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1

The evolution of transmembrane helix kinks and the structural diversity of G protein-coupled receptors  

Microsoft Academic Search

One of the hallmarks of membrane protein structure is the high frequency of transmembrane helix kinks, which commonly occur at proline residues. Because the proline side chain usually precludes normal helix geometry, it is reasonable to expect that proline residues generate these kinks. We observe, however, that the three prolines in bacteriorhodopsin transmembrane helices can be changed to alanine with

Sarah Yohannan; Salem Faham; Duan Yang; Julian P. Whitelegge; James U. Bowie

2004-01-01

2

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

3

TMPad: an integrated structural database for helix-packing folds in transmembrane proteins.  

PubMed

?-helical transmembrane (TM) proteins play an important role in many critical and diverse biological processes, and specific associations between TM helices are important determinants for membrane protein folding, dynamics and function. In order to gain insights into the above phenomena, it is necessary to investigate different types of helix-packing modes and interactions. However, such information is difficult to obtain because of the experimental impediment and a lack of a well-annotated source of helix-packing folds in TM proteins. We have developed the TMPad (TransMembrane Protein Helix-Packing Database) which addresses the above issues by integrating experimentally observed helix-helix interactions and related structural information of membrane proteins. Specifically, the TMPad offers pre-calculated geometric descriptors at the helix-packing interface including residue backbone/side-chain contacts, interhelical distances and crossing angles, helical translational shifts and rotational angles. The TMPad also includes the corresponding sequence, topology, lipid accessibility, ligand-binding information and supports structural classification, schematic diagrams and visualization of the above structural features of TM helix-packing. Through detailed annotations and visualizations of helix-packing, this online resource can serve as an information gateway for deciphering the relationship between helix-helix interactions and higher levels of organization in TM protein structure and function. The website of the TMPad is freely accessible to the public at http://bio-cluster.iis.sinica.edu.tw/TMPad. PMID:21177659

Lo, Allan; Cheng, Cheng-Wei; Chiu, Yi-Yuan; Sung, Ting-Yi; Hsu, Wen-Lian

2011-01-01

4

The evolution of transmembrane helix kinks and the structural diversity of G protein-coupled receptors.  

PubMed

One of the hallmarks of membrane protein structure is the high frequency of transmembrane helix kinks, which commonly occur at proline residues. Because the proline side chain usually precludes normal helix geometry, it is reasonable to expect that proline residues generate these kinks. We observe, however, that the three prolines in bacteriorhodopsin transmembrane helices can be changed to alanine with little structural consequences. This finding leads to a conundrum: if proline is not required for helix bending, why are prolines commonly present at bends in transmembrane helices? We propose an evolutionary hypothesis in which a mutation to proline initially induces the kink. The resulting packing defects are later repaired by further mutation, thereby locking the kink in the structure. Thus, most prolines in extant proteins can be removed without major structural consequences. We further propose that nonproline kinks are places where vestigial prolines were later removed during evolution. Consistent with this hypothesis, at 14 of 17 nonproline kinks in membrane proteins of known structure, we find prolines in homologous sequences. Our analysis allows us to predict kink positions with >90% reliability. Kink prediction indicates that different G protein-coupled receptor proteins have different kink patterns and therefore different structures. PMID:14732697

Yohannan, Sarah; Faham, Salem; Yang, Duan; Whitelegge, Julian P; Bowie, James U

2004-01-19

5

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

PubMed Central

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

Shen, L; Bassolino, D; Stouch, T

1997-01-01

6

Transmembrane helix-helix interactions are modulated by the sequence context and by lipid bilayer properties.  

PubMed

Folding of polytopic transmembrane proteins involves interactions of individual transmembrane helices, and multiple TM helix-helix interactions need to be controlled and aligned to result in the final TM protein structure. While defined interaction motifs, such as the GxxxG motif, might be critically involved in transmembrane helix-helix interactions, the sequence context as well as lipid bilayer properties significantly modulate the strength of a sequence specific transmembrane helix-helix interaction. Structures of 11 transmembrane helix dimers have been described today, and the influence of the sequence context as well as of the detergent and lipid environment on a sequence specific dimerization is discussed in light of the available structural information. This article is part of a Special Issue entitled: Protein Folding in Membranes. PMID:21827736

Cymer, Florian; Veerappan, Anbazhagan; Schneider, Dirk

2011-07-31

7

Residue specific vibrational echoes yield 3-dimensional structures of a transmembrane helix dimer  

PubMed Central

Two dimensional vibrational echo spectroscopy has previously been applied to structural determination of small peptides. Here we extend the technique to a more complex, biologically significant system: the homodimeric transmembrane dimer from the ?-chain of the integrin ?IIb?3. We prepared micelle suspensions of the pair of 30-residue chains that span the membrane in the native structure, with varying levels of heavy (13C=18O) isotopes substituted in the backbone of the central 10th through 20th positions. The constraints derived from vibrational coupling of the precisely spaced heavy residues led to determination of an optimized structure from a range of model candidates: Glycine residues at the 12th, 15th and 16th positions form a tertiary contact in parallel right handed helix dimers with crossing angles of ?58° ± 9° and interhelical distances of 7.7 ± 0.5 Å. The frequency correlation established the dynamical model used in the analysis and it indicated the absence of mobile water associated with labeled residues. Delocalization of vibrational excitations between the helices was also quantitatively established.

Remorino, Amanda; Korendovych, Ivan; Wu, Yibing; DeGrado, William F.; Hochstrasser, Robin M.

2012-01-01

8

Robust Driving Forces for Transmembrane Helix Packing  

PubMed Central

The packing structures of transmembrane helices are traditionally attributed to patterns in residues along the contact surface. In this view, besides keeping the helices confined in the membrane, the bilayer has only a minor effect on the helices structure. Here, we use two different approaches to show that the lipid environment has a crucial effect in determining the cross-angle distribution of packed helices. We analyzed structural data of a membrane proteins database. We show that the distribution of cross angles of helix pairs in this database is statistically indistinguishable from the cross-angle distribution of two noninteracting helices imbedded in the membrane. These results suggest that the cross angle is, to a large extent, determined by the tilt angle of the individual helices. We test this hypothesis using molecular simulations of a coarse-grained model that contains no specific residue interactions. These simulations reproduce the same cross-angle distribution as found in the database. As the tilt angle of a helix is dominated by hydrophobic mismatch between the protein and surrounding lipids, our results indicate that hydrophobic mismatch is the dominant factor guiding the transmembrane helix packing. Other short-range forces might then fine-tune the structure to its final configuration.

Benjamini, Ayelet; Smit, Berend

2012-01-01

9

Driving Forces for Transmembrane ?-Helix Oligomerization  

PubMed Central

Abstract We present what we believe to be a novel statistical contact potential based on solved structures of transmembrane (TM) ?-helical bundles, and we use this contact potential to investigate the amino acid likelihood of stabilizing helix-helix interfaces. To increase statistical significance, we have reduced the full contact energy matrix to a four-flavor alphabet of amino acids, automatically determined by our methodology, in which we find that polarity is a more dominant factor of group identity than is size, with charged or polar groups most often occupying the same face, whereas polar/apolar residue pairs tend to occupy opposite faces. We found that the most polar residues strongly influence interhelical contact formation, although they occur rarely in TM helical bundles. Two-body contact energies in the reduced letter code are capable of determining native structure from a large decoy set for a majority of test TM proteins, at the same time illustrating that certain higher-order sequence correlations are necessary for more accurate structure predictions.

Sodt, Alex J.; Head-Gordon, Teresa

2010-01-01

10

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

PubMed

Predicting the burial status (the residue exposure to the lipid bilayer or buried within the protein core) of transmembrane (TM) residues of ?-helix membrane protein (?HMP) is of great importance for genome-wide annotation and for experimental researchers to elucidate diverse physiological processes. In this work, we developed a new computational model that can be used for predicting the burial status of TM residues of ?HMP. By incorporating physicochemical scales and conservation index, an efficient prediction model using least squares support vector machine (LS-SVM) was developed. The model was developed from 43 protein chains and its prediction ability was evaluated by an independent test set of other non-redundant ten protein chains. The prediction accuracy of our method was much better than the results of the reported works. Our results demonstrate that the LS-SVM prediction model incorporating structural and physicochemical features derived from sequence information could greatly improve the prediction accuracy. PMID:20740371

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

2010-08-26

11

Role of Cysteine Residues in Structural Stability and Function of a Transmembrane Helix Bundle  

Microsoft Academic Search

To study the structural and functional roles of the cysteine residues at positions 36, 41, and 46 in the trans- membrane domain of phospholamban (PLB), we have used Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid- phase peptide synthesis to prepare -amino-n-butyric acid (Abu)-PLB, the analogue in which all three cysteine residues are replaced by Abu. Whereas previous studies have shown that replacement of the

Christine B. Karim; M. Germana Paterlini; Laxma G. Reddy; Gregory W. Hunter; George Barany; David D. Thomas

2001-01-01

12

Transmembrane helix stability: the effect of helix-helix interactions studied by Fourier transform infrared spectroscopy.  

PubMed Central

We have measured, using infrared spectroscopy, the hydrogen/deuterium exchange rates of the amide protons in the photosynthetic antenna of Rhodospirillum rubrum. These measurements were made not only on the intact protein in detergent solution but also on two dissociated forms (B820 and B777). We have, on the basis of our knowledge of the structure of this protein, been able to assign the various groups of amide protons that exchange with different time constants to distinct regions of the protein. The most protected group of protons that we observe exchanging with time constants near 6000 min we assign to the transmembrane helices. The slow exchange rates measured for the amide protons of the transmembrane helices of this protein in detergent solution may indicate a destabilization of the helices in detergent solution compared with the membrane. This group of protons is progressively destabilized by stepwise dissociation of the antenna protein, and this destabilization is greater than we can account for by increases in solvent accessibility. We suggest that the observed loss of amide proton protection in the transmembrane helices as they are dissociated might be due to an increase in the helix flexibility and breathing motions as interactions between helices are reduced.

Sturgis, J; Robert, B; Goormaghtigh, E

1998-01-01

13

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

PubMed Central

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

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

2011-01-01

14

Thrombopoietin receptor activation: transmembrane helix dimerization, rotation, and allosteric modulation  

PubMed Central

We report how rotational variations in transmembrane (TM) helix interactions participate in the activity states of the thrombopoietin receptor (TpoR), a type 1 cytokine receptor that controls the production of blood platelets. We also explore the mechanism of small-molecule agonists that do not mimic the natural ligand. We show, by a combination of cysteine cross-linking, alanine-scanning mutagenesis, and computational simulations, that the TpoR TM dimerizes strongly and can adopt 3 different stable, rotationally related conformations, which may correspond to specific states of the full-length receptor (active, inactive, and partially active). Thus, our data suggest that signaling and inactive states of the receptor are related by receptor subunit rotations, rather than a simple monomer-dimer transition. Moreover, results from experiments with and without agonists in vitro and in cells allow us to propose a novel allosteric mechanism of action for a class of small molecules, in which they activate TpoR by binding to the TM region and by exploiting the rotational states of the dimeric receptor. Overall, our results support the emerging view of the participation of mutual rotations of the TM domains in cytokine receptor activation.—Matthews, E. E., Thévenin, D., Rogers, J. M., Gotow, L., Lira, P. D., Reiter, L. A., Brissette, W. H., Engelman, D. M. Thrombopoietin receptor activation: transmembrane helix dimerization, rotation, and allosteric modulation.

Matthews, Erin E.; Thevenin, Damien; Rogers, Julia M.; Gotow, Lisa; Lira, Paul D.; Reiter, Lawrence A.; Brissette, William H.; Engelman, Donald M.

2011-01-01

15

Detergent properties influence the stability of the glycophorin A transmembrane helix dimer in lysophosphatidylcholine micelles.  

PubMed

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

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

2012-12-18

16

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

17

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

PubMed Central

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

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

1995-01-01

18

Light-induced movement of the transmembrane helix?b in channelrhodopsin-2.  

PubMed

A protein photoswitch: Site-directed spin labeling was applied to monitor conformational changes of channelrhodopsin-2. Double electron electron resonance spectroscopy reveals interspin distance changes upon light activation which can be attributed to a displacement of the cytoplasmic part of the transmembrane helix?B. This helix movement could be part of the channel opening mechanism. PMID:23893661

Sattig, Thomas; Rickert, Christian; Bamberg, Ernst; Steinhoff, Heinz-Jürgen; Bamann, Christian

2013-07-26

19

Molecular Dynamics Simulation of Membranes and a Transmembrane Helix  

NASA Astrophysics Data System (ADS)

Three molecular dynamics (MD) simulations of 1.5-ns length were carried out on fully hydrated patches of dimyristoyl phosphatidylcholine (DMPC) bilayers in the liquid-crystalline phase. The simulations were performed using different ensembles and electrostatic conditions: a microcanonical ensemble or constant pressure-temperature ensemble, with or without truncated electrostatic interactions. Calculated properties of the membrane patches from the three different protocols were compared to available data from experiments. These data include the resulting overall geometrical dimensions, the order characteristics of the lipid hydrocarbon chains, as well as various measures of the conformations of the polar head groups. The comparisons indicate that the simulation carried out within the microcanonical ensemble with truncated electrostatic interactions yielded results closest to the experimental data, provided that the initial equilibration phase preceding the production run was sufficiently long. The effects of embedding a non-ideal helical protein domain in the membrane patch were studied with the same MD protocols. This simulation was carried out for 2.5 ns. The protein domain corresponds to the seventh transmembrane segment (TMS7) of the human serotonin 5HT2Areceptor. The peptide is composed of two ?-helical segments linked by a hinge domain around a perturbing Asn-Pro motif that produces at the end of the simulation a kink angle of nearly 80° between the two helices. Several aspects of the TMS7 structure, such as the bending angle, backbone ? and ? torsion angles, the intramolecular hydrogen bonds, and the overall conformation, were found to be very similar to those determined by NMR for the corresponding transmembrane segment of the tachykinin NK-1 receptor. In general, the simulations were found to yield structural and dynamic characteristics that are in good agreement with experiment. These findings support the application of simulation methods to the study of the complex biomolecular systems at the membrane interface of cells.

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

1999-05-01

20

Oligomerization State of Photosynthetic Core Complexes is Correlated with the Dimerization Affinity of a Transmembrane Helix  

PubMed Central

In the Rhodobacter (Rba.) species of photosynthetic purple bacteria, a single transmembrane ?-helix, PufX, is found within the core complex, an essential photosynthetic macromolecular assembly that performs the absorption and the initial processing of light energy. Despite its structural simplicity, many unresolved questions surround PufX, the most important of which is its location within the photosynthetic core complex. One proposed placement of PufX is at the center of a core complex dimer, where two PufX helices associate in the membrane and form a homodimer. Inability for PufX of certain Rba. species to form a homodimer is thought to lead to monomeric core complexes. In the present study, we employ a combination of computational and experimental techniques to test the hypothesized homodimerization of PufX. We carry out a systematic investigation to measure the dimerization affinity of PufX from four Rba. species, Rba. blasticus, Rba. capsulatus, Rba. sphaeroides, and Rba. veldkampii, using a molecular dynamics-based free-energy method, as well as experimental TOXCAT assays. We found that the four PufX helices have substantially different dimerization affinities. Both computational and experimental techniques demonstrate that species with dimeric core complexes have PufX that can potentially form a homodimer, whereas the one species with monomeric core complexes has a PufX with little to no dimerization propensity. Our analysis of the helix-helix interface revealed a number of positions that may be important for PufX dimerization and the formation of a hydrogen bond network between these GxxxG-containing helices. Our results suggest that the different oligomerization states of core complexes in various Rba. species can be attributed, among other factors, to the different propensity of its PufX helix to homodimerize.

Hsin, Jen; LaPointe, Loren M.; Kazy, Alla; Chipot, Christophe; Senes, Alessandro; Schulten, Klaus

2011-01-01

21

A predictor of transmembrane ?-helix domains of proteins based on neural networks  

Microsoft Academic Search

Back-propagation, feed-forward neural networks are used to predict a-helical transmembrane segments of proteins. The networks are trained on the few membrane proteins whose transmembrane a-helix domains are known to atomic or nearly atomic resolution. When testing is performed with a jackknife procedure on the proteins of the training set, the fraction of total correct assignments is as high as 0.87,

Rita Casadio; Piero Fariselli; Chiara Taroni; Mario Compiani

1996-01-01

22

Use of molecular dynamics data in biochemistry courses: An amphipathy scale to determine protein ?-helix transmembrane segments.  

PubMed

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 ?-helix of proteins without the need of knowing anything besides the protein primary structure. In addition, it allows the students to correlate concepts of protein structure and function, energy minimization, molecular dynamics simulations, and protein location. PMID:21591177

Mazzé, Fernanda M; Fuzo, Carlos A; Degrève, Léo; Ciancaglini, Pietro

2008-03-01

23

Simultaneous prediction of protein secondary structure and transmembrane spans.  

PubMed

Prediction of transmembrane spans and secondary structure from the protein sequence is generally the first step in the structural characterization of (membrane) proteins. Preference of a stretch of amino acids in a protein to form secondary structure and being placed in the membrane are correlated. Nevertheless, current methods predict either secondary structure or individual transmembrane states. We introduce a method that simultaneously predicts the secondary structure and transmembrane spans from the protein sequence. This approach not only eliminates the necessity to create a consensus prediction from possibly contradicting outputs of several predictors but bears the potential to predict conformational switches, i.e., sequence regions that have a high probability to change for example from a coil conformation in solution to an ?-helical transmembrane state. An artificial neural network was trained on databases of 177 membrane proteins and 6048 soluble proteins. The output is a 3 × 3 dimensional probability matrix for each residue in the sequence that combines three secondary structure types (helix, strand, coil) and three environment types (membrane core, interface, solution). The prediction accuracies are 70.3% for nine possible states, 73.2% for three-state secondary structure prediction, and 94.8% for three-state transmembrane span prediction. These accuracies are comparable to state-of-the-art predictors of secondary structure (e.g., Psipred) or transmembrane placement (e.g., OCTOPUS). The method is available as web server and for download at www.meilerlab.org. PMID:23349002

Leman, Julia Koehler; Mueller, Ralf; Karakas, Mert; Woetzel, Nils; Meiler, Jens

2013-04-10

24

One face of a transmembrane helix is crucial in mechanosensitive channel gating  

PubMed Central

MscL is a mechanosensitive channel in bacteria that responds directly to membrane tension by opening a large conductance pore. To determine functionally important residues within this molecule, we have randomly mutagenized mscL, expressed the genes in living bacteria, and screened for gain-of-function mutants with hampered growth. Expression of these genes caused leakage of cytoplasmic solutes on little or no hypo-osmotic stress. In excised patches, the mutant channels gated at membrane tensions that are less than that required for the gating of the wild-type MscL. Hence, the data suggest that the slowed or no-growth phenotype is caused by solute loss because of inappropriate gating of the channel. Most of the mutations mapped to the first transmembrane domain. When this domain is modeled as an ?-helix, the most severe mutations are substitutions of smaller amino acids (three glycines and one valine) on one facet, suggesting an important role for this structure in MS channel gating.

Ou, Xiaorong; Blount, Paul; Hoffman, Robert J.; Kung, Ching

1998-01-01

25

Polar substitutions in helix 3 of the prion protein produce transmembrane isoforms that disturb vesicle trafficking.  

PubMed

Prion diseases encompass a diverse group of neurodegenerative conditions characterized by the accumulation of misfolded prion protein (PrP) isoforms. Other conformational variants of PrP have also been proposed to contribute to neurotoxicity in prion diseases, including misfolded intermediates as well as cytosolic and transmembrane isoforms. To better understand PrP neurotoxicity, we analyzed the role of two highly conserved methionines in helix 3 on PrP biogenesis, folding and pathogenesis. Expression of the PrP-M205S and -M205,212S mutants in Drosophila led to hyperglycosylation, intracellular accumulation and widespread conformational changes due to failure of oxidative folding. Surprisingly, PrP-M205S and -M205,212S acquired a transmembrane topology (Ctm) previously linked to mutations in the signal peptide (SP) and the transmembrane domain (TMD). PrP-M205,212S also disrupted the accumulation of key neurodevelopmental proteins in lipid rafts, resulting in shortened axonal projections. These results uncover a new role for the hydrophobic domain in promoting oxidative folding and preventing the formation of neurotoxic Ctm PrP, mechanisms that may be relevant in the pathogenesis of both inherited and sporadic prion diseases. PMID:23771030

Sanchez-Garcia, Jonatan; Arbelaez, Daniela; Jensen, Kurt; Rincon-Limas, Diego E; Fernandez-Funez, Pedro

2013-06-13

26

Unfolding a transmembrane helix dimer: A FRET study in mixed micelles  

Microsoft Academic Search

The exact nature of membrane protein folding and assembly is not understood in detail yet. Addition of SDS to a membrane protein dissolved in mild, non-polar detergent results in formation of mixed micelles and in subsequent denaturation of higher ordered membrane protein structures. The exact nature of this denaturation event is, however, enigmatic, and separation of an individual helix pair

Veerappan Anbazhagan; Florian Cymer; Dirk Schneider

2010-01-01

27

Large lateral movement of transmembrane helix S5 is not required for substrate access to the active site of rhomboid intramembrane protease.  

PubMed

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

2013-04-22

28

Agonist-specific requirement for a glutamate in transmembrane helix 1 of the oxytocin receptor.  

PubMed

Defining key differences between agonist and antagonist binding to hormone receptors is important and will aid rational drug design. Glu(1.35) in transmembrane helix 1 (TM1) of the human oxytocin receptor (OTR) is absolutely conserved in all OTRs cloned to date. We establish that Glu(1.35) is critical for high affinity binding of agonists (full and partial) but is not required for antagonist binding (peptide or non-peptide). Consequently, the mutant receptor [E1.35A]OTR exhibited markedly decreased OT affinity (>1200-fold) and disrupted second messenger generation. Substitutions of Glu(1.35) by Asp, Gln or Arg were incapable of supporting wild-type OTR agonist binding or signaling. Molecular modeling revealed that Glu(1.35) projects into the receptor's central binding crevice and provides agonist-specific contacts not utilized by antagonists. This study explains why Glu is absolutely conserved at residue-1.35 in all receptors binding OT and related peptides, and provides molecular insight into key differences between agonist-receptor and antagonist-receptor binding modes. PMID:21130837

Wootten, Denise L; Simms, John; Massoura, Amelia J; Trim, Julie E; Wheatley, Mark

2010-12-03

29

Structural characterizations of oligopyridyl foldamers, ?-helix mimetics.  

PubMed

Protein-protein interactions are central to many biological processes, from intracellular communication to cytoskeleton assembly, and therefore represent an important class of targets for new therapeutics. The most common secondary structure in natural proteins is an ?-helix. Small molecules seem to be attractive candidates for stabilizing or disrupting protein-protein interactions based on ?-helices. In our study, we assessed the ability of oligopyridyl scaffolds to mimic the ?-helical twist. The theoretical as well as experimental studies (X-ray diffraction and NMR) on conformations of bipyridines in the function of substituent and pyridine nitrogen positions were carried out. Furthermore, the experimental techniques showed that the conformations observed in bipyridines are maintained within a longer oligopyridyl scaffold (quaterpyridines). The alignment of the synthesized quaterpyridine with two methyl substituents showed that it is an ?-helix foldamer; their methyl groups overlap very well with side chain positions, i and i + 3, of an ideal ?-helix. PMID:22196240

Santos, Jana Sopkova-de Oliveira; Voisin-Chiret, Anne Sophie; Burzicki, Gregory; Sebaoun, Laure; Sebban, Muriel; Lohier, Jean-François; Legay, Rémi; Oulyadi, Hassan; Bureau, Ronan; Rault, Sylvain

2012-01-17

30

Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants.  

PubMed

Transmembrane proteins serve important biological functions, yet precise information on their secondary and tertiary structure is very limited. The boundaries and structures of membrane-embedded domains in integral membrane proteins can be determined by a method based on a combination of site-specific mutagenesis and nitroxide spin labeling. The application to one polypeptide segment in bacteriorhodopsin, a transmembrane chromoprotein that functions as a light-driven proton pump is described. Single cysteine residues were introduced at 18 consecutive positions (residues 125 to 142). Each mutant was reacted with a specific spin label and reconstituted into vesicles that were shown to be functional. The relative collision frequency of each spin label with freely diffusing oxygen and membrane-impermeant chromium oxalate was estimated with power saturation EPR (electron paramagnetic resonance) spectroscopy. The results indicate that residues 129 to 131 form a short water-exposed loop, while residues 132 to 142 are membrane-embedded. The oxygen accessibility for positions 131 to 138 varies with a periodicity of 3.6 residues, thereby providing a striking demonstration of an alpha helix. The orientation of this helical segment with respect to the remainder of the protein was determined. PMID:2160734

Altenbach, C; Marti, T; Khorana, H G; Hubbell, W L

1990-06-01

31

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

PubMed Central

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

Herzyk, P; Hubbard, R E

1995-01-01

32

Structural alignment of RNA with triple helix structure.  

PubMed

Structural alignment is useful in identifying members of ncRNAs. Existing tools are all based on the secondary structures of the molecules. There is evidence showing that tertiary interactions (the interaction between a single-stranded nucleotide and a base-pair) in triple helix structures are critical in some functions of ncRNAs. In this article, we address the problem of structural alignment of RNAs with the triple helix. We provide a formal definition to capture a simplified model of a triple helix structure, then develop an algorithm of O(mn(3)) time to align a query sequence (of length m) with known triple helix structure with a target sequence (of length n) with an unknown structure. The resulting algorithm is shown to be useful in identifying ncRNA members in a simulated genome. PMID:22468707

Wong, Thomas K F; Yiu, S M

2012-04-01

33

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

PubMed Central

Background Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the ?-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the ?1-subunit. Results Preparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020) successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT) and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME), can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the ?1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT. Conclusion We propose that progesterone binding to the first external loop of the ?1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions.

2010-01-01

34

A sequence-based computational model for the prediction of the solvent accessible surface area for ?-helix and ?-barrel transmembrane residues.  

PubMed

Predicting the solvent accessible surface area (ASA) of transmembrane (TM) residues is of great importance for experimental researchers to elucidate diverse physiological processes. TM residues fall into two major structural classes (?-helix membrane protein and ?-barrel membrane protein). The reported solvent ASA prediction models were developed for these two types of TM residues respectively. However, this prevents the general use of these methods because one cannot determine which model is suitable for a given TM residue without information of its type. To conquer this limitation, we developed a new computational model that can be used for predicting the ASA of both TM ?-helix and ?-barrel residues. The model was developed from 78 ?-helix membrane protein chains and 24 ?-barrel membrane protein. Its prediction ability was evaluated by cross validation method and its prediction result on an independent test set of 20 membrane protein chains. The results show that our model performs well for both types of TM residues and outperforms other prediction model which was developed for the specific type of TM residues. The prediction results also proved that the random forest model incorporating conservation score is an effective sequence-based computational approach for predicting the solvent ASA of TM residues. PMID:21935968

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

2011-09-21

35

Polar Residues in Transmembrane Helices can Decrease Electrophoretic Mobility in Polyacrylamide Gels Without Causing Helix Dimerization  

PubMed Central

There are only a few available methods to study lateral interactions and self assembly of transmembrane helices. One of the most frequently used methods is sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) which can report on strong interactions between peptides in SDS solution. Here we offer a cautionary tale about studying the folding and assembly of membrane proteins using peptides and SDS-PAGE experiments as membrane mimetic systems. At least for the specific peptide and detergent systems studied here, we show that a polar asparagine residue in the 12th position of an otherwise hydrophobic helical segment of 20 amino acids causes a peptide to migrate on SDS-PAGE gels with an apparent molecular weight that is twice its true molecular weight, suggesting dimerization. However when examined carefully in SDS solutions and in situ in the polyacrylamide gel itself using Forster resonance energy transfer no interaction can be detected. Instead we show evidence suggesting that differential interactions between peptide and detergent drive the differences in electrophoretic mobility without any interaction between peptides. These results emphasize the need to apply multiple independent techniques to the study of membrane protein folding, and they highlight the usefulness of studying folding and structure of membrane proteins in lipid membranes rather than in detergents.

Walkenhorst, William F.; Merzlyakov, Mikhail; Hristova, Kalina; Wimley, William C.

2010-01-01

36

Transmembrane beta-barrel protein structure prediction  

NASA Astrophysics Data System (ADS)

Transmembrane ?-barrel (TMB) proteins are embedded in the outer membranes of mitochondria, Gram-negative bacteria, and chloroplasts. These proteins perform critical functions, including active ion-transport and passive nutrient intake. Therefore, there is a need for accurate prediction of secondary and tertiary structures of TMB proteins. A variety of methods have been developed for predicting the secondary structure and these predictions are very useful for constructing a coarse topology of TMB structure; however, they do not provide enough information to construct a low-resolution tertiary structure for a TMB protein. In addition, while the overall structural architecture is well conserved among TMB proteins, the amino acid sequences are highly divergent. Thus, traditional homology modeling methods cannot be applied to many putative TMB proteins. Here, we describe the TMBpro: a pipeline of methods for predicting TMB secondary structure, ?-residue contacts, and finally tertiary structure. The tertiary prediction method relies on the specific construction rules that TMB proteins adhere to and on the predicted ?-residue contacts to dramatically reduce the search space for the model building procedure.

Randall, Arlo; Baldi, Pierre

37

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.

2011-01-01

38

Network analysis of transmembrane protein structures  

NASA Astrophysics Data System (ADS)

Most studies have shown that globular proteins exist in small-world networks. The present study is an attempt to determine differences in network parameters between transmembrane and globular proteins. Each protein is represented as an undirected graph, where residues represent nodes and inter-residue interactions as the edges. This was then compared to the degree-preserved random controls, to observe if any variation existed. Results indicate that there is a significant difference in shortest path lengths between transmembrane and globular proteins. Hydrophobic amino acids were found to be more spatially distributed in the transmembrane than in globular proteins causing significantly higher values of shortest path lengths (L). Assortativity values too, were found to be significantly higher in the transmembrane than in globular proteins which is due to the highly connected amino acid residues being exposed to the solvent in transmembrane proteins. On analyzing the closeness centrality, it was found that globular proteins have significantly higher values than the transmembrane proteins. We therefore conclude that transmembrane proteins possess small-world characteristics similar to that of globular proteins.

Emerson, I. Arnold; Gothandam, K. M.

