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1

Structural comparison and classification of alpha-helical transmembrane domains based on helix interaction patterns.  

PubMed

Structural classification of membrane proteins is still in its infancy due to the relative paucity of available three-dimensional structures compared with soluble proteins. However, recent technological advances in protein structure determination have led to a significant increase in experimentally known membrane protein folds, warranting exploration of the structural universe of membrane proteins. Here, a new and completely membrane protein specific structural classification system is introduced that classifies alpha-helical membrane proteins according to common helix architectures. Each membrane protein is represented by a helix interaction graph depicting transmembrane helices with their pairwise interactions resulting from individual residue contacts. Subsequently, proteins are clustered according to similarities among these helix interaction graphs using a newly developed structural similarity score called HISS. As HISS scores explicitly disregard structural properties of loop regions, they are more suitable to capture conserved transmembrane helix bundle architectures than other structural similarity scores. Importantly, we are able to show that a classification approach based on helix interaction similarity closely resembles conventional structural classification databases such as SCOP and CATH implying that helix interactions are one of the major determinants of alpha-helical membrane protein folds. Furthermore, the classification of all currently available membrane protein structures into 20 recurrent helix architectures and 15 singleton proteins demonstrates not only an impressive variability of membrane helix bundles but also the conservation of common helix interaction patterns among proteins with distinctly different sequences. PMID:20552684

Fuchs, Angelika; Frishman, Dmitrij

2010-09-01

2

A Ser residue influences the structure and stability of a Pro-kinked transmembrane helix dimer.  

PubMed

When localized adjacent to a Pro-kink, Thr and Ser residues can form hydrogen bonds between their polar hydroxyl group and a backbone carbonyl oxygen and thereby modulate the actual bending angle of a distorted transmembrane ?-helix. We have used the homo-dimeric transmembrane cytochrome b(559)' to analyze the potential role of a highly conserved Ser residue for assembly and stabilization of transmembrane proteins. Mutation of the conserved Ser residue to Ala resulted in altered heme binding properties and in increased stability of the holo-protein, most likely by tolerating subtle structural rearrangements upon heme binding. The results suggest a crucial impact of an intrahelical Ser hydrogen bond in defining the structure of a Pro-kinked transmembrane helix dimer. PMID:22525600

Weber, Mathias; Tome, Lydia; Otzen, Daniel; Schneider, Dirk

2012-09-01

3

A simple method for modeling transmembrane helix oligomers.  

PubMed

We describe an effective procedure for modeling the structures of simple transmembrane helix homo-oligomers. The method differs from many previous approaches in that the only structural constraint we use to help select the correct model is the oligomerization state of the protein. The method involves the following steps: (1) perform 100-250 independent Monte Carlo energy minimizations of helix pairs to produce a large collection of well-packed structures; (2) filter the minimized structures to find those that are consistent with the expected symmetry of the oligomer; (3) cluster the structures that pass the symmetry filter; and (4) select a representative of the most populous cluster as the final prediction. We applied the method to the transmembrane helices of five proteins and compare our results to the available experimental data. Our predictions of glycophorin A, neu, the M2 channel and phospholamban resulted in a single model for each protein that agreed with the experimental results. In the case of erbB-2, however, we obtained three structurally distinct clusters of approximately equal sizes, so it was not possible to identify a clearly favored structure. This may reflect a real heterogeneity of packing modes for erbB-2, which is known to interact with different receptor subunits. Our method should be useful for obtaining structural models of transmembrane domains, improving our understanding of structure/function relationships for particular membrane proteins. PMID:12787681

Kim, Sanguk; Chamberlain, Aaron K; Bowie, James U

2003-06-13

4

Transmembrane helix-helix interactions involved in ErbB receptor signaling  

PubMed Central

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

Cymer, Florian

2010-01-01

5

Methods for Transmembrane Protein Topology and Alpha Helix Prediction  

E-print Network

Methods for Transmembrane Protein Topology and Alpha Helix Prediction Kristen Carnohan BIOC 218 to instead accurately predict the topology of transmembrane proteins is inherently useful. Any paper on the prediction of transmembrane protein topology will begin by agreeing that the problem of characterizing

6

Structure of the Integrin ?IIb Transmembrane Segment*S?  

PubMed Central

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

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

2008-01-01

7

Terminal residue hydrophobicity modulates transmembrane helix-helix interactions.  

PubMed

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

Ng, Derek P; Deber, Charles M

2014-06-17

8

Structural features of transmembrane helices.  

PubMed

A total of 160 transmembrane helices of 15 non-homologous high-resolution X-ray protein structures have been analyzed in respect of their structural features. The dihedral angles and hydrogen bonds of the helical sections that span the hydrophobic interior of the lipid bilayer have been investigated. The Ramachandran plot of protein channels and solute transporters exhibit a significant shift Delta (phi- and psi-angles) of Delta mean (+4.5 degrees and -5.4 degrees ), compared to a reference group of 151 alpha-helices of the same average length derived from water-soluble globular proteins. At the C-termini of transmembrane helices structural motifs equivalent to the Gly-caps of helices in globular proteins have been found, with two third of the transmembrane Gly-caps taking up a primary structure that is typically not found at helix termini exposed to a polar solvent. The structural particularities reported here are relevant for the three-dimensional modelling of membrane protein structures. PMID:14960323

Hildebrand, Peter Werner; Preissner, Robert; Frömmel, Cornelius

2004-02-13

9

Transmembrane helix assembly and the role of salt bridges.  

PubMed

Transmembrane helix-helix interactions mediate the folding and assembly of membrane proteins. Recognition motifs range from GxxxG and leucine zippers to polar side chains and salt bridges. Some canonical membrane proteins contain local charge clusters that are important for folding and function, and which have to be compatible with a stable insertion into the bilayer via the translocon. Recently, the electrostatic "charge zipper" has been described as another kind of assembly motif. The protein sequences exhibit a quasi-symmetrical pattern of complementary charges that can form extended ladders of salt bridges. Such segments can insert reversibly into membranes, or even translocate across them. Nature uses charge zippers in transport processes, and they can also be adapted in the design of cell-penetrating carriers. PMID:24907460

Walther, Torsten H; Ulrich, Anne S

2014-08-01

10

Implications of threonine hydrogen bonding in the glycophorin A transmembrane helix dimer.  

PubMed Central

The transmembrane helix of glycophorin A contains a seven-residue motif, LIxxGVxxGVxxT, that mediates protein dimerization. Threonine is the only polar amino acid in this motif with the potential to stabilize the dimer through hydrogen-bonding interactions. Polarized Fourier transform infrared spectroscopy is used to establish a robust protocol for incorporating glycophorin A transmembrane peptides into membrane bilayers. Analysis of the dichroic ratio of the 1655-cm(-1) amide I vibration indicates that peptides reconstituted by detergent dialysis have a transmembrane orientation with a helix crossing angle of <35 degrees. Solid-state nuclear magnetic resonance spectroscopy is used to establish high resolution structural restraints on the conformation and packing of Thr-87 in the dimer interface. Rotational resonance measurement of a 2.9-A distance between the gamma-methyl and backbone carbonyl carbons of Thr-87 is consistent with a gauche- conformation for the chi1 torsion angle. Rotational-echo double-resonance measurements demonstrate close packing (4.0 +/- 0.2 A) of the Thr-87 gamma-methyl group with the backbone nitrogen of Ile-88 across the dimer interface. The short interhelical distance places the beta-hydroxyl of Thr-87 within hydrogen-bonding range of the backbone carbonyl of Val-84 on the opposing helix. These results refine the structure of the glycophorin A dimer in membrane bilayers and highlight the complementary role of small and polar residues in the tight association of transmembrane helices in membrane proteins. PMID:11964235

Smith, Steven O; Eilers, Markus; Song, David; Crocker, Evan; Ying, Weiwen; Groesbeek, Michel; Metz, Guenter; Ziliox, Martine; Aimoto, Saburo

2002-01-01

11

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

PubMed Central

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

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

2014-01-01

12

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

ERIC Educational Resources Information Center

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…

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

2008-01-01

13

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

Microsoft Academic Search

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

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

2007-01-01

14

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

PubMed

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

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

2014-12-17

15

Second transmembrane helix (M2) and long range coupling in Ca²?-ATPase.  

PubMed

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

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

2014-11-01

16

Thermodynamic and kinetic characterization of transmembrane helix association.  

PubMed

The transient dimerization of transmembrane proteins is an important event in several cellular processes and computational methods are being increasingly used to quantify their underlying energetics. Here, we probe the thermodynamics and kinetics of a simple transmembrane dimer to understand membrane protein association. A multi-step framework has been developed in which the dimerization profiles are calculated from coarse-grain molecular dynamics simulations, followed by meso-scale simulations using parameters calculated from the coarse-grain model. The calculated value of ?Gassoc is approx. -20 kJ mol(-1) and is consistent between three methods. Interestingly, the meso-scale stochastic model reveals low dimer percentages at physiologically-relevant concentrations, despite a favorable ?Gassoc. We identify generic driving forces arising from the protein backbone and lipid bilayer and complementary factors, such as protein density, that govern self-interactions in membranes. Our results provide an important contribution in understanding membrane protein organization and linking molecular, nano-scale computational studies to meso-scale experimental data. PMID:25427292

Pawar, Aiswarya B; Deshpande, Sneha A; Gopal, Srinivasa M; Wassenaar, Tsjerk A; Athale, Chaitanya A; Sengupta, Durba

2014-12-10

17

Lessons from Free Energy Simulations of ?-Opioid Receptor Homodimers Involving the Fourth Transmembrane Helix  

PubMed Central

Several G protein-coupled receptors (GPCRs), including opioid receptors ?OR, ?OR, and ?OR, have been reported to form stable dimers or oligomers in lipid bilayers and cell membranes. This notion has been recently challenged by imaging data supporting a transient nature of GPCR association. Here we use umbrella sampling reconstructed free energies of ?OR homodimers involving the fourth transmembrane helix to predict their association constant. The results of these simulations, combined with estimates of diffusion-limited association rates, suggest a short lifetime for ?OR homodimers in the membrane, in agreement with recent trends. PMID:20617813

2010-01-01

18

Transmembrane helix 6 observed at the interface of ?2AR homodimers in blind docking studies.  

PubMed

Peptide- and protein-protein dockings were carried out on ?2-adrenergic receptor (?2AR) to confirm the presence of transmembrane helix 6 (TM6) at the interface region between two ?2AR monomers, thereby its possible role in dimerization as suggested in numerous experimental and computational studies. Initially, a portion of TM6 was modeled as a peptide consisting of 23 residues and blindly docked to ?2AR monomer using a rigid body approach. Interestingly, all highest score conformations preferred to be near TM5 and TM6 regions of the receptor. Furthermore, longer peptides generated from a whole TM region were blindly docked to ?2AR using the same rigid body approach. This yielded a total of seven docked peptides, each derived from one TM helix. Most interestingly, for each peptide, TM6 was among the most preferred binding site region in the receptor. Besides the peptide dockings, two ?2AR monomers were blindly docked to each other using a full rigid-body search of docking orientations, which yielded a total of 16,000 dimer conformations. Each dimer was then filtered according to a fitness value based on the membrane topology. Among 149 complexes that met the topology requirements, 102 conformers were composed of two monomers oriented in opposite directions, whereas in the remaining 47, the monomers were arranged in parallel. Lastly, all 149 conformers were clustered based on a root mean-squared distance value of 6 Å. In agreement with the peptide results, the clustering yielded the largest population of conformers with the highest Z-score value having TM6 at the interface region. PMID:25262920

Koroglu, Ayca; Akten, Ebru Demet

2014-09-29

19

Large Lateral Movement of Transmembrane Helix S5 Is Not Required for Substrate Access to the Active Site of Rhomboid Intramembrane Protease*  

PubMed Central

Rhomboids represent an evolutionarily ancient protease family. Unlike most other proteases, they are polytopic membrane proteins and specialize in cleaving transmembrane protein substrates. The polar active site of rhomboid protease is embedded in the membrane and normally closed. For the bacterial rhomboid GlpG, it has been proposed that one of the transmembrane helices (S5) of the protease can rotate to open a lateral gate, enabling substrate to enter the protease from inside the membrane. Here, we studied the conformational change in GlpG by solving the cocrystal structure of the protease with a mechanism-based inhibitor. We also examined the lateral gating model by cross-linking S5 to a neighboring helix (S2). The crystal structure shows that inhibitor binding displaces a capping loop (L5) from the active site but causes only minor shifts in the transmembrane helices. Cross-linking S5 and S2, which not only restricts the lateral movement of S5 but also prevents substrate from passing between the two helices, does not hinder the ability of the protease to cleave a membrane protein substrate in detergent solution and in reconstituted membrane vesicles. Taken together, these data suggest that a large lateral movement of the S5 helix is not required for substrate access to the active site of rhomboid protease. PMID:23609444

Xue, Yi; Ha, Ya

2013-01-01

20

Influence of the g- conformation of Ser and Thr on the structure of transmembrane helices.  

PubMed

In order to study the influence of Ser and Thr on the structure of transmembrane helices we have analyzed a database of helix stretches extracted from crystal structures of membrane proteins and an ensemble of model helices generated by molecular dynamics simulations. Both complementary analyses show that Ser and Thr in the g- conformation induce and/or stabilize a structural distortion in the helix backbone. Using quantum mechanical calculations, we have attributed this effect to the electrostatic repulsion between the side chain Ogamma atom of Ser and Thr and the backbone carbonyl oxygen at position i-3. In order to minimize the repulsive force between these negatively charged oxygens, there is a modest increase of the helix bend angle as well as a local opening of the helix turn preceding Ser/Thr. This small distortion can be amplified through the helix, resulting in a significant displacement of the residues located at the other side of the helix. The crystal structures of aquaporin Z and the beta(2)-adrenergic receptor are used to illustrate these effects. Ser/Thr-induced structural distortions can be implicated in processes as diverse as ligand recognition, protein function and protein folding. PMID:19766191

Deupi, Xavier; Olivella, Mireia; Sanz, Arantxa; Dölker, Nicole; Campillo, Mercedes; Pardo, Leonardo

2010-01-01

21

Predicting Transmembrane Helix Packing Arrangements using Residue Contacts and a Force-Directed Algorithm  

Microsoft Academic Search

Alpha-helical transmembrane proteins constitute roughly 30% of a typical genome and are involved in a wide variety of important biological processes including cell signalling, transport of membrane-impermeable molecules and cell recognition. Despite significant efforts to predict transmembrane protein topology, comparatively little attention has been directed toward developing a method to pack the helices together. Here, we present a novel approach

Timothy Nugent; David T. Jones

2010-01-01

22

The Effect of Point Mutations on the Free Energy of Transmembrane aaa-Helix Dimerization  

E-print Network

enable a wide range of studies of molecular association events in membrane proteins. We ®nd as transmembrane a-helices, where they may rep- resent autonomous folding domains. Energetic con- siderations

23

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

PubMed Central

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

2012-01-01

24

Characterization and sequence prediction of structural variations in ?-helix  

PubMed Central

Background The structure conservation in various ?-helix subclasses reveals the sequence and context dependent factors causing distortions in the ?-helix. The sequence-structure relationship in these subclasses can be used to predict structural variations in ?-helix purely based on its sequence. We train support vector machine(SVM) with dot product kernel function to discriminate between regular ?-helix and non-regular ?-helices purely based on the sequences, which are represented with various overall and position specific propensities of amino acids. Results We characterize the structural distortions in five ?-helix subclasses. The sequence structure correlation in the subclasses reveals that the increased propensity of proline, histidine, serine, aspartic acid and aromatic amino acids are responsible for the distortions in regular ?-helix. The N-terminus of regular ?-helix prefers neutral and acidic polar amino acids, while the C-terminus prefers basic polar amino acid. Proline is preferred in the first turn of regular ?-helix , while it is preferred to produce kinked and curved subclasses. The SVM discriminates between regular ?-helix and the rest with precision of 80.97% and recall of 88.05%. Conclusions The correlation between structural variation in helices and their sequences is manifested by the performance of SVM based on sequence features. The results presented here are useful for computational design of helices. The results are also useful for prediction of structural perturbations in helix sequence purely based on its sequence. PMID:21342550

2011-01-01

25

Transmembrane helix 1 contributes to substrate translocation and protein stability of bile acid transporter SLC10A2.  

PubMed

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) plays a critical role in the enterohepatic circulation of bile acids, as well as in cholesterol homeostasis. ASBT reclaims bile acids from the distal ileum via active sodium co-transport, in a multistep process, orchestrated by key residues in exofacial loop regions, as well as in membrane-spanning helices. Here, we unravel the functional contribution of highly conserved transmembrane helix 1 (TM1) on the hASBT transport cycle. Consecutive cysteine substitution of individual residues along the TM1 helix (Ile(29)-Gly(50)), as well as exofacial Asn(27) and Asn(28), resulted in functional impairment of ?70% of mutants, despite appreciable cell surface expression for all but G50C. Cell surface expression of G50C and G50A was rescued upon MG132 treatment as well as cyclosporine A, but not by FK506 or bile acids, suggesting that Gly(50) is involved in hASBT folding. TM1 accessibility to membrane-impermeant MTSET remains confined to the exofacial half of the helix along a single, discrete face. Substrate protection from MTSET labeling was temperature-dependent for L34C, T36C, and L38C, consistent with conformational changes playing a role in solvent accessibility for these mutants. Residue Leu(30) was shown to be critical for both bile acid and sodium affinity, while Asn(27), Leu(38), Thr(39), and Met(46) participate in sodium co-transport. Combined, our data demonstrate that TM1 plays a pivotal role in ASBT function and stability, thereby providing further insight in its dynamic transport mechanism. PMID:21646357

da Silva, Tatiana Claro; Hussainzada, Naissan; Khantwal, Chandra M; Polli, James E; Swaan, Peter W

2011-08-01

26

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

PubMed Central

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

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

2014-01-01

27

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

PubMed

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

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

2013-11-01

28

Functional investigation of transmembrane helix 3 in H?-translocating pyrophosphatase.  

PubMed

H?-translocating pyrophosphatase (H?-PPase, EC 3.6.1.1) plays an important role in acidifying vacuoles by transporting protons across membranes at the expense of pyrophosphate (PP(i)) hydrolysis. Vigna radiata H?-PPase (VrH?-PPase) contains 16 transmembrane helices (TMs). The hydrophobicity of TM3 is relatively lower than that of most other TMs, and the amino acids in this TM are highly conserved in plants. Furthermore, TM5 and -6, which are the core TMs involving in H?-PPase functions, are near TM3. It is thus proposed that TM3 is associated with H?-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrH?-PPase. Upon alanine/serine substitution, T138 and S142, whose side chains face toward the center TMs, were found to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the crucial termini of the two GxxxG-like motifs are indispensable in maintaining enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is pivotal for efficient expression. S153, M161 and A164 are critical for the K?-mediated stimulation of H?-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PP(i) hydrolysis, proton transport, expression, and K? stimulation of H?-PPase. PMID:24121627

Lee, Ching-Hung; Chen, Yen-Wei; Huang, Yun-Tzu; Pan, Yih-Jiuan; Lee, Chien-Hsien; Lin, Shih-Ming; Huang, Lin-Kun; Lo, Yueh-Yu; Huang, Yu-Fen; Hsu, Yu-Di; Yen, Shih-Chung; Hwang, Jenn-Kang; Pan, Rong-Long

2013-12-01

29

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

PubMed

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

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

2014-12-19

30

Eukaryotic CTR Copper Uptake Transporters Require Two Faces of the Third Transmembrane Domain for Helix Packing, Oligomerization, and Function*  

PubMed Central

Members of the copper uptake transporter (CTR) family from yeast, plants, and mammals including human are required for cellular uptake of the essential metal copper. Based on biochemical data, CTRs have three transmembrane domains and have been shown to oligomerize in the membrane. Among individual members of the family, there is little amino acid sequence identity, raising questions as to how these proteins adopt a common fold, oligomerize, and participate in copper transport. Using site-directed mutagenesis, tryptophan scanning, genetic complementation, subcellular localization, chemical cross-linking, and the yeast unfolded protein response, we demonstrated that at least half of the third transmembrane domain (TM3) plays a vital role in CTR structure and function. The results of our analysis showed that TM3 contains two functionally distinct faces. One face bears a highly conserved Gly-X-X-X-Gly (GG4) motif, which we showed to be essential for CTR oligomerization. Moreover, we showed that steric constraints reach past the GG4-motif itself including amino acid residues that are not conserved throughout the CTR family. A second face of TM3 contains three amino acid positions that, when mutated to tryptophan, cause predominantly abnormal localization but are still partially functional in growth complementation experiments. These mutations cluster on the face opposite to the GG4-bearing face of TM3 where they may mediate interactions with the remaining two transmembrane domains. Taken together, our data support TM3 as being buried within trimeric CTR where it plays an essential role in CTR assembly. PMID:15385536

Aller, Stephen G.; Eng, Edward T.; De Feo, Christopher J.; Unger, Vinzenz M.

2005-01-01

31

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

PubMed Central

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

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

2011-01-01

32

Structure elucidation of dimeric transmembrane domains of bitopic proteins  

PubMed Central

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

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

2010-01-01

33

Structural Role of the Conserved Cysteines in the Dimerization of the Viral Transmembrane Oncoprotein E5  

PubMed Central

The E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. This 44-residue transmembrane protein can interact with the platelet-derived growth factor receptor ?, leading to ligand-independent activation and cell transformation. For productive interaction, E5 needs to dimerize via a C-terminal pair of cysteines, though a recent study suggested that its truncated transmembrane segment can dimerize on its own. To analyze the structure of the full protein in a membrane environment and elucidate the role of the Cys-Ser-Cys motif, we produced recombinantly the wild-type protein and four cysteine mutants. Comparison by circular dichroism in detergent micelles and lipid vesicular dispersion and by NMR in trifluoroethanol demonstrates that the absence of one or both cysteines does not influence the highly ?-helical secondary structure, nor does it impair the ability of E5 to dimerize, observations that are further supported by sodium dodecylsulfate polyacrylamide gel electrophoresis. We also observed assemblies of higher order. Oriented circular dichroism in lipid bilayers shows that E5 is aligned as a transmembrane helix with a slight tilt angle, and that this membrane alignment is also independent of any cysteines. We conclude that the Cys-containing motif represents a disordered region of the protein that serves as an extra covalent connection for stabilization. PMID:20858420

Windisch, Dirk; Hoffmann, Silke; Afonin, Sergii; Vollmer, Stefanie; Benamira, Soraya; Langer, Birgid; Bürck, Jochen; Muhle-Goll, Claudia; Ulrich, Anne S.

2010-01-01

34

Contact-Induced Structure Transformation in Transmembrane Prion Propagation  

E-print Network

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

Chen, Chi-Ming

35

Structural basis for LEAFY floral switch function and similarity with helix-turn-helix proteins  

PubMed Central

The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA-binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven-helix fold that binds DNA as a cooperative dimer, forming base-specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFY's effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix-turn-helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants. PMID:18784751

Hamès, Cécile; Ptchelkine, Denis; Grimm, Clemens; Thevenon, Emmanuel; Moyroud, Edwige; Gérard, Francine; Martiel, Jean-Louis; Benlloch, Reyes; Parcy, François; Müller, Christoph W

2008-01-01

36

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

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

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

2012-01-01

37

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

PubMed

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

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

2014-11-18

38

Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7) Structure  

PubMed Central

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

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

2013-01-01

39

Structural fragment clustering reveals novel structural and functional motifs in ?-helical transmembrane proteins  

PubMed Central

Background A large proportion of an organism's genome encodes for membrane proteins. Membrane proteins are important for many cellular processes, and several diseases can be linked to mutations in them. With the tremendous growth of sequence data, there is an increasing need to reliably identify membrane proteins from sequence, to functionally annotate them, and to correctly predict their topology. Results We introduce a technique called structural fragment clustering, which learns sequential motifs from 3D structural fragments. From over 500,000 fragments, we obtain 213 statistically significant, non-redundant, and novel motifs that are highly specific to ?-helical transmembrane proteins. From these 213 motifs, 58 of them were assigned to function and checked in the scientific literature for a biological assessment. Seventy percent of the motifs are found in co-factor, ligand, and ion binding sites, 30% at protein interaction interfaces, and 12% bind specific lipids such as glycerol or cardiolipins. The vast majority of motifs (94%) appear across evolutionarily unrelated families, highlighting the modularity of functional design in membrane proteins. We describe three novel motifs in detail: (1) a dimer interface motif found in voltage-gated chloride channels, (2) a proton transfer motif found in heme-copper oxidases, and (3) a convergently evolved interface helix motif found in an aspartate symporter, a serine protease, and cytochrome b. Conclusions Our findings suggest that functional modules exist in membrane proteins, and that they occur in completely different evolutionary contexts and cover different binding sites. Structural fragment clustering allows us to link sequence motifs to function through clusters of structural fragments. The sequence motifs can be applied to identify and characterize membrane proteins in novel genomes. PMID:20420672

2010-01-01

40

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

PubMed Central

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

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

2013-01-01

41

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

PubMed Central

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

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

2006-01-01

42

Relationship between Side Chain Structure and 14-Helix Stability of ?3-Peptides in Water  

PubMed Central

Folded polymers are used in Nature for virtually every vital process. Nonnatural folded polymers, or foldamers, have the potential for similar versatility, and the design and refinement of such molecules is of considerable current interest. Here we report a complete and systematic analysis of the relationship between side chain structure and the 14-helicity of a well-studied class of foldamers, ?3-peptides, in water. Our experimental results (1) verify the importance of macrodipole stabilization for maintaining 14-helix structure, (2) provide comprehensive evidence that ?3-amino acids branched at the first side chain carbon are 14-helix-stabilizing, (3) suggest a novel role for side chain hydrogen bonding as an additional stabilizing force in ?3-peptides containing ?3-homoserine or ?3-homothreonine, and (4) demonstrate that diverse functionality can be incorporated into a stable 14-helix. Gas- and solution-phase calculations and Monte Carlo simulations recapitulate the experimental trends only in the context of oligomers, yielding insight into the mechanisms behind 14-helix folding. The 14-helix propensities of ?3-amino acids differ starkly from the ?-helix propensities of analogous ?-amino acids. This contrast informs current models for ?-helix folding, and suggests that 14-helix folding is governed by radically different biophysical forces than is ?-helix folding. The ability to modulate 14-helix structure through side chain choice will assist rational design of 14-helical ?-peptide ligands for macromolecular targets. PMID:15631466

Kritzer, Joshua A.; Tirado-Rives, Julian; Hart, Scott A.; Lear, James D.; Jorgensen, William L.; Schepartz, Alanna

2010-01-01

43

Analysis and prediction of helix-helix interactions in membrane channels and transporters.  

PubMed

Membrane proteins span a large variety of different functions such as cell-surface receptors, redox proteins, ion channels, and transporters. Proteins with functional pores show different characteristics of helix-helix packing as other helical membrane proteins. We found that the helix-helix contacts of 13 nonhomologous high-resolution structures of membrane channels and transporters are mainly accomplished by weakly polar amino acids (G > S > T > F) that preferably create contacts every fourth residue, typical for right-handed helix crossings. There is a strong correlation between the now available biological hydrophobicity scale and the propensities of the weakly polar and hydrophobic residues to be buried at helix-helix interfaces or to be exposed to the lipids in membrane channels and transporters. The polar residues, however, make no major contribution towards the packing of their transmembrane helices, and are therefore subsumed to be primarily exposed to the polar milieu during the folding process. The contact formation of membrane channels and transporters is therefore ruled by the solubility of the residues, which we suppose to be the driving force for the assembly of their transmembrane helices. By contrast, in 14 nonhomologous high-resolution structures of other membrane protein coils, also large and polar amino acids (D > S > M > Q) create characteristic contacts every 3.5th residues, which is a signature for left-handed helix crossings. Accordingly, it seems that dependent on the function, different concepts of folding and stabilization are realized for helical membrane proteins. Using a sequence-based matrix prediction method these differences are exploited to improve the prediction of buried and exposed residues of transmembrane helices significantly. When the sequence motifs typical for membrane channels and transporters were applied for the prediction of helix-helix contacts the quality of prediction rises by 16% to an average value of 76%, compared to the same approach when only single amino acid positions are taken into account. PMID:16555307

Hildebrand, Peter Werner; Lorenzen, Stephan; Goede, Andrean; Preissner, Robert

2006-07-01

44

Analysis of Helix Slow Wave Structure for High Efficiency Space TWT  

NASA Astrophysics Data System (ADS)

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

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

2009-03-01

45

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

PubMed

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

Raiber, Eun-Ang; Murat, Pierre; Chirgadze, Dimitri Y; Beraldi, Dario; Luisi, Ben F; Balasubramanian, Shankar

2015-01-01

46

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

SciTech Connect

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.

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

2011-12-22

47

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

NASA Astrophysics Data System (ADS)

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.

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

2013-03-01

48

Structural dynamics of the myosin relay helix by time-resolved EPR and FRET.  

