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1

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

2

Transmembrane helix: simple or complex.  

PubMed

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

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

2012-07-01

3

Mapping the energy surface of transmembrane helix-helix interactions.  

PubMed Central

Transmembrane helices are no longer believed to be just hydrophobic segments that exist solely to anchor proteins to a lipid bilayer, but rather they appear to have the capacity to specify function and structure. Specific interactions take place between hydrophobic segments within the lipid bilayer whereby subtle mutations that normally would be considered innocuous can result in dramatic structural differences. That such specificity takes place within the lipid bilayer implies that it may be possible to identify the most favorable interaction surface of transmembrane alpha-helices based on computational methods alone, as shown in this study. Herein, an attempt is made to map the energy surface of several transmembrane helix-helix interactions for several homo-oligomerizing proteins, where experimental data regarding their structure exist (glycophorin A, phospholamban, Influenza virus A M2, Influenza virus C CM2, and HIV vpu). It is shown that due to symmetry constraints in homo-oligomers the computational problem can be simplified. The results obtained are mostly consistent with known structural data and may additionally provide a view of possible alternate and intermediate configurations.

Torres, J; Kukol, A; Arkin, I T

2001-01-01

4

Active machine learning for transmembrane helix prediction  

PubMed Central

Background About 30% of genes code for membrane proteins, which are involved in a wide variety of crucial biological functions. Despite their importance, experimentally determined structures correspond to only about 1.7% of protein structures deposited in the Protein Data Bank due to the difficulty in crystallizing membrane proteins. Algorithms that can identify proteins whose high-resolution structure can aid in predicting the structure of many previously unresolved proteins are therefore of potentially high value. Active machine learning is a supervised machine learning approach which is suitable for this domain where there are a large number of sequences but only very few have known corresponding structures. In essence, active learning seeks to identify proteins whose structure, if revealed experimentally, is maximally predictive of others. Results An active learning approach is presented for selection of a minimal set of proteins whose structures can aid in the determination of transmembrane helices for the remaining proteins. TMpro, an algorithm for high accuracy TM helix prediction we previously developed, is coupled with active learning. We show that with a well-designed selection procedure, high accuracy can be achieved with only few proteins. TMpro, trained with a single protein achieved an F-score of 94% on benchmark evaluation and 91% on MPtopo dataset, which correspond to the state-of-the-art accuracies on TM helix prediction that are achieved usually by training with over 100 training proteins. Conclusion Active learning is suitable for bioinformatics applications, where manually characterized data are not a comprehensive representation of all possible data, and in fact can be a very sparse subset thereof. It aids in selection of data instances which when characterized experimentally can improve the accuracy of computational characterization of remaining raw data. The results presented here also demonstrate that the feature extraction method of TMpro is well designed, achieving a very good separation between TM and non TM segments.

2010-01-01

5

Mutations at position 125 in transmembrane helix III of rhodopsin affect the structure and signalling of the receptor.  

PubMed

Mutation of L125R in trasmembrane helix III of rhodopsin, associated with the retinal degenerative disease retinitis pigmentosa, was previously shown to cause structural misfolding of the mutant protein. Also, conservative mutations at this position were found to cause partial misfolding of the mutant receptors. We report here on a series of mutations at position 125 to further investigate the role of Leu125 in the correct folding and function of rhodopsin. In particular, the effect of the size of the substituted amino-acid side chain in the functionality of the receptor, measured as the ability of the mutant rhodopsins to activate the G protein transducin, has been analysed. The following mutations have been studied: L125G, L125N, L125I, L125H, L125P, L125T, L125D, L125E, L125Y and L125W. Most of the mutant proteins, expressed in COS-1 cells, showed reduced 11-cis-retinal binding, red-shifts in the wavelength of the visible absorbance maximum, and increased reactivity towards hydroxylamine in the dark. Thermal stability in the dark was reduced, particularly for L125P, L125Y and L125W mutants. The ability of the mutant rhodopsins to activate the G protein transducin was significantly reduced in a size dependent manner, especially in the case of the bulkier L125Y and L125W substitutions, suggesting a steric effect of the substituted amino acid. On the basis of the present and previous results, Leu125 in transmembrane helix III of rhodopsin, in the vicinity of the beta-ionone ring of 11-cis-retinal, is proposed to be an important residue in maintaining the correct structure of the chromophore binding pocket. Thus, bulky substitutions at this position may affect the structure and signallling of the receptor by altering the optimal conformation of the retinal binding pocket, rather than by direct interaction with the chromophore, as seen from the recent crystallographic structure of rhodopsin. PMID:11722553

Andrés, A; Kosoy, A; Garriga, P; Manyosa, J

2001-11-01

6

Transmembrane helix-helix interactions involved in ErbB receptor signaling  

PubMed Central

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

Cymer, Florian

2010-01-01

7

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

8

Transmembrane Helix-helix Association: Relative Stabilities at Low pH†  

PubMed Central

We have previously studied the unfolding equilibrium of bacterio-opsin in a single phase solvent, using Förster mechanism fluorescence resonance energy transfer (FRET) as a probe, from tryptophan donors to a dansyl acceptor. We observed an apparent unfolding transition in bacterio-opsin perturbed by increasing ethanol concentrations [Nannepaga et al.(2004) Biochemistry 43, 50–59]. We have further investigated this transition and find the unfolding is pH-dependent. We have now measured the apparent pK of acid-induced unfolding of bacterio-opsin in 90% ethanol. When the acceptor is on helix B (Lys 41), the apparent pK for unfolding is 4.75; on the EF connecting loop (Cys 163), 5.15; and on helix G (Cys 222), 5.75. Five-helix proteolytic fragments are less stable. The apparent unfolding pKs are, for residues 72-248 (Cys 163), 5.46; and 1-166 (Lys 41), 7.36. When interpreted in terms of a simple equilibrium model for unfolding, the apparent pKs give relative free energies of unfolding, in the range of ?0.54 to ?3.5 kcal/mol. The results suggest that the C-terminal helix of bacterio-opsin is less stably folded than the N-terminal helices. We analyzed the pair-wise helix-helix interaction surfaces of bacteriorhodopsin and three other 7-transmembrane helix proteins, based on crystal structures. The results show that the interaction surfaces are smoother and the helix axis separations are closer in the amino-terminal two-thirds of the proteins compared with the carboxyl terminal one-third. However, the F helix is important in stabilizing the folded structure, as shown by the instability of the 1-166 fragment. Considering the high resolution crystal structure of bacteriorhodopsin, there are no obvious helix-helix interactions involving protein side chains which would be destabilized by protonation at the estimated pH of the unfolding transitions. However, a number of helix-bridging water molecules could become protonated, thereby weakening the helix-helix interactions.

Valluru, Neelima; Silva, Frances; Dhage, Manmath; Rodriguez, Gustavo; Alloor, Srinivas R.; Renthal, Robert

2008-01-01

9

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

Microsoft Academic Search

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

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

1999-01-01

10

Terminal residue hydrophobicity modulates transmembrane helix-helix interactions.  

PubMed

Central to the formation of tertiary structure in membrane protein folding is the presence of amino acid sequence motifs (such as "small-XXX-small" segments) in the TM segments that promote interaction-compatible surfaces through which the TM ?-helices interact. Here, we sought to elucidate additional factors that may work in tandem to dictate the ultimate interaction fate of TM-embedded segments. In this context, we used proteolipid protein (PLP), the major protein from central nervous system myelin for which mutant-dependent non-native oligomerization has been implicated in neurological disorders, to explore the specific effects of TM boundary residues (the membrane entry and exit points), keying on the secondary structure and self-association of peptides corresponding to the PLP TM2 ?-helix (wild-type sequence (66)AFQYVIYGTASFFFLYGALLLAEGF(90)). Using gel electrophoresis, circular dichroism, and Förster resonance energy transfer in the membrane-mimetic detergent sodium dodecyl sulfate (SDS), we found that mutation of F90 to residues such as A, I, L, or V maintains the onset of TM2-TM2 dimerization, whereas mutation to E, G, Q, N, S, or T abrogates dimer formation. We attribute this sensitivity to changes in local hydrophobicity, viz., a decrease in hydrophobicity reduces local lipid-peptide interactions, which in turn disrupts peptide ?-helicity and hence the effectiveness of an incipient interaction-compatible surface. Our results show that the secondary structure and oligomeric state of PLP TM2 Lys-tagged peptides are significantly modulated by the specific nature of their C-terminal boundary residue, thus providing insight as to how point mutations, particularly where they produce disease states, can compromise the folding process. PMID:24857611

Ng, Derek P; Deber, Charles M

2014-06-17

11

3D structural models of transmembrane proteins  

PubMed Central

Summary Transmembrane proteins are macromolecules implicated in major biological process and diseases. Due to their specific neighborhood, few transmembrane protein structures are nowadays available. The building of structural models of transmembrane proteins is a major research area. Due to the lack of available 3D structures, automatic homology modeling is not an efficient way to propose pertinent structural models. Hence, most of the structural models of transmembrane proteins are done through a more complex protocol. This latter comprises the use of secondary structure prediction to complete the comparative modeling process. Then, refinement and assessment steps are performed go often to a novel comparative modeling process. Nowadays, it is also possible to take attention to the helixhelix and helix – lipid interactions, and, to build even quaternary structures. In all cases, the taking into account of experimental data is the most important factor to proceed to correct structural models.

De Brevern, Alexandre G.

2010-01-01

12

An essential sensor histidine kinase controlled by transmembrane helix interactions with its auxiliary proteins  

PubMed Central

Two-component signal transduction systems with membrane-embedded sensor histidine kinases are believed to recognize environmental signals and transduce this information over the cellular membrane to influence the activity of a transcription factor to which they are mated. The YycG sensor kinase of Bacillus subtilis, containing two transmembrane helices, is subject to a complicated activity-control circuit involving two other proteins with N-terminal transmembrane helices, YycH and YycI. Truncation studies of YycH and YycI demonstrated that the individual transmembrane helices of these proteins are sufficient to adjust YycG activity, indicating that this control is achieved at the membrane level. A replica exchange molecular dynamics computational approach generated in silico structural models of the transmembrane helix complex that informed mutagenesis studies of the YycI transmembrane helix supporting the accuracy of the in silico model. The results predict that signal recognition by any of the extracellular domains of the sensor histidine kinase YycG or the associated proteins YycH and YycI is transmitted across the cellular membrane by subtle alterations in the positions of the helices within the transmembrane complex of the three proteins.

Szurmant, Hendrik; Bu, Lintao; Brooks, Charles L.; Hoch, James A.

2008-01-01

13

An essential sensor histidine kinase controlled by transmembrane helix interactions with its auxiliary proteins.  

PubMed

Two-component signal transduction systems with membrane-embedded sensor histidine kinases are believed to recognize environmental signals and transduce this information over the cellular membrane to influence the activity of a transcription factor to which they are mated. The YycG sensor kinase of Bacillus subtilis, containing two transmembrane helices, is subject to a complicated activity-control circuit involving two other proteins with N-terminal transmembrane helices, YycH and YycI. Truncation studies of YycH and YycI demonstrated that the individual transmembrane helices of these proteins are sufficient to adjust YycG activity, indicating that this control is achieved at the membrane level. A replica exchange molecular dynamics computational approach generated in silico structural models of the transmembrane helix complex that informed mutagenesis studies of the YycI transmembrane helix supporting the accuracy of the in silico model. The results predict that signal recognition by any of the extracellular domains of the sensor histidine kinase YycG or the associated proteins YycH and YycI is transmitted across the cellular membrane by subtle alterations in the positions of the helices within the transmembrane complex of the three proteins. PMID:18408157

Szurmant, Hendrik; Bu, Lintao; Brooks, Charles L; Hoch, James A

2008-04-15

14

Helix Packing and Orientation in the Transmembrane Dimer of gp55-P of the Spleen Focus Forming Virus  

PubMed Central

gp55-P is a dimeric membrane protein with a single transmembrane helix that is coded by the env gene of the polycythemic strain of the spleen focus forming virus. gp55-P activates the erythropoietin (Epo) receptor through specific transmembrane helix interactions, leading to Epo-independent growth of erythroid progenitors and eventually promoting erythroleukemia. We describe the use of magic angle spinning deuterium NMR to establish the structure of the transmembrane dimer of gp55-P in model membranes. Comparison of the deuterium lineshapes of leucines in the center (Leu396–399) and at the ends (Leu385, Leu407) of the transmembrane sequence shows that gp55-P has a right-handed crossing angle with Leu399 packed in the dimer interface. We discuss the implications of the structure of the gp55-P transmembrane dimer for activation of the Epo receptor.

Liu, Wei; Crocker, Evan; Constantinescu, Stefan N.; Smith, Steven O.

2005-01-01

15

Modulation of the transmembrane helix insertion pathway by polar cargo  

NASA Astrophysics Data System (ADS)

In an earlier study, we found a series of kinetic steps in the pH-triggered insertion of the pHLIP (pH (Low) Insertion Peptide). In the present work we observe that the polarity of the inserting end, including its cargo, modulates the number of intermediates, and that insertion can be described as a two state process for a simple case. Each investigated pHLIP variant preserve the pH-dependent properties of surface binding to membrane at neutral pH and insertion at low pH to form a transmembrane helix. However, there are thermodynamic and kinetic properties that are determined by the degree of cargo polarity. The presence of a polar cargo at the peptide's inserting end leads to the appearance of two additional intermediate states on the insertion pathway of the pHLIP-2E peptide, which itself (when no cargo is attached) shows an all-or-none transition from the partially unstructured membrane-surface to the transmembrane state described well by the two-state model at 800 ms timescale. We discuss the utility of our observations for the design of new delivery agents for the direct translocation of polar therapeutic and diagnostic cargo molecules across cellular membranes. The work is supported by NIH grants CA133890 to OAA, DME, YRK.

Wijesinghe, Dayanjali; Karabadzhak, Alexander; Markin, Vladislav; Engelman, Donald; Andreev, Oleg; Reshetnyak, Yana

2012-02-01

16

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

PubMed Central

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

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

2013-01-01

17

Helix Capping in RNA Structure  

PubMed Central

Helices are an essential element in defining the three-dimensional architecture of structured RNAs. While internal basepairs in a canonical helix stack on both sides, the ends of the helix stack on only one side and are exposed to the loop side, thus susceptible to fraying unless they are protected. While coaxial stacking has long been known to stabilize helix ends by directly stacking two canonical helices coaxially, based on analysis of helix-loop junctions in RNA crystal structures, herein we describe helix capping, topological stacking of a helix end with a basepair or an unpaired nucleotide from the loop side, which in turn protects helix ends. Beyond the topological protection of helix ends against fraying, helix capping should confer greater stability onto the resulting composite helices. Our analysis also reveals that this general motif is associated with the formation of tertiary structure interactions. Greater knowledge about the dynamics at the helix-junctions in the secondary structure should enhance the prediction of RNA secondary structure with a richer set of energetic rules and help better understand the folding of a secondary structure into its three-dimensional structure. These together suggest that helix capping likely play a fundamental role in driving RNA folding.

Lee, Jung C.; Gutell, Robin R.

2014-01-01

18

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

19

Interaction of a two-transmembrane-helix peptide with lipid bilayers and dodecyl sulfate micelles  

PubMed Central

To probe structural changes that occur when a membrane protein is transferred from lipid bilayers to SDS micelles, a fragment of bacteriorhodopsin containing transmembrane helical segments A and B was studied by fluorescence spectroscopy, molecular dynamics (MD) simulation, and stopped flow kinetics. In lipid bilayers, Förster resonance energy transfer (FRET) was observed between tyrosine 57 on helix B and tryptophans 10 and 12 on helix A. FRET efficiency decreased substantially when the peptide was transferred to SDS. MD simulation showed no evidence for significant disruption of helix-helix interactions in SDS micelles. However, a cluster of water molecules was observed to form a hydrogen-bonded network with the phenolic hydroxyl group of tyrosine 57, which probably causes the disappearance of tyrosine-to-tryptophan FRET in SDS. The tryptophan quantum yield decreased in SDS, and the change occurred at nearly the same rate as membrane solubilization. The results provide a clear example of the importance of corroborating distance changes inferred from FRET by using complementary methods.

Renthal, Robert; Brancaleon, Lorenzo; Pena, Isaac; Silva, Frances; Chen, Liao Y.

2011-01-01

20

Role of Side-Chain Conformational Entropy in Transmembrane Helix Dimerization of Glycophorin A  

Microsoft Academic Search

Dimerization of the transmembrane domain of glycophorin A is mediated by a seven residue motif LIxxGVxxGVxxT through a combination of van der Waals and hydrogen bonding interactions. One of the unusual features of the motif is the large number of ?-branched amino acids that may limit the entropic cost of dimerization by restricting side-chain motion in the monomeric transmembrane helix.

Wei Liu; Evan Crocker; David J. Siminovitch; Steven O. Smith

2003-01-01

21

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

PubMed

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

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

2014-08-12

22

Reversible transition between ?-helix and ?-sheet conformation of a transmembrane domain  

Microsoft Academic Search

Despite the important functions of protein transmembrane domains, their structure and dynamics are often scarcely known. The SNARE proteins VAMP\\/synaptobrevin and syntaxin 1 are implicated in membrane fusion. Using different spectroscopic approaches we observed a marked sensitivity of their transmembrane domain structure in regard to the lipid\\/peptide ratio. In the dilute condition, peptides corresponding to the complete transmembrane domain fold

Wissam Yassine; Nada Taib; Silvina Federman; Alexandra Milochau; Sabine Castano; Walid Sbi; Claude Manigand; Michel Laguerre; Bernard Desbat; Reiko Oda; Jochen Lang

2009-01-01

23

Structure and function in rhodopsin: correct folding and misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125-->Arg in the transmembrane helix C.  

PubMed

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore (lambda max 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed lambda max at 500 nm, whereas that from L125A showed lambda max at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain. PMID:8643443

Garriga, P; Liu, X; Khorana, H G

1996-05-14

24

Structure and function in rhodopsin: correct folding and misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125-->Arg in the transmembrane helix C.  

PubMed Central

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore (lambda max 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed lambda max at 500 nm, whereas that from L125A showed lambda max at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain. Images Fig. 2

Garriga, P; Liu, X; Khorana, H G

1996-01-01

25

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

PubMed Central

Summary TMpro is a transmembrane (TM) helix prediction algorithm that uses language processing methodology for TM segment identification. It is primarily based on the analysis of statistical distributions of properties of amino acids in transmembrane segments. This article describes the availability of TMpro on the internet via a web interface. The key features of the interface are: (i) output is generated in multiple formats including a user-interactive graphical chart which allows comparison of TMpro predicted segment locations with other labeled segments input by the user, such as predictions from other methods. (ii) Up to 5000 sequences can be submitted at a time for prediction. (iii) TMpro is available as a web server and is published as a web service so that the method can be accessed by users as well as other services depending on the need for data integration.

Ganapathiraju, Madhavi; Jursa, Christopher Jon; Karimi, Hassan A.

2011-01-01

26

Role of Side-Chain Conformational Entropy in Transmembrane Helix Dimerization of Glycophorin A  

PubMed Central

Dimerization of the transmembrane domain of glycophorin A is mediated by a seven residue motif LIxxGVxxGVxxT through a combination of van der Waals and hydrogen bonding interactions. One of the unusual features of the motif is the large number of ?-branched amino acids that may limit the entropic cost of dimerization by restricting side-chain motion in the monomeric transmembrane helix. Deuterium NMR spectroscopy is used to characterize the dynamics of fully deuterated Val80 and Val84, two essential amino acids of the dimerization motif. Deuterium spectra of the glycophorin A transmembrane dimer were obtained using synthetic peptides corresponding to the transmembrane sequence containing either perdeuterated Val80 or Val84. These data were compared with spectra of monomeric glycophorin A peptides deuterated at Val84. In all cases, the deuterium line shapes are characterized by fast methyl group rotation with virtually no motion about the C?-C? bond. This is consistent with restriction of the side chain in both the monomer and dimer due to intrahelical packing interactions involving the ?-methyl groups, and indicates that there is no energy cost associated with dimerization due to loss of conformational entropy. In contrast, deuterium NMR spectra of Met81 and Val82, in the lipid interface, reflected greater motional averaging and fast exchange between different side-chain conformers.

Liu, Wei; Crocker, Evan; Siminovitch, David J.; Smith, Steven O.

2003-01-01

27

Role of side-chain conformational entropy in transmembrane helix dimerization of glycophorin A.  

PubMed

Dimerization of the transmembrane domain of glycophorin A is mediated by a seven residue motif LIxxGVxxGVxxT through a combination of van der Waals and hydrogen bonding interactions. One of the unusual features of the motif is the large number of beta-branched amino acids that may limit the entropic cost of dimerization by restricting side-chain motion in the monomeric transmembrane helix. Deuterium NMR spectroscopy is used to characterize the dynamics of fully deuterated Val80 and Val84, two essential amino acids of the dimerization motif. Deuterium spectra of the glycophorin A transmembrane dimer were obtained using synthetic peptides corresponding to the transmembrane sequence containing either perdeuterated Val80 or Val84. These data were compared with spectra of monomeric glycophorin A peptides deuterated at Val84. In all cases, the deuterium line shapes are characterized by fast methyl group rotation with virtually no motion about the C(alpha)-C(beta) bond. This is consistent with restriction of the side chain in both the monomer and dimer due to intrahelical packing interactions involving the beta-methyl groups, and indicates that there is no energy cost associated with dimerization due to loss of conformational entropy. In contrast, deuterium NMR spectra of Met81 and Val82, in the lipid interface, reflected greater motional averaging and fast exchange between different side-chain conformers. PMID:12547806

Liu, Wei; Crocker, Evan; Siminovitch, David J; Smith, Steven O

2003-02-01

28

Channels formed by the transmembrane helix of phospholamban: a simulation study.  

PubMed

Phospholamban is a small membrane protein which can form cation selective ion channels in lipid bilayers. Each subunit contains a single, largely hydrophobic transmembrane helix. The helices are thought to assemble as a pentameric and approximately parallel bundle surrounding a central pore. A model of this assembly (PDB code IPSL) has been used as the starting point for molecular dynamics (MD) simulations of a system consisting of the pentameric helix bundle, plus 217 water molecules located within and at either mouth of the pore. Interhelix distance restraints were employed to maintain the integrity of the helix bundle during a 500 ps MD simulation. Water molecules within the pore exhibited reduced diffusional and rotational mobility. Interactions between the alpha-helix dipoles and the water dipoles, the latter aligned anti-parallel to the former, contribute to the stability of the system. Analysis of the potential energy of interaction of a K+ ion as it was moved through the pore suggested that unfavourable interactions of the cation with the aligned helix dipoles at the N-terminal mouth were overcome by favourable ion-water interactions. Comparable analysis for a Cl ion revealed that the ion-(pore + water) interactions were unfavourable along the whole of the pore, increasingly so from the N- to the C-terminal mouth. Overall, the interaction energy profiles were consistent with a pore selective for cations over anions. Pore radius profiles were used to predict a channel conductance of 50 to 70 ps in 0.2 M KCl, which compares well with an experimental value of 100 ps. PMID:9474759

Sansom, M S; Smith, G R; Smart, O S; Smith, S O

1997-12-01

29

An Indel in Transmembrane Helix 2 Helps to Trace the Molecular Evolution of Class A G-Protein-Coupled Receptors  

Microsoft Academic Search

Class A G-protein-coupled receptors (GPCRs) constitute a large family of transmembrane receptors. Helical distortions play\\u000a a major role in the overall fold of these receptors. Most are related to conserved proline residues. However, in transmembrane\\u000a helix 2, the proline pattern is not conserved, and when present, proline may be located at position 2.58, 2.59, or 2.60. Sequence\\u000a analysis, three-dimensional data

Julie Devillé; Julien Rey; Marie Chabbert

2009-01-01

30

The Roles of Transmembrane Domain Helix-III during Rhodopsin Photoactivation  

PubMed Central

Background Rhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11-cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear. Principal Findings We herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 4?-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108–135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees. Significance Accessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III.