2012-02-01

39

Proline-induced disruption of a transmembrane ?-helix in its natural environment 1 1 Edited by F. Cohen  

Microsoft Academic Search

?-Helix formation in globular proteins has been studied both theoretically and experimentally for decades, while a lack of both high-resolution structures and suitable experimental techniques has hampered the study of helices in membrane proteins. We have developed a new experimental approach, glycosylation mapping, where the active site of the lumenally exposed endoplasmic reticulum enzyme oligosaccharyl transferase is used as a

IngMarie Nilsson; Annika Sääf; Paul Whitley; Guro Gafvelin; Cecilia Waller; Gunnar von Heijne

1998-01-01

40

Molecular code for transmembrane-helix recognition by the Sec61 translocon  

Microsoft Academic Search

Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a

Tara Hessa; Nadja M. Meindl-Beinker; Andreas Bernsel; Hyun Kim; Yoko Sato; Mirjam Lerch-Bader; Ingmarie Nilsson; Stephen H. White; Gunnar von Heijne

2007-01-01

41

Requirement of ? and ? subunit transmembrane helix separation for integrin outside-in signaling  

PubMed Central

Adhesion to extracellular ligands through integrins regulates cell shape, migration, growth, and survival. How integrins transmit signals in the outside-to-in direction remains unknown. Whereas in resting integrins the ? and ? subunit transmembrane domains are associated, ligand binding promotes dissociation and separation of these domains. Here we address whether such separation is required for outside-in signaling. By introduction of an intersubunit disulfide bond, we generated mutant integrin ?IIb?3 with blocked transmembrane separation that binds ligand, mediates adhesion, adopts an extended conformation after ligand binding, and forms antibody-induced macroclusters on the cell surface similarly to wild type. However, the mutant integrin exhibits a profound defect in adhesion-induced outside-in signaling as measured by cell spreading, actin stress-fiber and focal adhesion formation, and focal adhesion kinase activation. This defect was rescued by reduction of the disulfide bond. Our results demonstrate that the separation of transmembrane domains is required for integrin outside-in signal transduction.

Zhu, Jieqing; Carman, Christopher V.; Kim, Minsoo; Shimaoka, Motomu; Springer, Timothy A.

2007-01-01

42

beta-Barrel transmembrane proteins: Geometric modelling, detection of transmembrane region, and structural properties.  

PubMed

The location of the membrane lipid bilayer relative to a transmembrane protein structure is important in protein engineering. Since it is not present on the determined structures, it is essential to automatically define the membrane embedded protein region in order to test mutation effects or to design potential drugs. beta-Barrel transmembrane proteins, present in nature as outer membrane proteins (OMPs), comprise one of the two transmembrane protein fold classes. Lately, the number of their determined structures has increased and this enables the implementation and evaluation of structure-based annotation methods and their more comprehensive study. In this paper, we propose two new algorithms for (i) the geometric modelling of beta-barrels and (ii) the detection of the transmembrane region of a beta-barrel transmembrane protein. The geometric modelling algorithm combines a non-linear least square minimization method and a genetic algorithm in order to find the characteristics (axis, radius) of a shape with axial symmetry which best models a beta-barrel. The transmembrane region is detected by profiling the external residues of the beta-barrel along its axis in terms of hydrophobicity and existence of aromatic and charged residues. TbB-Tool implements these algorithms and is available in . A non-redundant set of 22 OMPs is used in order to evaluate the algorithms implemented and the results are very satisfying. In addition, we quantify the abundance of all amino acids and the average hydrophobicity for external and internal beta-stranded residues along the axis of beta-barrel, thus confirming and extending other researchers' results. PMID:17097352

Valavanis, Ioannis K; Bagos, Pantelis G; Emiris, Ioannis Z

2006-11-09

43

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

PubMed Central

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

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

2003-01-01

44

Transmembrane helix I and periplasmic loop 1 of Escherichia coli ProP are involved in osmosensing and osmoprotectant transport.  

PubMed

Osmoregulatory transporters stimulate bacterial growth by mediating osmoprotectant uptake in response to increasing osmotic pressure. The ProP protein of Escherichia coli transports proline and other osmoprotectants. Like LacY, ProP is a member of the major facilitator superfamily and a H(+)-solute symporter. ProP is regulated by osmotic pressure via a membrane potential-dependent mechanism. A homology model predicts that ionizable and polar residues, highly conserved among ProP homologues, cluster deep within the N-terminal helix bundle of ProP. Chemical labeling of introduced cysteine (Cys) residues supported the homology model by confirming the predicted positions of transmembrane helix I (TMI) and periplasmic loop 1. Replacements of residues in the putative polar cluster impaired or altered ProP function, suggesting that they are important for osmosensing and may interact with the transport substrates. Asn34, Glu37, Phe41, Tyr44, and Ala48 line the most polar face of TMI; Tyr44 is on the periplasmic side of the putative polar cluster, and Ala59 is in periplasmic loop 1. The N-ethylmaleimide reactivities of Cys introduced at positions 41, 44, 48, and 59 increased with osmotic pressure, whereas the reactivities of those at cytoplasm-proximal positions 34 and 37 did not. Replacements of polar cluster residues that blocked transport also affected the NEM reactivity of Cys44 and its osmolality dependence. This report and previous work suggest that conformational changes associated with osmosensing may shift the equilibria between outward- and inward-facing transport pathway intermediates. PMID:20828170

Keates, Robert A B; Culham, Doreen E; Vernikovska, Yaroslava I; Zuiani, Adam J; Boggs, Joan M; Wood, Janet M

2010-10-19

45

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

2013-06-15

46

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

PubMed Central

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

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

1999-01-01

47

Twisting of the second transmembrane alpha-helix of the mitochondrial ADP/ATP carrier during the transition between two carrier conformational states.  

PubMed

To investigate the structural and functional features of the second alpha-helical transmembrane segment (TM2) of the mitochondrial ADP/ATP carrier (AAC), we adopted cysteine scanning mutagenesis analysis. Single-cysteine mutations of yeast AAC were systematically introduced at residues 98-106 in TM2, and the mutants were treated with the fluorescent SH reagent eosin-5-maleimide (EMA). EMA modified different amino acid residues of alpha-helical TM2 between the two distinct carrier conformations, called the m-state and the c-state, in which the substrate recognition site faces the matrix and cytosol, respectively. When amino acids in the helix were projected on a wheel plot, these EMA-modified amino acids were observed at distinct sides of the wheel. Since the SH reagent specifically modified cysteine in the water-accessible environment, these results indicate that distinct helical surfaces of TM2 faced the water-accessible space between the two conformations, possibly as a result of twisting of this helix. In the recently reported crystal structure of bovine AAC, several amino acids faced cocrystallized carboxyatractyloside (CATR), a specific inhibitor of the carrier. These residues correspond to those modified with EMA in the yeast carrier in the c-state. Since the binding site of CATR is known to overlap that of the transport substrate, the water-accessible space was thought to be a substrate transport pathway, and hence, the observed twisting of TM2 between the m-state and the c-state may be involved in the process of substrate translocation. On the basis of the results, the roles of TM2 in the transport function of AAC were discussed. PMID:15568812

Kihira, Yoshitaka; Iwahashi, Akihiro; Majima, Eiji; Terada, Hiroshi; Shinohara, Yasuo

2004-12-01

48

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.

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

2005-01-01

49

Predicting Residue-Residue Contacts and Helix-Helix Interactions in Transmembrane Proteins Using an Integrative Feature-Based Random Forest Approach  

PubMed Central

Integral membrane proteins constitute 25–30% of genomes and play crucial roles in many biological processes. However, less than 1% of membrane protein structures are in the Protein Data Bank. In this context, it is important to develop reliable computational methods for predicting the structures of membrane proteins. Here, we present the first application of random forest (RF) for residue-residue contact prediction in transmembrane proteins, which we term as TMhhcp. Rigorous cross-validation tests indicate that the built RF models provide a more favorable prediction performance compared with two state-of-the-art methods, i.e., TMHcon and MEMPACK. Using a strict leave-one-protein-out jackknifing procedure, they were capable of reaching the top L/5 prediction accuracies of 49.5% and 48.8% for two different residue contact definitions, respectively. The predicted residue contacts were further employed to predict interacting helical pairs and achieved the Matthew's correlation coefficients of 0.430 and 0.424, according to two different residue contact definitions, respectively. To facilitate the academic community, the TMhhcp server has been made freely accessible at http://protein.cau.edu.cn/tmhhcp.

Wang, Xiao-Feng; Chen, Zhen; Wang, Chuan; Yan, Ren-Xiang; Zhang, Ziding; Song, Jiangning

2011-01-01

50

Lipid Bilayer Topology of the Transmembrane ?-Helix of M13 Major Coat Protein and Bilayer Polarity Profile by Site-Directed Fluorescence Spectroscopy  

PubMed Central

This article presents a new formalism to perform a quantitative fluorescence analysis using the Stokes shift of AEDANS-labeled cysteine mutants of M13 major coat protein incorporated in lipid bilayers. This site-directed fluorescence spectroscopy approach enables us to obtain the topology of the bilayer-embedded transmembrane ?-helix from the orientation and tilt angles, and relative bilayer location. Both in pure dioleoylphosphatidylcholine and dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (4:1 mol/mol) bilayers, which have a similar bilayer thickness, the tilt angle of the transmembrane helix of the coat protein turns out to be 23° ± 4. Upon decreasing the hydrophobic thickness on going from dieicosenoylphosphatidylcholine to dimyristoylphosphatidylcholine, the tilt angle and orientation angle of the transmembrane ?-helix change. The protein responds to an increase of hydrophobic stress by increasing the tilt angle so as to keep much of its hydrophobic part inside the bilayer. At the same time, the transmembrane helix rotates at its long axis so as to optimize the hydrophobic and electrostatic interactions of the C-terminal phenylalanines and lysines, respectively. The increase of tilt angle cannot completely keep the hydrophobic protein section within the bilayer, but the C-terminal part remains anchored at the acyl-chain/glycerol backbone interface at the cost of the N-terminal section. In addition, our analysis results in the profile of the dielectric constant of the hydrophobic domain of the bilayer. For all phospholipid bilayers studied the profile has a concave shape, with a value of the dielectric constant of 4.0 in the center of the bilayer. The dielectric constant increases on approaching the headgroup region with a value of 12.4 at the acyl-chain/glycerol backbone interface for the various phosphatidylcholines with different chain lengths. For dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (4:1 mol/mol) bilayers the value of the dielectric constant at the acyl-chain/glycerol backbone interface is 18.6. In conclusion, the consistency of our analysis shows that the applied cysteine-scanning mutagenesis method with AEDANS labeling of a helical transmembrane protein in combination with a quantitative formalism offers a reliable description of the lipid bilayer topology of the protein and bilayer properties. This also indicates that the spacer link between the protein and AEDANS label is long enough to monitor the local polarity of the lipid environment and not that of the amino-acid residues of the protein, and short enough to have the topology of the protein imposing on the fluorescence properties of the AEDANS label.

Koehorst, Rob B. M.; Spruijt, Ruud B.; Vergeldt, Frank J.; Hemminga, Marcus A.

2004-01-01

51

Structure of hemocyanin from garden snail Helix lucorum  

Microsoft Academic Search

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthropods with different quaternary structure. They are represented in the hemolymph of molluscs with one, two or three isoforms, as decameric, didecameric, multidecameric and tubules aggregates. We describe here the structure of the hemocyanin Helix lucorum (HlH), species in the series of molluscan hemocyanins. In contrast with

Ludmila Velkova; Ivan Dimitrov; Heinz Schwarz; Stefan Stevanovic; Wolfgang Voelter; Benedeto Salvato; Pavlina Dolashka-Angelova

2010-01-01

52

Towards genome-scale structure prediction for transmembrane proteins  

PubMed Central

In this paper we briefly review some of the recent progress made by ourselves and others in developing methods for predicting the structures of transmembrane proteins from amino acid sequence. Transmembrane proteins are an important class of proteins involved in many diverse biological functions, many of which have great impact in terms of disease mechanism and drug discovery. Despite their biological importance, it has proven very difficult to solve the structures of these proteins by experimental techniques, and so there is a great deal of pressure to develop effective methods for predicting their structure. The methods we discuss range from methods for transmembrane topology prediction to new methods for low resolution folding simulations in a knowledge-based force field. This potential is designed to reproduce the properties of the lipid bilayer. Our eventual aim is to apply these methods in tandem so that useful three-dimensional models can be built for a large fraction of the transmembrane protein domains in whole proteomes.

Hurwitz, Naama; Pellegrini-Calace, Marialuisa; Jones, David T

2006-01-01

53

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

PubMed Central

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

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

2012-01-01

54

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

NASA Astrophysics Data System (ADS)

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

Harrington, Susan E.; Ben-Tal, Nir

55

Using protein structural information in evolutionary inference: transmembrane proteins.  

PubMed

We present a model of amino acid sequence evolution based on a hidden Markov model that extends to transmembrane proteins previous methods that incorporate protein structural information into phylogenetics. Our model aims to give a better understanding of processes of molecular evolution and to extract structural information from multiple alignments of transmembrane sequences and use such information to improve phylogenetic analyses. This should be of value in phylogenetic studies of transmembrane proteins: for example, mitochondrial proteins have acquired a special importance in phylogenetics and are mostly transmembrane proteins. The improvement in fit to example data sets of our new model relative to less complex models of amino acid sequence evolution is statistically tested. To further illustrate the potential utility of our method, phylogeny estimation is performed on primate CCR5 receptor sequences, sequences of l and m subunits of the light reaction center in purple bacteria, guinea pig sequences with respect to lagomorph and rodent sequences of calcitonin receptor and K-substance receptor, and cetacean sequences of cytochrome b. PMID:10605112

Liò, P; Goldman, N

1999-12-01

56

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

PubMed

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure. PMID:23967136

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

2013-08-15

57

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

The housekeeping sarco(endo)plasmic reticulum Ca(2+) ATPase SERCA2b transports Ca(2+) across the endoplasmic reticulum membrane maintaining a vital Ca(2+) 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 Ca(2+) affinity and lower V(max). 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 V(max) and higher Ca(2+) 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 Ca(2+) 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 Ca(2+) 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

2012-04-23

58

Structure of Staphylococcal alpha-Hemolysin, a Heptameric Transmembrane Pore  

Microsoft Academic Search

The structure of the Staphylococcus aureus alpha-hemolysin pore has been determined to 1.9 overset{circ}{mathrm A} resolution. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 overset{circ}{mathrm A} in length, that runs along the sevenfold axis and ranges from 14 overset{circ}{mathrm A} to 46 overset{circ}{mathrm A} in diameter. The lytic, transmembrane domain comprises the lower half of a 14-strand

Langzhou Song; Michael R. Hobaugh; Christopher Shustak; Stephen Cheley; Hagan Bayley; J. Eric Gouaux

1996-01-01

59

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

PubMed Central

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

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

2002-01-01

60

The Extension of the Fourth Transmembrane Helix of the Sensor Kinase KdpD of Escherichia coli Is Involved in Sensing?  

PubMed Central

The KdpD sensor kinase and the KdpE response regulator control expression of the kdpFABC operon coding for the KdpFABC high-affinity K+ transport system of Escherichia coli. In search of a distinct part of the input domain of KdpD which is solely responsible for K+ sensing, sequences of kdpD encoding the transmembrane region and adjacent N-terminal and C-terminal extensions were subjected to random mutagenesis. Nine KdpD derivatives were identified that had lost tight regulation of kdpFABC expression. They all carried single amino acid replacements located in a region encompassing the fourth transmembrane helix and the adjacent arginine cluster of KdpD. All mutants exhibited high levels of kdpFABC expression regardless of the external K+ concentration. However, 3- to 14-fold induction was observed under extreme K+-limiting conditions and in response to an osmotic upshift when sucrose was used as an osmolyte. These KdpD derivatives were characterized by a reduced phosphatase activity in comparison to the autokinase activity in vitro, which explains constitutive expression. Whereas for wild-type KdpD the autokinase activity and also, in turn, the phosphotransfer activity to KdpE were inhibited by increasing concentrations of K+, both activities were unaffected in the KdpD derivatives. These data clearly show that the extension of the fourth transmembrane helix encompassing the arginine cluster is mainly involved in sensing both K+ limitation and osmotic upshift, which may not be separated mechanistically.

Zimmann, Petra; Steinbrugge, Anne; Schniederberend, Maren; Jung, Kirsten; Altendorf, Karlheinz

2007-01-01

61

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

SciTech Connect

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

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

2005-01-01

62

Polyproline-II helix in proteins: structure and function.  

PubMed

The poly-l-proline type II (PPII) helix in recent years has emerged clearly as a structural class not only of fibrillar proteins (in collagen, PPII is a dominant conformation) but also of the folded and unfolded proteins. Although much less abundant in folded proteins than the ?-helix and ?-structure, the left-handed, extended PPII helix represents the only frequently occurring regular structure apart from these two structure classes. Natively unfolded proteins have a high content of the PPII helices identified by spectroscopic methods. Apart from the structural function, PPII is favorable for protein-protein and protein-nucleic acid interactions and plays a major role in signal transduction and protein complex assembly, as this structure is often found in binding sites, specifically binding sites of widely spread SH3 domains. PPII helices do not necessarily contain proline, but proline has high PPII propensity. Commonly occurring proline-rich regions, serving as recognition sites, are likely to have PPII structure. PPII helices are involved in transcription, cell motility, self-assembly, elasticity, and bacterial and viral pathogenesis, and has an important structural role in amyloidogenic proteins. However, PPII helices are not always assigned in experimentally solved structures, and they are rarely used in protein structure modeling. We aim to give an overview of this structural class and of the place it holds in our current understanding of protein structure and function. This review is subdivided into three main parts: the first part covers PPII helices in unfolded peptides and proteins, the second part includes studies of the PPII helices in folded proteins, and the third part discusses the functional role of the PPII. PMID:23507311

Adzhubei, Alexei A; Sternberg, Michael J E; Makarov, Alexander A

2013-03-16

63

The Structure of Barmah Forest Virus as Revealed by Cryo-Electron Microscopy at a 6-Angstrom Resolution Has Detailed Transmembrane Protein Architecture and Interactions ? †  

PubMed Central

Barmah Forest virus (BFV) is a mosquito-borne alphavirus that infects humans. A 6-Å-resolution cryo-electron microscopy three-dimensional structure of BFV exhibits a typical alphavirus organization, with RNA-containing nucleocapsid surrounded by a bilipid membrane anchored with the surface proteins E1 and E2. The map allows details of the transmembrane regions of E1 and E2 to be seen. The C-terminal end of the E2 transmembrane helix binds to the capsid protein. Following the E2 transmembrane helix, a short ?-helical endodomain lies on the inner surface of the lipid envelope. The E2 endodomain interacts with E1 transmembrane helix from a neighboring E1-E2 trimeric spike, thereby acting as a spacer and a linker between spikes. In agreement with previous mutagenesis studies, the endodomain plays an important role in recruiting other E1-E2 spikes to the budding site during virus assembly. The E2 endodomain may thus serve as a target for antiviral drug design.

Kostyuchenko, Victor A.; Jakana, Joanita; Liu, Xiangan; Haddow, Andrew D.; Aung, Myint; Weaver, Scott C.; Chiu, Wah; Lok, Shee-Mei

2011-01-01

64

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.

2010-01-01

65

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

PubMed

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 nuclear magnetic resonance (NMR) spectroscopy and electron micrographs. 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 distinctive features, including a shorter, pore-lining TM2 helix 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. Our structures provide additional templates for identifying potential drug targets in GlyRs and other mammalian pLGICs. PMID:23994010

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

2013-08-29

66

A novel mutation within a transmembrane helix of the bile salt export pump (BSEP, ABCB11) with delayed development of cirrhosis.  

PubMed

BACKGROUND & AIMS: The bile salt export pump (BSEP, ABCB11) is essential for bile salt secretion at the canalicular membrane of liver cells. Clinical phenotypes associated with BSEP mutations are commonly categorized as benign recurrent intrahepatic cholestasis (BRIC-2) or progressive familial intrahepatic cholestasis (PFIC-2). METHODS: The molecular basis of BSEP-associated liver disease in a sibling pair was characterized by immunostaining, gene sequencing, bile salt analysis and recombinant expression in mammalian cells and yeast for localization and in vitro activity studies respectively. RESULTS: Benign recurrent intrahepatic cholestasis was considered in a brother and sister who both suffered from intermittent cholestasis since childhood. Gene sequencing of ABCB11 identified the novel missense mutation p.G374S, which is localized in the putative sixth transmembrane helix of BSEP. Liver fibrosis was present in the brother at the age of 18 with progression to cirrhosis within 3 years. Immunofluorescence of liver tissue showed clear canalicular BSEP expression; however, biliary concentration of bile salts was drastically reduced. In line with these in vivo findings, HEK293 cells showed regular membrane targeting of human BSEP(G374S) , whereas in vitro transport measurements revealed a strongly reduced transport activity. CONCLUSIONS: The novel mutation p.G374S impairs transport function without disabling membrane localization of BSEP. While all other known BSEP mutations within transmembrane helices are associated with PFIC-2, the new p.G374S mutation causes a transitional phenotype between BRIC-2 and PFIC-2. PMID:23758865

Stindt, Jan; Ellinger, Philipp; Weissenberger, Katrin; Dröge, Carola; Herebian, Diran; Mayatepek, Ertan; Homey, Bernhard; Braun, Stephan; Schulte Am Esch, Jan; Horacek, Michael; Canbay, Ali; Schmitt, Lutz; Häussinger, Dieter; Kubitz, Ralf

2013-05-16

67

Anesthetic Effects on the Structure and Dynamics of the Second Transmembrane Domains of nAChR ?4?2  

PubMed Central

Channel functions of the neuronal ?4?2 nicotinic acetylcholine receptor (nAChR), one of the most widely expressed subtypes in the brain, can be inhibited by volatile anesthetics. Our Na+ flux experiments confirmed that the second transmembrane domains (TM2) of ?4 and ?2 in 2:3 stoichiometry, (?4)2(?2)3, could form pentameric channels, whereas the ?4 TM2 alone could not. The structure, topology, and dynamics of the ?4 TM2 and (?4)2(?2)3 TM2 in magnetically aligned phospholipid bicelles were investigated using solid-state NMR spectroscopy in the absence and presence of halothane and isoflurane, two clinically used volatile anesthetics. 2H NMR demonstrated that anesthetics increased lipid conformational heterogeneity. Such anesthetic effects on lipids became more profound in the presence of transmembrane proteins. PISEMA experiments on the selectively 15N-labeled ?4 TM2 showed that the TM2 formed transmembrane helices with tilt angles of 12° ± 1° and 16° ± 1° relative to the bicelle normal for the ?4 and (?4)2(?2)3 samples, respectively. Anesthetics changed the tilt angle of the ?4 TM2 from 12° ± 1° to 14° ± 1°, but had only a subtle effect on the tilt angle of the (?4)2(?2)3 TM2. A small degree of wobbling motion of the helix axis occurred in the (?4)2(?2)3 TM2. In addition, a subset of the (?4)2(?2)3 TM2 exhibited counterclockwise rotational motion around the helix axis on a time scale slower than 10?4 s in the presence of anesthetics. Both helical tilting and rotational motions have been identified computationally as critical elements for ion channel functions. This study suggested that anesthetics could alter these motions to modulate channel functions.

Cui, Tanxing; Canlas, Christian G.; Xu, Yan; Tang, Pei

2009-01-01

68

Relationship between side chain structure and 14-helix stability of beta3-peptides in water.  

PubMed

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, beta(3)-peptides, in water. Our experimental results (1) verify the importance of macrodipole stabilization for maintaining 14-helix structure, (2) provide comprehensive evidence that beta(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 beta(3)-peptides containing beta(3)-homoserine or beta(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 beta(3)-amino acids differ starkly from the alpha-helix propensities of analogous alpha-amino acids. This contrast informs current models for alpha-helix folding, and suggests that 14-helix folding is governed by different biophysical forces than is alpha-helix folding. The ability to modulate 14-helix structure through side chain choice will assist rational design of 14-helical beta-peptide ligands for macromolecular targets. PMID:15631466

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

2005-01-12

69

Energy-based classification and structure prediction of transmembrane beta-barrel proteins  

Microsoft Academic Search

Transmembrane ?? -barrel (TMB) proteins are a special class of transmembrane proteins which play several key roles in human body and diseases. Due to experimental difficulties, the number of TMB proteins with known structures is very small. Over the years, a number of learning-based methods have been introduced for recognition and structure prediction of TMB proteins. Most of these methods

Van Du Tran; Philippe Chassignet; Saad Sheikh; Jean-Marc Steyaert

2011-01-01

70

Structural organization and interactions of transmembrane domains in tetraspanin proteins  

PubMed Central

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

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

2005-01-01

71

Structural Plasticity in Self-Assembling Transmembrane ?-Sheets  

PubMed Central

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

Bishop, Christopher M.; Wimley, William C.

2011-01-01

72

Transmembrane and Juxtamembrane Structure of ?L Integrin in Bicelles  

PubMed Central

The accepted model for the interaction of ? and ? integrins in the transmembrane (TM) domain is based on the pair ?IIb?3. This involves the so-called outer and inner membrane association clasps (OMC and IMC, respectively). In the ? chain, the OMC involves a GxxxG-like motif, whereas in the IMC a conserved juxtamembrane GFFKR motif experiences a backbone reversal that partially fills the void generated by TM separation towards the cytoplasmic half. However, the GFFKR motif of several ? integrin cytoplasmic tails in non-bicelle environments has been shown to adopt an ?-helical structure that is not membrane-embedded and which was shown to bind a variety of cytoplasmic proteins. Thus it is not known if a membrane-embedded backbone reversal is a conserved structural feature in ? integrins. We have studied the system ?L?2 because of its importance in leukocytes, where integrin deactivation is particularly important. Herein we show that the backbone reversal feature is not only present in ?IIb but also in ?L-TM when reconstituted in bicelles. Additionally, titration with ?2 TM showed eight residues clustering along one side of ?L-TM, forming a plausible interacting face with ?2. The latter orientation is consistent with a previously predicted reported polar interaction between ?L Ser-1071 and ?2 Thr-686.

Millet, Oscar; Diercks, Tammo; Torres, Jaume

2013-01-01

73

Transmembrane and Juxtamembrane Structure of ?L Integrin in Bicelles.  

PubMed

The accepted model for the interaction of ? and ? integrins in the transmembrane (TM) domain is based on the pair ?IIb?3. This involves the so-called outer and inner membrane association clasps (OMC and IMC, respectively). In the ? chain, the OMC involves a GxxxG-like motif, whereas in the IMC a conserved juxtamembrane GFFKR motif experiences a backbone reversal that partially fills the void generated by TM separation towards the cytoplasmic half. However, the GFFKR motif of several ? integrin cytoplasmic tails in non-bicelle environments has been shown to adopt an ?-helical structure that is not membrane-embedded and which was shown to bind a variety of cytoplasmic proteins. Thus it is not known if a membrane-embedded backbone reversal is a conserved structural feature in ? integrins. We have studied the system ?L?2 because of its importance in leukocytes, where integrin deactivation is particularly important. Herein we show that the backbone reversal feature is not only present in ?IIb but also in ?L-TM when reconstituted in bicelles. Additionally, titration with ?2 TM showed eight residues clustering along one side of ?L-TM, forming a plausible interacting face with ?2. The latter orientation is consistent with a previously predicted reported polar interaction between ?L Ser-1071 and ?2 Thr-686. PMID:24069290

Surya, Wahyu; Li, Yan; Millet, Oscar; Diercks, Tammo; Torres, Jaume

2013-09-12

74

An unusual transmembrane helix in the endoplasmic reticulum ubiquitin ligase Doa10 modulates degradation of its cognate E2 enzyme.  

PubMed

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

Kreft, Stefan G; Hochstrasser, Mark

2011-04-05

75

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

PubMed Central

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

Kreft, Stefan G.; Hochstrasser, Mark

2011-01-01

76

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

PubMed Central

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

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

2013-01-01

77

patGPCR: a multitemplate approach for improving 3D structure prediction of transmembrane helices of G-protein-coupled receptors.  

PubMed

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

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

2013-03-11

78

Transformation of a design peptide between the ?-helix and ?-hairpin structures using a helix-strand replica-exchange molecular dynamics simulation.  

PubMed

We investigated the transformation between the ?-helix and ?-hairpin structures of an 18-residue design peptide, whose sequence is INYWLAHAKAGYIVHWTA. This peptide has both ?-helix and ?-hairpin structures in aqueous solution. For this purpose, we proposed the helix-strand replica-exchange method. This is one of the Hamiltonian replica-exchange methods in which we exchange parameters for umbrella potentials to enhance the ?-helix or ?-strand structure formation. We performed an all-atom helix-strand replica-exchange molecular dynamics (MD) simulation of this peptide in explicit water solvent with five replicas. Because the suitable umbrella potential was applied, the helix-strand replica-exchange MD simulation reproduced conformations closer to experimental conformations than a temperature replica-exchange MD simulation when the same numbers of the replicas were used, while the temperature replica-exchange MD simulation does not require bias along any specific order parameter. We calculated its free-energy landscape and revealed the transformation pathways between the ?-helix and ?-hairpin structures and the folding pathways from an extended structure. Although the fractions of the ?-helix and ?-hairpin structures are less than those obtained by the experiment, the free-energy difference between the two structures is calculated to be almost zero, which agrees with the experimental results. PMID:23839056

Okumura, Hisashi; Itoh, Satoru G

2013-07-10

79

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

80

Structure of hemocyanin from garden snail Helix lucorum.  

PubMed

Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthropods with different quaternary structure. They are represented in the hemolymph of molluscs with one, two or three isoforms, as decameric, didecameric, multidecameric and tubules aggregates. We describe here the structure of the hemocyanin Helix lucorum (HlH), species in the series of molluscan hemocyanins. In contrast with other molluscan hemocyanins, three different hemocyanin isopolypeptides were isolated from the hemolymph of the garden snail H. lucorum, named as beta-HlH, alpha(D)-HlH and alpha(N)-HlH. Their molecular masses were determined by size exclusion chromatography to be 1068 kDa (beta-HlH) and 1079 kDa (alpha(D)-HlH, and alpha(N)-HlH). Native HlH exhibits a predominant didecameric structure as revealed by electron microscopy and additionally few tridecamers are shown in the electron micrographs of HlH resulting from the association of a further decamer with one didecamer. The three isoforms are represented mainly as homogeneous didecamers, but they have different behaviour after dissociation and reassociation in the pH-stabilizing buffer, containing 20 mM CaCl(2). All isoforms were reassociated into didecamers and tubules with different length, but in contrast to alpha(D)-HlH isoform, longer tubules were observed in beta-HlH. Moreover the structure of beta-HlH was analysed after limited proteolysis with trypsin followed by FPLC and HPLC separation of the cleavage products. Eight different functional units were identified by their N-terminal sequences and molecular masses. The protein characteristics, including UV absorption at 340 nm, fluorescence and CD spectra of the native molecule and its units confirmed the structure of multimer protein complexes. PMID:20433940

Velkova, Ludmila; Dimitrov, Ivan; Schwarz, Heinz; Stevanovic, Stefan; Voelter, Wolfgang; Salvato, Benedeto; Dolashka-Angelova, Pavlina

2010-04-28

81

The residue composition of the aromatic anchor of the second transmembrane helix determines the signaling properties of the aspartate/maltose chemoreceptor Tar of Escherichia coli.  

