degradation.

    PubMed

    De Candia, Cristian; Espada, Constanza; Duette, Gabriel; Salomón, Horacio; Carobene, Mauricio

    2013-04-01

    We previously reported a naturally occurring BF intersubtype recombinant viral protein U (Vpu) variant with an augmented capacity to enhance viral replication. Structural analysis of this variant revealed that its transmembrane domain and ?-helix I in the cytoplasmic domain (CTD) corresponded to subtype B, whereas the ?-helix II in the CTD corresponded to subtype F1. In this study, we aimed to evaluate the role of the Vpu cytoplasmic ?-helix II domain in viral release enhancement and in the down-modulation of BST-2 and CD4 from the cell surface. In addition, as serine residues in Vpu amino acid positions 61 or 64 have been shown to regulate Vpu intracellular half-life, which in turn could influence the magnitude of viral release, we also studied the impact of these residues on the VpuBF functions, since S61 and S64 are infrequently found among BF recombinant Vpu variants. Our results showed that the exchange of Vpu ?-helix II between subtypes (B?F) directly correlated with the enhancement of viral release and, to a lesser extent, with changes in the capacity of the resulting chimera to down-modulate BST-2 and CD4. No differences in viral release and BST-2 down-modulation were observed between VpuBF and VpuBF-E61S. On the other hand, VpuBF-A64S showed a slightly reduced capacity to enhance viral production, but was modestly more efficient than VpuBF in down-modulating BST-2. In summary, our observations clearly indicate that ?-helix II is actively involved in Vpu viral-release-promoting activity and that intersubtype recombination between subtypes B and F1 created a protein variant with a higher potential to boost the spread of the recombinant strain that harbours it. PMID:23223624

  19. Global fold and backbone dynamics of the hepatitis C virus E2 glycoprotein transmembrane domain determined by NMR.

    PubMed

    Shalom-Elazari, Hila; Zazrin-Greenspon, Hadas; Shaked, Hadassa; Chill, Jordan H

    2014-11-01

    E1 and E2 are two hepatitis C viral envelope glycoproteins that assemble into a heterodimer that is essential for membrane fusion and penetration into the target cell. Both extracellular and transmembrane (TM) glycoprotein domains contribute to this interaction, but study of TM-TM interactions has been limited because synthesis and structural characterization of these highly hydrophobic segments present significant challenges. In this NMR study, by successful expression and purification of the E2 transmembrane domain as a fusion construct we have determined the global fold and characterized backbone motions for this peptide incorporated in phospholipid micelles. Backbone resonance frequencies, relaxation rates and solvent exposure measurements concur in showing this domain to adopt a helical conformation, with two helical segments spanning residues 717-726 and 732-746 connected by an unstructured linker containing the charged residues D728 and R730 involved in E1 binding. Although this linker exhibits increased local motions on the ps timescale, the dominating contribution to its relaxation is the global tumbling motion with an estimated correlation time of 12.3ns. The positioning of the helix-linker-helix architecture within the mixed micelle was established by paramagnetic NMR spectroscopy and phospholipid-peptide cross relaxation measurements. These indicate that while the helices traverse the hydrophobic interior of the micelle, the linker lies closer to the micelle perimeter to accommodate its charged residues. These results lay the groundwork for structure determination of the E1/E2 complex and a molecular understanding of glycoprotein heterodimerization. PMID:25109935

  20. A structurally dynamic N-terminal helix is a key functional determinant in staphylococcal complement inhibitor (SCIN) proteins.

    PubMed

    Garcia, Brandon L; Summers, Brady J; Ramyar, Kasra X; Tzekou, Apostolia; Lin, Zhuoer; Ricklin, Daniel; Lambris, John D; Laity, John H; Geisbrecht, Brian V

    2013-01-25

    Complement is a network of interacting circulatory and cell surface proteins that recognizes, marks, and facilitates clearance of microbial invaders. To evade complement attack, the pathogenic organism Staphylococcus aureus expresses a number of secreted proteins that interfere with activation and regulation of the complement cascade. Staphylococcal complement inhibitors (SCINs) are one important class of these immunomodulators and consist of three active members (SCIN-A/-B/-C). SCINs inhibit a critical enzymatic complex, the alternative pathway C3 convertase, by targeting a functional "hot spot" on the central opsonin of complement, C3b. Although N-terminal truncation mutants of SCINs retain complement inhibitory properties, they are significantly weaker binders of C3b. To provide a structural basis for this observation, we undertook a series of crystallographic and NMR dynamics studies on full-length SCINs. This work reveals that N-terminal SCIN domains are characterized by a conformationally dynamic helical motif. C3b binding and functional experiments further demonstrate that this sequence-divergent N-terminal region of SCINs is both functionally important and context-dependent. Finally, surface plasmon resonance data provide evidence for the formation of inhibitor·enzyme·substrate complexes ((SCIN·C3bBb)·C3). Similar to the (SCIN·C3bBb)(2) pseudodimeric complexes, ((SCIN·C3bBb)·C3) interferes with the interaction of complement receptors and C3b. This activity provides an additional mechanism by which SCIN couples convertase inhibition to direct blocking of phagocytosis. Together, these data suggest that tethering multi-host protein complexes by small modular bacterial inhibitors may be a global strategy of immune evasion used by S. aureus. The work presented here provides detailed structure-activity relationships and improves our understanding of how S. aureus circumvents human innate immunity. PMID:23233676

  1. Modelling Sodium Channel Kinetics using the Sliding Helix Concept

    NASA Astrophysics Data System (ADS)

    Chancey, C. C.; George, S. A.

    1998-03-01

    The activation of sodium channels is central to all excitable cell function. The S4 segment of the transmembrane channel molecule is the putative voltage sensor for voltage-gated channels. Elementary physical models have recently been successful in modelling several aspects BTX-modified channels: the activation curve and dwell time histograms, in particular. Such models have failed, however, in reproducing the channels' sensitivity to temperature. This report discusses recent attempts at adding a temperature dependence in a model based on the sliding helix concept.

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

    NASA Astrophysics Data System (ADS)

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

    2013-05-01

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

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

    PubMed Central

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

    2012-01-01

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

  4. Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations.

    PubMed

    Choi, Mei Y; Cardarelli, Lia; Therien, Alex G; Deber, Charles M

    2004-06-29

    Polar residues comprise about 15% of the transmembrane (TM) domains of proteins, where they can stabilize structure via native side chain-side chain interhelical hydrogen bonds between TM helices. However, non-native H-bonds may be implicated in disease states, through limiting protein dynamics during transport and/or misfolding the protein by inducing non-native rotational positions about TM helical axes. Here we have undertaken an investigation of the presence and strength of H-bond interactions within a series of helix-loop-helix ("hairpin") constructs derived from TM helices 3 and 4 (italic) of the cystic fibrosis transmembrane conductance regulator (CFTR) (prototypic sequence G(194)LALAHFVWIAPLQ(207)VALLMGLIWELLQASAFAGLGFLIV(232)LALFQ(237)AGLG(241)) in which wild-type Q207 in TM3 forms an interhelical H-bond with CF-phenotypic mutant V232D in TM4 [Therien, A. G., Grant, F. E., and Deber, C. M. (2001) Nat. Struct. Biol 8, 597-601]. In the present work, a library of 21 TM3/4 constructs was prepared, where Asp residues were placed individually at TM4 positions 221-241. Using gel shift assays-in which H-bond-linked hairpins (closed conformation) migrate faster than the elongated forms (open conformation)-we found that Q207 in TM3 is able to "capture" all 21 TM4 D mutations into measurable populations of interhelical H-bonds. A similar library of TM4 D mutants-but also containing Q207L-reverted to wild-type migration rates, confirming Q207 as the polar partner for TM4 D residues. In view of the broad capture range of Q207, these results emphasize the potential consequences to folding and dynamics of introducing polar mutations into the TM domains of membrane proteins in the vicinity of a native polar TM residue. PMID:15209503

  5. ?-Helix nucleation by a calcium-binding peptide loop

    PubMed Central

    Siedlecka, Monika; Goch, Gra?yna; Ejchart, Andrzej; Sticht, Heinrich; Bierzy?ski, Andrzej

    1999-01-01

    A 12-residue peptide AcDKDGDGYISAAENH2 analogous to the third calcium-binding loop of calmodulin strongly coordinates lanthanide ions (K = 105 M?1). When metal saturated, the peptide adopts a very rigid structure, the same as in the native protein, with three last residues AAE fixed in the ?-helical conformation. Therefore, the peptide provides an ideal helix nucleation site for peptide segments attached to its C terminus. NMR and CD investigations of peptide AcDKDGDGYISAAEAAAQNH2 presented in this paper show that residues A13-Q16 form an ?-helix of very high stability when the La3+ ion is bound to the D1-E12 loop. In fact, the lowest estimates of the helix content in this segment give values of at least 80% at 1°C and 70% at 25°C. This finding is not compatible with existing helix-coil transition theories and helix propagation parameters, s, reported in the literature. We conclude, therefore, that the initial steps of helix propagation are characterized by much larger s values, whereas helix nucleation is even more unfavorable than is believed. In light of our findings, thermodynamics of the nascent ?-helices is discussed. The problem of CD spectra of very short ?-helices is also addressed. PMID:9927666

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

    PubMed

    Maddox, Brenda

    2003-01-23

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

  7. DNA-like double helix formed by peptide nucleic acid

    Microsoft Academic Search

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

    1994-01-01

    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.

  8. Thermodynamics of an Intramolecular DNA Triple Helix: A Calorimetric and Spectroscopic Study of the pH and Salt Dependence of Thermally Induced Structural Transitions

    Microsoft Academic Search

    Eric G. Plum; Kenneth J. Breslauer

    1995-01-01

    We have characterized thermodynamically the melting transitions of a DNA 31-mer oligonucleotide (5?-GAAGAGGTTTTTCCTCTTCTTTTTCTTCTCC-3?) which is designed to fold into an intramolecular triple helix. The first 19 residues fold back on themselves to form an antiparallel Watson-Crick hairpin duplex with a T5loop. The 3?-terminal seven residues, which are connected to the Watson-Crick hairpin duplex by a second T5loop, form Hoogsteen interactions

  9. Detection of competing DNA structures by thermal gradient gel electrophoresis: from self-association to triple helix formation by (G,A)-containing oligonucleotides

    Microsoft Academic Search

    Paola B. Arimondo; Thérèse Garestier; Claude Hélène; Jian-Sheng Sun

    2001-01-01

    Sequence-specific recognition of DNA can be achieved by triple helix-forming oligonucleotides that bind to the major groove of double-helical DNA. These oligonucleotides have been used as sequence-specific DNA ligands for various purposes, including sequence-specific gene regulation in the so-called 'antigene strategy'. In particular, (G,A)- containing oligonucleotides can form stable triple helices under physiological conditions. However, triplex formation may be in

  10. The Arabidopsis Basic\\/Helix-Loop-Helix Transcription Factor Family

    Microsoft Academic Search

    Gabriela Toledo-Ortiz; Enamul Huq; Peter H. Quail

    2003-01-01

    The basic\\/helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that bind as dimers to specific DNA tar- get sites and that have been well characterized in nonplant eukaryotes as important regulatory components in diverse bio- logical processes. Based on evidence that the bHLH protein PIF3 is a direct phytochrome reaction partner in the photore- ceptor's signaling network, we have

  11. Phylogenetic Analysis of Plant Basic Helix-Loop-Helix Proteins

    Microsoft Academic Search

    Michael J. Buck; William R. Atchley

    2003-01-01

    The basic helix-loop-helix (bHLH) family of proteins is a group of functionally diverse transcription factors found in both plants and animals. These proteins evolved early in eukaryotic cells before the split of animals and plants, but appear to function in ‘plant-specific’ or ‘animal-specific’ processes. In animals bHLH proteins are involved in regulation of a wide variety of essential developmental processes.

  12. Human Cannabinoid 1 GPCR C-Terminal Domain Interacts with Bilayer Phospholipids to Modulate the Structure of its Membrane Environment

    Microsoft Academic Search

    Elvis K. Tiburu; Sergiy Tyukhtenko; Han Zhou; David R. Janero; Jochem Struppe; Alexandros Makriyannis

    2011-01-01

    G protein-coupled receptors (GPCRs) play critical physiological and therapeutic roles. The human cannabinoid 1 GPCR (hCB1)\\u000a is a prime pharmacotherapeutic target for addiction and cardiometabolic disease. Our prior biophysical studies on the structural\\u000a biology of a synthetic peptide representing the functionally significant hCB1 transmembrane helix 7 (TMH7) and its cytoplasmic\\u000a extension, helix 8 (H8), [hCB1(TMH7\\/H8)] demonstrated that the helices are

  13. RF loss profile measurement for a high gain, broadband helix TWT

    Microsoft Academic Search

    Vikas Kumar; Anil Vohra; Vishnu Srivastava

    Helix type structure is widely used as slow wave structure in the travelling wave tube (TWT) due to its natural wide bandwidth. Reflections present in the structure may cause oscillations and hence instability in the device. In order to minimize the reflections, a coating of lossy material is done on the helix support rods in a TWT. The loss introduced

  14. Analysis of the dominant modes of a slotted-helix-loaded cylindrical waveguide for use in plasma production

    Microsoft Academic Search

    A. Ganguli; P. Appala Naidu

    1990-01-01

    This paper is concerned with a detailed analysis of the dominant modes of a slotted helix loaded in an empty waveguide, with a view to improving the efficiency of plasma production using such structures. To this end the dispersion relation for a slotted helix loaded inside a cylindrical waveguide is first derived. As in the case of a tape-helix-loaded waveguide,

  15. Principles of Helix-Helix Packing in Proteins: The Helical Lattice Superposition Model

    Microsoft Academic Search

    Dirk Walther; Frank Eisenhaber; Patrick Argos

    1996-01-01

    The geometry of helix-helix packing in globular proteins is comprehen sively analysed within the model of the superposition of two helix lattices which result from unrolling the helix cylinders onto a plane containing points representing each residue. The requirements for the helix geometry (the radiusR, the twist angle ? and the rise per residue ?) under perfect match of the

  16. Proline localized to the interaction interface can mediate self-association of transmembrane domains.

    PubMed

    Sal-Man, Neta; Gerber, Doron; Shai, Yechiel

    2014-09-01

    Assembly of transmembrane domains (TMDs) is a critical step in the function of membrane proteins. In recent years, the role of specific amino acids in TMD-TMD interactions has been better characterized, with more emphasis on polar and aromatic residues. Despite the high abundance of proline residues in TMDs, contribution of proline to TMD-TMD association has not been intensively studied. Here, we evaluated statistically the frequency of appearance, and experimentally the contribution of proline, compared to other hydrophobic amino acids (Gly, Ala, Val, Leu, Ile, and Met), with regard to TMD-TMD self-assembly. Our model system is the assembly motif ((22)QxxS(25)) found previously in TMDs of the Escherichia coli aspartate receptor (Tar-1). Statistically, our data revealed that all different motifs, except PxxS (P/S), have frequencies similar to their theoretical random expectancy within a database of 41916 sequences of TMDs, while PxxS motif is underrepresented. Experimentally, using the ToxR assembly system, the SDS-gel running pattern of biotin-conjugated TMD peptides, and FRET experiments between fluorescence-labeled peptides, we found that only the P/S motif preserves the dimerization ability of wild-type Tar-1 TMD. Although proline is known as a helix breaker in solution, Circular Dichroism spectroscopy revealed that the secondary structure of the P/S and the wild-type peptides are similar. All together, these data suggest that proline can stabilize TM self-assembly when localized to the interaction interface of a transmembrane oligomer. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. PMID:24841754

  17. A transmembrane segment mimic derived from Escherichia coli diacylglycerol kinase inhibits protein activity.

    PubMed

    Partridge, Anthony W; Melnyk, Roman A; Yang, Dawn; Bowie, James U; Deber, Charles M

    2003-06-13

    The function of membrane proteins is inextricably linked to the proper packing and assembly of their independently helical transmembrane (TM) segments. Here we examined whether an externally added TM peptide analogue could specifically inhibit the function of the membrane protein from which it is derived by competing for native TM helix packing sites, thereby producing a non-functional peptide-protein complex. This hypothesis was tested using Lys-tagged peptides synthesized with sequences corresponding to the three TM segments of the homotrimeric Escherichia coli diacylglycerol kinase (DGK). The peptide corresponding to wild-type DGK TM-2 inhibited the protein's enzymatic activity in a dose-dependent manner through formation of an inactive pseudo-complex, whereas peptides derived from TM-1 and TM-3 were benign toward DGK structure/function. Also, substitution of a conserved residue (Glu-69) within the TM-2 peptide abolished these effects, demonstrating the strict sequence requirements for TM-2-mediated association. This strategy, coupled with the practical advantages of the water solubility of Lys-tagged TM peptides, may constitute an attractive approach for the design of therapeutic membrane protein modulators even in the absence of a high resolution structure. PMID:12686537

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

    PubMed Central

    Berisio, Rita; Vitagliano, Luigi

    2012-01-01

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

  19. Modeling Transmembrane Domain Dimers/Trimers of Plexin Receptors: Implications for Mechanisms of Signal Transmission across the Membrane

    PubMed Central

    Zhang, Liqun; Polyansky, Anton; Buck, Matthias

    2015-01-01

    Single-pass transmembrane (TM) receptors transmit signals across lipid bilayers by helix association or by configurational changes within preformed dimers. The structure determination for such TM regions is challenging and has mostly been accomplished by NMR spectroscopy. Recently, the computational prediction of TM dimer structures is becoming recognized for providing models, including alternate conformational states, which are important for receptor regulation. Here we pursued a strategy to predict helix oligomers that is based on packing considerations (using the PREDDIMER webserver) and is followed by a refinement of structures, utilizing microsecond all-atom molecular dynamics simulations. We applied this method to plexin TM receptors, a family of 9 human proteins, involved in the regulation of cell guidance and motility. The predicted models show that, overall, the preferences identified by PREDDIMER are preserved in the unrestrained simulations and that TM structures are likely to be diverse across the plexin family. Plexin-B1 and –B3 TM helices are regular and tend to associate, whereas plexin-A1, -A2, –A3, -A4, -C1 and –D1 contain sequence elements, such as poly-Glycine or aromatic residues that distort helix conformation and association. Plexin-B2 does not form stable dimers due to the presence of TM prolines. No experimental structural information on the TM region is available for these proteins, except for plexin-C1 dimeric and plexin-B1 – trimeric structures inferred from X-ray crystal structures of the intracellular regions. Plexin-B1 TM trimers utilize Ser and Thr sidechains for interhelical contacts. We also modeled the juxta-membrane (JM) region of plexin-C1 and plexin-B1 and show that it synergizes with the TM structures. The structure and dynamics of the JM region and TM-JM junction provide determinants for the distance and distribution of the intracellular domains, and for their binding partners relative to the membrane. The structures suggest experimental tests and will be useful for the interpretation of future studies. PMID:25837709

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

    PubMed Central

    Hudson, Karen A.; Hudson, Matthew E.

    2015-01-01

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

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

    PubMed

    Hudson, Karen A; Hudson, Matthew E

    2015-01-01

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

  2. Molecular Content of the Helix Nebula

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

    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.

  3. Kinetic, mechanistic, and structural aspects of unliganded gating of acetylcholine receptor channels: a single-channel study of second transmembrane segment 12' mutants.

    PubMed

    Grosman, C; Auerbach, A

    2000-05-01

    The spontaneous activity of adult mouse muscle acetylcholine receptor channels, transiently expressed in HEK-293 cells, was studied with the patch-clamp technique. To increase the frequency of unliganded openings, mutations at the 12' position of the second transmembrane segment were engineered. Our results indicate that: (a) in both wild type and mutants, a C <--> O kinetic scheme provides a good description of spontaneous gating. In the case of some mutant constructs, however, additional states were needed to improve the fit to the data. Similar additional states were also needed in one of six patches containing wild-type acetylcholine receptor channels; (b) the delta12' residue makes a more pronounced contribution to unliganded gating than the homologous residues of the alpha, beta, and straightepsilon subunits; (c) combinations of second transmembrane segment 12' mutations in the four different subunits appear to have cumulative effects; (d) the volume of the side chain at delta12' is relevant because residues larger than the wild-type Ser increase spontaneous gating; (e) the voltage dependence of the unliganded gating equilibrium constant is the same as that of diliganded gating, but the voltage dependences of the opening and closing rate constants are opposite (this indicates that the reaction pathway connecting the closed and open states of the receptor changes upon ligation); (f) engineering binding-site mutations that decrease diliganded gating (alphaY93F, alphaY190W, and alphaD200N) reduces spontaneous activity as well (this suggests that even in the absence of ligand the opening of the channel is accompanied by a conformational change at the binding sites); and (g) the diliganded gating equilibrium constant is also increased by the 12' mutations. Such increase is independent of the particular ligand used as the agonist, which suggests that these mutations affect mostly the isomerization step, having little, if any, effect on the ligand-affinity ratio. PMID:10779319

  4. Strong dimerization of wild-type ErbB2/Neu transmembrane domain and the oncogenic Val664Glu mutant in mammalian plasma membranes.

    PubMed

    Placone, Jesse; He, Lijuan; Del Piccolo, Nuala; Hristova, Kalina

    2014-09-01

    Here, we study the homodimerization of the transmembrane domain of Neu, as well as an oncogenic mutant (V664E), in vesicles derived from the plasma membrane of mammalian cells. For the characterization, we use a Förster resonance energy transfer (FRET)-based method termed Quantitative Imaging-FRET (QI-FRET), which yields the donor and acceptor concentrations in addition to the FRET efficiencies in individual plasma membrane-derived vesicles. Our results demonstrate that both the wild-type and the mutant are 100% dimeric, suggesting that the Neu TM helix dimerizes more efficiently than other RTK TM domains in mammalian membranes. Furthermore, the data suggest that the V664E mutation causes a very small, but statistically significant change in dimer structure. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. PMID:24631664

  5. Receptor binding properties of four-helix-bundle growth factors deduced from electrostatic analysis.

    PubMed Central

    Demchuk, E.; Mueller, T.; Oschkinat, H.; Sebald, W.; Wade, R. C.