PubMed

We have used two complementary time-resolved spectroscopic techniques, dipolar electron-electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.BeF(x)) or ADP.P(i) (ADP.AlF(4)) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF(4) was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition. PMID:19966224

Agafonov, Roman V; Negrashov, Igor V; Tkachev, Yaroslav V; Blakely, Sarah E; Titus, Margaret A; Thomas, David D; Nesmelov, Yuri E

2009-12-22

49

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

SciTech Connect

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

Zhou, H.X.; Kallenbach, N.R. (New York Univ., NY (United States)); Lyu, P.C.; Wemmer, D.E. (Univ. of California, Berkeley, CA (United States))

1994-02-09

50

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

SciTech Connect

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.

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

2011-12-31

51

Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein  

PubMed Central

Light-oxygen-voltage (LOV) domains are blue light-activated signaling modules integral to a wide range of photosensory proteins. Upon illumination, LOV domains form internal protein-flavin adducts that generate conformational changes which control effector function. Here we advance our understanding of LOV regulation with structural, biophysical, and biochemical studies of EL222, a light-regulated DNA-binding protein. The dark-state crystal structure reveals interactions between the EL222 LOV and helix-turn-helix domains that we show inhibit DNA binding. Solution biophysical data indicate that illumination breaks these interactions, freeing the LOV and helix-turn-helix domains of each other. This conformational change has a key functional effect, allowing EL222 to bind DNA in a light-dependent manner. Our data reveal a conserved signaling mechanism among diverse LOV-containing proteins, where light-induced conformational changes trigger activation via a conserved interaction surface. PMID:21606338

Nash, Abigail I.; McNulty, Reginald; Shillito, Mary Elizabeth; Swartz, Trevor E.; Bogomolni, Roberto A.; Luecke, Hartmut; Gardner, Kevin H.

2011-01-01

52

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

PubMed

Membrane-spanning segments of numerous proteins (e.g. receptor tyrosine kinases) represent a novel class of pharmacologically important targets, whose activity can be modulated by specially designed artificial peptides, the so-called interceptors. Rational construction of such peptides requires understanding of the main factors driving peptide-peptide association in lipid membranes. Here we present a new method for rapid prediction of the spatial structure of transmembrane (TM) helix-helix complexes. It is based on computer simulations in membrane-like media and subsequent refinement/validation of the results using experimental studies of TM helix dimerization in a bacterial membrane by means of the ToxR system. The approach was applied to TM fragments of the ephrin receptor A1 (EphA1). A set of spatial structures of the dimer was proposed based on Monte Carlo simulations in an implicit membrane followed by molecular dynamics relaxation in an explicit lipid bilayer. The resulting models were employed for rational design of wild-type and mutant genetic constructions for ToxR assays. The computational and the experimental data are self-consistent and provide an unambiguous spatial model of the TM dimer of EphA1. The results of this work can be further used to develop new biologically active 'peptide interceptors' specifically targeting membrane domains of proteins. PMID:20228445

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

2010-01-01

53

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

PubMed

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

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

2015-01-30

54

Structural snapshots of conformational changes in a seven-helix membrane protein  

PubMed Central

Recent advances in crystallizing integral membrane proteins have led to atomic models for the structures of several seven-helix membrane proteins, including those in the G-protein coupled receptor family. Further steps towards exploring structure-function relationships will undoubtedly involve determination of the structural changes that occur during the various stages of receptor activation and de-activation. We expect that these efforts will bear many parallels to studies of conformational changes in bacteriorhodopsin, which still remains the best-studied seven-helix membrane protein (see Table 1 for a list of selected pdb depositions). Here, we provide a brief review of some of the lessons learned, the challenges faced, and the controversies over the last decade with determining conformational changes in bacteriorhodopsin. Our hope is that this analysis will be instructive for similar structural studies, especially of other seven-helix membrane proteins, in the coming decade. PMID:19643594

Hirai, Teruhisa; Subramaniam, Sriram; Lanyi, Janos K.

2009-01-01

55

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

E-print Network

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

Bertram, Richard

56

Neutron Diffraction Studies of Fluid Bilayers with Transmembrane Proteins: Structural Consequences of the Achondroplasia Mutation  

Microsoft Academic Search

Achondroplasia, the most common form of human dwarfism, is due to a G380R mutation in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) in >97% of the studied cases. While the molecular mechanism of pathology induction is under debate, the structural consequences of the mutation have not been studied. Here we use neutron diffraction to determine the disposition

Xue Han; Mihaela Mihailescu; Kalina Hristova

2006-01-01

57

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

E-print Network

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

Snapp, Erik Lee

58

Metapopulation genetic structure and migration pathways in the land snail Helix aspersa: influence of landscape heterogeneity  

Microsoft Academic Search

The spatial genetic structuring of the land snail Helix aspersa was investigated for 32 colonies within an intensive agricultural area, the polders of the Bay of Mont-Saint-Michel (France). Given the habitat patchiness and envi- ronmental instability, the setting of H. aspersa colonies meets the broader view of a metapopulation structure. The identification of extrinsic barriers to migration and their impact

Jean-François Arnaud

2003-01-01

59

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

PubMed Central

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

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

2014-01-01

60

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

NASA Astrophysics Data System (ADS)

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

Eisenberg, David

2003-09-01

61

Structure and Dynamics of Helix0 of the N-BAR Domain in Lipid Micelles and Bilayers  

Microsoft Academic Search

Bin\\/Amphiphysin\\/Rvs-homology (BAR) domains generate and sense membrane curvature by binding the negatively charged membrane to their positively charged concave surfaces. N-BAR domains contain an N-terminal extension (helix-0) predicted to form an amphipathic helix upon membrane binding. We determined the NMR structure and nano-to-picosecond dynamics of helix-0 of the human Bin1\\/Amphiphysin II BAR domain in sodium dodecyl sulfate and dodecylphosphocholine micelles.

Christian Löw; Ulrich Weininger; Hwankyu Lee; Kristian Schweimer; Ines Neundorf; Annette G. Beck-Sickinger; Richard W. Pastor; Jochen Balbach

2008-01-01

62

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

E-print Network

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

Granada, Universidad de

63

A Conserved Stable Core Structure in the Passenger Domain b-Helix of Autotransporter Virulence Proteins  

E-print Network

, Helicobacter pylori, Pseudomonas aeruginosa, and Neissera, and are often associated with virulence.4 Each-negative bacteria, a wide variety of virulence factors are secreted via the autotransporter (AT) pathwayA Conserved Stable Core Structure in the Passenger Domain b-Helix of Autotransporter Virulence

Clark, Patricia L.

64

Structural and functional characterization of the C-terminal transmembrane region of NBCe1-A.  

PubMed

NBCe1-A and AE1 both belong to the SLC4 HCO(3)(-) transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala(800)-Lys(967). Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met(858) accessible to both biotin maleimide and TAMRA and Thr(926)-Ala(929) only to TAMRA labeling. The intracellular surface contains a highly exposed (Met(813)-Gly(828)) region and a cryptic (Met(887)-Arg(904)) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp(960). Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro(868)-Leu(967) (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1. PMID:20837482

Zhu, Quansheng; Kao, Liyo; Azimov, Rustam; Abuladze, Natalia; Newman, Debra; Pushkin, Alexander; Liu, Weixin; Chang, Connie; Kurtz, Ira

2010-11-26

65

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

PubMed Central

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

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

2014-01-01

66

Structural parameters of the myelin transmembrane proteolipid in reverse micelles.  

PubMed Central

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

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

1989-01-01

67

Metapopulation genetic structure and migration pathways in the land snail Helix aspersa : influence of landscape heterogeneity  

Microsoft Academic Search

The spatial genetic structuring of the land snail Helix aspersa was investigated for 32 colonies within an intensive agricultural area, the polders of the Bay of Mont-Saint-Michel (France). Given the habitat patchiness and environmental instability, the setting of H. aspersa colonies meets the broader view of a metapopulation structure. The identification of extrinsic barriers to migration and their impact on the

Jean-François Arnaud

2003-01-01

68

Crystal structure of the collagen triple helix model [(Pro-Pro-Gly)10]3  

PubMed Central

The first report of the full-length structure of the collagen-like polypeptide [(Pro-Pro-Gly)10]3 is given. This structure was obtained from crystals grown in a microgravity environment, which diffracted up to 1.3 ?, using synchrotron radiation. The final model, which was refined to an Rfactor of 0.18, is the highest-resolution description of a collagen triple helix reported to date. This structure provides clues regarding a series of aspects related to collagen triple helix structure and assembly. The strict dependence of proline puckering on the position inside the Pro-Pro-Gly triplets and the correlation between backbone and side chain dihedral angles support the propensity-based mechanism of triple helix stabilization/destabilization induced by hydroxyproline. Furthermore, the analysis of [(Pro-Pro-Gly)10]3 packing, which is governed by electrostatic interactions, suggests that charges may act as locking features in the axial organization of triple helices in the collagen fibrils. PMID:11790836

Berisio, Rita; Vitagliano, Luigi; Mazzarella, Lelio; Zagari, Adriana

2002-01-01

69

Crystal structure of strychninium chloride dihydrate: Hidden helix in the water/anion tape  

NASA Astrophysics Data System (ADS)

The crystal structure of strychninium chloride dihydrate SH +Cl -·2H 2O was determined. The structure is composed of strychnine herring-bone bilayer sheets with channels occupied by the hydrogen bonded anion/water tape between them. Considering different amount of water molecules in isomorphous crystal of SH +Cl -·2H 2O and previously described crystal of strychninium chloride sesquihydrate, we found that water molecules and chloride anions form a left-handed helix. Similarly, anions and water molecules in the crystal of strychnine bromide dihydrate form a left-handed helix related by the two-fold screw axis symmetry. Contrary, in the crystals of strychninium chloride sesquihydrate and SH +Cl -·2H 2O, the helices are related by only translation vector and are stabilized by one or two water bridges, respectively.

Bia?o?ska, Agata; Ciunik, Zbigniew

2005-11-01

70

Local frustration determines molecular and macroscopic helix structures.  

PubMed

Decorative domains force amyloid fibers to adopt spiral ribbon morphologies, as opposed to the more common twisted ribbon. We model the effect of decorating domains as a perturbation to the relative orientation of ? strands in a bilayered extended ?-sheet. The model consists of minimal energy assemblies of rigid building blocks containing two anisotropic interacting ellipsoids. The relative orientation of the ellipsoids dictates the morphology of the resulting assembly. Amyloid structures derived from experiment are consistent with our model, and we use magnets to demonstrate that the frustration principle is scale and system independent. In contrast to other models of amyloid, our model isolates the effect of frustration from the fundamental interactions between building blocks to reveal the frustration rather than dependence of morphology on the physical interactions. Consequently, amyloid is viewed as a discrete molecular version of the more general macroscopic frustrated bilayer that is exemplified by Bauhinia seedpods. The model supports the idea that the interactions arising from an arbitrary peptide sequence can support an amyloid structure if a bilayer can form first, which suggests that supplementary protein sequences, such as chaperones or decorative domains, could play a significant role in stabilizing such bilayers and therefore in selecting morphology during nucleation. Our model provides a foundation for exploring the effects of frustration on higher-order superstructural polymorphic assemblies that may exhibit complex functional behavior. Two outstanding examples are the systematic kinking of decorated fibers and the nested frustration of the Bauhinia seedpod. PMID:23724893

Forman, Christopher J; Fejer, Szilard N; Chakrabarti, Dwaipayan; Barker, Paul D; Wales, David J

2013-07-01

71

Shielded helix traveling wave cathode ray tube deflection structure  

DOEpatents

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.

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

1992-01-01

72

Shielded helix traveling wave cathode ray tube deflection structure  

DOEpatents

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.

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

1992-12-15

73

Transmembrane Signaling Characterized in Bacterial Chemoreceptors by Using Sulfhydryl Cross-Linking in vivo  

NASA Astrophysics Data System (ADS)

Transmembrane signaling by bacterial chemoreceptors is thought to involve conformational changes within a stable homodimer. We investigated the functional consequences of constraining movement between pairs of helices in the four-helix structure of the transmembrane domain of chemoreceptor Trg. Using a family of cysteine-containing receptors, we identified oxidation treatments for intact cells that catalyzed essentially complete sulfhydryl cross-linking at selected positions and yet left flagellar and sensory functions largely unperturbed. Constraining movement by cross-links between subunits had little effect on tactic response, but constraining movement between transmembrane segments of the monomer drastically reduced function. We deduce that transmembrane signaling requires substantial movement between transmembrane helices of a monomer but not between interacting helices across the interface between subunits.

Lee, Geoffrey F.; Lebert, Michael R.; Lilly, Angela A.; Hazelbauer, Gerald L.

1995-04-01

74

Reactive molecular dynamics study of the pH-dependent dynamic structure of ?-helix.  

PubMed

We have studied the ?-helix to random coil transition using ReaxFF reactive molecular dynamics as a function of pH. Urea binding to peptides and associated interference with backbone H-bonds and charged side chains interactions, which can both denature the helices, have been studied previously using nonreactive force fields (Topol, I. A. J. Am. Chem. Soc. 2001, 123, 6054-6060). This study reveals new proton-transfer mechanisms related to the denaturation of ?-helical structures, which cannot be captured by nonreactive molecular dynamics. In addition, we show that proton transfer between the solution and the peptide can break the ?-helix hydrogen bonds, and consequently, at extreme pHs, a significant amount of helix will unravel. We also compare the effects of temperature in the denaturation mechanism. The ReaxFF findings are in significantly better agreement with ab initio calculations than previous nonreactive force field results, indicating the relevance of the reactive component on helical loss. PMID:25365332

Golkaram, M; Shin, Y K; van Duin, A C T

2014-11-26

75

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

SciTech Connect

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.

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

2009-05-26

76

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

PubMed

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

Samoylovich, Mikhail; Talis, Alexander

2014-03-01

77

Differential transmembrane domain GXXXG motif pairing impacts major histocompatibility complex (MHC) class II structure.  

PubMed

Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II ?? heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2(+) class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2(+) I-A(k) conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2(+) versus Ia.2(-) I-A(k) class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response. PMID:24619409

Dixon, Ann M; Drake, Lisa; Hughes, Kelly T; Sargent, Elizabeth; Hunt, Danielle; Harton, Jonathan A; Drake, James R

2014-04-25

78

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

PubMed

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

Mahajan, Mukesh; Bhattacharjya, Surajit

2015-02-01

79

Structure and regulation of the cystic fibrosis transmembrane conductance regulator ( CFTR) gene in killifish: A comparative genomics approach  

Microsoft Academic Search

The structure, regulation and evolution of the cystic fibrosis transmembrane conductance regulator (CFTR) gene were characterized in common killifish (Fundulus heteroclitus). Killifish CFTR (kfCFTR) structure was conserved with other CFTR homologues, but was more compact than those of mammals. A motif in intron 1 was conserved across all teleost CFTR homologues except zebrafish, and was similar to a functionally important

T. D. Singer; K. R. Keir; M. Hinton; G. R. Scott; R. S. McKinley; P. M. Schulte

2008-01-01

80

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

PubMed Central

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

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

2014-01-01

81

PolyprOnline: polyproline helix II and secondary structure assignment database  

PubMed Central

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

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

2014-01-01

82

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

SciTech Connect

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

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

1995-01-20

83

Structural Requirements in the Transmembrane Domain of GLIC Revealed by Incorporation of Noncanonical Histidine Analogs.  

PubMed

The cyanobacterial pentameric ligand-gated ion channel GLIC, a homolog of the Cys-loop receptor superfamily, has provided useful structural and functional information about its eukaryotic counterparts. X-ray diffraction data and site-directed mutagenesis have previously implicated a transmembrane histidine residue (His234) as essential for channel function. Here, we investigated the role of His234 via synthesis and incorporation of histidine analogs and ?-hydroxy acids using in vivo nonsense suppression. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell voltage-clamp electrophysiology was used to monitor channel activity. We show that an interhelix hydrogen bond involving His234 is important for stabilization of the open state, and that the shape and basicity of its side chain are highly sensitive to perturbations. In contrast, our data show that two other His residues are not involved in the acid-sensing mechanism. PMID:25525989

Rienzo, Matthew; Lummis, Sarah C R; Dougherty, Dennis A

2014-12-18

84

Observational Evidence for a Double-Helix Structure in CMEs and Magnetic Clouds  

NASA Astrophysics Data System (ADS)

We compare recent observations of a solar eruptive prominence as seen in extreme-UV light on 30 March 2010 by the Solar Dynamics Observatory (SDO) with the multi-tube model for interplanetary magnetic clouds (Osherovich, Fainberg, Stone, Geophys. Res. Lett. 26, 2597, 1999). Our model is based on an exact analytical solution of the plasma equilibrium with magnetic force balanced by a gradient of scalar gas pressure. Topologically, this solution describes two magnetic helices with opposite magnetic polarity embedded in a cylindrical magnetic flux tube that creates magnetic flux inequality between the two helices by enhancing one helix and suppressing the other. The magnetic field in this model is continuous everywhere and has a finite magnetic energy per unit length of the tube. These configurations have been introduced as MHD bounded states (Osherovich, Soln. Dannye 5, 70, 1975). Apparently, the SDO observations depict two non-equal magnetically interacting helices described by this analytical model. We consider magnetic and thermodynamic signatures of multiple magnetic flux ropes inside the same magnetic cloud, using in situ observations. The ratio of magnetic energy density to bulk speed solar wind energy density has been defined as a solar wind quasi-invariant (QI). We analyze the structure of the QI profile to probe the topology of the internal structure of magnetic clouds. From the superposition of 12 magnetically isolated clouds observed by Ulysses, we have found that the corresponding QI is consistent with our double helix model.

Osherovich, Vladimir; Fainberg, Joseph; Webb, Alla

2013-05-01

85

Observational Evidence for Double Helix Structure in CMEs and Magnetic Clouds  

NASA Astrophysics Data System (ADS)

We compare recent observations of a solar eruptive prominence as seen in extreme UV light on March 30, 2010 by the Solar Dynamics Observatory (SDO) with the multi-tube model for interplanetary magnetic clouds (Osherovich et al., GRL 26, 2597, 1999). Our model is based on an exact analytical solution of the plasma equilibrium with magnetic force balanced by a gradient of scalar gas pressure. Topologically this solution describes two magnetic helices with equal absolute value of magnetic flux, but with opposite magnetic polarity embedded in a cylindrical magnetic flux tube which creates magnetic flux inequality between the two helices by enhancing one helix and suppressing the other. This also leads to a difference in the gas pressure deficit associated with each helix. The magnetic field in this model is continuous everywhere and has finite magnetic energy per unit length of the cylindrical tube. Such configurations have been introduced by Osherovich (Soln. Dann., 5, 70, 1975) as MHD bounded states. It appears that the SDO observations depict two non-equal magnetically interacting helices described by the ground state solution of this analytical model. We consider magnetic and thermodynamic signatures of multiple magnetic flux ropes inside the same magnetic cloud, using electron and proton in situ observations. The ratio of magnetic energy density to bulk speed solar wind energy density has been defined as a solar wind quasi-invariant (QI) (Osherovich et al. 1999). For most of the solar wind, QI << 1, but for magnetic clouds, QI has anomalously large values of more than an order of magnitude greater than that of the average solar wind. In this paper, we analyze the structure of the QI profiles for 12 clouds observed by Ulysses to probe their internal topology with results consistent with our double helix model.

Osherovich, V.; Fainberg, J.; Webb, A.

2012-12-01

86

Helix coupling  

DOEpatents

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

Ginell, W.S.

1989-04-25

87

Structural Consequences of ?-Amino Acid Preorganization in a Self-Assembling ?/?-Peptide: Fundamental Studies of Foldameric Helix Bundles  

PubMed Central

We report high-resolution crystal structures of six new ?/?-peptide foldamers that have a regular ?-residue/?-residue/?-residue (???) backbone repeat pattern. All of these foldamers were crystallized from aqueous solution, and all display four-helix bundle quaternary structure in the crystalline state. These oligomers are based on the well-studied 33-residue ?-peptide GCN4-pLI, which is an engineered derivative of the dimerization domain of GCN4, a yeast transcription factor. GCN4-pLI forms a stable tetramer in solution and crystallizes as a four-helix bundle (Harbury et al. Science 1993, 262, 1401–1407). Previously we described a foldamer (designated 1 here) that was generated from GCN4-pLI by replacing every third ?-amino acid residue with the homologous ?3-amino acid residue; this ??? oligomer retains the side chain sequence of the original ?-peptide, but the backbone contains 11 additional CH2 units, which are evenly distributed (Horne et al. Proc. Natl. Acad. Sci. USA 2008, 105, 9151–9156). Despite the expanded backbone, 1 was found to retain the ability to form a tetrameric quaternary structure in which the individual molecules adopt an ?-helix-like conformation. Here we compare nine analogues of 1 that have the same ??? backbone but in which one or more of the flexible ?3-amino acid residues is/are replaced with an analogous cyclic ?-residue. The motivation for ?3?cyclic replacements is to enhance conformational stability; however, a crystal structure of the one previously reported example (designated 2 here) revealed a “stammer” distortion of the helix-bundle architecture relative to 1. The results reported here suggest that the stammer is a peculiarity of 2, because all six of the new ?/?-peptides display undistorted four-helix bundle quaternary structures. More broadly, our results indicate that ?3?cyclic replacements are generally well-accommodated in helix-bundle quaternary structure, but that such replacements can be destabilizing in certain instances. PMID:20718422

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

2010-01-01

88

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

PubMed Central

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

Studer, Gabriel; Biasini, Marco; Schwede, Torsten

2014-01-01

89

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

PubMed Central

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 Å and its C-terminal DNA-binding domain at 1.7 Å 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 (??)4 topology instead of the canonical (??)5 fold observed in other response regulators. The linker region adopts a unique conformation which 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 ?10 which provides 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 ?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. PMID:18353359

Wisedchaisri, Goragot; Wu, Meiting; Sherman, David R.; Hol, Wim G. J.

2008-01-01

90

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

Microsoft Academic Search

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,

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

1999-01-01

91

Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface  

PubMed Central

Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R*) and catalyze GDP/GTP exchange in the ?-subunit of intracellular G proteins (G???). Kinetic analysis of transducin (Gt???) activation shows that an intermediary R*·Gt???·GDP complex is formed that precedes GDP release and formation of the nucleotide-free R*·G protein complex. Based on this reaction sequence, we explore the dynamic interface between the proteins during formation of these complexes. We start from the R* conformation stabilized by a Gt? C-terminal peptide (G?CT) obtained from crystal structures of the GPCR opsin. Molecular modeling allows reconstruction of the fully elongated C-terminal ?-helix of Gt? (?5) and shows how ?5 can be docked to the open binding site of R*. Two modes of interaction are found. One of them – termed stable or S-interaction – matches the position of the G?CT peptide in the crystal structure and reproduces the hydrogen-bonding networks between the C-terminal reverse turn of G?CT and conserved E(D)RY and NPxxY(x)5,6F regions of the GPCR. The alternative fit – termed intermediary or I-interaction – is distinguished by a tilt (42°) and rotation (90°) of ?5 relative to the S-interaction and shows different ?5 contacts with the NPxxY(x)5,6F region and the second cytoplasmic loop of R*. From the 2 ?5 interactions, we derive a “helix switch” mechanism for the transition of R*·Gt???·GDP to the nucleotide-free R*·G protein complex that illustrates how ?5 might act as a transmission rod to propagate the conformational change from the receptor-G protein interface to the nucleotide binding site. PMID:19541654

Scheerer, Patrick; Heck, Martin; Goede, Andrean; Park, Jung Hee; Choe, Hui-Woog; Ernst, Oliver P.; Hofmann, Klaus Peter; Hildebrand, Peter W.

2009-01-01

92

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

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

93

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

E-print Network

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 ...

Kim, Taehoon

2011-12-31

94

Solution Structure of an Alternate Conformation of Helix 27 from Escherichia coli 16S rRNA  

E-print Network

Solution Structure of an Alternate Conformation of Helix 27 from Escherichia coli 16S rRNA Meredith Newby Spano,* Nils G. Walter Department of Chemistry, University of Michigan, 930 N. University Ave ribosomal subunit, comprising nucleotides (nt) 885-912 in E. coli 16S ribosomal (r)RNA (Figure 1A

Walter, Nils G.

95

Functional states and fine structure of the contractile apparatus of the penis retractor muscle (PRM) of Helix pomatia L  

Microsoft Academic Search

The ultrastructure of the isolated glycerinated penis retractor muscle (PRM) of Helix pomatia was investigated. The diameter distributions of thick myofilaments from fibre cross sections in the relaxed, phasic contracted, tonic contracted, and in the catch states show that a characteristic filament spectrum is formed in the catch state and its preceding active state. The significant structural differences are discussed

Rudolf W. Wabnitz

1975-01-01

96

Predicting residue and helix contacts in membrane proteins  

NASA Astrophysics Data System (ADS)

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.

Fuchs, Angelika; Kirschner, Andreas; Frishman, Dmitrij

97

Phosphorylation by cAMP-dependent Protein Kinase Modulates the Structural Coupling Between the Transmembrane and Cytosolic Domains of Phospholamban  

SciTech Connect

We have used frequency-domain fluorescence spectroscopy to investigate the structural linkage between the transmembrane and cytosolic domains of the regulatory protein phospholamban (PLB). Using an engineered PLB having a single cysteine (Cys24) derivatized with the fluorophore N-(1-pyrenyl) maleimide (PMal), we have used fluorescence resonance energy transfer (FRET) to measure the average spatial separation and conformational heterogeneity between PMal bound to Cys24 in the transmembrane domain and Tyr6 in the cytosolic domain near the amino-terminus of PLB. In these measurements, PMal serves as an FRET donor and Tyr6 serves as a FRET acceptor following its nitration by tetranitromethane. The native structure of PLB is retained following site-directed mutagenesis and chemical modification, as indicated by the ability of the derivatized PLB to fully regulate the Ca-ATPase following their co-reconstitution. To assess how phosphorylation modulates the structure of PLB, FRET measurements were made following reconstitution of PLB in membrane vesicles made from lipids extracted from sarcoplasmic reticulum vesicles in the absence of the Ca-ATPase. We find that the cytosolic domain of PLB assumes a wide range of structures relative to the transmembrane sequence, consistent with other structural data indicating the presence of a flexible hinge region between the transmembrane and cytosolic domains of PLB. Phosphorylation of Ser16 by PKA results in almost a two-fold decrease in conformational heterogeneity, suggesting a stabilization of the hinge region of PLB possibly through an electrostatic-linkage between phosphoserine16 and Arg13. These results suggest that the stabilization of the structure of PLB following phosphorylation of Ser16 is part of a switching mechanism, which functions to alter binding interactions between PLB and the nucleotide-binding domain of the Ca-ATPase that modulates enzyme inhibition.

Li, Jinhui; Bigelow, Diana J.; Squier, Thomas C.

2003-09-16

98

Structure, Vol. 13, 849855, June, 2005, 2005 Elsevier Ltd All rights reserved. DOI 10.1016/j.str.2005.03.010 The Helix Dipole: Screened Out?  

E-print Network

and G. Matthias Ullmann1,2, * effective dipole of a helix. The screening occurs due to1 IWR the effective dipole moment is unknown.2 Structural Biology/Bioinformatics For solvent-exposed helical bundles gested that the helix dipole plays little or no role in sta-95447 Bayreuth bilizing the observed bundle

Ullmann, G. Matthias

99

Utilizing the GAAA tetraloop/receptor to facilitate crystal packing and structure determination of a CUG RNA helix  

PubMed Central

Myotonic dystrophy type 1 (DM1) is a microsatellite expansion disorder caused by the aberrant expansion of CTG repeats in the 3’ untranslated region of the DMPK gene. When transcribed, the toxic RNA CUG repeats sequester RNA binding proteins, which leads to disease symptoms. The expanded CUG repeats can adopt a double-stranded structure, and targeting this helix is a therapeutic strategy for DM1. In order to better understand the 5’CUG/3’GUC motif, and how it may interact with proteins and small molecules, we designed a short CUG helix attached to a GAAA tetraloop/receptor in order to facilitate crystal packing. Here we report the highest resolution structure (1.95 Å) to date of a GAAA tetraloop/receptor and the CUG helix it was used to crystallize. Within the CUG helix, we identify two different forms of non-canonical U-U pairs and reconfirm that CUG repeats are essentially A-form. An analysis of all non-canonical U-U pairs in the context of CUG repeats revealed six different classes of conformations that the non-canonical U-U pairs are able to adopt. PMID:23025897

Coonrod, Leslie A.; Lohman, Jeremy R.; Berglund, J. Andrew

2012-01-01

100

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

PubMed Central

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

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

2014-01-01

101

Structural and Functional Analysis of Transmembrane Segment IV of the Salt Tolerance Protein Sod2*  

PubMed Central

Sod2 is the plasma membrane Na+/H+ exchanger of the fission yeast Schizosaccharomyces pombe. It provides salt tolerance by removing excess intracellular sodium (or lithium) in exchange for protons. We examined the role of amino acid residues of transmembrane segment IV (TM IV) (126FPQINFLGSLLIAGCITSTDPVLSALI152) in activity by using alanine scanning mutagenesis and examining salt tolerance in sod2-deficient S. pombe. Two amino acids were critical for function. Mutations T144A and V147A resulted in defective proteins that did not confer salt tolerance when reintroduced into S. pombe. Sod2 protein with other alanine mutations in TM IV had little or no effect. T144D and T144K mutant proteins were inactive; however, a T144S protein was functional and provided lithium, but not sodium, tolerance and transport. Analysis of sensitivity to trypsin indicated that the mutations caused a conformational change in the Sod2 protein. We expressed and purified TM IV (amino acids 125–154). NMR analysis yielded a model with two helical regions (amino acids 128–142 and 147–154) separated by an unwound region (amino acids 143–146). Molecular modeling of the entire Sod2 protein suggested that TM IV has a structure similar to that deduced by NMR analysis and an overall structure similar to that of Escherichia coli NhaA. TM IV of Sod2 has similarities to TM V of the Zygosaccharomyces rouxii Na+/H+ exchanger and TM VI of isoform 1 of mammalian Na+/H+ exchanger. TM IV of Sod2 is critical to transport and may be involved in cation binding or conformational changes of the protein. PMID:23836910

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

2013-01-01

102

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

PubMed

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 Ca(2+) and acts as a sensor for ATP, Ca(2+), and bicarbonate/CO2/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

Steegborn, Clemens

2014-12-01

103

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

PubMed

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

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

2000-07-20

104

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

PubMed

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

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

2006-07-21

105

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

SciTech Connect

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

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

2009-05-26

106

Effects of core-packing on the structure, function, and mechanics of a four-helix-bundle protein ROP.  