PubMed

Repositioning of the tandem aromatic residues (Trp-209 and Tyr-210) at the cytoplasmic end of the second transmembrane helix (TM2) modulates the signal output of the aspartate/maltose chemoreceptor of Escherichia coli (Tar(Ec)). Here, we directly assessed the effect of the residue composition of the aromatic anchor by studying the function of a library of Tar(Ec) variants that possess all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. We identified three important properties of the aromatic anchor. First, a Trp residue at position 209 was required to maintain clockwise (CW) signal output in the absence of adaptive methylation, but adaptive methylation restored the ability of all of the mutant receptors to generate CW rotation. Second, when the aromatic anchor was replaced with tandem Ala residues, signaling was less compromised than when an Ala residue occupied position 209 and an aromatic residue occupied position 210. Finally, when Trp was present at position 209, the identity of the residue at position 210 had little effect on baseline signal output or aspartate chemotaxis, although maltose taxis was significantly affected by some substitutions at position 210. All of the mutant receptors we constructed supported some level of aspartate and maltose taxis in semisolid agar swim plates, but those without Trp at position 209 were overmethylated in their baseline signaling state. These results show the importance of the cytoplasmic aromatic anchor of TM2 in maintaining the baseline Tar(Ec) signal output and responsiveness to attractant signaling. PMID:22339259

Adase, Christopher A; Draheim, Roger R; Manson, Michael D

2012-02-27

82

Coordinating the impact of structural genomics on the human ?-helical transmembrane proteome  

PubMed Central

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

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

2013-01-01

83

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

84

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

National Technical Information Service (NTIS)

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

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

2004-01-01

85

Protonatable residues at the cytoplasmic end of transmembrane helix-2 in the signal transducer HtrI control photochemistry and function of sensory rhodopsin I.  

PubMed Central

Neutral residue replacements were made of 21 acidic and basic residues within the N-terminal half of the Halobacterium salinarium signal transducer HtrI [the halobacterial transducer for sensory rhodopsin I (SRI)] by site-specific mutagenesis. The replacements are all within the region of HtrI that we previously concluded from deletion analysis to contain sites of interaction with the phototaxis receptor SRI. Immunoblotting shows plasmid expression of the htrI-sopI operon containing the mutations produces SRI and mutant HtrI in cells at near wild-type levels. Six of the HtrI mutations perturb photochemical kinetics of SRI and one reverses the phototaxis response. Substitution with neutral amino acids of Asp-86, Glu-87, and Glu-108 accelerate, and of Arg-70, Arg-84, and Arg-99 retard, the SRI photocycle. Opposite effects on photocycle rate cancel in double mutants containing one replaced acidic and one replaced basic residue. Laser flash spectroscopy shows the kinetic perturbations are due to alteration of the rate of reprotonation of the retinylidene Schiff base. All of these mutations permit normal attractant and repellent signaling. On the other hand, the substitution of Glu-56 with the isosteric glutamine converts the normally attractant effect of orange light to a repellent signal in vivo at neutral pH (inverted signaling). Low pH corrects the inversion due to Glu-56 -> Gln and the apparent pK of the inversion is increased when arginine is substituted at position 56. The results indicate that the cytoplasmic end of transmembrane helix-2 and the initial part of the cytoplasmic domain contain interaction sites with SRI. To explain these and previous results, we propose a model in which (i) the HtrI region identified here forms part of an electrostatic bonding network that extends through the SRI protein and includes its photoactive site; (ii) alteration of this network by photoisomerization-induced Schiff base deprotonation and reprotonation shifts HtrI between attractant and repellent conformations; and (iii) HtrI mutations and extracellular pH alter the equilibrium ratios of these conformations.

Jung, K H; Spudich, J L

1996-01-01

86

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

PubMed Central

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.BeFx) or ADP.Pi (ADP.AlF4) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF4 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.

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

2009-01-01

87

Helix-packing motifs in membrane proteins  

PubMed Central

The fold of a helical membrane protein is largely determined by interactions between membrane-imbedded helices. To elucidate recurring helix–helix interaction motifs, we dissected the crystallographic structures of membrane proteins into a library of interacting helical pairs. The pairs were clustered according to their three-dimensional similarity (rmsd ?1.5 Å), allowing 90% of the library to be assigned to clusters consisting of at least five members. Surprisingly, three quarters of the helical pairs belong to one of five tightly clustered motifs whose structural features can be understood in terms of simple principles of helix–helix packing. Thus, the universe of common transmembrane helix-pairing motifs is relatively simple. The largest cluster, which comprises 29% of the library members, consists of an antiparallel motif with left-handed packing angles, and it is frequently stabilized by packing of small side chains occurring every seven residues in the sequence. Right-handed parallel and antiparallel structures show a similar tendency to segregate small residues to the helix–helix interface but spaced at four-residue intervals. Position-specific sequence propensities were derived for the most populated motifs. These structural and sequential motifs should be quite useful for the design and structural prediction of membrane proteins.

Walters, R. F. S.; DeGrado, W. F.

2006-01-01

88

Unraveling the Helix Nebula: Its Structure and Knots  

NASA Astrophysics Data System (ADS)

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

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

2004-11-01

89

Shifting hydrogen bonds may produce flexible transmembrane helices  

PubMed Central

The intricate functions of membrane proteins would not be possible without bends or breaks that are remarkably common in transmembrane helices. The frequent helix distortions are nevertheless surprising because backbone hydrogen bonds should be strong in an apolar membrane, potentially rigidifying helices. It is therefore mysterious how distortions can be generated by the evolutionary currency of random point mutations. Here we show that we can engineer a transition between distinct distorted helix conformations in bacteriorhodopsin with a single-point mutation. Moreover, we estimate the energetic cost of the conformational transitions to be smaller than 1 kcal/mol. We propose that the low energy of distortion is explained in part by the shifting of backbone hydrogen bonding partners. Consistent with this view, extensive backbone hydrogen bond shifts occur during helix conformational changes that accompany functional cycles. Our results explain how evolution has been able to liberally exploit transmembrane helix bending for the optimization of membrane protein structure, function, and dynamics.

Cao, Zheng; Bowie, James U.

2012-01-01

90

Physical conditions and structure in NGC 7293, 'The Helix'  

Microsoft Academic Search

Results are presented for analyses of image-tube interference filter photographs and spectra of the 'Helix' planetary nebula, NGC 7293. Extinction values as well as electron densities and temperatures are derived for 12 points in the central region and the bright SW filament. Temperatures derived from N II forbidden line\\/H-alpha ratio are found to be higher than expected if efficient cooling

J. W. Warner; V. C. Rubin

1975-01-01

91

Distinct roles of the C-terminal 11th transmembrane helix and luminal extension in the partial reactions determining the high Ca2+ affinity of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b).  

PubMed

The molecular mechanism underlying the characteristic high apparent Ca(2+) affinity of SERCA2b relative to SERCA1a and SERCA2a isoforms was studied. The C-terminal tail of SERCA2b consists of an 11th transmembrane helix (TM11) with an associated 11-amino acid luminal extension (LE). The effects of each of these parts and their interactions with the SERCA environment were examined by transient kinetic analysis of the partial reaction steps in the Ca(2+) transport cycle in mutant and chimeric Ca(2+)-ATPase constructs. Manipulations to the LE of SERCA2b markedly increased the rate of Ca(2+) dissociation from Ca(2)E1. Addition of the SERCA2b tail to SERCA1a slowed Ca(2+) dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca(2)E1. The interaction of LE with L7/8 is also important for the low rate of the Ca(2)E1P ? E2P conformational transition. These findings can be rationalized in terms of stabilization of the Ca(2)E1 and Ca(2)E1P forms by docking of the LE near L7/8. By contrast, low rates of E2P dephosphorylation and E2 ? E1 transition in SERCA2b depend critically on TM11, particularly in a SERCA2 environment, but do not at all depend on the LE or L7/8. This indicates that interaction of TM11 with SERCA2-specific sequence element(s) elsewhere in the structure is critical in the Ca(2+)-free E2/E2P states. Collectively these properties ensure a higher Ca(2+) affinity of SERCA2b relative to other SERCA isoforms, not only on the cytosolic side, but also on the luminal side. PMID:23024360

Clausen, Johannes D; Vandecaetsbeek, Ilse; Wuytack, Frank; Vangheluwe, Peter; Andersen, Jens Peter

2012-09-28

92

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

PubMed Central

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

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

2012-01-01

93

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

NASA Astrophysics Data System (ADS)

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.

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

2010-03-01

94

Stabilization of the fibrous structure of an {alpha}-helix-forming peptide by sequence reversal  

SciTech Connect

The {alpha}3-peptide, which comprises three repeats of the sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala and forms an amphipathic {alpha}-helix, is unique among various {alpha}-helix-forming peptides in that it assembles into fibrous structures that can be observed by transmission electron microscopy. As part of our investigation of the structure-stability relationships of the {alpha}3-peptide, we synthesized the r3-peptide, whose amino acid sequence is the reverse of that of the {alpha}3-peptide, and we investigated the effects of sequence reversal on {alpha}-helix stability and the formation of fibrous structures. Unexpectedly, the r3-peptide formed a more-stable {alpha}-helix and longer fibers than did the {alpha}3-peptide. The stability of the r3-peptide helix decreased when the ionic strength of the buffer was increased and when the pH of the buffer was adjusted to 2 or 12. These results suggest that the r3-peptide underwent a 'magnet-like' oligomerization and that an increase in the charge-distribution inequality may be the driving force for the formation of fibrous structures.

Kojima, Shuichi [Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-8588 (Japan)]. E-mail: shuichi.kojima@gakushuin.ac.jp; Kuriki, Yukino [Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-8588 (Japan); Yazaki, Kazumori [Department of Ultrastructural Research, Tokyo Metropolitan Institute for Medical Science, Honkomagome, Tokyo 113-8613 (Japan); Miura, Kin-ichiro [Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-8588 (Japan)

2005-06-03

95

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

96

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

PubMed Central

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

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

2011-01-01

97

Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A*  

PubMed Central

NBCe1-A and AE1 both belong to the SLC4 HCO3? 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 Ala800–Lys967. 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 Met858 accessible to both biotin maleimide and TAMRA and Thr926–Ala929 only to TAMRA labeling. The intracellular surface contains a highly exposed (Met813–Gly828) region and a cryptic (Met887–Arg904) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp960. 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 Pro868–Leu967 (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.

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

2010-01-01

98

Differential structure-function requirements of the transmembranal domain of the B cell antigen receptor  

PubMed Central

By generating phosphorylcholine (PC)-specific, wild-type (mu), and chimeric (mu-I-A alpha) antigen receptor transfectants of mature B cells, we have shown that the COOH terminus of the mu heavy chain is essential for three major functions: immediate signal transduction (measured as changes in intracellular Ca2+), antigen presentation, and induction of immunoglobulin M secretion. A more detailed analysis of structural requirements of the COOH-terminal domains contributing to these functions was achieved by systematically replacing the spacer, cytoplasmic, and transmembranal domains of the mu-I-A alpha chimeric chain with those of mu. Using this rescue approach, we show that the carboxyl two-thirds of the transmembranal domain (proximal to the cytoplasmic domain) is required for induction of intracellular Ca2+, whereas the complete transmembranal domain is required for the function of antigen presentation but is dispensable for induction of antibody secretion.

1992-01-01

99

Pivotal role of the glycine-rich TM3 helix in gating the MscS mechanosensitive channel  

Microsoft Academic Search

The crystal structure of an open form of the Escherichia coli MscS mechanosensitive channel was recently solved. However, the conformation of the closed state and the gating transition remain uncharacterized. The pore-lining transmembrane helix contains a conserved glycine- and alanine-rich motif that forms a helix-helix interface. We show that introducing 'knobs' on the smooth glycine face by replacing glycine with

Michelle D Edwards; Yuezhou Li; Sanguk Kim; Samantha Miller; Wendy Bartlett; Susan Black; Sally Dennison; Irene Iscla; Paul Blount; James U Bowie; Ian R Booth

2005-01-01

100

Structural Analysis of a Peptide Fragment of Transmembrane Transporter Protein Bilitranslocase  

PubMed Central

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

Zuperl, Spela; Sikorska, Emilia; Zhukov, Igor; Solmajer, Tom; Novic, Marjana

2012-01-01

101

Solution Structure of LC4 Transmembrane Segment of CCR5  

Microsoft Academic Search

CC-chemokine receptor 5 (CCR5) is a specific co-receptor allowing the entry of human immunodeficiency virus type 1 (HIV-1). The LC4 region in CCR5 is required for HIV-1 entry into the cells. In this study, the solution structure of LC4 in SDS micelles was elucidated by using standard 1H two-dimensional NMR spectroscopy, circular dichroism, and fluorescdence quenching. The LC4 structure adopts

Kazuhide Miyamoto; Kayo Togiya; John J. Rossi

2011-01-01

102

Thermal properties and power capability of helix structures for millimeter waves  

Microsoft Academic Search

The thermal properties of materials, that can be used for high power helix structures, were evaluated experimentally with equipment that incorporated an infrared microscope for temperature measurements. The interface heat transfer properties between such materials were measured as a function of contact pressure and for different surface finishes. In addition the thermal conductivity of suitable ceramic materials, such as anisotropic

O. Sauseng; A. E. Manoly; A. Hall; J. R. Mente

1978-01-01

103

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

Microsoft Academic Search

The introduction of disulfide crosslinks is a generally useful method by which to identify regions of a protein that are close together in space. Here we describe the use of disulfide crosslinks to investigate the structure and flexi- bility of a family of designed 4-helix bundle proteins. The results of these analyses lend support to our working model of the

Lynne Regan; Arlene Rockwell; Zelda Wasserman; William Degrado

1994-01-01

104

Origin and evolution of transmembrane Chl-binding proteins: hydrophobic cluster analysis suggests a common one-helix ancestor for prokaryotic (Pcb) and eukaryotic (LHC) antenna protein superfamilies  

Microsoft Academic Search

All chlorophyll (Chl)-binding proteins constituting the photosynthetic apparatus of both prokaryotes and eukaryotes possess hydrophobic domains, corresponding to membrane-spanning ?-helices (MSHs). Hydrophobic cluster analysis of representative members of the different Chl protein superfamilies revealed that all Chl proteins except the five-helix reaction center II proteins and the small subunits of photosystem I possess related domains. As a major conclusion, we

Laurence Garczarek; Anne Poupon

2003-01-01

105

Fe2+-Tetracycline-Mediated Cleavage of the Tn10 Tetracycline Efflux Protein TetA Reveals a Substrate Binding Site near Glutamine 225 in Transmembrane Helix 7  

Microsoft Academic Search

TetA specified by Tn10 is a class B member of a group of related bacterial transport proteins of 12 transmembrane alpha helices that mediate resistance to the antibiotic tetracycline. A tetracycline-divalent metal cation complex is expelled from the cell in exchange for a entering proton. The site(s) where tetracycline binds to this export pump is not known. We found that,

Laura M. McMurry; Mila L. Aldema-Ramos; Stuart B. Levy

2002-01-01

106

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

107

High salt solution structure of a left-handed RNA double helix  

Microsoft Academic Search

Right-handed RNA duplexes of (CG)n sequence undergo salt-induced helicity reversal, forming left- handed RNA double helices (Z-RNA). In contrast to the thoroughly studied Z-DNA, no Z-RNA structure of natural origin is known. Here we report the NMR structure of a half-turn, left-handed RNA helix (CGCGCG)2 determined in 6 M NaClO4. This is the first nucleic acid motif determined at such

Mariusz Popenda; Jan Milecki; Ryszard W. Adamiak

2004-01-01

108

Shielded helix traveling wave cathode ray tube deflection structure  

DOEpatents

This invention relates to deflection structures for cathode ray tubes. It is an object of this invention to provide an improved helical coil deflection structure for a cathode ray tube wherein each turn in the coil is separated from adjacent turns by conductive shielding means which surrounds at least three sides of each turn of the helical coil. (GHH)

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

1991-01-22

109

Effect of pressure on the ?-helix structure of tetrameric coiled-coil peptides  

NASA Astrophysics Data System (ADS)

The effect of pressure on the ?-helix structure of tetrameric coiled-coil peptides has been investigated using Fourier-transform infrared spectroscopy. To examine the influence of the hydrophobic core on pressure stability of the ?-helices, the present study targeted GCN4-pLI and its variants (L9S, L9A, and L9G), which have different cavity in size. The amide II band was used to examine the stability of the hydrophobic core. From monitoring the amide I' band, it was shown commonly for all the peptides that the solvent-inaccessible ?-helix decreases with increasing pressure while the solvent-accessible ?-helix increases with increasing pressure. It was strongly suggested that the hydration of the helices is a significant factor for the pressure-induced folding. From further detailed analyses of pressure dependence of the amide I' band intensities, it was found that there is a positive correlation between the cavity size and the pressure-induced unfolding of the solvent inaccessible ?-helix for the variants.

Segawa, Y.; Imamura, H.; Shimizu, A.; Kato, M.

2010-03-01

110

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

Microsoft Academic Search

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 (8.4kDa) composed of tandem 12-residue repeats (TCTxSxxCxxAx). Here we report its 1.4-Å resolution crystal structure, showing that this repetitive sequence translates into an exceptionally regular beta-helix. Not only are the 12-amino-acid

Yih-Cherng Liou; Ante Tocilj; Peter L. Davies; Zongchao Jia

2000-01-01

111

Microspatial genetic structure in the land snail Helix aspersa (Gastropoda: Helicidae)  

Microsoft Academic Search

The microspatial genetic structure of allele frequencies at seven isozyme loci was examined for 15 populations of the land snail Helix aspersa sampled in a village from Brittany (north-western France). Spatial heterogeneity of allele frequencies was highly significant (P < 0.001). Fixation indices reflected nonrandom mating within neighbourhoods and a slight but consistent differentiation between colonies (FST=0.044; P < 0.01).

J.-F. ARNAUD; L Madec; A Bellido; A Guiller

1999-01-01

112

Electric-field-induced transition from helical to planar smectic structures without helix unwinding  

NASA Astrophysics Data System (ADS)

Previously electric-field-induced transitions from helical to planar structures in bulk liquid crystals were considered to take place with unwinding of helix. We show that the transition to the planar structure in polar smectic with frustrating interlayer interaction can occur without unwinding but through the formation of a periodic modulated structure with successive left-hand and right-hand sense of rotation in every half period. With field the amplitude of the left-right rotation decreases and a planar structure is formed. This mechanism of transition can take place in the short-pitch SmC?* phase formed as a result of frustrating interaction.

Dolganov, P. V.; Zhilin, V. M.

2013-06-01

113

Electric-field-induced transition from helical to planar smectic structures without helix unwinding.  

PubMed

Previously electric-field-induced transitions from helical to planar structures in bulk liquid crystals were considered to take place with unwinding of helix. We show that the transition to the planar structure in polar smectic with frustrating interlayer interaction can occur without unwinding but through the formation of a periodic modulated structure with successive left-hand and right-hand sense of rotation in every half period. With field the amplitude of the left-right rotation decreases and a planar structure is formed. This mechanism of transition can take place in the short-pitch SmC(?)(*) phase formed as a result of frustrating interaction. PMID:23848703

Dolganov, P V; Zhilin, V M

2013-06-17

114

Predicting loop-helix tertiary structural contacts in RNA pseudoknots  

PubMed Central

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

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

2010-01-01

115

Helix probability profiles of denatured proteins and their correlation with native structures.  

PubMed

The Zimm-Bragg formulation for the one-dimensional Ising model is applied to denatured proteins in order to compute helix probability profiles with different sigma and s parameters for the various amino acids; the latter are in principle determinable from melting curves for helix-coil transitions in random copolymers of amino acids. Using a tentative assignment of sigma and s values, we found a correlation for the propensity of a residue to be helical in the denatured protein and its occurrence in a helical region in the globular structure of the corresponding native protein. Thus, these incipient helical regions in the denatured chain may serve to nucleate the folding to form the native protein. Short-range interactions appear to determine the tendency for a residue to be helical or not, whereas long-range interactions may serve to carry out the nucleation and refolding processes. PMID:5266152

Lewis, P N; Go, N; Go, M; Kotelchuck, D; Scheraga, H A

1970-04-01

116

Crystal structure of human thioredoxin revealing an unraveled helix and exposed S-nitrosation site  

PubMed Central

Thioredoxins reduce disulfide bonds and other thiol modifications in all cells using a CXXC motif. Human thioredoxin 1 is unusual in that it codes for an additional three cysteines in its 105 amino acid sequence, each of which have been implicated in other reductive activities. Cys 62 and Cys 69 are buried in the protein interior and lie at either end of a short helix (helix 3), and yet can disulfide link under oxidizing conditions. Cys 62 is readily S-nitrosated, giving rise to a SNO modification, which is also buried. Here, we present two crystal structures of the C69S/C73S mutant protein under oxidizing (1.5 Å) and reducing (1.1 Å) conditions. In the oxidized structure, helix 3 is unraveled and displays a new conformation that is stabilized by a series of new hydrogen bonds and a disulfide link with Cys 62 in a neighboring molecule. The new conformation provides an explanation for how a completely buried residue can participate in SNO exchange reactions.

Weichsel, Andrzej; Kem, Michelle; Montfort, William R

2010-01-01

117

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

PubMed

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

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

2013-04-04

118

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

PubMed Central

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

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

2012-01-01

119

Retention of a cis Golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain.  

PubMed Central

The first membrane-spanning domain (m1) of the model cis Golgi protein M (formerly called E1) from the avian coronavirus infectious bronchitis virus is required for targeting to the Golgi complex. When inserted in place of the membrane-spanning domain of a plasma membrane protein (vesicular stomatitis virus G protein), the chimeric protein ("Gm1") is retained in the Golgi complex of transfected cells. To determine the precise features of the m1 domain responsible for Golgi targeting, we produced single amino acid substitutions in m1 and analyzed their effects on localization of Gm1. Expression at the plasma membrane was used as the criterion for loss of Golgi retention. Rates of oligosaccharide processing were used as a measure of rate and efficiency of transport through the Golgi complex. We identified four uncharged polar residues that are critical for Golgi retention of Gm1 (Asn465, Thr469, Thr476, and Gln480). These residues line one face of a predicted alpha-helix. Interestingly, when the m1 domain of the homologous M protein from mouse hepatitis virus is inserted into the G protein reporter, the chimeric protein is not efficiently retained in the Golgi complex, but transported to the cell surface. Although it possesses three of the four residues we identified as important in the avian m1 sequence, other residues in the membrane-spanning domain from the mouse protein must prevent efficient recognition of the polar face within the lipid bilayer of the cis Golgi. Images

Machamer, C E; Grim, M G; Esquela, A; Chung, S W; Rolls, M; Ryan, K; Swift, A M

1993-01-01

120

Structure of an integrin alphaIIb beta3 transmembrane-cytoplasmic heterocomplex provides insight into integrin activation.  

PubMed

Heterodimeric integrin adhesion receptors regulate diverse biological processes including angiogenesis, thrombosis and wound healing. The transmembrane-cytoplasmic domains (TMCDs) of integrins play a critical role in controlling activation of these receptors via an inside-out signaling mechanism, but the precise structural basis remains elusive. Here, we present the solution structure of integrin alphaIIb beta3 TMCD heterodimer, which reveals a right-handed coiled-coil conformation with 2 helices intertwined throughout the transmembrane region. The helices extend into the cytoplasm and form a clasp that differs significantly from a recently published alphaIIb beta3 TMCD structure. We show that while a point mutation in the clasp interface modestly activates alphaIIb beta3, additional mutations in the transmembrane interface have a synergistic effect, leading to extensive integrin activation. Detailed analyses and structural comparison with previous studies suggest that extensive integrin activation is a highly concerted conformational transition process, which involves transmembrane coiled-coil unwinding that is triggered by the membrane-mediated alteration and disengagement of the membrane-proximal clasp. Our results provide atomic insight into a type I transmembrane receptor heterocomplex and the mechanism of integrin inside-out transmembrane signaling. PMID:19805198

Yang, Jun; Ma, Yan-Qing; Page, Richard C; Misra, Saurav; Plow, Edward F; Qin, Jun

2009-10-01

121

anthocyanin1 of Petunia Encodes a Basic Helix-Loop-Helix Protein That Directly Activates Transcription of Structural Anthocyanin Genes  

PubMed Central

The petunia loci anthocyanin1 (an1), an2, an4, and an11 are required for the transcription of anthocyanin biosynthetic genes in floral organs. The an2 and an11 loci were recently cloned and shown to encode a MYB-domain transcriptional activator and a cytosolic WD40 protein, respectively. Here, we report the isolation of an1 by transposon tagging. an1 encodes a new member of the basic helix-loop-helix family of transcription factors that is functionally and evolutionarily distinct from JAF13, the apparent petunia ortholog of maize RED1 and snapdragon DELILA. We provide genetic evidence that the transcription factors encoded by an1, an2, and an4 operate in an unexpectedly complex regulatory hierarchy. In leaves, ectopic expression of AN2 induces an1 expression, whereas in anthers, an1 expression depends on an4, encoding (or controlling) a MYB protein that is paralogous to AN2. Experiments with transgenic plants expressing a post-translationally controlled AN1–GLUCOCORTICOID RECEPTOR fusion protein indicated that independent of protein synthesis, AN1 directly activates the expression of the dfrA gene encoding the enzyme dihydroflavonol 4-reductase and of Pmyb27 encoding a MYB-domain protein of unknown function.

Spelt, Cornelis; Quattrocchio, Francesca; Mol, Joseph N. M.; Koes, Ronald

2000-01-01

122

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

123

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

PubMed Central

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

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

2009-01-01

124

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

PubMed Central

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

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

2008-01-01

125

Alternative primary structures in the transmembrane domain of the chicken erythroid anion transporter.  

PubMed Central

Isolation and characterization of the chicken erythroid anion transporter (band 3) cDNA clone, pCHB3-1, revealed that the chicken erythroid band 3 polypeptide is 844 amino acids in length with a predicted mass of 109,000 daltons. This polypeptide is composed of a hydrophilic N-terminal cytoplasmic domain and a hydrophobic C-terminal transmembrane domain. The approximately 90 N-terminal amino acids of the human and murine erythroid band 3 polypeptides are absent in the predicted sequence of the chicken erythroid band 3 polypeptide. The absence of this very acidic N-terminal region is consistent with the lack of binding of glyceraldehyde-3-phosphate dehydrogenase to chicken erythroid band 3, as well as the relatively basic isoelectric point observed for this molecule. The remainder of the cytoplasmic domain shows little similarity to the cytoplasmic domain of the murine and human erythroid band 3, with the exception of the putative ankyrin-binding site, which is highly conserved. In contrast, the transmembrane domain of the chicken band 3 polypeptide is very similar to that of the murine erythroid and human nonerythroid band 3 polypeptides. The transmembrane domain contains 10 hydrophobic regions that could potentially traverse the membrane 12 to 14 times. In addition, a variant of chicken erythroid band 3, pCHB3-2, was cloned in which one of the hydrophobic regions of pCHB3-1 is lacking. The transcript complementary to pCHB3-2 accumulated in chicken erythroid cells in a similar manner as the transcript complementary to pCHB3-1 during embryonic development. This is the first example of a transporter protein or ion channel with alternative primary structures in its membrane-spanning segments. Images

Cox, J V; Lazarides, E

1988-01-01

126

Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines.  

PubMed

Site-directed mutagenesis was used to produce mutants of bacteriorhodopsin where either glycine-72, threonine-90, leucine-92, or serine-169 was replaced by a cysteine. Two different spin labels were then covalently attached to these sites. The selection of attachment sites covered two postulated loops (72,169) and a membrane-spanning segment (90,92). It was not possible to properly refold the protein labeled at position 90, presumably due to steric problems, but the EPR spectra of the other mutants that were successfully reconstituted in phospholipid vesicles provided information on the dynamics of protein side chains in the vicinity of the label site. A power saturation approach was used to investigate the spin relaxation times, which in turn can be influenced by collisions with paramagnetic species. The differential effect of oxygen and a water-soluble chromium complex on the power-saturation behavior of the spin-labeled mutants was used to obtain topographical information on the sites in the membrane-bound protein. The results are consistent with residues 72 and 169 being located in structured loops exposed to the aqueous phase and residue 92 being localized in the membrane interior, possibly near a helix-helix contact region. PMID:2558712

Altenbach, C; Flitsch, S L; Khorana, H G; Hubbell, W L

1989-09-19

127

Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines  

SciTech Connect

Site-directed mutagenesis was used to produce mutants of bacteriorhodopsin where either glycine-72, threonine-90, leucine-92, or serine-169 was replaced by a cysteine. Two different spin labels were then covalently attached to these sites. The selection of attachment sites covered two postulated loops (72,169) and a membrane-spanning segment (90,92). It was not possible to properly refold the protein labeled at position 90, presumably due to steric problems, but the EPR spectra of the other mutants that were successfully reconstituted in phospholipid vesicles provided information on the dynamics of protein side chains in the vicinity of the label site. A power saturation approach was used to investigate the spin relaxation times, which in turn can be influenced by collisions with paramagnetic species. The differential effect of oxygen and a water-soluble chromium complex on the power-saturation behavior of the spin-labeled mutants was used to obtain topographical information on the sites in the membrane-bound protein. The results are consistent with residues 72 and 169 being located in structured loops exposed to the aqueous phase and residue 92 being localized in the membrane interior, possibly near a helix-helix contact region.

Altenbach, C.; Flitsch, S.L.; Khorana, H.G.; Hubbell, W.L. (Univ. of California, Los Angeles (USA))

1989-09-19

128

The primary structure of neuropeptide F (NPF) from the garden snail, Helix aspersa.  

PubMed

Neuropeptide F (NPF), originally isolated from the sheep tapeworm, Moniezia expansa, consists of 39 amino acid residues terminating in a phenylalaninamide. An analogous neuropeptide has been isolated and sequenced from extracts of circumoesophageal ganglia of the garden snail, Helix aspersa. This neuropeptide exhibits partial primary structural similarity to members of the vertebrate neuropeptide Y (NPY)/pancreatic polypeptide (PP) superfamily. NPF is thus of widespread occurrence in the nervous systems of invertebrates from different phyla and may represent the phylogenetic precursor of the vertebrate NPY/PP superfamily. PMID:1472263

Leung, P S; Shaw, C; Maule, A G; Thim, L; Johnston, C F; Irvine, G B

1992-09-01

129

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

PubMed

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

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

2004-09-14

130

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

NASA Astrophysics Data System (ADS)

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

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

2009-10-01

131

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

PubMed Central

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

2013-01-01

132

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

PubMed Central

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 of FGFR3 transmembrane domain in fluid lipid bilayers, and investigate whether the G380R mutation affects the topology of the protein in the bilayer. Our results demonstrate that, in a model system, the G380R mutation induces a shift in the segment that is embedded in the membrane. The center of the hydrocarbon core-embedded segment in the mutant is close to the midpoint between R380 and R397, supporting previous measurements of arginine insertion energetics into the endoplasmic reticulum. The presented results further our knowledge about basic amino-acid insertion into bilayers, and may lead to new insights into the mechanism of pathogenesis in achondroplasia.