    1994-01-01

    Hormones of the hematopoietin class mediate signal transduction by binding to specific transmembrane receptors. Structural data show that the human growth hormone (hGH) forms a complex with a homodimeric receptor and that hGH is a member of a class of hematopoietins possessing an antiparallel 4-alpha-helix bundle fold. Mutagenesis experiments suggest that electrostatic interactions may have an important influence on hormone-receptor recognition. In order to examine the specificity of hormone-receptor complexation, an analysis was made of the electrostatic potentials of hGH, interleukin-2 (IL-2), interleukin-4 (IL-4), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the hGH and IL-4 receptors. The binding surfaces of hGH and its receptor, and of IL-4 and its receptor, show complementary electrostatic potentials. The potentials of the hGH and its receptor display approximately 2-fold rotational symmetry because the receptor subunits are identical. In contrast, the potentials of GM-CSF and IL-2 lack such symmetry, consistent with their known high affinity for hetero-oligomeric receptors. Analysis of the electrostatic potentials supports a recently proposed hetero-oligomeric model for a high-affinity IL-4 receptor and suggests a possible new receptor binding mode for G-CSF; it also provides valuable information for guiding structural and mutagenesis studies of signal-transducing proteins and their receptors. PMID:7520794

  6. SignallingSeven-transmembrane receptors

    Microsoft Academic Search

    Kristen L. Pierce; Richard T. Premont; Robert J. Lefkowitz

    2002-01-01

    Seven-transmembrane receptors, which constitute the largest, most ubiquitous and most versatile family of membrane receptors, are also the most common target of therapeutic drugs. Recent findings indicate that the classical models of G-protein coupling and activation of second-messenger-generating enzymes do not fully explain their remarkably diverse biological actions.

  7. Helix Stabilization Precedes Aqueous and Bilayer-Catalyzed Fiber Formation in Islet

    E-print Network

    Miranker, Andrew

    Helix Stabilization Precedes Aqueous and Bilayer-Catalyzed Fiber Formation in Islet Amyloid, all--strand amyloid fiber. Although predominantly disordered, IAPP transiently samples -helical structures, yet all assemble into a common, highly ordered filamentous structure. Amyloid fiber structure

  8. Triple helix purification and sequencing

    DOEpatents

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

    1995-03-28

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

  9. Triple helix purification and sequencing

    DOEpatents

    Wang, Renfeng (Dublin, CA); Smith, Lloyd M. (Madison, WI); Tong, Xinchun E. (Madison, WI)

    1995-01-01

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

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

    PubMed Central

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

    2012-01-01

    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

  11. HUNT: launch of a full-length cDNA database from the Helix Research Institute.

    PubMed

    Yudate, H T; Suwa, M; Irie, R; Matsui, H; Nishikawa, T; Nakamura, Y; Yamaguchi, D; Peng, Z Z; Yamamoto, T; Nagai, K; Hayashi, K; Otsuki, T; Sugiyama, T; Ota, T; Suzuki, Y; Sugano, S; Isogai, T; Masuho, Y

    2001-01-01

    The Helix Research Institute (HRI) in Japan is releasing 4356 HUman Novel Transcripts and related information in the newly established HUNT database. The institute is a joint research project principally funded by the Japanese Ministry of International Trade and Industry, and the clones were sequenced in the governmental New Energy and Industrial Technology Development Organization (NEDO) Human cDNA Sequencing Project. The HUNT database contains an extensive amount of annotation from advanced analysis and represents an essential bioinformatics contribution towards understanding of the gene function. The HRI human cDNA clones were obtained from full-length enriched cDNA libraries constructed with the oligo-capping method and have resulted in novel full-length cDNA sequences. A large fraction has little similarity to any proteins of known function and to obtain clues about possible function we have developed original analysis procedures. Any putative function deduced here can be validated or refuted by complementary analysis results. The user can also extract information from specific categories like PROSITE patterns, PFAM domains, PSORT localization, transmembrane helices and clones with GENIUS structure assignments. The HUNT database can be accessed at http://www.hri.co.jp/HUNT. PMID:11125086

  12. VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S87), ...

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

    VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S-87), LOOKING WEST (without scale stick). - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

  13. VIEW OF SOUTH ELEVATION OF HELIX HOUSE NO. 2 (S87) ...

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

    VIEW OF SOUTH ELEVATION OF HELIX HOUSE NO. 2 (S-87) SHOWING MAIN ENTRY DOOR, LOOKING NORTH NORTHWEST. - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

  14. VIEW OF HELIX HOUSE NO. 2 (S87), WITH ANTENNA TOWER ...

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

    VIEW OF HELIX HOUSE NO. 2 (S-87), WITH ANTENNA TOWER CABLE SUPPORT IN FOREGROUND, LOOKING SOUTHEAST. - Naval Computer & Telecommunications Area Master Station, Eastern Pacific, Radio Transmitter Facility Lualualei, Helix House No. 2, Base of Radio Antenna Structure No. 427, Makaha, Honolulu County, HI

  15. VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S87), ...

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

    VIEW OF EAST ELEVATION OF HELIX HOUSE NO. 2 (S-87), LOOKING WEST (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

  16. New stochastic strategy to analyze helix folding.

    PubMed Central

    Moret, M A; Bisch, P M; Mundim, K C; Pascutti, P G

    2002-01-01

    We propose an alternative stochastic strategy to search secondary structures based on the generalized simulated annealing (GSA) algorithm, by using conformational preferences based on the Ramachandran map. We optimize the search for polypeptide conformational space and apply to peptides considered to be good alpha-helix promoters above a critical number of residues. Our strategy to obtain conformational energies consist in coupling a classical force field (THOR package) with the GSA procedure, biasing the Phi x Psi backbone angles to the allowed regions in the Ramachandran map. For polyalanines we obtained stable alpha-helix structures when the number of residues were equal or exceeded 13 amino acids residues. We also observed that the energy gap between the global minimum and the first local minimum tends to increase with the polypeptide size. These conformations were generated by performing 2880 stochastic molecular optimizations with a continuum medium approach. When compared with molecular dynamics or Monte Carlo methods, GSA can be considered the fastest. PMID:11867432

  17. DNA-The Double Helix - Nobel Prize Educational Game

    NSDL National Science Digital Library

    2009-01-01

    The 1962 Nobel Prize in Physiology or Medicine was awarded for the discovery of the molecular structure of DNA Â? the double helix. In this game your job is to first make exact copies of a double-stranded DNA molecule by correctly matching base pairs to each strand, and to then determine which organism the DNA belongs to.

  18. Identification of a cluster of residues in transmembrane segment 6 of domain III of the cockroach sodium channel essential for the action of pyrethroid insecticides.

    PubMed

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

    2009-04-15

    A phenylalanine residue (Phe1519) in the sixth transmembrane segment of domain III (IIIS6) of the cockroach BgNa(v) sodium channel is required for the binding and action of pyrethroids. However, whether or not other residues in IIIS6 participate in the action of pyrethroids remains to be determined. In the present study, we conducted a systematic analysis of 20 residues in IIIS6 of the BgNa(v) channel using alanine-scanning mutagenesis. Our results show that alanine substitutions of four residues, Ile1514, Gly1516, Phe1518 and Asn1522, altered sodium channel sensitivity to pyrethroid insecticides. Whereas the G1516A, F1518A and N1522A substitutions diminished sodium channel sensitivity to all seven pyrethroids examined, including four type I (lacking the alpha-cyano group at the phenoxybenzyl alcohol) and three type II (containing the alpha-cyano group) pyrethroids, the I1514A substitution enhanced sodium channel sensitivity to four type I and type II pyrethroids that contain the phenoxybenzyl alcohol only. We also show that alanine/lysine substitutions of Leu1521 and Ser1517 affected the action of BTX (batrachotoxin), but not pyrethroids. In the Kv1.2-based homology model of the open sodium channel, side chains of Ile1514, Phe1518 and Asn1522 are exposed towards helix IIS5 and linker IIS4-IIS5, which contain previously identified pyrethroid-interacting residues, whereas Ser1517 and Leu1521 face the inner pore where the BTX receptor is located. Thus the present study provides further evidence for structural models in which pyrethroids bind to the lipid-exposed interface formed by helices IIIS6, IIS5 and linker helix IIS4-IIS5, whereas BTX binds to the pore-exposed side of the IIIS6 helix. PMID:19154185

  19. The DNA double helix fifty years on.

    PubMed

    Macgregor, Robert B; Poon, Gregory M K

    2003-10-01

    This year marks the 50th anniversary of the proposal of a double helical structure for DNA by James Watson and Francis Crick. The place of this proposal in the history and development of molecular biology is discussed. Several other discoveries that occurred in the middle of the twentieth century were perhaps equally important to our understanding of cellular processes; however, none of these captured the attention and imagination of the public to the same extent as the double helix. The existence of multiple forms of DNA and the uses of DNA in biological technologies is presented. DNA is also finding increasing use as a material due to its rather unusual structural and physical characteristics as well as its ready availability. PMID:14642754

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

    E-print Network

    Springer, Timothy A.

    separation for integrin outside-in signaling Jieqing Zhu,1 Christopher V. Carman,2 Minsoo Kim,3 Motomu of Anesthesia, Harvard Medical School, Boston, MA Adhesion to extracellular ligands through integrins regulates cell shape, migration, growth, and survival. How integrins trans- mit signals in the outside

  1. The Effect of Point Mutations on the Free Energy of Transmembrane aaa-Helix Dimerization

    E-print Network

    of dimeri- zation in a non-denaturing detergent solution and have observed the changes in energy arising from two of the mutants previously studied. Use of the detergent pentaoxyethylene octyl ether (C8E5) is a great advan- tage, since its micelles are neutrally buoyant and the detergent allows a reversible

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

    E-print Network

    Funk, Geoffrey Alexander

    2013-02-22

    -automated chemical synthesis of peptides Reaction Reagents Reaction Time Deprotection Coupling Capping ? 20% Piperidine in NMP with 1% Triton X-100 ? 40 molar excess of desired amino acid residue - 552 mg HOBt - 1365 mg HBTU - 1. 260 mL DIEA in NMP with 1...

  3. Supplementary Information High-accuracy prediction of transmembrane inter-helix contacts and

    E-print Network

    Zhang, Yang

    a 48.2 7.8 79.4 SVM1 47.6 7.8 84.2 SVM2 48.7 8.2 85.2 SVM3 49.0 8.1 84.8 SVM4 46.9 7.7 84.0 SVM5 48.8 8.1. A sliding window covering neighboring residue pairs was used to encode the residue pair {i, j}, i.e., {i

  4. Structure of an atypical FeoB G-domain reveals a putative domain-swapped dimer

    PubMed Central

    Deshpande, Chandrika N.; McGrath, Aaron P.; Font, Josep; Guilfoyle, Amy P.; Maher, Megan J.; Jormakka, Mika

    2013-01-01

    FeoB is a transmembrane protein involved in ferrous iron uptake in prokaryotic organisms. FeoB comprises a cytoplasmic soluble domain termed NFeoB and a C-terminal polytopic transmembrane domain. Recent structures of NFeoB have revealed two structural subdomains: a canonical GTPase domain and a five-helix helical domain. The GTPase domain hydrolyses GTP to GDP through a well characterized mechanism, a process which is required for Fe2+ transport. In contrast, the precise role of the helical domain has not yet been fully determined. Here, the structure of the cytoplasmic domain of FeoB from Gallionella capsiferriformans is reported. Unlike recent structures of NFeoB, the G. capsiferriformans NFeoB structure is highly unusual in that it does not contain a helical domain. The crystal structures of both apo and GDP-bound protein forms a domain-swapped dimer. PMID:23545645

  5. Design of Tip Loss Profile on Support Rod for a Helix TWT

    NASA Astrophysics Data System (ADS)

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

    2009-07-01

    This paper describes the design of tip loss profile on support rod for a high gain, high efficiency helix TWT. The tip loss profile at sever ends for the input and the output section of the helix slow wave structure has been designed for return loss at severs ends better than -20 dB. The experimental results have been carried out in the input and the output section of the helix assemblies for the high gain helix TWT to find the return loss in the region of carbon coated tip loss on the support rods. The experimental result has been compared with the simulated performance for the return loss at the sever ends. The design of tip loss profile on the support rod for helix TWT has been carried out in real situation using Ansoft HFSS. A good agreement has been found in the simulated and experimental results.

  6. The Transition from a Neutral-pH Double Helix to a Low-pH Triple Helix Induces a Conformational Switch in the CCCG Tetraloop Closing a Watson-Crick Stem

    Microsoft Academic Search

    Maria J. P. van Dongen; Sybren S. Wijmenga; Gijs A. van der Marel; Jacques H. van Boom; Cornelis W. Hilbers

    1996-01-01

    The CCCG-loop in a DNA fragment, which is capable of forming an intramolecular triple helix as well as a hairpin structure, was investigated by NMR and molecular modeling studies. The structure of this loop is found as a type II conformation, one of the three commonly observed folding patterns of tetraloops, irrespective of the geometry of the underlying helix. In

  7. Differentiation of Arabidopsis Guard Cells: Analysis of the Networks Incorporating the Basic Helix-Loop-Helix Transcription Factor, FAMA1[C][W][OA

    PubMed Central

    Hachez, Charles; Ohashi-Ito, Kyoko; Dong, Juan; Bergmann, Dominique C.

    2011-01-01

    Nearly all extant land plants possess stomata, the epidermal structures that mediate gas exchange between the plant and the environment. The developmental pathways, cell division patterns, and molecules employed in the generation of these structures are simple examples of processes used in many developmental contexts. One specific module is a set of “master regulator” basic helix-loop-helix transcription factors that regulate individual consecutive steps in stomatal development. Here, we profile transcriptional changes in response to inducible expression of Arabidopsis (Arabidopsis thaliana) FAMA, a basic helix-loop-helix protein whose actions during the final stage in stomatal development regulate both cell division and cell fate. Genes identified by microarray and candidate approaches were then further analyzed to test specific hypothesis about the activity of FAMA, the shape of its regulatory network, and to create a new set of stomata-specific or stomata-enriched reporters. PMID:21245191

  8. Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal ?-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

    PubMed Central

    Morgan, Rhodri M. L.; Hernández-Ramírez, Laura C.; Trivellin, Giampaolo; Zhou, Lihong; Roe, S. Mark; Korbonits, Márta; Prodromou, Chrisostomos

    2012-01-01

    Mutations of the aryl hydrocarbon receptor interacting protein (AIP) have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal ?-7 helix (C?-7h) mutations, R304* (nonsense mutation), R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the C?-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the C?-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE) of TOMM20. PMID:23300914

  9. Structural correspondence between the ?-helix and the random-flight chain resolves how unfolded proteins can have native-like properties

    Microsoft Academic Search

    Bojan Zagrovic; Vijay S Pande

    2003-01-01

    Recently, we have proposed that, on average, the structure of the unfolded state of small, mostly ?-helical proteins may be similar to the native structure (the 'mean-structure' hypothesis). After examining thousands of simulations of both the folded and the unfolded states of five polypeptides in atomistic detail at room temperature, we report here a result that seems at odds with

  10. Type II transmembrane serine proteases.

    PubMed

    Bugge, Thomas H; Antalis, Toni M; Wu, Qingyu

    2009-08-28

    Analysis of genome and expressed sequence tag data bases at the turn of the millennium unveiled a new protease family named the type II transmembrane serine proteases (TTSPs) in a Journal of Biological Chemistry minireview (Hooper, J. D., Clements, J. A., Quigley, J. P., and Antalis, T. M. (2001) J. Biol. Chem. 276, 857-860). Since then, the number of known TTSPs has more than doubled, and more importantly, our understanding of the physiological functions of individual TTSPs and their contribution to human disease has greatly increased. Progress has also been made in identifying molecular substrates and endogenous inhibitors. This minireview summarizes the current knowledge of the rapidly advancing TTSP field. PMID:19487698

  11. Type II Transmembrane Serine Proteases*

    PubMed Central

    Bugge, Thomas H.; Antalis, Toni M.; Wu, Qingyu

    2009-01-01

    Analysis of genome and expressed sequence tag data bases at the turn of the millennium unveiled a new protease family named the type II transmembrane serine proteases (TTSPs) in a Journal of Biological Chemistry minireview (Hooper, J. D., Clements, J. A., Quigley, J. P., and Antalis, T. M. (2001) J. Biol. Chem. 276, 857–860). Since then, the number of known TTSPs has more than doubled, and more importantly, our understanding of the physiological functions of individual TTSPs and their contribution to human disease has greatly increased. Progress has also been made in identifying molecular substrates and endogenous inhibitors. This minireview summarizes the current knowledge of the rapidly advancing TTSP field. PMID:19487698

  12. The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

    SciTech Connect

    Hollier, Mark J. [Department of Biological Sciences, University of Warwick, Coventry CV4 7AL (United Kingdom); Dimmock, Nigel J. [Department of Biological Sciences, University of Warwick, Coventry CV4 7AL (United Kingdom)]. E-mail: n.j.dimmock@warwick.ac.uk

    2005-07-05

    In addition to the major ectodomain, the gp41 transmembrane glycoprotein of HIV-1 is now known to have a minor ectodomain that is part of the long C-terminal tail. Both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. However, data have so far been biologically based, and derived solely from T cell line-adapted (TCLA), B clade viruses. Here we have carried out sequence and theoretically based structural analyses of 357 gp41 C-terminal sequences of mainly primary isolates of HIV-1 clades A, B, C, and D. Data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, {beta}-sheet, membrane-spanning domains (MSDs). This means that the first (N-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. However, we suggest that only a minority of cell-associated gp41 molecules - those destined for incorporation into virions - has 3 MSDs and the minor ectodomain. Most intracellular gp41 has the conventional single MSD, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. The gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize HIV-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell.

  13. Structure of a bacterial homolog of vitamin K epoxide reductase

    PubMed Central

    Li, Weikai; Schulman, Sol; Dutton, Rachel J.; Boyd, Dana; Beckwith, Jon; Rapoport, Tom A.

    2010-01-01

    Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain ?-carboxylation of many blood coagulation factors. Here, we report the 3.6Å crystal structure of a bacterial homolog of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulfide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant. PMID:20110994

  14. The Origins of Transmembrane Ion Channels

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Wilson, Michael A.

    2012-01-01

    Even though membrane proteins that mediate transport of ions and small molecules across cell walls are among the largest and least understood biopolymers in contemporary cells, it is still possible to shed light on their origins and early evolution. The central observation is that transmembrane portions of most ion channels are simply bundles of -helices. By combining results of experimental and computer simulation studies on synthetic models and natural channels, mostly of non-genomic origin, we show that the emergence of -helical channels was protobiologically plausible, and did not require highly specific amino acid sequences. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. Specifically, we explain how the antiamoebin channels, which are made of identical helices, 16 amino acids in length, achieve efficiency comparable to that of highly evolved channels. We further show that antiamoebin channels are extremely flexible, compared to modern, genetically coded channels. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire stable rigid structures and mechanisms for precise regulation rather than improve efficiency. In general, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

  15. Manifestation of cystic fibrosis transmembrane regulator (CFTR) in hepatic ductal structures and renal tubules of female rats with experimental cholestasis of pregnancy.

    PubMed

    Aleksandrova, M I; Kushnareva, N S; Smirnova, O V

    2014-03-01

    Studies by the immunohistochemical method with semiquantitative analysis of images showed that hyperprolactinemia stimulated CFTR protein manifestation in the bile ducts of female rats, which was clearly expressed in experimental cholestasis of pregnancy. The expression of CFTR in the renal tubules was reduced in hyperprolactinemia under conditions of normal liver function and in cholestasis of pregnancy. Significant positive correlations between CFTR, prolactin receptor, and multiple drug resistance protein 3 were detected in the bile ducts, but not in the renal tubules. Presumably, prolactin has a direct effect on CFTR expression in the bile ducts and indirect effect in the renal tubules. Changes in CFTR protein manifestation in the hepatic ductal structures and renal tubules in experimental pregnancy cholestasis could aggravate the disease. PMID:24770751

  16. Helix Antennas Dr. Sandra Cruz-Pol

    E-print Network

    Cruz-Pol, Sandra L.

    , circular or linear (vertical), depending on which receiver is being used. Narrow Band Antennas Circularly11 Helix Antennas Dr. Sandra Cruz-Pol INEL 5305 UPRM Antenna Theory and Design Helix antenna Ioe- jkr 4r sin (lazo) (i) Si |E| = |E| tenemos polarización circular. (ii) En general la polarización

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

    NASA Astrophysics Data System (ADS)

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

    1998-09-01

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

  18. The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc

    E-print Network

    Tullius, Thomas D.

    stabilized by zinc Astrid P.A.M. Eijkelenboom*, Fusinita M.I. van den Ent, Arnold Vos, Jurgen F. Doreleijers domain with unknown structure thus far. This domain, which is known to bind zinc, contains a HHCC motif consists of three helices and a helical turn. The zinc is coordinated with His12 via the N2 atom, with His

  19. Discovering the double helix structure of DNA, James Watson, video with 3D animation and narrationSite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-10-06

    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.

  20. Alanine Scanning Mutagenesis of the ?-Helix 115-123 of Phage T4 Lysozyme: Effects on Structure, Stability and the Binding of Solvent

    Microsoft Academic Search

    Michael Blaber; Walter A. Baase; Nadine Gassner; Brian W. Matthews

    1995-01-01

    A series of individual alanine mutations has been constructed in the helical region 115 to 123 in phage T4 lysozyme in order to evaluate the contribution to protein stability of the different side-chains within this region. Pairwise alanine mutations and a combination mutant with seven alanine substitutions were constructed to evaluate the additive effects upon structure and stability. Only three

  1. Linus Pauling' s manuscript describing incorrect triple helix, Peter PaulingSite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-10-06

    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.

  2. 558 MISCELLANEOUS METHODS [34] site, and then covers a whole turn of the DNA helix on each end of the

    E-print Network

    OLIPHANT, and KEVIN STRUHL Introduction Since the advent of recombinant DNA technology, structure558 MISCELLANEOUS METHODS [34] site, and then covers a whole turn of the DNA helix on each end side of the DNA helix by TFIIIA, while at the ends of the control region (roughly positions 44

  3. Dispersion-shaping using an inhomogeneous dielectric support for the helix in a travelling-wave tube

    Microsoft Academic Search

    B. N. Basu; A. K. Sinha

    1981-01-01

    The employment of an inhomogeneous low-permittivity dielectric support for the helix in TWTs is suggested for obtaining flat dispersion characteristics of the slow-wave structure while ensuring a relatively high value of the interaction impedance. The analytical procedure adopted closely follows the equivalent circuit analysis developed by Basu (1979) for a helix surrounded by a homogeneous dielectric in an overall conducting

  4. The Mtr4 ratchet helix and arch domain both function to promote RNA unwinding.

    PubMed

    Taylor, Lacy L; Jackson, Ryan N; Rexhepaj, Megi; King, Alejandra Klauer; Lott, Lindsey K; van Hoof, Ambro; Johnson, Sean J

    2014-12-16

    Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3'-5' turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The 'ratchet helix' is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates. PMID:25414331

  5. The Mtr4 ratchet helix and arch domain both function to promote RNA unwinding

    PubMed Central

    Taylor, Lacy L.; Jackson, Ryan N.; Rexhepaj, Megi; King, Alejandra Klauer; Lott, Lindsey K.; van Hoof, Ambro; Johnson, Sean J.