PubMed

The effects of core-packing on the structure, function and mechanics of the RNA-binding 4-helix-bundle Rop have been studied by molecular dynamics simulations. The structural, dynamical and geometrical properties of the Rop homodimer, (formed by the antiparallel juxtaposition of two helix-turn-helix motifs), have been compared with those of three protein variants described by Munson et al. (Protein Sci, 5:1584-1593, 1996), where the core of the native protein has been systematically repacked using a two-amino acid alphabet: Ala(2)Leu(2)-8, Ala(2)Leu(2)-8-rev, and Leu(2)Ala(2)-8. The results showed that it was possible to readily distinguish the inactive protein Leu(2)Ala(2)-8 from the other functionally active systems based on tertiary and quaternary structure criteria. Structural properties such as native secondary structure content did not correlate with biological activity. Biological activity was related in part to the relative arrangement of the residues within the binding site. But, more global aspects, related to the overall topology of the helical bundle, accounted for the small functional differences between Ala(2)Leu(2)-8 and Ala(2)Leu(2)-8-rev. Mechanically, the 4-helix-bundle absorbed core mutations by altering the local structure at the sequence termini and in the turns that join the two helices of each monomer, and by changing the overall orientation and separation of the extremely rigid helices. Proteins 1999;36:436-446. PMID:10450085

Ceruso, M A; Grottesi, A; Di Nola, A

1999-09-01

107

Structure of rodent helix-destabilizing protein revealed by cDNA cloning.  

PubMed

A cDNA library of newborn rat brain poly(A+) RNA in lambda gt 11 was screened with a synthetic oligonucleotide probe corresponding to a five amino acid sequence in the N-terminal region of the calf helix-destabilizing protein, UP1. Six positive phage were isolated after testing 2 X 10(5) recombinants, and each phage was plaque purified. Four of these phage clones were positive with a second oligonucleotide probe corresponding to a 5 amino acid sequence in the C-terminal region of calf UP1; one of the clones positive with both probes was selected for detailed study. This phage, designated lambda HDP-182, contained a 1706-base pair cDNA insert corresponding to an mRNA with a poly(A) sequence at the 3' terminus and a single open reading frame starting 63 bases from the 5' terminus and extending 988 bases. The 3' untranslated region of the mRNA contained 718 bases, including an AAUAAA signal 21 bases from the poly(A) sequence and a 16-residue poly(U) sequence flanked on each side by oligonucleotide repeats. Primer extension analysis of newborn rat brain poly(A+) RNA suggested that the cDNA insert in lambda HDP-182 was full length except for about 35 nucleotide residues missing from the 5' end untranslated region, and Northern blot analysis revealed one relatively abundant mRNA species of approximately the same size as the cDNA insert. The 988-residue open reading frame in the cDNA predicted a 34,215-dalton protein of 320 amino acids. Residues 2 through 196 of this rat protein are identical to the 195-residue sequence of the calf helix-destabilizing protein, UP1. The 124-amino acid sequence in the C-terminal portion of the 34,215-dalton protein is not present in purified calf UP1. This 124-residue sequence has unusual amino acid content in that it is 11% asparagine, 15% serine, and 40% glycine and consists of 16 consecutive oligopeptide repeats. Computer-derived secondary structure predictions for the 34,215-dalton protein revealed two distinct domains consisting of residues 1 through approximately 196 and residues approximately 197 to 320, respectively. PMID:3005291

Cobianchi, F; SenGupta, D N; Zmudzka, B Z; Wilson, S H

1986-03-15

108

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

PubMed Central

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

Pabuwal, Vagmita; Li, Zhijun

2010-01-01

109

Structural regularities of helicoidally-like biopolymers in the framework of algebraic topology: II. ?-Helix and DNA structures  

NASA Astrophysics Data System (ADS)

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°. The arrangement of atoms in particular positions of this starting aggregation allows one to obtain a model of the ?-helix. This apparatus makes it possible to determine a priori the symmetry parameters of DNA double helices.

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

2013-09-01

110

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

SciTech Connect

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.

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

1991-06-25

111

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

Microsoft Academic Search

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

Renate Gessmann; Kyriacos Petratos

2003-01-01

112

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

NASA Technical Reports Server (NTRS)

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

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

1982-01-01

113

Toward beta-peptide tertiary structure: self-association of an amphiphilic 14-helix in aqueous solution.  

PubMed

A major frontier in foldamer research is creation of unnatural oligomers that adopt discrete tertiary structures; at present, only biopolymers are known to fold into such compact conformations. We report an initial step toward helix-bundle tertiary structure in the beta-peptide realm by showing that a 10-residue beta-peptide designed to adopt an amphiphilic helical conformation forms small soluble aggregates in water. Sedimentation equilibrium data indicate that the aggregated state falls in the tetramer-hexamer size range. [structure: see text] PMID:11720580

Raguse, T L; Lai, J R; LePlae, P R; Gellman, S H

2001-11-29

114

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

SciTech Connect

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

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

2011-09-28

115

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

PubMed Central

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

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

2012-01-01

116

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

Microsoft Academic Search

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

Cheng-fang Fu; Zhao Bo

2011-01-01

117

Mutation studies of Ser7.39 and Ser2.60 in the human CB1 cannabinoid receptor: evidence for a serine-induced bend in CB1 transmembrane helix 7.  

PubMed

Ligands of structurally diverse natures are able to bind at the CB(1) cannabinoid receptor, suggesting the existence of multiple binding sites on the receptor. Modeling studies have implicated Ser2.60(173) and Ser7.39(383) as possible interaction site(s) for CB(1) agonists. To test the importance of these residues for receptor recognition, recombinant human CB(1) receptors, stably expressed in human embryonic kidney 293 cells, were used to investigate the consequences of mutating Ser2.60 (to S2.60A) or Ser7.39 (to S7.39A) in radioligand binding and guanosine 5'-3-O-(thio)triphosphate functional assays. The S7.39A mutant resulted in a total ablation of [(3)H](-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP55,940) high-affinity binding. However, [(3)H](R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55,212-2) binding properties at S7.39A were comparable with those of the wild-type (WT) receptor. The binding affinity of (-)-11beta-hydroxy-3-(1',1'-dimethylheptyl)hexahydrocannabinol (AM4056) and (-)-11-hydroxydimethylheptyl-Delta(8)-tetrahydrocannabinol (HU210) were drastically reduced (50- to 100-fold) at the S7.39A mutant. Likewise, the EC(50) for HU210 and AM4056-mediated activation of the S7.39A receptor was increased by >200-fold. In contrast, the binding affinity and potency of WIN55,212-2, CP55,940, HU210, and AM4056 were unaltered at the S2.60A mutant compared with WT human CB(1) receptors. These results clearly suggest that Ser7.39, but not Ser2.60, plays a crucial role in mediating ligand specific interactions for CP55,940, HU210, and AM4056 at the human CB(1) receptor. Our modeling studies predict that Ser7.39 in a g-chi1 conformation may induce a helix bend in TMH7 that provides docking space for CP55,940 binding; the S7.39A mutation may alter this binding space, precluding CP55,940 binding. PMID:17384224

Kapur, Ankur; Hurst, Dow P; Fleischer, Daniel; Whitnell, Rob; Thakur, Ganesh A; Makriyannis, Alexandros; Reggio, Patricia H; Abood, Mary E

2007-06-01

118

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

NASA Astrophysics Data System (ADS)

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.

Javidpour, Leili; Sahimi, Muhammad

2011-09-01

119

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

PubMed Central

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

2011-01-01

120

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

PubMed Central

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

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

2013-01-01

121

Deletion of ?-Strand and ?-Helix Secondary Structure in Normal Prion Protein Inhibits Formation of Its Protease-Resistant Isoform  

PubMed Central

A fundamental event in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conversion of a normal, proteinase K-sensitive, host-encoded protein, PrP-sen, into its protease-resistant isoform, PrP-res. During the formation of PrP-res, PrP-sen undergoes conformational changes that involve an increase of ?-sheet secondary structure. While previous studies in which PrP-sen deletion mutants were expressed in transgenic mice or scrapie-infected cell cultures have identified regions in PrP-sen that are important in the formation of PrP-res, the exact role of PrP-sen secondary structures in the conformational transition of PrP-sen to PrP-res has not yet been defined. We constructed PrP-sen mutants with deletions of the first ?-strand, the second ?-strand, or the first ?-helix and tested whether these mutants could be converted to PrP-res in both scrapie-infected neuroblastoma cells (Sc+-MNB cells) and a cell-free conversion assay. Removal of the second ?-strand or the first ?-helix significantly altered both processing and the cellular localization of PrP-sen, while deletion of the first ?-strand had no effect on these events. However, all of the mutants significantly inhibited the formation of PrP-res in Sc+-MNB cells and had a greatly reduced ability to form protease-resistant PrP in a cell-free assay system. Thus, our results demonstrate that deletion of the ?-strands and the first ?-helix of PrP-sen can fundamentally affect PrP-res formation and/or PrP-sen processing. PMID:11581371

Vorberg, Ina; Chan, Kaman; Priola, Suzette A.

2001-01-01

122

Effect of polarization on the stability of a helix dimer  

NASA Astrophysics Data System (ADS)

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

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

2011-01-01

123

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

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

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

2014-01-01

124

Response of helical structures to high speed pulses or when can a helix be called an inductor  

SciTech Connect

Vacuum and dielectric cored loosely wound helices are widely used in pulse power systems. In one extreme, when the applied pulse length is shorter then the transit time in the helix, helices are used as transit time isolators, to lead signals from high voltage terminals to ground. In the other extreme, for pulse durations that are much longer than the transit time, helices act as conventional inductors and are used for high frequency isolation or as shields for trigger, signal or gas leads to pulse power components. In either case, the use of helices in the presence of fast pulses requires an understanding of their behavior in regimes where time delays associated with wave propagation in the helix are not negligible when compared to the length or risetime of the applied pulse and hence their description as lumped elements is no longer correct. This paper describes the results of analytical approximations, circuit simulations, 3-D time dependent electromagnetic modeling as well as laboratory measurements on helices surrounded by conducting structures. On the basis of these investigations their suitability as voltage measuring devices will be discussed.

Di Capua, M.S.; Molau, N.E.; Wheeler, P.C.

1987-06-29

125

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

PubMed Central

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

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

2014-01-01

126

Structural Dynamics Of The S4 Voltage-Sensor Helix In Lipid Bilayers Lacking Lipid Phosphates  

PubMed Central

Voltage-dependent K+ (Kv) channels require lipid phosphates for functioning. The S4 helix, which carries the gating charges in the voltage-sensing domain (VSD), inserts into membranes while being stabilized by a protein-lipid interface in which lipid phosphates play an essential role. To examine the physical basis of the protein-lipid interface in the absence of lipid phosphates, we performed molecular dynamics (MD) simulations of a KvAP S4 variant (S4mut) in bilayers with and without lipid phosphates. We find that in dioleoyltrimethylammoniumpropane (DOTAP) bilayers lacking lipid phosphates, the gating charges are solvated by anionic counterions and, hence, lack the bilayer support provided by phosphate-containing palmitoyloleoylglycerophosphocholine (POPC) bilayers. The result is a water-permeable bilayer with a significantly smaller deformations around the peptide. Together, these results provide an explanation for the non-functionality of VSDs in terms of a destabilizing protein-lipid interface. PMID:21692541

Andersson, Magnus; Freites, J. Alfredo; Tobias, Douglas J.; White, Stephen H.

2011-01-01

127

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

NASA Astrophysics Data System (ADS)

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

Kundu, Sourav; Karmakar, S. N.

2014-03-01

128

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

SciTech Connect

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.

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

2009-11-01

129

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

PubMed Central

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

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

2014-01-01

130

PoreWalker: A Novel Tool for the Identification and Characterization of Channels in Transmembrane Proteins from Their Three-Dimensional Structure  

PubMed Central

Transmembrane channel proteins play pivotal roles in maintaining the homeostasis and responsiveness of cells and the cross-membrane electrochemical gradient by mediating the transport of ions and molecules through biological membranes. Therefore, computational methods which, given a set of 3D coordinates, can automatically identify and describe channels in transmembrane proteins are key tools to provide insights into how they function. Herein we present PoreWalker, a fully automated method, which detects and fully characterises channels in transmembrane proteins from their 3D structures. A stepwise procedure is followed in which the pore centre and pore axis are first identified and optimised using geometric criteria, and then the biggest and longest cavity through the channel is detected. Finally, pore features, including diameter profiles, pore-lining residues, size, shape and regularity of the pore are calculated, providing a quantitative and visual characterization of the channel. To illustrate the use of this tool, the method was applied to several structures of transmembrane channel proteins and was able to identify shape/size/residue features representative of specific channel families. The software is available as a web-based resource at http://www.ebi.ac.uk/thornton-srv/software/PoreWalker/. PMID:19609355

Thornton, Janet M.

2009-01-01

131

Sequence and conformational preferences at termini of ?-helices in membrane proteins: role of the helix environment.  

PubMed

?-helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These ?-helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C-termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze ?-helices in a high-resolution dataset of integral ?-helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C-termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near-helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. PMID:25257385

Shelar, Ashish; Bansal, Manju

2014-12-01

132

Structure and regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in killifish: A comparative genomics approach.  

PubMed

The structure, regulation and evolution of the cystic fibrosis transmembrane conductance regulator (CFTR) gene were characterized in common killifish (Fundulus heteroclitus). Killifish CFTR (kfCFTR) structure was conserved with other CFTR homologues, but was more compact than those of mammals. A motif in intron 1 was conserved across all teleost CFTR homologues except zebrafish, and was similar to a functionally important site in human CFTR. The sequence of the CFTR promoter was highly conserved across nine species within the genus Fundulus, but contained additional glucocorticoid responsive elements in seawater species. The promoters of the seawater species also contained a putative osmotic responsive element that differed by a single base in the freshwater species. The kfCFTR promoter was only active in cell lines that express the endogenous CFTR gene. Transcription from the kfCFTR promoter was unaffected by application of dexamethasone or cortisol in cell culture, but increased by 1.5-fold in response to high osmolarity. Cortisol injection in vivo increased CFTR mRNA but there was no increase in luciferase expression driven by the kfCFTR promoter in transiently transgenic fish. Administration of the cortisol blocker RU486 resulted in a significant reduction in luciferase activity driven by the kfCFTR promoter in vivo. PMID:20483217

Singer, T D; Keir, K R; Hinton, M; Scott, G R; McKinley, R S; Schulte, P M

2008-06-01

133

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

PubMed Central

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

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

2014-01-01

134

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

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

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

2014-01-01

135

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

NASA Astrophysics Data System (ADS)

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.

Straub, John

2013-03-01

136

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

PubMed

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

Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

2014-05-01

137

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

PubMed Central

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

2014-01-01

138

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

PubMed Central

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

Marko, John F.; Neukirch, Sébastien

2014-01-01

139

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

PubMed

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

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

2014-05-01

140

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

PubMed Central

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

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

2014-01-01

141

Structural change in a B-DNA helix with hydrostatic pressure  

PubMed Central

Study of the effects of pressure on macromolecular structure improves our understanding of the forces governing structure, provides details on the relevance of cavities and packing in structure, increases our understanding of hydration and provides a basis to understand the biology of high-pressure organisms. A study of DNA, in particular, helps us to understand how pressure can affect gene activity. Here we present the first high-resolution experimental study of B-DNA structure at high pressure, using NMR data acquired at pressures up to 200 MPa (2 kbar). The structure of DNA compresses very little, but is distorted so as to widen the minor groove, and to compress hydrogen bonds, with AT pairs compressing more than GC pairs. The minor groove changes are suggested to lead to a compression of the hydration water in the minor groove. PMID:18515837

Wilton, David J.; Ghosh, Mahua; Chary, K. V. A.; Akasaka, Kazuyuki; Williamson, Mike P.

2008-01-01

142

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

SciTech Connect

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.

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

2014-10-02

143

The transition of alpha-helix to beta-structure of poly(L-lysine) induced by phosphatidic acid vesicles and its kinetics at alkaline pH.  

PubMed

Static and dynamic circular dichroism (CD) measurements were carried out for poly(L-lysine) in suspensions of dilauroylphosphatidic acid (DLPA) vesicles at alkaline pH (8-11.5). The static experiments demonstrated that the alpha-helix of poly(L-lysine) induced by deprotonation in alkaline solutions is transformed to beta-structure by the addition of DLPA vesicles. Stopped-flow CD measurements for such order-to-order transition revealed that the rate determining step is the unfolding process of alpha-helix to random coil. Previously, we have reported the conformational change of poly(L-lysine) induced by DLPA vesicles at neutral pH, where the beta-structure transition from random coil was observed. Thus two types of transition of poly(L-lysine) are observed depending on bulk pH, i.e., from random coil to beta-structure and from alpha-helix to beta-structure. So far the phospholipid-induced conformations of poly(L-lysine) were interpreted in terms of counterbalance between the positively charged terminals of the lysyl chains and the negative headgroups of the phospholipid in vesicle. However, present work indicates the direct interaction other than electrostatic interaction between the lysyl chain and phosphate groups of the lipid. PMID:8155666

Fukushima, K; Sakamoto, T; Tsuji, J; Kondo, K; Shimozawa, R

1994-04-20

144

Melatonin and 5-methoxytryptophol (5ML) in nervous and\\/or neurosensory structures of a gastropod mollusc ( Helix aspersa maxima): synthesis and diurnal rhythms  

Microsoft Academic Search

Daily patterns of melatonin and 5-methoxytryptophol (5-ML) concentrations and of aryl alkylamine N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities have been measured in the cerebroid ganglions, visceral ganglions, and ocular tentacles of the gastropod mollusc Helix aspersa maxima. Melatonin concentrations are very low in all the studied structures, except a small peak at the end of the night in the cerebroid

A. Blanc; B. Vivien-Roels; P. Pévet; J. Attia; B. Buisson

2003-01-01

145

Helix-helix packing and interfacial pairwise interactions of residues in membrane proteins.  

PubMed

Helix-helix packing plays a critical role in maintaining the tertiary structures of helical membrane proteins. By examining the overall distribution of voids and pockets in the transmembrane (TM) regions of helical membrane proteins, we found that bacteriorhodopsin and halorhodopsin are the most tightly packed, whereas mechanosensitive channel is the least tightly packed. Large residues F, W, and H have the highest propensity to be in a TM void or a pocket, whereas small residues such as S, G, A, and T are least likely to be found in a void or a pocket. The coordination number for non-bonded interactions for each of the residue types is found to correlate with the size of the residue. To assess specific interhelical interactions between residues, we have developed a new computational method to characterize nearest neighboring atoms that are in physical contact. Using an atom-based probabilistic model, we estimate the membrane helical interfacial pairwise (MHIP) propensity. We found that there are many residue pairs that have high propensity for interhelical interactions, but disulfide bonds are rarely found in the TM regions. The high propensity pairs include residue pairs between an aromatic residue and a basic residue (W-R, W-H, and Y-K). In addition, many residue pairs have high propensity to form interhelical polar-polar atomic contacts, for example, residue pairs between two ionizable residues, between one ionizable residue and one N or Q. Soluble proteins do not share this pattern of diverse polar-polar interhelical interaction. Exploratory analysis by clustering of the MHIP values suggests that residues similar in side-chain branchness, cyclic structures, and size tend to have correlated behavior in participating interhelical interactions. A chi-square test rejects the null hypothesis that membrane protein and soluble protein have the same distribution of interhelical pairwise propensity. This observation may help us to understand the folding mechanism of membrane proteins. PMID:11518538

Adamian, L; Liang, J

2001-08-24

146

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

PubMed Central

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

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

2002-01-01

147

Exploration of the Transition State for Tertiary Structure Formation between an RNA Helix and a  

E-print Network

into the pre-folded core of the Tetrahymena group I ribozyme exemplifies the formation of tertiary interactions in the context of a complex, structured RNA. We have applied F-analysis to P1 docking, which compares the effects of freedom between the P1 duplex and the ribozyme core accelerates docking, suggest a model in which

Das, Rhiju

148

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

SciTech Connect

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.

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

149

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

PubMed Central

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

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

2013-01-01

150

Entropically Driven Helix Formation  

Microsoft Academic Search

The helix is a ubiquitous motif for biopolymers. We propose a heuristic, entropically based model that predicts helix formation in a system of hard spheres and semiflexible tubes. We find that the entropy of the spheres is maximized when short stretches of the tube form a helix with a geometry close to that found in natural helices. Our model could

Yehuda Snir; Randall D. Kamien

2005-01-01

151

Detecting the Conformational Change of Transmembrane Signaling in a Bacterial Chemoreceptor by Measuring Effects on Disulfide Cross-Linking in vivo  

NASA Astrophysics Data System (ADS)

Transmembrane signaling by bacterial chemoreceptors is thought to involve relative movement among the four transmembrane helices of the homodimer. We assayed that movement by measuring effects of ligand occupancy on rates of oxidative cross-linking between cysteines introduced into neighboring helices of the transmembrane domain of chemoreceptor Trg from Escherichia coli. Measurements were done on chemoreceptors in their native environment, intact cells that were motile and chemotactically responsive. Receptor occupancy did not appear to cause drastic rearrangement of the four-helix structure since, among 67 cysteine pairs tested, the same 19 exhibited oxidative cross-linking in the presence or absence of saturating chemoattractant. However, occupancy did cause subtle changes that were detected as effects on rates of cross-linking. Among the seven disulfides appropriate for measurements of initial rates of formation, ligand occupancy had significant and different effects on all three cross-links that connected the two helices within a subunit but had minimal effects on the four that spanned the packing interface between subunits. This constitutes direct evidence that the conformational change of transmembrane signaling involves significant movement within a subunit and minimal movement between subunits, a pattern deduced from several previous studies and now documented directly. Among possible modes of movement between the two helices of a subunit, axial sliding of one helix relative to the other was the conformational change that best accounted for the observed effects on cross-linking.

Hughson, Andrew G.; Hazelbauer, Gerald L.

1996-10-01

152

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

SciTech Connect

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

Storrs, R.W.

1992-08-01

153

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

SciTech Connect

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

Storrs, R.W.

1992-08-01

154

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

SciTech Connect

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.

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

2001-10-30

155

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

PubMed

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

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

2014-10-01

156

The crystal structure of GXGD membrane protease FlaK  

SciTech Connect

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

Hu, Jian; Xue, Yi; Lee, Sangwon; Ha, Ya (Yale-MED)

2011-09-20

157

The Crystal Structure of GXGD Membrane Protease FlaK  

SciTech Connect

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

J Hu; Y Xue; S Lee; Y Ha

2011-12-31

158

RESEARCH ARTICLE Open Access Hydrophobic pulses predict transmembrane helix  

E-print Network

signaling and cell recog- nition. IMPs can be divided in two classes according to the characteristics (a in many aspects of cell physiology such as, for instance, transport of ions and solutes, cell-to-cell, while b-barrel IMPs are only located in the outer membrane of Gram-negative bacteria, mito- chondria

Paris-Sud XI, Université de

159

Transmembrane domain V plays a stabilizing role in the function of human bile acid transporter SLC10A2.  

PubMed

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2), primarily expressed in the ileum, is involved in both the recycling of bile acids and cholesterol homeostasis. In this study, the structure-function relationship of transmembrane domain 5 (TM5) residues involved in transport is elucidated. Cysteine scanning mutagenesis of each consecutive residue on TM5 resulted in 96% of mutants having a significantly decreased transport activity, although each was expressed at the cell surface. Specifically, G197 and I208 were no longer functional, and G201 and G212 functioned at a level of <10% upon cysteine mutation. Interestingly, each of these exists along one face of the helix. Studies suggest that neither G201 nor G212 is on the substrate pathway. Conservative alanine mutations of the four residues displayed a higher activity in all but G197A, indicating its functional importance. G197 and G201 form a GxxxG motif, which has been found to be important in helix-helix interactions. According to our model, G197 and G201 face transmembrane domain 4 (TM4) residues G179 and P175, respectively. Similarly, G212 faces G237, which forms part of a GxxxG domain in transmembrane domain 6 (TM6). It is possible that these GxxxG domains and their interacting partners are responsible for maintaining the structure of the helices and their interactions with one another. I205 and I208 are both in positions to anchor the GxxxG domains and direct the change in interaction of TM5 from TM4 to TM6. Combined, the results suggest that residues along TM5 are critical for ASBT function but are not directly involved in substrate translocation. PMID:23815591

Moore, Robyn H; Chothe, Paresh; Swaan, Peter W

2013-07-30

160

Structurally conserved aromaticity of Tyr249 and Phe264 in helix 7 is important for toxicity of the Bacillus thuringiensis Cry4Ba toxin.  

PubMed

Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry delta- endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, Trp(243), Phe(246), Tyr(249) and Phe(264), in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at Tyr(249) and Phe(264) still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/ Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of Tyr(249) and Phe(264) within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin. PMID:17394765

Tiewsiri, Kasorn; Angsuthanasombat, Chanan

2007-03-31

161

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

PubMed

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

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

2015-02-27

162

Structural Waters in the Minor and Major Grooves of DNA-A Major Factor Governing Structural Adjustments of the A-T Mini-Helix.  

PubMed

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

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

2015-01-15

163

Entropically driven helix formation.  

PubMed

The helix is a ubiquitous motif for biopolymers. We propose a heuristic, entropically based model that predicts helix formation in a system of hard spheres and semiflexible tubes. We find that the entropy of the spheres is maximized when short stretches of the tube form a helix with a geometry close to that found in natural helices. Our model could be directly tested with wormlike micelles as the tubes, and the effect could be used to self-assemble supramolecular helices. PMID:15718461

Snir, Yehuda; Kamien, Randall D

2005-02-18

164

TOPDB: topology data bank of transmembrane proteins  

PubMed Central

The Topology Data Bank of Transmembrane Proteins (TOPDB) is the most complete and comprehensive collection of transmembrane protein datasets containing experimentally derived topology information currently available. It contains information gathered from the literature and from public databases available on the internet for more than a thousand transmembrane proteins. TOPDB collects details of various experiments that were carried out to learn about the topology of particular transmembrane proteins. In addition to experimental data from the literature, an extensive collection of structural data was also compiled from PDB and from PDBTM. Because topology information is often incomplete, for each protein in the database the most probable topology that is consistent with the collected experimental constraints was also calculated using the HMMTOP transmembrane topology prediction algorithm. Each record in TOPDB also contains information on the given protein sequence, name, organism and cross references to various other databases. The web interface of TOPDB includes tools for searching, relational querying and data browsing as well as for visualization. TOPDB is designed to bridge the gap between the number of transmembrane proteins available in sequence databases and the publicly accessible topology information of experimentally or computationally studied transmembrane proteins. TOPDB is available at http://topdb.enzim.hu. PMID:17921502

Tusnády, Gábor E.; Kalmár, Lajos; Simon, István

2008-01-01

165

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

SciTech Connect

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

Awe, T. J., E-mail: tjawe@sandia.gov; Jennings, C. A.; McBride, R. D.; Cuneo, M. E.; Lamppa, D. C.; Martin, M. R.; Rovang, D. C.; Sinars, D. B.; Slutz, S. A.; Owen, A. C.; Gomez, M. R.; Hansen, S. B.; Herrmann, M. C.; Jones, M. C.; McKenney, J. L.; Robertson, G. K.; Rochau, G. A.; Savage, M. E.; Stygar, W. A. [Sandia National Laboratories, P.O. Box 5800, Albuquerque, New Mexico 87185 (United States)] [Sandia National Laboratories, P.O. Box 5800, Albuquerque, New Mexico 87185 (United States); Tomlinson, K. [General Atomics, San Diego, California 92121 (United States)] [General Atomics, San Diego, California 92121 (United States); and others

2014-05-15

166

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

PubMed Central

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

Vostrikov, Vitaly V.; Koeppe, Roger E.

2011-01-01

167

Monoclinic uncomplexed double-stranded, antiparallel, left-handed beta 5.6-helix (increases decreases beta 5.6) structure of gramicidin A: alternate patterns of helical association and deformation.  