Ou, Wen-bin; Yi, Tingfang; Kim, Jong-Myoung; Khorana, H. Gobind

2011-01-01

31

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

PubMed Central

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

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

2014-01-01

32

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

PubMed Central

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

Xue, Yi; Ha, Ya

2013-01-01

33

Mapping Human Protease-activated Receptor 4 (PAR4) Homodimer Interface to Transmembrane Helix 4*  

PubMed Central

Thrombin activates platelets by binding and cleaving protease-activated receptors 1 and 4 (PAR1 and PAR4). Because of the importance of PAR4 activation on platelets in humans and mice and emerging roles for PAR4 in other tissues, experiments were done to characterize the interaction between PAR4 homodimers. Bimolecular fluorescence complementation and bioluminescence resonance energy transfer (BRET) were used to examine the PAR4 homodimer interface. In bimolecular fluorescence complementation experiments, PAR4 formed homodimers that were disrupted by unlabeled PAR4 in a concentration-dependent manner, but not by rhodopsin. In BRET experiments, the PAR4 homodimers showed a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression. PAR4 did not interact with rhodopsin in BRET assays. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, rhodopsin was unable to disrupt the BRET signal, indicating that the disruption of the PAR4 homodimer is not due to nonspecific interactions. A panel of rho-PAR4 chimeras and PAR4 point mutants has mapped the dimer interface to hydrophobic residues in transmembrane helix 4. Finally, mutations that disrupted dimer formation had reduced calcium mobilization in response to the PAR4 agonist peptide. These results link the loss of dimer formation to a loss of PAR4 signaling.

de la Fuente, Maria; Noble, Daniel N.; Verma, Sheetal; Nieman, Marvin T.

2012-01-01

34

The Gramicidin A Transmembrane Channel: A Proposed ?(L,D) Helix  

PubMed Central

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

Urry, D. W.

1971-01-01

35

Transmembrane helix prediction using amino acid property features and latent semantic analysis  

PubMed Central

Background Prediction of transmembrane (TM) helices by statistical methods suffers from lack of sufficient training data. Current best methods use hundreds or even thousands of free parameters in their models which are tuned to fit the little data available for training. Further, they are often restricted to the generally accepted topology "cytoplasmic-transmembrane-extracellular" and cannot adapt to membrane proteins that do not conform to this topology. Recent crystal structures of channel proteins have revealed novel architectures showing that the above topology may not be as universal as previously believed. Thus, there is a need for methods that can better predict TM helices even in novel topologies and families. Results Here, we describe a new method "TMpro" to predict TM helices with high accuracy. To avoid overfitting to existing topologies, we have collapsed cytoplasmic and extracellular labels to a single state, non-TM. TMpro is a binary classifier which predicts TM or non-TM using multiple amino acid properties (charge, polarity, aromaticity, size and electronic properties) as features. The features are extracted from sequence information by applying the framework used for latent semantic analysis of text documents and are input to neural networks that learn the distinction between TM and non-TM segments. The model uses only 25 free parameters. In benchmark analysis TMpro achieves 95% segment F-score corresponding to 50% reduction in error rate compared to the best methods not requiring an evolutionary profile of a protein to be known. Performance is also improved when applied to more recent and larger high resolution datasets PDBTM and MPtopo. TMpro predictions in membrane proteins with unusual or disputed TM structure (K+ channel, aquaporin and HIV envelope glycoprotein) are discussed. Conclusion TMpro uses very few free parameters in modeling TM segments as opposed to the very large number of free parameters used in state-of-the-art membrane prediction methods, yet achieves very high segment accuracies. This is highly advantageous considering that high resolution transmembrane information is available only for very few proteins. The greatest impact of TMpro is therefore expected in the prediction of TM segments in proteins with novel topologies. Further, the paper introduces a novel method of extracting features from protein sequence, namely that of latent semantic analysis model. The success of this approach in the current context suggests that it can find potential applications in other sequence-based analysis problems. Availability and

Ganapathiraju, Madhavi; Balakrishnan, N; Reddy, Raj; Klein-Seetharaman, Judith

2008-01-01

36

Structural Signatures and Membrane Helix 4 in GLUT1  

PubMed Central

Exon IV of SLC2A1, a multiple facilitator superfamily (MFS) transporter gene, is particularly susceptible to mutations that cause GLUT1 deficiency syndrome, a human encephalopathy that results from decreased glucose flux through the blood-brain barrier. Genotyping of 100 patients revealed that in a third of them who harbor missense mutations in the GLUT1 transporter, transmembrane domain 4 (TM4), encoded by SLC2A1 exon IV, contains mutant residues that have the periodicity of one face of a kinked ?-helix. Arg-126, located at the amino terminus of TM4, is the locus for most of the mutations followed by other arginine and glycine residues located elsewhere in the transporter but conserved among MFS proteins. The Arg-126 mutants were constructed and assayed for protein expression, targeting, and transport capacity in Xenopus oocytes. The role of charge at position 126, as well as its accessibility, was investigated in R126H by determining its activity as a function of extracellular pH. The results indicate that intracellular charges at the MFS TM2–3 and TM8–9 signature loops and flanking TMs 3, 5, and 6 are critical for the structure of GLUT1 as are TM glycines and that TM4, located at the catalytic core of MFS proteins, forms a helix that surfaces into the extracellular solution where another proton facilitates transport.

Pascual, Juan M.; Wang, Dong; Yang, Ru; Shi, Lei; Yang, Hong; De Vivo, Darryl C.

2008-01-01

37

Statistical models for discerning protein structures containing the DNA-binding helix-turn-helix motif.  

PubMed

A method for discerning protein structures containing the DNA-binding helix-turn-helix (HTH) motif has been developed. The method uses statistical models based on geometrical measurements of the motif. With a decision tree model, key structural features required for DNA binding were identified. These include a high average solvent-accessibility of residues within the recognition helix and a conserved hydrophobic interaction between the recognition helix and the second alpha helix preceding it. The Protein Data Bank was searched using a more accurate model of the motif created using the Adaboost algorithm to identify structures that have a high probability of containing the motif, including those that had not been reported previously. PMID:12818201

McLaughlin, William A; Berman, Helen M

2003-06-27

38

Production and initial structural characterization of the TM4TM5 helix-loop-helix domain of the translocator protein.  

PubMed

Mainly present in the mitochondria, the translocator protein, TSPO, previously known as the peripheral benzodiazepine receptor, is a small essential membrane protein, involved in the translocation of cholesterol across mitochondrial membranes, a rate determining step in steroids biosynthesis. We previously reported the structure of five fragments encompassing the five putative transmembrane helices and showed that each of these fragments constitutes an autonomous folding unit. To further characterize the structural determinants responsible for helix-helix association of this membrane protein, we now investigate the folding of double transmembrane domains in various detergent micelles. Herein, we present the successful biosynthesis of a double transmembrane domain encompassing the last two C-terminal helices (TM4TM5). For optimal production of this domain in Escherichia coli, the evaluation of various peptide constructs, including TM4TM5 fused to different purification tags or to solubilizing proteins, was necessary. The protocol of production of TM4TM5 with more than 95% purity is reported. This domain was further characterized using circular dichroism and solution state NMR. Far-UV circular dichroism studies indicate that the secondary structure of TM4TM5 is highly helical when solubilized in various detergent micelles including n-dodecyl-?-d-maltoside, n-octyl-?-d-glucoside, n-dodecylphosphocholine, 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol). In addition, the solubilization conditions of the domain were optimized for NMR experiments, and preliminary analysis indicates that TM4TM5 adopts a stable tertiary fold within the TM4TM5-DHPC complex. PMID:23315717

Galvagnion, C; Montaville, P; Coïc, Y-M; Jamin, N

2013-02-01

39

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

PubMed Central

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

2012-01-01

40

Thermodynamic measurements of bilayer insertion of a single transmembrane helix chaperoned by fluorinated surfactants.  

PubMed

Accurate determination of the free energy of transfer of a helical segment from an aqueous into a transmembrane (TM) conformation is essential for understanding and predicting the folding and stability of membrane proteins. Until recently, direct thermodynamically sound measurements of free energy of insertion of hydrophobic TM peptides were impossible due to peptide aggregation outside the lipid bilayer. Here, we overcome this problem by using fluorinated surfactants that are capable of preventing aggregation but, unlike detergents, do not themselves interact with the bilayer. We have applied the fluorescence correlation spectroscopy methodology to study surfactant-chaperoned insertion into preformed POPC (palmitoyloleoylphosphatidylcholine) vesicles of the two well-studied dye-labeled TM peptides of different lengths: WALP23 and WALP27. Extrapolation of the apparent free-energy values measured in the presence of surfactants to a zero surfactant concentration yielded free-energy values of -9.0±0.1 and -10.0±0.1 kcal/mol for insertion of WALP23 and WALP27, respectively. Circular dichroism measurements confirmed helical structure of peptides in lipid bilayer, in the presence of surfactants, and in aqueous mixtures of organic solvents. From a combination of thermodynamic and conformational measurements, we conclude that the partitioning of a four-residue L-A-L-A segment in the context of a continuous helical conformation from an aqueous environment into the hydrocarbon core of the membrane has a favorable free energy of 1 kcal/mol. Our measurements, combined with the predictions of two independent experimental hydrophobicity scales, indicate that the per-residue cost of transfer of the helical backbone from water to the hydrocarbon core of the lipid bilayer is unfavorable and is equal to +2.13±0.17 kcal/mol. PMID:22227387

Kyrychenko, Alexander; Rodnin, Mykola V; Posokhov, Yevgen O; Holt, Andrea; Pucci, Bernard; Killian, J Antoinette; Ladokhin, Alexey S

2012-02-24

41

Spin-labeled extracellular loop from a seven-transmembrane helix receptor: studies in solution and interaction with model membranes.  

PubMed

A spin-labeled pentadecapeptide was synthesized containing 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) as the N-terminal amino acid and residues 253-266 (EYWSTFGNLHHISL) of the mass oncogene receptor, a membrane-bound protein from the G-protein coupled receptors family. According to predictions, this protein folds into seven transmembrane helices connected by three extra- and three intracellular loops, and the peptide encompasses part of the third extracellular loop and part of the seventh helix. Electron paramagnetic resonance (EPR) spectra of the spin-labeled peptide (TOAC-14) were obtained in aqueous solution as a function of pH and temperature, in a secondary structure-inducing solvent [trifluoroethanol (TFE)], and in the presence of detergent micelles and phospholipid bilayers. The charged and uncharged amino groups of TOAC and TOAC-14 yielded spectra with different isotropic hyperfine splittings (aN). The slow exchange between protonated and unprotonated forms in the EPR time scale gave rise to composite spectra weighted by the Henderson-Hasselbalch equation. Plots of aN vs pH allowed the determination of the amino group pK values (8.4 and 4.5, for TOAC and TOAC-14, respectively). A small change in aN centered at pH 6.5 was ascribed to the titration of the histidines. Values of calculated rotational correlation times were indicative of a pH-induced conformational change. A conformational change was also observed in TFE. TOAC-14 bound to micelles irrespective of peptide and detergent head group charge. In contrast, the peptide bound to phospholipid bilayers only when both carried opposite charges. The slow exchange (in the EPR time scale) between membrane-bound and free TOAC-14 allowed the calculation of the peptide's partition coefficient. The spectral line shapes were affected by aggregate size and degree of packing of the constituent molecules. It is proposed that pH, polarity, and lipid environment can affect the conformation of water-exposed regions of membrane-bound receptors, thereby playing a role in the mechanism of signal transduction. PMID:10904553

Pertinhez, T A; Nakaie, C R; Paiva, A C; Schreier, S

1997-12-01

42

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

43

The Transmembrane Helix of the Escherichia coli Division Protein FtsI Localizes to the Septal Ring  

PubMed Central

FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of about a dozen division proteins that localize to the septal ring. FtsI comprises a short amino-terminal cytoplasmic domain, a single transmembrane helix (TMH), and a large periplasmic domain that encodes the catalytic (transpeptidase) activity. We show here that a 26-amino-acid fragment of FtsI is sufficient to direct green fluorescent protein to the septal ring in cells depleted of wild-type FtsI. This fragment extends from W22 to V47 and corresponds to the TMH. This is a remarkable finding because it is usual for a TMH to target a protein to a site more specific than the membrane. Alanine-scanning mutagenesis of the TMH identified several residues important for septal localization. These residues cluster on one side of an alpha-helix, which we propose interacts directly with another division protein to recruit FtsI to the septal ring.

Wissel, Mark C.; Wendt, Jennifer L.; Mitchell, Calista J.; Weiss, David S.

2005-01-01

44

Allosteric small molecules unveil a role of an extracellular E2/transmembrane helix 7 junction for G protein-coupled receptor activation.  

PubMed

G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M(2) acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M(2)Trp(422) in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists. PMID:17890226

Jäger, Dorothea; Schmalenbach, Caroline; Prilla, Stefanie; Schrobang, Jasmin; Kebig, Anna; Sennwitz, Matthias; Heller, Eberhard; Tränkle, Christian; Holzgrabe, Ulrike; Höltje, Hans-Dieter; Mohr, Klaus

2007-11-30

45

An essential sensor histidine kinase controlled by transmembrane helix interactions with its auxiliary proteins  

Microsoft Academic Search

Two-component signal transduction systems with membrane-embedded sensor histidine kinases are believed to recognize environmental signals and transduce this information over the cellular membrane to influence the activity of a transcription factor to which they are mated. The YycG sensor kinase of Bacillus subtilis, containing two transmembrane helices, is subject to a complicated activity-control circuit involving two other proteins with N-terminal

Hendrik Szurmant; Lintao Bu; Charles L. Brooks; James A. Hoch

2008-01-01

46

Detecting DNA-binding helix-turn-helix structural motifs using sequence and structure information  

PubMed Central

In this work, we analyse the potential for using structural knowledge to improve the detection of the DNA-binding helix–turn–helix (HTH) motif from sequence. Starting from a set of DNA-binding protein structures that include a functional HTH motif and have no apparent sequence similarity to each other, two different libraries of hidden Markov models (HMMs) were built. One library included sequence models of whole DNA-binding domains, which incorporate the HTH motif, the second library included shorter models of ‘partial’ domains, representing only the fraction of the domain that corresponds to the functionally relevant HTH motif itself. The libraries were scanned against a dataset of protein sequences, some containing the HTH motifs, others not. HMM predictions were compared with the results obtained from a previously published structure-based method and subsequently combined with it. The combined method proved more effective than either of the single-featured approaches, showing that information carried by motif sequences and motif structures are to some extent complementary and can successfully be used together for the detection of DNA-binding HTHs in proteins of unknown function.

Pellegrini-Calace, Marialuisa; Thornton, Janet M.

2005-01-01

47

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

PubMed Central

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

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

2012-01-01

48

A helix-turn-helix structure unit in human centromere protein B (CENP-B).  

PubMed Central

CENP-B has been suggested to organize arrays of centromere satellite DNA into a higher order structure which then directs centromere formation and kinetochore assembly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically binds to the CENP-B box sequence (17 bp) in centromere DNA. We determined the solution structure of human CENP-B DBD RP1 by multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these proteins was not found. The DBD of yeast RAP1, a telomere binding protein, is most similar to CENP-B DBD RP1. We studied the interaction between CENP-B DBD RP1 and the CENP-B box by the use of NMR chemical shift perturbation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B box DNA, mainly at the N-terminal basic region, the N-terminal portion of helix 2 and helix 3.

Iwahara, J; Kigawa, T; Kitagawa, K; Masumoto, H; Okazaki, T; Yokoyama, S

1998-01-01

49

A helix-turn-helix structure unit in human centromere protein B (CENP-B).  

PubMed

CENP-B has been suggested to organize arrays of centromere satellite DNA into a higher order structure which then directs centromere formation and kinetochore assembly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically binds to the CENP-B box sequence (17 bp) in centromere DNA. We determined the solution structure of human CENP-B DBD RP1 by multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these proteins was not found. The DBD of yeast RAP1, a telomere binding protein, is most similar to CENP-B DBD RP1. We studied the interaction between CENP-B DBD RP1 and the CENP-B box by the use of NMR chemical shift perturbation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B box DNA, mainly at the N-terminal basic region, the N-terminal portion of helix 2 and helix 3. PMID:9451007

Iwahara, J; Kigawa, T; Kitagawa, K; Masumoto, H; Okazaki, T; Yokoyama, S

1998-02-01

50

Resource for structure related information on transmembrane proteins  

NASA Astrophysics Data System (ADS)

Transmembrane proteins are involved in a wide variety of vital biological processes including transport of water-soluble molecules, flow of information and energy production. Despite significant efforts to determine the structures of these proteins, only a few thousand solved structures are known so far. Here, we review the various resources for structure-related information on these types of proteins ranging from the 3D structure to the topology and from the up-to-date databases to the various Internet sites and servers dealing with structure prediction and structure analysis. Abbreviations: 3D, three dimensional; PDB, Protein Data Bank; TMP, transmembrane protein.

Tusnády, Gábor E.; Simon, István

51

Relative transmembrane segment rearrangements during BK channel activation resolved by structurally assigned fluorophore-quencher pairing.  

PubMed

Voltage-activated proteins can sense, and respond to, changes in the electric field pervading the cell membrane by virtue of a transmembrane helix bundle, the voltage-sensing domain (VSD). Canonical VSDs consist of four transmembrane helices (S1-S4) of which S4 is considered a principal component because it possesses charged residues immersed in the electric field. Membrane depolarization compels the charges, and by extension S4, to rearrange with respect to the field. The VSD of large-conductance voltage- and Ca-activated K(+) (BK) channels exhibits two salient inconsistencies from the canonical VSD model: (1) the BK channel VSD possesses an additional nonconserved transmembrane helix (S0); and (2) it exhibits a "decentralized" distribution of voltage-sensing charges, in helices S2 and S3, in addition to S4. Considering these unique features, the voltage-dependent rearrangements of the BK VSD could differ significantly from the standard model of VSD operation. To understand the mode of operation of this unique VSD, we have optically tracked the relative motions of the BK VSD transmembrane helices during activation, by manipulating the quenching environment of site-directed fluorescent labels with native and introduced Trp residues. Having previously reported that S0 and S4 diverge during activation, in this work we demonstrate that S4 also diverges from S1 and S2, whereas S2, compelled by its voltage-sensing charged residues, moves closer to S1. This information contributes spatial constraints for understanding the BK channel voltage-sensing process, revealing the structural rearrangements in a non-canonical VSD. PMID:22802360

Pantazis, Antonios; Olcese, Riccardo

2012-08-01

52

Relative transmembrane segment rearrangements during BK channel activation resolved by structurally assigned fluorophore-quencher pairing  

PubMed Central

Voltage-activated proteins can sense, and respond to, changes in the electric field pervading the cell membrane by virtue of a transmembrane helix bundle, the voltage-sensing domain (VSD). Canonical VSDs consist of four transmembrane helices (S1–S4) of which S4 is considered a principal component because it possesses charged residues immersed in the electric field. Membrane depolarization compels the charges, and by extension S4, to rearrange with respect to the field. The VSD of large-conductance voltage- and Ca-activated K+ (BK) channels exhibits two salient inconsistencies from the canonical VSD model: (1) the BK channel VSD possesses an additional nonconserved transmembrane helix (S0); and (2) it exhibits a “decentralized” distribution of voltage-sensing charges, in helices S2 and S3, in addition to S4. Considering these unique features, the voltage-dependent rearrangements of the BK VSD could differ significantly from the standard model of VSD operation. To understand the mode of operation of this unique VSD, we have optically tracked the relative motions of the BK VSD transmembrane helices during activation, by manipulating the quenching environment of site-directed fluorescent labels with native and introduced Trp residues. Having previously reported that S0 and S4 diverge during activation, in this work we demonstrate that S4 also diverges from S1 and S2, whereas S2, compelled by its voltage-sensing charged residues, moves closer to S1. This information contributes spatial constraints for understanding the BK channel voltage-sensing process, revealing the structural rearrangements in a non-canonical VSD.

Pantazis, Antonios

2012-01-01

53

Structure and function in rhodopsin: packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain are coupled.  

PubMed

A previous study of the retinitis pigmentosa mutation L125R and two designed mutations at this site, L125A and L125F, showed that these mutations cause partial or total misfolding of the opsins expressed in COS cells from the corresponding mutant opsin genes. We now report on expression and characterization of the opsins from the following retinitis pigmentosa mutants in the transmembrane domain of rhodopsin that correspond to six of the seven helices: G51A and G51V (helix A), G89D (helix B), A164V (helix D), H211P (helix E), P267L and P267R (helix F), and T297R (helix G). All the mutations caused partial misfolding of the opsins as observed by the UV/visible absorption characteristics and by separation of the expressed opsins into fractions that bound 11-cis-retinal to form the corresponding mutant rhodopsins and those that did not bind 11-cis-retinal. Further, all the mutant rhodopsins prepared from the above mutants, except for G51A, showed strikingly abnormal bleaching behavior with abnormal metarhodopsin II photointermediates. The results show that retinitis pigmentosa mutations in every one of the transmembrane helices can cause misfolding of the opsin. Therefore, on the basis of these and previous results, we conclude that defects in the packing of the transmembrane helices resulting from these mutations are relayed to the intradiscal domain, where they cause misfolding of the opsin by inducing the formation of a disulfide bond other than the native Cys-110---Cys-187 disulfide bond. Thus, there is coupling between packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain. PMID:9380676

Hwa, J; Garriga, P; Liu, X; Khorana, H G

1997-09-30

54

Structure and function in rhodopsin: Packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain are coupled*  

PubMed Central

A previous study of the retinitis pigmentosa mutation L125R and two designed mutations at this site, L125A and L125F, showed that these mutations cause partial or total misfolding of the opsins expressed in COS cells from the corresponding mutant opsin genes. We now report on expression and characterization of the opsins from the following retinitis pigmentosa mutants in the transmembrane domain of rhodopsin that correspond to six of the seven helices: G51A and G51V (helix A), G89D (helix B), A164V (helix D), H211P (helix E), P267L and P267R (helix F), and T297R (helix G). All the mutations caused partial misfolding of the opsins as observed by the UV/visible absorption characteristics and by separation of the expressed opsins into fractions that bound 11-cis-retinal to form the corresponding mutant rhodopsins and those that did not bind 11-cis-retinal. Further, all the mutant rhodopsins prepared from the above mutants, except for G51A, showed strikingly abnormal bleaching behavior with abnormal metarhodopsin II photointermediates. The results show that retinitis pigmentosa mutations in every one of the transmembrane helices can cause misfolding of the opsin. Therefore, on the basis of these and previous results, we conclude that defects in the packing of the transmembrane helices resulting from these mutations are relayed to the intradiscal domain, where they cause misfolding of the opsin by inducing the formation of a disulfide bond other than the native Cys-110—Cys-187 disulfide bond. Thus, there is coupling between packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain.