    2014-01-01

    Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3?-5? turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The ‘ratchet helix’ is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates. PMID:25414331

  6. A genome-wide survey on basic helix-loop-helix transcription factors in giant panda.

    PubMed

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

    2011-01-01

    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

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

    PubMed Central

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

    2014-01-01

    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

  8. An exploration of alternative visualisations of the basic helix-loop-helix protein interaction network

    PubMed Central

    Holden, Brian J; Pinney, John W; Lovell, Simon C; Amoutzias, Grigoris D; Robertson, David L

    2007-01-01

    Background Alternative representations of biochemical networks emphasise different aspects of the data and contribute to the understanding of complex biological systems. In this study we present a variety of automated methods for visualisation of a protein-protein interaction network, using the basic helix-loop-helix (bHLH) family of transcription factors as an example. Results Network representations that arrange nodes (proteins) according to either continuous or discrete information are investigated, revealing the existence of protein sub-families and the retention of interactions following gene duplication events. Methods of network visualisation in conjunction with a phylogenetic tree are presented, highlighting the evolutionary relationships between proteins, and clarifying the context of network hubs and interaction clusters. Finally, an optimisation technique is used to create a three-dimensional layout of the phylogenetic tree upon which the protein-protein interactions may be projected. Conclusion We show that by incorporating secondary genomic, functional or phylogenetic information into network visualisation, it is possible to move beyond simple layout algorithms based on network topology towards more biologically meaningful representations. These new visualisations can give structure to complex networks and will greatly help in interpreting their evolutionary origins and functional implications. Three open source software packages (InterView, TVi and OptiMage) implementing our methods are available. PMID:17683601

  9. Structural Basis for TatA Oligomerization: An NMR Study of Escherichia coli TatA Dimeric Structure

    PubMed Central

    Li, Hongwei; Jin, Changwen

    2014-01-01

    Many proteins are transported across lipid membranes by protein translocation systems in living cells. The twin-arginine transport (Tat) system identified in bacteria and plant chloroplasts is a unique system that transports proteins across membranes in their fully-folded states. Up to date, the detailed molecular mechanism of this process remains largely unclear. The Escherichia coli Tat system consists of three essential transmembrane proteins: TatA, TatB and TatC. Among them, TatB and TatC form a tight complex and function in substrate recognition. The major component TatA contains a single transmembrane helix followed by an amphipathic helix, and is suggested to form the translocation pore via self-oligomerization. Since the TatA oligomer has to accommodate substrate proteins of various sizes and shapes, the process of its assembly stands essential for understanding the translocation mechanism. A structure model of TatA oligomer was recently proposed based on NMR and EPR observations, revealing contacts between the transmembrane helices from adjacent subunits. Herein we report the construction and stabilization of a dimeric TatA, as well as the structure determination by solution NMR spectroscopy. In addition to more extensive inter-subunit contacts between the transmembrane helices, we were also able to observe interactions between neighbouring amphipathic helices. The side-by-side packing of the amphipathic helices extends the solvent-exposed hydrophilic surface of the protein, which might be favourable for interactions with substrate proteins. The dimeric TatA structure offers more detailed information of TatA oligomeric interface and provides new insights on Tat translocation mechanism. PMID:25090434

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

    PubMed

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

    2012-02-01

    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

  11. Distinctly Different Interactions of Anesthetic and Nonimmobilizer with Transmembrane Channel Peptides

    Microsoft Academic Search

    Pei Tang; Jian Hu; Serguei Liachenko; Yan Xu

    1999-01-01

    Although it plays no clinical role in general anesthesia, gramicidin A, a transmembrane channel peptide, provides an excellent model for studying the specific interaction between volatile anesthetics and membrane proteins at the molecular level. We show here that a pair of structurally similar volatile anesthetic and nonimmobilizer (nonanesthetic), 1-chloro-1,2,2-trifluorocyclobutane (F3) and 1,2-dichlorohexafluorocyclobutane (F6), respectively, interacts differently with the transmembrane peptide.

  12. Rationally Designed Transmembrane Peptide Mimics of the Multidrug Transporter Protein Cdr1 Act as Antagonists to Selectively Block Drug Efflux and Chemosensitize Azole-resistant Clinical Isolates of Candida albicans*?

    PubMed Central

    Maurya, Indresh Kumar; Thota, Chaitanya Kumar; Verma, Sachin Dev; Sharma, Jyotsna; Rawal, Manpreet Kaur; Ravikumar, Balaguru; Sen, Sobhan; Chauhan, Neeraj; Lynn, Andrew M.; Chauhan, Virander Singh; Prasad, Rajendra

    2013-01-01

    Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo. PMID:23592791

  13. Glycines: Role in ?-Helical Membrane Protein Structures and a Potential Indicator for Native Conformation

    PubMed Central

    Dong, Hao; Sharma, Mukesh; Zhou, Huan-Xiang; Cross, Timothy A.

    2012-01-01

    Among the growing number of membrane protein structures in the Protein Data Bank, there are many transmembrane domains that appear to be native-like; at the same time there are others that appear to have less than complete native-like character. Hence there is an increasing need for validation tools that distinguish native-like from nonnative-like structures. Membrane mimetics used in protein structural characterizations differ in numerous physicochemical properties from native membranes and provide many opportunities for introducing nonnative-like features into membrane protein structures. One possible approach for validating membrane protein structures is based on the use of glycine residues in transmembrane domains. Here, we have reviewed the membrane protein structure database and identified a set of benchmark proteins that appear to be native like. In these structures, conserved glycine residues rarely face the lipid interstices, and many of them participate in close helix-helix packing. Glycine-based validation allowed the identification of nonnative-like features in several membrane proteins and also shows the potential for verifying the native-like character for numerous other membrane protein structures. PMID:22650985

  14. A hidden Markov model for predicting transmembrane helices in protein sequences.

    PubMed

    Sonnhammer, E L; von Heijne, G; Krogh, A

    1998-01-01

    A novel method to model and predict the location and orientation of alpha helices in membrane-spanning proteins is presented. It is based on a hidden Markov model (HMM) with an architecture that corresponds closely to the biological system. The model is cyclic with 7 types of states for helix core, helix caps on either side, loop on the cytoplasmic side, two loops for the non-cytoplasmic side, and a globular domain state in the middle of each loop. The two loop paths on the non-cytoplasmic side are used to model short and long loops separately, which corresponds biologically to the two known different membrane insertions mechanisms. The close mapping between the biological and computational states allows us to infer which parts of the model architecture are important to capture the information that encodes the membrane topology, and to gain a better understanding of the mechanisms and constraints involved. Models were estimated both by maximum likelihood and a discriminative method, and a method for reassignment of the membrane helix boundaries were developed. In a cross validated test on single sequences, our transmembrane HMM, TMHMM, correctly predicts the entire topology for 77% of the sequences in a standard dataset of 83 proteins with known topology. The same accuracy was achieved on a larger dataset of 160 proteins. These results compare favourably with existing methods. PMID:9783223

  15. Homolog Structure of the SLAC1 Anion Channel for Closing Stomata in Leaves

    PubMed Central

    Chen, Yuhang; Hu, Lei; Punta, Marco; Bruni, Renato; Hillerich, Brandan; Kloss, Brian; Rost, Burkhard; Love, James; Siegelbaum, Steven A.; Hendrickson, Wayne A.

    2012-01-01

    Summary The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating exchange of water vapor and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. We determined the crystal structure of a bacterial homolog of SLAC1 at 1.20Å resolution, and we have used structure-inspired mutagenesis to analyze the conductance properties of SLAC1 channels. SLAC1 is a symmetric trimer composed from quasi-symmetric 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 suggest a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics suggest that selectivity among different anions is largely a function of the energetic cost of ion dehydration. PMID:20981093

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

    SciTech Connect

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

    2011-12-31

    The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 {angstrom} resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

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

    PubMed Central

    Kinoshita, Mikitaka; Okada, Tetsuji

    2015-01-01

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

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

    PubMed

    Kinoshita, Mikitaka; Okada, Tetsuji

    2015-01-01

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

  19. Transmembrane Protein Alignment and Fold Recognition Based on Predicted Topology

    PubMed Central

    Wang, Han; He, Zhiquan; Zhang, Chao; Zhang, Li; Xu, Dong

    2013-01-01

    Background Although Transmembrane Proteins (TMPs) are highly important in various biological processes and pharmaceutical developments, general prediction of TMP structures is still far from satisfactory. Because TMPs have significantly different physicochemical properties from soluble proteins, current protein structure prediction tools for soluble proteins may not work well for TMPs. With the increasing number of experimental TMP structures available, template-based methods have the potential to become broadly applicable for TMP structure prediction. However, the current fold recognition methods for TMPs are not as well developed as they are for soluble proteins. Methodology We developed a novel TMP Fold Recognition method, TMFR, to recognize TMP folds based on sequence-to-structure pairwise alignment. The method utilizes topology-based features in alignment together with sequence profile and solvent accessibility. It also incorporates a gap penalty that depends on predicted topology structure segments. Given the difference between ?-helical transmembrane protein (?TMP) and ?-strands transmembrane protein (?TMP), parameters of scoring functions are trained respectively for these two protein categories using 58 ?TMPs and 17 ?TMPs in a non-redundant training dataset. Results We compared our method with HHalign, a leading alignment tool using a non-redundant testing dataset including 72 ?TMPs and 30 ?TMPs. Our method achieved 10% and 9% better accuracies than HHalign in ?TMPs and ?TMPs, respectively. The raw score generated by TMFR is negatively correlated with the structure similarity between the target and the template, which indicates its effectiveness for fold recognition. The result demonstrates TMFR provides an effective TMP-specific fold recognition and alignment method. PMID:23894534

  20. Primary structure of a novel subunit in ba3-cytochrome oxidase from Thermus thermophilus.

    PubMed Central

    Soulimane, T.; Than, M. E.; Dewor, M.; Huber, R.; Buse, G.

    2000-01-01

    The bax-type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix "subunit IIa" spanning the membrane. The hydrophobic N-terminally blocked protein was isolated in high yield using high-performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C-terminal sequencing of the native protein. The molecular mass of 3,794 Da as determined by MALDI-MS and by ESI requires the N-terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5-Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart, however, with opposite polarity. It is 35% identical to subunit IV of the ba3-cytochrome oxidase from Natronobacterium pharaonis. The open reading frame encoding this new subunit IIa (cbaD) is located upstream of cbaB in the same operon as the genes for subunit I (cbaA) and subunit II (cbaB). PMID:11152118

  1. pH-dependent helix folding dynamics of poly-glutamic acid

    NASA Astrophysics Data System (ADS)

    Gooding, Edward A.; Sharma, Sapna; Petty, Sarah A.; Fouts, Elizabeth A.; Palmer, Colin J.; Nolan, Brian E.; Volk, Martin

    2013-08-01

    Poly-L-glutamic acid (PGA) is an ideal model system for investigating the transition between ?-helical and random coil peptide conformations, since its secondary structure is highly sensitive not only to temperature, but also to pH. Laser pulse-induced temperature jumps were used to observe pH-dependent PGA helix-coil relaxation dynamics on the microsecond time scale. The relaxation was found to be non-exponential, particularly if only short helical segments are present before the temperature jump, indicating the multi-step nature of the process which involves helix nucleation and propagation. Helix-coil relaxation is slowest near the mid-point of the pH-induced helix-coil transition, in agreement with theoretical predictions.

  2. An analysis of oligomerization interfaces in transmembrane proteins

    PubMed Central

    2013-01-01

    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

  3. Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry

    NASA Technical Reports Server (NTRS)

    Wang, N.; Ingber, D. E.

    1995-01-01

    We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) beta 1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (beta 1, alpha V beta 3, alpha V, alpha 5, alpha 2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.

  4. Helix stabilization precedes aqueous and bilayer catalyzed fiber formation in islet amyloid polypeptide

    PubMed Central

    Williamson, Jessica A.; Loria, J. Patrick; Miranker, Andrew D.

    2012-01-01

    Islet amyloid polypeptide (IAPP) is an unstructured polypeptide hormone that is cosecreted with insulin. In patients with type 2 diabetes, IAPP undergoes a transition from its natively disordered state to a highly ordered, all ?-strand amyloid fiber. Although predominantly disordered, IAPP transiently samples ?-helical structure in solution. IAPP adopts a fully helical structure when bound to membrane surfaces in a process associated with catalysis of amyloid formation. Here, we use spectroscopic techniques to study the structure of full-length, monomeric IAPP under amyloidogenic conditions. We observe that the residues with helical propensity in solution (1-22) also form the membrane-associated helix. Additionally, reduction of the N-terminal disulfide bond (Cys2-Cys7) decreases the extent of helix formed throughout this region. Through manipulation of sample conditions to increase or decrease the amount of helix, we show that the degree of helix formed affects the rate of amyloid assembly. Formation of helical structure is directly correlated with enhanced amyloid formation both on the membrane surface and in solution. These observations support suggested mechanisms in which parallel helix associations bring together regions of the peptide that could nucleate ?-strand structure. Remarkably, stabilization of non-amyloid structure appears to be a key intermediate in assembly of IAPP amyloid. PMID:19647750

  5. Motifs in Protein SequencesMotifs in Protein Sequences Examples: Helix-Turn-Helix, Zinc-finger,

    E-print Network

    Narasimhan, Giri

    Motifs in Protein SequencesMotifs in Protein Sequences Examples: Helix-Turn-Helix, Zinc. Examples: Helix-Turn-Helix, Zinc-finger, Homeobox domain, Hairpin-beta motif, Calcium-binding motif, Beta of the motif M? Example: Zinc Finger Motif ...YYKCCGLCCERSFFVEKSALLSRHHORVHHKN... 3 6 19 23 Input

  6. Structures of ?-Amyloid Peptide 1–40, 1–42, and 1–55 -the 672–726 fragment of APP- in a membrane environment with implications for interactions with ?-secretase

    PubMed Central

    Miyashita, Naoyuki; Straub, John E.; Thirumalai, D.

    2009-01-01

    Aggregation Amyloid ? (A?) peptide has been linked to the neurodegenerative Alzheimer’s Disease and implicated in other amyloid diseases including cerebral amyloid angiopathy. A? peptide is generated by cleavage of the amyloid precursor protein (APP) by transmembrane proteases. It is crucial to determine the structures of ?-amyloid peptides in a membrane to provide a molecular basis for the cleavage mechanism. We report the structures of amyloid ? peptide (A?1–40 and A?1–42) as well as the 672–726 fragment of APP (referred to as A?1–55) in a membrane environment determined by replica-exchange molecular dynamics simulation. A?1–40 is found to have two helical domains A (13–22) and B(30–35) and a type I ? turn at 23–27. The peptide is localized at the interface between membrane and solvent. Substantial fluctuations in domain A are observed. The dominant simulated tertiary structure of A?1–40 is observed to be similar to the simulated A?1–42 structure. However, there are differences observed in the overall conformational ensemble as characterized by the two-dimensional free energy surfaces. The fragment of APP (A?1–55) is observed to have a long transmembrane helix. The position of the transmembrane region and ensemble of membrane structures are elucidated. The conformational transition between the transmembrane A?1–55 structure, prior to cleavage, and the A?1–40 structure, following cleavage, is proposed. PMID:19995075

  7. PDB_TM: selection and membrane localization of transmembrane proteins in the protein data bank

    Microsoft Academic Search

    Gábor E. Tusnády; Zsuzsanna Dosztányi; István Simon

    2005-01-01

    PDB_TM is a database for transmembrane proteins with known structures. It aims to collect all trans- membrane proteins that are deposited in the protein structure database (PDB) and to determine their membrane-spanning regions. These assignments are based on the TMDET algorithm, which uses only structural information to locate the most likely posi- tion of the lipid bilayer and to distinguish

  8. Linus Pauling's triple DNA helix model, 3D animation with basic narrationSite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-10-06

    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.

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

    PubMed

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

    2009-06-01

    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 alpha6 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

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

    PubMed Central

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

    2009-01-01

    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

  11. Folding of immunogenic peptide fragments of proteins in water solution. II. The nascent helix.

    PubMed

    Dyson, H J; Rance, M; Houghten, R A; Wright, P E; Lerner, R A

    1988-05-01

    1H nuclear magnetic resonance experiments indicate formation of secondary structures in water solutions of a synthetic immunogenic peptide of sequence EVVPHKKMHKDFLEKIGGL corresponding to the C-helix (residues 69 to 87) of myohemerythrin. The conformational ensemble consists of a set of turn-like structures, distributed over the C-terminal half of the peptide and rapidly interconverting by way of unfolded states. These structures, termed nascent helix, are stabilized into helical structure with long-range order in water/trifluorethanol mixtures. Circular dichroism measurements confirm the presence of 50% helix in water/trifluoroethanol but show no evidence of helicity in water solutions of the peptide. It is apparent that no one member of the transient set of helical conformations which constitutes the nascent helix is sufficiently long to be detectable by circular dichroism experiments. No preferred conformations could be detected by nuclear magnetic resonance in the N-terminal half of the peptide, either in water or water/trifluoroethanol mixtures. This region of the peptide is stabilized in helix by long-range interactions in the folded protein. The possible role of nascent secondary structure in induction of antipeptide antibodies and in initiation of protein folding is discussed. PMID:3418697

  12. Structure of CrgA, a cell division structural and regulatory protein from Mycobacterium tuberculosis, in lipid bilayers.

    PubMed

    Das, Nabanita; Dai, Jian; Hung, Ivan; Rajagopalan, Malini R; Zhou, Huan-Xiang; Cross, Timothy A

    2015-01-13

    The 93-residue transmembrane protein CrgA in Mycobacterium tuberculosis is a central component of the divisome, a large macromolecular machine responsible for cell division. Through interactions with multiple other components including FtsZ, FtsQ, FtsI (PBPB), PBPA, and CwsA, CrgA facilitates the recruitment of the proteins essential for peptidoglycan synthesis to the divisome and stabilizes the divisome. CrgA is predicted to have two transmembrane helices. Here, the structure of CrgA was determined in a liquid-crystalline lipid bilayer environment by solid-state NMR spectroscopy. Oriented-sample data yielded orientational restraints, whereas magic-angle spinning data yielded interhelical distance restraints. These data define a complete structure for the transmembrane domain and provide rich information on the conformational ensembles of the partially disordered N-terminal region and interhelical loop. The structure of the transmembrane domain was refined using restrained molecular dynamics simulations in an all-atom representation of the same lipid bilayer environment as in the NMR samples. The two transmembrane helices form a left-handed packing arrangement with a crossing angle of 24° at the conserved Gly39 residue. This helix pair exposes other conserved glycine and alanine residues to the fatty acyl environment, which are potential sites for binding CrgA's partners such as CwsA and FtsQ. This approach combining oriented-sample and magic-angle spinning NMR spectroscopy in native-like lipid bilayers with restrained molecular dynamics simulations represents a powerful tool for structural characterization of not only isolated membrane proteins, but their complexes, such as those that form macromolecular machines. PMID:25548160

  13. Tau could protect DNA double helix structure

    Microsoft Academic Search

    Qian Hua; Rong-qiao He

    2003-01-01

    The hyperchromic effect has been used to detect the effect of tau on the transition of double-stranded DNA to single-stranded DNA. It was shown that tau increased the melting temperature of calf thymus DNA from 67 to 81 °C and that of plasmid from 75 to 85 °C. Kinetically, rates of increase in absorbance at 260 nm of DNA incubated

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

    PubMed Central

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

    2014-01-01

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

  15. Controlling 3(10)-helix and alpha-helix of short peptides in the solid state.

    PubMed

    Demizu, Yosuke; Tanaka, Masakazu; Nagano, Masanobu; Kurihara, Masaaki; Doi, Mitsunobu; Maruyama, Tokumi; Suemune, Hiroshi

    2007-05-01

    L-Leu hexapeptide containing alpha-aminoisobutyric acid (Aib) forms a right-handed (P) 3(10)-helix, whereas that containing cyclic alpha,alpha-disubstituted amino acid Ac(5)c(dOM) assumes a right-handed (P) alpha-helix in the solid state. PMID:17473484

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

    PubMed Central

    Carr, C S; Sharp, P A

    1990-01-01

    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

  17. Evolutionary aspects of developmentally regulated helix-loop-helix transcription factors in striated muscle of jellyfish.

    PubMed

    Müller, Peter; Seipel, Katja; Yanze, Nathalie; Reber-Müller, Susanne; Streitwolf-Engel, Ruth; Stierwald, Michael; Spring, Jürg; Schmid, Volker

    2003-03-15

    The function of basic helix-loop-helix (bHLH) proteins in cell differentiation was shown to be conserved from Drosophila to vertebrates, exemplified by the function of MyoD in striated muscle differentiation. In phylogeny striated muscle tissue appears first in jellyfish and the question of its evolutionary position is controversially discussed. For this reason we have studied the developmental role of myogenic bHLH genes in medusa development. Based on their dimerization ability, four genes of the bHLH family of transcription factors were isolated from the hydrozoan jellyfish Podocoryne carnea. While the proteins Id and Ash group with cognate family members from bilaterians, Net-like and JellyD1 could not be unequivocally classified. Id is expressed during the medusa budding process and in the adult medusa, Ash and Net-like are expressed in all life cycle stages from egg to adult medusa and JellyD1 is expressed in the blastula and gastrula stages, the planula larva, and in late medusa bud stages. The dimerization specificity, the expression pattern, and the conservation of two residues specific for a MyoD bHLH domain suggest that JellyD1 is related to an ancestral MyoD gene. Id, Net-like, and JellyD1 are either expressed in the entocodon or its derived tissues, the striated and smooth muscle of the bell. These findings strengthen the hypothesis that the entocodon is a mesoderm-like structure and that the common ancestor of Cnidaria and Bilateria was more complex in cell-type architecture and body organization than commonly thought. PMID:12648485

  18. Helix VIII of NhaA Na(+)/H(+) antiporter participates in the periplasmic cation passage and pH regulation of the antiporter.