Han, Xue; Mihailescu, Mihaela; Hristova, Kalina

2006-01-01

133

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

NASA Astrophysics Data System (ADS)

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

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

2013-06-01

134

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

NASA Astrophysics Data System (ADS)

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

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

2012-08-01

135

Single amino acid substitution in the putative transmembrane helix V in KdpB of the KdpFABC complex of Escherichia coli uncouples ATPase activity and ion transport.  

PubMed

The KdpFABC complex, found in a variety of prokaryotes, is an emergency potassium uptake system which belongs to the family of P-type ATPases. Site-directed mutagenesis of the charged residues aspartate 583 and lysine 586 in the putative transmembrane helix V of subunit KdpB revealed that these charges are involved in the coupling of ATP hydrolysis and ion translocation. Phenotypic characterization of KdpFABC derivatives carrying alterations at either D583 or K586 demonstrated that only restoration of charges at these positions allowed growth on low potassium concentrations. Substitutions, which eliminated the negative charge at position 583, did not allow growth below 15 mM potassium on solid media. In contrast, substitutions of the positive charge at position 586 allowed growth down to 0.3 mM potassium. Purified KdpFABC complexes carrying these substitutions exhibited ATPase activity, which was, however, found to be comparatively resistant to o-vanadate. Furthermore, elimination of the charges led to a complete loss of ion-stimulated ATPase activity, though the rate of hydrolysis was comparable to wild-type activity, indicating an uncoupling between ATP hydrolysis and ion translocation. This fact was substantiated by reconstitution experiments, in which the D583A complex was unable to facilitate ion translocation, whereas the D583E mutant complex still exhibited such activity. On the basis of these results, a new transport model for the Kdp-ATPase is presented here, in which the amino acids D583 and K586 are supposed to play a role in the gating mechanism of the complex. Furthermore, movement of the charged side chains could have a direct influence on the free energy profile within the potassium transporting subunit KdpA, thereby facilitating ion transport against the concentration gradient into the cytosol. PMID:15938615

Bramkamp, Marc; Altendorf, Karlheinz

2005-06-14

136

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

PubMed Central

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

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

2012-01-01

137

NMR Structure and Dynamics of a Designed Water-soluble Transmembrane Domain of Nicotinic Acetylcholine Receptor  

PubMed Central

The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ?83% sequence identity to the transmembrane (TM) domain of the nAChR ?1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T2 dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.

Cui, Tanxing; Mowrey, David; Bondarenko, Vasyl; Tillman, Tommy; Ma, Dejian; Landrum, Elizabeth; Perez-Aguilar, Jose Manuel; He, Jing; Wang, Wei; Saven, Jeffery G.; Eckenhoff, Roderic G.; Tang, Pei; Xu, Yan

2011-01-01

138

Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors  

PubMed Central

A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments—most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting, and possibly for cell entry. In addition, many virally-encoded chemokine 7TM receptors have been suggested to be causally involved in pathogenic phenotypes like Kaposi sarcoma, atherosclerosis, HIV-infection and tumour development. The role of these receptors during the viral life cycle and in viral pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we highlight the emerging impact of these receptor on virus-mediated diseases.

Rosenkilde, M M; Smit, M J; Waldhoer, M

2008-01-01

139

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

PubMed Central

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

Palasingam, Paaventhan; Kolatkar, Prasanna R.

2012-01-01

140

Structural propensities and entropy effects in peptide helix-coil transitions  

NASA Astrophysics Data System (ADS)

The helix-coil transition in peptides is a critical structural transition leading to functioning proteins. Peptide chains have a large number of possible configurations that must be accounted for in statistical mechanical investigations. Using hydrogen bond and local helix propensity interaction terms, we develop a method for obtaining and incorporating the degeneracy factor that allows the exact calculation of the partition function for a peptide as a function of chain length. The partition function is used in calculations for engineered peptide chains of various lengths that allow comparison with a variety of different types of experimentally measured quantities, such as fraction of helicity as a function of both temperature and chain length, heat capacity, and denaturation studies. When experimental sensitivity in helicity measurements is properly accounted for in the calculations, the calculated curves fit well with the experimental curves. We determine values of interaction energies for comparison with known biochemical interactions, as well as quantify the difference in the number of configurations available to an amino acid in a random coil configuration compared to a helical configuration.

Chemmama, Ilan E.; Pelea, Adam Colt; Bhandari, Yuba R.; Chapagain, Prem P.; Gerstman, Bernard S.

2012-09-01

141

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.

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

2010-01-01

142

Crystal structure of the signaling helix coiled-coil domain of the ?1 subunit of the soluble guanylyl cyclase  

PubMed Central

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

2010-01-01

143

Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor  

SciTech Connect

Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. Here we present the 2.4 {angstrom} crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120{sup GAP} {center_dot}H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, we show that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation.

Tong, Yufeng; Hota, Prasanta K.; Penachioni, Junia Y.; Hamaneh, Mehdi B.; Kim, SoonJeung; Alviani, Rebecca S.; Shen, Limin; He, Hao; Tempel, Wolfram; Tamagnone, Luca; Park, Hee-Won; Buck, Matthias; (Torino); (Toronto); (Case Western U.-Med)

2010-02-11

144

Membrane Insertion of a Voltage Sensor Helix  

PubMed Central

Most membrane proteins contain a transmembrane (TM) domain made up of a bundle of lipid-bilayer-spanning ?-helices. TM ?-helices are generally composed of a core of largely hydrophobic amino acids, with basic and aromatic amino acids at each end of the helix forming interactions with the lipid headgroups and water. In contrast, the S4 helix of ion channel voltage sensor (VS) domains contains four or five basic (largely arginine) side chains along its length and yet adopts a TM orientation as part of an independently stable VS domain. Multiscale molecular dynamics simulations are used to explore how a charged TM S4 ?-helix may be stabilized in a lipid bilayer, which is of relevance in the context of mechanisms of translocon-mediated insertion of S4. Free-energy profiles for insertion of the S4 helix into a phospholipid bilayer suggest that it is thermodynamically favorable for S4 to insert from water to the center of the membrane, where the helix adopts a TM orientation. This is consistent with crystal structures of Kv channels, biophysical studies of isolated VS domains in lipid bilayers, and studies of translocon-mediated S4 helix insertion. Decomposition of the free-energy profiles reveals the underlying physical basis for TM stability, whereby the preference of the hydrophobic residues of S4 to enter the bilayer dominates over the free-energy penalty for inserting charged residues, accompanied by local distortion of the bilayer and penetration of waters. We show that the unique combination of charged and hydrophobic residues in S4 allows it to insert stably into the membrane.

Wee, Chze Ling; Chetwynd, Alan; Sansom, Mark S.P.

2011-01-01

145

A Web-Based Program (WHAT) for the Simultaneous Prediction of Hydropathy, Amphipathicity, Secondary Structure and Transmembrane Topology for a Single Protein Sequence  

Microsoft Academic Search

We designed a web-based program, WHAT, which uses a sliding window to determine and plot the hydropathy, amphipathicity, secondary structure and transmembrane topology along the length of any protein sequence. This method is based on programs designed by us for hydropathy and amphipathicity but on JNET and MEMSAT for secondary structure and transmembrane topology predictions, respectively. It has a user-friendly

Yufeng Zhai; Milton H. Saier

146

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

PubMed

Mechanical aspects of the cellular environment can influence cell function, and in this context hydrogels can serve as an instructive matrix. Here we report that physicochemical properties of hydrogels derived from polysaccharides (agarose, ?-carrageenan) having an ?-helical backbone can be tailored by inducing a switch in the secondary structure from ?-helix to ?-sheet through carboxylation. This enables the gel modulus to be tuned over four orders of magnitude (G' 6 Pa-3.6 × 10(4) Pa) independently of polymer concentration and molecular weight. Using carboxylated agarose gels as a screening platform, we demonstrate that soft-carboxylated agarose provides a unique environment for the polarization of endothelial cells in the presence of soluble and bound signals, which notably does not occur in fibrin and collagen gels. Furthermore, endothelial cells organize into freestanding lumens over 100 ?m in length. The finding that a biomaterial can modulate soluble and bound signals provides impetus for exploring mechanobiology paradigms in regenerative therapies. PMID:23886665

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

2013-07-25

147

Right- and left-handed three-helix proteins. I. Experimental and simulation analysis of differences in folding and structure.  

PubMed

Despite the large number of publications on three-helix protein folding, there is no study devoted to the influence of handedness on the rate of three-helix protein folding. From the experimental studies, we make a conclusion that the left-handed three-helix proteins fold faster than the right-handed ones. What may explain this difference? An important question arising in this paper is whether the modeling of protein folding can catch the difference between the protein folding rates of proteins with similar structures but with different folding mechanisms. To answer this question, the folding of eight three-helix proteins (four right-handed and four left-handed), which are similar in size, was modeled using the Monte Carlo and dynamic programming methods. The studies allowed us to determine the orders of folding of the secondary-structure elements in these domains and amino acid residues which are important for the folding. The obtained data are in good correlation with each other and with the experimental data. Structural analysis of these proteins demonstrated that the left-handed domains have a lesser number of contacts per residue and a smaller radius of cross section than the right-handed domains. This may be one of the explanations of the observed fact. The same tendency is observed for the large dataset consisting of 332 three-helix proteins (238 right- and 94 left-handed). From our analysis, we found that the left-handed three-helix proteins have some less-dense packing that should result in faster folding for some proteins as compared to the case of right-handed proteins.Proteins 2013; © 2013 Wiley Periodicals, Inc. PMID:23588993

Glyakina, Anna V; Pereyaslavets, Leonid B; Galzitskaya, Oxana V

2013-06-20

148

Intermolecular recognition revealed by the complex structure of human CLOCK-BMAL1 basic helix-loop-helix domains with E-box DNA.  

PubMed

CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1) are both transcription factors of the circadian core loop in mammals. Recently published mouse CLOCK-BMAL1 bHLH (basic helix-loop-helix)-PAS (period-ARNT-single-minded) complex structure sheds light on the mechanism for heterodimer formation, but the structural details of the protein-DNA recognition mechanisms remain elusive. Here we have elucidated the crystal structure of human CLOCK-BMAL1 bHLH domains bound to a canonical E-box DNA. We demonstrate that CLOCK and BMAL1 bHLH domains can be mutually selected, and that hydrogen-bonding networks mediate their E-box recognition. We identified a hydrophobic contact between BMAL1 Ile80 and a flanking thymine nucleotide, suggesting that CLOCK-BMAL1 actually reads 7-bp DNA and not the previously believed 6-bp DNA. To find potential non-canonical E-boxes that could be recognized by CLOCK-BMAL1, we constructed systematic single-nucleotide mutations on the E-box and measured their relevant affinities. We defined two non-canonical E-box patterns with high affinities, AACGTGA and CATGTGA, in which the flanking A7-T7' base pair is indispensable for recognition. These results will help us to identify functional CLOCK-BMAL1-binding sites in vivo and to search for clock-controlled genes. Furthermore, we assessed the inhibitory role of potential phosphorylation sites in bHLH regions. We found that the phospho-mimicking mutation on BMAL1 Ser78 could efficiently block DNA binding as well as abolish normal circadian oscillation in cells. We propose that BMAL1 Ser78 should be a key residue mediating input signal-regulated transcriptional inhibition for external cues to entrain the circadian clock by kinase cascade. PMID:23229515

Wang, Zixi; Wu, Yaling; Li, Lanfen; Su, Xiao-Dong

2012-12-11

149

Impact of histidine residues on the transmembrane helices of viroporins.  

PubMed

Abstract The role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the TM helices from Virus protein 'u' (Vpu) and the M2 proton channel. Both proteins are members of the viroporin family of small membrane proteins that exhibit ion channel activity, and have a single TM helix that is capable of forming oligomers. The TM helices from both proteins have a conserved tryptophan towards the C-terminus. Previously, alanine 18 of Vpu was mutated to histidine in order to artificially introduce the same HXXXW motif that is central to the proton channel activity of M2. Interestingly, the mutated Vpu TM resulted in an increase in helix tilt angle of 11° in lipid bilayers compared to the wild-type Vpu TM. Here, we find the reverse, when histidine 37 of the HXXXW motif in M2 was mutated to alanine, it decreased the helix tilt by 10° from that of wild-type M2. The tilt change is independent of both the helix length and the presence of tryptophan. In addition, compared to wild-type M2, the H37A mutant displayed lowered sensitivity to proton concentration. We also found that the solvent accessibility of histidine-containing M2 is greater than without histidine. This suggests that the TM helix may increase the solvent exposure by changing its tilt angle in order to accommodate a polar/charged residue within the hydrophobic membrane region. The comparative results of M2, Vpu and their mutants demonstrated the significance of histidine in a transmembrane helix and the remarkable plasticity of the function and structure of ion channels stemming from changes at a single amino acid site. PMID:24102567

Wang, Yan; Park, Sang Ho; Tian, Ye; Opella, Stanley J

2013-10-09

150

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

PubMed Central

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

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

2000-01-01

151

Crystal structure of the complete integrin ?V?3 ectodomain plus an ?/? transmembrane fragment  

PubMed Central

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

Xiong, Jian-Ping; Mahalingham, Bhuvaneshwari; Alonso, Jose Luis; Borrelli, Laura Ann; Rui, Xianliang; Anand, Saurabh; Hyman, Bradley T.; Rysiok, Thomas; Muller-Pompalla, Dirk; Goodman, Simon L.

2009-01-01

152

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

PubMed

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

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

2013-03-06

153

Regio-selective detection of dynamic structure of transmembrane alpha-helices as revealed from (13)C NMR spectra of [3-13C]Ala-labeled bacteriorhodopsin in the presence of Mn2+ ion.  

PubMed Central

13C Nuclear magnetic resonance (NMR) spectra of [3-(13)C]Ala-labeled bacteriorhodopsin (bR) were edited to give rise to regio-selective signals from hydrophobic transmembrane alpha-helices by using NMR relaxation reagent, Mn(2+) ion. As a result of selective suppression of (13)C NMR signals from the surfaces in the presence of Mn(2+) ions, several (13)C NMR signals of Ala residues in the transmembrane alpha-helices were identified on the basis of site-directed mutagenesis without overlaps from (13)C NMR signals of residues located near the bilayer surfaces. The upper bound of the interatomic distances between (13)C nucleus in bR and Mn(2+) ions bound to the hydrophilic surface to cause suppressed peaks by the presence of Mn(2+) ion was estimated as 8.7 A to result in the signal broadening to 100 Hz and consistent with the data based on experimental finding. The Ala C(beta) (13)C NMR peaks corresponding to Ala-51, Ala-53, Ala-81, Ala-84, and Ala-215 located around the extracellular half of the proton channel and Ala-184 located at the kink in the helix F were successfully identified on the basis of (13)C NMR spectra of bR in the presence of Mn(2+) ion and site-directed replacement of Ala by Gly or Val. Utilizing these peaks as probes to observe local structure in the transmembrane alpha-helices, dynamic conformation of the extracellular half of bR at ambient temperature was examined, and the local structures of Ala-215 and 184 were compared with those elucidated at low temperature. Conformational changes in the transmembrane alpha-helices induced in D85N and E204Q and its long-range transmission from the proton release site to the site around the Schiff base in E204Q were also examined.

Tuzi, S; Hasegawa, J; Kawaminami, R; Naito, A; Saito, H

2001-01-01

154

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

PubMed Central

Early investigation of the EPR spectra of bis-histidine-coordinated membrane-bound ferriheme proteins led to the description of a spectral signal that had only one resolved feature. These became known as “highly anisotropic low-spin” (HALS) or “large gmax” ferriheme centers. Extensive work with small-molecule model heme complexes showed that this spectroscopic signature occurs in bis-imidazole ferrihemes in which the planes of the imidazole ligands are nearly perpendicular, ?? = 57–90°. In the last decade protein crystallographic studies have revealed the atomic structures of a number of examples of bis-histidine heme proteins. A frequent characteristic of these large gmax ferrihemes in membrane-bound proteins is the occurrence of the heme within a four-helix bundle with a left-handed twist. The histidine ligands occur at the same level on two diametrically opposed helices of the bundle. These ligands have the same side chain conformation and ligate heme iron on the bundle axis, resulting in a quasi-2-fold symmetric structure. The two non-ligand-bearing helices also obey this symmetry, and have a conserved small residue, usually glycine, where the edge of the heme ring makes contact with the helix backbones. In many cases this small residue is preceded by a threonine or serine residue whose side chain hydroxyl oxygen acts as a hydrogen-bond acceptor from the N?1 atom of the heme-ligating histidine. The ?? angle is thus determined by the common histidine side-chain conformation and the crossing angle of the ligand-bearing helices, in some cases constrained by H-bonds to the Ser/Thr residues on the non-ligand-bearing helices.

Berry, Edward A.; Walker, F. Ann

2009-01-01

155

Terminating a macromolecular helix. Structural model for the minor proteins of bacteriophage M13.  

PubMed

Analysis of the results of X-ray diffraction, electron microscopy and s sequence studies of filamentous bacteriophage M13 are used to construct structural models for the minor proteins gp7 and gp9 at the end of the virus assembled first, and a portion of gp6 at the end of the virus that binds host. Comparison of the sequence of the major coat protein, gp8, with those of gp7, gp9 and gp6 indicates that significant portions of these three proteins have sequences similar to that of gp8. Assuming that sequence similarity is indicative of structural similarity, gp7, gp9 and portions of gp6 are modeled based on what is known about the structure of gp8. These molecular models are analyzed to predict the packing of the minor proteins with the terminal gp8 proteins (the last gp8 proteins at either end of the helix). This analysis indicates that the gp8 proteins integrated into the virus first may have a structure distinct from those in the body of the virus particle. The gp8 proteins at the end assembled last appear to have a conformation very similar to that of the integral coat proteins. These models place specific constraints on models for the process of viral assembly. PMID:1469721

Makowski, L

1992-12-01

156

Transmembrane glycine zippers: Physiological and pathological roles in membrane proteins  

NASA Astrophysics Data System (ADS)

We have observed a common sequence motif in membrane proteins, which we call a glycine zipper. Glycine zipper motifs are strongly overrepresented and conserved in membrane protein sequences, and mutations in glycine zipper motifs are deleterious to function in many cases. The glycine zipper has a significant structural impact, engendering a strong driving force for right-handed packing against a neighboring helix. Thus, the presence of a glycine zipper motif leads directly to testable structural hypotheses, particularly for a subclass of glycine zipper proteins that form channels. For example, we suggest that the membrane pores formed by the amyloid- peptide in vitro are constructed by glycine zipper packing and find that mutations in the glycine zipper motif block channel formation. Our findings highlight an important structural motif in a wide variety of normal and pathological processes. amyloid- | membrane channel | membrane protein structure | prion | transmembrane helix

Kim, Sanguk; Jeon, Tae-Joon; Oberai, Amit; Yang, Duan; Schmidt, Jacob J.; Bowie, James U.

2005-10-01

157

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)  

PubMed Central

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

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

2011-01-01

158

The topological structure and function of Echinococcus granulosus lactate dehydrogenase, a tegumental transmembrane protein.  

PubMed

Lactate dehydrogenase (LDH), a terminal glycolytic enzyme, is generally considered as a cytosolic protein. We cloned lactate dehydrogenase from Echinococcus granulosus (EgLDH) and predicted it may be a membrane protein with two transmembrane regions through bioinformatics analysis. Intact worm immunofluorescence with antibodies prepared against linear B cell epitopes predicted in the region inside or outside of the membrane demonstrated that EgLDH spans the tegumental membrane twice, with the N terminal and C terminal all outside, just consistent with the putative topological structure. Then, the enzymatic characteristics and kinetic parameters of recombinant EgLDH were surveyed and the results suggested that EgLDH is responsible for catalyzing the reduction of pyruvic acid into lactic acid under physiological conditions. The enzymatic activity of the recombinant protein was inhibited by antibodies directed against the intact protein or against epitopes that contain key residues in the catalytic center or substrate binding sites. EgLDH is a potential target for drugs and vaccines against E. granulosus. PMID:22542488

Gan, Wenjia; Zhang, Zhaoping; Lv, Gang; Xu, Hongxu; Zeng, Suxiang; Li, Yuzhe; Wu, Weiping; Hu, Xuchu

2012-04-20

159

Mouse interleukin-2 structure-function studies: substitutions in the first alpha-helix can specifically inactivate p70 receptor binding and mutations in the fifth alpha-helix can specifically inactivate p55 receptor binding.  

PubMed Central

The function of two alpha-helical regions of mouse interleukin-2 were analyzed by saturation substitution analysis. The functional parts of the first alpha-helix (A) was defined as residues 31-39 by the observation that proline substitutions within this region inactivate the protein. Four residues within alpha-helix A, Leu31, Asp34, Leu35 and Leu38, were found to be crucial for biological activity. Structural modeling suggested that these four residues are clustered on one face of alpha-helix A. Residues 31 and 35 had to remain hydrophobic for the molecule to be functional. At residue 38 there was a preference for hydrophobic side chain residues, while at residue 34 some small side chain residues as well as acidic or amide side chain residues were functionally acceptable. Inactivating changes at residue 34 had no effect upon the ability of the protein to interact with the p55 receptor. Disruption of the fifth alpha-helix (E), which had little effect upon biological activity, resulted in an inability of the protein to interact with the p55 receptor. Mutagenesis of the alpha-helix E region demonstrated that alpha-helicity and the nature of the side chain residues in this region were unimportant for biological activity. The region immediately proximal to alpha-helix E was important only for the single intramolecular disulfide linkage.

Zurawski, S M; Zurawski, G

1989-01-01

160

Structural mimicry of the ?-helix in aqueous solution with an isoatomic ?/?/?-peptide backbone.  

PubMed

Artificial mimicry of ?-helices offers a basis for development of protein-protein interaction antagonists. Here we report a new type of unnatural peptidic backbone, containing ?-, ?-, and ?-amino acid residues in an ?????? repeat pattern, for this purpose. This unnatural hexad has the same number of backbone atoms as a heptad of ? residues. Two-dimensional NMR data clearly establish the formation of an ?-helix-like conformation in aqueous solution. The helix formed by our 12-mer ?/?/?-peptide is considerably more stable than the ?-helix formed by an analogous 14-mer ?-peptide, presumably because of the preorganized ? and ? residues employed. PMID:21520956

Sawada, Tomohisa; Gellman, Samuel H

2011-04-26

161

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

PubMed

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

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

2013-07-08

162

A combined analysis of the cystic fibrosis transmembrane conductance regulator: implications for structure and disease models.  

PubMed

Over the past decade, nearly 1,000 variants have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in classic and atypical cystic fibrosis (CF) patients worldwide, and an enormous wealth of information concerning the structure and function of the protein has also been accumulated. These data, if evaluated together in a sequence comparison of all currently available CFTR homologs, are likely to refine the global structure-function relationship of the protein, which will, in turn, facilitate interpretation of the identified mutations in the gene. Based on such a combined analysis, we had recently defined a "functional R domain" of the CFTR protein. First, presenting two full-length cDNA sequences (termed sCFTR-I and sCFTR-II) from the Atlantic salmon (Salmo salar) and an additional partial coding sequence from the eastern gray kangaroo (Macropus giganteus), this study went further to refine the boundaries of the two nucleotide-binding domains (NBDs) and the COOH-terminal tail (C-tail), wherein NBD1 was defined as going from P439 to G646, NBD2 as going from A1225 to E1417, and the C-tail as going from E1418 to L1480. This approach also provided further insights into the differential roles of the two halves of CFTR and highlighted several well-conserved motifs that may be involved in inter- or intramolecular interactions. Moreover, a serious concern that a certain fraction of missense mutations identified in the CFTR gene may not have functional consequences was raised. Finally, phylogenetic analysis of all the full-length CFTR amino acid sequences and an extended set of exon 13--coding nucleotide sequences reinforced the idea that the rabbit may represent a better CF model than the mouse and strengthened the assertion that a long-branch attraction artifact separates the murine rodents from the rabbit and the guinea pig, the other Glires. PMID:11504857

Chen, J M; Cutler, C; Jacques, C; Boeuf, G; Denamur, E; Lecointre, G; Mercier, B; Cramb, G; Férec, C

2001-09-01

163

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

NASA Astrophysics Data System (ADS)

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

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

2013-02-01

164

Crystal structure of hormone-bound atrial natriuretic peptide receptor extracellular domain: rotation mechanism for transmembrane signal transduction.  

PubMed

A cardiac hormone, atrial natriuretic peptide (ANP), plays a major role in blood pressure and volume regulation. ANP activities are mediated by a single span transmembrane receptor carrying intrinsic guanylate cyclase activity. ANP binding to its extracellular domain stimulates guanylate cyclase activity by an as yet unknown mechanism. Here we report the crystal structure of dimerized extracellular hormone-binding domain in complex with ANP. The structural comparison with the unliganded receptor reveals that hormone binding causes the two receptor monomers to undergo an intermolecular twist with little intramolecular conformational change. This motion produces a Ferris wheel-like translocation of two juxtamembrane domains in the dimer with essentially no change in the interdomain distance. This movement alters the relative orientation of the two domains by a shift equivalent to counterclockwise rotation of each by 24 degrees. These results suggest that transmembrane signaling by the ANP receptor is initiated via a hormone-induced rotation mechanism. PMID:15117952

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

2004-04-26

165

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

166

Stabilization of protein structure by interaction of alpha-helix dipole with a charged side chain  

Microsoft Academic Search

The alpha-helix in proteins has a dipole moment resulting from the alignment of dipoles of the peptide bond which can perturb the pKas of ionizing groups. One of the two histidine residues (His18) in barnase, the small ribonuclease from Bacillus amyloliquefaciens, is located at the negatively charged end (C-terminal) of an alpha-helix. From NMR titrations of wild-type and engineered mutants

Das A. Sali; Mark Bycroft; Alan R. Fersht

1988-01-01

167

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

PubMed

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

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

1995-11-25

168

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

PubMed Central

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

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

1995-01-01

169

The biolabile 2'-O-pivaloyloxymethyl modification in an RNA helix: an NMR solution structure.  

PubMed

The pivaloyloxymethyl (PivOM) group is a biolabile 2'-O-ribose protection that is under development in a prodrug-based approach for siRNA applications. Besides an expected cellular uptake, nucleic acid sequences carrying PivOM showed also increased nuclease resistance and, in most cases, an affinity for complementary RNA. The r(CGCU*ACGC)dT:r(GCGUAGCG)dT model duplex containing a single modified residue (U*) was synthesized and its solution structure was determined by NMR. The duplex showed a maintained A-RNA helix. In U*, both 2'-O-acetal ester side chain and ring pucker presented a notable rigid conformation. The PivOM moiety was oriented with the carbonyl group turned outside the minor groove and with trans, -ac and -ac torsion angles around the C2'-O2', O2'-CA and CA-OB1 bonds respectively. Gauche effects and dipolar interactions between the PivOM and the backbone appeared to be the predominant factors influencing the PivOM conformation and the orientation of the two supplementary H acceptors suggested that hydration could also play a role in the duplex stability. PMID:23455628

Baraguey, Carine; Lescrinier, Eveline; Lavergne, Thomas; Debart, Françoise; Herdewijn, Piet; Vasseur, Jean-Jacques

2013-04-28

170

Structural Insight into a Molecular Switch in Tandem Winged-helix Motifs from Elongation Factor SelB  

Microsoft Academic Search

Elongation factor SelB is responsible for co-translational incorporation of selenocysteine (Sec) into proteins. The UGA stop codon is recoded as a Sec codon in the presence of a downstream mRNA hairpin. In prokaryotes, in addition to the EF-Tu-like N-terminal domains, a C-terminal extension containing four tandem winged-helix motifs (WH1-4) recognizes the mRNA hairpin. The 2.3–Å resolution crystal structure of the

Nicolas Soler; Dominique Fourmy; Satoko Yoshizawa

2007-01-01

171

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

172

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

PubMed Central

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

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

2011-01-01

173

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

PubMed Central

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

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

2012-01-01

174

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

PubMed

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

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

2011-09-13

175

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

NASA Astrophysics Data System (ADS)

In the framework of algebraic topology the closed sequence of 4-dimensional polyhedra (algebraic polytopes) was defined. This sequence is started by the polytope {240}, discovered by Coxeter, and is determined by the second coordination sphere of 8-dimensional lattice E8. The second polytope of sequence allows to determine a topologically stable rod substructure that appears during multiplication by a non-crystallographic axis 40/11 of the starting union of 4 tetrahedra with common vertex. When positioning the appropriate atoms tin positions of special symmetry of the staring 4 tetrahedra, such helicoid determines an {\\alpha}-helix. The third polytope of sequence allows to determine the helicoidally-like union of rods with 12-fold axis, which can be compare with Z-DNA structures. This model is defined as a local lattice rod packing, contained within a surface of helicoidally similar type, which ensures its topological stability, as well as possibility for it to be transformed into other forms of DNA structures. Formation of such structures corresponds to lifting a configuration degeneracy, and the stability of a state - to existence of a point of bifurcation. Furthermore, in the case of DNA structures, a second "security check" possibly takes place in the form of local lattice (periodic) property using the lattices other than the main ones.

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

2013-09-01

176

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

Microsoft Academic Search

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

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

1993-01-01

177

The membrane environment modulates self-association of the human GpA TM domain—Implications for membrane protein folding and transmembrane signaling  

Microsoft Academic Search

The influence of lipid bilayer properties on a defined and sequence-specific transmembrane helix–helix interaction is not well characterized yet. To study the potential impact of changing bilayer properties on a sequence-specific transmembrane helix–helix interaction, we have traced the association of fluorescent-labeled glycophorin A transmembrane peptides by fluorescence spectroscopy in model membranes with varying lipid compositions. The observed changes of the

Veerappan Anbazhagan; Dirk Schneider

2010-01-01

178

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

SciTech Connect

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

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

2009-05-18

179

Molecular dynamics investigations of the polysaccharide scleroglucan: first study on the triple helix structure  

Microsoft Academic Search

Explicit solvent molecular dynamics (MD) simulations on the triple helix of the polysaccharide Scleroglucan (Sclg) at two temperatures (273 and 300K) were carried out. Owing to the complexity of the system, a united-atom force field, based on the properly modified GROMACS parameters, was adopted. To test these parameters for our system, MD simulations of the two disaccharidic units, representing the

Antonio Palleschi; Gianfranco Bocchinfuso; Tommasina Coviello; Franco Alhaique

2005-01-01

180

The Amyloid Precursor Protein has a Flexible Transmembrane Domain and Binds Cholesterol  

PubMed Central

C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by ?-secretase to release the amyloid-? polypeptides, which are associated with Alzheimer’s disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded “N-helix” followed by a short “N-loop” connecting to the transmembrane domain (TMD). The TMD is a flexibly curved ? helix, making it well suited for processive cleavage by ?-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer’s therapeutics.

Barrett, Paul J.; Song, Yuanli; Van Horn, Wade D.; Hustedt, Eric J.; Schafer, Johanna M.; Hadziselimovic, Arina; Beel, Andrew J.; Sanders, Charles R.

2012-01-01

181

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.