Hwa, John; Garriga, Pere; Liu, Xun; Khorana, H. Gobind

1997-01-01

55

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

PubMed Central

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

Hames, Cecile; Ptchelkine, Denis; Grimm, Clemens; Thevenon, Emmanuel; Moyroud, Edwige; Gerard, Francine; Martiel, Jean-Louis; Benlloch, Reyes; Parcy, Francois; Muller, Christoph W

2008-01-01

56

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

PubMed Central

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

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

2011-01-01

57

Comparative molecular field analysis using selectivity fields reveals residues in the third transmembrane helix of the serotonin transporter associated with substrate and antagonist recognition  

PubMed Central

The human serotonin transporter (hSERT) regulates the spatial and temporal actions of serotonin (5-HT) neurotransmission by removing 5-HT from the synapse. Previous studies have identified residues in the third transmembrane helix (TMH) that may be important for substrate translocation or antagonist recognition. We identified hSERT residues in TMH III that are divergent from drosophila SERT (dSERT) and employed species-scanning mutagenesis to generate reciprocal mutants. Transport inhibition assays suggest that the potency of substituted amphetamines was decreased for the hSERT mutants A169D, I172M, and S174M. Additionally, there was a loss of potency for several antidepressants and 3-phenyltropane analogs for the I172M mutant. These results suggest that residues in TMH III may contribute to antagonist recognition. We carried out Comparative Molecular Field Analyses (CoMFA) using selectivity fields to directly visualize the mutation-induced effects of antagonist potency for antidepressants, 3-phenyltropane analogs, and amphetamines. The hSERT I172M selectivity field analysis for the 3-phenyltropane analogs revealed that electrostatic interactions resulted in decreased potency. The amphetamine and antidepressant selectivity field analyses reveal the observed decreases in potencies for the hSERT I172M mutant are due to a change in tertiary structure of the hSERT protein and are not due to disruption of a direct binding site. Finally, the hSERT mutant A169D displayed altered kinetics for sodium binding, indicating that this residue may lie near the putative sodium-binding site. A SERT homology model developed from the Aquifex aeolicus leucine transporter structure provides a structural context for further interpreting the results of the TMH III mutations.

Walline, Crystal C.; Nichols, David E.; Carroll, F. Ivy; Barker, Eric L.

2009-01-01

58

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

59

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

PubMed Central

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

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

2013-01-01

60

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

PubMed Central

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

Somasekharan, Suma; Tanis, Jessica; Forbush, Biff

2012-01-01

61

Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif.  

PubMed Central

Ets-1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets-1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C-terminus. The ETS domain folds into a helix-turn-helix motif on a four-stranded anti-parallel beta-sheet scaffold. This structure places Ets-1 in the winged helix-turn-helix (wHTH) family of DNA binding proteins and provides a model for interpreting the sequence conservation of the ETS domain and the specific interaction of Ets-1 with DNA. The C-terminal sequence of Ets-1, which is mutated in the v-Ets oncoprotein, forms an alpha-helix that packs anti-parallel to the N-terminal helix of the ETS domain. In this position, the C-terminal helix is poised to interact directly with an N-terminal inhibitory region in Ets-1 as well as the wHTH motif. This explains structurally the concerted role of residues flanking the ETS domain in the intramolecular inhibition of Ets-1 DNA binding. Images

Donaldson, L W; Petersen, J M; Graves, B J; McIntosh, L P

1996-01-01

62

Structural basis for high-affinity volatile anesthetic binding in a natural 4-helix bundle protein.  

PubMed

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(A) values of approximately 10(5) M(-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 A 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. PMID:15791007

Liu, Renyu; Loll, Patrick J; Eckenhoff, Roderic G

2005-04-01

63

Thermal Analysis of a Helix TWT Slow-Wave Structure  

Microsoft Academic Search

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

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

2008-01-01

64

Structure of the spectrin superfamily: A three-?-helix motif  

NASA Astrophysics Data System (ADS)

Spectrin, ?-actinin and dystrophin make up a closely related group, termed spectrin superfamily. We have carried out a preliminary analysis of the repeating amino acid sequences in the rod-shaped domains of these proteins and made use of structural constraints imposed by ?-helical coiled-coil-like packing. We show that the common conformation of these repeats may be described four-?-helix motif found in many globular proteins.

Parry, David A. D.; Cohen, Carolyn

1991-05-01

65

Structural and thermodynamic insight into the process of "weak" dimerization of the ErbB4 transmembrane domain by solution NMR.  

PubMed

Specific helix-helix interactions between the single-span transmembrane domains of receptor tyrosine kinases are believed to be important for their lateral dimerization and signal transduction. Establishing structure-function relationships requires precise structural-dynamic information about this class of biologically significant bitopic membrane proteins. ErbB4 is a ubiquitously expressed member of the HER/ErbB family of growth factor receptor tyrosine kinases that is essential for the normal development of various adult and fetal human tissues and plays a role in the pathobiology of the organism. The dimerization of the ErbB4 transmembrane domain in membrane-mimicking lipid bicelles was investigated by solution NMR. In a bicellar DMPC/DHPC environment, the ErbB4 membrane-spanning ?-helices (651-678)(2) form a right-handed parallel dimer through the N-terminal double GG4-like motif A(655)GxxGG(660) in a fashion that is believed to permit proper kinase domain activation. During helix association, the dimer subunits undergo a structural adjustment (slight bending) with the formation of a network of inter-monomeric polar contacts. The quantitative analysis of the observed monomer-dimer equilibrium provides insights into the kinetics and thermodynamics of the folding process of the helical transmembrane domain in the model environment that may be directly relevant to the process that occurs in biological membranes. The lipid bicelles occupied by a single ErbB4 transmembrane domain behave as a true ("ideal") solvent for the peptide, while multiply occupied bicelles are more similar to the ordered lipid microdomains of cellular membranes and appear to provide substantial entropic enhancement of the weak helix-helix interactions, which may be critical for membrane protein activity. PMID:22579757

Bocharov, Eduard V; Mineev, Konstantin S; Goncharuk, Marina V; Arseniev, Alexander S

2012-09-01

66

Cystic fibrosis transmembrane conductance regulator (ABCC7) structure.  

PubMed

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

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

2013-02-01

67

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

68

The effect of point mutations on the free energy of transmembrane ?-helix dimerization 1 1 Edited by M. F. Moody  

Microsoft Academic Search

Glycophorin A forms homodimers through interaction of the single, helical transmembrane domains of the monomers. The dimers are stable in sodium dodecylsulfate (SDS), permitting a number of studies that have identified a critical motif of residues that mediates dimer formation. We have used analytical ultracentrifugation to measure the energy of dimerization in a non-denaturing detergent solution and have observed the

Karen G. Fleming; Anne L. Ackerman; Donald M. Engelman

1997-01-01

69

Analysis of a novel brazed helix tape slow wave structure with high power capability  

Microsoft Academic Search

Summary form only given. In order to enhance the power capability of the helix traveling wave tube, a novel helical slow wave structure (SWS) supported by 8 wedge shaped discontinuous dielectric pillars which exist only at the helix tape in each pitch, is developed in this paper. Each pillar can be easily brazed with the helix tape and the metal

Yubin Gong; Yanyu Wei; Wenxiang Wang; Zhaoyun Duan

2003-01-01

70

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

Microsoft Academic Search

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

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

1984-01-01

71

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

PubMed Central

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

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

2013-01-01

72

Effects of aromatic residues at the ends of transmembrane alpha-helices on helix interactions with lipid bilayers.  

PubMed

We have studied the effects of aromatic residues at the ends of peptides of the type Ac-KKGL(n)()WL(m)()KKA-amide on their interactions with lipid bilayers as a function of lipid fatty acyl chain length, physical phase, and charge. Peptide Ac-KKGFL(6)WL(8)FKKA-amide (F(2)L(14)) incorporated into bilayers of phosphatidylcholines containing monounsaturated fatty acyl chains of lengths C14-C24 at a peptide:lipid molar ratio of 1:100 in contrast to Ac-KKGL(7)WL(9)KKA-amide (L(16)) which did not incorporate at all into dierucoylphosphatidylcholine [di(C24:1)PC]; Ac-KKGYL(6)WL(8)YKKA-amide (Y(2)L(14)) incorporated partly into di(C24:1)PC. Lipid-binding constants relative to that for dioleoylphosphatidylcholine (C18:1)PC were obtained using a fluorescence quenching method. For Y(2)L(14) and F(2)L(14), relative lipid-binding constants increased with increasing fatty acyl chain length from C14 to C24; strongest binding did not occur at the point where the hydrophobic length of the peptide equalled the hydrophobic thickness of the bilayer. For Ac-KKGYL(9)WL(11)YKKA-amide (Y(2)L(20)), increasing chain length from C18 to C24 had little effect on relative binding constants. Anionic phospholipids bound more strongly than zwitterionic phospholipids to Y(2)L(14) and Y(2)L(20) but effects of charge were relatively small. In two phase (gel and liquid crystalline) mixtures, all the peptides partitioned more strongly into liquid crystalline than gel phase; effects were independent of the structure of the peptide or of the lipid (dipalmitoylphosphatidylcholine or bovine brain sphingomyelin). Addition of cholesterol had little effect on incorporation of the peptides into lipid bilayers. It is concluded that the presence of aromatic residues at the ends of transmembrane alpha-helices effectively buffers them against changes in bilayer thickness caused either by an increase in the chain length of the phospholipid or by the presence of cholesterol. PMID:10684657

Mall, S; Broadbridge, R; Sharma, R P; Lee, A G; East, J M

2000-02-29

73

BiHelix: Towards de novo Structure Prediction of an Ensemble of G-Protein Coupled Receptor Conformations  

PubMed Central

G-Protein Coupled Receptors (GPCRs) play a critical role in cellular signal transduction pathways and are prominent therapeutic targets. Recently there has been major progress in obtaining experimental structures for a few GPCRs. Each GPCR, however, exhibits multiple conformations that play a role in their function and we have been developing methods aimed at predicting structures for all these conformations. Analysis of available structures shows that these conformations differ in relative helix tilts and rotations. The essential issue is, determining how to orient each of the 7 helices about its axis since this determines how it interacts with the other 6 helices. Considering all possible helix rotations to ensure that no important packings are overlooked, and using rotation angle increments of 30° about the helical axis would still lead to 127 or 35 million possible conformations each with optimal residue positions. We show in this paper how to accomplish this. The fundamental idea is to optimize the interactions between each pair of contacting helices while ignoring the other 5 and then to estimate the energies of all 35 million combinations using these pair-wise interactions. This BiHelix approach dramatically reduces the effort to examine the complete set of conformations and correctly identifies the crystal packing for the experimental structures plus other near-native packings we believe may play an important role in activation. This approach also enables a detailed structural analysis of functionally distinct conformations using helix-helix interaction energy landscapes and should be useful for other helical transmembrane proteins as well.

Abrol, Ravinder; Bray, Jenelle K.; Goddard, William A.

2011-01-01

74

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

PubMed

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

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

2014-01-01

75

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

SciTech Connect

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

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

2008-11-18

76

Sequence Specificity in the Dimerization of Transmembrane a-Helices?  

Microsoft Academic Search

While several reports have suggested a role for helix-helix interactions in membrane protein oligomerization, there are few direct biochemical data bearing on this subject. Here, using mutational analysis, we show that dimerization of the transmembrane a-helix of glycophorin A in a detergent environment is spontaneous and highly specific. Very subtle changes in the side-chain structure at certain sensitive positions disrupt

Mark A. Lemmon; John M. Flanagan; Herbert R. Treutlein; Jian Zhang; Donald M. Engelman

1992-01-01

77

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

PubMed

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

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

2014-07-01

78

F?rster resonance energy transfer in liposomes: Measurements of transmembrane helix dimerization in the native bilayer environment  

PubMed Central

The lipid bilayer vesicle is a model of the cellular membrane. Even in this simple system, however, measuring the thermodynamics of membrane protein association is a challenge. Here we discuss Förster resonance energy transfer (FRET) in liposomes as a method to probe the dimerization of transmembrane helices in a membrane environment. Although the measurements are labor intensive, FRET in liposomes can be measured accurately provided that attention is paid to sample homogeneity and sample equilibration. One must also take into account statistical expectations and the FRET that results from random colocalization of donors and acceptors in the bilayer. Without careful attention to these details, misleading results are easy to obtain in membrane FRET experiments. The results that we obtain in model systems are reproducible and depend solely on the concentration of the protein in the bilayer (i.e., on the protein-to-lipid ratio), thereby yielding thermodynamic parameters that are directly relevant to processes in biological membranes.

You, Min; Li, Edwin; Wimley, William C.; Hristova, Kalina

2008-01-01

79

Secondary Structure, Orientation, Oligomerization, and Lipid Interactions of the Transmembrane Domain of Influenza Hemagglutinin †  

Microsoft Academic Search

Influenza virus hemagglutinin (HA), the viral envelope glycoprotein that mediates fusion between the viral and cellular membranes, is a homotrimer of three subunits, each containing two disulfide- linked polypeptide chains, HA1 and HA2. Each HA2 chain spans the viral membrane with a single putative transmembrane R-helix near its C-terminus. Fusion experiments with recombinant HAs suggest that this sequence is required

Suren A. Tatulian; Lukas K. Tamm

2000-01-01

80

Low-coupling impedance double-helix structure for use in a ferrite kicker magnet  

SciTech Connect

In a machine such as the CBA, the ejection ferrite kicker magnet has a very large longitudinal and transverse coupling impedance which could destroy the beam. Using a double-helix structure that surrounds the beam, the beam-induced fields are confined within the helix and, therefore, decoupled from the kicker; but at the same time the helix is transparent to the external fields of the kicker. At first, this may seem paradoxical that the helix is opaque to the fields generated inside the structure by the beam and simultaneously transparent to the external fields generated by the kicker.

Giordano, S.

1983-01-01

81

Procaine, a state-dependent blocker, inhibits HERG channels by helix residue Y652 and F656 in the S6 transmembrane domain.  

PubMed

The article evaluated the inhibitory action of procaine on wild-type and mutated HERG potassium channel current (I(HERG)) to determine whether mutations in the S6 region are important for the inhibition of I(HERG) by procaine. HERG channels (WT, Y652A, and F656A) were expressed in Xenopus laevis oocytes and studied using the standard two-microelectrode voltage-clamp technique. The results revealed that WT HERG is blocked in a concentration-, voltage-, and state-dependent manner by procaine ([IC??] = 34.79 ?M). The steady state activation curves slightly move to the negative, while inactivation parameters move to the positive in the presence of procaine. Time-dependent test reveals that voltage-dependent I(HERG) blockade occurs extremely rapidly. Furthermore, the mutation to Ala of Y652 and F656 produce about 11-fold and 18-fold increases in IC?? for I(HERG) blockade, respectively. Simultaneously, for Y652A, the steady state activation and inactivation parameters are shifted to more positive values after perfusion of procaine. Conclusively, procaine state-dependently inhibits HERG channels (WT, Y652A, and F656A). The helix residues Y652 and F656 in the S6 transmembrane domain might play a role in interaction of the drug with the channel. PMID:24005047

Wang, Na; Ma, Ji Hua; Zhang, Pei Hua

2013-09-20

82

Design of helix slow-wave structures for high efficiency TWTs  

Microsoft Academic Search

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

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

2000-01-01

83

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

84

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

2013-01-01

85

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

86

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

87

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

NASA Astrophysics Data System (ADS)

A planar helix, constituted of a pair of unidirectionally conducting screens conducting in different directions, is suggested as a slow-wave structure for application in a traveling-wave tube (TWT). Circuit parameters, such as interaction impedance and space-charge parameter, are derived for the suggested planar-helix TWT. Computed results for the planar helix indicate a performance comparable with that of its circular helix counterpart. Also, the change in interaction impedance and the dispersion characteristics of the planar helix, are considered in the presence of dielectric substrates and a metal shield. Results are obtained for a few different possible configurations of the planar helix on dielectric substrates with or without a metal shield. The phase velocity and interaction impedance reduce, both as the substrate thickness increases and as the metal shields are brought closer to the planar helix. However, the resulting degradation in the characteristics of the structure with commonly used substrate materials, for example alumina and beryllia, is less severe than in the case of the circular helix.

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

1984-02-01

88

Revisiting cystic fibrosis transmembrane conductance regulator structure and function.  

PubMed

The cystic fibrosis transmembrane conductance regulator (CFTR) is a channel/enzyme which mediates passive diffusion of chloride and bicarbonate through epithelial cell membranes. It is expressed in many cell types throughout the body, but in the airways it is found mainly in secretory serous cells of the submucosal glands. CFTR belongs to a large super-family of ATP binding cassette transporters that have two nucleotide binding domains with characteristic sequences or "motifs". Although most other ATP binding cassette transporters consume ATP to actively transport various substrates, in CFTR the interactions of ATP with nucleotide binding domains control opening and closing of the channel pore (i.e., channel gating). Recent high resolution structures of bacterial nucleotide binding domains combined with new biochemical and electrophysiological studies of CFTR itself have led to major advances in our understanding of CFTR gating. For example, it is now clear that the ATPase activity of CFTR is not strictly required for its channel activity. CFTR has at least two distinct gating modes; one dependent on hydrolysis and the other requiring only stable ATP binding. In this article we discuss a working hypothesis for CFTR that incorporates these recent findings and discuss some interesting implications of the paradigm shift for other aspects of CFTR function and dysfunction. PMID:16113406

Hanrahan, John W; Wioland, My-Anh

2004-01-01

89

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

NASA Astrophysics Data System (ADS)

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

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

2013-03-01

90

Thermal analysis of PPM-focused rod-supported TWT helix structures  

Microsoft Academic Search

A thermal analysis, which can be implemented on a small computer system, has been developed for PPM (periodic permanent-magnet)-focused rod-supported TWT (traveling-wave tube) helix structures. The method fully accounts for the variable thermal conductivity of several of the materials used in helix assemblies and considers the nonuniform geometry of the various elements of the structure along the direction of heat

Roberto Crivello; Richard W. Grow

1988-01-01

91

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

PubMed

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

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

2009-12-22

92

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

93

Phosphorylation by cAMP-dependent protein kinase modulates the structural coupling between the transmembrane and cytosolic domains of phospholamban.  

PubMed

We have used frequency-domain fluorescence spectroscopy to investigate the structural linkage between the transmembrane and cytosolic domains of the regulatory protein phospholamban (PLB). Using an engineered PLB having a single cysteine (Cys(24)) derivatized with the fluorophore N-(1-pyrenyl)maleimide (PMal), we have used fluorescence resonance energy transfer (FRET) to measure the average spatial separation and conformational heterogeneity between PMal bound to Cys(24) in the transmembrane domain and Tyr(6) in the cytosolic domain near the amino terminus of PLB. In these measurements, PMal serves as a FRET donor, and Tyr(6) serves as a FRET acceptor following its nitration by tetranitromethane. The native structure of PLB is retained following site-directed mutagenesis and chemical modification, as indicated by the ability of the derivatized PLB to fully regulate the Ca-ATPase following their co-reconstitution. To assess how phosphorylation modulates the structure of PLB itself, FRET measurements were made following reconstitution of PLB in membrane vesicles made from extracted sarcoplasmic reticulum membrane lipids. We find that the cytosolic domain of PLB assumes a wide range of conformations relative to the transmembrane sequence, consistent with other structural data indicating the presence of a flexible hinge region between the transmembrane and cytosolic domains of PLB. Phosphorylation of Ser(16) by PKA results in a 3 A decrease in the spatial separation between PMal at Cys(24) and nitroTyr(6) and an almost 2-fold decrease in conformational heterogeneity, suggesting a stabilization of the hinge region of PLB possibly through an electrostatic linkage between phosphoSer(16) and Arg(13) that promotes a coil-to-helix transition. This structural transition has the potential to function as a conformational switch, since inhibition of the Ca-ATPase requires disruption of the secondary structure of PLB in the vicinity of the hinge element to permit association with the nucleotide binding domain at a site located approximately 50 A above the membrane surface. Following phosphorylation, the stabilization of the helical content in the hinge domain will disrupt this inhibitory interaction by reducing the maximal dimension of the cytosolic domain of PLB. Thus, stabilization of the structure of PLB following phosphorylation of Ser(16) is part of a switching mechanism, which functions to alter binding interactions between PLB and the nucleotide binding domain of the Ca-ATPase that modulates enzyme inhibition. PMID:12962492

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

2003-09-16

94

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

Microsoft Academic Search

The one-dimensional problem of selecting the triple helix with the highest volume fraction is solved and hence the condition\\u000a for a helix to be close-packed is obtained. The close-packed triple helix is shown to have a pitch angle of v\\u000a \\u000a CP\\u000a = 43.3°. Contrary to the conventional notion, we suggest that close packing form the underlying principle behind the structure\\u000a of

Jakob Bohr; Kasper Olsen

2010-01-01

95

Helix capping.  

PubMed Central

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

Aurora, R.; Rose, G. D.

1998-01-01

96

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

PubMed Central

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

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

2011-01-01

97

Structure of the Transmembrane Cysteine Residues in Phospholamban  

Microsoft Academic Search

.   Phospholamban, a 52-residue membrane protein, associates to form a pentameric complex of five long ?-helices traversing the\\u000a sarcoplasmic reticulum membrane of cardiac muscle cells. The transmembrane domain of the protein is largely hydrophobic, with\\u000a only three cysteine residues having polar side chains, yet it functions as a Ca2+-selective ion channel. In this report, infrared spectroscopy is used to probe

I. T. Arkin; P. D. Adams; A. T. Brünger; S. Aimoto; D. M. Engelman; S. O. Smith

1997-01-01

98

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

99

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

2012-11-16

100

3D structure and significance of the GPhiXXG helix packing motif in tetramers of the E1beta subunit of pyruvate dehydrogenase from the archeon Pyrobaculum aerophilum.  

PubMed

As part of a structural genomics project, we have determined the 2.0 A structure of the E1beta subunit of pyruvate dehydrogenase from Pyrobaculum aerophilum (PA), a thermophilic archaeon. The overall fold of E1beta from PA is closely similar to the previously determined E1beta structures from humans (HU) and P. putida (PP). However, unlike the HU and PP structures, the PA structure was determined in the absence of its partner subunit, E1alpha. Significant structural rearrangements occur in E1beta when its E1alpha partner is absent, including rearrangement of several secondary structure elements such as helix C. Helix C is buried by E1alpha in the HU and PP structures, but makes crystal contacts in the PA structure that lead to an apparent beta(4) tetramer. Static light scattering and sedimentation velocity data are consistent with the formation of PA E1beta tetramers in solution. The interaction of helix C with its symmetry-related counterpart stabilizes the tetrameric interface, where two glycine residues on the same face of one helix create a packing surface for the other helix. This GPhiXXG helix-helix interaction motif has previously been found in interacting transmembrane helices, and is found here at the E1alpha-E1beta interface for both the HU and PP alpha(2)beta(2) tetramers. As a case study in structural genomics, this work illustrates that comparative analysis of protein structures can identify the structural significance of a sequence motif. PMID:11724561

Kleiger, G; Perry, J; Eisenberg, D

2001-12-01

101

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

SciTech Connect

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

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

2011-12-31

102

A soluble mutant of the transmembrane receptor Af1503 features strong changes in coiled-coil periodicity.  