PubMed Central

A comparison of the monoclinic and orthorhombic crystal structures of the uncomplexed double-stranded, antiparallel, left-handed beta-helix (5.6 amino acid residues per turn) (increases decreases beta 5.6) conformers of gramicidin A reveals marked differences in the tryptophan side-chain orientations and the degree of helical uniformity of the dimer and in the manner in which these helical dimers associate with one another in the crystal. The helix of the orthorhombic dimer exhibits a regular pattern of bulges and constrictions that appears to be induced by crystal packing forces affecting tryptophan side chains that are aligned parallel to the helix axis. The monoclinic dimer is more uniform than the orthorhombic dimer as a consequence of pi stacking interactions between dimers in which orientation of tryptophan side chains is normal to the helix axis to relieve the lateral crystal packing forces that may locally twist and deform the helix. It may be inferred from these observations that lipid interactions may be expected to destabilize the increases decreases beta 5.6 helix when it is inserted into a membrane bilayer. PMID:1711230

Langs, D A; Smith, G D; Courseille, C; Précigoux, G; Hospital, M

1991-01-01

168

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

PubMed Central

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

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

2013-01-01

169

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

SciTech Connect

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

Yu,P.

2007-01-01

170

Schistosoma mansoni: Structural and biochemical characterization of two distinct Venus Kinase Receptors  

Microsoft Academic Search

Venus Kinase Receptors (VKRs) are atypical transmembrane proteins composed of an extracellular Venus FlyTrap module linked through a single helix to a tyrosine kinase domain similar to that of insulin receptors. This structure was first described in Schistosoma mansoni, then in a selected range of invertebrates, including many insects. The preferential expression of VKRs in larvae and gonads suggested their

Nadege Gouignard; Mathieu Vanderstraete; Katia Cailliau; Arlette Lescuyer; Edith Browaeys; Colette Dissous

171

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

PubMed Central

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

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

1992-01-01

172

Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement  

Microsoft Academic Search

Voltage-gated ion channels are responsible for generating electrical impulses in nerves and other excitable cells. The fourth transmembrane helix (S4) in voltage-gated channels is the primary voltage-sensing unit that mediates the response to a changing membrane electric field. The molecular mechanism of voltage sensing, particularly with respect to the magnitude of the transmembrane movement of S4, remains controversial. To determine

Baron Chanda; Osei Kwame Asamoah; Rikard Blunck; Benoît Roux; Francisco Bezanilla

2005-01-01

173

Structural Basis for a pH Sensitive Calcium Leak across Membranes  

PubMed Central

Calcium homeostasis balances passive calcium leak and active calcium uptake. Human Bax inhibitor 1 (hBI-1) is an anti-apoptotic protein that mediates a calcium leak and is representative of highly conserved and widely distributed family, the transmembrane Bax inhibitor motif (TMBIM) proteins. Here we present crystal structures of a bacterial homolog and characterize its calcium leak activity. The structure has a seven-transmembrane-helix fold that features two triple-helix sandwiches wrapped around a central C-terminal helix. Structures obtained in closed and open conformations are reversibly inter-convertible by change of pH. A hydrogen-bonded, pKa-perturbed pair of conserved aspartate residues explains the pH dependence of this equilibrium, and biochemical studies show that pH regulates calcium influx in proteoliposomes. Homology models for hBI-1 provide insights into TMBIM-mediated calcium leak and cytoprotective activity. PMID:24904158

Chang, Yanqi; Bruni, Renato; Kloss, Brian; Assur, Zahra; Kloppmann, Edda; Rost, Burkhard; Hendrickson, Wayne A.; Liu, Qun

2014-01-01

174

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

PubMed Central

Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3?Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional ?-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand ?C and helix ?C in IL-17RA SEFIR is mostly well ordered, displaying a helix (?CC?ins) and a flexible loop (CC?). The DD? loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12?Å to accommodate the ?CC?ins helix without forming any knots. Helix ?C was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix ?C is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix ?C could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling. PMID:24816115

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

2014-01-01

175

A Helix Replacement Mechanism Directs Metavinculin Functions  

SciTech Connect

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

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

2010-10-11

176

Entropically driven helix formation  

NASA Astrophysics Data System (ADS)

We investigate a purely entropic approach to understanding the folding of helices that exclusively relies on a local and homogeneous interaction with depleting spheres. We found that by decreasing the size of the depleting spheres for a given volume fraction the helix formed becomes tighter. In the limit of small spheres the helix becomes the optimally tight helix of pitch to radius ratio of 2.5122 often found in alpha helices of protiens. The depletion interaction can be used as a surrogate for hydrophobicity, polymer-polymer interactions, and for boundary layers in elastica and liquid crystals.

Snir, Yehuda

2005-03-01

177

Effects of side chains in helix nucleation differ from helix propagation  

PubMed Central

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

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

2014-01-01

178

SIMULATING THE DYNAMICS OF THE DNA DOUBLE HELIX IN SOLUTION  

E-print Network

on DNA double helix structure is very subtle. All double helices have comple- mentary base-pairs and look13 SIMULATING THE DYNAMICS OF THE DNA DOUBLE HELIX IN SOLUTION Miriam Hirshberg' and Michael Levitt important molecular structure in biology: its aesthetic double-helical structure has come to symbolize

Levitt, Michael

179

Revisting the Double Helix  

SciTech Connect

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

Ha, Taekjip (University of Illinois) [University of Illinois

2010-12-08

180

A Putative Transmembrane Leucine Zipper of Agrobacterium VirB10 Is Essential for T-Pilus Biogenesis but Not Type IV Secretion  

PubMed Central

The Agrobacterium tumefaciens VirB/VirD4 type IV secretion system is composed of a translocation channel and an extracellular T pilus. Bitopic VirB10, the VirB7 lipoprotein, and VirB9 interact to form a cell envelope-spanning structural scaffold termed the “core complex” that is required for the assembly of both structures. The related pKM101-encoded core complex is composed of 14 copies each of these VirB homologs, and the transmembrane (TM) ? helices of VirB10-like TraF form a 55-Å-diameter ring at the inner membrane. Here, we report that the VirB10 TM helix possesses two types of putative dimerization motifs, a GxxxA (GA4) motif and two leucine (Leu1, Leu2) zippers. Mutations in the Leu1 motif disrupted T-pilus biogenesis, but these or other mutations in the GA4 or Leu2 motif did not abolish substrate transfer. Replacement of the VirB10 TM domain with a nondimerizing poly-Leu/Ala TM domain sequence also blocked pilus production but not substrate transfer or formation of immunoprecipitable complexes with the core subunits VirB7 and VirB9 and the substrate receptor VirD4. The VirB10 TM helix formed weak homodimers in Escherichia coli, as determined with the TOXCAT assay, whereas replacement of the VirB10 TM helix with the strongly dimerizing TM helix from glycophorin A blocked T-pilus biogenesis in A. tumefaciens. Our findings support a model in which VirB10's TM helix contributes to the assembly or activity of the translocation channel as a weakly self-interacting membrane anchor but establishes a heteromeric TM-TM helix interaction via its Leu1 motif that is critical for T-pilus biogenesis. PMID:23625845

Garza, Isaac

2013-01-01

181

Structure of Transmembrane Domain of Lysosome-associated Membrane Protein Type 2a (LAMP-2A) Reveals Key Features for Substrate Specificity in Chaperone-mediated Autophagy.  

PubMed

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation. PMID:25342746

Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

2014-12-19

182

Transmembrane extension and oligomerization of the CLIC1 chloride intracellular channel protein upon membrane interaction.  

PubMed

Chloride intracellular channel proteins (CLICs) differ from most ion channels as they can exist in both soluble and integral membrane forms. The CLICs are expressed as soluble proteins but can reversibly autoinsert into the membrane to form active ion channels. For CLIC1, the interaction with the lipid bilayer is enhanced under oxidative conditions. At present, little evidence is available characterizing the structure of the putative oligomeric CLIC integral membrane form. Previously, fluorescence resonance energy transfer (FRET) was used to monitor and model the conformational transition within CLIC1 as it interacts with the membrane bilayer. These results revealed a large-scale unfolding between the C- and N-domains of CLIC1 as it interacts with the membrane. In the present study, FRET was used to probe lipid-induced structural changes arising in the vicinity of the putative transmembrane region of CLIC1 (residues 24-46) under oxidative conditions. Intramolecular FRET distances are consistent with the model in which the N-terminal domain inserts into the bilayer as an extended ?-helix. Further, intermolecular FRET was performed between fluorescently labeled CLIC1 monomers within membranes. The intermolecular FRET shows that CLIC1 forms oligomers upon oxidation in the presence of the membranes. Fitting the data to symmetric oligomer models of the CLIC1 transmembrane form indicates that the structure is large and most consistent with a model comprising approximately six to eight subunits. PMID:22082111

Goodchild, Sophia C; Angstmann, Christopher N; Breit, Samuel N; Curmi, Paul M G; Brown, Louise J

2011-12-20

183

Structure of a Ca(2+)/CaM:Kv7.4 (KCNQ4) B-helix complex provides insight into M current modulation.  

PubMed

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 I(KS) 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 Ca(2+)/CaM bind to the C-terminal tail of the neuronal channel Kv7.4 (KCNQ4), which is involved in 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 Ca(2+)/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 Ca(2+)/CaM:Kv7.4 B segment complex shows that Ca(2+)/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 Ca(2+)/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 Ca(2+)/CaM form bound to the B-helix. The structure presented here also provides a context for a number of disease-causing mutations and for further dissection of the mechanisms by which CaM controls Kv7 function. PMID:23178170

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

2013-01-23

184

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

SciTech Connect

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

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

2010-01-01

185

The Crystal Structure of Six-transmembrane Epithelial Antigen of the Prostate 4 (Steap4), a Ferri/Cuprireductase, Suggests a Novel Interdomain Flavin-binding Site*  

PubMed Central

Steap4 is a cell surface metalloreductase linked to obesity-associated insulin resistance. Initial characterization of its cell surface metalloreductase activity has been reported, but thorough biochemical characterization of this activity is lacking. Here, we report detailed kinetic analysis of the Steap4 cell surface metalloreductase activities. Steap4 shows physiologically relevant Km values for both Fe3+ and Cu2+ and retains activity at acidic pH, suggesting it may also function within intracellular organelles to reduce these metals. Flavin-dependent NADPH oxidase activity that was much greater than the equivalent Steap3 construct was observed for the isolated N-terminal oxidoreductase domain. The crystal structure of the Steap4 oxidoreductase domain was determined, providing a structural explanation for these differing activities. Structure-function work also suggested Steap4 utilizes an interdomain flavin-binding site to shuttle electrons between the oxidoreductase and transmembrane domains, and it showed that the disordered N-terminal residues do not contribute to enzymatic activity. PMID:23733181

Gauss, George H.; Kleven, Mark D.; Sendamarai, Anoop K.; Fleming, Mark D.; Lawrence, C. Martin

2013-01-01

186

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

PubMed Central

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

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

2001-01-01

187

An S-band fluid-cooled helix TWT  

Microsoft Academic Search

The use of moving fluids in a hollow helix structure supported by hollow ceramics is shown to simultaneously provide helix cooling, selective losses for suppressing backward wave oscillations, and low dielectric loading. The same structure using a different fluid is a high-power attenuator. The 50 PSI input fluid passes through parallel hollow ceramic supports into and through a half-turn of

H. D. Arnett; H. E. Brown; R. H. Kyser; P. I. Peterson; S. T. Smith; L. M. Winslow

1972-01-01

188

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

PubMed Central

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

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

1997-01-01

189

The Hubble Helix  

NASA Astrophysics Data System (ADS)

For the 14 hours of peak Leonid meteoroid flux in November 2002, the Hubble Space Telescope was pointed away from incoming meteoroids, and the solar arrays were oriented to minimize their cross-section. By coincidence, one of the most prominent planetary nebulae, the Helix Nebula, a.k.a. NGC 7293, was nearly opposite the incoming Leonids and could be observed. A ``Hubble Helix Team'' of volunteers led by M. Meixner (STScI) organized a nine-orbit campaign to observe the Helix with the ACS, WFPC2, NICMOS, and STIS. A contiguous 3 by 3 grid of 4kx4k-pixel ACS images covering much but not all of the Helix was exposed in two filters, H? +[N II] (F658N) and [O III] (F502N). A few of the WFPC2 images observed in parallel also observed the nebula in [O I] (F631N) or He II 4686 (F469N) or H? (F656N). NICMOS/NIC3 observations were obtained at two locations on the nebula and two off, in H2 (F212N) and Paschen-? (F187N). A few of the STIS parallel observations in [OII] (F28X0OII) were located on the nebula. The main purpose of this presentation is to advertise to all interested parties the availability of the non-proprietary data via the HST archive. Initial data analysis by the Hubble Helix Team will be presented in this poster.

McCullough, P. R.; Hubble Helix Team

2002-12-01

190

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)

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.

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

2000-01-01

191

Roquin binding to target mRNAs involves a winged helix-turn-helix motif.  

PubMed

Roquin proteins mediate mRNA deadenylation by recognizing a conserved class of stem-loop RNA degradation motifs via their Roquin domain. Here we present the crystal structure of a Roquin domain, revealing a mostly helical protein fold bearing a winged helix-turn-helix motif. By combining structural, biochemical and mutation analyses, we gain insight into the mode of RNA binding. We show that the winged helix-turn-helix motif is involved in the binding of constitutive decay elements-containing stem-loop mRNAs. Moreover, we provide biochemical evidence that Roquin proteins are additionally able to bind to duplex RNA and have the potential to be functional in different oligomeric states. PMID:25504471

Schuetz, Anja; Murakawa, Yasuhiro; Rosenbaum, Eva; Landthaler, Markus; Heinemann, Udo

2014-01-01

192

N-terminal transmembrane domain of lipase LipA from Pseudomonas protegens Pf-5: A must for its efficient folding into an active conformation.  

PubMed

LipA from Pseudomonas protegens Pf-5 has been proven not to be secreted into the extracytoplasmic space, proposing that it is a membrane protein in virtue of its N-terminal transmembrane domain predicted by the TMHMM 2.0. However, LipA was confirmed to be an intracellular protein through determining the effects of lipA deletion or overexpression on the lipase activities in the whole-cell, lysis supernatant and lysis pellet, even through its transmembrane domain being able to make heterologous LacZ locate on the cytoplasmic membrane via construction of ?-galactosidase reporter strains. Subsequently, lipase activity assays showed that the transmembrane domain played an indispensable role for the catalytic function of LipA through construction of the markerless deletion mutant of transmembrane domain sequence of lipA and the expression and purification of LipA and LipA?TMD. To further investigate why the transmembrane domain lost its membrane localization function and significantly affected the catalytic function of LipA, the 3D structures of LipA and LipA?TMD were constructed. The results indicated that the transmembrane domain, located in the interior of LipA, helped the ?-helical lid to form an open conformation by the mediation of ?5 helix. It seems to act as a kind of intramolecular chaperone like the ?-roll motif of subfamily I.3 lipases, which is novel and is the first to notify the intramolecular chaperone of a subfamily I.1 lipase. PMID:25038570

Zha, Daiming; Zhang, Huaidong; Zhang, Houjin; Xu, Li; Yan, Yunjun

2014-10-01

193

A bi-phasic pulling force acts on transmembrane helices during translocon-mediated membrane integration  

PubMed Central

Membrane proteins destined for insertion into the inner membrane of bacteria or the endoplasmic reticulum membrane in eukaryotic cells are synthesized by ribosomes bound to the bacterial SecYEG or the homologous eukaryotic Sec61 translocon. During co-translational membrane integration, transmembrane ?-helical segments in the nascent chain exit the translocon via a lateral gate that opens towards the surrounding membrane, but the mechanism of lateral exit is not well understood. In particular, little is known about how a transmembrane helix behaves when entering and exiting the translocon. Using translation-arrest peptides from bacterial SecM proteins and from the mammalian Xbp1 protein as force sensors, we show that substantial force is exerted on a transmembrane helix at two distinct points during its transit through the translocon channel, providing direct insight into the dynamics of membrane integration. PMID:23001004

Ismail, Nurzian; Hedman, Rickard; Schiller, Nina; von Heijne, Gunnar

2013-01-01

194

Transmembrane allosteric coupling of the gates in a potassium channel  

PubMed Central

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

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

2014-01-01

195

Transmembrane allosteric coupling of the gates in a potassium channel.  

PubMed

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

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

2014-01-01

196

Helix kinks are equally prevalent in soluble and membrane proteins  

PubMed Central

Helix kinks are a common feature of ?-helical membrane proteins, but are thought to be rare in soluble proteins. In this study we find that kinks are a feature of long ?-helices in both soluble and membrane proteins, rather than just transmembrane ?-helices. The apparent rarity of kinks in soluble proteins is due to the relative infrequency of long helices (?20 residues) in these proteins. We compare length-matched sets of soluble and membrane helices, and find that the frequency of kinks, the role of Proline, the patterns of other amino acid around kinks (allowing for the expected differences in amino acid distributions between the two types of protein), and the effects of hydrogen bonds are the same for the two types of helices. In both types of protein, helices that contain Proline in the second and subsequent turns are very frequently kinked. However, there are a sizeable proportion of kinked helices that do not contain a Proline in either their sequence or sequence homolog. Moreover, we observe that in soluble proteins, kinked helices have a structural preference in that they typically point into the solvent. PMID:24638929

Wilman, Henry R; Shi, Jiye; Deane, Charlotte M

2014-01-01

197

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

SciTech Connect

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

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

2010-07-19

198

Topology of NBCe1 protein transmembrane segment 1 and structural effect of proximal renal tubular acidosis (pRTA) S427L mutation.  

PubMed

In the kidney proximal tubule, NBCe1-A plays a critical role in absorbing HCO3(-) from cell to blood. NBCe1-A transmembrane segment 1 (TM1) is involved in forming part of the ion permeation pathway, and a missense mutation S427L in TM1 impairs ion transport, causing proximal renal tubular acidosis. In the present study, we examined the topology of NBCe1-A-TM1 in detail and its structural perturbation induced by S427L. We analyzed the N-terminal cytoplasmic region (Cys-389-Gln-424) of NBCe1-A-TM1 using the substituted cysteine scanning accessibility method combined with extensive chemical stripping, in situ chemical probing, and functional transport assays. NBCe1-A-TM1 was previously modeled on the anion exchanger 1 TM1 (AE1-TM1); however, our data demonstrated that the topology of AE1-TM1 differs significantly from NBCe1-A-TM1. Our findings revealed that NBCe1-A-TM1 is unusually long, consisting of 31 membrane-embedded amino acids (Phe-412 to Thr-442). The linker region (Arg-394-Pro-411) between the N terminus of TM1 and the cytoplasmic domain is minimally exposed to aqueous and is potentially folded in a helical structure that intimately interacts with the NBCe1-A cytoplasmic domain. In contrast, AE1-TM1 contains 25 amino acids connected to an aqueous-exposed cytoplasmic region. Based on our new NBCe1-A-TM1 model, Ser-427 resides in the middle of TM1. Leucine substitution at Ser-427 blocks the normal aqueous access to Thr-442, Ala-435, and Lys-404, implying a significant alteration of NBCe1-TM1 orientation. Our study provides novel structural insights into the pathogenic mechanism of S427L in mediating proximal renal tubular acidosis. PMID:23362273

Zhu, Quansheng; Liu, Weixin; Kao, Liyo; Azimov, Rustam; Newman, Debra; Abuladze, Natalia; Kurtz, Ira

2013-03-15

199

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

PubMed

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

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

1998-05-01

200

Crystal Structure of the Virulence Gene Activator AphA from Vibrio cholerae Reveals It Is a Novel Member of the Winged Helix Transcription Factor Superfamily*  

PubMed Central

AphA is a member of a new and largely uncharacterized family of transcriptional activators that is required for initiating virulence gene expression in Vibrio cholerae, the causative agent of the frequently fatal epidemic diarrheal disease cholera. AphA activates transcription by an unusual mechanism that appears to involve a direct interaction with the LysR-type regulator AphB at the tcpPH promoter. As a first step toward understanding the molecular basis for tcpPH activation by AphA and AphB, we have determined the crystal structure of AphA to 2.2 Å resolution. AphA is a dimer with an N-terminal winged helix DNA binding domain that is architecturally similar to that of the MarR family of transcriptional regulators. Unlike this family, however, AphA has a unique C-terminal antiparallel coiled coil domain that serves as its primary dimerization interface. AphA monomers are highly unstable by themselves and form a linked topology, requiring the protein to partially unfold to form the dimer. The structure of AphA also provides insights into how it cooperates with AphB to activate transcription, most likely by forming a heterotetrameric complex at the tcpPH promoter. PMID:15647287

De Silva, Rukman S.; Kovacikova, Gabriela; Lin, Wei; Taylor, Ronald K.; Skorupski, Karen; Kull, F. Jon

2009-01-01

201

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

PubMed

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

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

2015-01-27

202

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

PubMed

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

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

2015-01-16

203

Analysis of dihedral angle preferences for alanine and glycine residues in alpha and beta transmembrane regions.  

PubMed

For the past 50?years, the Ramachandran map has been used effectively to study the protein structure and folding. However, though extensive analysis has been done on dihedral angle preferences of residues in globular proteins, related studies and reports of membrane proteins are limited. It is of interest to explore the conformational preferences of residues in transmembrane regions of membrane proteins which are involved in several important and diverse biological processes. Hence, in the present work, a systematic comparative computational analysis has been made on dihedral angle preferences of alanine and glycine in alpha and beta transmembrane regions (the two major classes of transmembrane proteins) with the aid of the Ramachandran map. Further, the conformational preferences of residues in transmembrane regions were compared with the non-transmembrane regions. We have extracted cation-pi interacting residues present in transmembrane regions and explored the dihedral angle preferences. From our observations, we reveal the higher percentage of occurrences of glycine in alpha and beta transmembrane regions than other hydrophobic residues. Further, we noted a clear shift in ?-angle preferences of glycine residues from negative bins in alpha transmembrane regions to positive bins in beta transmembrane regions. Also, cation-pi interacting residues in beta transmembrane regions avoid preferring ?-angles in the range of -59° to -30°. In this article, we insist that the studies on preferences of dihedral angles in transmembrane regions, thorough understanding of structure and folding of transmembrane proteins, can lead to modeling of novel transmembrane regions towards designing membrane proteins. PMID:24625248

Saravanan, K M; Krishnaswamy, S

2015-03-01

204

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

PubMed Central

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

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

2012-01-01

205

Light stress-regulated two-helix proteins in Arabidopsis thaliana related to the chlorophyll a/b-binding gene family  

PubMed Central

The chlorophyll a/b, chlorophyll a/c, and chlorophyll a/a light-harvesting proteins are part of an extended gene family that also includes the transiently expressed stress proteins, the Elips (early light-induced proteins). Four Elip homologue proteins, encoded by single-copy nuclear genes, have been identified in the Arabidopsis thaliana database. These proteins were divided into two groups according to the expression pattern under light-stress conditions and the predicted secondary structure. Group one included two members of the Elip family with three predicted transmembrane helices and a gene expression strictly related to light stress. Group two included two proteins, the Seps (stress-enhanced proteins), which possessed two predicted transmembrane segments. The transcripts of Sep1 and Sep2 were present under low light conditions, but their level increased 4- to 10-fold during illumination of plants with high-intensity light. Preliminary data indicated that the induced transcripts were translated in vivo. Other physiological stress conditions, such as cold, heat, desiccation, salt, wounding, or oxidative stress, did not significantly influence the expression of Sep genes. In vitro import of radioactively labeled precursors of Seps into isolated chloroplasts confirmed the thylakoid membrane localization of these proteins. Considering the predicted protein structure and homology to other pigment-antenna proteins, the two-helix Seps might represent an evolutionary missing link between the one- and three-helix antenna proteins present in pro- and eukaryota. PMID:10725357

Heddad, Mounia; Adamska, Iwona

2000-01-01

206

Membrane helix orientation from linear dichroism of infrared attenuated total reflection spectra.  

PubMed Central

Oriented multilamellar systems containing phospholipids and peptides have been formed on a germanium internal reflection element. Attenuated total reflection infrared spectra have been recorded and the linear dichroism of peptide amide I and amide II bands measured. Using peptides for which the orientation had been previously studied under similar experimental conditions by 15N solid-state nuclear magnetic resonance spectroscopy, important conclusions were drawn on the approach to be used to derive secondary structure orientation in a membrane from dichroic ratios. In particular, it is shown that the influence of the film thickness and refractive index on the orientation determination can be evaluated from the value of RATRiso, i.e., the dichroic ratio of a dipole oriented at the magic angle or with isotropic mobility. A series of peptides was used to test the validity of our suggestions on various helix orientations in the membrane. These include magainin 2 and hydrophobic (hPhi20) model peptides, the transmembrane segment of glycophorin (GLY), and LAH4, a designed peptide antibiotic that changes between a transmembrane and an in-plane orientation in a pH-dependent manner. PMID:9876168

Bechinger, B; Ruysschaert, J M; Goormaghtigh, E

1999-01-01

207

Pemphigoid gestationis autoantigen, transmembrane collagen XVII, promotes the migration of cytotrophoblastic cells of placenta and is a structural component of fetal membranes.  

PubMed

In pemphigoid gestationis (PG), autoantibodies target collagen XVII, a hemidesmosomal transmembrane protein, which is an important element in cutaneous epithelial adhesion and signalling. We report that collagen XVII is expressed in the first trimester and term syncytial and cytotrophoblastic cells of normal placenta and in epithelial cells of amniotic membrane. Immunoelectron microscopy confirmed the localization of collagen XVII to the hemidesmosomes of amniotic epithelium. Examination of three PG placentas showed mild villitis, but there were no differences between collagen XVII expression levels or immunostaining signals as compared to normal placenta. Collagen XVII expression was also detected in cultured extravillous trophoblast HTR-8/SVneo cells, where collagen XVII expression was upregulated by PMA and TGF-beta1. Interestingly, the presence of Col15, the cell migration domain of collagen XVII, induced the migration of HTR-8/SVneo cells in transmigration assay. Analysis of amniotic fluid samples at different gestational weeks revealed that a large quantity of collagen XVII ectodomain was shed into amniotic fluid throughout pregnancy. Biochemical and immunoblotting analysis indicated that the ectodomain in amniotic fluid is structurally very similar to the ectodomain produced by cultured keratinocytes. Cultured cells from amniotic fluid samples also expressed collagen XVII. Our results suggest that collagen XVII may contribute to the invasion of extravillous trophoblasts during placental development and is also required for the integrity of amniotic basement membrane. Although the exact pathomechanism of PG is still largely unknown, the clinical symptoms of PG are initiated after the expression of collagen XVII in placenta during the first trimester of pregnancy. PMID:18055190

Huilaja, Laura; Hurskainen, Tiina; Autio-Harmainen, Helena; Hofmann, Silke C; Sormunen, Raija; Räsänen, Juha; Ilves, Mika; Franzke, Claus-Werner; Bruckner-Tuderman, Leena; Tasanen, Kaisa

2008-04-01

208

The crystal structures of the calcium-bound con-G and con-T[K7gamma] dimeric peptides demonstrate a metal-dependent helix-forming motif.  

PubMed

Short peptides that have the ability to form stable alpha-helices in solution are rare, and a number of strategies have been used to produce them, including the use of metal chelation to stabilize folding of the backbone. However, no example exists of a structurally well-defined helix stabilized exclusively through metal ion chelation. Conantokins (con)-G and -T are short peptides that are potent antagonists of N-methyl-D-aspartate receptor channels. While con-G exhibits no helicity alone, it undergoes a structural transition to a helical conformation in the presence of a variety of multivalent cations, especially Mg2+ and Ca2+. This complexation also results in antiparallel dimerization of two peptide helices in the presence of Ca2+, but not Mg2+. A con-T variant, con-T[K7gamma], displays very similar behavior. We have solved the crystal structures of both Ca2+/con-G and Ca2+/con-T [K7gamma] at atomic resolution. These structures clearly show the nature of the metal-dependent dimerization and helix formation and surprisingly also show that the con-G dimer interface is completely different from the con-T[K7gamma] interface, even though the metal chelation is similar in the two peptides. This represents a new paradigm in helix stabilization completely independent of the hydrophobic effect, which we define as the "metallo-zipper." PMID:17243678

Cnudde, Sara E; Prorok, Mary; Dai, Qiuyun; Castellino, Francis J; Geiger, James H

2007-02-14

209

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

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.

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

2014-10-02

210

Structural integrity of ?-helix H12 in translation initiation factor eIF5B is critical for 80S complex stability  

PubMed Central

Translation initiation factor eIF5B promotes GTP-dependent ribosomal subunit joining in the final step of the translation initiation pathway. The protein resembles a chalice with the ?-helix H12 forming the stem connecting the GTP-binding domain cup to the domain IV base. Helix H12 has been proposed to function as a rigid lever arm governing domain IV movements in response to nucleotide binding and as a molecular ruler fixing the distance between domain IV and the G domain of the factor. To investigate its function, helix H12 was lengthened or shortened by one or two turns. In addition, six consecutive residues in the helix were substituted by Gly to alter the helical rigidity. Whereas the mutations had minimal impacts on the factor's binding to the ribosome and its GTP binding and hydrolysis activities, shortening the helix by six residues impaired the rate of subunit joining in vitro and both this mutation and the Gly substitution mutation lowered the yield of Met-tRNAiMet bound to 80S complexes formed in the presence of nonhydrolyzable GTP. Thus, these two mutations, which impair yeast cell growth and enhance ribosome leaky scanning in vivo, impair the rate of formation and stability of the 80S product of subunit joining. These data support the notion that helix H12 functions as a ruler connecting the GTPase center of the ribosome to the P site where Met-tRNAiMet is bound and that helix H12 rigidity is required to stabilize Met-tRNAiMet binding. PMID:21335519

Shin, Byung-Sik; Acker, Michael G.; Kim, Joo-Ran; Maher, Kathryn N.; Arefin, Shamsul M.; Lorsch, Jon R.; Dever, Thomas E.