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

2012-01-01

182

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

PubMed

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

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

2005-04-22

183

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

PubMed

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. PMID:21974560

Javidpour, Leili; Sahimi, Muhammad

2011-09-28

184

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

PubMed

A number of disorders related to cystic fibrosis have been described since the cloning of the cystic fibrosis gene, including infertility due to the congenital bilateral absence of the vas deferens. We have identified, in several patients, complex cystic fibrosis transmembrane conductance regulator genotypes like double-mutant alleles. We have now analyzed the structure-function relationships of one of these mutants, R74W-D1270N cystic fibrosis transmembrane conductance regulator, expressed in HeLa cells, to evaluate the contribution of each mutation in the phenotype. We found that R74W cystic fibrosis transmembrane conductance regulator appears to be a polymorphism, while D1270N cystic fibrosis transmembrane conductance regulator could be responsible for the congenital bilateral absence of the vas deferens phenotype. The combination of the two produced a more severe effect on the chloride conductance pathway as well as on the phenotype. PMID:10386624

Fanen, P; Clain, J; Labarthe, R; Hulin, P; Girodon, E; Pagesy, P; Goossens, M; Edelman, A

1999-06-11

185

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

PubMed

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

2013-10-15

186

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.

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

2013-01-01

187

Conformation of a 16-residue zervamicin IIA analog peptide containing three different structural features: 3(10)-helix, alpha-helix, and beta-bend ribbon.  

PubMed Central

Boc-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-Ala-Aib-Pro-Aib-Pro-Phe-OMe (where Boc is t-butoxycarbonyl and Aib is alpha-aminoisobutyric acid), a synthetic apolar analog of the membrane-active fungal peptide antibiotic zervamycin IIA, crystallizes in space group P1 with Z = 1 and cell parameters a = 9.086 +/- 0.002 A, b = 10.410 +/- 0.002 A, c = 28.188 +/- 0.004 A, alpha = 86.13 +/- 0.01 degrees, beta = 87.90 +/- 0.01 degrees, and gamma = 89.27 +/- 0.01 degrees; overall agreement factor R = 7.3% for 7180 data (F0 greater than 3 sigma) and 0.91-A resolution. The peptide backbone makes a continuous spiral that begins as a 3(10)-helix at the N-terminus, changes to an alpha-helix for two turns, and ends in a spiral of three beta-bends in a ribbon. Each of the beta-bends contains a proline residue at one of the corners. The torsion angles phi i range from -51 degrees to -91 degrees (average value -64 degrees), and the torsion angles psi i range from -1 degree to -46 degrees (average value -31 degrees). There are 10 intramolecular NH...OC hydrogen bonds in the helix and two direct head-to-tail hydrogen bonds between successive molecules. Two H2O and two CH3OH solvent molecules fill additional space with appropriate hydrogen bonding in the head-to-tail region, and two additional H2O molecules form hydrogen bonds with carbonyl oxygens near the curve in the helix at Pro-10. Since there is only one peptide molecule per cell in space group P1, the molecules repeat only by translation, and consequently the helices pack parallel to each other.

Karle, I L; Flippen-Anderson, J; Sukumar, M; Balaram, P

1987-01-01

188

Helix Antenna.  

National Technical Information Service (NTIS)

A helix antenna includes a base portion for containing a feed network including a power input, a 90 degree power splitter in communication with the power inlet, and first and second 180 degree power splitters in communication with the 90 degree power spli...

M. J. Josypenko

1999-01-01

189

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

Microsoft Academic Search

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

Bostjan Kobe; Bruce E. Kemp; Pantelis Poumbourios

1999-01-01

190

Transmembrane orientation and receptor-like structure of the Rhizobium meliloti common nodulation protein NodC  

PubMed Central

The 46.8-kd NodC protein of Rhizobium meliloti is a membrane protein, essential for nodule formation. Gene fusions of nodC to a portion of the ? cI repressor gene were used to define the membrane-anchor domain which is necessary for membrane insertion of the NodC protein into the membrane. The transmembrane orientation of NodC was confirmed by surface-specific radiolabeling and proteolysis experiments. A highly hydrophobic transmembrane-anchor domain was found near the carboxyl terminus, separating a large extracellular domain which contains an unusual cysteine-rich cluster from a short putative intracellular domain. Cross-linking studies showed that the NodC protein exists in the membrane probably as a dimer. The domain structure of the NodC protein shows striking similiarities with cell surface receptors. In nodules of various legumes a truncated form of the NodC protein was detected. The processed NodC was associated with the bacteroids and the amount of this protein increased during nodule development. Images

John, Michael; Schmidt, Jurgen; Wieneke, Ursula; Krussmann, Heinz-Dieter; Schell, Jeff

1988-01-01

191

Analysis of the contribution of an amphiphilic alpha-helix to the structure and to the function of ricin A chain.  

PubMed Central

The A chain of ricin is a cytotoxic RNA N-glycosidase that inactivates eukaryotic ribosomes. The contribution of the amphiphilic helix D, which is distant from the active site, to the catalysis of the depurination of the adenosine at position 4324 in 28S rRNA has been examined by systematic deletion of amino acids. Two sets of consecutive two- or three-amino acid deletions of the 12 residues in helix D, a total of 20 mutants, were constructed. All 12 of the amino acids could be deleted in one mutant or another without loss of activity; however, mutations that disrupted the amphiphilicity of the helix led to inactivation of the enzyme. Thus, the minimum contribution of helix D to the structure of the ricin A chain is to provide hydrophobic and hydrophilic surfaces to shield helix E, which has the active-site residues; moreover, no amino acid side chain in helix D makes a specific contribution to the recognition of the RNA substrate or to catalysis; and, finally, phasing of the amino acid deletions can be important to the phenotype of mutants. Images

Morris, K N; Wool, I G

1994-01-01

192

Molecular dynamics investigations of the polysaccharide scleroglucan: first study on the triple helix structure.  

PubMed

Explicit solvent molecular dynamics (MD) simulations on the triple helix of the polysaccharide Scleroglucan (Sclg) at two temperatures (273 and 300 K) were carried out. Owing to the complexity of the system, a united-atom force field, based on the properly modified GROMACS parameters, was adopted. To test these parameters for our system, MD simulations of the two disaccharidic units, representing the main chain and the side-chain linkages of the Sclg repeating unit, were performed and the results were compared with the literature data. The simulated triple helix of Sclg retained the main experimentally determined features of the polymer. The residence times of the solvent molecules at 273 and 300 K were analyzed. The results show that the more internal water molecules, interacting with the core of the Sclg triplex are not influenced substantially by changing the temperature, on the contrary the water molecules, interacting with the side-chain glucose residues show more significant differences. These data suggest that the more external water molecules, interacting with the side chain, play a major role in the conformational transition experimentally observed at low temperature. PMID:16043160

Palleschi, Antonio; Bocchinfuso, Gianfranco; Coviello, Tommasina; Alhaique, Franco

2005-09-26

193

Synergistic transmembrane alignment of the antimicrobial heterodimer PGLa/magainin.  

PubMed

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

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

2006-07-28

194

Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix.  

PubMed

Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Förster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics of poly-l-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale. PMID:17956989

Doose, Sören; Neuweiler, Hannes; Barsch, Hannes; Sauer, Markus

2007-10-23

195

Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix  

PubMed Central

Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Förster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics of poly-l-prolines with 1–10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.

Doose, Soren; Neuweiler, Hannes; Barsch, Hannes; Sauer, Markus

2007-01-01

196

A stable and switchable uniform lying helix structure in cholesteric liquid crystals  

NASA Astrophysics Data System (ADS)

This investigation demonstrates an electrically switchable uniformly lying helix (ULH) stable state in cholesteric liquid crystal. A stable ULH state can be achieved by applying the low-frequency (30 Hz) pulse electrical field via an electro-hydrodynamatic effect. The ULH state can be stably maintained with a helical pitch in the visible range (450 nm-630 nm) and exhibit a tunable uniaxial crystal wave plate property under 1 kHz electrical field. The study examines the electro-optical property of ULH state and driving scheme for switching among the three stable states. A multi-stable and electrically switchable cholesteric liquid crystal can provide various optical properties and has extensive potential applications.

Wang, Chun-Ta; Wang, Wei-Yuan; Lin, Tsung-Hsien

2011-07-01

197

Protein-protein interactions among West Nile non-structural proteins and transmembrane complex formation in mammalian cells.  

PubMed

To study the membrane orientation of flavivirus non-structural proteins (NSPs) in the replication complex, the seven major West Nile (WN) NSPs were separately expressed in monkey cells, and their subcellular localization was investigated by imaging-based techniques. First, we observed by confocal microscopy that four small transmembrane proteins (TP) (NS2A, NS2B, NS4A, and NS4B) were located to the endoplasmic reticulum (ER), whereas the largest NSPs, NS1, NS3, and NS5 were not. We then analyzed the colocalization and the association of WN NSPs using the methods of confocal microscopy, fluorescence resonance energy transfer (FRET), and biologic fluorescence complementation (BiFC). Through these combined imaging techniques, protein-protein interactions (PPI) among WNNSPs were detected. Our data demonstrate that there are interactions between NS2A and NS4A, and interactions of NS2B with three other TPs (NS2A, NS4A, and NS4B) as well as the expected interaction with NS3. PPI between NS2A and NS4B or between NS4A and NS4B were not detected. By the criteria of these techniques, NS5 interacted only with NS3, and NS1 was not shown to be in close proximity with other NSPs. In addition, homo-oligomerization of some NSPs was observed and three-way interactions between NS2A, NS4A, and NA4B with NS2B-NS3 were also observed, respectively. Our results suggest that the four TPs are required for formation of transmembrane complex. NS2B protein seems to play a key role in bringing the TPs together on the ER membrane and in bridging the TPs with non-membrane-associated proteins (NS3 and NS5). PMID:24074601

Yu, Li; Takeda, Kazuyo; Markoff, Lewis

2013-09-13

198

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

NASA Astrophysics Data System (ADS)

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

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

2013-06-01

199

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

PubMed

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

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

2013-07-27

200

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

201

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

PubMed Central

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

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

2009-01-01

202

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

NSDL National Science Digital Library

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

2008-10-06

203

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

SciTech Connect

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

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

2010-02-22

204

The Conformational Transitions of Uncharged Poly-L-Lysine. Alpha Helix-Random Coil-beta Structure.  

National Technical Information Service (NTIS)

The heat-induced transition of poly-L-lysine, alpha helix-random coil-beta form, was studied, mainly by optical rotatory dispersion (ORD). Below T-beta the transition has the properties of a reversible alpha-helix-random coil equilibrium. Above T-beta, th...

B. Davidson G. D. Fasman

1967-01-01

205

Structure-microbicidal activity relationship of synthetic fragments derived from the antibacterial alpha-helix of human lactoferrin.  

PubMed

There is a need for new microbicidal agents with therapeutic potential due to antibiotic resistance in bacteria and fungi. In this study, the structure-microbicidal activity relationship of amino acid residues 14 to 31 (sequence 14-31) from the N-terminal end, corresponding to the antibacterial alpha-helix of human lactoferrin (LF), was investigated by downsizing, alanine scanning, and substitution of amino acids. Microbicidal analysis (99% killing) was performed by a microplate assay using Escherichia coli, Staphylococcus aureus, and Candida albicans as test organisms. Starting from the N-terminal end, downsizing of peptide sequence 14-31 showed that the peptide sequence 19-31 (KCFQWQRNMRKVR, HL9) was the optimal length for antimicrobial activity. Furthermore, HL9 bound to lipid A/lipopolysaccharide, as shown by neutralizing endotoxic activity in a Limulus assay. Alanine scanning of peptide sequence 20-31 showed that Cys20, Trp23, Arg28, Lys29, or Arg31 was important for expressing full killing activity, particularly against C. albicans. Substituting the neutral hydrophilic amino acids Gln24 and Asn26 for Lys and Ala (HLopt2), respectively, enhanced microbicidal activity significantly against all test organisms compared to the amino acids natural counterpart, also, in comparison with HL9, HLopt2 had more than 10-fold-stronger fungicidal activity. Furthermore, HLopt2 was less affected by metallic salts than HL9. The microbicidal activity of HLopt2 was slightly reduced only at pH 7.0, as tested in the pH range of 4.5 to 7.5. The results showed that the microbicidal activity of synthetic peptide sequences, based on the antimicrobial alpha-helix region of LF, can be significantly enhanced by optimizing the length and substitution of neutral amino acids at specific positions, thus suggesting a sequence lead with therapeutic potential. PMID:19917761

Håversen, L; Kondori, N; Baltzer, L; Hanson, L A; Dolphin, G T; Dunér, K; Mattsby-Baltzer, I

2009-11-16

206

Structure and action of heteronemertine polypeptide toxins: importance of amphipathic helix for activity of Cerebratulus lacteus toxin A-III.  

PubMed

The marine heteronemertine Cerebratulus lacteus produces a family of protein cytolysins designated as A-toxins. Limited subtilisin digests of the most abundant homolog, toxin A-III, yield two major products which may be purified by high-performance liquid chromatography. One product is shown to represent residues 1-86 and the other contains the entire toxin sequence (1-95). Both polypeptides are shown to lack internal protease nicks. The 1-95 polypeptide retains full cytolytic activity in comparison to native toxin, whereas 1-86 has an activity that is approximately four times lower. Extensive treatment of A-III with carboxypeptidase Y yields a polypeptide containing residues 1-75 which is totally devoid of hemolytic activity. Residues 63-95 of native A-III have been predicted to form a relatively hydrophobic alpha-helix which is potentially important for activity. The circular dichroism spectrum of 1-95 is in excellent agreement with both experimental and Chou-Fasman-predicted secondary structures of native A-III, while the spectra of 1-86 and 1-75 indicate a loss of helicity quantitatively consistent with the removal of residues 87-95 and 76-95, respectively. Combined with our earlier data on bilayer penetration by N-terminal sequences (K. M. Blumenthal (1982) Biochemistry 21, 4229-4233], the current results indicate a direct involvement of both ends of A-III in lytic activity. The C-terminal region may function by contributing a membrane binding site in the form of an amphipathic helix. PMID:3881082

Dumont, J A; Blumenthal, K M

1985-01-01

207

Pressure-induced transformation of alpha-helix to beta-sheet in the secondary structures of amyloid beta (1-40) peptide exacerbated by temperature.  

PubMed

The effect of pressure on the conformational structure of amyloid beta (1-40) peptide (A beta(1-40)), exacerbated with or without temperature, was determined by Fourier transform infrared (FT-IR) microspectroscopy. The result indicates the shift of the maximum peak of amide I band of intact solid A beta(1-40) from 1655 cm(-1) (alpha-helix) to 1647-1643 cm(-1) (random coil) with the increase of the mechanical pressure. A new peak at 1634 cm(-1) assigned to beta-antiparallel sheet structure was also evident. Furthermore, the peak at 1540 cm(- 1) also shifted to 1527 (1529) cm(-1) in amide II band. The former was assigned to the combination of alpha-helix and random coil structures, and the latter was due to beta-sheet structure. Changes in the composition of each component in the deconvoluted and curve-fitted amide I band of the compressed A beta(1-40) samples were obtained from 33% to 22% for alpha-helix/random coil structures and from 47% to 57% for beta-sheet structure with the increase of pressure, respectively. This demonstrates that pressure might induce the conformational transition from alpha-helix to random coil and to beta- sheet structure. The structural transformation of the compressed A beta(1-40) samples was synergistically influenced by the combined effects of pressure and temperature. The thermal-induced formation of beta-sheet structure was significantly dependent on the pressures applied. The smaller the pressure applied the faster the beta-sheet structure transformed. The thermal-dependent transition temperatures of solid A beta(1-40) prepared by different pressures were near 55-60 degrees C. PMID:11843623

Lin, Shan-Yang; Chu, Horng-Lun; Wei, Yen-Shan

2002-02-01

208

Molecular mechanism of transmembrane signaling by the aspartate receptor: a model.  

PubMed Central

The aspartate receptor of bacterial chemotaxis is representative of a large class of membrane-spanning receptors found in prokaryotic and eukaryotic organisms. These receptors, which regulate histidine kinase pathways and possess two putative transmembrane helices per subunit, appear to control a wide variety of cellular processes. The best characterized subgroup of the two-helix receptor class is the homologous family of chemosensory receptors from Escherichia coli and Salmonella typhimurium, including the aspartate receptor. This receptor binds aspartate, an attractant, in the periplasmic compartment and undergoes an intramolecular, transmembrane conformational change, thereby modulating the autophosphorylation rate of a bound histidine kinase in the cytoplasm. Here, we analyze recent results from x-ray crystallographic, solution 19F NMR, and engineered disulfide studies probing the aspartate-induced structural change within the periplasmic and transmembrane regions of the receptor. Together, these approaches provide evidence that aspartate binding triggers a "swinging-piston" displacement of the second membrane-spanning helix, which is proposed to communicate the signal across the bilayer. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

Chervitz, S A; Falke, J J

1996-01-01

209

Structural Studies on Nucleic Acids: From Nucleotides to the Double Helix  

Microsoft Academic Search

In the field of nucleic acids the nucleoside and nucleotide units represent the lowest level of structural sophistication. Since they play a role in a number of metablic processes as regulators and as coenzymes, their structural properties are as important as those of the polymeric nucleic acids which form single, double, triple and quadruple he1 ices, depending on their nucleotide

Wolfram Saenger

1990-01-01

210

CRYSTAL STRUCTURE OF PHENYLALANINE AMMONIA LYASE: MULTIPLE HELIX DIPOLES IMPLICATED IN CATALYSIS  

Technology Transfer Automated Retrieval System (TEKTRAN)

The first three-dimensional structure of phenylalanine ammonia lyase (PAL) has been determined at 2.1 angstrom resolution using PAL from Rhodosporidium toruloides. The enzyme is structurally similar to the mechanistically-related histidine ammonia lyase (HAL), with PAL having an additional ~160 res...

211

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.

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

2008-01-01

212

Stable interactions between the transmembrane domains of the adenosine A2A receptor  

PubMed Central

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

Thevenin, Damien; Lazarova, Tzvetana

2008-01-01

213

High-level expression and purification of the second transmembrane domain of wild-type and mutant human melanocortin-4 receptor for solid-state NMR structural studies.  

PubMed

It has been demonstrated that human melanocortin-4 receptor (hMC4R) plays an important role in the control of energy homeostasis, and heterozygous mutations in the hMC4R gene are the most frequent genetic cause of severe human obesity. In order to obtain additional insight into the structure and function, we cloned, expressed, and purified the second transmembrane domain of the wild-type hMC4R (wt-TM2) and D90N mutant hMC4R (m-TM2). To facilitate structural studies of these hMC4R by solid-state NMR, efficient methods for the production of milligram quantities of isotopically labeled protein are necessary. However, large-scale production of most transmembrane proteins has been limited by experimental adversities due to insufficient yields and low solubility of protein. Nevertheless, through the optimization of the expression and purification approach, we could obtain uniformly or selectively labeled fusion proteins in yields as high as 200-250 mg per liter M9 minimal medium. These proteins were overexpressed in inclusion bodies as a fusion protein with ketosteroid isomerase (KSI) in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography under denaturing conditions. wt-/m-TM2 peptides were released from the fusion by cyanogen bromide cleavage at the Met residue and separated from the carrier KSI by size exclusion chromatography. Initial structural data obtained by solution NMR measurements of wt-/m-TM2 is also presented. The successful application to the production of the second transmembrane domain of human MC4R indicates that the method can be applied to other transmembrane proteins as well and also enable its structural and functional studies using solid-state NMR spectroscopy. PMID:18809499

Park, Tae-Joon; Choi, Sung-Sub; Gang, Ga-Ae; Kim, Yongae

2008-09-03

214

The structure of a stable intermediate in the A <-> B DNA helix transition  

PubMed Central

The DNA dodecamer CATGGGCCCATG in a crystal structure of resolution 1.3 ? has a conformation intermediate between A and B DNA. This trapping of a stable intermediate suggests that the A and B DNA families are not discrete, as previously believed. The structure supports a base-centered rather than a backbone-centered mechanism for the A ? B transition mediated by guanine tracts. Interconversion between A and B DNA provides another means for regulating protein–DNA recognition.

Ng, Ho-Leung; Kopka, Mary L.; Dickerson, Richard E.

2000-01-01

215

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)

2012-02-28

216

Toward a designed, functioning genetic system with expanded-size base pairs: solution structure of the eight-base xDNA double helix.  

PubMed

We describe the NMR-derived solution structure of the double-helical form of a designed eight-base genetic pairing system, termed xDNA. The benzo-homologous xDNA design contains base pairs that are wider than natural DNA pairs by ca. 2.4 A (the width of a benzene ring). The eight component bases of this xDNA helix are A, C, G, T, xA, xT, xC, and xG. The structure was solved in aqueous buffer using 1D and 2D NMR methods combined with restrained molecular dynamics. The data show that the decamer duplex is right-handed and antiparallel, and hydrogen-bonded in a way analogous to that of Watson-Crick DNA. The sugar-phosphate backbone adopts a regular conformation similar to that of B-form DNA, with small dihedral adjustments due to the larger circumference of the helix. The grooves are much wider and more shallow than those of B-form DNA, and the helix turn is slower, with ca. 12 base pairs per 360 degrees turn. There is an extensive intra- and interstrand base stacking surface area, providing an explanation for the greater stability of xDNA relative to natural DNA. There is also evidence for greater motion in this structure compared to a previous two-base-expanded helix; possible chemical and structural reasons for this are discussed. The results confirm paired self-assembly of the designed xDNA system. This suggests the possibility that other genetic system structures besides the natural one might be functional in encoding information and transferring it to new complementary strands. PMID:17090058

Lynch, Stephen R; Liu, Haibo; Gao, Jianmin; Kool, Eric T

2006-11-15

217

Expanded turn conformations: Characterization and sequence-structure correspondence in ?-turns with implications in helix folding  

Microsoft Academic Search

Like the-turns, which are charac- terized by a limiting distance between residues two positions apart (i, i3), a distance criterion (involv- ing residues at positions i and i4) is used here to identify -turns from a database of known protein structures. At least 15 classes of -turns have been enumerated based on the location in the , space of the

Bhaskar Dasgupta; Lipika Pal; Gautam Basu; Pinak Chakrabarti

2004-01-01

218

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

Microsoft Academic Search

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, ‚

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

2005-01-01

219

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

NASA Astrophysics Data System (ADS)

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

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

2010-03-01

220

Methionine Adenosyltransferase ?-Helix Structure Unfolds at Lower Temperatures than ?-Sheet: A 2D-IR Study  

PubMed Central

Two-dimensional infrared spectroscopy has been used to characterize rat liver methionine adenosyltransferase and the events taking place during its thermal unfolding. Secondary structure data have been obtained for the native recombinant enzyme by fitting the amide I band of infrared spectra. Thermal denaturation studies allow the identification of events associated with individual secondary-structure elements during temperature-induced unfolding. They are correlated to the changes observed in enzyme activity and intrinsic fluorescence. In all cases, thermal denaturation proved to be an irreversible process, with a Tm of 47–51°C. Thermal profiles and two-dimensional infrared spectroscopy show that unfolding starts with ?-helical segments and turns, located in the outer part of the protein, whereas extended structure, associated with subunit contacts, unfolds at higher temperatures. The data indicate a good correlation between the denaturation profiles obtained from activity measurements, fluorescence spectroscopy, and the behavior of the infrared bands. A study of the sequence of events that takes place is discussed in light of the previous knowledge on methionine adenosyltransferase structure and oligomerization pathway.

Iloro, Ibon; Chehin, Rosana; Goni, Felix M.; Pajares, Maria A.; Arrondo, Jose-Luis R.

2004-01-01

221

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

PubMed

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

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

2007-04-19

222

A'-form RNA double helix in the single crystal structure of r(UGAGCUUCGGCUC).  

PubMed Central

Here we demonstrate the presence of the A'-RNA conformation using the single crystal structure of a tridecamer: r(UGAGCUUCGGCUC). The average A'-RNA conformation deduced from X-ray fiber diffraction data had only been available previously, but now the presence of the A'-RNA conformation has been found in a single crystal structure for the first time. Statistical analysis showed that the A'-RNA conformation is distinguishable from the A-RNA conformation in a plot of the major groove width against the base pair inclination angle. The major groove of the A'-RNA conformation is wide enough to accommodate a protein or peptide while that of the A-RNA conformation is too narrow to do so. The presence of the A'-RNA conformation is significant for protein-RNA interaction.

Tanaka, Y; Fujii, S; Hiroaki, H; Sakata, T; Tanaka, T; Uesugi, S; Tomita, K; Kyogoku, Y

1999-01-01

223

Ab initio folding of extended ?-helix: A theoretical study about the role of electrostatic polarization in the folding of helical structures.  

PubMed

In this work, we report the ab initio folding of three different extended helical peptides namely 2khk, N36, and C34 through conventional molecular dynamics simulation at room temperature using implicit solvation model. Employing adaptive hydrogen bond specific charge (AHBC) scheme to account for the polarization effect of hydrogen bonds established during the simulation, the effective folding of the three extended helices were observed with best backbone RMSDs in comparison to the experimental structures over the helical region determined to be 1.30 Å for 2khk, 0.73 Å for N36 and 0.72 Å for C34. In this study, 2khk will be used as a benchmark case serving as a means to compare the ability of polarized (AHBC) and nonpolarized force field in the folding of an extended helix. Analyses conducted revealed the ability of the AHBC scheme in effectively folding the extended helix by promoting helix growth through the stabilization of backbone hydrogen bonds upon formation during the folding process. Similar observations were also noted when AHBC scheme was employed during the folding of C34 and N36. However, under Amber03 force field, helical structures formed during the folding of 2khk was not accompanied by stabilization thus highlighting the importance of electrostatic polarization in the folding of helical structures. Proteins 2013. © 2013 Wiley Periodicals, Inc. PMID:23670702

Lazim, Raudah; Wei, Caiyi; Sun, Tiedong; Zhang, Dawei

2013-06-17

224

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

PubMed

An optically inactive poly(4-carboxyphenyl isocyanide) (poly-1-H) changed its structure into the prevailing, one-handed helical structure upon complexation with optically active amines in dimethylsulfoxide (DMSO) and water, and the complexes show a characteristic induced circular dichroism in the polymer backbone region. Moreover, the macromolecular helicity induced in water and aqueous organic solutions containing more than 50 vol % water could be "memorized" even after complete removal of the chiral amines (h-poly-1b-H), while that induced in DMSO and DMSO-water mixtures containing less than 30 vol % water could not maintain the optical activity after removal of the chiral amines (poly-1a-H). We now report fully detailed studies of the helix induction mechanism with chiral amines and the memory of the macromolecular helicity in water and a DMSO-water mixture by various spectroscopic measurements, theoretical calculations, and persistence length measurements together with X-ray diffraction (XRD) measurements. From the spectroscopic results, such as circular dichroism (CD), absorption, IR, vibrational CD, and NMR of poly-1a-H, h-poly-1b-H, and original poly-1-H, we concluded that the specific configurational isomerization around the C horizontal lineN double bonds occurs during the helicity induction process in each solvent. In order to obtain the structural information, XRD measurements were done on the uniaxially oriented films of the corresponding methyl esters (poly-1-Me, poly-1a-Me, and h-poly-1b-Me) prepared from their liquid crystalline polymer solutions. On the basis of the XRD analyses, the most plausible helical structure of poly-1a-Me was proposed to be a 9-unit/5-turn helix with two monomer units as a repeating unit, and that of h-poly-1b-Me was proposed to be a 10-unit/3-turn helix consisting of one repeating monomer unit. The density functional theory calculations of poly(phenyl isocyanide), a model polymer of h-poly-1b-Me, afforded a 7-unit/2-turn helix as the most possible helical structure, which is in good agreement with the XRD results. Furthermore, the persistence length measurements revealed that these structural changes accompany a significant change in the main-chain stiffness. The mechanism of helix induction in poly-1-H and the memory of the macromolecular helicity are discussed on the basis of these results. PMID:19580322

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

2009-08-01

225

Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability.  

PubMed Central

In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within alpha-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the alpha-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions. All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (< or = 0.4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133-->Ala made alpha-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino- and carboxy-terminal domains changed by 3.6 degrees. This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.

Zhang, X. J.; Baase, W. A.; Matthews, B. W.

1992-01-01

226

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

227

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

228

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

PubMed

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

2011-01-01

229

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

PubMed

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

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

2011-12-09

230

Double helix  

NSDL National Science Digital Library

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

Dennis Myts (None;)

2007-02-19

231

Global structure and dynamics of human apolipoprotein CII in complex with micelles: evidence for increased mobility of the helix involved in the activation of lipoprotein lipase.  

PubMed

Apolipoprotein CII (apoCII), a surface constituent of plasma lipoproteins, is the activator for lipoprotein lipase (LPL) and is therefore central for lipid transport in blood. The three-dimensional structure of (13)C-, (15)N-enriched human full-length apoCII in complex with sodium dodecyl sulfate (SDS) micelles is reported. In addition to the structure determination, (15)N-relaxation measurements have been performed at two magnetic fields to characterize the dynamics of the backbone of apoCII in the complex. The relaxation data also provided global structural constraints, viz. the orientation of helices in the complex. In addition, global constraints were derived from the fact that apoCII helices are attached to the surface of the SDS micelle and that the hydrophobic moments of each helix faces the interior of the micelle. These three categories of global constraints, together with the local classical NMR constraints, were sufficient to define the 3D structure of the apoCII-SDS micelle complex. To our knowledge, this presents the first example in which the global structure of a protein-SDS micelle complex has been determined. The C-terminal helix of apoCII is known to be responsible for the activation of LPL. This helix is distinguished from the other helices by a higher degree of internal motion on the nanosecond time scale as shown by the relaxation data. The overall structure and the internal dynamics, combined with previous mutation data, give important clues toward a possible mechanism for the activation of LPL by apoCII. PMID:12590574

Zdunek, J; Martinez, G V; Schleucher, J; Lycksell, P O; Yin, Y; Nilsson, S; Shen, Y; Olivecrona, G; Wijmenga, S

2003-02-25

232

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

SciTech Connect

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

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

2007-01-01

233

Multiwalled ice helixes and ice nanotubes  

PubMed Central

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

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

2006-01-01

234

The effect of secondary structures on the NLO properties of single chain oligopeptides: a comparison between ?-strand and ?-helix polyglycines.  