PubMed

Structures of full-length, membrane-bound proteins are essential for understanding transmembrane signaling mechanisms. However, in prokaryotic receptors no such structure has been reported, despite active research for many years. Here we present results of an alternative strategy, whereby a transmembrane receptor is made soluble by selective mutations to the membrane-spanning region, chosen by analysis of helix geometry in the transmembrane regions of chemotaxis receptors. We thus converted the receptor Af1503 from Archaeoglobus fulgidus to a soluble form by deleting transmembrane helix 1 and mutating the surface residues of transmembrane helix 2 to hydrophilic amino acids. Crystallization of this protein resulted in the structure of a tetrameric proteolytic fragment representing the modified transmembrane helices plus the cytoplasmic HAMP domain, a ubiquitous domain of prokaryotic signal transducers. The protein forms a tetramer via native parallel dimerization of the HAMP domain and non-native antiparallel dimerization of the modified transmembrane helices. The latter results in a four-helical coiled coil, characterized by unusually large changes in helix periodicity. The structure offers the first view of the junction between the transmembrane region and HAMP and explains the conservation of a key sequence motif in HAMP domains. PMID:24568954

Hartmann, Marcus D; Dunin-Horkawicz, Stanislaw; Hulko, Michael; Martin, Jörg; Coles, Murray; Lupas, Andrei N

2014-06-01

103

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

PubMed Central

We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of ?-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in ?-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of ?-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide.

del Val, Coral; White, Stephen H.

2014-01-01

104

Genomic organization and regulation of a human 7-helix transmembrane receptor which is expressed in pulmonary epithelial cells and induced in hypoxia.  

PubMed

The genetic, regulatory, and tissue-specific characterization of G-protein-coupled receptors is substantial since it contributes to the identification of the natural ligand which may influence basic physiological processes and cell function. Here we explored the genomic structure of a human orphan seven-transmembrane receptor which presents the human homologue of a receptor which has been controversially identified as a rat adrenomedullin receptor subtype. Based on the cDNA sequence a 3.4 kb genomic DNA fragment was isolated. Sequencing of the fragment and comparison studies revealed an intron of 544 bp in the 5' untranslated region, followed by a second exon encoding the receptor protein of 404 amino acids. The gene is localized on chromosome 12q. The 5' regulatory region contains several SP1, AP2, and CAAT sites as well as hypoxia responsive elements (HRE) both in the 5' and 3' regulatory region. RT-PCR with intron spanning primers demonstrated mRNA signals in various tissues, especially in lung. Characterizing the histological expression pattern in lung sections by nonisotopic in situ hybridization, a strong signal of receptor mRNA was identified in pulmonary epithelial cells of bronchi and alveoli. Analysis of the two human pulmonary epithelial cell lines, H23 and A549, showed significant mRNA induction of this receptor subtype in hypoxia. PMID:11883938

Hänze, Jörg; Groneberg, David A; Rose, Frank; Hanisch, Anika; Dötsch, Jörg; Peiser, Christian; Seeger, Werner; Rascher, Wolfgang; Fischer, Axel; Grimminger, Friedrich

2002-03-15

105

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

106

Three-dimensional structure and antigenicity of transmembrane-protein peptides of the human immunodeficiency virus type 1. Effects of a neutralization-escape substitution.  

PubMed

A point mutation (Ala-589 to Thr) in the transmembrane protein of the human immunodeficiency virus type 1 (HIV-1) has been shown to decrease the sensitivity of the virus to the neutralizing effect of human HIV-1 specific antibodies [(1990) J. Virol. 64, 3240-3248]. Here 17-residue peptides with the parental and mutant sequences were compared: the parental peptide bound antibodies of sera from HIV-1 infected persons more frequently and with higher affinity than the mutant peptide. However, according to circular dichroism (CD), NMR spectroscopy and molecular modelling the peptides have indistinguishable backbone conformations under a variety of experimental conditions. These techniques showed for both peptides that no ordered helix was present in water solution. However, for both peptides in alcohol-water solutions approximately 60% alpha-helix could be induced. The three-dimensional structures of these peptides provide a basis for understanding how this mutation in the transmembrane protein may affect the interaction with both the outer envelope glycoprotein and with antibodies. PMID:8495750

Klasse, J; Pipkorn, R; Blomberg, J; Han, K H; Hilton, B; Ferretti, J A

1993-05-24

107

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

NASA Astrophysics Data System (ADS)

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

Eisenberg, David

2003-09-01

108

Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase.  

PubMed

Flavocytochrome b(558) (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22(phox) and gp91(phox). The latter subunit, also known as Nox2, has a cytosolic C-terminal "dehydrogenase domain" containing FAD/NADPH-binding sites. The N-terminal half of Nox2 contains six predicted transmembrane ?-helices coordinating two hemes. We studied the role of the second transmembrane ?-helix, which contains a "hot spot" for mutations found in rare X(+) and X(-) chronic granulomatous disease. By site-directed mutagenesis and transfection in X-CGD PLB-985 cells, we examined the functional and structural impact of seven missense mutations affecting five residues. P56L and C59F mutations drastically influence the level of Nox2 expression indicating that these residues are important for the structural stability of Nox2. A53D, R54G, R54M, and R54S mutations do not affect spectral properties of oxidized/reduced cytb, oxidase complex assembly, FAD binding, nor iodonitrotetrazolium (INT) reductase (diaphorase) activity but inhibit superoxide production. This suggests that Ala-53 and Arg-54 are essential in control of electron transfer from FAD. Surprisingly, the A57E mutation partially inhibits FAD binding, diaphorase activity, and oxidase assembly and affects the affinity of immunopurified A57E cytochrome b(558) for p67(phox). By competition experiments, we demonstrated that the second transmembrane helix impacts on the function of the first intracytosolic B-loop in the control of diaphorase activity of Nox2. Finally, by comparing INT reductase activity of immunopurified mutated and wild type cytb under aerobiosis versus anaerobiosis, we showed that INT reduction reflects the electron transfer from NADPH to FAD only in the absence of superoxide production. PMID:21659519

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

2011-08-12

109

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

PubMed Central

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

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

2011-01-01

110

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

PubMed

The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22-46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45-62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21-61). (13)C-(2)H distance measurements of (13)C-labeled protein and (2)H-labeled amantadine showed that in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers, the first equivalent of drug bound S31 inside the M2(21-61) pore, similar to the behavior of M2 transmembrane peptide (M2TM) in DMPC bilayers. The nonspecific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21-61) is S31 in the TM pore. Interestingly, when M2(21-61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, whereas M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state but did not rescue drug binding to M2(21-61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helix to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of this protein. PMID:21661724

Cady, Sarah; Wang, Tuo; Hong, Mei

2011-08-01

111

Conserved polar residues in the transmembrane domain of the human tachykinin NK2 receptor: functional roles and structural implications.  

PubMed Central

We have studied the effects of agonist and antagonist binding, agonist-induced activation and agonist-induced desensitization of the human tachykinin NK2 receptor mutated at polar residues Asn-51 [in transmembrane helix 1 (TM1)], Asp-79 (TM2) and Asn-303 (TM7), which are highly conserved in the transmembrane domain in the rhodopsin family of G-protein-coupled receptors. Wild-type and mutant receptors were expressed in both COS-1 cells and Xenopus oocytes. The results show that the N51D mutation results in a receptor which, in contrast with the wild-type receptor, is desensitized by the application of a concentration of 1 microM of the partial agonist GR64349, indicating that the mutant is more sensitive to agonist activation than is the wild-type receptor. In addition, we show that, whereas the D79E mutant displayed activation properties similar to those of the wild-type receptor, the D79N and D79A mutants displayed a severely impaired ability to activate the calcium-dependent chloride current. This suggests that it is the negative charge at Asn-79, rather than the ability of this residue to hydrogen-bond, that is critical for the activity of the receptor. Interestingly, the placement of a negative charge at position 303 could compensate for the removal of the negative charge at position 79, since the double mutant D79N/N303D displayed activation properties similar to those of the wild-type receptor. This suggests that these two residues are functionally coupled, and may even be in close proximity in the three-dimensional structure of the human tachykinin NK2 receptor. A three-dimensional model of the receptor displaying this putative interaction is presented.

Donnelly, D; Maudsley, S; Gent, J P; Moser, R N; Hurrell, C R; Findlay, J B

1999-01-01

112

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

NASA Astrophysics Data System (ADS)

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

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

2013-06-01

113

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

114

Structural features that modulate the transmembrane migration of a hydrophobic peptide in lipid vesicles.  

PubMed Central

Two approaches employing nuclear magnetic resonance (NMR) were used to investigate the transmembrane migration rate of the C-terminal end of native alamethicin and a more hydrophobic analog called L1. Native alamethicin exhibits a very slow transmembrane migration rate when bound to phosphatidylcholine vesicles, which is no greater than 1 x 10(-4) min(-1). This rate is much slower than expected, based on the hydrophobic partition energies of the amino acid side chains and the backbone of the exposed C-terminal end of alamethicin. The alamethicin analog L1 exhibits crossing rates that are at least 1000 times faster than that of native alamethicin. A comparison of the equilibrium positions of these two peptides shows that L1 sits approximately 3-4 A deeper in the membrane than does native alamethicin (Barranger-Mathys and Cafiso. 1996. Biochemistry. 35:489). The slow rate of alamethicin crossing can be explained if the peptide helix is irregular at its C-terminus and hydrogen bonded to solvent or lipid. We postulate that L1 does not experience as large a barrier to transport because its C-terminus is already buried within the membrane interface. This difference is most easily explained by conformational differences between L1 and alamethicin rather than differences in hydrophobicity. The results obtained here demonstrate that side-chain hydrophobicity alone cannot account for the energy barriers to peptide and protein transport across membranes.

Jayasinghe, S; Barranger-Mathys, M; Ellena, J F; Franklin, C; Cafiso, D S

1998-01-01

115

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

116

A Combined Analysis of the Cystic Fibrosis Transmembrane Conductance Regulator: Implications for Structure and Disease Models  

Microsoft Academic Search

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

Jian-Min Chen; Chris Cutler; Caroline Jacques; Erick Denamur; Guillaume Lecointre; Bernard Mercier; Gordon Cramb; Claude Ferec

117

Structural aspects of oligomerization taking place between the transmembrane ?-helices of bitopic membrane proteins  

Microsoft Academic Search

Recent advances in biophysical methods have been able to shed more light on the structures of helical bundles formed by the transmembrane segments of bitopic membrane proteins. In this manuscript, I attempt to review the biological importance and diversity of these interactions, the energetics of bundle formation, motifs capable of inducing oligomerization and methods capable of detecting, solving and predicting

Isaiah T. Arkin

2002-01-01

118

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

PubMed

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

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

2014-07-01

119

Structure of a Novel Winged-Helix Like Domain from Human NFRKB Protein  

PubMed Central

The human nuclear factor related to kappa-B-binding protein (NFRKB) is a 1299-residue protein that is a component of the metazoan INO80 complex involved in chromatin remodeling, transcription regulation, DNA replication and DNA repair. Although full length NFRKB is predicted to be around 65% disordered, comparative sequence analysis identified several potentially structured sections in the N-terminal region of the protein. These regions were targeted for crystallographic studies, and the structure of one of these regions spanning residues 370–495 was determined using the JCSG high-throughput structure determination pipeline. The structure reveals a novel, mostly helical domain reminiscent of the winged-helix fold typically involved in DNA binding. However, further analysis shows that this domain does not bind DNA, suggesting it may belong to a small group of winged-helix domains involved in protein-protein interactions.

Kumar, Abhinav; Mocklinghoff, Sabine; Yumoto, Fumiaki; Jaroszewski, Lukasz; Farr, Carol L.; Grzechnik, Anna; Nguyen, Phuong; Weichenberger, Christian X.; Chiu, Hsiu-Ju; Klock, Heath E.; Elsliger, Marc-Andre; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Conklin, Bruce R.; Fletterick, Robert J.; Wilson, Ian A.

2012-01-01

120

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

121

Structure of the pore-helix of the hERG K + channel  

Microsoft Academic Search

The hERG K+ channel undergoes rapid inactivation that is mediated by ‘collapse’ of the selectivity filter, thereby preventing ion conduction.\\u000a Previous studies have suggested that the pore-helix of hERG may be up to seven residues longer than that predicted by homology\\u000a with channels with known crystal structures. In the present work, we determined structural features of a peptide from the

Guilhem Pages; Allan M. Torres; Pengchu Ju; Paramjit S. Bansal; Paul F. Alewood; Philip W. Kuchel; Jamie I. Vandenberg

2009-01-01

122

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

123

Dimeric structure of the transmembrane domain of glycophorin a in lipidic and detergent environments.  

PubMed

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

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

2011-04-01

124

Shielded helix traveling wave cathode ray tube deflection structure  

SciTech Connect

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

125

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

PubMed

MgtR, a highly hydrophobic peptide expressed in Salmonella enterica serovar Typhimurium, inhibits growth in macrophages through binding to the membrane protein MgtC that has been identified as essential for replication in macrophages. While the Mycobacterium tuberculosis MgtC is highly homologous to its S. Typhi analogue, there does not appear to be an Mtb homologue for MgtR, raising significant pharmacological interest in this system. Here, solid-state NMR and EPR spectroscopy in lipid bilayer preparations were used to demonstrate the formation of a heterodimer between S. Typhi MgtR and the transmembrane helix 4 of Mtb MgtC. Based on the experimental restraints, a structural model of this heterodimer was developed using computational techniques. The result is that MgtR appears to be ideally situated in the membrane to influence the functionality of MgtC. PMID:24140750

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

2014-01-23

126

Impact of Histidine Residues on the Transmembrane Helices of Viroporins  

PubMed Central

The role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the transmembrane 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 transmembrane 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.

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

2014-01-01

127

A secondary structural transition in the C-helix promotes gating of cyclic nucleotide-regulated ion channels.  

PubMed

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

Puljung, Michael C; Zagotta, William N

2013-05-01

128

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

PubMed Central

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

Puljung, Michael C.; Zagotta, William N.

2013-01-01

129

Structural parameters of the myelin transmembrane proteolipid in reverse micelles.  

PubMed Central

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

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

1989-01-01

130

Predicting loop-helix tertiary structural contacts in RNA pseudoknots.  

PubMed

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 (DeltaH) of -14 kcal/mol and a contact entropy (DeltaS) of -38 cal/mol/K for a protonated C(+)*(G-C) base triple at pH 7.0, and (DeltaH = -7 kcal/mol, DeltaS = -19 cal/mol/K) for an unprotonated base triple. Tests of the model for a series of pseudoknots show good theory-experiment agreement. Based on the extracted energy parameters for the tertiary structural contacts, the model enables predictions for the structure, stability, and folding pathways for RNA pseudoknots with known or postulated loop-stem tertiary contacts from the nucleotide sequence alone. PMID:20100813

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

2010-03-01

131

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

PubMed

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

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

2005-05-01

132

Shielded helix traveling wave cathode ray tube deflection structure  

DOEpatents

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

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

1992-12-15

133

Shielded helix traveling wave cathode ray tube deflection structure  

DOEpatents

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

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

1992-01-01

134

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

135

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

NASA Astrophysics Data System (ADS)

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

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

2013-10-01

136

Structure-based thermodynamic scale of alpha-helix propensities in amino acids.  

PubMed

A structural parameterization of the folding energetics has been used to predict the effect of single amino acid mutations at exposed locations in alpha-helices. The results have been used to derive a structure-based thermodynamic scale of alpha-helix propensities for amino acids. The structure-based thermodynamic analysis was performed for four different systems for which structural and experimental thermodynamic data are available: T4 lysozyme [Blaber et al (1994) J. Mol. Biol.235, 600-624], barnase [Horovitz et al. (1992) J.Mol.Biol.227,560-568], a synthetic leucine zipper [O'Neil & Degrado (1990) Science 250, 646-651], and a synthetic peptide [Lyu et al. (1990) Science 250, 669-673]. These studies have permitted the optimization of the set of solvent-accessible surface areas (ASA) for all amino acids in the unfolded state. It is shown that a single set of structure/thermodynamic parameters accounts well for all the experimental data sets of helix propensities. For T4 lysozyme, the average value of the absolute difference between predicted and experimental delta G values is 0.09 kcal/mol, for barnase 0.14 kcal/mol, for the synthetic coiled-coil 0.11 kcal/mol, and for the synthetic peptide 0.08 kcal/mol. In addition, this approach predicts well the overall stability of the proteins and rationalizes the differences in alpha-helix propensities between amino acids. The excellent agreement observed between predicted and experimental delta G values for all amino acids validates the use of this structural parameterization in free energy calculations for folding or binding. PMID:8885848

Luque, I; Mayorga, O L; Freire, E

1996-10-22

137

Role of helix 8 in G protein-coupled receptors based on structure-function studies on the type 1 angiotensin receptor.  

PubMed

G protein-coupled receptors (GPCRs) are transmembrane receptors that convert extracellular stimuli to intracellular signals. The type 1 angiotensin II receptor is a widely studied GPCR with roles in blood pressure regulation,water and salt balance and cell growth. The complex molecular and structural changes that underpin receptor activation and signaling are the focus of intense research. Increasingly, there is an appreciation that the plasma membrane participates in receptor function via direct, physical interactions that reciprocally modulate both lipid and receptor and provide microdomains for specialized activities. Reversible protein:lipid interactions are commonly mediated by amphipathic -helices in proteins and one such motif - a short helix, referred to as helix VIII/8 (H8), located at the start of the carboxyl (C)-terminus of GPCRs - is gaining recognition for its importance to GPCR function. Here, we review the identification of H8 in GPCRs and examine its capacity to sense and interact with diverse proteins and lipid environment, most notably with acidic lipids that include phosphatidylinositol phosphates. PMID:19418628

Huynh, John; Thomas, Walter Glen; Aguilar, Marie-Isabel; Pattenden, Leonard Keith

2009-04-29

138

Biophysical investigations of structural and stability changes in helix 69 of Escherichia coli 23S rRNA  

Microsoft Academic Search

Helix 69 is a region of significant interest, which is located in the 50S subunit at the subunit interface of the ribosome. This research focused on identifying structural changes in this region upon changes in solution conditions such as pH and Mg2+ concentration. Furthermore, the roles of naturally occurring, highly conserved modified nucleotides in helix 69, namely pseudouridine and 3-methylpseudouridine,

Sanjaya Chinthaka Abeysirigunawardena

2008-01-01

139

Role of Basic Residues within or near the Predicted Transmembrane Helix 2 of the Human Breast Cancer Resistance Protein in Drug Transport  

PubMed Central

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics out of cells. In this study, we investigated the role of five basic residues within or near transmembrane (TM) 2 of BCRP in transport activity. Lys452, Lys453, His457, Arg465, and Lys473 were replaced with Ala or Asp. K452A, K453D, H457A, R465A, and K473A were stably expressed in human embryonic kidney (HEK) cells, and their plasma membrane expression and transport activities were examined. All of the mutants were expressed predominantly on the plasma membrane of HEK cells. After normalization to BCRP levels, the activities of K452A and H457A in effluxing mitoxantrone, boron-dipyrromethene-prazosin, and Hoechst33342 were increased approximately 2- to 6-fold compared with those of wild-type BCRP, whereas the activities of K453D and R465A were decreased by 40 to 60%. Likewise, K452A and H457A conferred increased resistance to mitoxantrone and 7-ethyl-10-hydroxy-camptothecin (SN-38), and K453D and R465A exhibited lower resistance. The transport activities and drug-resistance profiles of K473A were not changed. These mutations also differentially affected BCRP ATPase activities with a 2- to 4-fold increase in Vmax/Km for K452A and H457A and a 40 to 70% decrease for K453D and R465A. These mutations may induce conformational changes as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin and altered trypsin digestion. Molecular modeling and docking calculations indicated that His457 and Arg465 might be directly involved in substrate binding. In conclusion, we have identified several basic residues within or near TM2 that may be important for interaction of substrates with BCRP.

Cai, Xiaokun; Bikadi, Zsolt; Ni, Zhanglin; Lee, Eun-Woo; Wang, Honggang; Rosenberg, Mark F.

2010-01-01

140

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

PubMed

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

Samoylovich, Mikhail; Talis, Alexander

2014-03-01

141

Atomistic account of structural and dynamical changes induced by small binders in the double helix of a short DNA.  

PubMed

Nucleic acids are flexible molecules and their dynamical properties play a key role in molecular recognition events. Small binders interacting with DNA fragments induce both structural and dynamical changes in the double helix. We study the dynamics of a DNA dodecamer and of its complexes with Hoechst 33258, which is a minor groove binder, and with the ethidium cation, which is an intercalator, by molecular dynamics simulation. The thermodynamics of DNA-drug interaction is evaluated in connection with the structure and the dynamics of the resulting complexes. We identify and characterize the relevant changes in the configurational distribution of the DNA helix and relate them to the corresponding entropic contributions to the binding free energy. The binder Hoechst locks the breathing motion of the minor groove inducing a reduction of the configurational entropy of the helix, which amounts to 20 kcal mol(-1). In contrast, intercalations with the ethidium cation enhance the flexibility of the double helix. We show that the balance between the energy required to deform the helix for the intercalation and the gain in configurational entropy is the origin of cooperativity in the binding of a second ethidium and of anti-cooperativity in the binding of a third one. The results of our study provide an understanding of the relation between structure, dynamics and energetics in the interaction between DNA fragments and small binders, highlighting the role of dynamical changes and consequent variation of the configurational entropy of the DNA double helix for both types of binders. PMID:24902052

Fresch, Barbara; Remacle, F

2014-07-21

142

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

NASA Astrophysics Data System (ADS)

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

Karpusenka, Vadzim

143

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

PubMed Central

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

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

2013-01-01

144

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

2013-04-16

145

Solution structure and dynamics of a de novo designed three-helix bundle protein  

PubMed Central

Although de novo protein design is an important endeavor with implications for understanding protein folding, until now, structures have been determined for only a few 25- to 30-residue designed miniproteins. Here, the NMR solution structure of a complex 73-residue three-helix bundle protein, ?3D, is reported. The structure of ?3D was not based on any natural protein, and yet it shows thermodynamic and spectroscopic properties typical of native proteins. A variety of features contribute to its unique structure, including electrostatics, the packing of a diverse set of hydrophobic side chains, and a loop that incorporates common capping motifs. Thus, it is now possible to design a complex protein with a well defined and predictable three-dimensional structure.

Walsh, Scott T. R.; Cheng, Hong; Bryson, James W.; Roder, Heinrich; DeGrado, William F.

1999-01-01

146

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

PubMed Central

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

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

2010-01-01

147

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

148

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

PubMed Central

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

Skerra, A; Brickmann, J

1987-01-01

149

Structure of a double transmembrane fragment of a G-protein-coupled receptor in micelles.  