2011-01-01

211

Gap Junction Channel Structure in the Early 21st Century: Facts and Fantasies  

PubMed Central

Gap junction channels connect the cytoplasms of adjacent cells through the end-to-end docking of single-membrane structures called connexons, formed by a ring of six connexin monomers. Each monomer contains 4 transmembrane ?-helices, for a total of 24 ?-helices in a connexon. The fundamental structure of the connexon pore is probably similar in unpaired connexons and junctional channels, and for channels formed by different connexin isoforms. Nevertheless, variability in results from structurally-focused mutagenesis and electrophysiological studies raise uncertainty about the specific assignments of the transmembrane helices. Mapping of human mutations onto a suggested C? model predicts that mutations that disrupt helix-helix packing impair channel function. An experimentally determined structure at atomic resolution will be essential to confirm and resolve these concepts. PMID:17945477

Harris, Andrew L.

2009-01-01

212

A deterministic algorithm for constrained enumeration of transmembrane protein folds.  

SciTech Connect

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

Brown, William Michael; Young, Malin M. (Sandia National Laboratories, Livermore, California); Sale, Kenneth L. (Sandia National Laboratories, Livermore, California); Faulon, Jean-Loup Michel; Schoeniger, Joseph S. (Sandia National Laboratories, Livermore, California)

2004-07-01

213

Noncanonical structures and their thermodynamics of DNA and RNA under molecular crowding: beyond the Watson-Crick double helix.  

PubMed

How does molecular crowding affect the stability of nucleic acid structures inside cells? Water is the major solvent component in living cells, and the properties of water in the highly crowded media inside cells differ from that in buffered solution. As it is difficult to measure the thermodynamic behavior of nucleic acids in cells directly and quantitatively, we recently developed a cell-mimicking system using cosolutes as crowding reagents. The influences of molecular crowding on the structures and thermodynamics of various nucleic acid sequences have been reported. In this chapter, we discuss how the structures and thermodynamic properties of nucleic acids differ under various conditions such as highly crowded environments, compartment environments, and in the presence of ionic liquids, and the major determinants of the crowding effects on nucleic acids are discussed. The effects of molecular crowding on the activities of ribozymes and riboswitches on noncanonical structures of DNA- and RNA-like quadruplexes that play important roles in transcription and translation are also described. PMID:24380597

Sugimoto, Naoki

2014-01-01

214

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

PubMed Central

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

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

2014-01-01

215

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

PubMed

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

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

2009-08-28

216

Role of amphipathic helix of a herpesviral protein in membrane deformation and T cell receptor downregulation.  

PubMed

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 deformation. In turn, this motif directs Tip's lysosomal trafficking and selective TCR downregulation. The amphipathic helix binds to the negatively charged lipids and induces liposome tubulation, the TM domain mediates oligomerization, and cooperation of the membrane-proximal helix with the TM domain is sufficient for localization to lipid rafts and lysosomal compartments, especially the mutivesicular bodies. These findings suggest that the membrane-proximal amphipathic helix and TM domain provide HVS Tip with the unique ability to deform the cellular membranes in lipid rafts and to downregulate TCRs potentially through MVB formation. PMID:19023411

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

2008-11-01

217

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

PubMed Central

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 deformation. In turn, this motif directs Tip's lysosomal trafficking and selective TCR downregulation. The amphipathic helix binds to the negatively charged lipids and induces liposome tubulation, the TM domain mediates oligomerization, and cooperation of the membrane-proximal helix with the TM domain is sufficient for localization to lipid rafts and lysosomal compartments, especially the mutivesicular bodies. These findings suggest that the membrane-proximal amphipathic helix and TM domain provide HVS Tip with the unique ability to deform the cellular membranes in lipid rafts and to downregulate TCRs potentially through MVB formation. PMID:19023411

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

2008-01-01

218

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

PubMed

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

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

219

Into the Eye of the Helix  

NASA Astrophysics Data System (ADS)

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

2009-02-01

220

Folding energetics and oligomerization of polytopic ?-helical transmembrane proteins.  

PubMed

While interactions of single-span transmembrane helices have been studied to a significant extent in the past years, the folding of polytopic ?-helical transmembrane proteins, as well as their oligomerization, are far less analyzed and understood. The goal of the few thus far performed thermodynamic studies, in which unfolding of polytopic TM proteins was described, was to achieve a mild, potentially reversible unfolding process, to finally derive thermodynamic parameters for the reverse folding pathway. In the first part of this review, we summarize the studies analyzing the thermodynamic stability and folding pathways of polytopic transmembrane proteins. Based on these studies, we deduce some common principles, guiding transmembrane protein unfolding and folding, important for the design of future folding/unfolding studies. Furthermore, the discussed observations can conceptually guide an experimental search for proper in vitro transmembrane protein refolding conditions. In many of the resolved membrane protein structures, individual monomers interact to form higher ordered oligomers. In most cases, oligomerization of those monomeric units appears to be intimately linked to the protein function, and folding of the individual protomers might even occur only after interaction. In the second part of this review, we discuss folding pathways of oligomeric ?-helical transmembrane proteins as well as causes and consequences of ?-helical transmembrane protein oligomerization. PMID:25057769

Neumann, Jennifer; Klein, Noreen; Otzen, Daniel E; Schneider, Dirk

2014-12-15

221

Transmembrane Domain V Plays a Stabilizing Role in the Function of Human Bile Acid Transporter SLC10A2  

PubMed Central

The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2), primarily expressed in the ileum, is involved in both the recycling of bile acids and cholesterol homeostasis. In this study, the structure-function relationship of transmembrane domain 5 (TM5) residues involved in transport is elucidated. Cysteine scanning mutagenesis of each consecutive residue on TM5 resulted in 96% of mutants with a significantly decreased transport activity although each was expressed at the cell surface. Specifically, G197 and I208 were no longer functional and G201 and G212 functioned at less than 10% upon cysteine mutation. Interestingly, each of these exists along one face of the helix. Studies suggest that neither G201 nor G212 are on the substrate pathway. Conservative alanine mutations of the four residues displayed a higher activity in all but G197A, indicating its functional importance. G197 and G201 form a GxxxG motif, which has been found to be important in helix-helix interactions. According to our model, G197 and G201 face TM4 residues G179 and P175 respectively. Similarly, G212 faces G237, which forms part of a GxxxG domain in TM6. It is possible that these GxxxG domains and their interacting partners are responsible for maintaining the structure of the helices and their interactions with one another. I205 and I208 are both in positions to anchor the GxxxG domains and direct the change in interaction of TM5 from TM4 to TM6. Combined, the results suggest that residues along TM5 are critical for ASBT function but are not directly involved in substrate translocation. PMID:23815591

Moore, Robyn H.; Chothe, Paresh; Swaan, Peter W.

2013-01-01

222

The infrared dichroism of transmembrane helical polypeptides.  

PubMed Central

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

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

1995-01-01

223

Endocytic sorting of transmembrane protein cargo.  

PubMed

The accurate distribution and recycling of transmembrane proteins amongst the membrane-bound organelles of the cell is vital to ensure its correct functioning. Transmembrane protein cargo destined for clathrin-mediated endocytosis and transport along the endocytic pathway is sorted into transport vesicles by interactions with adaptors, which simultaneously link clathrin to the membrane. Clathrin adaptors recognize a variety of signals present in the cytoplasmic portions of cargo proteins; recent structural, biophysical and cell biological studies have elucidated new types of cargo-adaptor interactions and probed the molecular mechanisms regulating cargo selection and vesicle maturation. Here, we review this recent progress in the context of our existing knowledge of endocytic sorting mechanisms. PMID:21450449

Kelly, Bernard T; Owen, David J

2011-08-01

224

Salvador-warts-hippo pathway in a developmental checkpoint monitoring helix-loop-helix proteins.  

PubMed

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

Wang, Lan-Hsin; Baker, Nicholas E

2015-01-26

225

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

PubMed Central

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

2014-01-01

226

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

PubMed

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

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

2014-09-01

227

Molecular-mechanics modelling of drug-DNA structures; the effects of differing dielectric treatment on helix parameters and comparison with a fully solvated structural model.  

PubMed

This study analyses the influence that the nature of the dielectric constant has on the final structures obtained from in vacuo molecular mechanics calculations on a drug-DNA complex and compares these structures with the energy minimised complex including explicit solvent molecules. Minimisations have been performed on a proflavine-decanucleotide structure, where the drug was intercalated at the d(CpG) site of the d(GpApTpApCpGpApTpApC) decamer duplex, using two expressions for the dielectric constant: a distance-independent, epsilon ij = EPS, and a distance-dependent, epsilon ij = EPS*Rij, form and for values of EPS from 1 to 8. Significantly different structures are obtained for the distance-independent and the distance-dependent expressions of the dielectric constant. The use of a distance-independent dielectric constant leads to distorted structures, which are very sensitive to slight changes in the value of EPS. The use of a distance-dependent dielectric constant leads to less distorted and more stable structures. The effects on helical parameters are analysed in detail. The structures obtained for different values of EPS (within the distance-dependent formalism) seem to converge for values of EPS equal to 4 or greater. Based on these results a distance-dependent form of the dielectric with an EPS value of 4 is recommended in order to produce reliable refined nucleic acid structures by molecular mechanics. These conclusions have been supported by molecular-mechanics minimisation of the same structure with the inclusion of explicit water molecules and counter-ions. PMID:2176506

Orozco, M; Laughton, C A; Herzyk, P; Neidle, S

1990-10-01

228

Single-spanning transmembrane domains in cell growth and cell-cell interactions  

PubMed Central

As a whole, integral membrane proteins represent about one third of sequenced genomes, and more than 50% of currently available drugs target membrane proteins, often cell surface receptors. Some membrane protein classes, with a defined number of transmembrane (TM) helices, are receiving much attention because of their great functional and pharmacological importance, such as G protein-coupled receptors possessing 7 TM segments. Although they represent roughly half of all membrane proteins, bitopic proteins (with only 1 TM helix) have so far been less well characterized. Though they include many essential families of receptors, such as adhesion molecules and receptor tyrosine kinases, many of which are excellent targets for biopharmaceuticals (peptides, antibodies, et al.). A growing body of evidence suggests a major role for interactions between TM domains of these receptors in signaling, through homo and heteromeric associations, conformational changes, assembly of signaling platforms, etc. Significantly, mutations within single domains are frequent in human disease, such as cancer or developmental disorders. This review attempts to give an overview of current knowledge about these interactions, from structural data to therapeutic perspectives, focusing on bitopic proteins involved in cell signaling. PMID:20543559

Sawma, Paul; Duneau, Jean-Pierre; Khao, Jonathan; Hénin, Jélerôme; Bagnard, Dominique; Sturgis, James

2010-01-01

229

The Other Double Helix--The Fascinating Chemistry of Starch  

NASA Astrophysics Data System (ADS)

Current textbooks deal only briefly with the chemistry of starch. A short review with 21 references is presented, describing the structure of starch and indicating the double helix structure of A-type and B-type starch. The structure of the starch granule is examined, pointing out the existence of growth rings of alternating crystalline and noncrystalline starch, with growing amylopectin molecules extending from the hilum (point of origin) to the surface of the starch granule. The swelling of starch granules in water, above the gelatinization temperature of about 60 °C, is discussed. The process of gelatinization involves unraveling of the starch helix and a manyfold increase in volume of the starch granule as water is imbibed and bound to the unraveled starch polymer by hydrogen bonding. Baking bread or pastries causes unraveling of the starch helix, and the process by which these products become stale corresponds primarily to the re-forming of the starch helix. The importance of this phenomenon in food science is discussed. The absorption of nonpolar linear molecules such as I2, or linear nonpolar portions of molecules such as n-butanol or fats and phospholipids, by the C-type helix of starch is examined. The way in which starch is structurally modified to retard staling is discussed in relation to food technology.

Hancock, Robert D.; Tarbet, Bryon J.

2000-08-01

230

Conservative mutations in the immunosuppressive region of the bovine leukemia virus transmembrane protein affect fusion but not infectivity in vivo.  

PubMed

Many retroviruses, including bovine leukemia virus (BLV), contain a highly conserved region located about 40 amino acids downstream from the fusion peptide within the sequence of the external domain of the transmembrane (TM) protein. This region is notably thought to be involved in the presentation of the NH2-terminal peptide to allow cell fusion. By using hydrophobic cluster analysis and by analogy with the influenza A hemagglutinin structures, the core of the TM structure including this particular region was predicted to consist, in the BLV and other retroviral envelope proteins, of an alpha-helix followed by a loop region, both docked against a subsequent alpha-helix that forms a triple-stranded coiled coil. The loop region could undergo, as in hemagglutinin, a major refolding into an alpha-helix integrating the coiled coil structure and putting the fusion peptide to one tip of the molecule. Based on this model, we have identified amino acids that may be essential to the BLV TM structure, and a series of mutations were introduced in the BLV env gene of an infectious molecular clone. A first series of mutations was designed to disturb the coiled coil structure (substitutions with proline residues), whereas others would maintain the general TM structure. When expressed by Semliki Forest virus recombinants, all the mutated envelope proteins were stable and efficiently synthesized in baby hamster kidney cells. Both proline-substituted and conservative mutants were strongly affected in their capacity to fuse to CC81 indicator cells. In addition, it appeared that the integrity of the TM coiled coil structure is essential for envelope protein multimerization, as analyzed by metrizamide gradient centrifugation. Finally, to gain insight into the role of this coiled coil in the infectious potential of BLV in vivo, the mutated TM genes were introduced in an infectious and pathogenic molecular clone and injected into sheep. It appeared that only the conservative mutations (A60V and A64S) allowed maintenance of viral infectivity in vivo. Since these mutations destroyed the ability to induce syncytia, we conclude that efficient fusion capacity of the recombinant envelopes is not a prerequisite for the infectious potential of BLV in vivo. Viral propagation of these mutants was strongly affected in some of the infected sheep. However, the proviral loads within half of the infected animals (2 out of 2 for A60V and 1 out of 4 for A64S) were close to the wild-type levels. In these sheep, it thus appears that the A60V and A64S mutants propagate efficiently despite being unable to induce syncytia in cell culture. PMID:9582317

Gatot, J S; Callebaut, I; Mornon, J P; Portetelle, D; Burny, A; Kerkhofs, P; Kettmann, R; Willems, L

1998-05-22

231

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

PubMed

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

Beisswenger, Michael; Cabrele, Chiara

2014-09-01

232

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

PubMed Central

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

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

2012-01-01

233

Structural-functional analysis of the third transmembrane domain of the corticotropin-releasing factor type 1 receptor: role in activation and allosteric antagonism.  

PubMed

The corticotropin-releasing factor (CRF) type 1 receptor (CRF1R) for the 41-amino acid peptide CRF is a class B G protein-coupled receptor, which plays a key role in the response of our body to stressful stimuli and the maintenance of homeostasis by regulating neural and endocrine functions. CRF and related peptides, such as sauvagine, bind to the extracellular regions of CRF1R and activate the receptor. In contrast, small nonpeptide antagonists, which are effective against stress-related disorders, such as depression and anxiety, have been proposed to interact with the helical transmembrane domains (TMs) of CRF1R and allosterically antagonize peptide binding and receptor activation. Here, we aimed to elucidate the role of the third TM (TM3) in the molecular mechanisms underlying activation of CRF1R. TM3 was selected because its tilted orientation, relative to the membrane, allows its residues to establish key interactions with ligands, other TM helices, and the G protein. Using a combination of pharmacological, biochemical, and computational approaches, we found that Phe-203(3.40) and Gly-210(3.47) in TM3 play an important role in receptor activation. Our experimental findings also suggest that Phe-203(3.40) interacts with nonpeptide antagonists. PMID:24838244

Spyridaki, Katerina; Matsoukas, Minos-Timotheos; Cordomi, Arnau; Gkountelias, Kostas; Papadokostaki, Maria; Mavromoustakos, Thomas; Logothetis, Diomedes E; Margioris, Andrew N; Pardo, Leonardo; Liapakis, George

2014-07-01

234

Integrin ?IIb?3 Transmembrane Domain Separation Mediates Bi-Directional Signaling across the Plasma Membrane  

PubMed Central

Integrins play an essential role in hemostasis, thrombosis, and cell migration, and they transmit bidirectional signals. Transmembrane/cytoplasmic domains are hypothesized to associate in the resting integrins; whereas, ligand binding and intracellular activating signals induce transmembrane domain separation. However, how this conformational change affects integrin outside-in signaling and whether the ? subunit cytoplasmic domain is important for this signaling remain elusive. Using Chinese Hamster Ovary (CHO) cells that stably expressed different integrin ?IIb?3 constructs, we discovered that an ?IIb cytoplasmic domain truncation led to integrin activation but not defective outside-in signaling. In contrast, preventing transmembrane domain separation abolished both inside-out and outside-in signaling regardless of removing the ?IIb cytoplasmic tail. Truncation of the ?IIb cytoplasmic tail did not obviously affect adhesion-induced outside-in signaling. Our research revealed that transmembrane domain separation is a downstream conformational change after the cytoplasmic domain dissociation in inside-out activation and indispensable for ligand-induced outside-in signaling. The result implicates that the ? TM helix rearrangement after dissociation is essential for integrin transmembrane signaling. Furthermore, we discovered that the PI3K/Akt pathway is not essential for cell spreading but spreading-induced Erk1/2 activation is PI3K dependent implicating requirement of the kinase for cell survival in outside-in signaling. PMID:25617834

Hu, Ping; Luo, Bing-Hao

2015-01-01

235

Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement.  

PubMed

Voltage-gated ion channels are responsible for generating electrical impulses in nerves and other excitable cells. The fourth transmembrane helix (S4) in voltage-gated channels is the primary voltage-sensing unit that mediates the response to a changing membrane electric field. The molecular mechanism of voltage sensing, particularly with respect to the magnitude of the transmembrane movement of S4, remains controversial. To determine the extent of this transmembrane movement, we use fluorescent resonance energy transfer between the S4 domain and a reference point in the lipid bilayer. The lipophilic ion dipicrylamine distributes on either side of the lipid bilayer depending on the membrane potential, and is used here as a resonance-energy-transfer acceptor from donor molecules attached to several positions in the Shaker K+ channel. A voltage-driven transmembrane movement of the donor should produce a transient fluorescence change because the acceptor also translocates as a function of voltage. In Shaker K+ channels no such transient fluorescence is observed, indicating that the S4 segment does not translocate across the lipid bilayer. Based on these observations, we propose a molecular model of voltage gating that can account for the observed 13e gating charge with limited transmembrane S4 movement. PMID:16094369

Chanda, Baron; Asamoah, Osei Kwame; Blunck, Rikard; Roux, Benoît; Bezanilla, Francisco

2005-08-11

236

Point mutations in the herpes simplex virus type 1 Vmw110 RING finger helix affect activation of gene expression, viral growth, and interaction with PML-containing nuclear structures.  

PubMed Central

Herpes simplex virus type 1 immediate-early protein Vmw110 (also known as ICP0) has been implicated in the control of the balance between the lytic and latent states, but the precise mechanisms by which it exerts its effects are unknown. Vmw110 includes a characteristic zinc binding domain, termed the C3HC4 domain or RING finger, which is essential for its function. The solution structure of a related herpesvirus RING finger domain suggested that an amphipathic alpha helix might be an important functional component of the RING finger. In this paper, we show that the equivalent region of Vmw110 is important for virus growth in tissue culture and for the normal interaction of Vmw110 with nuclear structures which include the PML protein. PMID:7474166

Everett, R; O'Hare, P; O'Rourke, D; Barlow, P; Orr, A

1995-01-01

237

Spontaneous and Deterministic Three-dimensional Curling of Pre-strained Elastomeric Strips: From Hemi-helix to Helix  

NASA Astrophysics Data System (ADS)

A variety of three dimensional curls are produced by a simple generic process consisting of pre-straining one elastomeric strip, joining it to another and then releasing the bi-strip. The hemi-helix, one kind of three dimensional curls, consists of multiple, alternating helical sections of half wavelength in opposite chiralities and separated by perversions. The hemi-helix wavelength and the number of perversions are determined by the strip cross-section, the constitutive behavior of the elastomer and the value of the pre-strain. Topologically, the perversions also separate regions of the helix deforming principally by bending from those where twisting dominates. Changing the prestrain and the ratio between the thickness and the width induce a phase separation of hemi-helical structure, helical structure and hybrid structure which have similarities to coiled polymer molecules and plant tendrils.

Huang, Jiangshui; Liu, Jia; Kroll, Benedikt; Bertoldi, Katia; Suo, Zhigang; Clarke, David

2012-02-01

238

pH jump induced ?-helix folding.  

NASA Astrophysics Data System (ADS)

pH can be used to impact the folding equilibrium of peptides and proteins. This fact is utilized, similarly to temperature jumps, in pH jump experiments employing laser time-resolved spectroscopy to study the function and structural dynamics of these molecules. Here the application of pH jumps in folding experiments was investigated. Experiments with poly-L-glutamic acid alpha-helix formation shown the critical aspects of pH jump experiments and yielded direct information about the folding kinetics monitored with the amide I IR band.

Donten, M. L.; Hamm, P.

2013-03-01

239

A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies  

Microsoft Academic Search

Membrane proteins with transmembrane domains (TMDs) that contain polar residues exposed to the lipid bilayer are selectively sorted into multivesicular bodies (MVBs) and delivered to the yeast vacuole. Sorting of some, although not all, proteins into these structures is mediated by ubiquitination. We have identified a transmembrane ubiquitin ligase, Tul1, that is resident in the Golgi apparatus and is required

Fulvio Reggiori; Hugh R. B. Pelham

2002-01-01

240

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

PubMed

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

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

2015-01-01

241

Probing ?-3(10) transitions in a voltage-sensing S4 helix.  

PubMed

The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between ? and 310 conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 310 conformations and remains mostly ?-helical. Although the S4 of NavAb displays a fully extended 310 conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended ?-to-310 helical conversions of the S4 in Ci-VSP associated with voltage activation. PMID:25185547

Kubota, Tomoya; Lacroix, Jérôme J; Bezanilla, Francisco; Correa, Ana M

2014-09-01

242

X-ray structure of dopamine transporter elucidates antidepressant mechanism.  

PubMed

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

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

2013-11-01

243

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

PubMed Central

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

Faingold, Omri; Cohen, Tomer; Shai, Yechiel

2012-01-01

244

The cytosolic half of helix III forms the substrate exit route during permeation events of the sodium/bile acid cotransporter ASBT.  

PubMed

Site-directed alkylation of consecutively introduced cysteines was employed to probe the solvent-accessible profile of highly conserved transmembrane helix 3 (TM3), spanning residues V127-T149 of the apical sodium-dependent bile acid transporter (ASBT), a key membrane protein involved in cholesterol homeostasis. Sequence alignment of SLC10 family members has previously identified a signature motif (ALGMMPL) localized to TM3 of ASBT with as yet undetermined function. Cysteine mutagenesis of this motif resulted in severe decreases in uptake activity only for mutants M141C and P142C. Additional conservative and nonconservative replacement of P142 suggests its structural and functional importance during the ASBT transport cycle. Significant decreases in transport activity were also observed for three cysteine mutants clustered along the exofacial half of the helix (M129C, T130C, S133C) and five mutants consecutively lining the cytosolic half of TM3 (L145C-T149C). Measurable surface expression was detected for all TM3 mutants. Using physicochemically different alkylating reagents, sites predominantly lining the cytosolic half of the TM3 helix were found to be solvent accessible (i.e., S128C, L143C-T149C). Analysis of substrate kinetics for select TM3 mutants demonstrates significant loss of taurocholic acid affinity for mutants S128C and L145C-T149C. Overall, we conclude (i) the functional and structural importance of P142 during the transport cycle and (ii) the presence of a large hydrophilic cleft region lining the cytosolic half of TM3 that may form portions of the substrate exit route during permeation. Our studies provide unique insight into molecular mechanisms guiding the ASBT transport cycle with respect to substrate binding and translocation events. PMID:19653651

Hussainzada, Naissan; Claro Da Silva, Tatiana; Swaan, Peter W

2009-09-15

245

Suppressed decay of a laterally confined persistent spin helix  

NASA Astrophysics Data System (ADS)

We experimentally investigate the dynamics of a persistent spin helix in etched GaAs wire structures of 2 -80 ? m width. Using magneto-optical Kerr rotation with high spatial resolution, we determine the lifetime of the spin helix. A few nanoseconds after locally injecting spin polarization into the wire, the polarization is strongly enhanced as compared to the two-dimensional case. This is mostly attributed to a transition to one-dimensional diffusion, strongly suppressing diffusive dilution of spin polarization. The intrinsic lifetime of the helical mode is only weakly increased, which indicates that the channel confinement can only partially suppress the cubic Dresselhaus spin-orbit interaction.

Altmann, P.; Walser, M. P.; Reichl, C.; Wegscheider, W.; Salis, G.

2014-11-01

246

Cytochromes b561: Ascorbate-Mediated Trans-Membrane Electron Transport  

PubMed Central

Abstract Significance: Cytochromes b561 (CYB561s) constitute a family of trans-membrane (TM), di-heme proteins, occurring in a variety of organs and cell types, in plants and animals, and using ascorbate (ASC) as an electron donor. CYB561s function as monodehydroascorbate reductase, regenerating ASC, and as Fe3+-reductases, providing reduced iron for TM transport. A CYB561-core domain is also associated with dopamine ?-monooxygenase redox domains (DOMON) in ubiquitous CYBDOM proteins. In plants, CYBDOMs form large protein families. Physiological functions supported by CYB561s and CYBDOMs include stress defense, cell wall modifications, iron metabolism, tumor suppression, and various neurological processes, including memory retention. CYB561s, therefore, significantly broaden our view on the physiological roles of ASC. Recent Advances: The ubiquitous nature of CYB561s is only recently being recognized. Significant advances have been made through the study of recombinant CYB561s, revealing structural and functional properties of a unique “two-heme four-helix” protein configuration. In addition, the DOMON domains of CYBDOMs are suggested to contain another heme b. Critical Issues: New CYB561 proteins are still being identified, and there is a need to provide an insight and overview on the various roles of these proteins and their structural properties. Future Directions: Mutant studies will reveal in greater detail the mechanisms by which CYB561s and CYBDOMs participate in cell metabolism in plants and animals. Moreover, the availability of efficient heterologous expression systems should allow protein crystallization, more detailed (atomic-level) structural information, and insights into the intra-molecular mechanism of electron transport. Antioxid. Redox Signal. 19, 1026–1035. PMID:23249217

Barbaro, Raffaella; Trost, Paolo; Bérczi, Alajos

2013-01-01

247

Design of Coaxial Couplers for High Efficiency Helix TWT  

NASA Astrophysics Data System (ADS)

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

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

2008-12-01

248

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

NASA Astrophysics Data System (ADS)

Gramicidin A is a linear hydrophobic 15-residue peptide which consists of alternating D- and L-amino acids and forms a unique tertiary structure, called the ?6.3-helix, to act as a cation-selective ion channel in the natural conditions. In order to investigate the intrinsic ability of the gramicidin A monomer to form secondary structures, we performed the folding simulation of gramicidin A using a simulated annealing molecular dynamics (MD) method in vacuum mimicking the low-dielectric, homogeneous membrane environment. The initial conformation was a fully extended one. From the 200 different MD runs, we obtained a right-handed ?4.4-helix as the lowest-potential-energy structure, and left-handed ?4.4-helix, right-handed and left-handed ?6.3-helix as local-minimum energy states. These results are in accord with those of the experiments of gramicidin A in homogeneous organic solvent. Our simulations showed a slight right-hand sense in the lower-energy conformations and a quite ?-sheet-forming tendency throughout almost the entire sequence. In order to examine the stability of the obtained right-handed ?6.3-helix and ?4.4-helix structures in more realistic membrane environment, we have also performed all-atom MD simulations in explicit water, ion, and lipid molecules, starting from these ?-helix structures. The results suggested that ?6.3-helix is more stable than ?4.4-helix in the inhomogeneous, explicit membrane environment, where the pore water and the hydrogen bonds between Trp side-chains and lipid-head groups have a role to further stabilize the ?6.3-helix conformation.

Mori, Takaharu; Okamoto, Yuko

2009-10-01

249

Circular dichroism, molecular modeling, and serology indicate that the structural basis of antigenic variation in foot-and-mouth disease virus is [alpha]-helix formation  

SciTech Connect

Seven antigenic variants obtained from a single field isolate of foot-and-mouth disease virus, serotype A12, differ only at residues 148 and 153 in the immunodominant loop of viral protein VP1. Synthetic peptides corresponding to the region 141-160 are highly immunogenic. UV circular dichroism shows that (i) in aqueous solution of the peptides are nearly identical, but in 100% trifluoroethanol they display helix-forming properties which correlate well with their serological crossreactivities for anti-peptide sera, and (ii) these properties are insensitive to substitutions at position 153, except for proline, but are highly sensitive to substitutions at position 148. This pattern can be explained by the effects of these substitutions on the amphiphilic character and positions of helices postulated in the region 146-156. Molecular models indicate that residues 147, 148, 150, 151, 153-155, and 157 are most likely to interact with residues of the antibody paratopes. The data are consistent with the existence of an inverse [gamma]-turn around Pro-153, and a [beta]-turn at the cell-attachment site at residues 145-147. 31 refs., 5 figs.