PubMed

The evolution of the electronic first-order longitudinal hyperpolarizability (?zzz) and the hyperpolarizability aligned along the direction of the dipole moment (??) of the ?-helix and ?-strand single chain H2N-(CH2-CO-NH)n-CH2-COOH (n = 1-9) oligoglycines, were investigated. For this purpose we have used Hartree-Fock, second-order Møller-Plesset perturbation theory and Coulomb-attenuating Density Functional Theory computations. For the longest chain, ??(?-strand) is one order of magnitude greater than ??(?-helix), due to the cooperative effect of the ?-helices being unfavourable for the NLO properties. The ?zzz and ?? values per unit cell of the ?-strand conformation were determined, extrapolating the properties in the limit of the polymer. The calculated ?zzz values were elucidated using the two-state model involving the characteristic ?-?* NV1 electronic transition of peptides. Single chain ?-strand polyglycines can be discriminated from the ?-helices using second-order NLO effects. PMID:23817555

Alparone, Andrea

2013-08-21

235

Population genetic structure in a human-disturbed environment: a case study in the land snail Helix aspersa (Gastropoda: Pulmonata)  

Microsoft Academic Search

Local patterns of genetic variation were analysed in the land snail Helix aspersa for 32 populations sampled within a patchy agricultural landscape: the polders of the Bay of Mont-Saint-Michel (France). This investigation examined the allele frequencies at four enzymatic markers and five microsatellite loci through the genotyping of 580 individuals. A strongly significant population genetic substructuring (mean FST=0.088, P<0.001) was

J-F Arnaud; L Madec; A Guiller; J Deunff

2003-01-01

236

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

PubMed Central

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

2013-01-01

237

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

PubMed

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

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

2012-04-01

238

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

PubMed Central

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

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

2012-01-01

239

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

SciTech Connect

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

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

1990-05-23

240

Structure of the purine-pyrimidine alternating RNA double helix, r(GUAUAUA)d(C), with a 3'-terminal deoxy residue.  

PubMed

The crystal structure of the purine-pyrimidine alternating octameric RNA helix, r(GUAUAUA)d(C), carrying a 3'-terminal deoxycytidine residue, has been determined at 2.2 A resolution. The molecule crystallizes in the rhombohedral space group R3 (hexagonal cell constants: a = b = 43.07,c = 59.36 A;alpha = beta = 90,gamma = 120 degrees )with one duplex in an asymmetric unit. The structure was solved by molecular replacement and refined with 83 and 2/3 solvent molecules and 2/3 sodium ions to a final R factor of 15.6% using 1775 reflections (86%). The duplexes are approximately linear, their global helix axes are inclined by 10 degrees with respect to the 3(2)-screw axes, and they are stacked on top of each other in a head-to-tail fashion. The twist between the junction base pairs of the stacked duplexes is negligible resulting in a discontinuity of the helix backbones and grooves. The sodium ions on the threefold axis play a significant role in the organization of the packing network. The helical parameters, particularly the twist and the roll, of this alternating sequence are in accord with Calladine's rules. Almost all the 2'-hydroxyl groups are involved in specific hydrogen-bonding interactions, either directly to the sugar ring oxygens O4' on the 3' side, or, through water bridges, to the sugars, phosphates, or bases. This hydrogen bonding of the 2'-hydroxyl groups restrains the conformation of the sugar-phosphate backbone and the glycosidic torsion angles of this RNA fragment. The lack of intermolecular packing contacts in the grooves provides a clear picture of the groove solvation. PMID:15299629

Wahl, M C; Ban, C; Sekharudu, C; Ramakrishnan, B; Sundaralingam, M

1996-07-01

241

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

PubMed Central

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

1994-01-01

242

Measuring the Double Helix  

SciTech Connect

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

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

2009-05-26

243

?-Helix mimicry with ?/?-peptides.  

PubMed

We describe a general strategy for creating peptidic oligomers that have unnatural backbones but nevertheless adopt a conformation very similar to the ?-helix. These oligomers contain both ?- and ?-amino acid residues (?/?-peptides). If the ? content reaches 25-30% of the residue total, and the ? residues are evenly distributed along the backbone, then substantial resistance to proteolytic degradation is often observed. These ?/?-peptides can mimic the informational properties of ?-helices involved in protein-protein recognition events, as documented in numerous crystal structures. Thus, these unnatural oligomers can be a source of antagonists of undesirable protein-protein interactions that are mediated by natural ?-helices, or agonists of receptors for which the natural polypeptide ligands are ?-helical. Successes include mimicry of BH3 domains found in proapoptotic proteins, which leads to ligands for antiapoptotic Bcl-2 family proteins, and mimicry of the gp41 CHR domain, which leads to inhibition of HIV infection in cell-based assays. PMID:23422441

Johnson, Lisa M; Gellman, Samuel H

2013-01-01

244

Rosalind Franklin and the Double Helix  

NASA Astrophysics Data System (ADS)

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

Elkin, Lynne Osman

2003-03-01

245

Rosalind Franklin and the Double Helix  

Microsoft Academic Search

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

Lynne Osman Elkin

2003-01-01

246

Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: the Fab is directed against an intermediate in the helix-coil dynamics of the enzyme.  

PubMed

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a gamma turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains. PMID:9770525

Murali, R; Sharkey, D J; Daiss, J L; Murthy, H M

1998-10-13

247

Engineering a high-affinity anti-IL-15 antibody: crystal structure reveals an ?-helix in VH CDR3 as key component of paratope.  

PubMed

Interleukin (IL) 15 is an inflammatory cytokine that plays an essential role in the activation, proliferation, and maintenance of specific natural killer cell and T-cell populations, and has been implicated as a mediator of inflammatory diseases. An anti-IL-15 antibody that blocked IL-15-dependent cellular responses was isolated by phage display and optimised via mutagenesis of the third complementarity-determining regions (CDRs) of variable heavy (VH) and variable light chains. Entire repertoires of improved variants were recombined with each other to explore the maximum potential sequence space. DISC0280, the most potent antibody isolated using this comprehensive strategy, exhibits a 228-fold increase in affinity and a striking 40,000-fold increase in cellular potency compared to its parent. Such a wholesale recombination strategy therefore represents a useful method for exploiting synergistic potency gains as part of future antibody engineering efforts. The crystal structure of DISC0280 Fab (fragment antigen binding), in complex with human IL-15, was determined in order to map the structural epitope and paratope. The most remarkable feature revealed lies within the paratope and is a novel six-amino-acid ?-helix that sits within the VH CDR3 loop at the center of the antigen binding site. This is the first report to describe an ?-helix as a principal component of a naturally derived VH CDR3 following affinity maturation. PMID:21167836

Lowe, David C; Gerhardt, Stefan; Ward, Alison; Hargreaves, David; Anderson, Malcolm; Ferraro, Franco; Pauptit, Richard A; Pattison, Debbie V; Buchanan, Catriona; Popovic, Bojana; Finch, Donna K; Wilkinson, Trevor; Sleeman, Matthew; Vaughan, Tristan J; Mallinder, Philip R

2010-12-16

248

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.

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

2013-01-01

249

Transmembrane signaling and assembly of the cytochrome b6f-lipidic charge transfer complex.  

PubMed

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

Hasan, S Saif; Yamashita, Eiki; Cramer, William A

2013-03-16

250

Conversion of a beta-strand to an alpha-helix induced by a single-site mutation observed in the crystal structure of Fis mutant Pro26Ala.  

PubMed Central

The conversion from an alpha-helix to a beta-strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self-assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a beta-strand to an alpha-helix by a single-site mutation as observed in the crystal structure of Fis mutant Pro26Ala determined at 2.0 A resolution. Pro26 in Fis occurs at the point where a flexible extended beta-hairpin arm leaves the core structure. Thus it can be classified as a "hinge proline" located at the C-terminal end of the beta2-strand and the N-terminal cap of the A alpha-helix. The replacement of Pro26 to alanine extends the A alpha-helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted from a beta-strand to an alpha-helix. This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant's reduced ability to activate Hin-catalyzed DNA inversion. The peptide (residues 20 to 26) in the second monomer subunit presumably retains its beta-strand conformation in the crystal; therefore, this peptide shows a "chameleon-like" character since it can adopt either an alpha-helix or a beta-strand structure in different environments. The structure of Pro26Ala provides an additional example where not only the protein sequence, but also non-local interactions determine the secondary structure of proteins.

Yang, W. Z.; Ko, T. P.; Corselli, L.; Johnson, R. C.; Yuan, H. S.

1998-01-01

251

A c subunit with four transmembrane helices and one ion (Na+)-binding site in an archaeal ATP synthase: implications for c ring function and structure.  

PubMed

The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na(+)-DCCD competition experiments revealed only one binding site for DCCD and Na(+), indicating that the mature c subunit of this A(1)A(O) ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na(+)-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na(+)-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na(+)-specific under in vivo conditions, comparable with the Na(+)-dependent V(1)V(O) ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na(+)-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A(1)A(O) ATP synthases. PMID:23007388

Mayer, Florian; Leone, Vanessa; Langer, Julian D; Faraldo-Gómez, José D; Müller, Volker

2012-09-24

252

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

NASA Astrophysics Data System (ADS)

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

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

2013-08-01

253

TMP21 Transmembrane Domain Regulates ?-Secretase Cleavage*  

PubMed Central

TMP21 has been shown to be associated with the ?-secretase complex and can specifically regulate ?-cleavage without affecting ?-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of ?-secretase activity was investigated independent of ?-cleavage using an amyloid-? precursor protein? (APP?) construct which lacks the amyloid intracellular domain domain. The APP? construct behaves similarly to the full-length precursor protein with respect to ?- and ?-cleavages and is able to undergo normal ?-processing. Co-expression of APP? and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight A?-positive fragments, consistent with an inhibition of ?-secretase cleavage. The APP? system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the ?-secretase complex and could decrease ?-secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce A? production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased ?-cleavage activity.

Pardossi-Piquard, Raphaelle; Bohm, Christopher; Chen, Fusheng; Kanemoto, Soshi; Checler, Frederic; Schmitt-Ulms, Gerold; St. George-Hyslop, Peter; Fraser, Paul E.

2009-01-01

254

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

255

A Helix Replacement Mechanism Directs Metavinculin Functions  

PubMed Central

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 ?Leu954 metavinculin deletion mutant. These structures reveal that an ?-helix (H1?) and extended coil of the metavinculin insert replace ?-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 ?Leu954 and Arg975Trp metavinculin mutants reside on the replaced extended coil and the H1? ?-helix, respectively. Thus, a helix replacement mechanism directs metavinculin's unique functions.

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

2010-01-01

256

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

257

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.

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

1992-01-01

258

Interleukin 32 (IL-32) Contains a Typical ?-Helix Bundle Structure That Resembles Focal Adhesion Targeting Region of Focal Adhesion Kinase-1*  

PubMed Central

IL-32 can be expressed in several isoforms. The amino acid sequences of the major IL-32 isoforms were used to predict the secondary and tertiary protein structure by I-TASSER software. The secondary protein structure revealed coils and ?-helixes, but no ? sheets. Furthermore, IL-32 contains an RGD motif, which potentially activates procaspase-3 intracellular and or binds to integrins. Mutation of the RGD motif did not result in inhibition of the IL-32?- or IL-32?-induced cytotoxicity mediated through caspase-3. Although IL-32? interacted with the extracellular part of ?V?3 and ?V?6 integrins, only the ?V?3 binding was inhibited by small RGD peptides. Additionally, IL-32? was able to bind to ?V?3 integrins, whereas this binding was not inhibited by small RGD peptides. In addition to the IL-32/integrin interactions, we observed that IL-32 is also able to interact with intracellular proteins that are involved in integrin and focal adhesion signaling. Modeling of IL-32 revealed a distinct ?-helix protein resembling the focal adhesion targeting region of focal adhesion kinase (FAK). Inhibition of FAK resulted in modulation of the IL-32?- or IL-32?-induced cytotoxicity. Interestingly, IL-32? binds to paxillin without the RGD motif being involved. Finally, FAK inhibited IL-32?/paxillin binding, whereas FAK also could interact with IL-32?, demonstrating that IL-32 is a member of the focal adhesion protein complex. This study demonstrates for the first time that IL-32 binds to the extracellular domain of integrins and to intracellular proteins like paxillin and FAK, suggesting a dual role for IL-32 in integrin signaling.

Heinhuis, Bas; Koenders, Marije I.; van den Berg, Wim B.; Netea, Mihai G.; Dinarello, Charles A.; Joosten, Leo A. B.

2012-01-01

259

Interleukin 32 (IL-32) contains a typical ?-helix bundle structure that resembles focal adhesion targeting region of focal adhesion kinase-1.  

PubMed

IL-32 can be expressed in several isoforms. The amino acid sequences of the major IL-32 isoforms were used to predict the secondary and tertiary protein structure by I-TASSER software. The secondary protein structure revealed coils and ?-helixes, but no ? sheets. Furthermore, IL-32 contains an RGD motif, which potentially activates procaspase-3 intracellular and or binds to integrins. Mutation of the RGD motif did not result in inhibition of the IL-32?- or IL-32?-induced cytotoxicity mediated through caspase-3. Although IL-32? interacted with the extracellular part of ?V?3 and ?V?6 integrins, only the ?V?3 binding was inhibited by small RGD peptides. Additionally, IL-32? was able to bind to ?V?3 integrins, whereas this binding was not inhibited by small RGD peptides. In addition to the IL-32/integrin interactions, we observed that IL-32 is also able to interact with intracellular proteins that are involved in integrin and focal adhesion signaling. Modeling of IL-32 revealed a distinct ?-helix protein resembling the focal adhesion targeting region of focal adhesion kinase (FAK). Inhibition of FAK resulted in modulation of the IL-32?- or IL-32?-induced cytotoxicity. Interestingly, IL-32? binds to paxillin without the RGD motif being involved. Finally, FAK inhibited IL-32?/paxillin binding, whereas FAK also could interact with IL-32?, demonstrating that IL-32 is a member of the focal adhesion protein complex. This study demonstrates for the first time that IL-32 binds to the extracellular domain of integrins and to intracellular proteins like paxillin and FAK, suggesting a dual role for IL-32 in integrin signaling. PMID:22203669

Heinhuis, Bas; Koenders, Marije I; van den Berg, Wim B; Netea, Mihai G; Dinarello, Charles A; Joosten, Leo A B

2011-12-27

260

Translocation of ?-helix chains through a nanopore  

NASA Astrophysics Data System (ADS)

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

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

2010-10-01

261

Population genetic structure in a human-disturbed environment: a case study in the land snail Helix aspersa (Gastropoda: Pulmonata).  

PubMed

Local patterns of genetic variation were analysed in the land snail Helix aspersa for 32 populations sampled within a patchy agricultural landscape: the polders of the Bay of Mont-Saint-Michel (France). This investigation examined the allele frequencies at four enzymatic markers and five microsatellite loci through the genotyping of 580 individuals. A strongly significant population genetic substructuring (mean F(ST)=0.088, P<0.001) was found at the scale of the whole polders area (3050 ha) and both categories of markers displayed a similar magnitude of spatial genetic differentiation. We did not find any obvious effects of habitat fragmentation on the distribution of genetic variability. Despite the reality of habitat patchiness and environmental instability (related to farming practices), an isolation by distance process was clearly depicted, although selective pressures cannot be ruled out for one enzymatic locus. Overall, genetic drift, along with occasional long-distance episodes of gene flow, was presumably the most likely evolutionary force that shaped the observed pattern of genetic variation. PMID:12764420

Arnaud, J-F; Madec, L; Guiller, A; Deunff, J

2003-06-01

262

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

PubMed Central

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

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

2010-01-01

263

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

264

Addition of side-chain interactions to 3(10)-helix/coil and alpha-helix/3(10)-helix/coil theory.  

PubMed Central

An increasing number of experimental and theoretical studies have demonstrated the importance of the 3(10)-helix/ alpha-helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side-chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the alpha-helix, will favor the alpha-helix over 3(10), while an i, i + 4 repulsion will favor the 3(10)-helix over alpha. To quantify the 3(10)/alpha/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 3(10)-helix/coil and 3(10)-helix/alpha-helix/ coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side-chain interaction energies to the models. The theory is based on classifying residues into alpha-helical, 3(10)-helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side-chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side-chain interactions favor alpha-helix formation, while the 3(10)-helix population is maximized when weaker i, i + 4 side-chain interactions are present.

Sun, J. K.; Doig, A. J.

1998-01-01

265

Microfibrillar structure of PGG-glucan in aqueous solution as triple-helix aggregates by small angle x-ray scattering.  

PubMed

The conformation of polysaccharide PGG-Glucan, isolated from yeast cell walls, in aqueous solution was investigated by small angle x-ray scattering (SAXS) and multidetector gel permeation chromatography coupled with postcolumn delivery (GPC/PCD) techniques in comparison with scleroglucan. It was shown that both polysaccharides exhibit a rigid rod-like conformation in aqueous solution by SAXS experiments. The mass per unit length (M/L) and radius (R) of rod cross section of PGG-Glucan were measured to be 6300 daltons/nm and 1.89 nm, while those of scleroglucan are 2300 and 0.83, respectively. Utilizing a GPC/light scattering technique, the average aggregation number of PGG-Glucan is 9, while that of scleroglucan is around 3. From the comparison of the M/L and R of the respective rod cross sections as well as their aggregation number data, it is concluded that PGG-Glucan is composed of triple helices, which tend to aggregate as triplets in solution, whereas scleroglucan is composed of a single triple helix. The aggregation number distribution of PGG-Glucan was found to range from 1 to about 25 determined by GPC/PCD. From the observation of a Debye-Scherrer ring type of peak in the macroscopic scattering cross section of PGG-Glucan by SAXS, the existence of a small amount of ordered clusters of PGG-Glucan can be deduced. The "lattice parameter" of these ordered fasces-like clusters is consistent with the radius of the individual triple-helical rods forming a microfibrillar superstructure. These results indicate that higher aggregated forms of PGG-Glucan containing up to 8 triple helices behave as ordered fasces-like clusters. We conclude that PGG-Glucan is triple-helix aggregates formed by rigid rods stacking together side by side. We propose a molecular structural model for PGG-Glucan conformations. PMID:10508959

Gawronski, M; Park, J T; Magee, A S; Conrad, H

1999-11-01

266

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

SciTech Connect

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

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

2008-01-01

267

The high resolution crystal structure of the human tumor suppressor maspin reveals a novel conformational switch in the G-helix.  

PubMed

Maspin is a serpin that acts as a tumor suppressor in a range of human cancers, including tumors of the breast and lung. Maspin is crucial for development, because homozygous loss of the gene is lethal; however, the precise physiological role of the molecule is unclear. To gain insight into the function of human maspin, we have determined its crystal structure in two similar, but non-isomorphous crystal forms, to 2.1- and 2.8-A resolution, respectively. The structure reveals that maspin adopts the native serpin fold in which the reactive center loop is expelled fully from the A beta-sheet, makes minimal contacts with the core of the molecule, and exhibits a high degree of flexibility. A buried salt bridge unique to maspin orthologues causes an unusual bulge in the region around the D and E alpha-helices, an area of the molecule demonstrated in other serpins to be important for cofactor recognition. Strikingly, the structural data reveal that maspin is able to undergo conformational change in and around the G alpha-helix, switching between an open and a closed form. This change dictates the electrostatic character of a putative cofactor binding surface and highlights this region as a likely determinant of maspin function. The high resolution crystal structure of maspin provides a detailed molecular framework to elucidate the mechanism of function of this important tumor suppressor. PMID:15760906

Law, Ruby H P; Irving, James A; Buckle, Ashley M; Ruzyla, Katya; Buzza, Marguerite; Bashtannyk-Puhalovich, Tanya A; Beddoe, Travis C; Nguyen, Kim; Worrall, D Margaret; Bottomley, Stephen P; Bird, Phillip I; Rossjohn, Jamie; Whisstock, James C

2005-03-10

268

Structural determinants of nitroxide motion in spin-labeled proteins: Solvent-exposed sites in helix B of T4 lysozyme  

PubMed Central

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

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

2008-01-01

269

A Small Nonerythropoietic Helix B Surface Peptide Based upon Erythropoietin Structure Is Cardioprotective against Ischemic Myocardial Damage  

PubMed Central

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

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

2011-01-01

270

HELANAL: a program to characterize helix geometry in proteins.  

PubMed

A detailed analysis of structural and position dependent characteristic features of helices will give a better understanding of the secondary structure formation in globular proteins. Here we describe an algorithm that quantifies the geometry of helices in proteins on the basis of their C alpha atoms alone. The Fortran program HELANAL can extract the helices from the PDB files and then characterises the overall geometry of each helix as being linear, curved or kinked, in terms of its local structural features, viz. local helical twist and rise, virtual torsion angle, local helix origins and bending angles between successive local helix axes. Even helices with large radius of curvature are unambiguously identified as being linear or curved. The program can also be used to differentiate a kinked helix and other motifs, such as helix-loop-helix or a helix-turn-helix (with a single residue linker) with the help of local bending angles. In addition to these, the program can also be used to characterise the helix start and end as well as other types of secondary structures. PMID:10798526

Bansal, M; Kumar, S; Velavan, R

2000-04-01

271

The orientation of helix 4 in apolipoprotein A-I-containing reconstituted high density lipoproteins.  

PubMed

The three-dimensional structure of the high density lipoprotein (HDL) component apolipoprotein (apo) A-I and the molecular basis for its protection against coronary artery disease are unknown. In terms of discoidal HDL particles, there has been a debate as to the orientation of the apoA-I alpha-helices around the disc edge. The "picket fence" model states that the alpha-helical repeats, separated by turns, are arranged parallel to the phospholipid acyl chains of the enclosed lipid bilayer. On the other hand, the "belt" model states that the helical segments run perpendicular to the acyl chains. To distinguish between these models, we used nitroxide spin labels present at various depths in the bilayer of reconstituted HDL (rHDL) to measure the position of Trp residues in single Trp mutants of human proapoA-I. Two mutants were studied; the first contained a Trp at position 108, which was located near the center of helix 4. The second contained a Trp at position 115, two turns along the same helix. The picket fence model predicts that these Trp residues should be at different depths in the bilayer, whereas the belt model predicts that they should be at similar depths. Different sized rHDL particles were produced that contained 2, 3, and >4 molecules of proapoA-I per complex. In each case, parallax analysis indicated that Trp-108 and Trp-115 were present at similar depths of about 6 A from the center of the bilayer, consistent with helix 4 being oriented perpendicular to the acyl chains (in agreement with the belt model). Similar experiments showed that control transmembrane peptides were oriented parallel to the acyl chains in vesicles, demonstrating that the method was capable of distinguishing between the two models. This study provides one of the first experimental measurements of the location of an apoA-I helix with respect to the bilayer edge. PMID:10751383

Maiorano, J N; Davidson, W S

2000-06-01

272

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

PubMed Central

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

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

1994-01-01

273

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

274

Deciphering the folding kinetics of transmembrane helical proteins  

PubMed Central

Nearly a quarter of genomic sequences and almost half of all receptors that are likely to be targets for drug design are integral membrane proteins. Understanding the detailed mechanisms of the folding of membrane proteins is a largely unsolved, key problem in structural biology. Here, we introduce a general model and use computer simulations to study the equilibrium properties and the folding kinetics of a C?-based two-helix bundle fragment (comprised of 66 aa) of bacteriorhodopsin. Various intermediates are identified and their free energy are calculated together with the free energy barrier between them. In 40% of folding trajectories, the folding rate is considerably increased by the presence of nonobligatory intermediates acting as traps. In all cases, a substantial portion of the helices is rapidly formed. This initial stage is followed by a long period of consolidation of the helices accompanied by their correct packing within the membrane. Our results provide the framework for understanding the variety of folding pathways of helical transmembrane proteins.

Orlandini, Enzo; Seno, Flavio; Banavar, Jayanth R.; Laio, Alessandro; Maritan, Amos

2000-01-01

275

Structural Models of the Transmembrane Region of Voltage-Gated and Other K +Channels in Open, Closed, and Inactivated Conformations  

Microsoft Academic Search

A large collaborative, multidisciplinary effort involving many research laboratories continues which uses indirect methods of molecular biology and membrane biophysics to analyze the three-dimensional structures and functional mechanisms of K+channels. This work also extends to the distant relatives of these channels, including the voltage-gated Na+and Ca2+channels. The role that our group plays in this process is to combine the information

Stewart R. Durell; Yili Hao; H. Robert Guy

1998-01-01

276

Double-helix stellarator  

SciTech Connect

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

Moroz, P.E.

1997-09-01

277

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

278

Critical amino acid residues in transmembrane span 7 of the serotonin transporter identified by random mutagenesis.  

PubMed

Transmembrane span 7 of the rat brain serotonin transporter was subjected to random mutagenesis. Of the 27 amino acid residues mutated, six were identified as functionally important by their sensitivity to nonconservative mutations. These residues were Asn-368 and Tyr-385, where substitutions that retained hydrogen-bonding ability were preferred; Gly-376 and Gly-384, where only glycine was accepted; Phe-380, where a phenyl ring was preferred; and Met-386, where hydrophobic substitutions were preferred. Mutations that did not preserve these structural characteristics were highly detrimental to serotonin transport activity. These six residues form a stripe that runs at an angle down the side of the putative alpha-helix, lending support to this structural prediction. Mutations at some of these positions also specifically impaired transport activity under low Na+ conditions. Other mutations at nearby positions in transmembrane span 7 also impaired activity in low Na+, although the activity of the mutants in high Na+ was similar to wild type. These results suggest that at least some of the six critical residues play a role in Na+ binding or perhaps in the coupling of Na+ binding to later steps in the transport cycle. These residues may be important in other aspects of the transporter's function as well. PMID:9774428

Penado, K M; Rudnick, G; Stephan, M M

1998-10-23

279

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

PubMed Central

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

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

2009-01-01

280

Insights into avian influenza virus pathogenicity: the hemagglutinin precursor HA0 of subtype H16 has an alpha-helix structure in its cleavage site with inefficient HA1/HA2 cleavage.  

PubMed

With a new serotype (H17) of hemagglutinin (HA) recently being discovered, there are now 17 serotypes (H1 to H17) of influenza A viruses in total. It is believed that HA is initially expressed as a precursor of HA0 and then cleaved into HA1 and HA2, forming a disulfide bond-linked complex, for its full function. Structural data show that a loop structure exists in the cleavage site between HA1 and HA2, and this flexible loop is crucial for the efficient cleavage of HA0. Here, the crystal structures of H16 (a low-pathogenicity avian influenza virus) in their HA0 form (H16HA0) have been solved at 1.7-? and 2.0-? resolutions. To our surprise, an ?-helix element in the cleavage site which inserts into the negatively charged cavity with the key residue R329 hidden behind the helix was observed. In vitro trypsin cleavage experiments demonstrated inefficient cleavage of H16HA0 under both neutral and low-pH conditions. The results provide new insights into influenza A virus pathogenicity; both the relatively stable ?-helix structure in the flexible cleavage loop and inaccessibility of the cleavage site likely contribute to the low pathogenicity of avian influenza A virus. Furthermore, compared to all of the HAs whose structures have been solved, H16 is a good reference for assigning the HA subtypes into two groups on the basis of the three-dimensional structure, which is consistent with the phylogenetic grouping. We conclude that in light of the current H16HA0 structure, the natural ?-helix element might provide a new opportunity for influenza virus inhibitor design. PMID:22993148

Lu, Xishan; Shi, Yi; Gao, Feng; Xiao, Haixia; Wang, Ming; Qi, Jianxun; Gao, George F

2012-09-19

281

Transmembrane Domain Mutations Influence the Cellular Distribution of Lysosomal Membrane Glycoprotein A  

Microsoft Academic Search

The lgp\\/LAMP family of mammalian and avian lysosomal type I membrane glycoproteins features short, conserved, cytosolic tails that possess lysosomal targeting information. The sequences of the adjacent transmembrane domains are also highly conserved, with six amino acids identical in all sixteen known lgp variants. These six residues are found along one side of a hypothetical alpha-helix that may comprise this

Susan Wimer-Mackin; Bruce L. Granger

1996-01-01

282

Spicula in Helix  

Microsoft Academic Search

WHILST dissecting, a few days ago, a common garden snail (Helix aspersa). I came across two calcareous spicula, lying immediately under the ``albummiparous gland,'' which I cannot find mentioned in any of the text-books. I at once dissected three other snails of the same species, and in two I found no spicula, while in the third I found one lying

Edwd. B. Parfitt

1879-01-01

283

Multiple oligomeric states regulate the DNA binding of helix-loop-helix peptides.  

PubMed Central

To study the protein-protein interactions that allow Id, a negative regulator of cell differentiation, to inhibit the DNA-binding activities of MyoD and E47, we have synthesized peptides corresponding to the helix-loop-helix domains of MyoD, E47, and Id. We show that Id preferentially inhibits the sequence-specific DNA-binding activity of MyoD, a muscle-specific protein, as compared to E47, a more ubiquitous protein. The Id helix-loop-helix domain itself forms stable tetramers, and its inhibitory activity arises from the formation of a heterotetrameric structure with MyoD. The formation of this higher order complex provides a general mechanism by which inhibitory proteins can generate sufficient interaction free energy to overcome the large DNA-binding free energy of dimeric DNA-binding proteins. Images Fig. 2 Fig. 3

Fairman, R; Beran-Steed, R K; Anthony-Cahill, S J; Lear, J D; Stafford, W F; DeGrado, W F; Benfield, P A; Brenner, S L

1993-01-01

284

Reversible ?-helix formation controlled by a hydrogen bond surrogate.  

PubMed

Strategically placed covalent linkages have been shown to stabilize helical conformations in short peptide sequences. Here we report the synthesis of a stabilized ?-helix that utilizes an internal disulfide linkage. Structural analysis indicates that the dynamic nature of the disulfide bridge allows for the reversible formation of an ?-helix through oxidation and reduction reactions. PMID:23144512

Miller, Stephen E; Kallenbach, Neville R; Arora, Paramjit S

2011-12-29

285

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

NASA Astrophysics Data System (ADS)

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

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

2013-09-01

286

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

PubMed Central

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

Ma, Kan; Wang, Kuan

2003-01-01

287

A Combination of Compositional Index and Genetic Algorithm for Predicting Transmembrane Helical Segments  

PubMed Central

Transmembrane helix (TMH) topology prediction is becoming a focal problem in bioinformatics because the structure of TM proteins is difficult to determine using experimental methods. Therefore, methods that can computationally predict the topology of helical membrane proteins are highly desirable. In this paper we introduce TMHindex, a method for detecting TMH segments using only the amino acid sequence information. Each amino acid in a protein sequence is represented by a Compositional Index, which is deduced from a combination of the difference in amino acid occurrences in TMH and non-TMH segments in training protein sequences and the amino acid composition information. Furthermore, a genetic algorithm was employed to find the optimal threshold value for the separation of TMH segments from non-TMH segments. The method successfully predicted 376 out of the 378 TMH segments in a dataset consisting of 70 test protein sequences. The sensitivity and specificity for classifying each amino acid in every protein sequence in the dataset was 0.901 and 0.865, respectively. To assess the generality of TMHindex, we also tested the approach on another standard 73-protein 3D helix dataset. TMHindex correctly predicted 91.8% of proteins based on TM segments. The level of the accuracy achieved using TMHindex in comparison to other recent approaches for predicting the topology of TM proteins is a strong argument in favor of our proposed method. Availability: The datasets, software together with supplementary materials are available at: http://faculty.uaeu.ac.ae/nzaki/TMHindex.htm.