PubMed

The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for alpha-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 [(15)N], [(15)N, (13)C], [(15)N, (13)C, (2)H]-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 alpha-helices encompassing residues 39-47, 49-72, and 80-103, with higher flexibility around the internal Arg(58) site of TM1. RMSD values of individually superimposed helical segments 39-47, 49-72, and 80-103 were 0.25 +/- 0.10 A, 0.40 +/- 0.13 A, and 0.57 +/- 0.19 A, 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 G(56)VRSG(60) region. (15)N-relaxation and hydrogen-deuterium exchange data support a stable fold for the TM parts of TM1-TM2, whereas the solvent-exposed segments are more flexible. The NMR structure is consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor. PMID:19383463

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

2009-04-22

150

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

151

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

152

Genomic structure and embryonic expression of the Xenopus winged helix factors XFD-13/13'.  

PubMed

We have isolated the gene, its corresponding cDNA and a closely related cDNA encoding the Xenopus winged helix factors XFD-13' and XFD-13, respectively. XFD-13/13' are regarded as pseudo-alleles and, based upon a comparison of sequences and genomic structures, represent the Xenopus orthologues to mammalian FREAC-1/HFH-8. XFD-13/13' genes are not transcribed during oogenesis, zygotic transcription starts at late gastrula/early neurula and transcripts persist throughout embryogenesis. Expression is found within head derived neural crest cells and the dorsolateral plate (DLP). At later developmental stages, cell populations of the DLP migrate to the ventral region but exclude the most posterior part. Since they are subsequently found to accumulate in vessel like structures, we suggest that these cells represent hematopoietic/endothelial progenitor cells. PMID:10525191

Köster, M; Dillinger, K; Knöchel, W

1999-10-01

153

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

PubMed Central

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 high salt. Sequential assignments of non-exchangeable proton resonances of the Z-form were based on the hitherto unreported NOE connectivity path [H6(n)-H5?/H5?(n)-H8(n+1)-H1?(n+1)-H6(n+2)] found for left-handed helices. Z-RNA structure shows several conformational features significantly different from Z-DNA. Intra-strand but no inter-strand base stacking was observed for both CpG and GpC steps. Helical twist angles for CpG steps have small positive values (4–7°), whereas GpC steps have large negative values (?61°). In the full-turn model of Z-RNA (12.4 bp per turn), base pairs are much closer to the helix axis than in Z-DNA, thus both the very deep, narrow minor groove with buried cytidine 2?-OH groups, and the major groove are well defined. The 2?-OH group of cytidines plays a crucial role in the Z-RNA structure and its formation; 2?-O-methylation of cytidine, but not of guanosine residues prohibits A to Z helicity reversal.

Popenda, Mariusz; Milecki, Jan; Adamiak, Ryszard W.

2004-01-01

154

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

PubMed

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

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

2014-04-25

155

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

PubMed Central

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

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

2014-01-01

156

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

PubMed

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

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

2014-05-01

157

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

Microsoft Academic Search

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

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

2008-01-01

158

Structures of Two Arabidopsis thaliana Major Latex Proteins Represent Novel Helix-Grip Folds  

PubMed Central

The major latex proteins (MLP) are a protein family first identified in the latex of opium poppy. They are found only in plants and have 24 identified members in Arabidopsis alone as well as in other plants such as peach, strawberry, melon, cucumber, and soybean. While the function of the MLPs is unknown, they have been associated with fruit and flower development and in pathogen defense responses. Based on modest sequence similarity, they have been characterized as members of the Bet v 1 protein superfamily; however, no structures have yet been reported. As part of an ongoing structural genomics effort, we determined the structures of two Arabidopsis thaliana MLPs: the solution structure of MLP28 (gene product of At1g70830.1) and the crystal structure of At1g24000.1. The structures revealed distinct differences when compared to one another and to the typical Bet v 1 fold. Nevertheless, NMR titration experiments demonstrated that the characteristic Bet v 1 hydrophobic binding pocket of At1g24000.1 is able to bind a ligand, suggesting that it plays a role in the function of the MLPs. A structure-based sequence analysis identified conserved hydrophobic residues in the long alpha helix that contribute to the binding cavity and may specify preferred ligands for the MLP family.

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

2010-01-01

159

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

PubMed Central

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 × 104 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.

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

2013-01-01

160

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

NASA Astrophysics Data System (ADS)

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

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

2014-02-01

161

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

SciTech Connect

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

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

1995-01-20

162

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

SciTech Connect

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

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

2006-01-01

163

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

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

164

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

PubMed

Resonant X-ray diffraction (RXD) uses X-rays in the vicinity of a specific atomic absorption edge and is a powerful technique for studying symmetry breaking by motifs of various multipole moments, such as electric monopoles (charge), magnetic dipoles (spin) and electric quadrupoles (orbital). Using circularly polarized X-rays, this technique has been developed to verify symmetry breaking effects arising from chirality, the asymmetry of an object upon its mirroring. Chirality plays a crucial role in the emergence of functionalities such as optical rotatory power and multiferroicity. Here we apply spatially resolved RXD to reveal the helix chirality of Dy 4f electric quadrupole orientations and its domain structure in DyFe3(BO3)4, which shows a reversible phase transition into an enantiomorphic space-group pair. The present study provides evidence for a helix chiral motif of quadrupole moments developed in crystallographic helix chirality. PMID:24705382

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

2014-06-01

165

Mutational analysis of designed peptides that undergo structural transition from ? helix to ? sheet and amyloid fibril formation  

Microsoft Academic Search

Background: Conformational alteration and fibril formation of proteins have a key role in a variety of amyloid diseases. A simplified model peptide would lead to a better understanding of underlying mechanisms whereby protein misfolding and aggregation occur. Recently, we reported the design of peptides that undergo a self-initiated structural transition from an ? helix to a ? sheet and form

Yuta Takahashi; Akihiko Ueno; Hisakazu Mihara

2000-01-01

166

Electric Field-Driven Disruption of a Native ?-Sheet Protein Conformation and Generation of a Helix-Structure  

PubMed Central

Abstract We demonstrate that an external constant electric field is able to modify the secondary structure of a protein and induce a transition from a ?-sheet into a helix-like conformation. This dramatic change is driven by a global rearrangement of the dipole moments at the amide planes. We also predict electric-field-induced modifications of the intermediate states of the protein.

Ojeda-May, Pedro; Garcia, Martin E.

2010-01-01

167

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

Microsoft Academic Search

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

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

2003-01-01

168

How important are transmembrane helices of bitopic membrane proteins?  

Microsoft Academic Search

The topology of a bitopic membrane protein consists of a single transmembrane helix connecting two extra-membranous domains. As opposed to helices from polytopic proteins, the transmembrane helices of bitopic proteins were initially considered as merely hydrophobic anchors, while more recent studies have begun to shed light on their role in the protein's function. Herein the overall importance of transmembrane helices

Moti Zviling; Uzi Kochva; Isaiah T. Arkin

2007-01-01

169

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

170

Structure and dynamics of E. coli 16S rRNA helix 23 and human 18S rRNA helix 24 by NMR spectroscopy  

Microsoft Academic Search

The ribosome is responsible for translation of mRNA into proteins using the genetic code. This process depends upon the proper folding, dynamics, and molecular interactions of RNA and proteins within the ribosome. Helix 23 and Helix 24a are found in the central domain of the small subunit in the platform region of the subunit interface. Biochemical studies of this region

Fredrick Sijenyi

2008-01-01

171

Structure of the Cdt1 C-terminal domain: Conservation of the winged helix fold in replication licensing factors  

PubMed Central

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2-7 complex onto the origin of chromosome. The C-terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2-7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X-ray crystallography and solution NMR spectroscopy, respectively. While the N-terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172–368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.

Khayrutdinov, Bulat I; Bae, Won Jin; Yun, Young Mi; Lee, Jie Hye; Tsuyama, Takashi; Kim, Jung Joo; Hwang, Eunha; Ryu, Kyoung-Seok; Cheong, Hae-Kap; Cheong, Chaejoon; Ko, Jung-Soon; Enomoto, Takemi; Karplus, P Andrew; Guntert, Peter; Tada, Shusuke; Jeon, Young Ho; Cho, Yunje

2009-01-01

172

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

PubMed Central

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

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

2012-01-01

173

Crystal structure of a DNA decamer showing a novel pseudo four-way helix-helix junction.  

PubMed

The crystal structure of the decanucleotide d(CGCAATTGCG)2 has been solved by a combination of molecular replacement and heavy-atom procedures and has been refined to an R factor of 20.2% at 2.7 A. It is not a fully base-paired duplex but has a central core of eight Watson-Crick base pairs flanked by unpaired terminal guanosines and cytosines. These participate in hydrogen-bonding arrangements with adjacent decamer duplexes in the crystal lattice. The unpaired guanosines are bound in the G+C regions of duplex minor grooves. The cytosines have relatively high mobility, even though they are constrained to be in one region where they are involved in base-paired triplets with G.C base pairs. The 5'-AATT sequence in the duplex region has a narrow minor groove, providing further confirmation of the sequence-dependent nature of groove width. PMID:7479880

Spink, N; Nunn, C M; Vojtechovsky, J; Berman, H M; Neidle, S

1995-11-01

174

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

Microsoft Academic Search

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

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

2009-01-01

175

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

Microsoft Academic Search

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

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

2009-01-01

176

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

Microsoft Academic Search

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

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

2009-01-01

177

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

PubMed Central

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

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

2014-01-01

178

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

PubMed Central

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

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

2010-01-01

179

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

PubMed Central

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

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

2012-01-01

180

Structure, topology and assembly of a 32-mer peptide corresponding to the loop 3 and transmembrane domain 4 of divalent metal transporter (DMT1) in membrane-mimetic environments.  

PubMed

Divalent metal transporter (DMT1) belongs to the family of Nramp proteins. The fourth transmembrane domain (TM4) housing the disease-causing mutations both in Nramp1 and Nramp2 at the conserved two adjacent glycine residues, was implicated to serve an important biological function. In the present study, we have characterized structurally and topologically a 32-mer synthetic peptide, corresponding to the sequence of the loop 3 and the fourth transmembrane domain of rat DMT1 in membrane-mimetic environments (e.g. TFE, SDS micelles) using both CD and NMR spectroscopic techniques. Solution structures derived from NMR and molecular dynamic/simulated annealing calculation demonstrated that the peptide exhibits a highly defined alpha-helice in the middle portion of the peptide, flanked by a highly flexible N-terminus and a relatively ordered C-terminus. Paramagnetic broadening on peptide signals by spin-labels and Mn2+ suggested that both the N-terminus and helical core of the peptide were embedded into the SDS micelles. The peptide exhibited amphipathic characteristics, with hydrophilic residues (Thr189, Asp192, Thr193 and Asp200) lying in one side of the helix which provides a basis for the formation of water-filled channel architectures through self-associations. Diffusion-ordered spectroscopy (DOSY) indicated that the peptide exhibits mixtures of hexamers, trimers and monomers, in contrast to the fourth transmembrane peptide (24-mer) being aggregated as a trimer only. This appears to be the first report on the effects of loops on aggregation behavior of transmembrane domains in membrane-mimetic environments. PMID:18243325

Li, Hongyan; Gu, Ji-Dong; Sun, Hongzhe

2008-01-01

181

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

182

Influence of the C-terminus of the glycophorin A transmembrane fragment on the dimerization process.  

PubMed Central

The monomer-dimer equilibrium of the glycophorin A (GpA) transmembrane (TM) fragment has been used as a model system to investigate the amino acid sequence requirements that permit an appropriate helix-helix packing in a membrane-mimetic environment. In particular, we have focused on a region of the helix where no crucial residues for packing have been yet reported. Various deletion and replacement mutants in the C-terminal region of the TM fragment showed that the distance between the dimerization motif and the flanking charged residues from the cytoplasmic side of the protein is important for helix packing. Furthermore, selected GpA mutants have been used to illustrate the rearrangement of TM fragments that takes place when leucine repeats are introduced in such protein segments. We also show that secondary structure of GpA derivatives was independent from dimerization, in agreement with the two-stage model for membrane protein folding and oligomerization.

Orzaez, M.; Perez-Paya, E.; Mingarro, I.

2000-01-01

183

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

PubMed

Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R*) and catalyze GDP/GTP exchange in the alpha-subunit of intracellular G proteins (Galphabetagamma). Kinetic analysis of transducin (G(t)alphabetagamma) activation shows that an intermediary R*xG(t)alphabetagamma.GDP complex is formed that precedes GDP release and formation of the nucleotide-free R*xG protein complex. Based on this reaction sequence, we explore the dynamic interface between the proteins during formation of these complexes. We start from the R* conformation stabilized by a G(t)alpha C-terminal peptide (GalphaCT) obtained from crystal structures of the GPCR opsin. Molecular modeling allows reconstruction of the fully elongated C-terminal alpha-helix of G(t)alpha (alpha5) and shows how alpha5 can be docked to the open binding site of R*. Two modes of interaction are found. One of them--termed stable or S-interaction--matches the position of the GalphaCT peptide in the crystal structure and reproduces the hydrogen-bonding networks between the C-terminal reverse turn of GalphaCT and conserved E(D)RY and NPxxY(x)(5,6)F regions of the GPCR. The alternative fit--termed intermediary or I-interaction--is distinguished by a tilt (42 degrees ) and rotation (90 degrees ) of alpha5 relative to the S-interaction and shows different alpha5 contacts with the NPxxY(x)(5,6)F region and the second cytoplasmic loop of R*. From the 2 alpha5 interactions, we derive a "helix switch" mechanism for the transition of R*xG(t)alphabetagamma.GDP to the nucleotide-free R*xG protein complex that illustrates how alpha5 might act as a transmission rod to propagate the conformational change from the receptor-G protein interface to the nucleotide binding site. PMID:19541654

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

2009-06-30

184

Predicting residue and helix contacts in membrane proteins  

NASA Astrophysics Data System (ADS)

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

Fuchs, Angelika; Kirschner, Andreas; Frishman, Dmitrij

185

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

186

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

PubMed Central

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

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

2014-01-01

187

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

SciTech Connect

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

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

2008-06-24

188

Structure-function analysis of the Z-DNA-binding domain Zalpha of dsRNA adenosine deaminase type I reveals similarity to the (alpha + beta) family of helix-turn-helix proteins.  

PubMed Central

RNA editing alters pre-mRNA through site-selective adenosine deamination, which results in codon changes that lead to the production of novel proteins. An enzyme that catalyzes this reaction, double-stranded RNA adenosine deaminase (ADAR1), contains two N-terminal Z-DNA-binding motifs, Zalpha and Zbeta, the function of which is as yet unknown. In this study, multidimensional NMR spectroscopy was used to show that the topology of Zalpha is alpha1beta1alpha2alpha3beta2beta3. Long-range NOEs indicate that beta1 and beta3 interact with each other. Site-directed mutagenesis was used to identify residues in alpha3, beta3 and the loop connecting beta2 to beta3 that affect Z-DNA binding. Also identified were 11 hydrophobic residues that are essential for protein stability. Comparison with known structures reveals some similarity between Zalpha and (alpha + beta) helix-turn-helix proteins, such as histone 5 and the family of hepatocyte nuclear factor-3 winged-helix-turn-helix transcription factors. Taken together, the structural and functional data suggest that recognition of Z-DNA by Zalpha involves residues in both the alpha3 helix and the C-terminal beta-sheet.

Schade, M; Turner, C J; Lowenhaupt, K; Rich, A; Herbert, A

1999-01-01

189

Structural basis for endosomal trafficking of diverse transmembrane cargos by PX-FERM proteins.  

PubMed

Transit of proteins through the endosomal organelle following endocytosis is critical for regulating the homeostasis of cell-surface proteins and controlling signal transduction pathways. However, the mechanisms that control these membrane-transport processes are poorly understood. The Phox-homology (PX) domain-containing proteins sorting nexin (SNX) 17, SNX27, and SNX31 have emerged recently as key regulators of endosomal recycling and bind conserved Asn-Pro-Xaa-Tyr-sorting signals in transmembrane cargos via an atypical band, 4.1/ezrin/radixin/moesin (FERM) domain. Here we present the crystal structure of the SNX17 FERM domain bound to the sorting motif of the P-selectin adhesion protein, revealing both the architecture of the atypical FERM domain and the molecular basis for recognition of these essential sorting sequences. We further show that the PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases, and propose a model for their coordinated molecular interactions with membrane, cargo, and regulatory proteins. PMID:23382219

Ghai, Rajesh; Bugarcic, Andrea; Liu, Huadong; Norwood, Suzanne J; Skeldal, Sune; Coulson, Elizabeth J; Li, Shawn Shun-Cheng; Teasdale, Rohan D; Collins, Brett M

2013-02-19

190

The transmembrane domain sequence affects the structure and function of the Newcastle disease virus fusion protein.  

PubMed

The role of specific sequences in the transmembrane (TM) domain of Newcastle disease virus (NDV) fusion (F) protein in the structure and function of this protein was assessed by replacing this domain with the F protein TM domains from two other paramyxoviruses, Sendai virus (SV) and measles virus (MV), or the TM domain of the unrelated glycoprotein (G) of vesicular stomatitis virus (VSV). Mutant proteins with the SV or MV F protein TM domains were expressed, transported to cell surfaces, and proteolytically cleaved at levels comparable to that of the wild-type protein, while mutant proteins with the VSV G protein TM domain were less efficiently expressed on cell surfaces and proteolytically cleaved. All mutant proteins were defective in all steps of membrane fusion, including hemifusion. In contrast to the wild-type protein, the mutant proteins did not form detectable complexes with the NDV hemagglutinin-neuraminidase (HN) protein. As determined by binding of conformation-sensitive antibodies, the conformations of the ectodomains of the mutant proteins were altered. These results show that the specific sequence of the TM domain of the NDV F protein is important for the conformation of the preactivation form of the ectodomain, the interactions of the protein with HN protein, and fusion activity. PMID:21270151

Gravel, Kathryn A; McGinnes, Lori W; Reitter, Julie; Morrison, Trudy G

2011-04-01

191

The secondary structure of the ets domain of human Fli-1 resembles that of the helix-turn-helix DNA-binding motif of the Escherichia coli catabolite gene activator protein.  

PubMed Central

The ets family of eukaryotic transcription factors is characterized by a conserved DNA-binding domain of approximately 85 amino acids for which the three-dimensional structure is not known. By using multidimensional NMR spectroscopy, we have determined the secondary structure of the ets domain of one member of this gene family, human Fli-1, both in the free form and in a complex with a 16-bp cognate DNA site. The secondary structure of the Fli-1 ets domain consists of three alpha-helices and a short four-stranded antiparallel beta-sheet. This secondary structure arrangement resembles that of the DNA-binding domain of the catabolite gene activator protein of Escherichia coli, as well as those of several eukaryotic DNA-binding proteins including histone H5, HNF-3/fork head, and the heat shock transcription factor. Differences in chemical shifts of backbone resonances and amide exchange rates between the DNA-bound and free forms of the Fli-1 ets domain suggest that the third helix is the DNA recognition helix, as in the catabolite gene activator protein and other structurally related proteins. These results suggest that the ets domain is structurally similar to the catabolite gene activator protein family of helix-turn-helix DNA-binding proteins. Images

Liang, H; Olejniczak, E T; Mao, X; Nettesheim, D G; Yu, L; Thompson, C B; Fesik, S W

1994-01-01

192

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

SciTech Connect

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

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

2013-09-15

193

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

NASA Astrophysics Data System (ADS)

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

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

2013-09-01

194

A common motif organizes the structure of multi-helix loops in 16 S and 23 S ribosomal RNAs.  

PubMed

Phylogenetic and chemical probing data indicate that a modular RNA motif, common to loop E of eucaryotic 5 S ribosomal RNA (rRNA) and the alpha-sarcin/ricin loop of 23 S rRNA, organizes the structure of multi-helix loops in 16 S and 23 S ribosomal RNAs. The motif occurs in the 3' domain of 16 S rRNA at positions 1345-1350/1372-1376 (Escherichia coli numbering), within the three-way junction loop, which binds ribosomal protein S7, and which contains nucleotides that help to form the binding site for P-site tRNA in the ribosome. The motif also helps to structure a three-way junction within domain I of 23 S, which contains many universally conserved bases and which lies close in the primary and secondary structure to the binding site of r-protein L24. Several other highly conserved hairpin, internal, and multi-helix loops in 16 S and 23 S rRNA contain the motif, including the core junction loop of 23 S and helix 27 in the core of 16 S rRNA. Sequence conservation and range of variation in bacteria, archaea, and eucaryotes as well as chemical probing and cross-linking data, provide support for the recurrent and autonomous existence of the motif in ribosomal RNAs. Besides its presence in the hairpin ribozyme, the loop E motif is also apparent in helix P10 of bacterial RNase P, in domain P7 of one sub-group of group I introns, and in domain 3 of one subgroup of group II introns. PMID:9784367

Leontis, N B; Westhof, E

1998-10-30

195

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

NASA Technical Reports Server (NTRS)

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

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

1982-01-01

196

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

Microsoft Academic Search

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

Pradip K. Jain; B. N. Basu

1992-01-01

197

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

PubMed Central

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

Pabuwal, Vagmita; Li, Zhijun

2010-01-01

198

Thermal-structural reliability assessment of helix TWT interaction circuit using finite element analysis  

Microsoft Academic Search

Finite element analysis (FEA) techniques were used to assess the mechanical performance due to thermal loading of a high power, wide band, helix traveling wave tube's interaction (slow wave) circuit. A steady state heat transfer analysis was performed using calculated heat dissipations and boundary temperatures that were obtained through data supplied by the TWT's manufacturer. The resulting temperatures from this

Peter J. Rocci

1993-01-01

199

Structural implications of a Val-->Glu mutation in transmembrane peptides from the EGF receptor.  

PubMed Central

Certain specific point mutations within the transmembrane domains of class I receptor tyrosine kinases are known to induce altered behavior in the host cell. An internally controlled pair of peptides containing the transmembrane portion of the human epidermal growth factor (EGF) receptor (ErbB-1) was examined in fluid, fully hydrated lipid bilayers by wide-line 2H-NMR for insight into the physical basis of this effect. One member of the pair encompassed the native transmembrane sequence from ErbB-1, while in the other the valine residue at position 627 was replaced by glutamic acid to mimic a substitution that produces a transformed phenotype in cells. Heteronuclear probes having a defined relationship to the peptide backbone were incorporated by deuteration of the methyl side chains of natural alanine residues. 2H-NMR spectra were recorded in the range 35 degrees C to 65 degrees C in membranes composed of 1-palmitoyl-2-oleoyl phosphatidylcholine. Narrowed spectral components arising from species rotating rapidly and symmetrically within the membrane persisted to very high temperature and appeared to represent monomeric peptide. Probes at positions 623 and 629 within the EGF receptor displayed changes in quadrupole splitting when Val(627) was replaced by Glu, while probes downstream at position 637 were relatively unaffected. The results demonstrate a measurable spatial reorientation in the region of the 5-amino acid motif (residues 624-628) often suggested to be involved in side-to-side interactions of the receptor transmembrane domain. Spectral changes induced by the Val-->Glu mutation in ErbB-1 were smaller than those induced by the analogous oncogenic mutation in the homologous human receptor, ErbB-2 (Sharpe, S., K. R. Barber, and C. W. M. Grant. 2000. Biochemistry. 39:6572-6580). Quadrupole splittings at probe sites examined were only modestly sensitive to temperature, suggesting that each transmembrane peptide behaved as a motionally ordered unit possessing considerable conformational stability.