France, L.L.; Piatti, P.G.; Newman, J.F.E.; Brown, F. (Plum Island Animal Disease Center, Greenport, NY (United States)); Toth, I.; Gibbons, W.A. (Univ. of London (United Kingdom))

1994-08-30

250

DNA-The Double Helix - Nobel Prize Educational Game  

NSDL National Science Digital Library

The 1962 Nobel Prize in Physiology or Medicine was awarded for the discovery of the molecular structure of DNA ÃÂ 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.

2009-01-01

251

Unravelling the therapeutic potential of transmembrane peptides.  

E-print Network

??In eukaryotic cells, membrane spanning proteins are tethered to the lipid bilayer mostly through ?-helical transmembrane (TM) domains. These membrane-embedded sequences are capable of interacting… (more)

De Zan, E.

2014-01-01

252

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

PubMed

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

Wang, Wuyang; Linsdell, Paul

2012-03-23

253

Identification of Amino Acids Essential for Estrone-3-Sulfate Transport within Transmembrane Domain 2 of Organic Anion Transporting Polypeptide 1B1  

PubMed Central

As an important structure in membrane proteins, transmembrane domains have been found to be crucial for properly targeting the protein to cell membrane as well as carrying out transport functions in transporters. Computer analysis of OATP sequences revealed transmembrane domain 2 (TM2) is among those transmembrane domains that have high amino acid identities within different family members. In the present study, we identify four amino acids (Asp70, Phe73, Glu74, and Gly76) that are essential for the transport function of OATP1B1, an OATP member that is specifically expressed in the human liver. A substitution of these four amino acids with alanine resulted in significantly reduced transport activity. Further mutagenesis showed the charged property of Asp70 and Glu74 is critical for proper function of the transporter protein. Comparison of the kinetic parameters indicated that Asp70 is likely to interact with the substrate while Glu74 may be involved in stabilizing the binding site through formation of a salt-bridge. The aromatic ring structure of Phe73 seems to play an important role because substitution of Phe73 with tyrosine, another amino acid with a similar structure, led to partially restored transport function. On the other hand, replacement of Gly76 with either alanine or valine could not recover the function of the transporter. Considering the nature of a transmembrane helix, we proposed that Gly76 may be important for maintaining the proper structure of the protein. Interestingly, when subjected to transport function analysis of higher concentration of esteone-3-sulfate (50 µM) that corresponds to the low affinity binding site of OATP1B1, mutants of Phe73, Glu74, and Gly76 all showed a transport function that is comparable to that of the wild-type, suggesting these amino acids may have less impact on the low affinity component of esteone-3-sulfate within OATP1B1, while Asp 70 seems to be involved in the interaction of both sites. PMID:22574206

Li, Nan; Hong, Weifang; Huang, Hong; Lu, Hanping; Lin, Guangyun; Hong, Mei

2012-01-01

254

A Specific Interface between Integrin Transmembrane Helices and Affinity for Ligand  

PubMed Central

Conformational communication across the plasma membrane between the extracellular and intracellular domains of integrins is beginning to be defined by structural work on both domains. However, the role of the ? and ? subunit transmembrane domains and the nature of signal transmission through these domains have been elusive. Disulfide bond scanning of the exofacial portions of the integrin ?II? and ?3 transmembrane domains reveals a specific heterodimerization interface in the resting receptor. This interface is lost rather than rearranged upon activation of the receptor by cytoplasmic mutations of the ? subunit that mimic physiologic inside-out activation, demonstrating a link between activation of the extracellular domain and lateral separation of transmembrane helices. Introduction of disulfide bridges to prevent or reverse separation abolishes the activating effect of cytoplasmic mutations, confirming transmembrane domain separation but not hinging or piston-like motions as the mechanism of transmembrane signaling by integrins. PMID:15208712

2004-01-01

255

Position of transmembrane helix 6 determines receptor G protein coupling specificity.  

PubMed

G protein coupled receptors (GPCRs) transmit extracellular signals into the cell by binding and activating different intracellular signaling proteins, such as G proteins (G???, families Gi, Gs, Gq, G12/13) or arrestins. To address the issue of Gs vs Gi coupling specificity, we carried out molecular dynamics simulations of lipid-embedded active ?2-adrenoceptor (?2AR*) in complex with C-terminal peptides derived from the key interaction site of G? (G?CT) as surrogate of G???. We find that Gi?CT and Gs?CT exploit distinct cytoplasmic receptor conformations that coexist in the uncomplexed ?2AR*. The slim Gi?CT stabilizes a ?2AR* conformation, not accessible to the bulkier Gs?CT, which requires a larger TM6 outward tilt for binding. Our results suggest that the TM6 conformational heterogeneity regulates the catalytic activity of ?2AR* toward Gi or Gs. PMID:25046433

Rose, Alexander S; Elgeti, Matthias; Zachariae, Ulrich; Grubmüller, Helmut; Hofmann, Klaus Peter; Scheerer, Patrick; Hildebrand, Peter W

2014-08-13

256

Supplementary Information High-accuracy prediction of transmembrane inter-helix contacts and  

E-print Network

;Table S3. Performance comparisons of feature level fusion versus decision level fusion. In the feature level fusion, we treat correlated mutation scores (CMs) as the input features for OET1 and SVM1 Feature level fusion; CMs are encoded as feature vectors fed

Zhang, Yang

257

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

E-print Network

% Triton X-100 - 5% Pyridine - 5% Acetic anhydride in NMP with 1% Triton X-100 5 minutes, flip stack, 5 minutes 2-6 hours 15 minutes, flip stack, 15 minutes This series of three reactions is repeated for every residue of the peptide. Cleavage from...

Funk, Geoffrey Alexander

2013-02-22

258

Evidence for an alpha-helix --> pi-bulge helicity modulation for the neu/erbB-2 membrane-spanning segment. A 1H NMR and circular dichroism study.  

PubMed

The 35-residue peptide corresponding to the very hydrophobic transmembrane region of the tyrosine kinase receptor neu, Neu(TM35), has been synthesized. The peptide can be solubilized in millimolar concentrations in TFE or incorporated into an SDS-water micellar solution or into well-hydrated DMPC/DCPC bicelles. In all these media, circular dichroism demonstrated that the peptide adopts a helical structure for about 80% of its amino acids. The peptide is monomeric below 2 mM in TFE, as also determined by variable concentration experiments. The three-dimensional solution structure in TFE has been obtained by homonuclear proton NMR and shows a well-defined alpha-helix from residues 4 to 21, then a pi-bulge from Ile(22) to Gly(28), and a final short alpha-helix from positions 29 to 32. This experimental finding is in agreement with structures predicted recently by molecular dynamics calculations in a vacuum [Sajot, N., and Genest, M. (2000) Eur. Biophys. J. 28, 648-662]. The biological implications of a possible retention of this structure in a membrane environment are finally discussed. PMID:11371217

Goetz, M; Carlotti, C; Bontems, F; Dufourc, E J

2001-05-29

259

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

PubMed Central

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

Salwi?ski, L.; Hubbell, W. L.

1999-01-01

260

Modeling of Peptides Connecting the Ligand Binding and Transmembrane Domains in the GluR2 Glutamate Receptor  

PubMed Central

Ligand-gated Glutamate receptors (GluR) mediate synaptic signals in the nervous system. Ionotropic GluRs of AMPA type, the subject of the present study, are tetrameric assemblies of monomer subunits, each of which is constructed in a modular fashion from functional subdomains. The extracellular ligand binding domain (LBD) changes its conformation upon binding of an agonist ligand followed by opening of a transmembrane (TM) ion channel. Peptides connecting the LBD and TM domains facilitate gating of the channel, and their structure and composition are important for the receptor functioning. In this study we used Replica Exchange Molecular Dynamics (REMD) simulations to model S1M1 and S2M3 connecting peptides of the GluR2 receptor in two implicit solvents, water and interfacial water/lipid medium characterized by lower polarity. Propensity of these peptides to form helical structures was analyzed using helicity measure derived from the free energy of the simulated ensembles of structures. The S1M1 and S2M3 connecting peptides were not helical in our simulations in both dielectric environments in the absence of the rest of the protein. The structures of the LBD fragment with known high resolution ?-helical structure and of the TM3 helix were successfully predicted in the simulations, which in part validate our results. The S2M3 peptide which is important in gating formed a well-defined coil structure and salt-bridges with the S2 domain. The S1M1 peptide formed a loop structure via formation of internal salt-bridges. Potential implications of these structures on function of the receptor are discussed. PMID:19205024

Speranskiy, K.; Kurnikova, M. G.

2010-01-01

261

Dispersion relations for a plasma-filled helix-loaded-waveguide  

SciTech Connect

The propagation of waves on bounded, magnetized plasma columns arises in connection with a variety of applications. To this end dispersion relations axe developed for a variety of multi-region circularly symmetric configurations. These include, a sheath helix in free space, a plasma column in free space, a plasma filled conducting tube, a plasma filled sheath-helix in free space, a sheath helix within a conducting cylinder, a plasma filled sheath-helix within a conducting cylinder, and a plasma column within a sheath-helix contained within a conducting cylinder. The latter configuration is of the most interest for whistler wave excitation for plasma thruster applications, since it includes the effect of a vacuum region separating the plasma column from the helical excitation structure.

Makowski, M.A.; Hooper, E.B.; Stallard, B.W.

1994-01-01

262

Type II Transmembrane Serine Proteases*  

PubMed Central

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

Bugge, Thomas H.; Antalis, Toni M.; Wu, Qingyu

2009-01-01

263

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

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.

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

264

Structure of a bacterial homologue of vitamin K epoxide reductase  

SciTech Connect

Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain {gamma}-carboxylation of many blood coagulation factors. Here, we report the 3.6 {angstrom} crystal structure of a bacterial homologue of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from cysteines of newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulphide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.

Li, Weikai; Schulman, Sol; Dutton, Rachel J.; Boyd, Dana; Beckwith, Jon; Rapoport, Tom A. (Harvard-Med); (HHMI)

2010-03-19

265

The first transmembrane domain (TM1) of ?2-subunit binds to the transmembrane domain S1 of ?-subunit in BK potassium channels  

PubMed Central

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming ?-subunit is associated to an auxiliary ?-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in ?-subunits that are important for the physical association with the ?-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the ?2-subunit are dispensable for association with the ?-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the ?2-subunit physically binds to the transmembrane S1 domain of the ?-subunit. PMID:22710124

Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

2012-01-01

266

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

PubMed

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

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

2009-04-01

267

Light Signal Transduction Pathway from Flavin Chromophore to the J? Helix of Arabidopsis Phototropin1  

PubMed Central

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

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

2009-01-01

268

Critical Exponents of Kappa Carrageenan in the Coil-Helix and Helix-Coil Hysteresis Loops  

Microsoft Academic Search

The steady-state fluorescence technique was used to study coil-helix (sol-gel) and helix-coil (gel-sol) transitions of the kappa carrageenan-water system with various carrageenan contents. Fluorescence (I) and scattered light (Isc) intensities were measured against temperature to determine critical phase transition temperatures and exponents. It was observed that the coil-helix transition temperatures, Tch were much lower than the helix-coil (Thc) transition temperatures

Özlem Tari; Selim Kara; Önder Pekcan

2009-01-01

269

Roles of carboxyl groups in the transmembrane insertion of peptides  

PubMed Central

We have used the pHLIP® peptide to study the roles of carboxyl groups in transmembrane peptide insertion. The pH (low) insertion peptide (pHLIP) binds to the surface of a lipid bilayer as a disordered peptide at neutral pH, and when the pH is lowered it inserts across the membrane to form a transmembrane helix. Peptide insertion is reversed when the pH is raised above the characteristic pKa (6.0). A key event facilitating the membrane insertion is the protonation of aspartic (Asp) and/or glutamic (Glu) acid residues, since at neutral pH their negatively charged side chains hinder membrane insertion. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ~pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. A further pH drop leads to the protonation of His residues in the TM part of peptide, which induces peptide exit from the bilayer. We also find that the number of ionizable residues that undergo a change in protonation during membrane insertion correlates with the pH-dependent insertion into and exit from the lipid bilayer, and that cooperativity increases with their number. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP peptides. PMID:21888917

Barrera, Francisco N.; Weerakkody, Dhammika; Anderson, Michael; Andreev, Oleg A.; Reshetnyak, Yana K.; Engelman, Donald M.

2011-01-01

270

The Origins of Transmembrane Ion Channels  

NASA Technical Reports Server (NTRS)

Even though membrane proteins that mediate transport of ions and small molecules across cell walls are among the largest and least understood biopolymers in contemporary cells, it is still possible to shed light on their origins and early evolution. The central observation is that transmembrane portions of most ion channels are simply bundles of -helices. By combining results of experimental and computer simulation studies on synthetic models and natural channels, mostly of non-genomic origin, we show that the emergence of -helical channels was protobiologically plausible, and did not require highly specific amino acid sequences. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. Specifically, we explain how the antiamoebin channels, which are made of identical helices, 16 amino acids in length, achieve efficiency comparable to that of highly evolved channels. We further show that antiamoebin channels are extremely flexible, compared to modern, genetically coded channels. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire stable rigid structures and mechanisms for precise regulation rather than improve efficiency. In general, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

Pohorille, Andrew; Wilson, Michael A.

2012-01-01

271

Crystal structures of an Extracytoplasmic Solute Receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimer requiring a cation for ?-keto acid binding  

PubMed Central

Background The import of solutes into the bacterial cytoplasm involves several types of membrane transporters, which may be driven by ATP hydrolysis (ABC transporters) or by an ion or H+ electrochemical membrane potential, as in the tripartite ATP-independent periplasmic system (TRAP). In both the ABC and TRAP systems, a specific periplasmic protein from the ESR family (Extracytoplasmic Solute Receptors) is often involved for the recruitment of the solute and its presentation to the membrane complex. In Rhodobacter sphaeroides, TakP (previously named SmoM) is an ESR from a TRAP transporter and binds ?-keto acids in vitro. Results We describe the high-resolution crystal structures of TakP in its unliganded form and as a complex with sodium-pyruvate. The results show a limited "Venus flytrap" conformational change induced by substrate binding. In the liganded structure, a cation (most probably a sodium ion) is present and plays a key role in the association of the pyruvate to the protein. The structure of the binding pocket gives a rationale for the relative affinities of various ligands that were tested from a fluorescence assay. The protein appears to be dimeric in solution and in the crystals, with a helix-swapping structure largely participating in the dimer formation. A 30 Å-long water channel buried at the dimer interface connects the two ligand binding cavities of the dimer. Conclusion The concerted recruitment by TakP of the substrate group with a cation could represent a first step in the coupled transport of both partners, providing the driving force for solute import. Furthermore, the unexpected dimeric structure of TakP suggests a molecular mechanism of solute uptake by the dimeric ESR via a channel that connects the binding sites of the two monomers. PMID:17362499

Gonin, Sophie; Arnoux, Pascal; Pierru, Bénédicte; Lavergne, Jérôme; Alonso, Béatrice; Sabaty, Monique; Pignol, David

2007-01-01

272

Effective Dielectric Constant Method for a Planar Helix With StraightEdge Connections  

Microsoft Academic Search

A new planar slow-wave structure consisting of a planar helix with straight-edge connections is presented. It is shown that the phase velocity of the proposed structure can be much slower than that for a rectangular helix with identical cross-sectional dimensions. Further, the dispersion characteristics are obtained simply by using the effective dielectric constant method; it is shown that the method

Ciersiang Chua; Sheel Aditya; Zhongxiang Shen

2009-01-01

273

Dynamics of alpha helix formation in the CSAW model  

NASA Astrophysics Data System (ADS)

We study the folding dynamics of polyalanine ( Ala20, a protein fragment with 20 residues whose native state is a single alpha helix. We use the CSAW model (conditioned self-avoiding walk), which treats the protein molecule as a chain in Brownian motion, with interactions that include hydrophobic force and internal hydrogen bonding. We find that large-scale structures form before small-scale structures, and obtain the relevant relaxation times. We find that helix nucleation occurs at two separate points on the protein chain, one near each end. The evolution of small- and large-scale structures involves different mechanisms. While the former can be described by rate equations that govern the growth of helical content, the latter is akin to the relaxation of an elastic solid. Supplementary material in the form of a pdf file available from the journal web page at 10.1140/epje/i2007-10371-4 and are accessible for authorised users.

Lei, J.; Huang, K.

2008-10-01

274

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

E-print Network

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

Apgar, James R. (James Reasoner)

2008-01-01

275

C11/C9 helices in crystals of ?? hybrid peptides and switching structures between helix types by variation in the ?-residue.  

PubMed

Close-packed helices with mixed hydrogen bond directionality are unprecedented in the structural chemistry of ?-polypeptides. While NMR studies in solution state provide strong evidence for the occurrence of mixed helices in (??)n and (??)n sequences, limited information is currently available in crystals. The peptide structures presented show the occurrence of C11/C9 helices in (??)n peptides. Transitions between C11 and C11/C9 helices are observed upon varying the ?-amino acid residue. PMID:25144906

Basuroy, Krishnayan; Karuppiah, Vasantham; Balaram, Padmanabhan

2014-09-01

276

Crystal Structure of the Interferon Gamma Receptor Alpha Chain from Chicken Reveals an Undetected Extra Helix Compared with the Human Counterparts  

PubMed Central

Interferon gamma (IFN-?) is an important cytokine that induces antiviral, antiproliferative, and immunomodulatory effects on target cells, and is also crucial in the early defense against intracellular parasites, such as Listeria monocytogenes and Toxoplasma gondii. The biological activity of IFN-? relies upon the formation of a complex with its 2 receptors, the interferon gamma alpha chain (IFNGR1) and beta chain (IFNGR2), which are type II cytokine receptors. Structural models of ligand–receptor interaction and complex structure of chicken IFNs with their receptors have remained elusive. Here we report the first structure of Gallus gallus (chicken) IFNGR1 (chIFNGR1) at 2.0 Å by molecule replacement according to the structure of selenomethionine substituted chIFNGR1. The structural comparison reveals its structural similarities with other class II cytokine receptors, despite divergent primary sequences. We further investigate the ligand–receptor interaction properties of chicken IFN-? (chIFN-?) and chIFNGR1 using size-exclusion chromatography and surface plasmon resonance techniques. These data aid in the understanding of the interaction of chicken (avian) IFN-? with its receptors and its signal transduction. PMID:24283193

Ping, Zhiguang; Qi, Jianxun; Sun, Yanling; Lu, Guangwen; Shi, Yi; Wang, Xiaojia

2014-01-01

277

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

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

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

278

Partitivirus structure reveals a 120-subunit, helix-rich capsid with distinctive surface arches formed by quasisymmetric coat-protein dimers.  

PubMed

Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3 A resolution. The capsid, approximately 350 A in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is, in turn, formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of alpha helices and consistent with secondary structure predictions for the coat protein. This three-dimensional structure of a virus from the family Partitiviridae exhibits both similarities to and differences from the so-called "T = 2" capsids of other dsRNA viruses. PMID:18462682

Ochoa, Wendy F; Havens, Wendy M; Sinkovits, Robert S; Nibert, Max L; Ghabrial, Said A; Baker, Timothy S

2008-05-01

279

Linus Pauling' s manuscript describing incorrect triple helix, Peter PaulingSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

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

2008-10-06

280

The Mtr4 ratchet helix and arch domain both function to promote RNA unwinding  

PubMed Central

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

Taylor, Lacy L.; Jackson, Ryan N.; Rexhepaj, Megi; King, Alejandra Klauer; Lott, Lindsey K.; van Hoof, Ambro; Johnson, Sean J.

2014-01-01

281

The Mtr4 ratchet helix and arch domain both function to promote RNA unwinding.  

PubMed

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

Taylor, Lacy L; Jackson, Ryan N; Rexhepaj, Megi; King, Alejandra Klauer; Lott, Lindsey K; van Hoof, Ambro; Johnson, Sean J

2014-12-16

282

The solution structure of the amino-terminal HHCC domain of HIV-2 integrase: a three-helix bundle stabilized by zinc  

E-print Network

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

Tullius, Thomas D.

283

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

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.

2008-10-06

284

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

PubMed Central

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

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

2014-01-01

285

Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1  

SciTech Connect

Cell proliferation and differentiation are precisely coordinated during the development and maturation of the mammary gland, and this balance invariably is disrupted during carcinogenesis. Little is known about the cell-specific transcription factors that regulate these processes in the mammary gland. The mouse mammary epithelial cell line SCp2 grows well under standard culture conditions but arrests growth, forms alveolus-like structures, and expresses {beta}-casein, a differentiation marker, 4 to 5 days after exposure to basement membrane and lactogenic hormones (differentiation signals). The authors show that this differentiation entails a marked decline in the expression of Id-1, a helix-loop-helix (HLH) protein that inactivates basic HLH transcription factors in other cell types. SCp2 cells stably transfected with an Id-1 expression vector grew more rapidly than control cells under standard conditions, but in response to differentiation signals, they lost three-dimensional organization, invaded the basement membrane, and then resumed growth. SCp2 cells expressing an Id-1 antisense vector grew more slowly than controls; in response to differentiation signals, they remained stably growth arrested and fully differentiated, as did control cells. The authors suggest that Id-1 renders cells refractory to differentiation signals and receptive to growth signals by inactivating one or more basic HLH proteins that coordinate growth and differentiation in the mammary epithelium. 53 refs., 6 figs.

Desprez, P.; Hara, E.; Bissell, M.J. [Lawrence Berkeley Lab., CA (United States)] [and others

1995-06-01

286

SOMRuler: a novel interpretable transmembrane helices predictor.  

PubMed

Transmembrane helices (TMH) identification is one of the most important steps in membrane protein structure prediction. Existing TMH predictors tend to pursue accurate computational models without carefully considering the interpretability of these models and thus act as a black box. In this paper, a novel TMH predictor called SOMRuler with excellent interpretability while possessing high prediction accuracy is presented. The SOMRuler uses a self-organizing map (SOM) to learn helices distribution knowledge, which is encoded in the codebook vectors of the trained SOM, from the training samples. Human interpretable fuzzy rules are then extracted from the codebook vectors of the trained SOM. By extracting fuzzy rules from the learned knowledge rather than the original training samples, on the one hand, the computational burden of extracting fuzzy rules can be greatly reduced; on the other hand, the reliability of the extracted rules can also be enhanced since noise contained in the original samples can be smoothened by the learning procedure of SOM. The validity of the fuzzy rules extracted by SOMRuler is qualitatively and quantitatively analyzed. Experimental results on the benchmark dataset show that the SOMRuler outperforms most existing popular TMH predictors and is flexible to suite for a wide variety of problems in bioinformatics. The SOMRuler software is implemented by Java and Matlab and is available for academic use at: http://www.csbio.sjtu.edu.cn/bioinf/SOMRuler/. PMID:21742571

Yu, Dongjun; Shen, Hongbin; Yang, Jingyu

2011-06-01

287

Partitivirus Structure Reveals a 120-Subunit, Helix-Rich Capsid with Distinctive Surface Arches Formed by Quasisymmetric Coat-Protein Dimers  

PubMed Central

SUMMARY Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3-Å resolution. The capsid, ~350 Å in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is in turn formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of ?-helices and consistent with secondary-structure predictions for the coat protein. This is the first three-dimensional structure of a virus from the family Partitiviridae and exhibits both similarities to and differences from the so-called “T=2” capsids of other dsRNA viruses. PMID:18462682

Ochoa, Wendy F.; Havens, Wendy M.; Sinkovits, Robert S.; Nibert, Max L.; Ghabrial, Said A.; Baker, Timothy S.

2008-01-01

288

Comparison of Class A and D G Protein-Coupled Receptors: Common Features in Structure and Activation†  

PubMed Central

All G protein-coupled receptors (GPCRs) share a common seven TM helix architecture and the ability to activate heterotrimeric G proteins. Nevertheless, these receptors have widely divergent sequences with no significant homology. We present a detailed structure–function comparison of the very divergent Class A and D receptors to address whether there is a common activation mechanism across the GPCR superfamily. The Class A and D receptors are represented by the vertebrate visual pigment rhodopsin and the yeast ?-factor pheromone receptor Ste2, respectively. Conserved amino acids within each specific receptor class and amino acids where mutation alters receptor function were located in the structures of rhodopsin and Ste2 to assess whether there are functionally equivalent positions or regions within these receptors. We find several general similarities that are quite striking. First, strongly polar amino acids mediate helix interactions. Their mutation generally leads to loss of function or constitutive activity. Second, small and weakly polar amino acids facilitate tight helix packing. Third, proline is essential at similar positions in transmembrane helices 6 and 7 of both receptors. Mapping the specific location of the conserved amino acids and sites of constitutively active mutations identified conserved microdomains on transmembrane helices H3, H6, and H7, suggesting that there are underlying similarities in the mechanism of the widely divergent Class A and Class D receptors. PMID:15966721

Eilers, Markus; Hornak, Viktor; Smith, Steven O.; Konopka, James B.

2005-01-01

289

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

SciTech Connect

The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 {angstrom} resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

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

2011-12-31

290

Primary structure of a novel subunit in ba3-cytochrome oxidase from Thermus thermophilus.  

PubMed Central

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

Soulimane, T.; Than, M. E.; Dewor, M.; Huber, R.; Buse, G.

2000-01-01

291

Splice isoform estrogen receptors as integral transmembrane proteins.  

PubMed

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

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

2011-11-01

292

pH-dependent helix folding dynamics of poly-glutamic acid  

NASA Astrophysics Data System (ADS)

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.

Gooding, Edward A.; Sharma, Sapna; Petty, Sarah A.; Fouts, Elizabeth A.; Palmer, Colin J.; Nolan, Brian E.; Volk, Martin

2013-08-01

293

Macroscopic control of helix orientation in films dried from cholesteric liquid-crystalline cellulose nanocrystal suspensions.  

PubMed

The intrinsic ability of cellulose nanocrystals (CNCs) to self-organize into films and bulk materials with helical order in a cholesteric liquid crystal is scientifically intriguing and potentially important for the production of renewable multifunctional materials with attractive optical properties. A major obstacle, however, has been the lack of control of helix direction, which results in a defect-rich, mosaic-like domain structure. Herein, a method for guiding the helix during film formation is introduced, which yields dramatically improved uniformity, as confirmed by using polarizing optical and scanning electron microscopy. By raising the CNC concentration in the initial suspension to the fully liquid crystalline range, a vertical helix orientation is promoted, as directed by the macroscopic phase boundaries. Further control of the helix orientation is achieved by subjecting the suspension to a circular shear flow during drying. PMID:24677344

Park, Ji Hyun; Noh, JungHyun; Schütz, Christina; Salazar-Alvarez, German; Scalia, Giusy; Bergström, Lennart; Lagerwall, Jan P F

2014-05-19

294

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

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

Miyashita, Naoyuki; Straub, John E.; Thirumalai, D.

2009-01-01

295

Motifs in Protein SequencesMotifs in Protein Sequences Examples: Helix-Turn-Helix, Zinc-finger,  

E-print Network

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

Narasimhan, Giri

296

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

PubMed Central

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

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

2009-01-01

297

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

PubMed Central

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

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

2014-01-01

298

Linus Pauling's triple DNA helix model, 3D animation with basic narrationSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

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

2008-10-06

299

Light Stress-Induced One-Helix Protein of the Chlorophyll a/b-Binding Family Associated with Photosystem I1  

PubMed Central

The superfamily of light-harvesting chlorophyll a/b-binding (Lhc) proteins in higher plants and green algae is composed of more than 20 different antenna proteins associated either with photosystem I (PSI) or photosystem II (PSII). Several distant relatives of this family with conserved chlorophyll-binding residues and proposed photoprotective functions are induced transiently under various stress conditions. Whereas “classical” Lhc proteins contain three-transmembrane ?-helices, their distant relatives span the membrane with between one and four transmembrane segments. Here, we report the identification and isolation of a novel member of the Lhc family from Arabidopsis with one predicted transmembrane ?-helix closely related to helix I of Lhc protein from PSI (Lhca4) that we named Ohp2 (for a second one-helix protein of Lhc family described from higher plants). We showed that the Ohp2 gene expression is triggered by light stress and that the Ohp2 transcript and protein accumulated in a light intensity-dependent manner. Other stress conditions did not up-regulate the expression of the Ohp2 gene. Localization studies revealed that Ohp2 is associated with PSI under low- or high-light conditions. Because all stress-induced Lhc relatives reported so far were found in PSII, we propose that the accumulation of Ohp2 might represent a novel photoprotective strategy induced within PSI in response to light stress. PMID:12805611

Andersson, Ulrica; Heddad, Mounia; Adamska, Iwona

2003-01-01

300

Structure of CrgA, a cell division structural and regulatory protein from Mycobacterium tuberculosis, in lipid bilayers.  