    PubMed

    Diab, Mohammad; Rimon, Abraham; Tzubery, Tzvi; Padan, Etana

    2011-10-28

    The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na(+)/H(+) antiporter. However, because NhaA is active at physiological pH (pH 6.5-8.5), many questions related to the active state of NhaA have remained unanswered. Our Cys scanning of the highly conserved transmembrane VIII at physiological pH reveals that (1) the Cys replacement G230C significantly increases the apparent K(m) of the antiporter to both Na(+) (10-fold) and Li(+) (6-fold). (2) Variants G223C and G230C cause a drastic alkaline shift of the pH profile of NhaA by 1 pH unit. (3) Residues Gly223-Ala226 line a periplasmic funnel at physiological pH as they do at pH 4. Both were modified by membrane-impermeant negatively charged 2-sulfonatoethyl methanethiosulfonate and positively charged 2-(trimethyl ammonium)-ethylmethanethiosulfonate sulfhydryl reagents that could reach Cys replacements from the periplasm via water-filled funnels only, whereas other Cys replacements on helix VIII were not accessible/reactive to the reagents. (4) Remarkably, the modification of variant V224C by 2-sulfonatoethyl methanethiosulfonate or 2-(trimethyl ammonium)-ethylmethanethiosulfonate totally inhibited antiporter activity, while N-ethyl maleimide modification had a very small effect on NhaA activity. Hence, the size-rather than the chemical modification or the charge-of the larger reagents interferes with the passage of ions through the periplasmic funnel. Taken together, our results at physiological pH reveal that amino acid residues in transmembrane VIII contribute to the cation passage of NhaA and its pH regulation. PMID:21907722

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

    SciTech Connect

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

    2010-01-20

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

  20. The C-Terminal RpoN Domain of sigma54 Forms an unpredictedHelix-Turn-Helix Motif Similar to domains of sigma70

    SciTech Connect

    Doucleff, Michaeleen; Malak, Lawrence T.; Pelton, Jeffrey G.; Wemmer, David E.

    2005-11-01

    The ''{delta}'' subunit of prokaryotic RNA-polymerase allows gene-specific transcription initiation. Two {sigma} families have been identified, {sigma}{sup 70} and {sigma}{sup 54}, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the {sigma}{sup 70}-type factors have been well characterized structurally by x-ray crystallography, no high-resolution structural information is available for the {sigma}{sup 54}-type factors. Here we present the NMR derived structure of the C-terminal domain of {sigma}{sup 54} from Aquifex aeolicus. This domain (Thr323 to Gly389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the {sigma}{sup 70}-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. This structure's homology with other DNA binding proteins, combined with previous biochemical data, suggest how the C-terminal domain of {sigma}{sup 54} binds to DNA.

  1. Dynamic behavior of helix interaction circuits

    NASA Astrophysics Data System (ADS)

    Rocci, Peter J.

    1992-12-01

    The dynamic behavior of a high power traveling wave tube's (TWT) helix interaction circuit was determined while operating in its system environment. The purpose of this effort was to develop a fundamental knowledge base of material interactions under thermal loading, develop an understanding of long-term thermal effects on materials and to develop a relationship to TWT performance and reliability. The interaction circuit for the TWT analyzed consists of a variable pitch tungsten helix, three beryllia support rods spaced at equal 120 degree intervals around the helix, and a stainless steel barrel which encloses the vacuum envelope. A three-dimensional finite element model was constructed that represented the entire interaction circuit (89 helix turns in length). There was a great deal of complexity associated with the development of this model due to the irregular 'T-shaped' cross-section of the support rods and variation in pitch of the helix. Upon completion, the model contained approximately 18,000 elements and 45,000 nodes.

  2. 5-HT3 receptor ion size selectivity is a property of the transmembrane channel, not the cytoplasmic vestibule portals

    PubMed Central

    McKinnon, Nicole K.; Reeves, David C.

    2011-01-01

    5-HT3A receptors select among permeant ions based on size and charge. The membrane-associated (MA) helix lines the portals into the channel’s cytoplasmic vestibule in the 4-Å resolution structure of the homologous acetylcholine receptor. 5-HT3A MA helix residues are important determinants of single-channel conductance. It is unknown whether the portals into the cytoplasmic vestibule also determine the size selectivity of permeant ions. We sought to determine whether the portals form the size selectivity filter. Recently, we showed that channels functioned when the entire 5-HT3A M3–M4 loop was replaced by the heptapeptide M3–M4 loop sequence from GLIC, a bacterial Cys-loop neurotransmitter gated ion channel homologue from Gloebacter violaceus. We used homomeric 5-HT3A receptors with either a wild-type (WT) M3–M4 loop or the chimeric heptapeptide (5-HT3A–glvM3M4) loop, i.e., with or without portals. In Na+-containing buffer, the WT receptor current–voltage relationship was inwardly rectifying. In contrast, the 5-HT3A–glvM3M4 construct had a negative slope conductance region at voltages less than ?80 mV. Glutamine substitution for the heptapeptide M3–M4 loop arginine eliminated the negative slope conductance region. We measured the relative permeabilities and conductances of a series of inorganic and organic cations ranging from 0.9 to 4.5 Å in radius (Li+, Na+, ammonium, methylammonium, ethanolammonium, 2-methylethanolammonium, dimethylammonium, diethanolammonium, tetramethylammonium, choline, tris [hydroxymethyl] aminomethane, and N-methyl-d-glucamine). Both constructs had measurable conductances with Li+, ammonium, and methylammonium (size range of 0.9–1.8-Å radius). Many of the organic cations >2.4 Å acted as competitive antagonists complicating measurement of conductance ratios. Analysis of the permeability ratios by excluded volume theory indicates that the minimal pore radius for 5-HT3A and 5-HT3–glvM3M4 receptors was similar, ?5 Å. We infer that the 5-HT3A size selectivity filter is located in the transmembrane channel and not in the portals into the cytoplasmic vestibule. Thus, the determinants of size selectivity and conductance are located in physically distinct regions of the channel protein. PMID:21948949

  3. MeMotif: a database of linear motifs in alpha-helical transmembrane proteins.

    PubMed

    Marsico, Annalisa; Scheubert, Kerstin; Tuukkanen, Anne; Henschel, Andreas; Winter, Christof; Winnenburg, Rainer; Schroeder, Michael

    2010-01-01

    Membrane proteins are important for many processes in the cell and used as main drug targets. The increasing number of high-resolution structures available makes for the first time a characterization of local structural and functional motifs in alpha-helical transmembrane proteins possible. MeMotif (http://projects.biotec.tu-dresden.de/memotif) is a database and wiki which collects more than 2000 known and novel computationally predicted linear motifs in alpha-helical transmembrane proteins. Motifs are fully described in terms of several structural and functional features and editable. Motifs contained in MeMotif can be used in different biological applications, from the identification of biochemically important functional residues which are candidates for mutagenesis experiments to the improvement of tools for transmembrane protein modeling. PMID:19910368

  4. Targeting of Drosophila Rhodopsin Requires Helix 8 but Not the Distal C-Terminus

    PubMed Central

    Kock, Ines; Bulgakova, Natalia A.; Knust, Elisabeth; Sinning, Irmgard; Panneels, Valérie

    2009-01-01

    Background The fundamental role of the light receptor rhodopsin in visual function and photoreceptor cell development has been widely studied. Proper trafficking of rhodopsin to the photoreceptor membrane is of great importance. In human, mutations in rhodopsin involving its intracellular mislocalization, are the most frequent cause of autosomal dominant Retinitis Pigmentosa, a degenerative retinal pathology characterized by progressive blindness. Drosophila is widely used as an animal model in visual and retinal degeneration research. So far, little is known about the requirements for proper rhodopsin targeting in Drosophila. Methodology/Principal Findings Different truncated fly-rhodopsin Rh1 variants were expressed in the eyes of Drosophila and their localization was analyzed in vivo or by immunofluorescence. A mutant lacking the last 23 amino acids was found to properly localize in the rhabdomeres, the light-sensing organelle of the photoreceptor cells. This constitutes a major difference to trafficking in vertebrates, which involves a conserved QVxPA motif at the very C-terminus. Further truncations of Rh1 indicated that proper localization requires the last amino acid residues of a region called helix 8 following directly the last transmembrane domain. Interestingly, the very C-terminus of invertebrate visual rhodopsins is extremely variable but helix 8 shows conserved amino acid residues that are not conserved in vertebrate homologs. Conclusions/Significance Despite impressive similarities in the folding and photoactivation of vertebrate and invertebrate visual rhodopsins, a striking difference exists between mammalian and fly rhodopsins in their requirements for proper targeting. Most importantly, the distal part of helix 8 plays a central role in invertebrates. Since the last amino acid residues of helix 8 are dispensable for rhodopsin folding and function, we propose that this domain participates in the recognition of targeting factors involved in transport to the rhabdomeres. PMID:19572012

  5. Green laser light (532nm) activates a chloride current in the C1 neuron of Helix aspersa.

    PubMed

    Reece, Peter J; Dholakia, Kishan; Thomas, Roger C; Cottrell, Glen A

    2008-03-15

    Five hundred and thirty-two nanometers laser light evokes neuron-specific electrical responses in identified neurons of Helix ganglia. Such responses are intensity-dependent over the range 25-1500 mW, readily reversible and repeatable. Detailed experiments on the C1 neuron, which is inhibited by 532 nm light, showed that inhibition results from a selective increase in transmembrane Cl(-) ion conductance. Experiments with calcium-sensitive microelectrodes suggest that the response does not result from an increase in [Ca(2+)](i). The change in Cl(-) ion conductance probably occurs in the extensive plasmalemma infoldings of the proximal axon. PMID:18258365

  6. Introduction The cystic fibrosis transmembrane conductance regu-

    E-print Network

    Engelhardt, John F.

    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

  7. Mutational Analysis of Synaptobrevin Transmembrane Domain Oligomerization †

    Microsoft Academic Search

    Mark E. Bowen; Donald M. Engelman; Axel T. Brunger

    2002-01-01

    Synaptobrevin 2 is thought to facilitate fusion of synaptic vesicles with the presynaptic membrane through formation of a soluble NSF attachment protein receptor complex (SNARE) with syntaxin 1a and a synaptosomal associated protein of 25 kDa (SNAP-25). Previous reports have described a homodimer of synaptobrevin that is dependent on the transmembrane domain. However, these reports disagree about the magnitude of

  8. 50?Hz Electromagnetic Field Produced Changes in FTIR Spectroscopy Associated with Mitochondrial Transmembrane Potential Reduction in Neuronal-Like SH-SY5Y Cells

    PubMed Central

    Calabrò, Emanuele; Condello, Salvatore; Currò, Monica; Ferlazzo, Nadia; Vecchio, Mercurio; Caccamo, Daniela; Magazù, Salvatore; Ientile, Riccardo

    2013-01-01

    SH-SY5Y neuroblastoma cells were used as an experimental model to study the effects of 50?Hz electromagnetic field, in the range from 50?µT to 1.4?mT. Fourier transform infrared spectroscopy analysis evidenced a reduction in intensity of the amide A band and a slight increase of vibration bands at 2921?cm?1 and 2853?cm?1 corresponding to methylene groups. A further increase of the magnetic field intensity of exposure up to 0.8?mT and 1.4?mT produced a clear increase in intensity of CH2 vibration bands. Moreover, it has been observed some alterations in the amide I region, such as a shifted peak of the amide I band to a smaller wavenumber, probably due to protein conformational changes. These results suggested that exposure to extremely low electromagnetic fields influenced lipid components of cellular membrane and the N–H in-plane bending and C–N stretching vibrations of peptide linkages, modifying the secondary structures of ?-helix and ?-sheet contents and producing unfolding process in cell membrane proteins. The observed changes after exposure to 50?Hz electromagnetic field higher than 0.8?mT were associated with a significant reduction of cell viability and reduced mitochondrial transmembrane potential. PMID:23970948

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

    PubMed

    Baker, Michael E; Uh, Kayla Y

    2012-10-01

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

  10. Epicyclic Twin-Helix Ionization Cooling Simulations

    SciTech Connect

    Vasiliy Morozov, Yaroslav Derbenev, A. Afanaciev, R.P. Johnson

    2011-04-01

    Parametric-resonance Ionization Cooling (PIC) is proposed as the final 6D cooling stage of a highluminosity muon collider. For the implementation of PIC, we earlier developed an epicyclic twin-helix channel with correlated behavior of the horizontal and vertical betatron motions and dispersion. We now insert absorber plates with short energy-recovering units located next to them at the appropriate locations in the twin-helix channel. We first demonstrate conventional ionization cooling in such a system with the optics uncorrelated. We then adjust the correlated optics state and induce a parametric resonance to study ionization cooling under the resonant condition.

  11. Bacterial morphogenesis and the enigmatic MreB helix.

    PubMed

    Errington, Jeff

    2015-04-01

    Work over the past decade has highlighted the pivotal role of the actin-like MreB family of proteins in the determination and maintenance of rod cell shape in bacteria. Early images of MreB localization revealed long helical filaments, which were suggestive of a direct role in governing cell wall architecture. However, several more recent, higher-resolution studies have questioned the existence or importance of the helical structures. In this Opinion article, I navigate a path through these conflicting reports, revive the helix model and summarize the key questions that remain to be answered. PMID:25578957

  12. PD-118057 contacts the pore helix of hERG1 channels to attenuate inactivation and enhance K+ conductance

    PubMed Central

    Perry, Matthew; Sachse, Frank B.; Abbruzzese, Jennifer; Sanguinetti, Michael C.

    2009-01-01

    Human ether-a-go-go-related gene 1 (hERG1) K+ channels mediate repolarization of cardiac action potentials. Unintended block of hERG1 channels by some drugs can prolong the QT interval and induce arrhythmia. Recently, hERG1 channel agonists were discovered and, based on their mechanisms of action can be classified into two types. RPR260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5 trifluorophenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid], a type 1 agonist, binds to residues located near the intracellular end of S5 and S6 transmembrane segments and activates hERG1 channels by a dual mechanism of slowed deactivation and attenuated P-type inactivation. As defined here, type 2 agonists such as PD-118057 [2-(4-[2-(3,4-dichloro-phenyl)-ethyl]-phenylamino)-benzoic acid] attenuate inactivation but do not slow deactivation. At 10 ?M, PD-118057 shifted the half-point for inactivation of wild-type hERG1 channels by +19 mV and increased peak outward current by 136%. Scanning mutagenesis and functional characterization of 44 mutant channels expressed in Xenopus oocytes was used to identify the major structural determinants of the binding site for PD-118057. Single mutations of residues in the pore helix (F619) or the S6 segment (L646) of hERG1 eliminated agonist activity. Mutation of a nearby residues in the S6 segment (C643, M645) enhanced drug activity, presumably by reducing steric hindrance for drug binding. Molecular modeling indicates that PD-118057 binds to a hydrophobic pocket formed by L646 of one hERG1 subunit and F619 of an adjacent subunit. We conclude that direct interaction of PD-118057 with the pore helix attenuates fast P-type inactivation and increases open probability of hERG1 channels. PMID:19892732

  13. A model for the coupling of alpha-helix and tertiary contact formation.

    PubMed

    Hausrath, Andrew C

    2006-09-01

    Peptides corresponding to excised alpha-helical segments of natural proteins can spontaneously form helices in solution. However, peptide helices are usually substantially less stable in solution than in the structural context of a folded protein, because of the additional interactions possible between helices in a protein. Such interactions can be thought of as coupling helix formation and tertiary contact formation. The relative energetic contributions of the two processes to the total energy of the folded state of a protein is a matter of current debate. To investigate this balance, an extended helix-coil model (XHC) that incorporates both effects has been constructed. The model treats helix formation with the Lifson-Roig formalism, which describes helix initiation and propagation through cooperative local interactions. The model postulates an additional parameter representing participation of a site in a tertiary contact. In the model, greater helix stability can be achieved through combinations of these short-range and long-range interactions. For instance, stronger tertiary contacts can compensate for helices with little intrinsic stability. By varying the strength of the nonlocal interactions, the model can exhibit behavior consistent with a variety of qualitative models describing the relative importance of secondary and tertiary structure. Moreover, the model is explicit in that it can be used to fit experimental data to individual peptide sequences, providing a means to quantify the two contributions on a common energetic basis. PMID:16882994

  14. Cytotoxic Helix-Rich Oligomer Formation by Melittin and Pancreatic Polypeptide

    PubMed Central

    Singh, Pradeep K.; Ghosh, Dhiman; Tewari, Debanjan; Mohite, Ganesh M.; Carvalho, Edmund; Jha, Narendra Nath; Jacob, Reeba S.; Sahay, Shruti; Banerjee, Rinti; Bera, Amal K.; Maji, Samir K.

    2015-01-01

    Conversion of amyloid fibrils by many peptides/proteins involves cytotoxic helix-rich oligomers. However, their toxicity and biophysical studies remain largely unknown due to their highly dynamic nature. To address this, we chose two helical peptides (melittin, Mel and pancreatic polypeptide, PP) and studied their aggregation and toxicity. Mel converted its random coil structure to oligomeric helical structure upon binding to heparin; however, PP remained as helix after oligomerization. Interestingly, similar to Parkinson’s associated ?-synuclein (AS) oligomers, Mel and PP also showed tinctorial properties, higher hydrophobic surface exposure, cellular toxicity and membrane pore formation after oligomerization in the presence of heparin. We suggest that helix-rich oligomers with exposed hydrophobic surface are highly cytotoxic to cells irrespective of their disease association. Moreover as Mel and PP (in the presence of heparin) instantly self-assemble into stable helix-rich amyloidogenic oligomers; they could be represented as models for understanding the biophysical and cytotoxic properties of helix-rich intermediates in detail. PMID:25803428

  15. September 2000 ALPHA HELIX CRUISE HX235

    E-print Network

    Washington at Seattle, University of

    1 September 2000 ALPHA HELIX CRUISE HX235 Friday 25th August 2000 - Thursday 7th September 2000 BERING STRAIT CRUISE REPORT FUNDING SOURCE: NSF-OPP-9815707 (Kelly Falkner, OSU) CHIEF SCIENTIST: Rebecca PURPOSE: This cruise has two scientific goals. The first (and foremost) is the recovery and redeployment

  16. High velocity knot in the Helix nebula

    SciTech Connect

    Meaburn, J.; Walsh, J.R.

    1980-01-01

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

  17. Helix–loop–helix/basic helix–loop–helix transcription factor network represses cell elongation in Arabidopsis through an apparent incoherent feed-forward loop

    PubMed Central

    Zhiponova, Miroslava K.; Morohashi, Kengo; Vanhoutte, Isabelle; Machemer-Noonan, Katja; Revalska, Miglena; Van Montagu, Marc; Grotewold, Erich; Russinova, Eugenia

    2014-01-01

    Cell elongation is promoted by different environmental and hormonal signals, involving light, temperature, brassinosteroid (BR), and gibberellin, that inhibit the atypical basic helix–loop–helix (bHLH) transcription factor INCREASED LEAF INCLINATION1 BINDING bHLH1 (IBH1). Ectopic accumulation of IBH1 causes a severe dwarf phenotype, but the cell elongation suppression mechanism is still not well understood. Here, we identified a close homolog of IBH1, IBH1-LIKE1 (IBL1), that also antagonized BR responses and cell elongation. Genome-wide expression analyses showed that IBH1 and IBL1 act interdependently downstream of the BRASSINAZOLE-RESISTANT1 (BZR1)–PHYTOCHROME-INTERACTING FACTOR 4 (PIF4)–DELLA module. Although characterized as non-DNA binding, IBH1 repressed direct IBL1 transcription, and they both acted in tandem to suppress the expression of a common downstream helix–loop–helix (HLH)/bHLH network, thus forming an incoherent feed-forward loop. IBH1 and IBL1 together repressed the expression of PIF4, known to stimulate skotomorphogenesis synergistically with BZR1. Strikingly, PIF4 bound all direct and down-regulated HLH/bHLH targets of IBH1 and IBL1. Additional genome-wide comparisons suggested a model in which IBH1 antagonized PIF4 but not the PIF4–BZR1 dimer. PMID:24505057

  18. A triple helix-loop-helix/basic helix-loop-helix cascade controls cell elongation downstream of multiple hormonal and environmental signaling pathways in Arabidopsis.

    PubMed

    Bai, Ming-Yi; Fan, Min; Oh, Eunkyoo; Wang, Zhi-Yong

    2012-12-01

    Environmental and endogenous signals, including light, temperature, brassinosteroid (BR), and gibberellin (GA), regulate cell elongation largely by influencing the expression of the paclobutrazol-resistant (PRE) family helix-loop-helix (HLH) factors, which promote cell elongation by interacting antagonistically with another HLH factor, IBH1. However, the molecular mechanism by which PREs and IBH1 regulate gene expression has remained unknown. Here, we show that IBH1 interacts with and inhibits a DNA binding basic helix-loop-helix (bHLH) protein, HBI1, in Arabidopsis thaliana. Overexpression of HBI1 increased hypocotyl and petiole elongation, whereas dominant inactivation of HBI1 and its homologs caused a dwarf phenotype, indicating that HBI1 is a positive regulator of cell elongation. In vitro and in vivo experiments showed that HBI1 directly bound to the promoters and activated two EXPANSIN genes encoding cell wall-loosening enzymes; HBI1's DNA binding and transcriptional activities were inhibited by IBH1, but the inhibitory effects of IBH1 were abolished by PRE1. The results indicate that PREs activate the DNA binding bHLH factor HBI1 by sequestering its inhibitor IBH1. Altering each of the three factors affected plant sensitivities to BR, GA, temperature, and light. Our study demonstrates that PREs, IBH1, and HBI1 form a chain of antagonistic switches that regulates cell elongation downstream of multiple external and endogenous signals. PMID:23221598

  19. Localized Lipid Packing of Transmembrane Domains Impedes Integrin Clustering

    PubMed Central

    Mehrbod, Mehrdad; Mofrad, Mohammad R. K.