Sharpe, S; Grant, C W; Barber, K R; Giusti, J; Morrow, M R

2001-01-01

200

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

PubMed

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

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

2008-08-01

201

Spare parts for helix-helix interaction.  

PubMed

About 6000 contact regions (patches) of helix-to-helix packing from 300 well-resolved non-homologous protein structures were considered. The patches were defined by the spatial helical neighbors and were estimated in atomic detail using a variable distance criterion. The following questions are addressed. (1) Are the amino acid preferences and atomic composition of distinct types of helical patches indicative for the type of their neighbor? Distributions of size, atomic composition and packing density are compared for different types of helical interfaces. Thereby contact preferences are derived for parts of secondary structures adjoining each other or pointing towards the solvent. (2) Is it possible to cluster helical patches according to their structural similarity? For these purposes the patches were classified with an automatic sequence-independent superposition procedure which yields a distinctively reduced set of representative interfaces. On this basis, the methodology for finding exchangeable patches in different proteins is demonstrated. PMID:10556242

Preissner, R; Goede, A; Frömmel, C

1999-10-01

202

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

203

Helix Project.  

National Technical Information Service (NTIS)

The Helix project is a multi-year investigation of the demographics and effectiveness of systems engineers in the US Department of Defense (DoD) and the defense community. This project is sponsored by the DoD and the National Defense Industrial Associatio...

A. Pyster D. Henry K. Lasfer N. Hutchsion S. Rifkin

2013-01-01

204

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

205

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

PubMed Central

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

Spano, Meredith Newby; Walter, Nils G.

2011-01-01

206

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

207

Yummy Gummy Double Helix  

NSDL National Science Digital Library

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

Institute, Wellcome T.

2013-10-10

208

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

PubMed Central

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

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

2007-01-01

209

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

PubMed Central

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

2011-01-01

210

Structure of a Virulence Regulatory Factor CvfB Reveals a Novel Winged-helix RNA Binding Module  

PubMed Central

SUMMARY CvfB is a conserved regulatory protein important for the virulence of Staphylococcus aureus. We show here that CvfB binds RNA. The crystal structure of the CvfB ortholog from Streptococcus pneumoniae at 1.4 Å resolution reveals a unique RNA binding protein that is formed from a concatenation of well-known structural modules that bind nucleic acids: three consecutive S1 RNA-binding domains and a winged-helix (WH) domain. The third S1 and the WH domains are required for cooperative RNA binding and form a continuous surface that likely contributes to the RNA interaction. The WH domain is critical to CvfB function and contains a unique structural motif. Thus CvfB represents a novel assembly of modules for binding RNA.

Matsumoto, Yasuhiko; Xu, Qingping; Miyazaki, Shinya; Kaito, Chikara; Farr, Carol L.; Axelrod, Herbert L.; Chiu, Hsiu-Ju; Klock, Heath E.; Knuth, Mark W.; Miller, Mitchell D.; Elsliger, Marc-Andre; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Sekimizu, Kazuhisa; Wilson, Ian A.

2010-01-01

211

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

2007-10-30

212

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

213

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

214

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

NASA Astrophysics Data System (ADS)

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

Kundu, Sourav; Karmakar, S. N.

2014-03-01

215

Pro-Gly mediated conformational switch of mycobacteriophage D29 holin transmembrane domain I is lipid concentration driven.  

PubMed

Biophysical and spectroscopic analysis of synthetic transmembrane domain I (1) of mycobacteriophage D29 holin shows a lipid concentration dependent conformational switch from an ?-helix to a ?-sheet structure. The reversibility of this switch, upon change in the lipid-to-peptide ratio, requires a central Pro-Gly segment, and is abolished upon mutation to Ala-Ala or (D)Pro-Gly. PMID:24018990

Lella, Muralikrishna; Mahalakshmi, Radhakrishnan

2013-10-25

216

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

2006-10-27

217

Structure and computational analysis of a novel protein with metallopeptidase-like and circularly permuted winged-helix-turn-helix domains reveals a possible role in modified polysaccharide biosynthesis  

PubMed Central

Background CA_C2195 from Clostridium acetobutylicum is a protein of unknown function. Sequence analysis predicted that part of the protein contained a metallopeptidase-related domain. There are over 200 homologs of similar size in large sequence databases such as UniProt, with pairwise sequence identities in the range of ~40-60%. CA_C2195 was chosen for crystal structure determination for structure-based function annotation of novel protein sequence space. Results The structure confirmed that CA_C2195 contained an N-terminal metallopeptidase-like domain. The structure revealed two extra domains: an ?+? domain inserted in the metallopeptidase-like domain and a C-terminal circularly permuted winged-helix-turn-helix domain. Conclusions Based on our sequence and structural analyses using the crystal structure of CA_C2195 we provide a view into the possible functions of the protein. From contextual information from gene-neighborhood analysis, we propose that rather than being a peptidase, CA_C2195 and its homologs might play a role in biosynthesis of a modified cell-surface carbohydrate in conjunction with several sugar-modification enzymes. These results provide the groundwork for the experimental verification of the function.

2014-01-01

218

Ion-Mediated Nucleic Acid Helix-Helix Interactions  

PubMed Central

Salt ions are essential for the folding of nucleic acids. We use the tightly bound ion (TBI) model, which can account for the correlations and fluctuations for the ions bound to the nucleic acids, to investigate the electrostatic free-energy landscape for two parallel nucleic acid helices in the solution of added salt. The theory is based on realistic atomic structures of the helices. In monovalent salt, the helices are predicted to repel each other. For divalent salt, while the mean-field Poisson-Boltzmann theory predicts only the repulsion, the TBI theory predicts an effective attraction between the helices. The helices are predicted to be stabilized at an interhelix distance ?26–36 Å, and the strength of the attractive force can reach ?0.37 kBT/bp for helix length in the range of 9–12 bp. Both the stable helix-helix distance and the strength of the attraction are strongly dependent on the salt concentration and ion size. With the increase of the salt concentration, the helix-helix attraction becomes stronger and the most stable helix-helix separation distance becomes smaller. For divalent ions, at very high ion concentration, further addition of ions leads to the weakening of the attraction. Smaller ion size causes stronger helix-helix attraction and stabilizes the helices at a shorter distance. In addition, the TBI model shows that a decrease in the solvent dielectric constant would enhance the ion-mediated attraction. The theoretical findings from the TBI theory agree with the experimental measurements on the osmotic pressure of DNA array as well as the results from the computer simulations.

Tan, Zhi-Jie; Chen, Shi-Jie

2006-01-01

219

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

220

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

221

Membrane interaction and structure of the transmembrane domain of influenza hemagglutinin and its fusion peptide complex  

PubMed Central

Background To study the organization and interaction with the fusion domain (or fusion peptide, FP) of the transmembrane domain (TMD) of influenza virus envelope glycoprotein for its role in membrane fusion which is also essential in the cellular trafficking of biomolecules and sperm-egg fusion. Results The fluorescence and gel electrophoresis experiments revealed a tight self-assembly of TMD in the model membrane. A weak but non-random interaction between TMD and FP in the membrane was found. In the complex, the central TMD oligomer was packed by FP in an antiparallel fashion. FP insertion into the membrane was altered by binding to TMD. An infrared study exhibited an enhanced membrane perturbation by the complex formation. A model was built to illustrate the role of TMD in the late stages of influenza virus-mediated membrane fusion reaction. Conclusion The TMD oligomer anchors the fusion protein in the membrane with minimal destabilization to the membrane. Upon associating with FP, the complex exerts a synergistic effect on the membrane perturbation. This effect is likely to contribute to the complete membrane fusion during the late phase of fusion protein-induced fusion cascade. The results presented in the work characterize the nature of the interaction of TMD with the membrane and TMD in a complex with FP in the steps leading to pore initiation and dilation during virus-induced fusion. Our data and proposed fusion model highlight the key role of TMD-FP interaction and have implications on the fusion reaction mediated by other type I viral fusion proteins. Understanding the molecular mechanism of membrane fusion may assist in the design of anti-viral drugs.

Chang, Ding-Kwo; Cheng, Shu-Fang; Kantchev, Eric Aseen B; Lin, Chi-Hui; Liu, Yu-Tsan

2008-01-01

222

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

PubMed

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

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

2014-05-01

223

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

PubMed Central

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

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

2014-01-01

224

Drug binding to higher ordered DNA structures: netropsin complexation with a nucleic acid triple helix.  

PubMed Central

We have used a combination of spectroscopic and calorimetric techniques to characterize how netropsin, a ligand that binds in the minor groove of DNA, influences the properties of a DNA triple helix. Specifically, our data allow us to reach the following conclusions: (i) netropsin binds to the triplex without displacing the major-groove-bound third strand; (ii) netropsin binding to the triplex exhibits a lower saturation binding density (7.0 base triplets per netropsin bound) than netropsin binding to the corresponding duplex (5.5 base pairs per netropsin bound); (iii) the netropsin-free and the netropsin-bound triplexes each melt in two well-resolved transitions, initial conversion of the triplex to the duplex state followed by duplex melting to the component single-stranded states; (iv) netropsin remains bound to DNA as the triplex melts to the duplex state; (v) netropsin binding thermally destabilizes the triplex in equilibrium with duplex equilibrium dramatically, while thermally stabilizing the duplex to single-strand equilibrium; (vi) netropsin binding to the triplex is enthalpically 4 times more favorable (more exothermic) than netropsin binding to the corresponding duplex; (vii) netropsin binding to the triplex decreases the cooperativity of the triplex----duplex melting event. These results demonstrate that occupancy of the minor groove of a triplex by a ligand such as netropsin can exert a profound impact on the properties of the host triplex, particularly with regard to the equilibrium in which the third strand is expelled from the major groove. Thus, our results reveal considerable major groove/minor groove crosstalk. Such knowledge may prove of practical importance by providing an approach for modulating the affinity and specificity of major-groove-binding third strands in triplex-forming protocols designed to target specific duplex domains. Fundamentally, our results provide insights into the crosstalk that can result when ligands bind to the two major receptor sites of duplex DNA--namely, the major and minor grooves.

Park, Y W; Breslauer, K J

1992-01-01

225

Crystal structure of the human FOXK1a-DNA complex and its implications on the diverse binding specificity of winged helix/forkhead proteins.  

PubMed

Interleukin enhancer binding factor (ILF) is a human transcription factor and a new member of the winged helix/forkhead family. ILF can bind to purine-rich regulatory motifs such as the human T-cell leukemia virus-long terminal region and the interleukin-2 promoter. Here we report the 2.4 A crystal structure of two DNA binding domains of ILF (FOXK1a) binding to a 16-bp DNA duplex containing a promoter sequence. Electrophoretic mobility shift assay studies demonstrate that two ILF-DNA binding domain molecules cooperatively bind to DNA. In addition to the recognition helix recognizing the core sequences through the major groove, the structure shows that wing 1 interacts with the minor groove of DNA, and the H2-H3 loop region makes ionic bonds to the phosphate group, which permits the recognition of DNA. The structure also reveals that the presence of the C-terminal alpha-helix in place of a typical wing 2 in a member of this family alters the orientation of the C-terminal basic residues (RKRRPR) when binding to DNA outside the core sequence. These results provide a new insight into how the DNA binding specificities of winged helix/forkhead proteins may be regulated by their less conserved regions. PMID:16624804

Tsai, Kuang-Lei; Huang, Cheng-Yang; Chang, Chia-Hao; Sun, Yuh-Ju; Chuang, Woei-Jer; Hsiao, Chwan-Deng

2006-06-23

226

Role of the sequence surrounding predicted transmembrane helix M4 in membrane association and function of the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor isoform 1).  

PubMed

The role of the sequence surrounding M4 in ryanodine receptors (RyR) in membrane association and function was investigated. This sequence contains a basic, 19-amino acid M3/M4 loop, a hydrophobic 44-49 amino acid sequence designated M4 (or M4a/M4b), and a hydrophilic M4/M5 loop. Enhanced green fluorescent protein (EGFP) was inserted into RyR1 and truncated just after the basic sequence, just after M4, within the M4/M5 loop, just before M5 and just after M5. The A52 epitope was inserted into RyR2 and truncated just after M4a. Analysis of these constructs ruled out a M3/M4 transmembrane hairpin and narrowed the region of membrane association to M4a/M4b. EGFP inserted between M4a and M4b in full-length RyR2 was altered conformationally, losing fluorescence and gaining trypsin sensitivity. Although it was accessible to an antibody from the cytosolic side, tryptic fragments were membrane-bound. The expressed protein containing EGFP retained caffeine-induced Ca(2+) release channel function. These results suggest that M4a/M4b either forms a transmembrane hairpin or associates in an unorthodox fashion with the cytosolic leaflet of the membrane, possibly involving the basic M3/M4 loop. The expression of a mutant RyR1, Delta4274-4535, deleted in the sequence surrounding both M3 and M4, restored robust, voltage-gated L-type Ca(2+) currents and Ca(2+) transients in dyspedic myotubes, demonstrating that this sequence is not required for either orthograde (DHPR activation of sarcoplasmic reticulum Ca(2+) release) or retrograde (RyR1 increase in DHPR Ca(2+) channel activity) signals of excitation-contraction coupling. Maximal amplitudes of L-currents and Ca(2+) transients with Delta4274-4535 were larger than with wild-type RyR1, and voltage-gated sarcoplasmic reticulum Ca(2+) release was more sensitive to activation by sarcolemmal voltage sensors. Thus, this region may act as a negative regulatory module that increases the energy barrier for Ca(2+) release channel opening. PMID:15226293

Du, Guo Guang; Avila, Guillermo; Sharma, Parveen; Khanna, Vijay K; Dirksen, Robert T; MacLennan, David H

2004-09-01

227

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

2013-11-01

228

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

PubMed Central

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

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

2003-01-01

229

Crystal Structure of F-93 from Sulfolobus Spindle-Shaped Virus 1, a Winged-Helix DNA Binding Protein  

PubMed Central

Sulfolobus spindle-shaped viruses (SSVs), or Fuselloviridae, are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs around the world (pH ?4.0; temperature of ?70°C). Because they are relatively easy to isolate, they represent the best studied of the crenarchaeal viruses. This is particularly true for the type virus, SSV1, which contains a double-stranded DNA genome of 15.5 kilobases, encoding 34 putative open reading frames. Interestingly, the genome shows little sequence similarity to organisms other than its SSV homologues. Together, sequence similarity and biochemical analyses have suggested functions for only 6 of the 34 open reading frames. Thus, even though SSV1 is the best-studied crenarchaeal virus, functions for most (28) of its open reading frames remain unknown. We have undertaken biochemical and structural studies for the gene product of open reading frame F-93. We find that F-93 exists as a homodimer in solution and that a tight dimer is also present in the 2.7-Å crystal structure. Further, the crystal structure reveals a fold that is homologous to the SlyA and MarR subfamilies of winged-helix DNA binding proteins. This strongly suggests that F-93 functions as a transcription factor that recognizes a (pseudo-)palindromic DNA target sequence.

Kraft, Paul; Oeckinghaus, Andrea; Kummel, Daniel; Gauss, George H.; Gilmore, John; Wiedenheft, Blake; Young, Mark; Lawrence, C. Martin

2004-01-01

230

The functional topography of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor, revealed by scanning mutagenesis.  

PubMed

Alanine-scanning mutagenesis has been applied to residues 100-121 in transmembrane domain 3 of the M1 muscarinic acetylcholine receptor. This study complements a previous investigation of the triad Asp122-Arg123-Tyr124 (Lu, Z-L., Curtis, C. A., Jones, P. G., Pavia, J., and Hulme, E. C. (1997) Mol. Pharmacol. 51, 234-241). The results demonstrate the alpha-helical secondary structure of the domain and suggest its orientation with respect to the other transmembrane domains. The C-terminal part of the helix appears to be largely buried within the receptor structure. On its surface, there is a patch of three residues, Val113, Leu116, and Ser120, which may form intramolecular contacts that help to stabilize the inactive ground state of the receptor. Mutagenic disruption of these increased agonist affinity and signaling efficacy. In two cases (L116A and S120A), this led to constitutive activation of the receptor. Parallel to the helix axis and spanning the whole transmembrane region, a distinct strip of residues on one face of transmembrane domain 3 forms intermolecular (acetylcholine-receptor, receptor-G protein) or intrareceptor bonds that contribute to the activated state. The binding of acetylcholine may destabilize the first set of contacts while favoring the formation of the second. PMID:10066794

Lu, Z L; Hulme, E C

1999-03-12

231

From 1D Helix to 0D Loop: Nitrite Anion Induced Structural Transformation Associated with Unexpected N-Nitrosation of Amine Ligand.  

PubMed

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

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

2014-06-01

232

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

PubMed

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

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

1997-10-13

233

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

PubMed

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

Marko, John F; Neukirch, Sébastien

2013-12-01

234

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

235

Structural Disorder of the CD3? Transmembrane Domain Studied with 2D IR Spectroscopy and Molecular Dynamics Simulations  

PubMed Central

In a recently reported study [Mukherjee, et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3528], we used 2D IR spectroscopy and 1-13C?18O isotope labeling to measure the vibrational dynamics of 11 amide I modes in the CD3? transmembrane domain. We found that the homogeneous line widths and population relaxation times were all nearly identical, but that the amount of inhomogeneous broadening correlated with the position of the amide group inside the membrane. In this study, we use molecular dynamics simulations to investigate the structural and dynamical origins of these experimental observations. We use two models to convert the simulations to frequency trajectories from which the mean frequencies, standard deviations, frequency correlation functions, and 2D IR spectra are calculated. Model 1 correlates the hydrogen-bond length to the amide I frequency, whereas model 2 uses an ab initio-based electrostatic model. We find that the structural distributions of the peptidic groups and their environment are reflected in the vibrational dynamics of the amide I modes. Environmental forces from the water and lipid headgroups partially denature the helices, shifting the infrared frequencies and creating larger inhomogeneous distributions for residues near the ends. The least inhomogeneously broadened residues are those located in the middle of the membrane where environmental electrostatic forces are weakest and the helices are most ordered. Comparison of the simulations to experiment confirms that the amide I modes near the C-terminal are larger than at the N-terminal because of the asymmetric structure of the peptide bundle in the membrane. The comparison also reveals that residues at a kink in the ?-helices have broader line widths than more helical parts of the peptide because the peptide backbone at the kink exhibits a larger amount of structural disorder. Taken together, the simulations and experiments reveal that infrared line shapes are sensitive probes of membrane protein structural and environmental heterogeneity.

Mukherjee, Prabuddha; Kass, Itamar; Arkin, Isaiah T.; Zanni, Martin T.

2009-01-01

236

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

PubMed Central

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

Thornton, Janet M.

2009-01-01

237

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

PubMed Central

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

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

2013-01-01

238

Conformational Changes in Talin on Binding to Anionic Phospholipid Membranes Facilitate Signaling by Integrin Transmembrane Helices  

PubMed Central

Integrins are heterodimeric (??) cell surface receptors that are activated to a high affinity state by the formation of a complex involving the ?/? integrin transmembrane helix dimer, the head domain of talin (a cytoplasmic protein that links integrins to actin), and the membrane. The talin head domain contains four sub-domains (F0, F1, F2 and F3) with a long cationic loop inserted in the F1 domain. Here, we model the binding and interactions of the complete talin head domain with a phospholipid bilayer, using multiscale molecular dynamics simulations. The role of the inserted F1 loop, which is missing from the crystal structure of the talin head, PDB:3IVF, is explored. The results show that the talin head domain binds to the membrane predominantly via cationic regions on the F2 and F3 subdomains and the F1 loop. Upon binding, the intact talin head adopts a novel V-shaped conformation which optimizes its interactions with the membrane. Simulations of the complex of talin with the integrin ?/? TM helix dimer in a membrane, show how this complex promotes a rearrangement, and eventual dissociation of, the integrin ? and ? transmembrane helices. A model for the talin-mediated integrin activation is proposed which describes how the mutual interplay of interactions between transmembrane helices, the cytoplasmic talin protein, and the lipid bilayer promotes integrin inside-out activation.

Kalli, Antreas C.; Campbell, Iain D.; Sansom, Mark S. P.

2013-01-01

239

Structure and dynamics of the membrane-bound form of Pf1 coat protein: implications of structural rearrangement for virus assembly.  

PubMed

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

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

2010-09-01

240

HFIP-induced structures and assemblies of the peptides from the transmembrane domain 4 of membrane protein Nramp1.  

PubMed

Membrane protein Nramp1 (natural resistance-associated macrophage protein 1) is a pH-dependent divalent metal cation transporter that regulates macrophage activation in infectious and autoimmune diseases. A naturally occurring glycine to aspartic acid substitution at position 169 (G169D) within the transmembrane domain 4 (TM4) of Nramp1 makes mice susceptible to Leishmania donovani, Salmonella typhimurium, and Mycobacterium bovis. Here we present a structural and self-assembling study on two synthetic 24-residue peptides, corresponding to TM4 of mouse Nramp1 and its G169D mutant, respectively, in 1,1,1,3,3,3-hexafluoroisopropanol-d(2) (HFIP-d(2)) aqueous solution by nuclear magnetic resonance (NMR) spectroscopy. The results show that amphipathic alpha-helical structures are formed from residue Ile173 to Tyr187 for the wild-type peptide and from Trp168 to Tyr187 for the G169D mutant, respectively. The segment of the N-terminus from Leu167 to Leu172 is poorly structured for the wild-type peptide, whereas it is well defined for the G169D mutant. Both peptides aggregate to form a tetramer and the monomeric peptides in peptide bundles are structurally and orientationally similar. The intermolecular interactions in assemblies could be stronger in the C-terminal regions related to residues Phe180-Leu184 than those in the central helical segments for both peptides. The G169D mutation may change the size of the opening on the termini of assembly. PMID:16479587

Xue, Rong; Wang, Shuo; Wang, Chunyu; Zhu, Tao; Li, Fei; Sun, Hongzhe

2006-01-01

241

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

NASA Astrophysics Data System (ADS)

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

Straub, John

2013-03-01

242

Structure in solution of a four-helix lipid binding protein.  