PubMed

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

Das, Nabanita; Dai, Jian; Hung, Ivan; Rajagopalan, Malini R; Zhou, Huan-Xiang; Cross, Timothy A

2015-01-13

301

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

PubMed Central

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

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

2010-01-01

302

Essential molecular determinants for thyroid hormone transport and first structural implications for monocarboxylate transporter 8.  

PubMed

Monocarboxylate transporter 8 (MCT8, SLC16A2) is a thyroid hormone (TH) transmembrane transport protein mutated in Allan-Herndon-Dudley syndrome, a severe X-linked psychomotor retardation. The neurological and endocrine phenotypes of patients deficient in MCT8 function underscore the physiological significance of carrier-mediated TH transmembrane transport. MCT8 belongs to the major facilitator superfamily of 12 transmembrane-spanning proteins and mediates energy-independent bidirectional transport of iodothyronines across the plasma membrane. Structural information is lacking for all TH transmembrane transporters. To gain insight into structure-function relations in TH transport, we chose human MCT8 as a paradigm. We systematically performed conventional and liquid chromatography-tandem mass spectrometry-based uptake measurements into MCT8-transfected cells using a large number of compounds structurally related to iodothyronines. We found that human MCT8 is specific for L-iodothyronines and requires at least one iodine atom per aromatic ring. Neither thyronamines, decarboxylated metabolites of iodothyronines, nor triiodothyroacetic acid and tetraiodothyroacetic acid, TH derivatives lacking both chiral center and amino group, are substrates for MCT8. The polyphenolic flavonoids naringenin and F21388, potent competitors for TH binding at transthyretin, did not inhibit T(3) transport, suggesting that MCT8 can discriminate its ligand better than transthyretin. Bioinformatic studies and a first molecular homology model of MCT8 suggested amino acids potentially involved in substrate interaction. Indeed, alanine mutation of either Arg(445) (helix 8) or Asp(498) (helix 10) abrogated T(3) transport activity of MCT8, supporting their predicted role in substrate recognition. The MCT8 model allows us to rationalize potential interactions of amino acids including those mutated in patients with Allan-Herndon-Dudley syndrome. PMID:20628049

Kinne, Anita; Kleinau, Gunnar; Hoefig, Carolin S; Grüters, Annette; Köhrle, Josef; Krause, Gerd; Schweizer, Ulrich

2010-09-01

303

Fluctuations in the DNA double helix  

NASA Astrophysics Data System (ADS)

DNA is not the static entity suggested by the famous double helix structure. It shows large fluctuational openings, in which the bases, which contain the genetic code, are temporarily open. Therefore it is an interesting system to study the effect of nonlinearity on the physical properties of a system. A simple model for DNA, at a mesoscopic scale, can be investigated by computer simulation, in the same spirit as the original work of Fermi, Pasta and Ulam. These calculations raise fundamental questions in statistical physics because they show a temporary breaking of equipartition of energy, regions with large amplitude fluctuations being able to coexist with regions where the fluctuations are very small, even when the model is studied in the canonical ensemble. This phenomenon can be related to nonlinear excitations in the model. The ability of the model to describe the actual properties of DNA is discussed by comparing theoretical and experimental results for the probability that base pairs open an a given temperature in specific DNA sequences. These studies give us indications on the proper description of the effect of the sequence in the mesoscopic model.

Peyrard, M.; López, S. C.; Angelov, D.

2007-08-01

304

Unprecedented chain-length-dependent conformational conversion between 11/9 and 18/16?helix in ?/?-hybrid peptides.  

PubMed

?,?-Hybrid oligomers of varying lengths with alternating proteogenic ?-amino acid and the rigid ?(2,3,3) -trisubstituted bicyclic amino acid ABOC residues were studied using both X-ray crystal and NMR solution structures. While only an 11/9?helix was obtained in the solid state regardless of the length of the oligomers, conformational polymorphism as a chain-length-dependent phenomenon was observed in solution. Consistent with DFT calculations, we established that short oligomers adopted an 11/9?helix, whereas an 18/16?helix was favored for longer oligomers in solution. A rapid interconversion between the 11/9?helix and the 18/16?helix occurred for oligomers of intermediate length. PMID:25258017

Legrand, Baptiste; André, Christophe; Moulat, Laure; Wenger, Emmanuel; Didierjean, Claude; Aubert, Emmanuel; Averlant-Petit, Marie Christine; Martinez, Jean; Calmes, Monique; Amblard, Muriel

2014-11-24

305

Seven-helix bundles: molecular modeling via restrained molecular dynamics.  

PubMed Central

Simulated annealing via restrained molecular dynamics (SA/MD) has been used to model compact bundles of seven approximately (anti)parallel alpha-helices. Seven such helix bundles occur, e.g., in bacteriorhodopsin, in rhodopsin, and in the channel-forming N-terminal domain of Bacillus thuringiensis delta-endotoxin. Two classes of model are considered: (a) those consisting of seven Ala20 peptide chains; and (b) those containing a single polypeptide chain, made up of seven Ala20 helices linked by GlyN interhelix loops (where N = 5 or 10). Three different starting C alpha templates for SA/MD are used, in which the seven helices are arranged (a) on a left-handed circular template, (b) on a bacteriorhodopsin-like template, or (c) on a zig-zag template. The ensembles of models generated by SA/MD are analyzed in terms of their geometry and energetics, and the most stable structures from each ensemble are examined in greater detail. Structures resembling bacteriorhodopsin and structures resembling delta-endotoxin are both represented among the most stable structures. delta-Endotoxin-like structures arise from both circular and bacteriorhodopsin-like C alpha templates. A third helix-packing mode occurs several times among the stable structures, regardless of the C alpha template and of the presence or absence of interhelix loops. It is characterized by a "4 + 1" core, in which four helices form a distorted left-handed supercoil around a central, buried helix. The remaining two helices pack onto the outside of the core. This packing mode is comparable with that proposed for rhodopsin on the basis of two-dimensional electron crystallographic and sequence analysis studies. Images FIGURE 1 FIGURE 4 FIGURE 6 PMID:7787019

Sansom, M S; Son, H S; Sankararamakrishnan, R; Kerr, I D; Breed, J

1995-01-01

306

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

Microsoft Academic Search

In this study we evaluated the differences between concentrations of copper, iron, zinc, manganese, lead and cadmium in three terrestrial gastropods: Helix pomatia, Helix aspersa and Arion rufus, collected in a semi-rural location in Northern Italy. Metal concentrations in the foot and in the digestive gland were measured. In the hepatopancreas, copper and zinc did not differ significantly in the

C Menta; V Parisi

2001-01-01

307

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

NASA Astrophysics Data System (ADS)

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

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

2003-12-01

308

Introduction The cystic fibrosis transmembrane conductance regu-  

E-print Network

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

Engelhardt, John F.

309

Expression of the helix-loop-helix genes Id-1 and NSCL-1 during cerebellar development.  

PubMed

Neurons throughout the central nervous system (CNS) undergo proliferation, migration, and differentiation during their histogenesis. Although numerous regulatory molecules are expressed in developing neurons, it is unknown whether most of these molecules have the same function throughout the CNS or play different roles in different neuronal populations. Previous studies have shown that Id-1 and NSCL-1 are expressed at high levels in the ventricular and subependymal zones, respectively, of the embryonic brain. In the present study, the expression of Id-1 and NSCL-1 was further investigated during postnatal development of the cerebellum. By Northern blot hybridization analysis, the expression levels of Id-1 and NSCL-1 mRNA were developmentally regulated in the cerebellum, with the highest mRNA levels coinciding with the time of maximal granule cell histogenesis. By in situ hybridization, NSCL-1 mRNA was found in the premigratory zone of the external granule layer (EGL), a structure developmentally analogous to the subependymal zone of the embryonic brain. In normal mice, Id-1 mRNA was found to be transiently expressed in the upper internal granule layer (IGL), a population of cells that recently completed their migration from the EGL. In the mouse mutant weaver, Id mRNA was only seen in granule cells that have reached their normal positions in the IGL. No Id-1 hybridization signal was observed in the large numbers of granule cells remaining in the EGL of weaver mice, indicating that Id-1 expression is controlled by spatial cues. The lack of Id-1 expression in ectopic weaver granule cells is compatible with previous suggestions of arrested differentiation. These results support the idea that transcriptional regulators of the helix-loop-helix gene family play important roles in neuronal development, exhibiting region-specific expression and function. PMID:8989525

Duncan, M K; Bordas, L; Dicicco-Bloom, E; Chada, K K

1997-01-01

310

The C-Terminal RpoN Domain of sigma54 Forms an unpredictedHelix-Turn-Helix Motif Similar to domains of sigma70  

SciTech Connect

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.

Doucleff, Michaeleen; Malak, Lawrence T.; Pelton, Jeffrey G.; Wemmer, David E.

2005-11-01

311

Targeting of Drosophila Rhodopsin Requires Helix 8 but Not the Distal C-Terminus  

PubMed Central

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

Kock, Ines; Bulgakova, Natalia A.; Knust, Elisabeth; Sinning, Irmgard; Panneels, Valérie

2009-01-01

312

50?Hz Electromagnetic Field Produced Changes in FTIR Spectroscopy Associated with Mitochondrial Transmembrane Potential Reduction in Neuronal-Like SH-SY5Y Cells  

PubMed Central

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

Calabrò, Emanuele; Condello, Salvatore; Currò, Monica; Ferlazzo, Nadia; Vecchio, Mercurio; Caccamo, Daniela; Magazù, Salvatore; Ientile, Riccardo

2013-01-01

313

The Transmembrane Domain C of AMPA Receptors is Critically Involved in Receptor Function and Modulation  

PubMed Central

Ionotropic glutamate receptors are major players in synaptic transmission and are critically involved in many cognitive events. Although receptors of different subfamilies serve different functions, they all show a conserved domain topology. For most of these domains, structure–function relationships have been established and are well understood. However, up to date the role of the transmembrane domain C in receptor function has been investigated only poorly. We have constructed a series of receptor chimeras and point mutants designed to shed light on the structural and/or functional importance of this domain. We here present evidence that the role of transmembrane domain C exceeds that of a mere scaffolding domain and that several amino acid residues located within the domain are crucial for receptor gating and desensitization. Furthermore, our data suggest that the domain may be involved in receptor interaction with transmembrane AMPA receptor regulatory proteins. PMID:21206529

Terhag, Jan; Gottschling, Kevin; Hollmann, Michael

2010-01-01

314

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

PubMed

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

Baker, Michael E; Uh, Kayla Y

2012-10-01

315

Epicyclic Twin-Helix Ionization Cooling Simulations  

SciTech Connect

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.

Vasiliy Morozov, Yaroslav Derbenev, A. Afanaciev, R.P. Johnson

2011-04-01

316

Nucleic acid helix-coil transitions mediated by helix-unwinding proteins from calf thymus.  

PubMed

We have studied nucleic acid double helix destabilization mediated by purified calf helix-unwinding proteins, measuring ultraviolet hyperchromicity to detect helix melting. Both calf unwinding protein 1 (UP1) and a high salt eluting protein fraction are found to depress strongly the helix melting temperature (Tm) of the synthetic alternating copolymers poly[d(AT)] and poly[r(AU)], indicating that both DNA and RNA are recognized by these proteins. UP1 also destabilizes natural, GC-containing DNA helices, but to a smaller extent than observed with the above polymers. A simple model is presented to aid in the qualitative interpretation of the data, outlining the expected effect on the helix-coil transition of a protein ligand with differential affinity for the helix or coil form of nucleic acid. The observed helix-destabilizing effect of UP1 is dependent on the protein to nucleic acid ratio in an expected manner. Competition studies demonstrate a low, but appreciable affinity of UP1 for native DNA, opening the possibility that protein-mediated denaturation might be initiated by protein binding to the double helix. "Hairpin" helical regions of denatured DNA are strongly destabilized by UP1. Despite the fact that removal of these hairpin helices might greatly facilitate DNA renaturation, we failed to observe renaturation from the UP1-DNA complex after a switch to helix-stabilizing conditions. Thus, UP1 shows an important difference from its presumed prokaryotic analogue, T4 gene 32-protein. Possible in vivo functions of the calf proteins are discussed in light of these observations. PMID:1270426

Herrick, G; Alberts, B

1976-04-10

317

PD-118057 contacts the pore helix of hERG1 channels to attenuate inactivation and enhance K+ conductance  

PubMed Central

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

Perry, Matthew; Sachse, Frank B.; Abbruzzese, Jennifer; Sanguinetti, Michael C.

2009-01-01

318

Localized Lipid Packing of Transmembrane Domains Impedes Integrin Clustering  

PubMed Central

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

Mehrbod, Mehrdad; Mofrad, Mohammad R. K.

2013-01-01

319

Heat conductivity of the DNA double helix  

NASA Astrophysics Data System (ADS)

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

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

2011-06-01

320

Modeling Mechanotunable Transmembrane Transport in Lipid Vesicles  

NASA Astrophysics Data System (ADS)

Using Dissipative Particle Dynamics approach, we study the effects of applied stress on transmembrane transport in lipid vesicles. The lipids comprising the vesicle are composed of a hydrophilic head group and two hydrophobic tails. The vesicle is immersed into the hydrophilic solution and initially contains a number of amphiphilic species inside its cavity. We show that such enclosed species can be released ``on demand'' by vesicle's stretching. We find that the magnitude of the external force required to release the vesicle's content depends on the chemical nature and volume fraction of the enclosed species. Furthermore, we isolate the scenarios where the stretching of the lipid vesicle depleted of the enclosed species results in its ``refilling'' with the fresh species from the outer solution. Our results illustrate that applied mechanical stress provides an effective means to fine tune the transmembrane transport in lipid vesicles.

Salib, Isaac G.; Kuksenok, Olga; Balazs, Anna C.

2012-02-01

321

Tunable single photonic defect-mode in cholesteric liquid crystals with laser-induced local modifications of helix  

SciTech Connect

The authors demonstrate a tunable single photonic defect-mode in a single cholesteric liquid crystal material based on a structural defect introduced by local modification of the helix. An unpolymerized region of cholesteric liquid crystal acting as the defect was left between two polymerized regions via a two-photon excitation laser-lithography process. Upon polymerization, the cholesteric liquid crystal helix elongated and became thermally stable, and a single photonic defect mode was exhibited due to the contrast in the helix pitch at the defect. The defect mode showed tunability upon heating, and a 36 nm redshift was seen over a temperature range of 30 deg. C.

Yoshida, Hiroyuki; Lee, Chee Heng; Fujii, Akihiko; Ozaki, Masanori [Department of Electrical, Electronic and Informational Engineering, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871 (Japan)

2006-12-04

322

Transmembrane Domain II of the Human Bile Acid Transporter SLC10A2 Coordinates Sodium Translocation*  

PubMed Central

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

Sabit, Hairat; Mallajosyula, Sairam S.; MacKerell, Alexander D.; Swaan, Peter W.

2013-01-01

323

Probing the non-native H helix translocation in apomyoglobin folding intermediates.  

PubMed

Apomyoglobin folds via sequential helical intermediates that are formed by rapid collapse of the A, B, G, and H helix regions. An equilibrium molten globule with a similar structure is formed near pH 4. Previous studies suggested that the folding intermediates are kinetically trapped states in which folding is impeded by non-native packing of the G and H helices. Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates. Förster resonance energy transfer measurements show that a 5-({2-[(acetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid acceptor coupled to K140C (helix H) is closer to Trp14 (helix A) in the equilibrium molten globule than in the native state, by a distance that is consistent with sliding of the H helix in an N-terminal direction by approximately one helical turn. Formation of an S108C-L135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register. By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate. The apomyoglobin folding landscape is highly rugged, with several energetic bottlenecks that frustrate folding; relief of any one of the major identified bottlenecks is insufficient to speed progression to the transition state. PMID:24857522

Aoto, Phillip C; Nishimura, Chiaki; Dyson, H Jane; Wright, Peter E

2014-06-17

324

A Triple Helix-Loop-Helix/Basic Helix-Loop-Helix Cascade Controls Cell Elongation Downstream of Multiple Hormonal and Environmental Signaling Pathways in Arabidopsis[C][W  

PubMed Central

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

Bai, Ming-Yi; Fan, Min; Oh, Eunkyoo; Wang, Zhi-Yong

2012-01-01

325

Helix–loop–helix/basic helix–loop–helix transcription factor network represses cell elongation in Arabidopsis through an apparent incoherent feed-forward loop  

PubMed Central

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

Zhiponova, Miroslava K.; Morohashi, Kengo; Vanhoutte, Isabelle; Machemer-Noonan, Katja; Revalska, Miglena; Van Montagu, Marc; Grotewold, Erich; Russinova, Eugenia

2014-01-01

326

A triple helix-loop-helix/basic helix-loop-helix cascade controls cell elongation downstream of multiple hormonal and environmental signaling pathways in Arabidopsis.  

PubMed

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

Bai, Ming-Yi; Fan, Min; Oh, Eunkyoo; Wang, Zhi-Yong

2012-12-01

327

MODELS OF THE ACTIN-LIKE MREB HELIX IN PROKARYOTES  

E-print Network

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

Allard, Jun

328

Helix Nebula (NGC 7293) www.nasa.gov  

E-print Network

Helix Nebula (NGC 7293) www.nasa.gov National Aeronautics and Space Administration #12;Helix Nebula (NGC 7293) www.nasa.gov This Galaxy Evolution Explorer (GALEX) ultraviolet image shows the Helix Nebula (also called NGC 7293), one of the largest and oldest planetary nebulae known. It is quite close to us

329

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

Microsoft Academic Search

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

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

2007-01-01

330

Autoimmunogenicity of the helix-loop-helix DNA-binding domain  

Microsoft Academic Search

Nonimmunogenic character of native DNA, and its high immunogenicity when presented in complex with the DNA-binding proteins indicate that the latter might contain molecular triggers of anti-DNA response. To find if this is the case, we have evaluated the autoimmunogenic potential of the main DNA-binding domain of HIV-1 reverse transcriptase that belongs to the canonical helix-loop-helix type. BALB\\/c mice were

Natalia Petrakova; Lindvi Gudmundsdotter; Maryna Yermalovich; Sergey Belikov; Lars Eriksson; Pawan Pyakurel; Olle Johansson; Peter Biberfeld; Sören Andersson; Maria Isaguliants

2009-01-01

331

Evolutionary aspects of developmentally regulated helix-loop-helix transcription factors in striated muscle of jellyfish  

Microsoft Academic Search

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

Peter Müller; Katja Seipel; Nathalie Yanze; Susanne Reber-Müller; Ruth Streitwolf-Engel; Michael Stierwald; J. ürg Spring; Volker Schmid

2003-01-01

332

DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy  

PubMed Central

O6-alkylguanine-DNA alkyltransferase (AGT) is a crucial target both for the prevention of cancer and for chemotherapy, since it repairs mutagenic lesions in DNA, and it limits the effectiveness of alkylating chemotherapies. AGT catalyzes the unique, single-step, direct damage reversal repair of O6-alkylguanines by selectively transferring the O6-alkyl adduct to an internal cysteine residue. Recent crystal structures of human AGT alone and in complex with substrate DNA reveal a two-domain a/? fold and a bound zinc ion. AGT uses its helix-turn-helix motif to bind substrate DNA via the minor groove. The alkylated guanine is then flipped out from the base stack into the AGT active site for repair by covalent transfer of the alkyl adduct to Cys145. An asparagine hinge (Asn137) couples the helix-turn-helix DNA binding and active site motifs. An arginine finger (Arg128) stabilizes the extrahelical DNA conformation. With this newly improved structural understanding of AGT and its interactions with biologically relevant substrates, we can now begin to unravel the role it plays in preserving genetic integrity and discover how it promotes resistance to anticancer therapies. PMID:17485252

Tubbs, Julie L.; Pegg, Anthony E.; Tainer, John A.

2007-01-01

333

Porous silicon membrane for investigation of transmembrane proteins  

NASA Astrophysics Data System (ADS)

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

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

2013-06-01

334

Helix bending as a factor in protein/DNA recognition.  

PubMed

Normal vectors perpendicular to individual base pairs are a powerful tool for studying the bending behavior of B-DNA, both in the form of normal vector plots and in matrices that list angles between vectors for all possible base pair combinations. A new analysis program, FREEHELIX, has been written for this purpose, and applied to 86 examples of sequence-specific protein/DNA complexes whose coordinates are on deposit in the Nucleic Acid Data Base. Bends in this sample of 86 structures almost invariably follow from roll angles between adjacent base pairs; tilt makes no net contribution. Roll in a direction compressing the broad major groove is much more common than that which compresses the minor groove. Three distinct types of B-DNA bending are observed, each with a different molecular origin: (1) Localized kinking is produced by large roll at single steps or at two steps separated by one turn of helix. (2) Smooth, planar curvature is produced by positive and negative roll angles spaced a half-turn apart, with random side-to-side zigzag roll at intermediate points, rather than a tilt contribution that might have been expected theoretically. (3) Three-dimensional writhe results from significant roll angles at a continuous series of steps. Writhe need not change the overall direction of helix axis, if it is continued indefinitely or for an integral number of helical turns. A-DNA itself can be formally considered as possessing uniform, continuous writhe that yields no net helix bending. Smooth curvature is the most intricate deformation of the three, and is least common. Writhe is the simplest deformation and is most common; indeed, a low level of continuous writhe is the normal condition of an otherwise unbent B-DNA helix of general sequence. With one exception, every example of major kinking in this sample of 86 structures involves a pyrimidine-purine step: C-A/T-G, T-A, or C-G. Purine-purine steps, especially A-A, show the least tendency toward roll deformations. PMID:9782776

Dickerson, R E; Chiu, T K

1997-01-01

335

Low complexity and disordered regions of proteins have different structural and amino acid preferences.  

PubMed

Low complexity regions (LCRs) or non-random regions of a few amino acids are abundantly present in proteins. LCRs are traditionally considered as floppy structures with high solvent accessibility. Thus little attention was paid to them for structural studies. However LCRs have been found to contain information relevant to protein structure and various important functions. The present study is an attempt to understand the structural trend of LCRs. Here we report a study conducted to understand the structural trend, solvent accessibility and amino acid preferences of LCRs. The results show that LCRs might attain any type of secondary structure; however, the helix is frequently seen, whereas sheets occur rarely. We also found that LCRs are not always exposed on the surface. We found insignificant contribution of trans-membrane helices to the overall helix content. The LCRs having a secondary structure have different enrichment and depletion of amino acids from LCRs without a secondary structure and disordered protein sequences. However, LCRs of NMR structures showed compositional and functional similarity to the disordered regions of proteins. We also noted that in ?3/4 LCRs, the entire amino acid did not have a single structural class, but rather an ensemble of more than one secondary structure, which indicates that they are found at places where structure transition occurs. Overall analysis suggests that the overall protein sequence has a greater influence on the structural and sequence enrichment rather than only the local amino acid composition of LCRs. PMID:25468592

Kumari, Bandana; Kumar, Ravindra; Kumar, Manish

2015-02-20

336

Two helix DNA binding motif of CAP found in lac repressor and gal repressor.  

PubMed Central

Comparison of both the DNA and protein sequences of catabolite gene activator protein (CAP) with the sequences of lac and gal repressors shows significant homologies between a sequence that forms a two alpha-helix motif in CAP and sequences near the amino terminus of both repressors. This two-helix motif is thought to be involved in specific DNA sequence recognition by CAP. The region in lac repressor to which CAP is homologous contains many i-d mutations that are defective in DNA binding. Less significant sequence homologies between CAP and phage repressors and activators are also shown. The amino acid residues that are critical to the formation of the two-helix motif are conserved, while those residues expected to interact with DNA are variable. These observations suggest the lac and gal repressors also have a two alpha-helix structural motif which is involved in DNA binding and that this two helix motif may be generally found in many bacterial and phage repressors. We conclude that one major mechanism by which proteins can recognize specific base sequences in double stranded DNA is via the amino acid side chains of alpha-helices fitting into the major groove of B-DNA. PMID:6897114

Weber, I T; McKay, D B; Steitz, T A

1982-01-01

337

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

PubMed

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

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

2014-10-01

338

Left handed ? helix models for mammalian prion fibrils  

PubMed Central

We propose models for in vitro grown mammalian prion protein fibrils based upon left handed beta helices formed both from the N-terminal and C-terminal regions of the proteinase resistant infectious prion core. The C-terminal threading onto a ?-helical structure is almost uniquely determined by fixing the cysteine disulfide bond on a helix corner. In comparison to known left handed helical peptides, the resulting model structures have similar stability attributes including relatively low root mean square deviations in all atom molecular dynamics, substantial side-chain-to-side-chain hydrogen bonding, good volume packing fraction, and low hydrophilic/hydrophobic frustration. For the N-terminus, we propose a new threading of slightly more than two turns, which improves upon the above characteristics relative to existing three turn ?-helical models. The N-terminal and C-terminal beta helices can be assembled into eight candidate models for the fibril repeat units, held together by large hinge (order 30 residues) domain swapping, with three amenable to fibril promoting domain swapping via a small (five residue) hinge on the N-terminal side. Small concentrations of the metastable C-terminal ? helix in vivo might play a significant role in templating the infectious conformation and in enhancing conversion kinetics for inherited forms of the disease and explain resistance (for canines) involving hypothesized coupling to the methionine 129 sulfur known to play a role in human disease. PMID:19098440

Kunes, Kay C; Clark, Scott C; Singh, Rajiv RP

2008-01-01

339

Role of solvation in pressure-induced helix stabilization  

NASA Astrophysics Data System (ADS)

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.

Best, Robert B.; Miller, Cayla; Mittal, Jeetain

2014-12-01

340

Role of solvation in pressure-induced helix stabilization.  

PubMed

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. PMID:25494793

Best, Robert B; Miller, Cayla; Mittal, Jeetain

2014-12-14

341

DNA helix, still imageSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

Image depicting DNA helix model and table. In 1953, Watson and Crick published a paper: A Structure for Deoxyribose Nucleic Acid. Based on available data from X-ray diffraction patterns and 3D conceptualization through model building, Watson and Crick proposed that DNA is a double helix ��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.

2008-10-06

342

The double helix and immunology  

NASA Astrophysics Data System (ADS)

The immune system can recognize and produce antibodies to virtually any molecule in the Universe. This enormous diversity arises from the ingenious reshuffling of DNA sequences encoding components of the immune system. Immunology is an example of a field completely transformed during the past 50 years by the discovery of the structure of DNA and the emergence of DNA technologies that followed.

Nossal, Gustav J. V.

2003-01-01

343

An Implicit Membrane Generalized Born Theory for the Study of Structure, Stability, and Interactions of Membrane Proteins  

PubMed Central

Exploiting recent developments in generalized Born (GB) electrostatics theory, we have reformulated the calculation of the self-electrostatic solvation energy to account for the influence of biological membranes. Consistent with continuum Poisson-Boltzmann (PB) electrostatics, the membrane is approximated as an solvent-inaccessible infinite planar low-dielectric slab. The present membrane GB model closely reproduces the PB electrostatic solvation energy profile across the membrane. The nonpolar contribution to the solvation energy is taken to be proportional to the solvent-exposed surface area (SA) with a phenomenological surface tension coefficient. The proposed membrane GB/SA model requires minor modifications of the pre-existing GB model and appears to be quite efficient. By combining this implicit model for the solvent/bilayer environment with advanced computational sampling methods, like replica-exchange molecular dynamics, we are able to fold and assemble helical membrane peptides. We examine the reliability of this model and approach by applications to three membrane peptides: melittin from bee venom, the transmembrane domain of the M2 protein from Influenza A (M2-TMP), and the transmembrane domain of glycophorin A (GpA). In the context of these proteins, we explore the role of biological membranes (represented as a low-dielectric medium) in affecting the conformational changes in melittin, the tilt of transmembrane peptides with respect to the membrane normal (M2-TMP), helix-to-helix interactions in membranes (GpA), and the prediction of the configuration of transmembrane helical bundles (GpA). The present method is found to perform well in each of these cases and is anticipated to be useful in the study of folding and assembly of membrane proteins as well as in structure refinement and modeling of membrane proteins where a limited number of experimental observables are available. PMID:14581194

Im, Wonpil; Feig, Michael; Brooks, Charles L.

2003-01-01

344

PAG--a multipurpose transmembrane adaptor protein.  

PubMed

Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), also known as Csk-binding protein (Cbp), is a ubiquitously expressed transmembrane adaptor protein present in lipid rafts and involved in a number of signaling pathways. It helps recruit cytoplasmic C-terminal Src kinase (Csk) to lipid raft-associated Src kinases, mediates a link to actin cytoskeleton and interacts with several other important cytoplasmic and plasma membrane-associated proteins. In recent years, PAG has been implicated in various aspects of cancer cell biology. Our review covers all so far published data on this interesting protein. PMID:24213579

Hrdinka, M; Horejsi, V

2014-10-01

345

Calculations of the Mueller scattering matrix for a DNA plectonemic helix  

SciTech Connect

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

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

1994-09-01

346

Astronomical Journal revision September 2007 Interpretation of the Helix Planetary Nebula using  

E-print Network

Astronomical Journal revision September 2007 Interpretation of the Helix Planetary Nebula using nebulae (PNe) within PGCs. The dying central star of a PNe slowly accretes JPP mass to grow the white-planet atmospheres. Subject headings: ISM: structure ­ Planetary Nebula: general ­ Cosmology: theory ­ Galaxy: halo

Wang, Deli

347

Molecular dynamics simulation of polymer helix formation using rigid-link methods D. C. Rapaport*  

E-print Network

Molecular dynamics simulation of polymer helix formation using rigid-link methods D. C. Rapaport July 2002 Molecular dynamics simulations are used to study structure formation in simple model polymer. The computational techniques span an equally broad range; they include molecular dynamics MD simulation employing

Rapaport, Dennis C.