    2013-01-01

    Integrin clustering plays a pivotal role in a host of cell functions. Hetero-dimeric integrin adhesion receptors regulate cell migration, survival, and differentiation by communicating signals bidirectionally across the plasma membrane. Thus far, crystallographic structures of integrin components are solved only separately, and for some integrin types. Also, the sequence of interactions that leads to signal transduction remains ambiguous. Particularly, it remains controversial whether the homo-dimerization of integrin transmembrane domains occurs following the integrin activation (i.e. when integrin ectodomain is stretched out) or if it regulates integrin clustering. This study employs molecular dynamics modeling approaches to address these questions in molecular details and sheds light on the crucial effect of the plasma membrane. Conducting a normal mode analysis of the intact ?llb?3 integrin, it is demonstrated that the ectodomain and transmembrane-cytoplasmic domains are connected via a membrane-proximal hinge region, thus merely transmembrane-cytoplasmic domains are modeled. By measuring the free energy change and force required to form integrin homo-oligomers, this study suggests that the ?-subunit homo-oligomerization potentially regulates integrin clustering, as opposed to ?-subunit, which appears to be a poor regulator for the clustering process. If ?-subunits are to regulate the clustering they should overcome a high-energy barrier formed by a stable lipid pack around them. Finally, an outside-in activation-clustering scenario is speculated, explaining how further loading the already-active integrin affects its homo-oligomerization so that focal adhesions grow in size. PMID:23516344

  20. MODELS OF THE ACTIN-LIKE MREB HELIX IN PROKARYOTES

    E-print Network

    Allard, Jun

    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

  1. Structure of a ?1-adrenergic G protein-coupled receptor

    PubMed Central

    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.

    2010-01-01

    Summary G protein-coupled receptors play a major role in transmembrane signalling in higher organisms and many are important drug targets. We report the 2.7 Å resolution crystal structure of a ?1-adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey receptor had been 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 ?-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilised by two disulphide bonds and a sodium ion. Cyanopindolol binding to the ?1-adrenergic receptor and carazolol binding to the ?2-adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the ?2-adrenergic receptor, directly interacts via a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation. PMID:18594507

  2. Phylogenetic analysis of the human basic helix-loop-helix proteins

    Microsoft Academic Search

    Valérie Ledent; Odier Paquet; Michel Vervoort

    2002-01-01

    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

  3. Evolutionary aspects of developmentally regulated helix-loop-helix transcription factors in striated muscle of jellyfish

    Microsoft Academic Search

    Peter Müller; Katja Seipel; Nathalie Yanze; Susanne Reber-Müller; Ruth Streitwolf-Engel; Michael Stierwald; J. ürg Spring; Volker Schmid

    2003-01-01

    The function of basic helix-loop-helix (bHLH) proteins in cell differentiation was shown to be conserved from Drosophila to vertebrates, exemplified by the function of MyoD in striated muscle differentiation. In phylogeny striated muscle tissue appears first in jellyfish and the question of its evolutionary position is controversially discussed. For this reason we have studied the developmental role of myogenic bHLH

  4. Gene Replacement Strategies to Test the Functional Redundancy of Basic Helix–Loop–Helix Transcription Factor

    Microsoft Academic Search

    Anthony B. FirulliBeth; Beth A. Firulli; Jian Wang; Rhonda H. Rogers; Simon J. Conway

    2010-01-01

    Basic helix–loop–helix (bHLH) transcription factors control developmental decisions for a wide range of embryonic cell types.\\u000a Hand1 and Hand2 are closely related bHLH proteins that control cardiac, craniofacial, and limb development. Within the developing\\u000a 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

  5. Ion fluxes through nanopores and transmembrane channels.

    PubMed

    Bordin, J R; Diehl, A; Barbosa, M C; Levin, Y

    2012-03-01

    We introduce an implicit solvent Molecular Dynamics approach for calculating ionic fluxes through narrow nanopores and transmembrane channels. The method relies on a dual-control-volume grand-canonical molecular dynamics (DCV-GCMD) simulation and the analytical solution for the electrostatic potential inside a cylindrical nanopore recently obtained by Levin [Europhys. Lett. 76, 163 (2006)]. The theory is used to calculate the ionic fluxes through an artificial transmembrane channel which mimics the antibacterial gramicidin A channel. Both current-voltage and current-concentration relations are calculated under various experimental conditions. We show that our results are comparable to the characteristics associated to the gramicidin A pore, especially the existence of two binding sites inside the pore and the observed saturation in the current-concentration profiles. PMID:22587130

  6. Orientation of gramicidin A transmembrane channel. Infrared dichroism study of gramicidin in vesicles.

    PubMed Central

    Nabedryk, E; Gingold, M P; Breton, J

    1982-01-01

    Polarized infrared spectroscopy has been used to investigate the orientation of gramicidin A incorporated in dimyristoylphosphatidylcholine liposomes. Dichroism measurements of the major lipid (C = O ester, PO2-, CH2) and peptide (amide A, I, II) bands were performed on liposomes (with or without gramicidin) oriented by air-drying. The mean orientation of the lipid groups and of the pi LD helix chain in the gramicidin has been determined. It can be inferred from infrared frequencies of gramicidin that the dominant conformation of the peptide in liposomes cannot be identified to the antiparallel double-helical dimer found in organic solution. No shift in lipid frequencies was observed upon incorporation of gramicidin in the liposomes. However, a slight reorganization of the lipid hydrocarbon chains which become oriented more closely to the normal to the bilayer is evidenced by a change in the dichroism of the CH2 vibrations. The infrared dichroism results of gramicidin imply a perpendicular orientation of the gramicidin transmembrane channel with the pi LD helix axis at less than 15 degrees with respect to the normal to the bilayer. PMID:6179549

  7. Estimating the length of transmembrane helices using Z-coordinate predictions.

    PubMed

    Papaloukas, Costas; Granseth, Erik; Viklund, Håkan; Elofsson, Arne

    2008-02-01

    Zpred2 is an improved version of ZPRED, a predictor for the Z-coordinates of alpha-helical membrane proteins, that is, the distance of the residues from the center of the membrane. Using principal component analysis and a set of neural networks, Zpred2 analyzes data extracted from the amino acid sequence, the predicted topology, and evolutionary profiles. Zpred2 achieves an average accuracy error of 2.18 A (2.17 A when an independent test set is used), an improvement by 15% compared to the previous version. We show that this accuracy is sufficient to enable the predictions of helix lengths with a correlation coefficient of 0.41. As a comparison, two state-of-the-art HMM-based topology prediction methods manage to predict the helix lengths with a correlation coefficient of less than 0.1. In addition, we applied Zpred2 to two other problems, the re-entrant region identification and model validation. Re-entrants were able to be detected with a certain consistency, but not better than with previous approaches, while incorrect models as well as mispredicted helices of transmembrane proteins could be distinguished based on the Z-coordinate predictions. PMID:18096645

  8. Transmembrane transforming growth factor-? tethers to the PDZ domain-containing, Golgi membrane-associated protein p59/GRASP55

    PubMed Central

    Kuo, Alfred; Zhong, Cuiling; Lane, William S.; Derynck, Rik

    2000-01-01

    Transforming growth factor-? (TGF-?) and related proteins represent a family of transmembrane growth factors with representatives in flies and worms. Little is known about the transport of TGF-? and other transmembrane growth factors to the cell surface and its regulation. p59 was purified as a cytoplasmic protein, which at endogenous levels associates with transmembrane TGF-?. cDNA cloning of p59 revealed a 452 amino acid sequence with two PDZ domains. p59 is myristoylated and palmitoylated, and associates with the Golgi system, where it co-localizes with TGF-?. Its first PDZ domain interacts with the C-terminus of transmembrane TGF-? and select transmembrane proteins. p59 is the human homolog of GRASP55, which is structurally related to GRASP65. GRASP55 and GRASP65 have been shown to play a role in stacking of the Golgi cisternae in vitro. C-terminal mutations of transmembrane TGF-?, which decrease or abolish the interaction with p59, also strongly impair cell surface expression of TGF-?. Our observations suggest a role for membrane tethering of p59/GRASP55 to select transmembrane proteins, including TGF-?, in maturation and transport to the cell surface. PMID:11101516

  9. Exploring Transmembrane Diffusion Pathways with Molecular Dynamics

    PubMed Central

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

    2013-01-01

    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

  10. Exploring Transmembrane Diffusion Pathways With Molecular Dynamics

    NSDL National Science Digital Library

    Yi Wang (University of Illinois at Urbana-Champaign)

    2010-06-01

    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.

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

    PubMed Central

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

    1992-01-01

    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

  12. Transmembrane Domain II of the Human Bile Acid Transporter SLC10A2 Coordinates Sodium Translocation*

    PubMed Central

    Sabit, Hairat; Mallajosyula, Sairam S.; MacKerell, Alexander D.; Swaan, Peter W.

    2013-01-01

    Human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is responsible for intestinal reabsorption of bile acids and plays a key role in cholesterol homeostasis. We used a targeted and systematic approach to delineate the role of highly conserved transmembrane helix 2 on the expression and function of hASBT. Cysteine mutation significantly depressed transport activity for >60% of mutants without affecting cell surface localization of the transporter. All mutants were inaccessible toward chemical modification by membrane-impermeant MTSET reagent, strongly suggesting that transmembrane 2 (TM2) plays an indirect role in bile acid substrate translocation. Both bile acid uptake and sodium dependence of TM2 mutants revealed a distinct ?-helical periodicity. Kinetic studies with conservative and non-conservative mutants of sodium sensitive residues further underscored the importance of Gln75, Phe76, Met79, Gly83, Leu86, Phe90, and Asp91 in hASBT function. Computational analysis indicated that Asp91 may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr134, Leu138, and Thr149) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln75, Met79, Thr82, and Leu86 from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBTNM), suggesting a universal mechanism for the SLC10A transporter family. PMID:24045943

  13. Folding Dynamics of an ? Helix and a ? Hairpin

    NASA Astrophysics Data System (ADS)

    Hofrichter, James

    1998-03-01

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

  14. HELIX 3 IS NECESSARY AND SUFFICIENT FOR PRION PROTEIN’S ANTI-BAX FUNCTION

    PubMed Central

    Laroche-Pierre, Stéphanie; Jodoin, Julie; LeBlanc, Andréa C.

    2009-01-01

    To identify the structural elements of the prion protein (PrP) necessary for its protective function against Bax, we performed structure-function analyses of the anti-Bax function of cytosolic PrP (CyPrP) in MCF-7 cells. Deletions of 1, 2, or 3 N-terminal BH2-like octapeptide repeats (BORs), but not deletion of all 4 BORs, abolish CyPrP’s anti-Bax function. Deletion of ?-helix 3 (PrP23-199) or further C-terminal deletions of ?-helix 1 and 2, and ?-strand 1 and 2 (PrP23-172, PrP23-160, PrP23-143, PrP23-127) eliminates CyPrP’s protection against Bax-mediated cell death. The substitution of helix 3 amino acid residues K204, V210, and E219 by proline inhibits the anti-Bax function of CyPrP. The substitution of K204, but not V210 and E219, by alanine residues also prevents CyPrP’s anti-Bax function. Expression of PrP’s helix 3 displays anti-Bax activity in MCF-7 cells and in human neurons. Together, these results indicate that although the BOR domain has an influence on PrP’s anti-Bax function, the helix 3 is necessary and sufficient for the anti-Bax function of CyPrP. Identification of helix 3 as the structural element for the anti-Bax function thus provides a molecular target to modulate PrP’s anti-Bax function in cancer and neurodegeneration. PMID:19196429

  15. Role of solvation in pressure-induced helix stabilization

    NASA Astrophysics Data System (ADS)

    Best, Robert B.; Miller, Cayla; Mittal, Jeetain

    2014-12-01

    In contrast to the well-known destabilization of globular proteins by high pressure, recent work has shown that pressure stabilizes the formation of isolated ?-helices. However, all simulations to date have obtained a qualitatively opposite result within the experimental pressure range. We show that using a protein force field (Amber03w) parametrized in conjunction with an accurate water model (TIP4P/2005) recovers the correct pressure-dependence and an overall stability diagram for helix formation similar to that from experiment; on the other hand, we confirm that using TIP3P water results in a very weak pressure destabilization of helices. By carefully analyzing the contributing factors, we show that this is not merely a consequence of different peptide conformations sampled using TIP3P. Rather, there is a critical role for the solvent itself in determining the dependence of total system volume (peptide and solvent) on helix content. Helical peptide structures exclude a smaller volume to water, relative to non-helical structures with both the water models, but the total system volume for helical conformations is higher than non-helical conformations with TIP3P water at low to intermediate pressures, in contrast to TIP4P/2005 water. Our results further emphasize the importance of using an accurate water model to study protein folding under conditions away from standard temperature and pressure.

  16. The double helix and immunology

    NASA Astrophysics Data System (ADS)

    Nossal, Gustav J. V.

    2003-01-01

    The immune system can recognize and produce antibodies to virtually any molecule in the Universe. This enormous diversity arises from the ingenious reshuffling of DNA sequences encoding components of the immune system. Immunology is an example of a field completely transformed during the past 50 years by the discovery of the structure of DNA and the emergence of DNA technologies that followed.

  17. DNA helix, still imageSite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-10-06

    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 ��ì a twisted ladder ��ì 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.

  18. Screening Helix-threading Peptides for RNA Binding Using a Thiazole Orange Displacement Assay

    PubMed Central

    Krishnamurthy, Malathy; Schirle, Nicole T.; Beal, Peter A.

    2008-01-01

    The fluorescent intercalator displacement assay using thiazole orange has been adapted to the study of RNA-binding helix-threading peptides (HTPs). This assay is highly sensitive with HTP-binding RNAs and provides binding affinity data in good agreement with quantitative ribonuclease footprinting without the need for radiolabeling or gel electrophoresis. The FID assay was used to define structure activity relationships for a small library of helix-threading peptides. Results of these studies indicate their RNA binding is dependent on peptide sequence, ?-amino acid stereochemistry, and cyclization (versus linear peptides), but independent of macrocyclic ring size for the penta-, tetra- and tri peptides analyzed. PMID:18789700

  19. Core structure of the envelope glycoprotein GP2 from Ebola virus at 1.9-? resolution

    PubMed Central

    Malashkevich, Vladimir N.; Schneider, Brian J.; McNally, Margaret L.; Milhollen, Michael A.; Pang, James X.; Kim, Peter S.

    1999-01-01

    Ebola virions contain a surface transmembrane glycoprotein (GP) that is responsible for binding to target cells and subsequent fusion of the viral and host-cell membranes. GP is expressed as a single-chain precursor that is posttranslationally processed into the disulfide-linked fragments GP1 and GP2. The GP2 subunit is thought to mediate membrane fusion. A soluble fragment of the GP2 ectodomain, lacking the fusion-peptide region and the transmembrane helix, folds into a stable, highly helical structure in aqueous solution. Limited proteolysis studies identify a stable core of the GP2 ectodomain. This 74-residue core, denoted Ebo-74, was crystallized, and its x-ray structure was determined at 1.9-? resolution. Ebo-74 forms a trimer in which a long, central three-stranded coiled coil is surrounded by shorter C-terminal helices that are packed in an antiparallel orientation into hydrophobic grooves on the surface of the coiled coil. Our results confirm the previously anticipated structural similarity between the Ebola GP2 ectodomain and the core of the transmembrane subunit from oncogenic retroviruses. The Ebo-74 structure likely represents the fusion-active conformation of the protein, and its overall architecture resembles several other viral membrane-fusion proteins, including those from HIV and influenza. PMID:10077567

  20. Structure and Selectivity in Bestrophin Ion Channels

    PubMed Central

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

    2015-01-01

    Human bestrophin 1 (hBest1) is a calcium-activated chloride channel from the retinal pigment epithelium, where it can suffer mutations 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 subtle control of ion selectivity in the bestrophins, including reversal of anion/cation selectivity, and dramatic activation by mutations at the exit restriction. A homology model of hBest1 shows the locations of disease-causing mutations and suggests possible roles in regulation. PMID:25324390

  1. Structure and selectivity in bestrophin ion channels.

    PubMed

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

    2014-10-17

    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

  2. Distribution of the axial electric field in a helix travelling wave tube filled with plasma

    Microsoft Academic Search

    Xie Hongquan; Liu Pukun

    2004-01-01

    A plasma loaded helix type slow wave structure is immersed in a strong longitudinal magnetic field. Taking into account the effect of the plasma and the dielectric, the system is separated along radial into three regions. By means of sheath model and the Maxwell's equation, the distribution of the electromagnetic field is established. Using the boundary conditions of each region,

  3. Analysis of the axial electric field in a plasma-loaded-helix travelling wave tube

    Microsoft Academic Search

    Hong-Quan Xie; Pu-Kun Liu

    2006-01-01

    A helix type slow wave structure filled with plasma is immersed in a strong longitudinal magnetic field. Taking into account the effect of the plasma and the dielectric, the system is separated radially into three regions. By means of the sheath model and Maxwell equation, the distribution of the electromagnetic field is established. Using the boundary conditions of each region,

  4. Parallel helix bundles and ion channels: molecular modeling via simulated annealing and restrained molecular dynamics.

    PubMed Central

    Kerr, I D; Sankararamakrishnan, R; Smart, O S; Sansom, M S

    1994-01-01

    A parallel bundle of transmembrane (TM) alpha-helices surrounding a central pore is present in several classes of ion channel, including the nicotinic acetylcholine receptor (nAChR). We have modeled bundles of hydrophobic and of amphipathic helices using simulated annealing via restrained molecular dynamics. Bundles of Ala20 helices, with N = 4, 5, or 6 helices/bundle were generated. For all three N values the helices formed left-handed coiled coils, with pitches ranging from 160 A (N = 4) to 240 A (N = 6). Pore radius profiles revealed constrictions at residues 3, 6, 10, 13, and 17. A left-handed coiled coil and a similar pattern of pore constrictions were observed for N = 5 bundles of Leu20. In contrast, N = 5 bundles of Ile20 formed right-handed coiled coils, reflecting loosened packing of helices containing beta-branched side chains. Bundles formed by each of two classes of amphipathic helices were examined: (a) M2a, M2b, and M2c derived from sequences of M2 helices of nAChR; and (b) (LSSLLSL)3, a synthetic channel-forming peptide. Both classes of amphipathic helix formed left-handed coiled coils. For (LSSLLSL)3 the pitch of the coil increased as N increased from 4 to 6. The M2c N = 5 helix bundle is discussed in the context of possible models of the pore domain of nAChR. Images FIGURE 1 FIGURE 3 PMID:7529585

  5. Transmembrane topology of mammalian planar cell polarity protein Vangl1.

    PubMed

    Iliescu, Alexandra; Gravel, Michel; Horth, Cynthia; Apuzzo, Sergio; Gros, Philippe

    2011-03-29

    Vangl1 and Vangl2 are membrane proteins that play an important role in neurogenesis, and Vangl1/Vangl2 mutations cause neural tube defects in mice and humans. At the cellular level, Vangl proteins regulate the establishment of planar cell polarity (PCP), a process requiring membrane assembly of asymmetrically distributed multiprotein complexes that transmit polarity information to neighboring cells. The membrane topology of Vangl proteins and the protein segments required for structural and functional aspects of multiprotein membrane PCP complexes is unknown. We have used epitope tagging and immunofluorescence to establish the secondary structure of Vangl proteins, including the number, position, and polarity of transmembrane domains. Antigenic hemagglutinin A (HA) peptides (YYDVPDYS) were inserted in predicted intra- or extracellular loops of Vangl1 at positions 18, 64, 139, 178, 213, and 314, and individual mutant variants were stably expressed at the membrane of MDCK polarized cells. The membrane topology of the exofacial HA tag was determined by a combination of immunofluorescence in intact (extracellular epitopes) and permeabilized (intracellular epitopes) cells and by surface labeling. Results indicate that Vangl proteins have a four-transmembrane domain structure with the N-terminal portion (HA18 and HA64) and the large C-terminal portion (HA314) of the protein being intracellular. Topology mapping and hydropathy profiling show that the loop delineated by TMD1-2 (HA139) and TMD3-4 (HA213) is extracellular while the segment separating predicted TMD2-3 (HA178) is intracellular. This secondary structure reveals a compact membrane-associated portion with very short predicted extra- and intracellular loops, while the protein harbors a large intracellular domain. PMID:21291170

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

    PubMed Central

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

    2012-01-01

    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

  7. Mutational analyses define helix organization and key residues of a bacterial membrane energy-transducing complex.

    PubMed

    Goemaere, Emilie L; Cascales, Eric; Lloubès, Roland

    2007-03-01

    In Gram-negative bacteria, many biological processes are coupled to inner membrane ion gradients. Ions transit at the interface of helices of integral membrane proteins, generating mechanical energy to drive energetic processes. To better understand how ions transit through these channels, we used a model system involved in two different processes, one of which depends on inner membrane energy. The Tol machinery of the Escherichia coli cell envelope is dedicated to maintaining outer membrane stability, a process driven by the proton-motive force. The Tol system is parasitized by bacterial toxins called colicins, which are imported through the outer membrane using an energy-independent process. Herein, we mutated TolQ and TolR transmembrane residues, and we analyzed the mutants for outer membrane stability, colicin import and protein complex formation. We identified residues involved in the assembly of the complex, and a new class of discriminative mutations that conferred outer membrane destabilization identical to a tol deletion mutant, but which remained fully sensitive to colicins. Further genetic approaches revealed transmembrane helix interactions and organization in the bilayer, and suggested that most of the discriminative residues are located in a putative aqueous ion channel. We discuss a model for the function of related bacterial molecular motors. PMID:17222427

  8. Four-Armed Spiral-Helix Antenna

    Microsoft Academic Search

    Matthew J. Radway; Dejan S. Filipovic

    2012-01-01

    An antenna formed by the union of a four-armed, reflective-backed spiral antenna with a top-fed quadrifilar helix, operating over the 0.35–2.6-GHz band, is presented. The antenna features a novel dual-layering scheme that allows the modal impedance of the four-armed spiral to be greatly reduced while maintaining the desirable self-complementary metallization. A reconfigurable end-loading scheme is also presented that allows the

  9. New triple-helix DNA stabilizing agents.

    PubMed

    Strekowski, Lucjan; Hojjat, Maryam; Wolinska, Ewa; Parker, Alesia N; Paliakov, Ekaterina; Gorecki, Tadeusz; Tanious, Farial A; Wilson, W David