Zaki, Nazar; Bouktif, Salah; Lazarova-Molnar, Sanja

2011-01-01

288

Direct Assessment of the ?-Helix Nucleation Time  

PubMed Central

The nucleation event in ?-helix formation is a fundamental process in protein folding. However, determining how quickly it takes place based on measurements of the relaxation dynamics of helical peptides is difficult because such relaxations invariably contain contributions from various structural transitions such as, from helical to non-helical states and helical to partial-helical conformations. Herein we measure the temperature-jump (T-jump) relaxation kinetics of three model peptides that fold into a single-turn ?-helix, using time-resolved infrared spectroscopy, aiming to provide a direct assessment of the helix nucleation rate. The ?-helical structure of these peptides is stabilized by a covalent cross-linker formed between the sidechains of two residues at the i and i+4 positions. If we assume that this cross-linker mimics the structural constraint arising from a strong sidechain-sidechain interaction (e.g., a salt-bridge) in proteins, these peptides would represent good models for studying the nucleation process of an ?-helix in a protein environment. Indeed, we find that the T-jump induced relaxation rate of these peptides is approximately (0.6 ?s)?1 at room temperature, which is slower than that of commonly studied alanine-based helical peptides but faster than that of a naturally occurring ?-helix whose folded state is stabilized by a series of sidechain-sidechain interactions. Taken together, our results put an upper limit of about 1 ?s for the helix nucleation time at 20 °C and suggest that the subsequent propagation steps occur with a time constant of about 240 ns.

Serrano, Arnaldo L.; Tucker, Matthew J.; Gai, Feng

2011-01-01

289

Direct assessment of the ?-helix nucleation time.  

PubMed

The nucleation event in ?-helix formation is a fundamental process in protein folding. However, determining how quickly it takes place based on measurements of the relaxation dynamics of helical peptides is difficult because such relaxations invariably contain contributions from various structural transitions such as from helical to nonhelical states and helical to partial-helical conformations. Herein, we measure the temperature-jump (T-jump) relaxation kinetics of three model peptides that fold into a single-turn ?-helix, using time-resolved infrared spectroscopy, aiming to provide a direct assessment of the helix nucleation rate. The ?-helical structure of these peptides is stabilized by a covalent cross-linker formed between the side chains of two residues at the i and i + 4 positions. If we assume that this cross-linker mimics the structural constraint arising from a strong side chain-side chain interaction (e.g., a salt bridge) in proteins, these peptides would represent good models for studying the nucleation process of an ?-helix in a protein environment. Indeed, we find that the T-jump induced relaxation rate of these peptides is approximately (0.6 ?s)(-1) at room temperature, which is slower than that of commonly studied alanine-based helical peptides but faster than that of a naturally occurring ?-helix whose folded state is stabilized by a series of side chain-side chain interactions. Taken together, our results put an upper limit of about 1 ?s for the helix nucleation time at 20 °C and suggest that the subsequent propagation steps occur with a time constant of about 240 ns. PMID:21568273

Serrano, Arnaldo L; Tucker, Matthew J; Gai, Feng

2011-05-13

290

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.

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

2013-01-01

291

STRUCTURE OF THE LA MOTIF: A WINGED HELIX DOMAIN MEDIATES RNA BINDING VIA A CONSERVED AROMATIC PATCH  

Microsoft Academic Search

The La protein is a ubiquitous nuclear phosphoprotein that recognizes the 30 uridylates found in all newly synthe- sized RNA polymerase III transcripts. La binding stabilizes these transcripts from exonucleases and may also assist their folding. Here we present the first structural insights into how the La protein specifically interacts with its RNA substrates. The most conserved region of the

Gang Dong; Ghadiyaram Chakshusmathi; Sandra L Wolin; Karin M Reinisch

2004-01-01

292

Structural integrity of {alpha}-helix H12 in translation initiation factor eIF5B is critical for 80S complex stability.  

PubMed

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-tRNA(i)(Met) 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-tRNA(i)(Met) is bound and that helix H12 rigidity is required to stabilize Met-tRNA(i)(Met) binding. PMID:21335519

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

2011-02-18

293

GxxxG Motifs, Phenylalanine, and Cholesterol Guide the Self-Association of Transmembrane Domains of ErbB2 Receptors  

PubMed Central

GxxxG motifs are common in transmembrane domains of membrane proteins and are often introduced to artificial peptides to inhibit or promote association to stable structures. The transmembrane domain of ErbB2 presents two separate such motifs that are proposed to be connected to stability and activity of the dimer. Using molecular simulations, we show that these sequences play a critical role during the recognition stage, forming transient complexes that lead to stable dimers. In pure phospholipid bilayers association occurs by contacts formed at the C-terminus promoted by the presence of phenylalanine residues. Helices subsequently rotate to eventually pack at short separations favored by lipid entropic contributions. In contrast, at intermediate cholesterol concentrations, a different pathway is followed that involves dimers with a weaker interface toward the N-terminus. However, at high cholesterol content, a switch toward the C-terminus is observed with an overall nonmonotonic change of the dimerization affinity. This conformational switch modulated by cholesterol has important implications on the thermodynamic, structural, and kinetic characteristics of helix-helix association in lipid membranes.

Prakash, Anupam; Janosi, Lorant; Doxastakis, Manolis

2011-01-01

294

The Solution Structure of Human Mitochondria Fission Protein Fis1 Reveals a Novel TPR-like Helix Bundle  

Microsoft Academic Search

Fis1 in yeast localizes to the outer mitochondrial membrane and facilitates mitochondrial fission by forming protein complexes with Dnm1 and Mdv1. Fis1 orthologs exist in higher eukaryotes, suggesting that they are functionally conserved. In the present study, we cloned the human Fis1 ortholog that was predicted in a database, and determined the protein structure using NMR spectroscopy. Following a flexible

Motoshi Suzuki; Seon-Yong Jeong; Mariusz Karbowski; Richard J. Youle; Nico Tjandra

2003-01-01

295

Six-Helix and Eight-Helix DNA Nanotubes Assembled from Half-Tubes  

PubMed Central

DNA nanotubes are cylinder-like structures formed from DNA double helical molecules whose helix axes are fused at least twice by crossovers. It is potentially useful to use such tubes as sheaths around rod-like species that arise in biological systems and in nanotechnology. It seems easiest to obtain such sheathing by joining two or more components around an object, rather than attempting to thread the object through a cavity in the tube. We report two examples of tubes containing a specific number of helices that are assembled from half-tube components. These tubes are a six-helix bundle and an eight-helix bundle, constructed respectively from a two bent triple crossover (BTX) molecules and from two 4-helix arched motifs. Both species contain single strands in one molecule that are missing in its mate. The six-helix bundle is formed from two different BTX molecules, whereas the 8-helix species is a closed cyclic dimer of the same molecule. We demonstrate the formation of these species by gel electrophoresis, and we examine their arrangement into long one-dimensional arrays by means of atomic force microscopy.

Kuzuya, Akinori; Wang, Risheng; Sha, Ruojie; Seeman, Nadrian C.

2008-01-01

296

Helix advancement meatoplasty.  

PubMed

We describe a new technique of helix advancement meatoplasty. This technique is useful in both mastoid surgery and some cases of otitis externa. The technique is designed to avoid the problems of (1) inferior positioning of the meatoplasty at the time of surgery, and (2) later inferior migration of the pinna (as can occur when the suspensory ligaments of the pinna have been cut or weakened). Such outcomes can result in a mastoid cavity which is difficult to clean as the approach to it is awkward; in such cases, it is common to have to look up into the cavity rather than directly into it. Helix advancement meatoplasty improves post-operative visualisation and aeration. It eases cleaning of the cavity by creating a more superiorly placed meatoplasty, which is supported by the tragus and is therefore less likely to drop. PMID:22643206

Goodyear, P W A; Reddy, C E; Lesser, T H J

2012-06-01

297

Influence of proline on the thermostability of the active site and membrane arrangement of transmembrane proteins.  

PubMed

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

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

2008-07-25

298

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.

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

1999-01-01

299

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.

Heddad, Mounia; Adamska, Iwona

2000-01-01

300

Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1).  

PubMed Central

We have examined the functional co-assembly of non-complementary pairs of N- and C-terminal polypeptide fragments of the anion transport domain (b3mem) of human red-cell band 3. cDNA clones encoding non-contiguous pairs of fragments with one transmembrane (TM) region omitted, or overlapping pairs of fragments with between one and ten TM regions duplicated, were co-expressed in Xenopus oocytes and a cell-free translation system. Stilbene disulphonate-sensitive chloride uptake assays in oocytes revealed that the omission of any single TM region of b3mem except spans 6 and 7 caused a complete loss of functional expression. In contrast, co-expressed pairs of fragments overlapping a single TM region 5, 6, 7, 8, 9-10 or 11-12 retained a high level of functionality, whereas fragments overlapping the clusters of TM regions 2-5, 4-5, 5-8 and 8-10 also mediated some stilbene disulphonate-sensitive uptake. The co-assembly of N- or C-terminal fragments with intact band 3, b3mem or other fragments was examined by co-immunoprecipitation in non-denaturing detergent solutions by using monoclonal antibodies against the termini of b3mem. All the fragments, except for TM spans 13-14, co-immunoprecipitated with b3mem. The medium-sized N-terminal fragments comprising spans 1-6, 1-7 or 1-8 co-immunoprecipitated particularly strongly with the C-terminal fragments containing spans 8-14 or 9-14. The fragments comprising spans 1-4 or 1-12 co-immunoprecipitated less extensively than the other N-terminal fragments with either b3mem or C-terminal fragments. There is sufficient flexibility in the structure of b3mem to allow the inclusion of at least one duplicated TM span without a loss of function. We propose a working model for the organization of TM spans of dimeric band 3 based on current evidence.

Groves, J D; Tanner, M J

1999-01-01

301

Crystallizing Transmembrane Peptides in Lipidic Mesophases  

SciTech Connect

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

Höfer, Nicole; Aragão, David; Caffrey, Martin (Trinity)

2011-09-28

302

Energetic Frustration of Apomyoglobin Folding: Role of the B Helix  

PubMed Central

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

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

2010-01-01

303

NMR Structures of the Second Transmembrane Domain of the Human Glycine Receptor ? 1 Subunit: Model of Pore Architecture and Channel Gating  

Microsoft Academic Search

Glycine receptors (GlyR) are the primary inhibitory receptors in the spinal cord and belong to a superfamily of ligand-gated ion channels (LGICs) that are extremely sensitive to low-affinity neurological agents such as general anesthetics and alcohols. The high-resolution pore architecture and the gating mechanism of this superfamily, however, remain unclear. The pore-lining second transmembrane (TM2) segments of the GlyR ?1

Pei Tang; Pravat K. Mandal; Yan Xu

2002-01-01

304

The conserved third transmembrane segment of YidC contacts nascent Escherichia coli inner membrane proteins.  

PubMed

Escherichia coli YidC is a polytopic inner membrane protein that plays an essential and versatile role in the biogenesis of inner membrane proteins. YidC functions in Sec-dependent membrane insertion but acts also independently as a separate insertase for certain small membrane proteins. We have used a site-specific cross-linking approach to show that the conserved third transmembrane segment of YidC contacts the transmembrane domains of both nascent Sec-dependent and -independent substrates, indicating a generic recognition of insertion intermediates by YidC. Our data suggest that specific residues of the third YidC transmembrane segment alpha-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis. PMID:18840604

Yu, Zhong; Koningstein, Gregory; Pop, Ana; Luirink, Joen

2008-10-06

305

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

306

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

307

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

PubMed Central

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

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

2007-01-01

308

Functional characterization of amphipathic ?-helix in the osmoregulatory ABC transporter OpuA.  

PubMed

The ATP-binding-cassette transporter OpuA from Lactococcus lactis is composed of two ATPase subunits (OpuAA) and two subunits (OpuABC) with the transmembrane domain fused to an extracellular substrate-binding protein. Of the almost 1900 homologues of OpuA known to date, a subset has an amino-terminal amphipathic helix (plus extra transmembrane segment) fused to the core of the transmembrane domain of the OpuABC subunit. FRET measurements indicate that the amphipathic ?-helix is located close to the membrane surface, where its hydrophobic face interacts with the transport protein rather than the membrane lipids. Next, we determined the functional role of this accessory region by engineering the amphipathic ?-helix. We analyzed the consequence of the mutations in intact cells by monitoring growth and transport of glycine betaine under normal and osmotic stress conditions. More detailed studies were performed in hybrid membrane vesicles, proteoliposomes, and bilayer nanodisks. We show that the amphipathic ?-helix of OpuA is necessary for high activity of OpuA but is not critical for the biogenesis of the protein or the ionic regulation of transport. PMID:22656643

Gul, Nadia; Schuurman-Wolters, Gea; Karasawa, Akira; Poolman, Bert

2012-06-13

309

Analysis and prediction of helix shift errors in homology modeling.  

PubMed

High sequence identity between two proteins (e.g. > 60%) is a strong evidence for high structural similarity. However, internal shifts in one of the two proteins can sometimes give rise to unexpectedly high structural differences. This, in turn, causes unreliable structure predictions when two such proteins are used in homology modeling. Here, we perform a computational analysis of helix shifts and we show that their occurrence can be predicted with statistical learning methods. Our results indicate that helix shifts increase the RMS error by factor 2.6 compared to those protein pairs without a helix shift. Although helix shifts are rare (1.6% of helices and a commensurately higher number of proteins are affected), they therefore pose a significant problem for reliable structure prediction systems. In this paper, we prototype a new approach for model quality assessment and demonstrate that it can successfully warn against helix shifts. A support vector machine trained on a wide range of sequence and structure properties predicts the occurrence of helix shifts with a sensitivity of 74.2% and a specificity of 83.6%. On an equalized test dataset, this corresponds to an accuracy of 78.9%. Projected to the full dataset, it translates to an accuracy of 83.4%. Our analysis shows that helix shift detection is a valuable building block for highly reliable structure prediction systems. Furthermore, the statistical learning based approach to helix shift detection that we employ here is orthogonal to well-established model quality assessment methods (which use geometric constraint checking or mean force potentials). Therefore, a further increase of prediction accuracy is expected from the combination of these methods. PMID:16789920

Bock, Christoph; Hesser, Jürgen

2006-01-01

310

Comparative Analysis of the Heptahelical Transmembrane Bundles of G Protein-Coupled Receptors  

PubMed Central

Background G protein-coupled receptors represent a large family of eukaryotic membrane proteins, and are involved in almost all physiological processes in humans. Recent advances in the crystallographic study of these receptors enable a detailed comparative analysis of the commonly shared heptahelical transmembrane bundle. Systematic comparison of the bundles from a variety of receptors is indispensable for understanding not only of the structural diversification optimized for the binding of respective ligands but also of the structural conservation required for the common mechanism of activation accompanying the interaction changes among the seven helices. Methodology/Principal Findings We have examined the bundles of 94 polypeptide chains from almost all available structures of 11 receptors, which we classified into either inactivated chain or activated chain, based on the type of bound ligand. For the inactivated chains, superposition of 200 residue bundles by secondary structure matching demonstrated that the bound ligands share a laterally limited cavity in the extracellular section of the bundle. Furthermore, a distinct feature was found for helix III of bovine rhodopsin, which might have evolved to lower its activity in the presence of 11-cis-retinal, to a level that other receptors could hardly achieve with any currently available ligands. Conclusions/Significance Systematic analysis described here would be valuable for understanding of the rearrangement of seven helices which depends on the ligand specificity and activation state of the receptors.

Okada, Tetsuji

2012-01-01

311

Chemical Modifications of Triple Helix Forming Oligonucleotides  

Microsoft Academic Search

\\u000a Ever since its initial discovery, the triple-helix structure (1), because of possible applications in biotechnology, diagnostics, and therapeutics, has attracted considerable attention\\u000a (2–5). It has been demonstrated that homopurine-homopyrimidine tracts of DNA can be targeted by third strand oligonucleotides\\u000a which bind to the major groove of DNA, and held in place to purine bases by specific hydrogen bonds. A homopyrimidine

Ulysse Asseline

312

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

PubMed Central

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

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

2012-01-01

313

The transmembrane domain of CEACAM1-4S is a determinant of anchorage independent growth and tumorigenicity.  

PubMed

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

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

2012-01-03

314

?-Helix nucleation by a calcium-binding peptide loop  

PubMed Central

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

Siedlecka, Monika; Goch, Grazyna; Ejchart, Andrzej; Sticht, Heinrich; Bierzynski, Andrzej

1999-01-01

315

Metallic helix array as a broadband wave plate.  

PubMed

This study demonstrates theoretically and experimentally that a metallic helix array can operate as a highly transparent broadband wave plate in propagation directions perpendicular to the axis of helices. The functionality arises from a special property of the helix array, namely, that two branches of elliptically right-handed and left-handed polarized states are nearly rigidly shifted in frequency and their dispersions are controlled by different mechanisms that can be independently tuned by structural parameters. PMID:22107579

Wu, Chao; Li, Hongqiang; Yu, Xing; Li, Fang; Chen, Hong; Chan, C T

2011-10-18

316

Four complete turns of a curved 3??-helix at atomic resolution: the crystal structure of the peptaibol trichovirin I-4A in a polar environment suggests a transition to ?-helix for membrane function.  

PubMed

The first crystal structure of a member of peptaibol antibiotic subfamily 4, trichovirin I-4A (14 residues), has been determined by direct methods and refined at atomic resolution. The monoclinic unit cell has two molecules in the asymmetric unit. Both molecules assume a 3?? right-handed helical conformation and are significantly bent. The molecules pack loosely along the crystallographic twofold axis, forming two large tunnels between symmetry-related molecules in which no ordered solvent could be located. Carbonyl O atoms which are not involved in intramolecular hydrogen bonding participate in close van der Waals interactions with apolar groups. The necessary amphipathicity for biological activity of peptaibols is not realised in the crystal structure. Hence, a structural change of trichovirin to an ?-helical conformation is proposed for membrane integration and efficient water/ion transportation across the lipid bilayer. PMID:22281739

Gessmann, Renate; Axford, Danny; Owen, Robin L; Brückner, Hans; Petratos, Kyriacos

2012-01-06

317

Differentiation of Arabidopsis guard cells: analysis of the networks incorporating the basic helix-loop-helix transcription factor, FAMA.  

PubMed

Nearly all extant land plants possess stomata, the epidermal structures that mediate gas exchange between the plant and the environment. The developmental pathways, cell division patterns, and molecules employed in the generation of these structures are simple examples of processes used in many developmental contexts. One specific module is a set of "master regulator" basic helix-loop-helix transcription factors that regulate individual consecutive steps in stomatal development. Here, we profile transcriptional changes in response to inducible expression of Arabidopsis (Arabidopsis thaliana) FAMA, a basic helix-loop-helix protein whose actions during the final stage in stomatal development regulate both cell division and cell fate. Genes identified by microarray and candidate approaches were then further analyzed to test specific hypothesis about the activity of FAMA, the shape of its regulatory network, and to create a new set of stomata-specific or stomata-enriched reporters. PMID:21245191

Hachez, Charles; Ohashi-Ito, Kyoko; Dong, Juan; Bergmann, Dominique C

2011-01-18

318

Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment.  

PubMed

MN is a transmembrane glycoprotein that has been detected in HeLa cells and in some human carcinomas. The expression of MN protein in HeLa cells is regulated by cell density. In HeLa x fibroblast cell hybrids its expression correlates with tumorigenicity. Using a specific monoclonal antibody we have identified a cDNA clone coding for MN. Analysis of the deduced amino acid sequence revealed strong structural homology between the central region of the MN protein and carbonic anhydrases (CA). MN sequence retains the conserved zinc-binding site as well as the enzyme's active center. In accord with these findings, MN protein from HeLa cells was found to bind zinc and to have carbonic anhydrase activity. The N-terminal region of MN shares some similarity with DNA binding proteins of the helix-loop-helix (HLH) family, and the protein was found to have affinity for DNA by DNA-cellulose chromatography. The region between the CA-like domain and the putative HLH domain is rich in imperfect repeats of serine, proline, glycine and acidic residues with few hydrophobic amino acids, resembling thus an activation region of transcription factors. The fact that MN protein is detectable in several types of human carcinomas, but not in corresponding non-cancerous tissues, suggests its possible role in neoplasia. In addition, the analysis of biological consequences of MN expression of NIH3T3 cells provides the evidence in favour of MN protein involvement in control of cell proliferation and transformation. PMID:8084592

Pastorek, J; Pastoreková, S; Callebaut, I; Mornon, J P; Zelník, V; Opavský, R; Zat'ovicová, M; Liao, S; Portetelle, D; Stanbridge, E J

1994-10-01

319

Automatic ?-helix identification in Patterson maps.  

PubMed

?-Helices are peculiar atomic arrangements characterizing protein structures. Their occurrence can be used within crystallographic methods as minimal a priori information to drive the phasing process towards solution. Recently, brute-force methods have been developed which search for all possible positions of ?-helices in the crystal cell by molecular replacement and explore all of them systematically. Knowing the ?-helix orientations in advance would be a great advantage for this kind of approach. For this purpose, a fully automatic procedure to find ?-helix orientations within the Patterson map has been developed. The method is based on Fourier techniques specifically addressed to the identification of helical shapes and operating on Patterson maps described in spherical coordinates. It supplies a list of candidate orientations, which are then refined by using a figure of merit based on a rotation function calculated for a template polyalanine helix oriented along the current direction. The orientation search algorithm has been optimized to work at 3 Å resolution, while the candidates are refined against all measured reflections. The procedure has been applied to a large number of protein test structures, showing an overall efficiency of 77% in finding ?-helix orientations, which decreases to 48% on limiting the number of candidate solutions (to 13 on average). The information obtained may be used in many aspects in the framework of molecular-replacement phasing, as well as to constrain the generation of models in computational modelling programs. The procedure will be accessible through the next release of IL MILIONE and could be decisive in the solution of new unknown structures. PMID:22194328

Caliandro, Rocco; Dibenedetto, Domenica; Cascarano, Giovanni Luca; Mazzone, Annamaria; Nico, Giovanni

2011-12-09

320

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

Microsoft Academic Search

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

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

2003-01-01

321

The Drosophila Pipsqueak protein defines a new family of helix-turn-helix DNA-binding proteins  

Microsoft Academic Search

Many prokaryotic and eukaryotic DNA-binding proteins use a helix-turn-helix (HTH) structure for DNA recognition. Here we describe a new family of eukaryotic HTH proteins, the Pipsqueak (Psq) family, which includes proteins from fungi, sea urchins, nematodes, insects, and vertebrates. Three subgroups of the Psq family can be distinguished. Like the HTH proteins of the prokaryotic resolvase family, members of the

Thomas Siegmund; Michael Lehmann

2002-01-01

322

Nhlh1 , a basic helix-loop-helix transcription factor, is very tightly linked to the mouse looptail ( Lp ) mutation  

Microsoft Academic Search

Looptail (Lp) is a mutation on the distal portion of mouse Chromosome (Chr) 1 that affects neurulation in mouse and is phenotypically expressed by appearance of an open neural tube along the entire antero-posterior axis of the embryo (craniorachischisis). Nhlh1, a member of the basic helix-loop-helix family of transcription factors, is expressed in the developing neural tube in structures affected

A. Mullick; N. Groulx; D. Trasler; P. Gros

1995-01-01

323

Structural characterization reveals that viperin is a radical S-adenosyl- l-methionine (SAM) enzyme  

Microsoft Academic Search

Viperin is an interferon-inducible protein inhibiting many DNA and RNA viruses. It contains an N-terminal transmembrane helix, a highly conserved C-terminus and a middle region carrying a CX3CX2C motif, characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. So far no structural characterization has been reported and reconstitution of the [4Fe–4S] cluster in viperin all failed. Here, by dissecting the 361-residue human viperin

Goyal Shaveta; Jiahai Shi; Vincent T. K. Chow; Jianxing Song

2010-01-01

324

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

PubMed

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

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

2009-10-09

325

Penetration of Lipid Chains into Transmembrane Surfaces of Membrane Proteins: Studies with MscL  

PubMed Central

The transmembrane surface of a multi-helix membrane protein will be rough with cavities of various sizes between the transmembrane ?-helices. Efficient solvation of the surface by the lipid molecules that surround the protein in a membrane requires that the lipid fatty acyl chains be able to enter the cavities. This possibility has been investigated using fluorescence quenching methods. Trp residues have been introduced into lipid-facing sites in the first transmembrane ?-helix (M1) of the mechanosensitive channel of large-conductance MscL; lipid-facing residues at the N-terminal end of M1 are buried below the transmembrane surface of the protein. Fluorescence emission maxima for lipid-facing Trp residues in M1 vary with position in the bilayer comparably to those for Trp residues in the second transmembrane ?-helix (M2) despite the fact that lipid-facing residues in M2 are on the surface of the protein. Fluorescence emission spectra for most Trp residues on the periplasmic sides of M1 and M2 fit well to a model proposing a trough-like variation of dielectric constant across the membrane, but the relationship between location and fluorescence emission maximum on the cytoplasmic side of the membrane is more complex. The fluorescence of Trp residues in M1 is quenched efficiently by phospholipids with bromine-containing fatty acyl chains, showing that the lipid chains must be able to enter the Trp-containing cavities on the surface of MscL, resulting in efficient solvation of the surface.

Carney, Joanne; East, J. Malcolm; Lee, Anthony G.

2007-01-01

326

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

PubMed Central

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

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

1996-01-01

327

Reduction of membrane protein hydrophobicity by site-directed mutagenesis: introduction of multiple polar residues in helix D of bacteriorhodopsin.  

PubMed

Introduction of polar and charged residues on the lipid-exposed face of transmembrane proteins using site-directed mutagenesis represents a novel approach to render membrane proteins more soluble in aqueous solution. We have sequentially introduced as many as five polar and charged amino acids onto the lipid-exposed face of helix D of bacteriorhodopsin from Halobacterium salinarium. The most polar mutant (Q4D) has four glutamine residues at positions 113, 116, 120 and 124 and an aspartate at position 117. In combination with wild-type residues Gln105, Thr107, Thr121 and Thr128, the Q4D mutant has a nearly uninterrupted stripe of polar residues on the surface of helix D. All of the mutants refold, bind retinal and the resulting pigments exhibit light- and dark-adapted UV and visible spectroscopic properties that are similar to the wild-type pigment, indicating that the secondary, tertiary and active site structures are similar to the wild-type protein. These results demonstrate that micelle-solubilized bacteriorhodopsin can tolerate multiple non-conservative substitution of amino acids that face the non-polar portion of the lipid bilayer in vivo, thus lending credence to the notion of partial or complete solubilization of integral membrane proteins by site-directed mutagenesis. PMID:9464570

Chen, G Q; Gouaux, E

1997-09-01

328

The specificity of interaction of archaeal transducers with their cognate sensory rhodopsins is determined by their transmembrane helices.  

PubMed

Chimeras of the Halobacterium salinarum transducers HtrI and HtrII were constructed to study the structural determinants for their specific interaction with the phototaxis receptors sensory rhodopsins I and II (SRI and SRII), respectively. Interaction of receptors and transducers was assessed by two criteria: phototaxis responses by the cells and transducer-modulation of receptor photochemical reaction kinetics in membranes. Coexpression of HtrI with SRII or HtrII with SRI did not result in interaction by either criterion. Each receptor was coexpressed with chimeric transducers in which various domains of the two transducers were interchanged. The results show that the presence of the two transmembrane helices of HtrI in a chimera is necessary and sufficient for functional transducer complexation with SRI, i.e., for wild-type SRI photoreactions and attractant and 2-photon repellent phototaxis responses. Additionally, a previously demonstrated chaperone-like facilitation of SRI folding or stability by HtrI was shown to depend only on the two transmembrane helices of HtrI in chimeric transducers. Similarly, the two transmembrane helices of HtrII specify interaction with the repellent receptor SRII according to motility analysis and laser-flash spectroscopy. The results support a model in which the membrane domains of the receptor/transducer complexes, consisting of the seven helices of the receptor interacting with the four-helix bundle of the transducer dimer, produce SRI- and SRII-specific signals to the flagellar motor by means of interchangeable cytoplasmic domains. PMID:9927658

Zhang, X N; Zhu, J; Spudich, J L

1999-02-01

329

Time-Resolved EPR Immersion Depth Studies of a Transmembrane Peptide Incorporated into Bicelles  

PubMed Central

The reduction in EPR signal intensity of nitroxide spin-labels by ascorbic acid has been measured as a function of time to investigate the immersion depth of the spin-labeled M2? AChR peptide incorporated into a bicelle system utilizing EPR spectroscopy. The corresponding decay curves of n-DSA (n = 5, 7, 12, and 16) EPR signals have been used to (1) calibrate the depth of the bicelle membrane and (2) establish a calibration curve for measuring the depth of spin-labeled transmembrane peptides. The kinetic EPR data of CLS, n-DSA (n = 5, 7, 12, and 16), and M2? AChR peptide spin-labeled at Glu-1 and Ala-12 revealed excellent exponential and linear fits. For a model M2? AChR peptide, the depth of immersion was calculated to be 5.8 Å and 3 Å for Glu-1, and 21.7 Å and 19 Å for Ala-12 in the gel-phase (298 K) and L?-phases (318 K), respectively. The immersion depth values are consistent with the pitch of an ?–helix and the structural model of M2? AChR incorporated into the bicelle system is in a good agreement with previous studies. Therefore, this EPR time-resolved kinetic technique provides a new reliable method to determine the immersion depth of membrane-bound peptides, as well as, explore the structural characteristics of the M2? AChR peptide.

Nusair, Nisreen A.; Mayo, Daniel J.; Dorozenski, Tia D.; Cardon, Thomas B.; Inbaraj, Johnson J.; Karp, Ethan S.; Newstadt, Justin P.; Grosser, Stuart M.; Lorigan, Gary A.

2011-01-01

330

THE ROLE OF ARRESTIN ?-HELIX I IN RECEPTOR BINDING  

PubMed Central

Arrestins rapidly bind phosphorylated activated forms of their cognate G protein-coupled receptors (GPCRs), thereby preventing G protein coupling and often switching the signaling to other pathways. Amphipathic ?-helix I (residues 100–111) has been implicated in receptor binding, but the mechanism of its action is not yet determined. Here we show that several mutations in the helix itself and adjacent hydrophobic residues in the body of the N-domain reduce arrestin1 binding to phosphorylated light-activated rhodopsin (PRh*). On the background of phosphorylation-independent mutants that bind with high affinity to both P-Rh* and light-activated unphosphorylated rhodopsin (Rh*), these mutations reduce the stability of the arrestin complex with P-Rh*, but not with Rh*. Using site-directed spin labeling we found that the local structure around ? helix I changes upon binding to rhodopsin. However, the intra-molecular distances between ?-helix I and adjacent ?-strand I, or the rest of the N-domain, measured using double electron-electron resonance, do not change, ruling out relocation of the helix due to receptor binding. Collectively, these data demonstrate that ?-helix I plays an indirect role in receptor binding, likely keeping ?-strand I, carrying several phosphate-binding residues, in a position favorable for its interaction with receptor-attached phosphates.