PubMed Central

Because of the low solubility of lipids in water, intercellular and intracellular pathways of lipid transfer are necessary, e.g., for membrane formation. The mechanism by which lipids in vivo are transported from their site of biogenesis (endoplasmatic reticulum and the chloroplasts) to their place of action is unknown. Several small plant proteins with the ability to mediate transfer of radiolabeled phospholipids in vitro from liposomal donor membranes to mitochondrial and chloroplast acceptor membranes have been isolated, and a protein with this ability, the nonspecific lipid transfer protein (nsLTP) isolated from barley seeds (bLTP), has been studied here. The structure and the protein lipid interactions of lipid transfer proteins are relevant for the understanding of their function, and here we present the three-dimensional structure in solution of bLTP as determined by NMR spectroscopy. The 1H NMR spectrum of the 91-residue protein was assigned for more than 97% of the protein 1H atoms, and the structure was calculated on the basis of 813 distance restraints from 1H-1H nuclear Overhauser effects, four disulfide bond restraints, from dihedral angle restraints for 66 phi-angles, 61 chi 1 angles, and 2 chi 2 angles, and from 31 sets of hydrogen bond restraints. The solution structure of bLTP consists of four well-defined alpha-helices A-D (A, Cys 3-Gly 19; B, Gly 25-Ala 38; C, Arg 44-Gly 57; D, Leu 63-Cys 73), separated by three short loops that are less well defined and concluded by a well defined C-terminal peptide segment with no observable regular secondary structure. For the 17 structures that are used to represent the solution structure of bLTP, the RMS deviation to an average structure is 0.63 A +/- 0.04 A for backbone atoms and 0.93 A +/- 0.06 A for all heavy atoms. The secondary structure elements and their locations in the sequence resemble those of nsLTP from two other plant species, wheat and maize, whose structures were previously determined (Gincel E et al, 1995, Eur J Biochem 226:413-422; Shin DH et al, 1995, Structure 3:189-199). In bLTP, the residues analogous to those in maize nsLTP that constitute the palmitate binding site are forming a similar hydrophobic cavity and a potential acyl group binding site. Analysis of the solution structure of bLTP and bLTP in complex with a ligand might provide information on the conformational changes in the protein upon ligand binding and subsequently provide information on the mode of ligand uptake and release. In this work, we hope to establish a foundation for further work of determining the solution structure of bLTP in complex with palmitoyl coenzyme A, which is a suitable ligand, and subsequently to outline the mode of ligand binding.

Heinemann, B.; Andersen, K. V.; Nielsen, P. R.; Bech, L. M.; Poulsen, F. M.

1996-01-01

243

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

PubMed

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

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

2013-01-01

244

Structural and Dynamic Study of the Tetramerization Region of Non-Erythroid ?-Spectrin: A Frayed Helix Revealed by Site-Directed Spin Labeling EPR†  

PubMed Central

The N-terminal region of ?-spectrin is responsible for its association with ?-spectrin in a heterodimer to form functional tetramers. Non-erythroid alpha spectrin (?II-spectrin) has significantly higher association affinity with ?-spectrin than the homologous erythroid alpha spectrin (?I-spectrin). We have previously determined the solution structure of the N-terminal region of ?I-spectrin by NMR methods, but currently no structural information is available for ?II-spectrin. We have now used cysteine scanning, spin labeling EPR and isothermal titration calorimetry (ITC) methods to study the tetramerization region of ?II-spectrin. EPR data clearly showed that, in ?II-spectrin, the first 9 N-terminal residues were unstructured, followed by an irregular helix (Helix C?), frayed at the N-terminal end, but rigid at the C-terminal end, which merges into the putative triple helical structural domain. The region corresponding to the important unstructured junction region linking Helix C? to the first structural domain in ?I-spectrin was clearly structured. Based on the published model for aligning Helices A?, B? and C?, important interactions amongst residues in Helix C? of ?I/II-spectrin and Helices A? and B? of ?I/II-spectrin are identified, suggesting similar coiled coil helical bundling for spectrin I and II in forming tetramers. The differences in affinities are likely due to the differences in the conformation of the junction regions. Equilibrium dissociation constants of spin labeled ?II and ?I complexes from ITC measurements indicate that residues 15, 19, 37 and 40 are functionally important residues in ?II-spectrin. Interestingly, all four corresponding homologous residues in ?I-spectrin (residues 24, 28, 46 and 49) have been reported to be clinically significant residues involved in hematological diseases.

Li, Qufei; Fung, L. W.-M.

2009-01-01

245

Lipid exposure prediction enhances the inference of rotational angles of transmembrane helices  

PubMed Central

Background Since membrane protein structures are challenging to crystallize, computational approaches are essential for elucidating the sequence-to-structure relationships. Structural modeling of membrane proteins requires a multidimensional approach, and one critical geometric parameter is the rotational angle of transmembrane helices. Rotational angles of transmembrane helices are characterized by their folded structures and could be inferred by the hydrophobic moment; however, the folding mechanism of membrane proteins is not yet fully understood. The rotational angle of a transmembrane helix is related to the exposed surface of a transmembrane helix, since lipid exposure gives the degree of accessibility of each residue in lipid environment. To the best of our knowledge, there have been few advances in investigating whether an environment descriptor of lipid exposure could infer a geometric parameter of rotational angle. Results Here, we present an analysis of the relationship between rotational angles and lipid exposure and a support-vector-machine method, called TMexpo, for predicting both structural features from sequences. First, we observed from the development set of 89 protein chains that the lipid exposure, i.e., the relative accessible surface area (rASA) of residues in the lipid environment, generated from high-resolution protein structures could infer the rotational angles with a mean absolute angular error (MAAE) of 46.32?. More importantly, the predicted rASA from TMexpo achieved an MAAE of 51.05?, which is better than 71.47? obtained by the best of the compared hydrophobicity scales. Lastly, TMexpo outperformed the compared methods in rASA prediction on the independent test set of 21 protein chains and achieved an overall Matthew’s correlation coefficient, accuracy, sensitivity, specificity, and precision of 0.51, 75.26%, 81.30%, 69.15%, and 72.73%, respectively. TMexpo is publicly available at http://bio-cluster.iis.sinica.edu.tw/TMexpo. Conclusions TMexpo can better predict rASA and rotational angles than the compared methods. When rotational angles can be accurately predicted, free modeling of transmembrane protein structures in turn may benefit from a reduced complexity in ensembles with a significantly less number of packing arrangements. Furthermore, sequence-based prediction of both rotational angle and lipid exposure can provide essential information when high-resolution structures are unavailable and contribute to experimental design to elucidate transmembrane protein functions.

2013-01-01

246

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

SciTech Connect

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

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

2009-06-02

247

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

PubMed

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

Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

2014-05-01

248

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

Microsoft Academic Search

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

Neocles B. Leontis; Eric Westhof

1998-01-01

249

Double-helix stellarator.  

National Technical Information Service (NTIS)

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 characterist...

P. E. Moroz

1997-01-01

250

The structural basis for the specificity of retinoid-X receptor-selective agonists: new insights into the role of helix H12.  

PubMed

Ligands that specifically target retinoid-X receptors (RXRs) are emerging as potentially powerful therapies for cancer, diabetes, and the lowering of circulatory cholesterol. To date, RXR has only been crystallized in the absence of ligand or with the promiscuous ligand 9-cis retinoic acid, which also activates retinoic acid receptors. Here we present the structure of hRXRbeta in complex with the RXR-specific agonist LG100268 (LG268). The structure clearly reveals why LG268 is specific for the RXR ligand binding pocket and will not activate retinoic acid receptors. Intriguingly, in the crystals, the C-terminal "activation" helix (AF-2/helix H12) is trapped in a novel position not seen in other nuclear receptor structures such that it does not cap the ligand binding cavity. Mammalian two-hybrid assays indicate that LG268 is unable to release co-repressors from RXR unless co-activators are also present. Together these findings suggest that RXR ligands may be inefficient at repositioning helix H12. PMID:11782480

Love, James D; Gooch, John T; Benko, Szilvia; Li, Chuan; Nagy, Laszlo; Chatterjee, V Krishna K; Evans, Ronald M; Schwabe, John W R

2002-03-29

251

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. PMID:23670702

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

2013-09-01

252

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

PubMed

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 glycophorin A dimerization propensities in different lipid bilayers suggest that the lipid bilayer thickness severely influences the monomer-dimer equilibrium of this transmembrane domain, and dimerization was most efficient under hydrophobic matching conditions. Moreover, cholesterol considerably promotes self-association of transmembrane helices in model membranes by affecting the lipid acyl chain ordering. In general, the order of the lipid acyl chains appears to be an important factor involved in determining the strength and stability of transmembrane helix-helix interactions. As discussed, the described influences of membrane properties on transmembrane helix-helix interactions are highly important for understanding the mechanism of transmembrane protein folding and functioning as well as for gaining a deeper insight into the regulation of signal transduction via membrane integral proteins by bilayer properties. PMID:20603102

Anbazhagan, Veerappan; Schneider, Dirk

2010-10-01

253

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

254

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

255

Membrane assembly of M13 major coat protein: evidence for a structural adaptation in the hinge region and a titled transmembrane domain  

Microsoft Academic Search

New insights into the low-resolution structure of the hinge region and the transmembrane domain of the membrane-bound major coat protein of the bacteriophage M13 are deduced from a single cysteine-scanning approach using fluorescence spectroscopy. New mutant coat proteins are labeled and reconstituted into phospholipid bilayers with varying headgroup compositions (PC, PE, and PG) and thicknesses (14:1PC, 18:1PC, and 22:1PC). Information

Ruud B. Spruijt; Cor J. A. M. Wolfs; Marcus A. Hemminga

2004-01-01

256

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

PubMed

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

Papadakis, George; Tsortos, Achilleas; Gizeli, Electra

2009-12-15

257

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

SciTech Connect

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

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

2001-10-30

258

Infrared Observations of the Helix Planetary Nebula  

Microsoft Academic Search

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 mum IRAC bands is dominated by the pure rotational

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

2006-01-01

259

Molecular Architecture of a Sodium Channel S6 Helix  

PubMed Central

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

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

2013-01-01

260

Structure of the Membrane Reconstituted Transmembrane?Juxtamembrane Peptide EGFR(622?660) and Its Interaction with Ca 2+ \\/Calmodulin †  

Microsoft Academic Search

The transmembrane (TM) and juxtamembrane (JM) regions of the epidermal growth factor receptor (EGFR) couple ligand binding in the extracellular domain to activation of the kinase domain. Solid-state NMR and polarized FTIR measurements of peptides corresponding to the TM plus JM regions of EGFR (residues 622-660) reconstituted in model phospholipid membranes are presented to address the role of the short

Takeshi Sato; Payal Pallavi; Urszula Golebiewska; Stuart McLaughlin; Steven O. Smith

2006-01-01

261

Transmembrane helices predicted at 95% accuracy.  

PubMed Central

We describe a neural network system that predicts the locations of transmembrane helices in integral membrane proteins. By using evolutionary information as input to the network system, the method significantly improved on a previously published neural network prediction method that had been based on single sequence information. The input data were derived from multiple alignments for each position in a window of 13 adjacent residues: amino acid frequency, conservation weights, number of insertions and deletions, and position of the window with respect to the ends of the protein chain. Additional input was the amino acid composition and length of the whole protein. A rigorous cross-validation test on 69 proteins with experimentally determined locations of transmembrane segments yielded an overall two-state per-residue accuracy of 95%. About 94% of all segments were predicted correctly. When applied to known globular proteins as a negative control, the network system incorrectly predicted fewer than 5% of globular proteins as having transmembrane helices. The method was applied to all 269 open reading frames from the complete yeast VIII chromosome. For 59 of these, at least two transmembrane helices were predicted. Thus, the prediction is that about one-fourth of all proteins from yeast VIII contain one transmembrane helix, and some 20%, more than one.

Rost, B.; Casadio, R.; Fariselli, P.; Sander, C.

1995-01-01

262

The structural and functional essentiality of the N-terminal alpha-helix in the phospholipase A2 of the Taiwan banded krait.  

PubMed

In order to identify the structural and functional essentiality of the N-terminal alpha-helix of Bungarus multicinctus PLA2 for its enzymatic activity, comparative studies of the biochemical properties of native and recombinant PLA2 were made. It was found that the appearance of a Met residue preceding the N-terminus Asn-1 of the recombinant protein appreciably affected PLA2 activity and the hydrophobic character of the ANS-binding site. Additionally, the charged state and the hydrophobicity of the molecular surface changed as well. However, removal of the N-terminal Met-1 from the recombinant PLA2 resulted in the production of a fully active PLA2, whose biochemical properties were indistinguishable from those of the native enzyme. These observations, together with the findings that the helical wheel plot of the N-terminal alpha-helix showed distinct hydrophobic and hydrophilic faces and that the ANS-binding site was the active site of PLA2 enzymes, suggest that the hydrophobic face of the N-terminal alpha-helix in native PLA2 should be in the interior of the enzyme molecule for binding with ANS and the phospholipid/substrate. PMID:9161720

Chang, L S; Chang, C C; Wu, P F

1997-05-01

263

Helix-helix interactions and their impact on protein motifs and assemblies.  

PubMed

Protein secondary structure elements are arranged in distinct structural motifs such as four-alpha-helix bundle, 8alpha/8beta TIM-barrel, Rossmann dinucleotide binding fold, assembly of a helical rod. Each structural motif is characterized by a particular type of helix-helix interactions. A unique pattern of contacts is formed by interacting helices of the structural motif. In each type of fold, edges of the helix surface, which participate in the formation of helix-helix contacts with preceding and following helices, differ. This work shows that circular arrangements of the four, eight, and sixteen alpha-helices, which are found in the four-alpha-helical motif, TIM-barrel 8alpha/8beta fold, and helical rod of 16.3 helices per turn correspondingly, can be associated with the mutual positioning of the edges of the helix surfaces. Edges (i, i+1)-(i+1, i+2) of the helix surface are central for the interhelical contacts in a four-alpha-helix bundle. Edges (i, i+1)-(i+2, i+3) are involved in the assembly of four-alpha-helix subunits into helical rod of a tobacco mosaic virus and a three-helix fragment of a Rossmann fold. In 8alpha/8beta TIM-barrel fold, edges (i, i+1)-(i+5, i+6) are involved in the octagon arrangement. Approximation of a cross section of each motif with a polygon (n-gon, n=4, 8, 16) shows that a good correlation exists between polygon interior angles and angles formed by the edges of helix surfaces. PMID:20202472

Kurochkina, Natalya

2010-05-21

264

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

PubMed Central

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

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

2013-01-01

265

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

Microsoft Academic Search

The upstream stimulatory factor 1 (USF1) belongs to the basic helix-loop-helix leucine zipper (b\\/HLH\\/Z) transcription factor family, recognizing the CACGTG DNA motive as a dimer and playing an important role in the regulation of transcription in a variety of cellular and viral promoters. In this study we investigate the USF1 b\\/HLH\\/Z domain and its complexes with DNA by small angle

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

2008-01-01

266

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

Microsoft Academic Search

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

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

2007-01-01

267

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

268

X-ray Structure of the T. Aquaticus Ftsy:GDP Complex Suggests Functional Roles for the C-Terminal Helix of the SRP GTPases  

PubMed Central

FtsY and Ffh are structurally similar prokaryotic Signal Recognition Particle GTPases that play an essential role in the Signal Recognition Particle (SRP)-mediated cotranslational targeting of proteins to the membrane. The two GTPases assemble in a GTP-dependent manner to form a heterodimeric SRP targeting complex. We report here the 2.1 Å X-ray structure of FtsY from T. aquaticus bound to GDP. The structure of the monomeric protein reveals, unexpectedly, canonical binding interactions for GDP. A comparison of the structures of the monomeric and complexed FtsY NG GTPase domain suggests that it undergoes a conformational change similar to that of Ffh NG during the assembly of the symmetric heterodimeric complex. However, in contrast to Ffh, in which the C-terminal helix shifts independently of the other subdomains, the C-terminal helix and N domain of T. aquaticus FtsY together behave as a rigid body during assembly, suggesting distinct mechanisms by which the interactions of the NG domain “module” are regulated in the context of the two SRP GTPases.

Gawronski-Salerno, Joseph; Coon, John S.; Focia, Pamela J.; Freymann, Douglas M.

2013-01-01

269

Stabilization of alpha-helix structure by polar side-chain interactions: complex salt bridges, cation-pi interactions, and C-H em leader O H-bonds.  

PubMed

It is generally understood that helical proteins are stabilized by a combination of hydrophobic and packing interactions, together with H-bonds and electrostatic interactions. Here we show that polar side-chain interactions on the surface can play an important role in helix formation and stability. We review studies on model helical peptides that reveal the effect of weak interactions between side chains on helix stability, focusing on some nonclassical side-chain-side-chain interactions: complex salt bridges, cation-pi, and C-H em leader O H-bonding interactions. Each of these can be shown to contribute to helix stability, and thus must be included in a comprehensive catalogue of helix stabilizing effects. The issue of the structure of the unfolded states of helical peptides is also discussed, in the light of recent experiments showing that these contain substantial amounts of polyproline II conformation. PMID:12115147

Shi, Z; Olson, C A; Bell, A J; Kallenbach, N R

2001-01-01

270

The Infrared Helix  

NASA Technical Reports Server (NTRS)

The Helix nebula exhibits complex structure on the smallest visible scales. It is composed of gaseous shells and disks puffed out by a dying sun-like star.

In this new image from NASA's Spitzer Space Telescope, 'cometary knots' show blue-green heads caused by excitation of their molecular material from shocks or ultraviolet radiation. The tails of the cometary knots appear redder due to being shielded from the central star's ultraviolet radiation and wind by the heads of the knots.

This image was captured by the telescope's infrared array camera. The false color composite depicts wavelengths of 3.6 microns (blue), 4.5 microns (green), and 8.0 microns (red). The color saturation has been increased to intensify hues.

2006-01-01

271

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

272

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

273

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

PubMed Central

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

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

2009-01-01

274

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

NASA Astrophysics Data System (ADS)

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

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

2014-05-01

275

The Crystal Structure of GXGD Membrane Protease FlaK  

SciTech Connect

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

J Hu; Y Xue; S Lee; Y Ha

2011-12-31

276

The crystal structure of GXGD membrane protease FlaK  

SciTech Connect

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

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

2011-09-20

277

Structure of Amantadine-Bound M2 Transmembrane Peptide of Influenza A in Lipid Bilayers from Magic-Angle-Spinning Solid-State NMR: The Role of Ser31 in Amantadine Binding  

Microsoft Academic Search

The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine, whose effectiveness has been abolished by a single-site mutation of Ser31 to Asn in the transmembrane domain of the protein. Recent high-resolution structures of the M2 transmembrane domain obtained from detergent-solubilized protein in solution and crystal environments gave conflicting drug binding sites. We

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

2009-01-01

278

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

PubMed

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

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

2013-07-30

279

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

PubMed Central

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

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

1991-01-01

280

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

PubMed

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

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

2005-08-01

281

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

PubMed

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

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

2014-01-01

282

The structure and function of the replication terminator protein of Bacillus subtilis: identification of the 'winged helix' DNA-binding domain.  

PubMed

The replication terminator protein (RTP) of Bacillus subtilis impedes replication fork movement in a polar mode upon binding as two interacting dimers to each of the replication termini. The mode of interaction of RTP with the terminus DNA is of considerable mechanistic significance because the DNA-protein complex not only localizes the helicase-blocking activity to the terminus, but also generates functional asymmetry from structurally symmetric protein dimers. The functional asymmetry is manifested in the polar impedance of replication fork movement. Although the crystal structure of the apoprotein has been solved, hitherto there was no direct evidence as to which parts of RTP were in contact with the replication terminus. Here we have used a variety of approaches, including saturation mutagenesis, genetic selection for DNA-binding mutants, photo cross-linking, biochemical and functional characterizations of the mutant proteins, and X-ray crystallography, to identify the regions of RTP that are either in direct contact with or are located within 11 angstroms of the replication terminus. The data show that the unstructured N-terminal arm, the alpha3 helix and the beta2 strand are involved in DNA binding. The mapping of amino acids of RTP in contact with DNA, confirms a 'winged helix' DNA-binding motif. PMID:8670817

Pai, K S; Bussiere, D E; Wang, F; Hutchison, C A; White, S W; Bastia, D

1996-06-17

283

The structure and function of the replication terminator protein of Bacillus subtilis: identification of the 'winged helix' DNA-binding domain.  

PubMed Central

The replication terminator protein (RTP) of Bacillus subtilis impedes replication fork movement in a polar mode upon binding as two interacting dimers to each of the replication termini. The mode of interaction of RTP with the terminus DNA is of considerable mechanistic significance because the DNA-protein complex not only localizes the helicase-blocking activity to the terminus, but also generates functional asymmetry from structurally symmetric protein dimers. The functional asymmetry is manifested in the polar impedance of replication fork movement. Although the crystal structure of the apoprotein has been solved, hitherto there was no direct evidence as to which parts of RTP were in contact with the replication terminus. Here we have used a variety of approaches, including saturation mutagenesis, genetic selection for DNA-binding mutants, photo cross-linking, biochemical and functional characterizations of the mutant proteins, and X-ray crystallography, to identify the regions of RTP that are either in direct contact with or are located within 11 angstroms of the replication terminus. The data show that the unstructured N-terminal arm, the alpha3 helix and the beta2 strand are involved in DNA binding. The mapping of amino acids of RTP in contact with DNA, confirms a 'winged helix' DNA-binding motif. Images

Pai, K S; Bussiere, D E; Wang, F; Hutchison, C A; White, S W; Bastia, D

1996-01-01

284

Helical Hairpin Structure of Influenza Hemagglutinin Fusion Peptide Stabilized by Charge-Dipole Interactions between the N-terminal Amino Group and the Second Helix  

PubMed Central

The fusion domain of the influenza coat protein hemagglutinin HA2, bound to dodecyl phosphocholine (DPC) micelles, was recently shown to adopt a structure consisting of two anti-parallel ?-helices, packed together in an exceptionally tight hairpin configuration. Four interhelical H? to C=O aliphatic H-bonds were identified as factors stabilizing this fold. Here, we report evidence for an additional stabilizing force: a strong charge dipole interaction between the N-terminal Gly1 amino group and the dipole moment of helix 2. pH titration of the amino-terminal 15N resonance, using a methylene-TROSY based 3D NMR experiment, and observation of Gly1 13C? show a strongly elevated pK value of 8.8, considerably higher than expected for an N-terminal amino group in a lipophilic environment. Chemical shifts of three C-terminal carbonyl carbons of helix 2 titrate with the protonation state of Gly1-N, indicative of a close proximity between the N-terminal amino group and the axis of helix 2, thereby providing an optimal charge-dipole stabilization of the antiparallel hairpin fold. pK values of the side chain carboxylate groups of Glu11 and Asp19 are higher by about one and 0.5 unit, respectively, than commonly seen for solvent-exposed side chains in water-soluble proteins, indicative of dielectric constants of ? = ~30 (Glu11) and ? = ~60 (Asp19), which places these groups in the headgroup region of the phospholipid micelle.

Lorieau, Justin L.; Louis, John M.; Bax, Ad

2011-01-01

285

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

286

Transformation between ?-helix and ?-sheet structures of one and two polyglutamine peptides in explicit water molecules by replica-exchange molecular dynamics simulations.  

PubMed

Aggregation of polyglutamine peptides with ?-sheet structures is related to some important neurodegenerative diseases such as Huntington's disease. However, it is not clear how polyglutamine peptides form the ?-sheets and aggregate. To understand this problem, we performed all-atom replica-exchange molecular dynamics simulations of one and two polyglutamine peptides with 10 glutamine residues in explicit water molecules. Our results show that two polyglutamine peptides mainly formed helix or coil structures when they are separated, as in the system with one-polyglutamine peptide. As the interpeptide distance decreases, the intrapeptide ?-sheet structure sometimes appear as an intermediate state, and finally the interpeptide ?-sheets are formed. We also find that the polyglutamine dimer tends to form the antiparallel ?-sheet conformations rather than the parallel ?-sheet, which is consistent with previous experiments and a coarse-grained molecular dynamics simulation. © 2014 Wiley Periodicals, Inc. PMID:24831733

Chiang, Hsin-Lin; Chen, Chun-Jung; Okumura, Hisashi; Hu, Chin-Kun

2014-07-15

287

Sequence-dependent crossed helix packing in the crystal structure of a B-DNA decamer yields a detailed model for the Holliday junction.  