    2005-02-15

    Several substituted quinolin-4-amines and heteroaromatic analogs were synthesized and evaluated for interaction with triplex polydA.2polydT and duplex polydA.polydT by using UV-thermal melting experiments. Excellent triple-helix DNA ligands with high affinity toward T.A.T triplets and triple/duplex selectivity were designed through a rational approach. PMID:15686920

  10. Molecular basis of transmembrane beta-barrel formation of staphylococcal pore-forming toxins.

    PubMed

    Yamashita, Daichi; Sugawara, Takaki; Takeshita, Miyu; Kaneko, Jun; Kamio, Yoshiyuki; Tanaka, Isao; Tanaka, Yoshikazu; Yao, Min

    2014-01-01

    Pathogenic bacteria secrete pore-forming toxins (PFTs) to attack target cells. PFTs are expressed as water-soluble monomeric proteins, which oligomerize into nonlytic prepore intermediates on the target cell membrane before forming membrane-spanning pores. Despite a wealth of biochemical data, the lack of high-resolution prepore structural information has hampered understanding of the ?-barrel formation process. Here, we report crystal structures of staphylococcal ?-haemolysin and leucocidin prepores. The structures reveal a disordered bottom half of the ?-barrel corresponding to the transmembrane region, and a rigid upper extramembrane half. Spectroscopic analysis of fluorescently labelled mutants confirmed that the prepore is distinct from the pore within the transmembrane region. Mutational analysis also indicates a pivotal role for the glycine residue located at the lipid-solvent interface as a 'joint' between the two halves of the ?-barrel. These observations suggest a two-step transmembrane ?-barrel pore formation mechanism in which the upper extramembrane and bottom transmembrane regions are formed independently. PMID:25263813

  11. 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

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

    2008-01-01

    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

  12. 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

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

    2014-01-01

    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

  13. Dramatic in situ conformational dynamics of the transmembrane protein bacteriorhodopsin.

    PubMed Central

    Draheim, J E; Gibson, N J; Cassim, J Y

    1991-01-01

    The conformational dynamic capabilities of the in situ bacteriorhodopsin (bR) can be studied by determination of the changes of the bR net helical segmental tilt angle (the angle between the polypeptide segments and the membrane normal) induced by various perturbations of the purple membrane (PM). The analysis of the far-UV oriented circular dichroism (CD) of the PM provides one means of achieving this. Previous CD studies have indicated that the tilt angle can change from approximately 10 degrees to 39 degrees depending on the perturbants used with no changes in the secondary structure of the bR. A recent study has indicated that the bleaching-induced tilt angle can be enhanced from approximately 24 degrees to 39 degrees by cross-linkage and papain-digestion perturbations which by themselves do not alter the tilt angle. To add further credence, this study has been repeated using midinfrared (IR) linear dichroic spectral analysis. In contrast to the CD method, analysis by the IR method depends on the orientation of the amide plane of the helix assumed. Excellent consistency is achieved between the two methods only when it is assumed that the structural characteristics of the alpha-helices of the bR are equally alpha I and alpha II in nature. Furthermore, the analysis of the IR data becomes essentially independent of the three amide transitions utilized. The net tilt angle of segments completely randomized relative to the incident light must be 54.736 in view of helix symmetry. A value of 54.735 degrees +/- 0.001 degree was achieved by the IR method for the ethanol-treated PM film, establishing this kind of film as an ideal random state standard and demonstrating the accuracy potential of the IR method. PMID:1883946

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

    PubMed

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

    2013-11-01

    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

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

    PubMed Central

    Hallsson, Jón H; Haflidadóttir, Benedikta S; Stivers, Chad; Odenwald, Ward; Arnheiter, Heinz; Pignoni, Francesca; Steingrímsson, Eiríkur

    2004-01-01

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

  16. A basic helix-loop-helix transcription factor DvIVS determines flower color intensity in cyanic dahlia cultivars.

    PubMed

    Ohno, Sho; Deguchi, Ayumi; Hosokawa, Munetaka; Tatsuzawa, Fumi; Doi, Motoaki

    2013-08-01

    The study was aimed to identify the factors that regulate the intensity of flower color in cyanic dahlia (Dahlia variabilis), using fifteen cultivars with different color intensities in their petals. The cultivars were classified into three groups based on their flavonoid composition: ivory white cultivars with flavones; purple and pink cultivars with flavones and anthocyanins; and red cultivars with flavones, anthocyanins, and chalcones. Among the purple, pink, and ivory white cultivars, an inverse relationship was detected between lightness, which was used as an indicator for color intensity and anthocyanin content. A positive correlation was detected between anthocyanin contents and the expression of some structural genes in the anthocyanin synthesis pathway that are regulated by DvIVS, a basic helix-loop-helix transcription factor. A positive correlation between anthocyanin content and expression of DvIVS was also found. The promoter region of DvIVS was classified into three types, with cultivars carrying Type 1 promoter exhibited deep coloring, those carrying Type 2 and/or Type 3 exhibited pale coloring, and those carrying Type 1 and Type 2 and/or Type 3 exhibited medium coloring. The transcripts of the genes from these promoters encoded full-length predicted proteins. These results suggested that the genotype of the promoter region in DvIVS is one of the key factors determining the flower color intensity. PMID:23689377

  17. Genome-wide identification and analysis of basic helix-loop-helix domains in dog, Canis lupus familiaris.

    PubMed

    Wang, Xu-Hua; Wang, Yong; Liu, A-Ke; Liu, Xiao-Ting; Zhou, Yang; Yao, Qin; Chen, Ke-Ping

    2015-04-01

    The basic helix-loop-helix (bHLH) domain is a highly conserved amino acid motif that defines a group of DNA-binding transcription factors. bHLH proteins play essential regulatory roles in a variety of biological processes in animal, plant, and fungus. The domestic dog, Canis lupus familiaris, is a good model organism for genetic, physiological, and behavioral studies. In this study, we identified 115 putative bHLH genes in the dog genome. Based on a phylogenetic analysis, 51, 26, 14, 4, 12, and 4 dog bHLH genes were assigned to six separate groups (A-F); four bHLH genes were categorized as ''orphans''. Within-group evolutionary relationships inferred from the phylogenetic analysis were consistent with positional conservation, other conserved domains flanking the bHLH motif, and highly conserved intron/exon patterns in other vertebrates. Our analytical results confirmed the GenBank annotations of 89 dog bHLH proteins and provided information that could be used to update the annotations of the remaining 26 dog bHLH proteins. These data will provide good references for further studies on the structures and regulatory functions of bHLH proteins in the growth and development of dogs, which may help in understanding the mechanisms that underlie the physical and behavioral differences between dogs and wolves. PMID:25403511

  18. Evolution of vertebrate interferon inducible transmembrane proteins

    PubMed Central

    2012-01-01

    Background Interferon inducible transmembrane proteins (IFITMs) have diverse roles, including the control of cell proliferation, promotion of homotypic cell adhesion, protection against viral infection, promotion of bone matrix maturation and mineralisation, and mediating germ cell development. Most IFITMs have been well characterised in human and mouse but little published data exists for other animals. This study characterised IFITMs in two distantly related marsupial species, the Australian tammar wallaby and the South American grey short-tailed opossum, and analysed the phylogeny of the IFITM family in vertebrates. Results Five IFITM paralogues were identified in both the tammar and opossum. As in eutherians, most marsupial IFITM genes exist within a cluster, contain two exons and encode proteins with two transmembrane domains. Only two IFITM genes, IFITM5 and IFITM10, have orthologues in both marsupials and eutherians. IFITM5 arose in bony fish and IFITM10 in tetrapods. The bone-specific expression of IFITM5 appears to be restricted to therian mammals, suggesting that its specialised role in bone production is a recent adaptation specific to mammals. IFITM10 is the most highly conserved IFITM, sharing at least 85% amino acid identity between birds, reptiles and mammals and suggesting an important role for this presently uncharacterised protein. Conclusions Like eutherians, marsupials also have multiple IFITM genes that exist in a gene cluster. The differing expression patterns for many of the paralogues, together with poor sequence conservation between species, suggests that IFITM genes have acquired many different roles during vertebrate evolution. PMID:22537233

  19. Flip of spin helix chirality and ferromagnetic state in Fe1 -xCoxGe compounds

    NASA Astrophysics Data System (ADS)

    Grigoriev, S. V.; Siegfried, S.-A.; Altynbayev, E. V.; Potapova, N. M.; Dyadkin, V.; Moskvin, E. V.; Menzel, D.; Heinemann, A.; Axenov, S. N.; Fomicheva, L. N.; Tsvyashchenko, A. V.

    2014-11-01

    We have synthesized the solid solutions of Fe1 -xCoxGe compounds with x running from 0.0 to 0.9. Small-angle neutron scattering and magnetization measurements have shown that these compounds are ordered into the spin helix structure below the critical temperature Tc. The helix is transformed into the ferromagnet by application of the magnetic field above the critical value Hc 2. It is shown that Tc decreases smoothly with concentration x from 280 K for FeGe to 0 for CoGe. The values of the helix wave vector ks and the critical field Hc 2 depend strongly on concentration x , firstly, decreasing from pure FeGe to its minimum (| ks|?0 , Hc 2?0 ) at xc?0.6 , and, then increasing again at higher x . Thus, we observe a transformation of the helix structure to the ferromagnet at x ?xc at zero field. We concluded that this transformation is caused by different signs of the spin helicity for the compounds with x >xc and x

  20. A conserved phenylalanine as a relay between the ?5 helix and the GDP binding region of heterotrimeric Gi protein ? subunit.

    PubMed

    Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, Tina M; Meiler, Jens; Hamm, Heidi E

    2014-08-29

    G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the ?5 helix in the G protein ? subunit plays a major role during this activation process. However, the precise signaling pathway between the ?5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the ?5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the ?2-?3 strands and ?1 helix, leading to high basal nucleotide exchange. However, mutations in ? strands ?5 and ?6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via ?2-?3/?1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the ?1 and ?5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal ?5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the ? subunit. Rather, destabilization of the backdoor region of the G? subunit is sufficient for triggering the activation process. PMID:25037222

  1. Intracellular segment between transmembrane helices S0 and S1 of BK channel ? subunit contains two amphipathic helices connected by a flexible loop

    SciTech Connect

    Shi, Pan [Hefei National Laboratory of Microscale Physical Sciences, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027 (China) [Hefei National Laboratory of Microscale Physical Sciences, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027 (China); High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui, 230031 (China); Li, Dong; Lai, Chaohua [Hefei National Laboratory of Microscale Physical Sciences, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027 (China)] [Hefei National Laboratory of Microscale Physical Sciences, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027 (China); Zhang, Longhua, E-mail: zlhustc@ustc.edu.cn [Hefei National Laboratory of Microscale Physical Sciences, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027 (China)] [Hefei National Laboratory of Microscale Physical Sciences, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027 (China); Tian, Changlin, E-mail: cltian@ustc.edu.cn [Hefei National Laboratory of Microscale Physical Sciences, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027 (China) [Hefei National Laboratory of Microscale Physical Sciences, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027 (China); High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui, 230031 (China)

    2013-08-02

    Highlights: •The loop between S0 and S1 of BK channel was overexpressed and purified in DPC. •NMR studies indicated BK-IS1 contained two helices connected by a flexible loop. •Mg{sup 2+} titration of BK-IS1 indicated two possible binding sites of divalent ions. -- Abstract: The BK channel, a tetrameric potassium channel with very high conductance, has a central role in numerous physiological functions. The BK channel can be activated by intracellular Ca{sup 2+} and Mg{sup 2+}, as well as by membrane depolarization. Unlike other tetrameric potassium channels, the BK channel has seven transmembrane helices (S0–S6) including an extra helix S0. The intracellular segment between S0 and S1 (BK-IS1) is essential to BK channel functions and Asp99 in BK-IS1 is reported to be responsible for Mg{sup 2+} coordination. In this study, BK-IS1 (44–113) was over-expressed using a bacterial system and purified in the presence of detergent micelles for multidimensional heteronuclear nuclear magnetic resonance (NMR) structural studies. Backbone resonance assignment and secondary structure analysis showed that BK-IS1 contains two amphipathic helices connected by a 36-residue loop. Amide {sup 1}H–{sup 15}N heteronuclear NOE analysis indicated that the loop is very flexible, while the two amphipathic helices are possibly stabilized through interaction with the membrane. A solution NMR-based titration assay of BK-IS1 was performed with various concentrations of Mg{sup 2+}. Two residues (Thr45 and Leu46) with chemical shift changes were observed but no, or very minor, chemical shift difference was observed for Asp99, indicating a possible site for binding divalent ions or other modulation partners.

  2. Computational de novo design of a four-helix bundle protein-DND_4HB.

    PubMed

    Murphy, Grant S; Sathyamoorthy, Bharatwaj; Der, Bryan S; Machius, Mischa C; Pulavarti, Surya V; Szyperski, Thomas; Kuhlman, Brian

    2015-04-01

    The de novo design of proteins is a rigorous test of our understanding of the key determinants of protein structure. The helix bundle is an interesting de novo design model system due to the diverse topologies that can be generated from a few simple ?-helices. Previously, noncomputational studies demonstrated that connecting amphipathic helices together with short loops can sometimes generate helix bundle proteins, regardless of the bundle's exact sequence. However, using such methods, the precise positions of helices and side chains cannot be predetermined. Since protein function depends on exact positioning of residues, we examined if sequence design tools in the program Rosetta could be used to design a four-helix bundle with a predetermined structure. Helix position was specified using a folding procedure that constrained the design model to a defined topology, and iterative rounds of rotamer-based sequence design and backbone refinement were used to identify a low energy sequence for characterization. The designed protein, DND_4HB, unfolds cooperatively (Tm >90°C) and a NMR solution structure shows that it adopts the target helical bundle topology. Helices 2, 3, and 4 agree very closely with the design model (backbone RMSD?=?1.11 Å) and >90% of the core side chain ?1 and ?2 angles are correctly predicted. Helix 1 lies in the target groove against the other helices, but is displaced 3 Å along the bundle axis. This result highlights the potential of computational design to create bundles with atomic-level precision, but also points at remaining challenges for achieving specific positioning between amphipathic helices. PMID:25287625

  3. Six-helix bundles designed from DNA.

    PubMed

    Mathieu, Frederick; Liao, Shiping; Kopatsch, Jens; Wang, Tong; Mao, Chengde; Seeman, Nadrian C

    2005-04-01

    We present a designed cyclic DNA motif that consists of six DNA double helices that are connected to each other at two crossover sites. DNA double helices with 10.5 nucleotide pairs per turn facilitate the programming of DNA double crossover molecules to form hexagonally symmetric arrangements when the crossover points are separated by seven or fourteen nucleotide pairs. We demonstrate by atomic force microscopy well-formed arrays of hexagonal six-helix bundle motifs both in 1D and in 2D. PMID:15826105

  4. Six-Helix Bundles Designed from DNA

    PubMed Central

    Mathieu, Frederick; Liao, Shiping; Kopatsch, Jens; Wang, Tong; Mao, Chengde; Seeman, Nadrian C.

    2012-01-01

    We present a designed cyclic DNA motif that consists of six DNA double helices that are connected to each other at two crossover sites. DNA double helices with 10.5 nucleotide pairs per turn facilitate the programming of DNA double crossover molecules to form hexagonally symmetric arrangements when the crossover points are separated by seven or fourteen nucleotide pairs. We demonstrate by atomic force microscopy well-formed arrays of hexagonal six-helix bundle motifs both in 1D and in 2D. PMID:15826105

  5. Photo-active collagen systems with controlled triple helix architecture

    E-print Network

    Tronci, Giuseppe; Wood, David J

    2013-01-01

    The design of photo-active collagen systems is presented as a basis for establishing biomimetic materials with varied network architecture and programmable macroscopic properties. Following in-house isolation of type I collagen, reaction with vinyl-bearing compounds of varied backbone rigidity, i.e. 4-vinylbenzyl chloride (4VBC) and glycidyl methacrylate (GMA), was carried out. TNBS colorimetric assay, 1H-NMR and ATR-FTIR confirmed covalent and tunable functionalization of collagen lysines. Depending on the type and extent of functionalization, controlled stability and thermal denaturation of triple helices were observed via circular dichroism (CD), whereby the hydrogen-bonding capability of introduced moieties was shown to play a major role. Full gel formation was observed following photo-activation of functionalized collagen solutions. The presence of a covalent network only slightly affected collagen triple helix conformation (as observed by WAXS and ATR-FTIR), confirming the structural organization of fun...

  6. pH (low) insertion peptide (pHLIP) inserts across a lipid bilayer as a helix and exits by a different path

    PubMed Central

    Andreev, Oleg A.; Karabadzhak, Alexander G.; Weerakkody, Dhammika; Andreev, Gregory O.; Engelman, Donald M.; Reshetnyak, Yana K.

    2010-01-01

    What are the molecular events that occur when a peptide inserts across a membrane or exits from it? Using the pH-triggered insertion of the pH low insertion peptide to enable kinetic analysis, we show that insertion occurs in several steps, with rapid (0.1 sec) interfacial helix formation, followed by a much slower (100 sec) insertion pathway to give a transmembrane helix. The reverse process of unfolding and peptide exit from the bilayer core, which can be induced by a rapid rise of the pH from acidic to basic, proceeds approximately 400 times faster than folding/insertion and through different intermediate states. In the exit pathway, the helix–coil transition is initiated while the polypeptide is still inside the membrane. The peptide starts to exit when about 30% of the helix is unfolded, and continues a rapid exit as it unfolds inside the membrane. These insights may guide understanding of membrane protein folding/unfolding and the design of medically useful peptides for imaging and drug delivery. PMID:20160113

  7. Evaluation of Pierce's small-signal parameters of helix, travelling-wave tubes for gain calculation

    Microsoft Academic Search

    P. C. V. Rao; P. K. Jain; B. N. Basu

    1992-01-01

    The slow-wave structure of a travelling-wave tube (TWT), consisting of a helix supported by a number of dielectric rods in a glass tube envelope, is modelled and theoretically analysed for the attenuation constant, phase propagation constant and interaction impedance of the structure. The expressions for these quantities are then interpreted to obtain the expressions for Pierce's velocity, loss and space-charge

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

    SciTech Connect

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

    2010-03-08

    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.

  9. Mathematical Characterization of Protein Transmembrane Regions

    PubMed Central

    Roy Choudhury, Amrita; Zhukov, Nikolay; Novi?, Marjana

    2013-01-01

    Graphical bioinformatics has paved a unique way of mathematical characterization of proteins and proteomic maps. The graphics representations and the corresponding mathematical descriptors have proved to be useful and have provided unique solutions to problems related to identification, comparisons, and analyses of protein sequences and proteomics maps. Based on sequence information alone, these descriptors are independent from physiochemical properties of amino acids and evolutionary information. In this work, we have presented invariants from amino acid adjacency matrix and decagonal isometries matrix as potential descriptors of protein sequences. Encoding protein sequences into amino acid adjacency matrix is already well established. We have shown its application in classification of transmembrane and nontransmembrane regions of membrane protein sequences. We have introduced the dodecagonal isometries matrix, which is a novel method of encoding protein sequences based on decagonal isometries group. PMID:23690747

  10. Molecular mechanisms for generating transmembrane proton gradients.

    PubMed

    Gunner, M R; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

    2013-01-01

    Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side. PMID:23507617

  11. Molecular mechanisms for generating transmembrane proton gradients

    PubMed Central

    Gunner, M.R.; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

    2013-01-01

    Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side. PMID:23507617

  12. Visualizing Water Molecules in Transmembrane Proteins Using Radiolytic Labeling Methods

    SciTech Connect

    Orban, T.; Gupta, S; Palczewski, K; Chance, M

    2010-01-01

    Essential to cells and their organelles, water is both shuttled to where it is needed and trapped within cellular compartments and structures. Moreover, ordered waters within protein structures often colocalize with strategically placed polar or charged groups critical for protein function, yet it is unclear if these ordered water molecules provide structural stabilization, mediate conformational changes in signaling, neutralize charged residues, or carry out a combination of all these functions. Structures of many integral membrane proteins, including G protein-coupled receptors (GPCRs), reveal the presence of ordered water molecules that may act like prosthetic groups in a manner quite unlike bulk water. Identification of 'ordered' waters within a crystalline protein structure requires sufficient occupancy of water to enable its detection in the protein's X-ray diffraction pattern, and thus, the observed waters likely represent a subset of tightly bound functional waters. In this review, we highlight recent studies that suggest the structures of ordered waters within GPCRs are as conserved (and thus as important) as conserved side chains. In addition, methods of radiolysis, coupled to structural mass spectrometry (protein footprinting), reveal dynamic changes in water structure that mediate transmembrane signaling. The idea of water as a prosthetic group mediating chemical reaction dynamics is not new in fields such as catalysis. However, the concept of water as a mediator of conformational dynamics in signaling is just emerging, because of advances in both crystallographic structure determination and new methods of protein footprinting. Although oil and water do not mix, understanding the roles of water is essential to understanding the function of membrane proteins.

  13. Orbital dynamics in the Tevatron double helix

    SciTech Connect

    Michelotti, L.; Saritepe, S.

    1989-03-01

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

  14. Characterization by immunocytochemistry of ionic channels in Helix aspersa suboesophageal brain ganglia neurons.

    PubMed

    Azanza, M J; Pérez-Castejón, C; Pes, N; Pérez-Bruzón, R N; Aisa, J; Junquera, C; Maestú, C; Lahoz, M; Martínez-Ciriano, C; Vera-Gil, A; Del Moral, A

    2008-04-01

    The aim of this work was to characterize several ionic channels in nervous cells of the suboesophageal visceral, left and right parietal, and left and right pleural brain ganglia complex of the snail Helix aspersa by immunocytochemistry. We have studied the immunostaining reaction for a wide panel of eleven polyclonal antibodies raised against mammal antigens as follows: voltage-gated-Na+ channel; voltage-gated-delayed-rectifier-K+ channel; SK2-small-conductance-Ca2+-dependent-K+ channel apamin sensitive; SK3 potassium channel; charybdotoxin-sensitive voltage-dependent potassium channel; BKCa-maxi-conductance-Ca2+-dependent-K+ channel; hyperpolarization-activated cyclic nucleotide-gated potassium channel 4; G-protein-activated inwardly rectifying potassium channel GIRK2 and voltage-gated-calcium of L, N and P/Q type channels. Our results show positive reaction in neurons, but neither in glia cells nor in processes in the Helix suboesophageal ganglia. Our results suggest the occurrence of molecules in Helix neurons sharing antigenic determinants with mammal ionic channels. The reaction density and distribution of immunoreactive staining within neurons is specific for each one of the antisera tested. The studies of co-localization of immunoreaction, on alternate serial sections of the anterior right parietal ganglion, have shown for several recognized mapped neurons that they can simultaneously be expressed among two and seven different ionic protein channels. These results are considered a key structural support for the interpretation of Helix aspersa neuron electrophysiological activity. PMID:18228196

  15. Deformation of cholesteric elastomers by uniaxial stress along the helix axis

    NASA Astrophysics Data System (ADS)

    Stille, W.