Vishnivetskiy, Sergey A.; Francis, Derek; Van Eps, Ned; Kim, Miyeon; Hanson, Susan M.; Klug, Candice S.; Hubbell, Wayne L.; Gurevich, Vsevolod V.

2009-01-01

331

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.

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

2012-01-01

332

Helix/Coil Nucleation: A Local Response to Global Demands  

PubMed Central

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

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

2009-01-01

333

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

334

Data-driven model for the prediction of protein transmembrane regions  

Microsoft Academic Search

We present a novel approach combining mathematical methods and artificial neural networks to predict the transmembrane regions of transmembrane proteins, considering protein sequence information alone. We have focused on developing a data-driven model based on a non-linear modelling method, the counter-propagation artificial neural network, and on mathematical descriptors defining the sequence information of transmembrane proteins with known three-dimensional structures. The

A. Roy Choudhury; M. Novi?

2009-01-01

335

Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor.  

PubMed Central

The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane alpha-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.

Sun, J.; Ember, J. A.; Chao, T. H.; Fukuoka, Y.; Ye, R. D.; Hugli, T. E.

1999-01-01

336

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

337

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels  

PubMed Central

P2X receptor channels open in response to the binding of extracellular ATP, a property that is essential for purinergic sensory signaling. Apo and ATP-bound X-ray structures of the detergent-solubilized zebrafish P2X4 receptor provide a blueprint for receptor mechanisms but unexpectedly showed large crevices between subunits within the transmembrane (TM) domain of the ATP-bound structure. Here we investigate both intersubunit and intrasubunit interactions between TM helices of P2X receptors in membranes using both computational and functional approaches. Our results suggest that intersubunit crevices found in the TM domain of the ATP-bound crystal structure are not present in membrane-embedded receptors but substantiate helix interactions within individual subunits and identify a hot spot at the internal end of the pore where both the gating and permeation properties of P2X receptors can be tuned. We propose a model for the structure of the open state that has stabilizing intersubunit interactions and that is compatible with available structural constraints from functional channels in membrane environments.

Heymann, Gabriel; Dai, Jian; Silberberg, Shai D.; Zhou, Huan-Xiang; Swartz, Kenton J.

2013-01-01

338

Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels.  

PubMed

P2X receptor channels open in response to the binding of extracellular ATP, a property that is essential for purinergic sensory signaling. Apo and ATP-bound X-ray structures of the detergent-solubilized zebrafish P2X4 receptor provide a blueprint for receptor mechanisms but unexpectedly showed large crevices between subunits within the transmembrane (TM) domain of the ATP-bound structure. Here we investigate both intersubunit and intrasubunit interactions between TM helices of P2X receptors in membranes using both computational and functional approaches. Our results suggest that intersubunit crevices found in the TM domain of the ATP-bound crystal structure are not present in membrane-embedded receptors but substantiate helix interactions within individual subunits and identify a hot spot at the internal end of the pore where both the gating and permeation properties of P2X receptors can be tuned. We propose a model for the structure of the open state that has stabilizing intersubunit interactions and that is compatible with available structural constraints from functional channels in membrane environments. PMID:24082111

Heymann, Gabriel; Dai, Jian; Li, Mufeng; Silberberg, Shai D; Zhou, Huan-Xiang; Swartz, Kenton J

2013-09-30

339

Multi-Tox: Application of the ToxR-transcriptional reporter assay to the study of multi-pass protein transmembrane domain oligomerization  

PubMed Central

ToxR-based transcriptional reporter assays allow the strength of transmembrane helix interactions in biological membranes to be measured. Previously, these assays have only been used to study single-pass transmembrane systems. To facilitate investigation of polytopic transmembrane domain (TMD) oligomerization, we applied the ToxR methodology to the study of multi-pass TMD oligomerization to give ‘Multi-Tox’. Association propensities of the viral oncoprotein, latent membrane protein-1 (LMP-1), and the E. coli membrane-integral diacylglycerol kinase (DAGK) were studied by Multi-Tox, highlighting residues of particular mechanistic importance. Both homo- and hetero-oligomerizations were studied.

Joce, Catherine; Wiener, Alyssa A.; Yin, Hang

2011-01-01

340

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

341

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.

Faingold, Omri; Cohen, Tomer; Shai, Yechiel

2012-01-01

342

Novel Scaffolds for Beta-Helix Mimcry.  

National Technical Information Service (NTIS)

Functionalized pyridazine derivatives having a low molecular weight and pharmaceutical compositions thereof are useful as alpha-helix mimetics and for treating conditions and/or disorders mediated by alpha-helix-binding receptors and proteins.

J. Rebek S. Biros S. Gu

2006-01-01

343

Transmembrane halogen-bonding cascades.  

PubMed

Halogen bonds have recently been introduced as ideal to transport anions across lipid bilayer membranes. However, activities obtained with small transporters were not impressive, and cyclic arrays of strong halogen-bond donors above a calix[4]arene scaffold gave even weaker activities. Here, we report that their linear alignment for anion hopping along transmembrane rigid-rod scaffolds gives excellent activities with an unprecedented cooperativity coefficient m = 3.37. PMID:23517007

Vargas Jentzsch, Andreas; Matile, Stefan

2013-03-26

344

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

PubMed Central

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 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. Images

France, L L; Piatti, P G; Newman, J F; Toth, I; Gibbons, W A; Brown, F

1994-01-01

345

Lac repressor with the helix-turn-helix motif of lambda cro binds to lac operator.  

PubMed Central

Lac repressor, lambda cro protein and their operator complexes are structurally, biochemically and genetically well analysed. Both proteins contain a helix-turn-helix (HTH) motif which they use to bind specifically to their operators. The DNA sequences 5'-GTGA-3' and 5'-TCAC-3' recognized in palindromic lac operator are the same as in lambda operator but their order is inverted form head to head to tail to tail. Different modes of aggregation of the monomers of the two proteins determine the different arrangements of the HTH motifs. Here we show that the HTH motif of lambda cro protein can replace the HTH motif of Lac repressor without changing its specificity. Such hybrid Lac repressor is unstable. It binds in vitro more weakly than Lac repressor but with the same specificity to ideal lac operator. It does not bind to consensus lambda operator. Images

Kolkhof, P; Teichmann, D; Kisters-Woike, B; von Wilcken-Bergmann, B; Muller-Hill, B

1992-01-01

346

A topologically conserved aliphatic residue in alpha-helix 6 stabilizes the hydrophobic core in domain II of glutathione transferases and is a structural determinant for the unfolding pathway.  

PubMed Central

A topologically conserved residue in alpha-helix 6 of domain II of human glutathione transferase (hGST) A1-1 was mutated to investigate its contribution to protein stability and the unfolding pathway. The replacement of Leu-164 with alanine (L164A) did not impact on the functional and gross structural properties of native hGST A1-1. The wild-type protein unfolds via a three-state pathway in which only folded dimer and unfolded monomer were highly populated at equilibrium; a native-like dimeric intermediate with partially dissociated domains I and II was detected using stopped-flow fluorescence studies [Wallace, Sluis-Cremer and Dirr (1998) Biochemistry 37, 5320-5328]. In the present study, urea-induced equilibrium unfolding of L164A hGST A1-1 indicated a destabilization of the native state and suggested the presence of a stable dimeric intermediate. The unfolding kinetic pathway for L164A hGST A1-1, like that for the wild type, is biphasic, with a fast and a slow unfolding event; the cavity-forming mutation has a substantially greater effect on the rate of unfolding of the fast event. The equilibrium and kinetic unfolding data for L164A hGST A1-1 suggest that a rapid pre-equilibrium is established between the native dimer and a dimeric intermediate before complete domain and subunit dissociation and unfolding. It is proposed that the topologically conserved bulky residue in alpha-helix 6 plays a role in specifying and stabilizing the core of domain II and the interface of domains I and II.

Wallace, L A; Blatch, G L; Dirr, H W

1998-01-01

347

Transmembrane domain oligomerization propensity determined by ToxR assay.  

PubMed

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action. One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers. Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association. Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve, direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays. The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1). In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay. The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where ?-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for ?-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by ?-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2). PMID:21654625

Joce, Catherine; Wiener, Alyssa; Yin, Hang

2011-05-26

348

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay  

PubMed Central

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action.1-3 One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers.4,5 Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association.6,7 Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve,8 direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays.9 The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1). In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay.10-13 The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where ?-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for ?-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by ?-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2).

Joce, Catherine; Wiener, Alyssa; Yin, Hang

2011-01-01

349

Morphology and Composition of the Helix Nebula  

NASA Astrophysics Data System (ADS)

We present new narrowband filter imagery in H? and [N II] lambda6584, along with UV and optical spectrophotometry measurements from 1200 to 9600 Å of NGC 7293, the Helix Nebula, a nearby, photogenic planetary nebula of large diameter and low surface brightness. Detailed models of the observable ionized nebula support the recent claim that the Helix is actually a flattened disk whose thickness is roughly one-third its diameter, with an inner region containing hot, highly ionized gas that is generally invisible in narrowband images. The outer visible ring structure is of lower ionization and temperature and is brighter because of a thickening in the disk. We also confirm a central star effective temperature and luminosity of 120,000 K and 100 L_solar, and we estimate a lower limit to the nebular mass to be 0.30 M_solar. Abundance measurements indicate the following values: He/H=0.12 (+/-0.017), O/H=4.60x10^-4 (+/-0.18), C/O=0.87 (+/-0.12), N/O=0.54 (+/-0.14), Ne/O=0.33 (+/-0.04), S/O=3.22x10^-3 (+/-0.26), and Ar/O=6.74x10^-3 (+/-0.76). Our carbon abundance measurements represent the first of their kind for the Helix Nebula. The S/O ratio that we derive is anomalously low; such values are found in only a few other planetary nebulae. The central star properties, the supersolar values of He/H and N/O, and a solar level of C/O are consistent with a 6.5 M_solar progenitor that underwent three phases of dredge-up and hot bottom burning before forming the planetary nebula.

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

1999-06-01

350

The helix bundle: A reversible lipid binding motif  

PubMed Central

Apolipoproteins are the protein components of lipoproteins that have the innate ability to inter convert between a lipid-free and a lipid-bound form in a facile manner, a remarkable property conferred by the helix bundle motif. Composed of a series of four or five amphipathic ?-helices that fold to form a helix bundle, this motif allows the en face orientation of the hydrophobic faces of the ?-helices in the protein interior in the lipid-free state. A conformational switch then permits helix-helix interactions to be substituted by helix-lipid interactions upon lipid binding interaction. This review compares the apolipoprotein high resolution structures and the factors that trigger this switch in insect apolipophorin III and the mammalian apolipoproteins, apolipoprotein E and apolipoprotein A-I, pointing out the commonalities and key differences in the mode of lipid interaction. Further insights into the lipid bound conformation of apolipoproteins are required to fully understand their functional role under physiological conditions.

Narayanaswami, Vasanthy; Kiss, Robert S.; Weers, Paul M.M.

2009-01-01

351

Alpha-helix stabilization by natural and unnatural amino acids with alkyl side chains.  

PubMed Central

Knowledge of the role of individual side chains in forming different secondary structures such as the alpha-helix would be useful for prediction of protein structure from sequence or de novo protein design. Experimental and theoretical studies on natural and synthetic peptides and proteins indicate that individual side chains differ in their helix-forming potential. Four aliphatic side chains occur in the standard complement of amino acids: alanine and leucine are helix stabilizing, whereas isoleucine and valine are weakly destabilizing. We have synthesized a series of helical peptides containing unnatural aliphatic side chains having two to four carbons to explore some of the factors involved in alpha-helix stabilization and the basis for selection of the natural set. We find that linear side chains with two, three, or four carbons are as strongly helix stabilizing as the single methyl in alanine and that all linear side chains are stronger helix promoters than leucine. In addition, a t-butyl side chain is significantly more helix destabilizing than the sec-butyl side chain of isoleucine, the isopropyl side chain of valine, or even the unrestricted side chain of glycine. These results provide experimental evidence that restriction in conformational freedom of a side chain imposed by alpha-helix formation is a major component of the role of a side chain in stabilizing helical structure.

Lyu, P C; Sherman, J C; Chen, A; Kallenbach, N R

1991-01-01

352

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

353

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.

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

2009-01-01

354

Fourier Transform Infrared Spectroscopy andSite-Directed Isotope Labeling as a ProbeofLocalSecondary Structure intheTransmembrane DomainofPhospholamban  

Microsoft Academic Search

transform infrared (ATR-FTIR) spectroscopy alongwithsite-directed isotope labeling to probe thelocal structure ofhPLB.Thefrequency anddichroism oftheamideIand11bandsappearing at1658cm-1and1544 cm-1,respectively, showthatdehydrated andhydrated hPLBreconstituted intodimyristoylphosphatidylcholine bilayer membranes ispredominantly a-helical andhasa nettransmembrane orientation. Specific local secondary structure ofhPLB was probedbyincorporating 13Cattwopositions intheprotein backbone. A small bandseen near1614cm-1isassigned totheamideImodeofthe13C-labeled amidecarbonyl group(s). Thefrequency anddichroism ofthisbandindicate that residues 39and46area-helical, with an axial orientation thatisapproximately 300relative tothemembranenormal. Upon exposure to

Cheryl F. C. Ludlam; Isaiah T. Arkin; Xiao-Mei Liu; Parshuram Rath

355

Dermal melanocytosis of the helix.  

PubMed

A 2-year-old Japanese boy had a congenital gray-blue macule involving the right helix along with a few melanotic spots on both sclerae. Histopathology showed dermal melanocytosis. Q-switched alexandrite laser treatment induced a good cosmetic response. This patient shows the overlap between Ota and Ito nevi, and we suggest dermal melanocytosis is better used as a generic term for these unusual pigmentations. PMID:20609156

Mizuashi, Masato; Suetake, Takaki; Aiba, Setsuya; Tagami, Hachiro

356

Expression of a chimeric helix-loop-helix gene, Id-SCL, in K562 human leukemic cells is associated with nuclear segmentation.  

PubMed Central

We have designed a chimeric gene, Id-SCL, in which the 3' helix-loop-helix encoding portion of the presumptive oncogene SCL/tal is joined to the 5' coding portion of Id, an inhibitory helix-loop-helix gene. The predicted protein product of this chimeric gene contains the helix-loop-helix dimerization domain of SCL/tal, but, lacking a basic DNA binding domain, is predicted to have the inhibitory function of the Id product. Expression of the Id-SCL fusion gene in stably transfected K562 cells reproducibly resulted in nuclear segmentation and depressed growth rates; both of these phenotypic effects demonstrated a dosage dependence on the levels of Id-SCL mRNA and protein expressed in the various clones. Electron microscopy of cells expressing high levels of Id-SCL mRNA showed a significant increase in cytoplasmic perinuclear thin filaments and diminution of marginal heterochromatin in the nuclei. No other changes in hematopoietic differentiation status were observed in association with Id-SCL expression. Expression of intact Id and SCL/tal genes, as well as deletion mutants of Id and SCL/tal, independently transfected into K562 cells, indicated that the nuclear segmentation effect is dependent on the presence of a protein possessing a helix-loop-helix domain but lacking a basic domain. Our studies suggest that the balance of transcriptional inhibitory and stimulatory helix-loop-helix proteins in cells may be important determinants of proliferation and of structural organization within cells. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 8 Figure 11

Goldfarb, A. N.; Wolf, M. L.; Greenberg, J. M.

1992-01-01

357

Infrared Observations of the Helix Planetary Nebula  

NASA Astrophysics Data System (ADS)

We have mapped the Helix (NGC 7293) planetary nebula (PN) with the IRAC instrument on the Spitzer Space Telescope. The Helix is one of the closest bright PNs and therefore provides an opportunity to resolve the small-scale structure in the nebula. The emission from this PN in the 5.8 and 8 ?m IRAC bands is dominated by the pure rotational lines of molecular hydrogen, with a smaller contribution from forbidden line emission such as [Ar III] in the ionized region. The IRAC images resolve the ``cometary knots,'' which have been previously studied in this PN. The ``tails'' of the knots and the radial rays extending into the outer regions of the PN are seen in emission in the IRAC bands. IRS spectra on the main ring and the emission in the IRAC bands are consistent with shock-excited H2 models, with a small (~10%) component from photodissociation regions. In the northeast arc, the H2 emission is located in a shell outside the H? emission.

Hora, Joseph L.; Latter, William B.; Smith, Howard A.; Marengo, Massimo

2006-11-01

358

Cooperative alpha-helix unfolding in a protein-DNA complex from hydrogen-deuterium exchange.  

PubMed

We present experimental evidence for a cooperative unfolding transition of an alpha-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen-deuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals in D2O, and of their mutual HN-HN NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that HN exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the HN exchange in helix 3 is dominated by random local structure fluctuations. PMID:16751603

Salinas, Roberto K; Diercks, Tammo; Kaptein, Robert; Boelens, Rolf

2006-06-02

359

Determination of alpha-helix N1 energies after addition of N1, N2, and N3 preferences to helix/coil theory.  

PubMed Central

Surveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the alpha-helix. We have therefore extended helix-coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N-cap, C-cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac-XAKAAAAKAAGY-CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol(-1) compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol(-1) less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1.

Sun, J. K.; Penel, S.; Doig, A. J.

2000-01-01

360

Crystal Structure of Taq DNA Polymerase in Complex with an Inhibitory Fab: The Fab is Directed against an Intermediate in the Helix-Coil Dynamics of the Enzyme  

Microsoft Academic Search

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a gamma turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate

R. Murali; D. J. Sharkey; J. L. Daiss; H. M. Krishna Murthy

1998-01-01

361

Multiple alignment of transmembrane protein sequences  

NASA Astrophysics Data System (ADS)

Multiple sequence alignment remains one of the most powerful tools for assessing evolutionary sequence relationships and for identifying structurally and functionally important protein regions. Membrane-bound proteins represent a special class of proteins. The regions that insert into the cell membrane have a profoundly different hydrophobicity pattern as compared with soluble proteins. Multiple alignment techniques employing scoring schemes tailored for sequences of soluble proteins are therefore in principle not optimal to align membrane-bound proteins. In this chapter we describe some of the characteristics leading transmembrane proteins to display differences at the sequence level. We will also cover computational strategies and methods developed over the years for aligning this special class of proteins, discuss some current bottlenecks, and suggest some avenues for improvement.

Pirovano, Walter; Abeln, Sanne; Feenstra, K. Anton; Heringa, Jaap

362

Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal ?-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition  

PubMed Central

Mutations of the aryl hydrocarbon receptor interacting protein (AIP) have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal ?-7 helix (C?-7h) mutations, R304* (nonsense mutation), R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the C?-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the C?-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE) of TOMM20.

Morgan, Rhodri M. L.; Hernandez-Ramirez, Laura C.; Trivellin, Giampaolo; Zhou, Lihong; Roe, S. Mark; Korbonits, Marta; Prodromou, Chrisostomos

2012-01-01

363

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

364

Structure of an atypical FeoB G-domain reveals a putative domain-swapped dimer.  

PubMed

FeoB is a transmembrane protein involved in ferrous iron uptake in prokaryotic organisms. FeoB comprises a cytoplasmic soluble domain termed NFeoB and a C-terminal polytopic transmembrane domain. Recent structures of NFeoB have revealed two structural subdomains: a canonical GTPase domain and a five-helix helical domain. The GTPase domain hydrolyses GTP to GDP through a well characterized mechanism, a process which is required for Fe(2+) transport. In contrast, the precise role of the helical domain has not yet been fully determined. Here, the structure of the cytoplasmic domain of FeoB from Gallionella capsiferriformans is reported. Unlike recent structures of NFeoB, the G. capsiferriformans NFeoB structure is highly unusual in that it does not contain a helical domain. The crystal structures of both apo and GDP-bound protein forms a domain-swapped dimer. PMID:23545645

Deshpande, Chandrika N; McGrath, Aaron P; Font, Josep; Guilfoyle, Amy P; Maher, Megan J; Jormakka, Mika

2013-03-29

365

Design of Tip Loss Profile on Support Rod for a Helix TWT  

NASA Astrophysics Data System (ADS)

This paper describes the design of tip loss profile on support rod for a high gain, high efficiency helix TWT. The tip loss profile at sever ends for the input and the output section of the helix slow wave structure has been designed for return loss at severs ends better than -20 dB. The experimental results have been carried out in the input and the output section of the helix assemblies for the high gain helix TWT to find the return loss in the region of carbon coated tip loss on the support rods. The experimental result has been compared with the simulated performance for the return loss at the sever ends. The design of tip loss profile on the support rod for helix TWT has been carried out in real situation using Ansoft HFSS. A good agreement has been found in the simulated and experimental results.

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

2009-07-01

366

Differentiation of Arabidopsis Guard Cells: Analysis of the Networks Incorporating the Basic Helix-Loop-Helix Transcription Factor, FAMA1[C][W][OA  

PubMed Central

Nearly all extant land plants possess stomata, the epidermal structures that mediate gas exchange between the plant and the environment. The developmental pathways, cell division patterns, and molecules employed in the generation of these structures are simple examples of processes used in many developmental contexts. One specific module is a set of “master regulator” basic helix-loop-helix transcription factors that regulate individual consecutive steps in stomatal development. Here, we profile transcriptional changes in response to inducible expression of Arabidopsis (Arabidopsis thaliana) FAMA, a basic helix-loop-helix protein whose actions during the final stage in stomatal development regulate both cell division and cell fate. Genes identified by microarray and candidate approaches were then further analyzed to test specific hypothesis about the activity of FAMA, the shape of its regulatory network, and to create a new set of stomata-specific or stomata-enriched reporters.

Hachez, Charles; Ohashi-Ito, Kyoko; Dong, Juan; Bergmann, Dominique C.

2011-01-01

367

Helix switching of a key active-site residue in the cytochrome cbb3 oxidases.  

PubMed

In the respiratory chains of mitochondria and many aerobic prokaryotes, heme-copper oxidases are the terminal enzymes that couple the reduction of molecular oxygen to proton pumping, contributing to the protonmotive force. The cbb(3) oxidases belong to the superfamily of enzymes that includes all of the heme-copper oxidases. Sequence analysis indicates that the cbb(3) oxidases are missing an active-site tyrosine residue that is absolutely conserved in all other known heme-copper oxidases. In the other heme-copper oxidases, this tyrosine is known to be subject to an unusual post-translational modification and to play a critical role in the catalytic mechanism. The absence of this tyrosine in the cbb(3) oxidases raises the possibility that the cbb(3) oxidases utilize a different catalytic mechanism from that of the other members of the superfamily. Using homology modeling, quantum chemistry, and molecular dynamics, a model of the structure of subunit I of a cbb(3) oxidase (Vibrio cholerae) was constructed. The model predicts that a tyrosine residue structurally analogous to the active-site tyrosine in other oxidases is present in the cbb(3) oxidases but that the tyrosine originates from a different transmembrane helix within the protein. The predicted active-site tyrosine is conserved in the sequences of all of the known cbb(3) oxidases. Mutagenesis of the tyrosine to phenylalanine in the V. cholerae oxidase resulted in a fully assembled enzyme with nativelike structure but lacking catalytic activity. These findings strongly suggest that all of the heme-copper oxidases utilize the same catalytic mechanism and provide an unusual example in which a critical active-site residue originates from different places within the primary sequence for different members of the same superfamily. PMID:16086579

Hemp, James; Christian, Caroline; Barquera, Blanca; Gennis, Robert B; Martínez, Todd J

2005-08-16

368

Structural snapshots of full-length Jak1, a transmembrane gp130/IL-6/IL-6R? cytokine receptor complex, and the receptor-Jak1 holocomplex  

PubMed Central

Summary The shared cytokine receptor gp130 signals as a homodimer or heterodimer through activation of Janus kinases (Jaks) associated with the receptor intracellular domains. Here we reconstitute, in parts and whole, the full-length gp130 homodimer in complex with the cytokine interleukin-6 (IL-6), its alpha receptor (IL-6R?) and Jak1, for electron microscopy imaging. We find that the full-length gp130 homodimer complex has intimate interactions between the trans- and juxtamembrane segments of the two receptors, appearing to rigidify the connection between the extra- and intracellular regions. 2D averages and 3D reconstructions of full-length Jak1 reveal a three-lobed structure comprised of FERM-SH2, pseudokinase and kinase modules possessing extensive inter-segmental flexibility that likely facilitates allosteric activation. Single-particle imaging of the gp130/IL-6/IL-6R?/Jak1 holocomplex shows Jak1 associated with the membrane proximal intracellular regions of gp130, abutting the would-be inner leaflet of the cell membrane. Jak1 association with gp130 is enhanced by the presence of a membrane environment.

Lupardus, Patrick J.; Skiniotis, Georgios; Rice, Amanda J.; Thomas, Christoph; Fischer, Suzanne; Walz, Thomas; Garcia, K. Christopher

2011-01-01

369

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

PubMed Central

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

Moin, Syed M; Urban, Sinisa

2012-01-01

370

Transmembrane signaling in kidney health and disease  

Microsoft Academic Search

Transmembrane signal transduction is the process whereby a ligand binds to the external surface of the cell membrane and elicits a physiological response specific for that ligand and cell type. It is now appreciated that numerous disease states represent disturbances in normal transmembrane signaling mechanisms. In the current paper, we focus our attention on the mesangial cell of the glomerular

Nashrudeen Hack; Aylit Schultz; Paula Clayman; Howard Goldberg; Karl L. Skorecki

1995-01-01

371

High-salt d(CpGpCpG), a left-handed Z' DNA double helix  

Microsoft Academic Search

The DNA tetramer d(CpGpCpG) or CGCG crystallizes from high-salt solution as a left-handed double helix, the Z' helix. Its structure differs from that of the other known left-handed helix, Z-DNA, by a Cl'-exo sugar pucker at deoxyguanosines rather than C3'-endo, and these represent two alternative solutions to the same steric constraint arising from the syn glycosyl bond orientation. The apparent

Horace Drew; Tsunehiro Takano; Shoji Tanaka; Keiichi Itakura; Richard E. Dickerson

1980-01-01

372

Oligophenylenaminones as scaffolds for ?-helix mimicry.  

PubMed

The design and synthesis of small molecule ?-helix mimetics has been a productive field over the past decade. These compounds have performed well in a variety of biological systems as functional disruptors of ?-helix-mediated protein-protein interactions. In our studies we have continued to develop novel, more biologically compatible scaffolds, which are often easier to assemble and capable of mimicking longer and/or more diverse helices. To this end, we have constructed a new series of i, i+4, i+7 ?-helix mimics based on the enaminone scaffold. These molecules represent a step forward in the pursuit of idealized monofacial ?-helix mimetics. PMID:21739975

Adler, Marc J; Hamilton, Andrew D

2011-08-03

373

A modular strategy for the semi-synthesis of a K+ channel: Investigating interactions of the pore helix  

PubMed Central

Chemical synthesis is a powerful method for precise modification of the structural and electronic properties of proteins. The difficulties in the synthesis and purification of peptides containing transmembrane segments have presented obstacles to the chemical synthesis of integral membrane proteins. Here, we present a modular strategy for the semi-synthesis of integral membrane proteins in which solid phase peptide synthesis is limited to the region of interest, while the rest of the protein is obtained by recombinant means. This modular strategy considerably simplifies the synthesis and purification steps that have previously hindered the chemical synthesis of integral membrane proteins. We develop a sumo-fusion and proteolysis approach for obtaining the N-terminal cysteine containing membrane spanning peptides required for the semi-synthesis. We demonstrate the feasibility of the modular approach by the semi-synthesis of full-length KcsA K+ channels in which only regions of interest, such as the selectivity filter or the pore helix, are obtained by chemical synthesis. The modular approach is used to investigate the hydrogen bond interactions of a tryptophan residue in the pore helix, tryptophan 68, by substituting it with the iso-steric analog, ?-(3-benzothienyl)-L-alanine (3BT). A functional analysis of the 3BT mutant channels indicates that the K+ conduction and selectivity of the 3BT mutant channels are similar to the wild type, but the mutant channels show a three-fold increase in Rb+ conduction. These results suggest that the hydrogen bond interactions of tryptophan 68 are essential for optimizing the selectivity filter for K+ conduction over Rb+ conduction.

Komarov, Alexander G.; Linn, Kellie M.; Devereaux, Jordan J.; Valiyaveetil, Francis I.

2009-01-01

374

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.

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

2009-01-01

375

Persistent interactions between the two transmembrane clusters dictate the targeting and functional assembly of adenylyl cyclase  

Microsoft Academic Search

Adenylyl cyclases possess complex structures like those of the ATP binding cassette (ABC) transporter family, which includes the cystic fibrosis transmembrane regulator, the P-glycoprotein, and ATP-sensitive K+ channels [1–4]. These structures comprise a cytosolic N terminus followed by two tandem six-transmembrane cassettes, each associated with a highly homologous (ATP binding) cytosolic loop [5–8]. The catalytic domains, which are located in

Chen Gu; Alexander Sorkin; Dermot M. F. Cooper

2001-01-01

376

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

377

Design and synthesis of DNA four-helix bundles  

NASA Astrophysics Data System (ADS)

The field of DNA nanotechnology has evolved significantly in the past decade. Researchers have succeeded in synthesizing tile-based structures and using them to form periodic lattices in one, two and three dimensions. Origami-based structures have also been used to create nanoscale structures in two and three dimensions. Design and construction of DNA bundles with fixed circumference has added a new dimension to the field. Here we report the design and synthesis of a DNA four-helix bundle. It was found to be extremely rigid and stable. When several such bundles were assembled using appropriate sticky-ends, they formed micrometre-long filaments. However, when creation of two-dimensional sheet-like arrays of the four-helix bundles was attempted, nanoscale rings were observed instead. The exact reason behind the nanoring formation is yet to be ascertained, but it provides an exciting prospect for making programmable circular nanostructures using DNA.

Rangnekar, Abhijit; Gothelf, Kurt V.; LaBean, Thomas H.

2011-06-01

378

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.

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

2011-01-01

379

In vitro dimerization of the bovine papillomavirus E5 protein transmembrane domain  

PubMed Central

The E5 protein from bovine papillomavirus is a type II membrane protein and the product of the smallest known oncogene. E5 causes tumor formation by binding and activating the platelet derived growth factor beta receptor (PDGF?R). In order to productively interact with the receptor it is thought that E5 binds as a dimer. However, wild-type E5 and various mutants have also been shown to form trimers, tetramers and even higher order oligomers. The residues in E5 that drive and stabilize a dimeric state are also still in question. At present, two different models for the E5 dimer exist in the literature, one symmetric and one asymmetric. There is universal agreement, however, that the transmembrane (TM) domain plays a vital role in stabilizing the functional oligomer, indeed mutation of various TM domain residues can abolish E5 function. In order to better resolve the role of the E5 TM domain in function, we have undertaken the first quantitative in vitro characterization of the E5 TM domain in detergent micelles and liposomes. Circular and linear dichroism analyses verify that the TM domain adopts a stable ?-helical structure and is able to partition efficiently across lipid bilayers. SDS-PAGE and analytical ultracentrifugation confirm for the first time that the T