348

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

PubMed Central

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

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

2012-01-01

349

Transmembrane Segment 11 Appears to Line the Purine Permeation Pathway of the Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1)*  

PubMed Central

Purine transport is essential for malaria parasites to grow because they lack the enzymes necessary for de novo purine biosynthesis. The Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is a member of the equilibrative nucleoside transporter (ENT) gene family. PfENT1 is a primary purine transport pathway across the P. falciparum plasma membrane because PfENT1 knock-out parasites are not viable at physiologic extracellular purine concentrations. Topology predictions and experimental data indicate that ENT family members have eleven transmembrane (TM) segments although their tertiary structure is unknown. In the current work, we showed that a naturally occurring polymorphism, F394L, in TM11 affects transport substrate Km. We investigated the structure and function of the TM11 segment using the substituted cysteine accessibility method. We showed that mutation to Cys of two highly conserved glycine residues in a GXXXG motif significantly reduces PfENT1 protein expression levels. We speculate that the conserved TM11 GXXXG glycines may be critical for folding and/or assembly. Small, cysteine-specific methanethiosulfonate (MTS) reagents reacted with four TM11 Cys substitution mutants, L393C, I397C, T400C, and Y403C. Larger MTS reagents do not react with the more cytoplasmic positions. Hypoxanthine, a transported substrate, protected L393C, I397C, and T400C from covalent modification by the MTS reagents. Plotted on an ?-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60° arc suggesting that TM11 is largely ? helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure. PMID:20335165

Riegelhaupt, Paul M.; Frame, I. J.; Akabas, Myles H.

2010-01-01

350

Transmembrane segment 11 appears to line the purine permeation pathway of the Plasmodium falciparum equilibrative nucleoside transporter 1 (PfENT1).  

PubMed

Purine transport is essential for malaria parasites to grow because they lack the enzymes necessary for de novo purine biosynthesis. The Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is a member of the equilibrative nucleoside transporter (ENT) gene family. PfENT1 is a primary purine transport pathway across the P. falciparum plasma membrane because PfENT1 knock-out parasites are not viable at physiologic extracellular purine concentrations. Topology predictions and experimental data indicate that ENT family members have eleven transmembrane (TM) segments although their tertiary structure is unknown. In the current work, we showed that a naturally occurring polymorphism, F394L, in TM11 affects transport substrate K(m). We investigated the structure and function of the TM11 segment using the substituted cysteine accessibility method. We showed that mutation to Cys of two highly conserved glycine residues in a GXXXG motif significantly reduces PfENT1 protein expression levels. We speculate that the conserved TM11 GXXXG glycines may be critical for folding and/or assembly. Small, cysteine-specific methanethiosulfonate (MTS) reagents reacted with four TM11 Cys substitution mutants, L393C, I397C, T400C, and Y403C. Larger MTS reagents do not react with the more cytoplasmic positions. Hypoxanthine, a transported substrate, protected L393C, I397C, and T400C from covalent modification by the MTS reagents. Plotted on an alpha-helical wheel, Leu-393, Ile-397, and Thr-400 lie on one face of the helix in a 60 degrees arc suggesting that TM11 is largely alpha helical. We infer that they line a water-accessible surface, possibly the purine permeation pathway. These results advance our understanding of the ENT structure. PMID:20335165

Riegelhaupt, Paul M; Frame, I J; Akabas, Myles H

2010-05-28

351

Interferon-induced Transmembrane Protein 3 Is a Type II Transmembrane Protein*  

PubMed Central

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

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

2013-01-01

352

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

PubMed

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

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

2013-11-01

353

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

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 (12)C-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

Walther, Frans J; Waring, Alan J; Hernández-Juviel, José M; Ruchala, Piotr; Wang, Zhengdong; Notter, Robert H; Gordon, Larry M

2014-01-01

354

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

PubMed Central

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

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

2014-01-01

355

Anchors Aweigh: Protein Traffic Mediated by Transmembrane Domains  

PubMed Central

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

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

2013-01-01

356

LINKIN, a new transmembrane protein necessary for cell adhesion.  

PubMed

In epithelial collective migration, leader and follower cells migrate while maintaining cell-cell adhesion and tissue polarity. We have identified a conserved protein and interactors required for maintaining cell adhesion during a simple collective migration in the developing C. elegans male gonad. LINKIN is a previously uncharacterized, transmembrane protein conserved throughout Metazoa. We identified seven atypical FG-GAP domains in the extracellular domain, which potentially folds into a ?-propeller structure resembling the ?-integrin ligand-binding domain. C. elegans LNKN-1 localizes to the plasma membrane of all gonadal cells, with apical and lateral bias. We identified the LINKIN interactors RUVBL1, RUVBL2, and ?-tubulin by using SILAC mass spectrometry on human HEK 293T cells and testing candidates for lnkn-1-like function in C. elegans male gonad. We propose that LINKIN promotes adhesion between neighboring cells through its extracellular domain and regulates microtubule dynamics through RUVBL proteins at its intracellular domain. PMID:25437307

Kato, Mihoko; Chou, Tsui-Fen; Yu, Collin Z; DeModena, John; Sternberg, Paul W

2014-01-01

357

Protein sequence and structure relationship ARMA spectral analysis: application to membrane proteins.  

PubMed Central

If it is assumed that the primary sequence determines the three-dimensional folded structure of a protein, then the regular folding patterns, such as alpha-helix, beta-sheet, and other ordered patterns in the three-dimensional structure must correspond to the periodic distribution of the physical properties of the amino acids along the primary sequence. An AutoRegressive Moving Average (ARMA) model method of spectral analysis is applied to analyze protein sequences represented by the hydrophobicity of their amino acids. The results for several membrane proteins of known structures indicate that the periodic distribution of hydrophobicity of the primary sequence is closely related to the regular folding patterns in a protein's three-dimensional structure. We also applied the method to the transmembrane regions of acetylcholine receptor alpha subunit and Shaker potassium channel for which no atomic resolution structure is available. This work is an extension of our analysis of globular proteins by a similar method. PMID:8075343

Sun, S; Parthasarathy, R

1994-01-01

358

Genome-wide identification and analysis of basic helix-loop-helix domains in dog, Canis lupus familiaris.  

PubMed

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

Wang, Xu-Hua; Wang, Yong; Liu, A-Ke; Liu, Xiao-Ting; Zhou, Yang; Yao, Qin; Chen, Ke-Ping

2014-11-18

359

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

PubMed Central

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

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

2006-01-01

360

Persistent expression of helix-loop-helix factor HES-1 prevents mammalian neural differentiation in the central nervous system.  

PubMed Central

In the developing mammalian central nervous system, neural precursor cells present in the ventricular zone determine their fate to become neurons or glial cells, migrate towards the outer layers and undergo terminal differentiation. The transcriptional repressor HES-1, a basic helix-loop-helix (bHLH) factor structurally related to the Drosophila hairy gene, is expressed at high levels throughout the ventricular zone, but the level decreases as neural differentiation proceeds. Because of this negative correlation, we tested whether continuous expression of HES-1 inhibits neural differentiation. A HES-1 and lacZ-transducing retrovirus (SG-HES1) and a control lacZ-transducing retrovirus (SG) were injected into the lateral ventricles of mouse embryos, and the fate of the infected neural precursor cells was examined by X-gal staining. The SG virus-infected cells migrated and differentiated into neurons and glial cells. In contrast, the cells infected with SG-HES1 virus remained in the ventricular/subventricular zone, decreased to approximately 10% in number as compared with that of the newborn during the postnatal 4-5 weeks and, when they survived, were present exclusively in the ependymal layer. Furthermore, whereas cultured neural precursor cells infected with SG virus became immunoreactive for neuronal and glial markers, the cells infected with SG-HES1 virus did not. These results show that persistent expression of HES-1 severely perturbs neuronal and glial differentiation. Images PMID:7909512

Ishibashi, M; Moriyoshi, K; Sasai, Y; Shiota, K; Nakanishi, S; Kageyama, R

1994-01-01

361

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

PubMed Central

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

Hallsson, Jón H; Haflidadóttir, Benedikta S; Stivers, Chad; Odenwald, Ward; Arnheiter, Heinz; Pignoni, Francesca; Steingrímsson, Eiríkur

2004-01-01

362

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

PubMed

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

Menta, C; Parisi, V

2001-01-01

363

Structural characterizations of the chloroplast translocon protein Tic110  

PubMed Central

Tic110 is a major component of the chloroplast protein import translocon. Two functions with mutually exclusive structures have been proposed for Tic110: a protein-conducting channel with six transmembrane domains and a scaffold with two N-terminal transmembrane domains followed by a large soluble domain for binding transit peptides and other stromal translocon components. To investigate the structure of Tic110, Tic110 from Cyanidioschyzon merolae (CmTic110) was characterized. We constructed three fragments, CmTic110A, CmTic110B and CmTic110C, with increasing N-terminal truncations, to perform small-angle X-ray scattering (SAXS) and X-ray crystallography analyses and Dali structural comparison. Here we report the molecular envelope of CmTic110B and CmTic110C determined by SAXS, and the crystal structure of CmTic110C at 4.2 Å. Our data indicate that the C-terminal half of CmTic110 possesses a rod-shaped helix-repeat structure that is too flattened and elongated to be a channel. The structure is most similar to the HEAT-repeat motif that functions as scaffolds for protein–protein interactions. PMID:23711301

Tsai, Jia-Yin; Chu, Chiung-Chih; Yeh, Yi-Hung; Chen, Lih-Jen; Li, Hsou-min; Hsiao, Chwan-Deng

2013-01-01

364

The role of context on alpha-helix stabilization: host-guest analysis in a mixed background peptide model.  

PubMed Central

The helix content of a series of peptides containing single substitutions of the 20 natural amino acids in a new designed host sequence, succinyl-YSEEEEKAKKAXAEEAEKKKK-NH2, has been determined using CD spectroscopy. This host is related to one previously studied, in which triple amino acid substitutions were introduced into a background of Glu-Lys blocks completely lacking alanine. The resulting free energies show that only Ala and Glu- prove to be helix stabilizing, while all other side chains are neutral or destabilizing. This agrees with results from studies of alanine-rich peptide modela, but not the previous Glu-Lys block oligomers in which Leu and Met also stabilize helix. The helix propensity scale derived from the previous block oligomers correlated well with the frequencies of occurrence of different side chains in helical sequences of proteins, whereas the values from the present series do not. The role of context in determining scales of helix propensity values is discussed, and the ability of algorithms designed to predict helix structure from sequence is compared. PMID:9194186

Yang, J.; Spek, E. J.; Gong, Y.; Zhou, H.; Kallenbach, N. R.

1997-01-01

365

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

SciTech Connect

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

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

1988-09-06

366

Cystic Fibrosis Transmembrane Regulator Correctors and Potentiators  

PubMed Central

Cystic fibrosis (CF) is caused by loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) protein, a cAMP-regulated anion channel expressed primarily at the apical plasma membrane of secretory epithelia. Nearly 2000 mutations in the CFTR gene have been identified that cause disease by impairing its translation, cellular processing, and/or chloride channel gating. The fundamental premise of CFTR corrector and potentiator therapy for CF is that addressing the underlying defects in the cellular processing and chloride channel function of CF-causing mutant CFTR alleles will result in clinical benefit by addressing the basic defect underlying CF. Correctors are principally targeted at F508del cellular misprocessing, whereas potentiators are intended to restore cAMP-dependent chloride channel activity to mutant CFTRs at the cell surface. This article reviews the discovery of CFTR potentiators and correctors, what is known regarding their mechanistic basis, and encouraging results achieved in clinical testing. PMID:23818513

Rowe, Steven M.; Verkman, Alan S.

2014-01-01

367

Transmembrane topology of the mammalian KDEL receptor.  

PubMed Central

The mammalian KDEL receptor is an integral membrane protein with seven hydrophobic regions. Fusion proteins comprising a 37-kDa N-glycosylation reporter fused downstream of amino-terminal fragments of the KDEL receptor with varying numbers of hydrophobic regions were synthesized in an in vitro translation system containing canine pancreatic microsomes. The luminal or cytosolic orientation of the reporter, and hence of the hydrophilic region to which it is fused, was inferred from the presence or absence of glycosylation, which occurs only in the lumen of the microsomes. The cytosolic orientation of the N and C termini was also confirmed immunocytochemically. Our results suggest that the KDEL receptor is inserted into the membrane with only six transmembrane domains and that both the amino and carboxy termini are located in the cytoplasm. Images PMID:8413242

Singh, P; Tang, B L; Wong, S H; Hong, W

1993-01-01

368

Transmembrane Protein Oxygen Content and Compartmentalization of Cells  

E-print Network

in the levels of atmospheric oxygen the size and number of communication-related transmembrane proteins that lead to a large-scale innovation in the origin of protein domains with roles in cellular communication that atmospheric oxygen levels influenced the composition of transmembrane proteins, and that older taxa (bacteria

Gerstein, Mark

369

Molecular mechanisms for generating transmembrane proton gradients  

PubMed Central

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

Gunner, M.R.; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

2013-01-01

370

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

PubMed Central

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

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

2014-01-01

371

Miniaturization of quadrifilar helix antenna for VHF band applications  

Microsoft Academic Search

This paper addresses the miniaturization of the VHF band quadrifilar helix antenna. The design and experimental validation are reported in this paper. Such a helix could be used, e.g., for telemetry link in Space-based Multi-band Variable Object Monitor satellite project. Compared to the reference quarter wavelength quadrifilar helix antenna, a significant height reduction from 48% to 62.5% is obtained. Moreover,

Alexandru Takacs; Nelson J. G. Fonseca; Hervé Aubert; Xavier Dollat

2009-01-01

372

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

PubMed

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

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

2013-04-01

373

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

SciTech Connect

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

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

2010-03-08

374

Structural plasticity and catalysis regulation of a thermosensor histidine kinase.  

PubMed

Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases. PMID:19805278

Albanesi, Daniela; Martín, Mariana; Trajtenberg, Felipe; Mansilla, María C; Haouz, Ahmed; Alzari, Pedro M; de Mendoza, Diego; Buschiazzo, Alejandro

2009-09-22

375

Multivalent helix mimetics for PPI-inhibition.  

PubMed

The exploitation of multivalent ligands for the inhibition of protein-protein interactions has not yet been explored as a supramolecular design strategy. This is despite the fact that protein-protein interactions typically occur within the context of multi-protein complexes and frequently exploit avidity effects or co-operative binding interactions to achieve high affinity interactions. In this paper we describe preliminary studies on the use of a multivalent N-alkylated aromatic oligoamide helix mimetic for inhibition of p53/hDM2 and establish that protein dimerisation is promoted, rather than enhanced binding resulting from a higher effective concentration of the ligand. PMID:25408290

Barnard, Anna; Miles, Jennifer A; Burslem, George M; Barker, Amy M; Wilson, Andrew J

2015-01-01

376

A Herschel study of the Helix nebula  

NASA Astrophysics Data System (ADS)

The Herschel Guaranteed Time Key Project “MESS” (Mass loss of Evolved StarS) has observed a wide variety of evolved stellar objects in spectroscopic and photometric mode using both the PACS and SPIRE instruments on board the Herschel satellite. The aims of the project are to study the nebular morphology at far infrared wavelengths, the mass loss history of the central star, and the properties of the dust that was formed in the ejecta. In the poster we presented the results of the Herschel PACS and SPIRE imaging of the dust shell of the Helix planetary nebula NGC 7293.

Van de Steene, G.

2014-04-01

377

Nucleic acid helices: I. Structure of M1 RNA from E. coli as determined bypsoralen crosslinking. II. Thermodynamics of the helix-coil transition of DNA oligonucleotides in solutions containing 3. 0 M tetramethylammonium chloride  

SciTech Connect

This work includes two different investigations examining nucleic acid helices. The first study discusses secondary and tertiary interactions in the RNA moiety of ribonuclease P from Escherichia coli. The second study discusses the thermodynamics of the helix-coil transition of DNA oligonucleotides in solutions containing tetramethylammonium chloride. The RNA moiety of ribonuclease P from Escherichia coli (M1 RNA) has been photoreacted with 4{prime}-hydroxymethyl-4,5{prime}8-trimethylpsoralen and long wave UV light (320-380 nm) in a buffer in which the M1 RNA alone acts as a true catalyst of tRNA processing. Limited specific digestion followed by two dimensional gel electrophoresis yields fragments crosslinked by HMT. The positions of the crosslinks have been determined to within {plus minus}15 nucleotides by photoreversal of the isolated crosslinked fragments and enzymatic sequencing of the resulting RNA. Further assignments of the exact locations of the crosslinks have been made on the known photoreactivity of the psoralen with different bases.

Lipson, S.E.

1987-01-01

378

Structure Modeling of All Identified G Protein–Coupled Receptors in the Human Genome  

PubMed Central

G protein–coupled receptors (GPCRs), encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER) method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C? root-mean-squared deviation from native of 4.6 Å, with a root-mean-squared deviation in the transmembrane helix region of 2.1 Å. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness and robustness of the in silico models for GPCR functional analysis. All predicted GPCR models are freely available for noncommercial users on our Web site (http://www.bioinformatics.buffalo.edu/GPCR). PMID:16485037

Zhang, Yang; DeVries, Mark E; Skolnick, Jeffrey

2006-01-01

379

Helix formation and capping energetics of arginine analogs with varying side chain length.  

PubMed

Arginine (Arg) has been used for recognizing negatively charged biological molecules, cell penetration, and oligosaccharide mass signal enhancement. The versatility of Arg has inspired the need to develop Arg analogs and to research the structural effects of incorporating Arg analogs. Accordingly, we investigated the effect of Arg side chain length on helix formation by studying 12 Ala-based peptides containing the Arg analogs (S)-2-amino-6-guanidino-hexanoic acid (Agh), (S)-2-amino-4-guanidinobutyric acid (Agb), and (S)-2-amino-3-guanidinopropionic acid (Agp). Solid phase guanidinylation with orthogonal protection strategies was necessary to synthesize Agb- and Agp-containing peptides using Fmoc-based chemistry. The fraction helix for the peptides was determined by circular dichroism spectroscopy, and used to derive the statistical mechanical parameters and energetics for N-capping, C-capping, and helix propagation (propensity). All four Arg analogs were unfavorable for N-capping. The C-cap parameter followed the trend Agphelix propensity followed the trend AgpAgh, highlighting the uniqueness of the Arg side chain length in helix formation. Molecular mechanics calculations and a survey on protein structures were consistent with the experimental results. Furthermore, calculations and survey both showed that the g- conformation for the ?1 dihedral was present for the first two residues at the N-terminus of helices, but not favored in the center or C-terminus of helices due to sterics. These results should serve as the foundation for developing Arg-related bioactive compounds and technologies. PMID:21922267

Cheng, Richard P; Weng, Yi-Jen; Wang, Wei-Ren; Koyack, Marc J; Suzuki, Yuta; Wu, Cheng-Hsun; Yang, Po-An; Hsu, Hao-Chun; Kuo, Hsiou-Ting; Girinath, Prashant; Fang, Chun-Jen

2012-07-01

380

Infrared Spectroscopic Studies of Cells and Tissues: Triple Helix Proteins as a Potential Biomarker for Tumors  

PubMed Central

In this work, the infrared (IR) spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection. Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM) of tissues. An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders. PMID:23526977

Stelling, Allison L.; Toher, Deirdre; Uckermann, Ortrud; Tavkin, Jelena; Leipnitz, Elke; Schweizer, Julia; Cramm, Holger; Steiner, Gerald; Geiger, Kathrin D.; Kirsch, Matthias

2013-01-01

381

The cellular Pax-Hox-helix connection.  

PubMed

Basic helix-loop-helix (bHLH) transcription factors are important regulators of lineage determination during embryogenesis. Initial experiments in Drosophila showed that early neural selection and specification are dependent on atonal (ato) and members of the achaete-scute complex (as-c). In mammals, transcription factors homologous to as-c and ato are causally involved during development of organs throughout the body. Development of subsets of lineages in intestine, stomach, pancreas, lung, thyroid and placenta have been shown to be regulated by members of the as-c and ato families. These functional studies show that an individual bHLH transcription factor can regulate multiple developmental processes throughout the mammalian body, which implicates that extant as-c and ato transcription factors play a distinct function dependent on their cellular context. Based on the synergistic activation of the insulin, POMC and Pax4 promotors by bHLH and homeobox (Hox) protein complexes, we hypothesize that the underlying cellular function-modulating factors include members of the Hox and paired box (Pax) multigene families. These examples indicate that unique combinations of bHLH and Hox proteins, mediated by protein-protein interactions, might be responsible for activating cell-specific sets of target genes. PMID:14522074

Westerman, Bart A; Murre, Cornelis; Oudejans, Cees B M

2003-10-01

382

The seventh transmembrane domains of the delta and kappa opioid receptors have different accessibility patterns and interhelical interactions.  

PubMed

We applied the substituted cysteine accessibility method (SCAM) to map the residues of the transmembrane helices (TMs) 7 of delta and kappa opioid receptors (deltaOR and kappaOR) that are on the water-accessible surface of the binding-site crevices. A total of 25 consecutive residues (except C7.38) in the TMs 7 were mutated to Cys, one at a time, and each mutant was expressed in HEK 293 cells. Most mutants displayed similar binding affinity for [(3)H]diprenorphine, an antagonist, as the wild types. Pretreatment with (2-aminoethyl)methanethiosulfonate (MTSEA) inhibited [(3)H]diprenorphine binding to eight deltaOR and eight kappaOR mutants. All mutants except deltaOR L7.52(317)C were protected by naloxone from the MTSEA effect, indicating that the side chains of V7.31(296), A7.34(299), I7.39(304), L7.41(306), G7.42(307), P7.50(315), and Y7.53(318) of deltaOR and S7.34(311), F7.37(314), I7.39(316), A7.40(317), L7.41(318), G7.42(319), Y7.43(320), and N7.49(326) of kappaOR are on the water-accessible surface of the binding pockets. Combining the SCAM data with rhodopsin-based molecular models of the receptors led to the following conclusions. (i) The residues of the extracellular portion of TM7 predicted to face TM1 are sensitive to MTSEA in kappaOR but are not in deltaOR. Thus, TM1 may be closer to TM7 in deltaOR than in kappaOR. (ii) MTSEA-sensitive mutants start at position 7.31(296) in deltaOR and at 7.34(311) in kappaOR, suggesting that TM7 in deltaOR may have an additional helical turn (from 7.30 to 7.33). (iii) There is a conserved hydrogen-bond network linking D2.50 of the NLxxxD motif in TM2 with W6.48 of the CWxP motif in TM6. (iv) The NPxxY motif in TM7 interacts with TM2, TM6, and helix 8 to maintain receptors in inactive states. To the best of our knowledge, this represents the first such comparison of the structures of two highly homologous GPCRs. PMID:16331961

Xu, Wei; Campillo, Mercedes; Pardo, Leonardo; Kim de Riel, J; Liu-Chen, Lee-Yuan

2005-12-13

383

System and methods for predicting transmembrane domains in membrane proteins and mining the genome for recognizing G-protein coupled receptors  

DOEpatents

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.

Trabanino, Rene J; Vaidehi, Nagarajan; Hall, Spencer E; Goddard, William A; Floriano, Wely

2013-02-05

384

A novel procedure for the efficient purification of the cystic fibrosis transmembrane conductance regulator (CFTR).  

PubMed Central

This report describes a novel, single-step strategy for the purification of the cystic fibrosis transmembrane conductance regulator from Sf9 cells, which will facilitate studies of the structure-function relationships of this clinically important molecule. The new method combines the use of the novel detergent sodium pentadecafluoro-octanoate with metal-affinity chromatography to produce a high yield of purified protein which can be functionally reconstituted as a chloride channel and an ATPase. PMID:9355728

Ramjeesingh, M; Li, C; Garami, E; Huan, L J; Hewryk, M; Wang, Y; Galley, K; Bear, C E

1997-01-01

385

Purification of transmembrane proteins from Saccharomyces cerevisiae for X-ray crystallography  

Microsoft Academic Search

To enhance the quantity and quality of eukaryotic transmembrane proteins (TMPs) available for structure determination by X-ray crystallography, we have optimized protocols for purification of TMPs expressed in the yeast Saccharomyces cerevisiae. We focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays. Genes encoding the target TMPs are transferred via ligation-independent cloning

Kathleen M. Clark; Nadia Fedoriw; Katrina Robinson; Sara M. Connelly; Joan Randles; Michael G. Malkowski; George T. DeTitta; Mark E. Dumont

2010-01-01

386

On the Role of Acylation of Transmembrane Proteins  

PubMed Central

Abstract Acylation is a frequent means to ensure membrane association of a variety of soluble proteins in living cells. However, many transmembrane proteins are palmitoylated, indicating that this posttranslational modification may also serve as a means to regulate protein trafficking. Based on coarse-grained membrane simulations, we find that protein acylation significantly alters the tilting of transmembrane proteins with respect to the bilayer normal. In addition, the proteins' partitioning behavior and cluster formation ability due to hydrophobic mismatching is strongly altered. Based on our results, we propose that acylation is a potent means to regulate the trafficking of transmembrane proteins along the early secretory pathway. PMID:20197033

Morozova, Diana; Weiss, Matthias

2010-01-01

387

Guided Transport of a Transmembrane Nanochannel  

NASA Astrophysics Data System (ADS)

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

Dutt, Meenakshi; Kuksenok, Olga; Balazs, Anna

2011-03-01

388

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

PubMed

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

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

2013-07-10

389

Structural Basis of Lipid-Driven Conformational Transitions in the KvAP Voltage Sensing Domain  

PubMed Central

Voltage-gated ion channels respond to transmembrane electric fields through reorientations of the positively charged S4 helix within the voltage-sensing domain (VSD). Despite a wealth of structural and functional data, the details of this conformational change remain controversial. Recent electrophysiological evidence showed that equilibrium between the resting (Down) and activated (Up) conformations of KvAP-VSD from Aeropyrum pernix can be biased through reconstitution in lipids with or without phosphate groups. We investigated the structural transition between these functional states using site-directed spin labeling and EPR spectroscopic methods. Solvent accessibility and inter-helical distance determinations suggest that KvAP gates through S4 movements involving a ~3 Å upward tilt and simultaneous ~2 Å axial shift. This motion leads to large accessibly changes in the intracellular water-filled crevice and supports a novel model of gating that combines structural rearrangements and electric field remodeling. PMID:24413055

Li, Qufei; Wanderling, Sherry; Somponspisut, Pornthep; Perozo, Eduardo

2013-01-01

390

Nucleotide-induced conformational motions and transmembrane gating dynamics in a bacterial ABC transporter  

E-print Network

ATP-binding cassette (ABC) transporters are integral membrane proteins that mediate the exchange of diverse substrates across membranes powered by ATP hydrolysis. We report results of coarse-grained dynamical simulations performed for the bacterial heme transporter HmuUV. Based on the nucleotide-free structure, we have constructed a ligand-elastic-network description for this protein and investigated ATP-induced conformational motions in structurally resolved computer experiments. As we found, interactions with nucleotides resulted in generic motions which are functional and robust. Upon binding of ATP-mimicking ligands the structure changed from a conformation in which the nucleotide-binding domains formed an open shape, to a conformation in which they were found in tight contact and the transmembrane domains were rotated. The heme channel was broadened in the ligand-bound complex and the gate to the cytoplasm, which was closed in the nucleotide-free conformation, was rendered open by a mechanism that involved tilting motions of essential transmembrane helices. Based on our findings we propose that the HmuUV transporter behaves like a `simple' mechanical device in which, induced by binding of ATP ligands, linear motions of the nucleotide-binding domains are translated into rotational motions and internal tilting dynamics of the transmembrane domains that control gating inside the heme pathway.

Holger Flechsig

2014-02-07

391

Isosteric substitutions of urea to thiourea and selenourea in aliphatic oligourea foldamers: site-specific perturbation of the helix geometry.  

PubMed

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

Nelli, Yella Reddy; Antunes, Stéphanie; Salaün, Arnaud; Thinon, Emmanuelle; Massip, Stéphane; Kauffmann, Brice; Douat, Céline; Guichard, Gilles

2015-02-01

392

Topological analysis of hedgehog acyltransferase, a multipalmitoylated transmembrane protein.  

PubMed

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

Konitsiotis, Antonio D; Jovanovi?, Biljana; Ciepla, Paulina; Spitaler, Martin; Lanyon-Hogg, Thomas; Tate, Edward W; Magee, Anthony I

2015-02-01

393

Comets Kick up Dust in Helix Nebula  

NASA Technical Reports Server (NTRS)

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

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

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

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

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

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

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

2007-01-01

394

Probing the Dynamics of the P1 Helix within the Tetrahymena Group I Intron  

PubMed Central

RNA conformational transformations are integral to RNA's biological functions. Further, structured RNA molecules exist as a series of dynamic intermediates in the course of folding or complexation with proteins. Thus, an understanding of RNA folding and function will require deep and incisive understanding of its dynamic behavior. However, existing tools to investigate RNA dynamics