    2009-01-01

    The deformation of cholesteric elastomers by mechanical stress applied parallel to the helix axis is studied by calculation of the free-energy density. The Frank-elasticity contribution is taken into account. A chiral solvent, present at cross-linking time, is in general considered to be replaced after cross-linking by a solvent with different chirality. Two special cases considered are zero and unchanged solvent chirality, the first known as that of imprinted cholesteric elastomers, the latter equivalent to intrinsic cholesteric elastomers with chemically attached chiral groups. Depending on material parameters and imposed strain, the director can show a tilt towards the helix axis up to the maximum tilt, corresponding to a nematic state. In case of intrinsic elastomers with low conformation anisotropy, direct transitions from untilted to nematic states can be induced by straining. The helix structure of the director field is coarsened with an average wave number different to that of the information inscribed in the network at cross-linking time, if this lowers the average free-energy density. Switching between different states can be achieved with electric fields of reasonable values applied parallel to the helix axis. Spectra of the reflection of polarized light are calculated.

  16. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    PubMed

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a functional unit. PMID:19181816

  17. Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT?

    PubMed Central

    Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

    2009-01-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a functional unit. PMID:19181816

  18. Doppler velocimetry of a current driven spin helix

    NASA Astrophysics Data System (ADS)

    Yang, Luyi

    2012-02-01

    We present direct observation of the translational motion of spin helices in GaAs quantum wells under the influence of applied electric fields. Previously, the lifetime of such helices was observed by time-resolving the amplitude of light diffracted from the periodic spin polarization [1]. This technique cannot be applied to tracking the motion of current-driven spin helices because diffraction amplitude is insensitive to translation of the center of mass of a periodic structure. In this talk, we describe a new experimental technique, Doppler spin velocimetry, capable of resolving displacements of spin polarization at the level of 1 nm on a picosecond time scale [2]. This is accomplished through the use of heterodyne detection to measure the optical phase of the diffracted light. We discuss experiments in which this technique is used to measure the motion of spin helices as a function of temperature, in-plane electric field, and photoinduced spin polarization amplitude. Several striking observations will be reported -- for example, the spin helix velocity changes sign as a function of wavevector and is zero at the wavevector that yields the largest spin lifetime. Another important observation is that the velocity of spin polarization packets becomes equal to the drift velocity of the high-mobility electron gas in the limit of small spin helix amplitude. Finally, we show that spin helices continue propagate at the same speed as the Fermi sea even when the electron drift velocity exceeds the Fermi velocity of 10^7 cm-s-1. In collaboration with J. D. Koralek and J. Orenstein, UC Berkeley and LBNL, D. R. Tibbetts, J. L. Reno, and M. P. Lilly, SNL. Supported by DOE under Contract No. DE-AC02-05CH11231 and DE-AC04-94AL85000. [4pt] [1] J. D. Koralek et al., ``Emergency of the persistent spin helix in semiconductor quantum wells,'' Nature 458, 610-613 (2009). [0pt] [2] L. Yang et al, ``Doppler velocimetry of spin propagation in a two-dimensional electron gas,'' to appear in Nature Physics.

  19. R E V I E W A R T I C L E A phylogenomic analysis of bacterial helix^turn^helix transcription

    E-print Network

    Thioulouse, Jean

    R E V I E W A R T I C L E A phylogenomic analysis of bacterial helix^turn^helix transcription adaptability; helix­turn­helix; transcriptional regulators. Abstract Perception by each individual organism for ecological adaptability. Transcription regulators are key decision-making proteins that mediate

  20. Functional Extension of Amino Acid Triads from the Fourth Transmembrane Segment (S4) into Its External Linker in Shaker K+ Channels*

    PubMed Central

    Yang, Ya-Chin; Lin, Shin; Chang, Po-Chun; Lin, Hsiao-Chun; Kuo, Chung-Chin

    2011-01-01

    The highly conserved fourth transmembrane segment (S4) is the primary voltage sensor of the voltage-dependent channel and would move outward upon membrane depolarization. S4 comprises repetitive amino acid triads, each containing one basic (presumably charged and voltage-sensing) followed by two hydrophobic residues. We showed that the triad organization is functionally extended into the S3–4 linker right external to S4 in Shaker K+ channels. The arginine (and lysine) substitutes for the third and the sixth residues (Ala-359 and Met-356, respectively) external to the outmost basic residue (Arg-362) in S4 dramatically and additively stabilize S4 in the resting conformation. Also, Leu-361 and Leu-358 play a very similar role in stabilization of S4 in the resting position, presumably by their hydrophobic side chains. Moreover, the double mutation A359R/E283A leads to a partially extruded position of S4 and consequently prominent closed-state inactivation, suggesting that Glu-283 in S2 may coordinate with the arginines in the extruded S4 upon depolarization. We conclude that the triad organization extends into the S3–4 linker for about six amino acids in terms of their microenvironment. These approximately six residues should retain the same helical structure as S4, and their microenvironment serves as part of the “gating canal” accommodating the extruding S4. Upon depolarization, S4 most likely moves initially as a sliding helix and follows the path that is set by the approximately six residues in the S3–4 linker in the resting state, whereas further S4 translocation could be more like, for example, a paddle, without orderly coordination from the contiguous surroundings. PMID:21900243

  1. Electrostatic free energy landscapes for nucleic acid helix assembly

    PubMed Central

    Tan, Zhi-Jie; Chen, Shi-Jie

    2006-01-01

    Metal ions are crucial for nucleic acid folding. From the free energy landscapes, we investigate the detailed mechanism for ion-induced collapse for a paradigm system: loop-tethered short DNA helices. We find that Na+ and Mg2+ play distinctive roles in helix–helix assembly. High [Na+] (>0.3 M) causes a reduced helix–helix electrostatic repulsion and a subsequent disordered packing of helices. In contrast, Mg2+ of concentration >1 mM is predicted to induce helix–helix attraction and results in a more compact and ordered helix–helix packing. Mg2+ is much more efficient in causing nucleic acid compaction. In addition, the free energy landscape shows that the tethering loops between the helices also play a significant role. A flexible loop, such as a neutral loop or a polynucleotide loop in high salt concentration, enhances the close approach of the helices in order to gain the loop entropy. On the other hand, a rigid loop, such as a polynucleotide loop in low salt concentration, tends to de-compact the helices. Therefore, a polynucleotide loop significantly enhances the sharpness of the ion-induced compaction transition. Moreover, we find that a larger number of helices in the system or a smaller radius of the divalent ions can cause a more abrupt compaction transition and a more compact state at high ion concentration, and the ion size effect becomes more pronounced as the number of helices is increased. PMID:17145719

  2. Genetic Conservation in gp36 Transmembrane Sequences of Indian HIV Type 2 Isolates.

    PubMed

    Jadhav, Sushama; Tripathy, Srikanth; Kulkarni, Smita; Chaturbhuj, Devidas; Ghare, Rucha; Bhattacharya, Jayanta; Paranjape, Ramesh

    2011-12-01

    HIV-2 group A is predominant in different parts of the world, especially Africa, Portugal, Spain, France, the United Kingdom, the United States, Korea, and India. Among the Asian countries, India accounts for about 95% of all HIV-2 infections. The prevalence of HIV-2 has been reported from various states of India such as Maharashtra, Goa, Tamil Nadu, West Bengal, and Uttar Pradesh. In the present study, we analyzed transmembrane region (gp36) sequences of 10 HIV-2 group A Indian strains, isolated from Indian HIV-2-seropositive individuals. HIV Blast analysis for the 1.0-Kb region of the gp36 transmembrane region has shown that all these sequences belong to HIV-2 group A. Phylogenetic analysis indicated that the sequences cluster with HIV-2 group A sequences of Cameroon and Senegal. The epitope found at position 645-656 (YELQKLNSWDVF), previously reported as a broadly neutralizing determinant, was very well conserved in all 10 study sequences. The percentage similarity between Indian and South African HIV-2 group A gp36 sequences was 90% (range 86-100, SD 2.8) and with other nonsubtype A clades was 84% (range 77-100, SD 6.06) indicating overall less variability among the reported HIV-2 sequences. Similarly, the consensus amino acid sequences of the envelope transmembrane region of HIV-1 (gp41) and HIV-2 (gp36) is quite synonymous, indicating 87% similarity; however, limited information is available about the gp36 transmembrane region of the prevalent HIV 2 group A Indian strain. The rate of synonymous substitutions reported in the gp105 region was significantly higher, suggesting lower virulence of HIV-2, which does translate into a lower rate of evolution, while the dN/dS ratio for the gp36 transmembrane region was less than one, indicating its conservation and significance (p<0.05) in structural and functional constraints. PMID:21453135

  3. Comets Kick up Dust in Helix Nebula

    NASA Technical Reports Server (NTRS)

    2007-01-01

    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.

  4. Isosteric substitutions of urea to thiourea and selenourea in aliphatic oligourea foldamers: site-specific perturbation of the helix geometry.

    PubMed

    Nelli, Yella Reddy; Antunes, Stéphanie; Salaün, Arnaud; Thinon, Emmanuelle; Massip, Stéphane; Kauffmann, Brice; Douat, Céline; Guichard, Gilles

    2015-02-01

    Nearly isosteric oxo to thioxo substitution was employed to interrogate the structure of foldamers with a urea backbone and explore the relationship between helical folding and hydrogen-bonding interactions. A series of oligomers with urea bonds substituted by thiourea bonds at discrete or all positions in the sequence have been prepared and their folding propensity was studied by using a combination of spectroscopic methods and X-ray diffraction. The outcome of oxo to thioxo replacements on the helical folding was found to depend on whether central or terminal ureas were modified. The canonical helix geometry was not affected upon insertion of thioureas close to the negative end of the helix dipole, whereas thioureas close to the positive pole were found to increase the terminal flexibility and cause helix fraying. Perturbation was amplified when a selenourea was incorporated instead, leading to a structure that is only partly folded. PMID:25529793

  5. Transmembrane topology of the sulfonylurea receptor SUR1.

    PubMed

    Conti, L R; Radeke, C M; Shyng, S L; Vandenberg, C A

    2001-11-01

    Sulfonylurea receptors (SURx) are multi-spanning transmembrane proteins of the ATP-binding cassette (ABC) family, which associate with Kir6.x to form ATP-sensitive potassium channels. Two models, with 13-17 transmembrane segments, have been proposed for SURx topologies. Recently, we demonstrated that the amino-terminal region of SUR1 contains 5 transmembrane segments, supporting the 17-transmembrane model. To investigate the topology of the complete full-length SUR1, two strategies were employed. Topology was probed by accessibility of introduced cysteines to a membrane-impermeable biotinylating reagent, biotin maleimide. Amino acid positions 6/26, 99, 159, 337, 567, 1051, and 1274 were accessible, therefore extracellular, whereas many endogenous and some introduced cysteines were inaccessible, thus likely cytoplasmic or intramembrane. These sites correspond to extracellular loops 1-3, 5-6, and 8 and the NH2 terminus, and intracellular loops 3-8 and COOH terminus in the 17-transmembrane model. Immunofluorescence was used to determine accessibility of epitope-tagged SUR1 in intact and permeabilized cells. Epitopes at positions 337 and 1050 (putative external loops 3 and 6) were labeled in intact cells, therefore external, whereas positions 485 and 1119 (putative internal loops 5 and 7) only were accessible after permeabilization and therefore internal. These results are compatible with the 17-transmembrane model with two pairs of transmembrane segments as possible reentrant loops. PMID:11546780

  6. System and methods for predicting transmembrane domains in membrane proteins and mining the genome for recognizing G-protein coupled receptors

    DOEpatents

    Trabanino, Rene J; Vaidehi, Nagarajan; Hall, Spencer E; Goddard, William A; Floriano, Wely

    2013-02-05

    The invention provides computer-implemented methods and apparatus implementing a hierarchical protocol using multiscale molecular dynamics and molecular modeling methods to predict the presence of transmembrane regions in proteins, such as G-Protein Coupled Receptors (GPCR), and protein structural models generated according to the protocol. The protocol features a coarse grain sampling method, such as hydrophobicity analysis, to provide a fast and accurate procedure for predicting transmembrane regions. Methods and apparatus of the invention are useful to screen protein or polynucleotide databases for encoded proteins with transmembrane regions, such as GPCRs.

  7. Crystal structure of a claudin provides insight into the architecture of tight junctions.

    PubMed

    Suzuki, Hiroshi; Nishizawa, Tomohiro; Tani, Kazutoshi; Yamazaki, Yuji; Tamura, Atsushi; Ishitani, Ryuichiro; Dohmae, Naoshi; Tsukita, Sachiko; Nureki, Osamu; Fujiyoshi, Yoshinori

    2014-04-18

    Tight junctions are cell-cell adhesion structures in epithelial cell sheets that surround organ compartments in multicellular organisms and regulate the permeation of ions through the intercellular space. Claudins are the major constituents of tight junctions and form strands that mediate cell adhesion and function as paracellular barriers. We report the structure of mammalian claudin-15 at a resolution of 2.4 angstroms. The structure reveals a characteristic ?-sheet fold comprising two extracellular segments, which is anchored to a transmembrane four-helix bundle by a consensus motif. Our analyses suggest potential paracellular pathways with distinctive charges on the extracellular surface, providing insight into the molecular basis of ion homeostasis across tight junctions. PMID:24744376

  8. Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers

    PubMed Central

    Feld, Geoffrey K.; Thoren, Katie L.; Kintzer, Alexander F.; Sterling, Harry J.; Tang, Iok I.; Greenberg, Shoshana G.; Williams, Evan R.; Krantz, Bryan A.

    2011-01-01

    The protein transporter, anthrax lethal toxin, is comprised of protective antigen (PA), a transmembrane translocase, and lethal factor (LF), a cytotoxic enzyme. Following assembly into holotoxin complexes, PA forms an oligomeric channel that unfolds LF and translocates it into the host cell. We report the crystal structure of the core of a lethal toxin complex to 3.1-Å resolution; the structure contains a PA octamer bound to four LF PA-binding domains (LFN). The first ? helix and ? strand of each LFN unfold and dock into a deep amphipathic cleft on the surface of the PA octamer, which we call the ? clamp. The ? clamp possesses nonspecific polypeptide binding activity and is functionally relevant to efficient holotoxin assembly, PA octamer formation, and LF unfolding and translocation. This structure provides insight on the mechanism of translocation-coupled protein unfolding. PMID:21037566

  9. Recognition of a Single Transmembrane Degron by Sequential Quality Control Checkpoints

    Microsoft Academic Search

    Laurence Fayadat; Ron R. Kopito

    2003-01-01

    To understand the relationship between conformational maturation and quality control-mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the -subunit of the T-cell antigen receptor (TCR) into the -helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as

  10. The Transmembrane Domain of the Acetylcholine Receptor: Insights from Simulations on Synthetic Peptide Models

    Microsoft Academic Search

    Leonor Saiz; Michael L. Klein

    2005-01-01

    We have studied the structure and properties of a bundle of ?-helical peptides embedded in a 1,2-dimyristoyl-3-phosphatidylcholine phospholipid bilayer by molecular dynamics simulations. The bundle of five transmembrane ?M2 segments constitutes the model for the pore region of the nicotinic acetylcholine receptor, which is the neurotransmitter-gated ion-channel responsible for the fast propagation of electrical signals between cells at the nerve-muscle

  11. Potential dual molecular interaction of the Drosophila 7-pass transmembrane cadherin Flamingo in dendritic morphogenesis

    Microsoft Academic Search

    Hiroshi Kimura; Tadao Usui; Asako Tsubouchi; Tadashi Uemura

    2006-01-01

    Seven-pass transmembrane cadherins (7-TM cadherins) play pleiotropic roles in epithelial planar cell polarity, shaping dendritic arbors and in axonal outgrowth. In contrast to their role in planar polarity, how 7-TM cadherins control dendritic and axonal outgrowth at the molecular level is largely unknown. Therefore, we performed extensive structure-function analysis of the Drosophila 7-TM cadherin Flamingo (Fmi) and investigated the activities

  12. Hexa Helix: Modified Quad Helix Appliance to Correct Anterior and Posterior Crossbites in Mixed Dentition

    PubMed Central

    Yaseen, Syed Mohammed; Acharya, Ravindranath

    2012-01-01

    Among the commonly encountered dental irregularities which constitute developing malocclusion is the crossbite. During primary and mixed dentition phase, the crossbite is seen very often and if left untreated during these phases then a simple problem may be transformed into a more complex problem. Different techniques have been used to correct anterior and posterior crossbites in mixed dentition. This case report describes the use of hexa helix, a modified version of quad helix for the management of anterior crossbite and bilateral posterior crossbite in early mixed dentition. Correction was achieved within 15 weeks with no damage to the tooth or the marginal periodontal tissue. The procedure is a simple and effective method for treating anterior and bilateral posterior crossbites simultaneously. PMID:23119188

  13. Sequence preference in DNA binding: de novo designed helix-turn-helix metallopeptides recognize a family of DNA target sites.

    PubMed

    Wong-Deyrup, Siu Wah; Kim, Youngbae; Franklin, Sonya J

    2006-01-01

    The DNA-binding behavior and target sequences of two designed metallopeptides have been investigated with an iterative electrophoresis mobility shift assay followed by PCR amplification, and by circular dichroism spectroscopy. Peptides P3W and P5b were designed based on the structural similarity of the helix-turn-helix motif of homeodomains and the EF-hand motifs of calmodulin, as previously described for P3W. Like P3W, P5b binds both Eu(III) (K(d) = 12.6 +/- 1.9 microM) and Ca(II) (K(d) = 70 +/- 8 microM) with reasonable affinity. Binding selection from a library of randomized 8-mer DNA oligonucleotide sequences identified one target family for CaP5b [5'-pur-T-pur-G-(G/C)-3'], and two target sites for CaP3W [5'-(A/T)-G-G-G-(T/C)-3' and 5'-A-T-(G/T)-T-G-3']. Circular dichroism studies indicate that unlike EuP3W, EuP5b is poorly folded in the absence of DNA. In the presence of DNA containing target-binding sites for both peptides, both EuP3W and EuP5b increase in helical content, in the latter case significantly. These results suggest that EuP5b binding to target DNA involves an induced-fit mechanism. These small chimeric metallopeptides have been found to bind selectively to DNA targets, analogous to natural protein-DNA interactions. This corroborates our earlier conclusions (J. Am. Chem. Soc. 125:6656, 2003) that sequence-preferential DNA cleavage by Ce(IV)P3W was due to sequence recognition. PMID:16292553

  14. A cellular factor stimulates ligand-dependent release of hsp90 from the basic helix-loop-helix dioxin receptor.

    PubMed Central

    McGuire, J; Whitelaw, M L; Pongratz, I; Gustafsson, J A; Poellinger, L

    1994-01-01

    In response to dioxin, the nuclear basic helix-loop-helix (bHLH) dioxin receptor forms a complex with the bHLH partner factor Arnt that regulates target gene transcription by binding to dioxin-responsive sequence motifs. Previously, we have demonstrated that the latent form of dioxin receptor present in extracts from untreated cells is stably associated with molecular chaperone protein hsp90, and Arnt is not a component of this complex. Here, we used a coimmunoprecipitation assay to demonstrate that the in vitro-translated dioxin receptor, but not Arnt, is stably associated with hsp90. Although it showed ligand-binding activity, the in vitro-translated dioxin receptor failed to dissociate from hsp90 upon exposure to ligand. Addition of a specific fraction from wild-type hepatoma cells, however, to the in vitro-expressed receptor promoted dioxin-dependent release of hsp90. This stimulatory effect was mediated via the bHLH dimerization and DNA-binding motif of the receptor. Moreover, ligand-dependent release of hsp90 from the receptor was not promoted by fractionated cytosolic extracts from mutant hepatoma cells which are deficient in the function of bHLH dioxin receptor partner factor Arnt. Thus, our results provide a novel model for regulation of bHLH factor activity and suggest that derepression of the dioxin receptor by ligand-induced release of hsp90 may require bHLH-mediated concomitant recruitment of an additional cellular factor, possibly the structurally related bHLH dimerization partner factor Arnt. In support of this model, addition of in vitro-expressed wild-type Arnt, but not a mutated form of Arnt lacking the bHLH motif, promoted release of hsp90 from the dioxin receptor in the presence of dioxin. Images PMID:8139547

  15. Molecular Distinction between Specification and Differentiation in the Myogenic Basic Helix-Loop-Helix Transcription Factor Family

    PubMed Central

    Bergstrom, Donald A.; Tapscott, Stephen J.

    2001-01-01

    The myogenic basic helix-loop-helix (bHLH) proteins regulate both skeletal muscle specification and differentiation: MyoD and Myf5 establish the muscle lineage, whereas myogenin mediates differentiation. Previously, we demonstrated that MyoD was more efficient than myogenin at initiating the expression of skeletal muscle genes, and in this study we present the molecular basis for this difference. A conserved amphipathic alpha-helix in the carboxy terminus of the myogenic bHLH proteins has distinct activities in MyoD and myogenin: the MyoD helix facilitates the initiation of endogenous gene expression, whereas the myogenin helix functions as a general transcriptional activation domain. Thus, the alternate use of a similar motif for gene initiation and activation provides a molecular basis for the distinction between specification and differentiation within the myogenic bHLH gene family. PMID:11259589

  16. Video imaging of cardiac transmembrane activity

    NASA Astrophysics Data System (ADS)

    Baxter, William T.; Davidenko, Jorge; Cabo, Candido; Jalife, Jose

    1994-05-01

    High resolution movies of transmembrane electrical activity in thin (0.5 mm) slices of sheep epicardial muscle were recorded by optical imaging with voltage-sensitive dyes and a CCD video camera. Activity was monitored at approximately 65,000 picture elements per 2 cm2 tissue for several seconds at a 16 msec sampling rate. Simple image processing operations permitted visualization and analysis of the optical signal, while isochrome maps depicted complex patterns of propagation. Maps of action potential duration and regional intermittent conduction block showed that even these small preparations may exhibit considerable spatial heterogeneity. Self-sustaining reentrant activity in the form of spiral waves was consistently initiated and observed either drifting across the tissue or anchored to small heterogeneities. The current limitations of video optical mappings are a low signal-to- noise ratio and low temporal resolution. The advantages include high spatial resolution and direct correlation of electrical activity with anatomy. Video optical mapping permits the analysis of the electrophysiological properties of any region of the preparation during both regular stimulation and reentrant activation, providing a useful tool for studying cardiac arrhythmias.

  17. Molecular archeological studies of transmembrane transport systems

    NASA Astrophysics Data System (ADS)

    Saier, Milton H.; Wang, Bin; Sun, Eric I.; Matias, Madeleine; Yen, Ming Ren

    We here review studies concerned with the evolutionary pathways taken for the appearance of complex transport systems. The transmembrane protein constituents of these systems generally arose by (1) intragenic duplications, (2) gene fusions, and (3) the superimposition of enzymes onto carriers. In a few instances, we have documented examples of “reverse” or “retrograde” evolution where complex carriers have apparently lost parts of their polypeptide chains to give rise to simpler channels. Some functional superfamilies of transporters that are energized by adenosine triphosphate (ATP) or phosphoenolpyruvate (PEP) include several independently evolving permease families. The ubiquitous ATP-binding cassette (ABC) superfamily couples transport to ATP hydrolysis where the ATPases are superimposed on at least three distinct, independently evolving families of permeases. The prokaryotic sugar transporting phosphotransferase system (PTS) uses homologous PEP-dependent general energy-coupling phosphoryl transfer enzymes superimposed on at least three independently arising families of permeases to give rise to complex group translocators that modify their sugar substrates during transport, releasing cytoplasmic sugar phosphates. We suggest that simple carriers evolved independently of the energizing enzymes, and that chemical energization of transport resulted from the physical and functional coupling of the enzymes to the carriers.

  18. Topological Analysis of Hedgehog Acyltransferase, a Multipalmitoylated Transmembrane Protein*

    PubMed Central

    Konitsiotis, Antonio D.; Jovanovi?, Biljana; Ciepla, Paulina; Spitaler, Martin; Lanyon-Hogg, Thomas; Tate, Edward W.; Magee, Anthony I.

    2015-01-01

    Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo. PMID:25505265

  19. Topological analysis of Hedgehog acyltransferase, a multipalmitoylated transmembrane protein.

    PubMed

    Konitsiotis, Antonio D; Jovanovi?, Biljana; Ciepla, Paulina; Spitaler, Martin; Lanyon-Hogg, Thomas; Tate, Edward W; Magee, Anthony I

    2015-02-01

    Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo. PMID:25505265

  20. Suppression of In-Band Power Holes in Helix Traveling-Wave Tubes

    Microsoft Academic Search

    Yubin Gong; Zhaoyun Duan; Yanmei Wang; Yanyu Wei; Hairong Yin; Wenxiang Wang

    2011-01-01

    Slow-wave structures employed in forward-wave lin- ear beam amplifiers such as wideband helix traveling-wave tubes (TWTs) always support backward waves. A backward-wave space harmonic of a signal at a harmonic of a drive signal can effectively interact with a slow space-charge wave of an electron beam. In this case, the backward wave can compete with a forward wave, which results

  1. [Histological and ultrastructural study of the gonad of Helix aspersa Müller at hatching].

    PubMed

    Griffond, B; Bride, J

    1981-01-01

    The gonad of young Helix aspersa, studied by light and electron microscopy during the first post-hatching week, showed a compact structure. Eventually, a lumen appeared as the cells moved apart. The gonad was composed of stem and germinal cells. The latter cells were represented by primordial germ cells and oocytes. With our method of rearing, the female germinal line differentiated first. PMID:6891485

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

    PubMed

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

    2014-05-15

    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

  3. DNA helix model closeup, still imageSite: DNA Interactive (www.dnai.org)

    NSDL National Science Digital Library

    2008-10-06

    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?

  4. A Lys-Trp cation-? interaction mediates the dimerization and function of the chloride intracellular channel protein 1 transmembrane domain.

    PubMed

    Peter, Bradley; Polyansky, Anton A; Fanucchi, Sylvia; Dirr, Heini W

    2014-01-14

    Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. The oligomerization of the transmembrane domain (TMD) remains speculative despite it being implicated in pore formation. The extent to which electrostatic and van der Waals interactions drive folding and association of the dimorphic TMD is unknown and is complicated by the requirement of interactions favorable in both aqueous and membrane environments. Here we report a putative Lys37-Trp35 cation-? interaction and show that it stabilizes the dimeric form of the CLIC1 TMD in membranes. A synthetic 30-mer peptide comprising a K37M TMD mutant was examined in 2,2,2-trifluoroethanol, sodium dodecyl sulfate micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes using far-ultraviolet (UV) circular dichroism, fluorescence, and UV absorbance spectroscopy. Our data suggest that Lys37 is not implicated in the folding, stability, or membrane insertion of the TMD peptide. However, removal of this residue impairs the formation of dimers and higher-order oligomers. This is accompanied by a 30-fold loss of chloride influx activity, suggesting that dimerization modulates the rate of chloride conductance. We propose that, within membranes, individual TMD helices associate via a Lys37-mediated cation-? interaction to form active dimers. The latter findings are also supported by results of modeling a putative TMD dimer conformation in which Lys37 and Trp35 form cation-? pairs at the dimer interface. Dimeric helix bundles may then associate to form fully active ion channels. Thus, within a membrane-like environment, aromatic interactions involving a polar lysine side chain provide a thermodynamic driving force for helix-helix association. PMID:24328417

  5. Cholesterol-induced lipophobic interaction between transmembrane helices using ensemble and single-molecule fluorescence resonance energy transfer.

    PubMed

    Yano, Yoshiaki; Kondo, Kotaro; Kitani, Ryota; Yamamoto, Arisa; Matsuzaki, Katsumi

    2015-02-17

    The solvent environment regulates the conformational dynamics and functions of solvated proteins. In cell membranes, cholesterol, a major eukaryotic lipid, can markedly modulate protein dynamics. To investigate the nonspecific effects of cholesterol on the dynamics and stability of helical membrane proteins, we monitored association-dissociation dynamics on the antiparallel dimer formation of two simple transmembrane helices (AALALAA)3 with single-molecule fluorescence resonance energy transfer (FRET) using Cy3B- and Cy5-labeled helices in lipid vesicles (time resolution of 17 ms). The incorporation of 30 mol % cholesterol into phosphatidylcholine bilayers significantly stabilized the helix dimer with average lifetimes of 450-170 ms in 20-35 °C. Ensemble FRET measurements performed at 15-55 °C confirmed the cholesterol-induced stabilization of the dimer (at 25 °C, ??G(a) = -9 kJ mol(-1) and ??Ha = -60 kJ mol(-1)), most of which originated from "lipophobic" interactions by reducing helix-lipid contacts and the lateral pressure in the hydrocarbon core region. The temperature dependence of the dissociation process (activation energy of 48 kJ) was explained by the Kramers-type frictional barrier in membranes without assuming an enthalpically unfavorable transition state. In addition to these observations, cholesterol-induced tilting of the helices, a positive ?C(p(a)), and slower dimer formation compared with the random collision rate were consistent with a hypothetical model in which cholesterol stabilizes the helix dimer into an hourglass shape to relieve the lateral pressure. Thus, the liposomal single-molecule approach highlighted the significance of the cholesterol-induced basal force for interhelical interactions, which will aid discussions of complex protein-membrane systems. PMID:25629582

  6. Structural models of the MscL gating mechanism

    NASA Technical Reports Server (NTRS)

    Sukharev, S.; Durell, S. R.; Guy, H. R.

    2001-01-01

    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.

  7. Structural models of the MscL gating mechanism.

    PubMed Central

    Sukharev, S; Durell, S R; Guy, H R

    2001-01-01

    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. PMID:11463635

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

    PubMed

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

    2013-04-12

    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

  9. Design, synthesis, and conformational analysis of trispyrimidonamides as ?-helix mimetics.

    PubMed

    Spanier, Lukas; Ciglia, Emanuele; Hansen, Finn K; Kuna, Krystina; Frank, Walter; Gohlke, Holger; Kurz, Thomas

    2014-02-21

    The straightforward synthesis of trispyrimidonamides as a new class of ?-helix mimetics is reported. Because of the versatility of our synthetic protocol, a variety of side chains including aliphatic, basic, aromatic, and heteroaromatic residues were included. A comprehensive conformational analysis revealed that in polar solvents a trimeric compound adopts conformations that can lead to i, i + 4, i + 8, or i, i + 8 patterns of side chain orientation. This suggests that trispyrimidonamides could be promising ?-helix mimetics to target hot spots that are distributed over a wider angular range of an ?-helix interface than in the classical i, i + 4, i + 7 case. PMID:24447208

  10. BET, a novel neuronal transmembrane protein with multiple EGF-like motifs.

    PubMed

    Nishizumi, Hirofumi; Komiyama, Takaki; Miyabayashi, Tomoyuki; Sakano, Seiji; Sakano, Hitoshi

    2002-05-01

    Using the signal sequence trap method, we have identified a cDNA clone coding for a type I transmembrane protein, BET, with 10 epidermal growth factor (EGF) motifs in the extracellular domain. In situ hybridization revealed that the bet mRNA is specifically expressed in the mitral/tufted cells in the olfactory bulb, Purkinje cells in the cerebellum, and pyramidal cells in the hippocampus. Using polyclonal antibodies, we have demonstrated that the BET protein is highly glycosylated and is localized in patches in the dendrites and in the somata of neurons. Since the predicted structure of BET shares many similarities with the Notch ligands, this novel protein may play crucial roles in establishing the neuronal networks in the olfactory system, cerebellum, and hippocampus. BET is the first transmembrane protein containing only multiple EGF-like repeats specifically expressed in the brain. PMID:11997712

  11. Targeting transmembrane TNF-? suppresses breast cancer growth.

    PubMed

    Yu, Mingxia; Zhou, Xiaoxi; Niu, Lin; Lin, Guohong; Huang, Jin; Zhou, Wenjing; Gan, Hui; Wang, Jing; Jiang, Xiaodan; Yin, Bingjiao; Li, Zhuoya

    2013-07-01

    TNF antagonists may offer therapeutic potential in solid tumors, but patients who have high serum levels of TNF-? fail to respond to infliximab, suggesting consumption of the circulating antibody and loss of transmembrane TNF-? (tmTNF-?) on tumors by ectodomain shedding. Addressing this possibility, we developed a monoclonal antibody (mAb) that binds both full-length tmTNF-? and its N-terminal truncated fragment on the membrane after tmTNF-? processing but does not cross-react with soluble TNF-?. We documented high levels of tmTNF-? expression in primary breast cancers, lower levels in atypical hyperplasia or hyperplasia, but undetectable levels in normal breast tissue, consistent with the notion that tmTNF-? is a potential therapeutic target. Evaluations in vitro and in vivo further supported this assertion. tmTNF-? mAb triggered antibody-dependent cell-mediated cytotoxicity against tmTNF-?-expressing cells but not to tmTNF-?-negative cells. In tumor-bearing mice, tmTNF-? mAb delayed tumor growth, eliciting complete tumor regressions in some mice. Moreover, tmTNF-? mAb inhibited metastasis and expression of CD44v6, a prometastatic molecule. However, the antibody did not activate tmTNF-?-mediated reverse signaling, which facilitates tumor survival and resistance to apoptosis, but instead inhibited NF-?B activation and Bcl-2 expression by decreasing tmTNF-?-positive cells. Overall, our results established that tmTNF-? mAb exerts effective antitumor activities and offers a promising candidate to treat tmTNF-?-positive tumors, particularly in patients that are nonresponders to TNF antagonists. PMID:23794706

  12. Molecular mechanisms of intercellular communication: transmembrane signaling

    SciTech Connect

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

    1982-01-01

    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.

  13. Stapling of a 3(10)-helix with click chemistry.

    PubMed

    Jacobsen, Øyvind; Maekawa, Hiroaki; Ge, Nien-Hui; Görbitz, Carl Henrik; Rongved, Pål; Ottersen, Ole Petter; Amiry-Moghaddam, Mahmood; Klaveness, Jo

    2011-03-01

    Short peptides are important as lead compounds and molecular probes in drug discovery and chemical biology, but their well-known drawbacks, such as high conformational flexibility, protease lability, poor bioavailability and short half-lives in vivo, have prevented their potential from being fully realized. Side chain-to-side chain cyclization, e.g., by ring-closing olefin metathesis, known as stapling, is one approach to increase the biological activity of short peptides that has shown promise when applied to 3(10)- and ?-helical peptides. However, atomic resolution structural information on the effect of side chain-to-side chain cyclization in 3(10)-helical peptides is scarce, and reported data suggest that there is significant potential for improvement of existing methodologies. Here, we report a novel stapling methodology for 3(10)-helical peptides using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction in a model aminoisobutyric acid (Aib) rich peptide and examine the structural effect of side chain-to-side chain cyclization by NMR, X-ray diffraction, linear IR and femtosecond 2D IR spectroscopy. Our data show that the resulting cyclic peptide represents a more ideal 3(10)-helix than its acyclic precursor and other stapled 3(10)-helical peptides reported to date. Side chain-to-side chain stapling by CuAAC should prove useful when applied to 3(10)-helical peptides and protein segments of interest in biomedicine. PMID:21275402

  14. Deformed helix ferroelectric liquid crystal display: A new electrooptic mode in ferroelectric chiral smectic C liquid crystals

    Microsoft Academic Search

    L. A. Beresnev; V. G. Chigrinov; D. I. Dergachev; E. P. Poshidaev; J. Fünfschilling; M. Schadt

    1989-01-01

    A new electrooptic mode of operation of ferroelectric chiral smectic C liquid crystal displays (LCDs) is proposed and demonstrated. The effect, which is called the deformed helical ferroelectric (DHF) effect, is based on the deformation of the helical structure by weak electric fields. In the unbiased device the smectic layers are arranged in the bookshelf geometry with the helix axis

  15. Hydrated and Dehydrated Tertiary Interactions–Opening and Closing–of a Four-Helix Bundle Peptide

    PubMed Central

    Lignell, Martin; Tegler, Lotta T.; Becker, Hans-Christian

    2009-01-01

    Abstract The structural heterogeneity and thermal denaturation of a dansyl-labeled four-helix bundle homodimeric peptide was studied with steady-state and time-resolved fluorescence spectroscopy and with circular dichroism (CD). At room temperature the fluorescence decay of the polarity-sensitive dansyl, located in the hydrophobic core region, can be described by a broad distribution of fluorescence lifetimes, reflecting the heterogeneous microenvironment. However, the lifetime distribution is nearly bimodal, which we ascribe to the presence of two major conformational subgroups. Since the fluorescence lifetime reflects the water content of the four-helix bundle conformations, we can use the lifetime analysis to monitor the change in hydration state of the hydrophobic core of the four-helix bundle. Increasing the temperature from 9°C to 23°C leads to an increased population of molten-globule-like conformations with a less ordered helical backbone structure. The fluorescence emission maximum remains constant in this temperature interval, and the hydrophobic core is not strongly affected. Above 30°C the structural dynamics involve transient openings of the four-helix bundle structure, as evidenced by the emergence of a water-quenched component and less negative CD. Above 60°C the homodimer starts to dissociate, as shown by the increasing loss of CD and narrow, short-lived fluorescence lifetime distributions. PMID:19619472

  16. Mixed-salt effects on the conformation of a short salt-bridge-forming ? helix: A simulation study

    NASA Astrophysics Data System (ADS)

    Shen, Hao; Cheng, Wei; Zhang, Feng-Shou

    2014-02-01

    The structure of a single alanine-based ACE-AEAAAKEAAAKA-NH2 peptide in explicit aqueous solutions with mixed inorganic salts (NaCl and KCl) is investigated by using molecular simulations. The concentration of Na+, cNa+, varies from 0.0M to 1.0M, whereas the concentration of K+ is 1-cNa+. The simulated peptide is very sensitive to the change of concentration ratio between Na+ and K+. When the concentration ratio between Na+ and K+ is changed from 0.5/0.5, the structure of the peptide becomes loose or disordered. This specific phenomenon is confirmed via checking the changes of helix parameters and mapping the free energy along different coordinates. The higher normalized probability of forming direct and indirect salt bridges between residues Glu7- and Lys11+ and the smallest probability of forming ringlike structures should be responsible for the stabilized helix structure in the 0.5 Na+/0.5 K+ solution. Furthermore, a noticeable conformational transition from an extended helix to an ? helix is found in the 0.5 Na+/0.5 K+ solution, where a local ion cloud shows that some Na+ ions in the inner shells are still directly binding with the peptide, while K+ in the outer shells are moving into the inner shells, keeping the peptide in the collapsed state.

  17. The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm

    PubMed Central

    Wang, Loo Chien; Morgan, Leslie K; Godakumbura, Pahan; Kenney, Linda J; Anand, Ganesh S

    2012-01-01

    Two-component systems mediate bacterial signal transduction, employing a membrane sensor kinase and a cytoplasmic response regulator (RR). Environmental sensing is typically coupled to gene regulation. Understanding how input stimuli activate kinase autophosphorylation remains obscure. The EnvZ/OmpR system regulates expression of outer membrane proteins in response to osmotic stress. To identify EnvZ conformational changes associated with osmosensing, we used HDXMS to probe the effects of osmolytes (NaCl, sucrose) on the cytoplasmic domain of EnvZ (EnvZc). Increasing osmolality decreased deuterium exchange localized to the four-helix bundle containing the autophosphorylation site (His243). EnvZc exists as an ensemble of multiple conformations and osmolytes favoured increased helicity. High osmolality increased autophosphorylation of His243, suggesting that these two events are linked. In-vivo analysis showed that the cytoplasmic domain of EnvZ was sufficient for osmosensing, transmembrane domains were not required. Our results challenge existing claims of robustness in EnvZ/OmpR and support a model where osmolytes promote intrahelical H-bonding enhancing helix stabilization, increasing autophosphorylation and downstream signalling. The model provides a conserved mechanism for signalling proteins that respond to diverse physical and mechanical stimuli. PMID:22543870

  18. A helix-breaking mutation in the epithelial Ca2+ channel TRPV5 leads to reduced Ca2+-dependent inactivation

    PubMed Central

    Lee, Kyu Pil; Nair, Anil V.; Grimm, Christian; van Zeeland, Femke; Heller, Stefan; Bindels, René J.M.; Hoenderop, Joost G.J.

    2013-01-01

    TRPV5, a member of transient receptor potential (TRP) superfamily of ion channels, plays a crucial role in epithelial calcium transport in the kidney. This channel has a high selectivity for Ca2+ and is tightly regulated by intracellular Ca2+ concentrations. Recently it was shown that the molecular basis of deafness in varitint-waddler mouse is the result of hair cell death caused by the constitutive activity of transient receptor potential mucolipin 3 (TRPML3) channel carrying a helix breaking mutation, A419P, at the intracellular proximity of the fifth transmembrane domain (TM5). This mutation significantly elevates intracellular Ca2+ concentration and causes rapid cell death. Here we show that substituting the equivalent location in TRPV5, the M490, to proline significantly modulates Ca2+-dependent inactivation of TRPV5. The single channel conductance, time constant of inactivation (?) and half maximal inhibition constant (IC50) of TRPV5(M490P) were increased compared to TRPV5(WT). Moreover TRPV5(M490P) showed lower Ca2+ permeability. Out of different point mutations created to characterize the importance of M490 in Ca2+-dependent inactivation, only TRPV5(M490P)-expressing cells showed apoptosis and extremely altered Ca2+-dependent inactivation. In conclusion, the TRPV5 channel is susceptible for helix breaking mutations and the proximal intracellular region of TM5 of this channel plays an important role in Ca2+-dependent inactivation. PMID:21035851

  19. Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis

    PubMed Central

    Pai, Vaibhav P.; Aw, Sherry; Shomrat, Tal; Lemire, Joan M.; Levin, Michael

    2012-01-01

    Uncovering the molecular mechanisms of eye development is crucial for understanding the embryonic morphogenesis of complex structures, as well as for the establishment of novel biomedical approaches to address birth defects and injuries of the visual system. Here, we characterize change in transmembrane voltage potential (Vmem) as a novel biophysical signal for eye induction in Xenopus laevis. During normal embryogenesis, a striking hyperpolarization demarcates a specific cluster of cells in the anterior neural field. Depolarizing the dorsal lineages in which these cells reside results in malformed eyes. Manipulating Vmem of non-eye cells induces well-formed ectopic eyes that are morphologically and histologically similar to endogenous eyes. Remarkably, such ectopic eyes can be induced far outside the anterior neural field. A Ca2+ channel-dependent pathway transduces the Vmem signal and regulates patterning of eye field transcription factors. These data reveal a new, instructive role for membrane voltage during embryogenesis and demonstrate that Vmem is a crucial upstream signal in eye development. Learning to control bioelectric initiators of organogenesis offers significant insight into birth defects that affect the eye and might have significant implications for regenerative approaches to ocular diseases. PMID:22159581

  20. A gatekeeper helix determines the substrate specificity of Sjögren–Larsson Syndrome enzyme fatty aldehyde dehydrogenase

    PubMed Central

    Keller, Markus A.; Zander, Ulrich; Fuchs, Julian E.; Kreutz, Christoph; Watschinger, Katrin; Mueller, Thomas; Golderer, Georg; Liedl, Klaus R.; Ralser, Markus; Kräutler, Bernhard; Werner, Ernst R.; Marquez, Jose A.

    2014-01-01

    Mutations in the gene coding for membrane-bound fatty aldehyde dehydrogenase (FALDH) lead to toxic accumulation of lipid species and development of the Sjögren–Larsson Syndrome (SLS), a rare disorder characterized by skin defects and mental retardation. Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase. The dimeric FALDH displays a previously unrecognized element in its C-terminal region, a ‘gatekeeper’ helix, which extends over the adjacent subunit, controlling the access to the substrate cavity and helping orientate both substrate cavities towards the membrane surface for efficient substrate transit between membranes and catalytic site. Activity assays demonstrate that the gatekeeper helix is important for directing the substrate specificity of FALDH towards long-chain fatty aldehydes. The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases. Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations. PMID:25047030