PubMed

The structure of the B-DNA decamer d(CGCAATTGCG)2 has been determined by X-ray diffraction analysis to a resolution of 2.3 A and an R-factor of 17.7%. The decamer crystallises in the monoclinic space group C2 and packs with a crossed arrangement of helices and a unique crossing contact distinct from all other decamer structures. This is believed to be a direct result of the sequence-dependent minor groove width of the duplex. Crossed helix structures of DNA are valuable starting points for modelling studies of the Holliday junction. Two unique sites are observed at the cross-over junction where strand exchange may occur. A Holliday junction model has been constructed for each case and modelled using molecular mechanics and dynamics techniques. One of these models was found to be fully consistent with the available physical data. PMID:9223644

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

1997-06-27

288

The helix located between the two domains of a mip-like peptidyl-prolyl cis-trans isomerase is crucial for its structure, stability, and protein folding ability.  

PubMed

FKBP22, a PPIase (peptidyl-prolyl cis-trans isomerase) produced by Escherichia coli, binds FK506 and rapamycin (both immunosuppressive drugs), shares significant homology with the Mip-like virulence factors, and has been thought to carry a long ?-helix (namely ?3) between its two domains. To understand whether the length of helix ?3 plays any role in the structure, function, and stability of FKBP22-like proteins, we studied a recombinant E. coli FKBP22 (rFKBP22) and its four helix ?3 mutant variants by various in vitro probes. Of the helix ?3 mutants, two were deletion mutants (rFKBP22D5 and rFKBP22D30), whereas the two others were insertion mutants (rFKBP22I3 and rFKBP22I6). Our investigations revealed that the molecular dimensions, dimerization efficiencies, secondary structures, tertiary structures, stabilities, and protein folding abilities of all mutant proteins are different from those of rFKBP22. Conversely, the rapamycin binding affinities of the mutant proteins were affected very little. Urea-induced unfolding of each protein followed a two-state mechanism and was reversible in nature. Interestingly, rFKBP22D30 was the least stable, whereas rFKBP22I3 appeared to be the most stable of the five proteins. The data together suggest that length of helix ?3 contributes significantly to the preservation of the structure, function, and stability of E. coli FKBP22. PMID:22989269

Jana, Biswanath; Sau, Subrata

2012-10-01

289

Solution NMR structures of the antimicrobial peptides phylloseptin-1, -2, and -3 and biological activity: the role of charges and hydrogen bonding interactions in stabilizing helix conformations.  

PubMed

Phylloseptins are antimicrobial peptides of 19-20 residues which are found in the skin secretions of the Phyllomedusa frogs that inhabit the tropical forests of South and Central Americas. The peptide sequences of PS-1, -2, and -3 carry an amidated C-terminus and they exhibit 74% sequence homology with major variations of only four residues close to the C-terminus. Here we investigated and compared the structures of the three phylloseptins in detail by CD- and two-dimensional NMR spectroscopies in the presence of phospholipid vesicles or in membrane-mimetic environments. Both CD and NMR spectroscopies reveal a high degree of helicity in the order PS-2> or =PS-1>PS-3, where the differences accumulate at the C-terminus. The conformational variations can be explained by taking into consideration electrostatic interactions of the negative ends of the helix dipoles with potentially cationic residues at positions 17 and 18. Whereas two are present in the sequence of PS-1 and -2 only one is present in PS-3. In conclusion, the antimicrobial phylloseptin peptides adopt alpha-helical conformations in membrane environments which are stabilized by electrostatic interactions of the helix dipole as well as other contributions such hydrophobic and capping interactions. PMID:18656510

Resende, Jarbas M; Moraes, Cléria Mendonça; Prates, Maura V; Cesar, Amary; Almeida, Fabio C L; Mundim, Nathália C C R; Valente, Ana Paula; Bemquerer, Marcelo P; Piló-Veloso, Dorila; Bechinger, Burkhard

2008-10-01

290

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

PubMed Central

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

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

2014-01-01

291

A novel 2D double helix cadmium(II) coordination polymer: synthesis, crystal structures and luminescence properties  

Microsoft Academic Search

A novel double helix coordination polymer, [Cd(2,2?-bpeec)I2]n (1) (2,2?-bpeec=3,6-bis[2-(2-pyridyl)ethenyl]-9-ethylcarbazole) has been prepared and characterized by elemental analyses, IR and luminescence spectra, single crystal X-ray diffraction. Complex 1 crystallizes in the space group P21\\/c with a=8.444(2), b=23.126(4), c=14.156(2), ?=97.927(2), Z=4, V=2737.8(7)Å3, ?=3.073mm?1. The 2D framework of the title compound is formed by C–H?I hydrogen bonds and ?–? interactions. The solid-state fluorescent

Hong-Ping Zhou; Ju-Zhou Zhang; Dong-Mei Li; Yong-Min Zhu; Zhang-Jun Hu; Jie-Ying Wu; Yi Xie; Min-Hua Jiang; Xu-Tang Tao; Yu-Peng Tian

2005-01-01

292

A helix replacement mechanism directs metavinculin functions.  

PubMed

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 DeltaLeu954 metavinculin deletion mutant. These structures reveal that an alpha-helix (H1') 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 DeltaLeu954 and Arg975Trp metavinculin mutants reside on the replaced extended coil and the H1' alpha-helix, respectively. Thus, a helix replacement mechanism directs metavinculin's unique functions. PMID:20502710

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

2010-01-01

293

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

294

Effects of side chains in helix nucleation differ from helix propagation.  

PubMed

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

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

2014-05-01

295

Double-helix stellarator  

Microsoft Academic Search

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 â 1--1.2. Other advantages include a high enclosed volume, appreciable rotational transform, and a

Moroz

1997-01-01

296

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

297

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

PubMed

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

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

1992-08-01

298

Hepatitis C Virus RNA Replication Requires a Conserved Structural Motif within the Transmembrane Domain of the NS5B RNA-Dependent RNA Polymerase ?  

PubMed Central

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), the viral RNA-dependent RNA polymerase (RdRp), is a tail-anchored protein with a highly conserved C-terminal transmembrane domain (TMD) that is required for the assembly of a functional replication complex. Here, we report that the TMD of the HCV RdRp can be functionally replaced by a newly identified analogous membrane anchor of the GB virus B (GBV-B) NS5B RdRp. Replicons with a chimeric RdRp consisting of the HCV catalytic domain and the GBV-B membrane anchor replicated with reduced efficiency. Compensatory amino acid changes at defined positions within the TMD improved the replication efficiency of these chimeras. These observations highlight a conserved structural motif within the TMD of the HCV NS5B RdRp that is required for RNA replication.

Brass, Volker; Gouttenoire, Jerome; Wahl, Anja; Pal, Zsuzsanna; Blum, Hubert E.; Penin, Francois; Moradpour, Darius

2010-01-01

299

Structural basis for a pH-sensitive calcium leak across membranes.  

PubMed

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

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

2014-06-01

300

Numerical study of helix photonic metamaterial  

NASA Astrophysics Data System (ADS)

Helix photonic metamaterials are attractive to many applications due to the unique properties of strong circular dichroism and gyrotropy. In this study, the optical properties of metallic helix metamaterial were systematically investigated. Such metamaterial is composed of three-dimensional metallic helical nanowires arranged in a two-dimensional array. 3D finite-difference time-domain (FDTD) method was adopted for simulating the spectral response under the excitation of circularly polarized light. We show that the spectral responses were correlated to the dimensions of the helix structures. Generally, the resonance wavelengths as well as optical properties were determined by the geometrical parameters and the composed materials of the structures. When the dimension scaled down, electromagnetic interactions between helices are pronounced, which consequently affect the optical responses of the structures. The dependency between structure dimension and the corresponding optical properties were discussed and presented in this report.

Li, Yu-Ray; Huang, Hsiang-Hung; Ling, Yi-Chun; Hung, Yu-Chueh

2012-02-01

301

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

302

Specific recognition of the collagen triple helix by chaperone HSP47: minimal structural requirement and spatial molecular orientation.  

PubMed

The unique folding of procollagens in the endoplasmic reticulum is achieved with the assistance of procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed "flying capstan model" (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319). PMID:16326708

Koide, Takaki; Asada, Shinichi; Takahara, Yoshifumi; Nishikawa, Yoshimi; Nagata, Kazuhiro; Kitagawa, Kouki

2006-02-10

303

THE FIRST TRANSMEMBRANE REGION OF THE BETA CHAIN STABILIZES THE TETRAMERIC Fc?RI COMPLEX  

PubMed Central

The family of activating immune receptors stabilizes via the 3-helix assembly principle. A charged basic transmembrane residue interacts with two charged acidic transmembrane residues and forms a 3-helix interface to stabilize receptor complexes in the lipid bilayer. One family member, the high affinity receptor for IgE, Fc?RI, is a key regulator of immediate allergic responses. Tetrameric Fc?RI consists of the IgE-binding ?-chain, the multimembrane spanning ?-chain and a dimer of the ?-subunit (Fc?R?). Comparative analysis of these seven transmembrane regions indicates that Fc?RI does not meet the charge requirements for the 3-helix assembly mechanism. We performed alanine mutagenesis to show that the only basic amino acid in the transmembrane regions, ?K97, is not involved in Fc?RI stabilization or surface up-regulation, a hallmark function of the ?-chain. Even a ?K97E mutant is functional despite four negatively charged acidic amino acids in the transmembrane regions. Using truncation mutants, we demonstrate that the first uncharged transmembrane domain of the ?-chain contains the interface for receptor stabilization. In vitro translation experiments depict the first transmembrane region as the internal signal peptide of the ?-chain. We also show that this ?-chain domain can function as a cleavable signal peptide when used as a leader peptide for a Type I protein. Our results provide evidence that tetrameric Fc?RI does not assemble according to the 3-helix assembly principle. We conclude that receptors formed with multispanning proteins use different mechanisms of shielding transmembrane charged amino acids.

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

2009-01-01

304

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

305

Triple Helix, Quadruple Helix and Quintuple Helix and How Do Knowledge, Innovation and the Environment Relate To Each Other? : A Proposed Framework for a Transdisciplinary Analysis of Sustainable Development and Social Ecology  

Microsoft Academic Search

This article develops an inter-disciplinary and trans-disciplinary framework of analysis that relates knowledge, innovation and the environment (natural environments) to each other. For that purpose the five-helix structure model of the Quintuple Helix is being introduced. The Triple Helix model, designed by Etzkowitz and Leydesdorff (2000), focuses on the relations of universities, industry and governments. The Quadruple Helix (Carayannis &

Elias G. Carayannis; David F. J. Campbell

2010-01-01

306

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

307

Hydrophobic matching controls the tilt and stability of the dimeric platelet-derived growth factor receptor (PDGFR) ? transmembrane segment.  

PubMed

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

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

2012-07-27

308

Crystallizing Transmembrane Peptides in Lipidic Mesophases  

PubMed Central

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 ?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.

Hofer, Nicole; Aragao, David; Caffrey, Martin

2010-01-01

309

Characterization of a helix-loop-helix (EF hand) motif of silver hake parvalbumin isoform B.  

PubMed Central

Parvalbumins are a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. It is suspected that these proteins evolved via intragene duplication from a single EF-hand. Silver hake parvalbumin (SHPV) consists of three EF-type helix-loop-helix regions, two of which have the ability to bind calcium. The three helix-loop-helix motifs are designated AB, CD, and EF, respectively. In this study, native silver hake parvalbumin isoform B (SHPV-B) has been sequenced by mass spectrometry. The sequence indicates that this parvalbumin is a beta-lineage parvalbumin. SHPV-B was cleaved into two major fragments, consisting of the ABCD and EF regions of the native protein. The 33-amino acid EF fragment (residues 76-108), containing one of the calcium ion binding sites in native SHPV-B, has been isolated and studied for its structural characteristics, ability to bind divalent and trivalent cations, and for its propensity to undergo metal ion-induced self-association. The presence of Ca2+ does not induce significant secondary structure in the EF fragment. However, NMR and CD results indicate significant secondary structure promotion in the EF fragment in the presence of the higher charge-density trivalent cations. Sedimentation equilibrium analysis results show that the EF fragment exists in a monomer-dimer equilibrium when complexed with La3+.

Revett, S. P.; King, G.; Shabanowitz, J.; Hunt, D. F.; Hartman, K. L.; Laue, T. M.; Nelson, D. J.

1997-01-01

310

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

311

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

312

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

PubMed

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

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

2011-12-20

313

Backfire bifilar helix antenna  

Microsoft Academic Search

A backfire bifilar helix antenna is disclosed having two helically wound conductors made of coaxial cable wherein the two conductors comprise the shield portion of the cable. The coaxial cable of one of the conductors serves as a transmission line for supplying signals to the feed end of the antenna and has its center conductor connected to the shield of

C. W. Webster

1984-01-01

314

Helix Translation Device.  

National Technical Information Service (NTIS)

This invention places a single flat shim, wound into a helical coil, above and below an object to be moved which has a 'transition area' between its upper and lower surface. This 'transition area' allows the helix shim to 'transition' from the top surface...

M. B. Milam P. Studer

1991-01-01

315

Gibbs sampling and helix-cap motifs  

PubMed Central

Protein backbones have characteristic secondary structures, including ?-helices and ?-sheets. Which structure is adopted locally is strongly biased by the local amino acid sequence of the protein. Accurate (probabilistic) mappings from sequence to structure are valuable for both secondary-structure prediction and protein design. For the case of ?-helix caps, we test whether the information content of the sequence–structure mapping can be self-consistently improved by using a relaxed definition of the structure. We derive helix-cap sequence motifs using database helix assignments for proteins of known structure. These motifs are refined using Gibbs sampling in competition with a null motif. Then Gibbs sampling is repeated, allowing for frameshifts of ±1 amino acid residue, in order to find sequence motifs of higher total information content. All helix-cap motifs were found to have good generalization capability, as judged by training on a small set of non-redundant proteins and testing on a larger set. For overall prediction purposes, frameshift motifs using all training examples yielded the best results. Frameshift motifs using a fraction of all training examples performed best in terms of true positives among top predictions. However, motifs without frameshifts also performed well, despite a roughly one-third lower total information content.

Kruus, Erik; Thumfort, Peter; Tang, Chao; Wingreen, Ned S.

2005-01-01

316

Importance of lipid-pore loop interface for potassium channel structure and function  

PubMed Central

Potassium (i.e., K+) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K+ channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K+ channel inactivation and exhibits a remarkable structural plasticity that correlates to K+ channel inactivation. The transmembrane helix 1 unwinds when the K+ channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K+ ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K+ channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K+ channel pore.

van der Cruijsen, Elwin A. W.; Nand, Deepak; Weingarth, Markus; Prokofyev, Alexander; Hornig, Sonke; Cukkemane, Abhishek Arun; Bonvin, Alexandre M. J. J.; Becker, Stefan; Hulse, Raymond E.; Perozo, Eduardo; Pongs, Olaf; Baldus, Marc

2013-01-01

317

CHUK, a conserved helix-loop-helix ubiquitous kinase, maps to human chromosome 10 and mouse chromosome 19  

SciTech Connect

Helix-loop-helix proteins contain stretches of DNA that encode two amphipathic {alpha}-helices joined by a loop structure and are involved in protein dimerization and transcriptional regulation essential to a variety of cellular processes. CHUK, a newly described conserved helix-loop-helix ubiquitous kinase, was mapped by somatic cell hybrid analyses to human Chr 10q24-q25. Chuk and a related sequence, Chuk-rs1, were mapped to mouse chromosomes 19 and 16, respectively, by a combination of somatic cell hybrid, recombinant inbred, and backcross analyses. 17 refs., 2 figs., 1 tab.

Mock, B.A.; McBride, O.W.; Kozak, C.A. [National Institutes of Health, Bethesda, MD (United States)] [and others] [National Institutes of Health, Bethesda, MD (United States); and others

1995-05-20

318

Analyses of the Effects That Disease-Causing Missense Mutations Have on the Structure and Function of the Winged-Helix Protein FOXC1  

PubMed Central

Five missense mutations of the winged-helix FOXC1 transcription factor, found in patients with Axenfeld-Rieger (AR) malformations, were investigated for their effects on FOXC1 structure and function. Molecular modeling of the FOXC1 forkhead domain predicted that the missense mutations did not alter FOXC1 structure. Biochemical analyses indicated that, whereas all mutant proteins correctly localize to the cell nucleus, the I87M mutation reduced FOXC1-protein levels. DNA-binding experiments revealed that, although the S82T and S131L mutations decreased DNA binding, the F112S and I126M mutations did not. However, the F112S and I126M mutations decrease the transactivation ability of FOXC1. All the FOXC1 mutations had the net effect of reducing FOXC1 transactivation ability. These results indicate that the FOXC1 forkhead domain contains separable DNA-binding and transactivation functions. In addition, these findings demonstrate that reduced stability, DNA binding, or transactivation, all causing a decrease in the ability of FOXC1 to transactivate genes, can underlie AR malformations.

Saleem, Ramsey A.; Banerjee-Basu, Sharmila; Berry, Fred B.; Baxevanis, Andreas D.; Walter, Michael A.

2001-01-01

319

Tryptophan scanning mutagenesis reveals distortions in the helical structure of the ?M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor  

PubMed Central

The lipid-protein interface is an important domain of the nicotinic acetylcholine receptor (nAChR) that has recently garnered increased relevance. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, there is still a need to gain insight into the mechanism by which lipid-protein interactions regulate the function and conformational transitions of the nAChR. In this study, we extended the tryptophan scanning mutagenesis (TrpScanM) approach to dissect secondary structure and monitor the conformational changes experienced by the ?M4 transmembrane domain (TMD) of the Torpedo californica nAChR, and to identify which positions on this domain are potentially linked to the regulation of ion channel kinetics. The difference in oscillation patterns between the closed- and open-channel states suggests a substantial conformational change along this domain as a consequence of channel activation. Furthermore, TrpScanM revealed distortions along the helical structure of this TMD that are not present on current models of the nAChR. Our results show that a Thr-Pro motif at positions 462–463 markedly bends the helical structure of the TMD, consistent with the recent crystallographic structure of the GluCl Cys-loop receptor which reveals a highly bent TMD4 in each subunit. This Thr-Pro motif acts as a molecular hinge that delineates two gating blocks in the ?M4 TMD. These results suggest a model in which a hinge-bending motion that tilts the helical structure is combined with a spring-like motion during transition between the closed- and open-channel states of the ?M4 TMD.

Caballero-Rivera, Daniel; Cruz-Nieves, Omar A.; Oyola-Cintron, Jessica; Torres-Nunez, David A.; Otero-Cruz, Jose D.; Lasalde-Dominicci, Jose A.

2012-01-01

320

Modulation of the intrinsic helix propensity of an intrinsically disordered protein reveals long-range helix-helix interactions.  

PubMed

Intrinsically disordered proteins (IDPs) are widespread and important in biology but defy the classical protein structure-function paradigm by being functional in the absence of a stable, folded conformation. Here we investigate the coupling between transient secondary and tertiary structure in the protein activator for thyroid hormone and retinoid receptors (ACTR) by rationally modulating the helical propensity of a partially formed ?-helix via mutations. Eight mutations predicted to affect the population of a transient helix were produced and investigated by NMR spectroscopy. Chemical shift changes distant to the mutation site are observed in regions containing other transient helices indicating that distant helices are stabilized through long-range hydrophobic helix-helix interactions and demonstrating the coupling of transient secondary and tertiary structure. The long-range structure of ACTR is also probed using paramagnetic relaxation enhancements (PRE) and residual dipolar couplings, which reveal an additional long-range contact between the N- and C-terminal segments. Compared to residual dipolar couplings and PRE, modulation of the helical propensity by mutagenesis thus reveals a different set of long-range interactions that may be obscured by stronger interactions that dominate other NMR measurements. This approach thus offers a complementary and generally applicable strategy for probing long-range structure in disordered proteins. PMID:23758617

Iešmantavi?ius, Vytautas; Jensen, Malene Ringkjøbing; Ozenne, Valéry; Blackledge, Martin; Poulsen, Flemming M; Kjaergaard, Magnus

2013-07-10

321

Role of Transmembrane Domain 4 in Ligand Permeation by Crithidia fasciculata Equilibrative Nucleoside Transporter 2 (CfNT2)*  

PubMed Central

Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability, indicating that this helix lines the water-filled ligand translocation channel. Additionally, the pattern of modification of transmembrane domain 4 suggested that it may deviate from helicity in the vicinity of residue 155. Position 155 was also protected from modification in the presence of ligand, suggesting that lysine 155 is in or near the ligand binding site. Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2.

Arendt, Cassandra S.; Ullman, Buddy

2010-01-01

322

Statistical Thermodynamic Analysis of Helix-Coil Transitions in Polypeptides  

Microsoft Academic Search

Synthetic polypeptides distinguish themselves from common polymer substances in the characteristic feature that a majority, though not all of them, have molecular chains capable of maintaining a partially or entirely helical secondary structure called a-helix in appropriate solvents over a certain range of temperature. Thus with a helix-forming polypeptide it is possible to observe (for example, by measurement of specific

Akio Teramoto; Hiroshi Fujita

1976-01-01

323

Magnesium Ion-Dependent TripleHelix Structure Formed by Homopurine-Homopyrimidine Sequences in Supercoiled Plasmid DNA  

Microsoft Academic Search

DNA can be chemically cleaved at the site of chloroacetaldehyde-modified residues by the chemicals used for Maxam-Gilbert sequencing reactions. Use of this technique facilitates fine structural analysis of unpaired DNA bases in DNA with non-B-DNA structure. This method was used to study the non-B-DNA structure adopted by the poly(dG)\\\\cdot poly(dC) sequence under torsional stress at various ionic conditions. In the

Yoshinori Kohwi; Terumi Kohwi-Shigematsu

1988-01-01

324

Theoretical Studies of the M2 Transmembrane Segment of the Glycine Receptor: Models of the Open Pore Structure and Current-Voltage Characteristics  

PubMed Central

The pentameric glycine receptor (GlyR), a member of the nicotinicoid superfamily of ligand-gated ion channels, is an inhibitory Cl? channel that is gated by glycine. Using recently published NMR data of the second transmembrane segment (M2) of the human ?1 GlyR, structural models of pentameric assemblies embedded in a lipid bilayer were constructed using a combination of experimentally determined constraints coupled with all-atom energy minimization. Based on this structure of the pentameric M2 “pore”, Brownian dynamics simulations of ion permeation through this putative conducting open state of the channel were carried out. Simulated I-V curves were in good agreement with published experimental current-voltage curves and the anion/cation permeability ratio, suggesting that our open-state model may be representative of the conducting channel of the full-length receptor. These studies also predicted regions of chloride occupancy and suggested residues critical to anion permeation. Calculations of the conductance of the cation-selective mutant A251E channel are also consistent with experimental data. In addition, both rotation and untilting of the pore helices of our model were found to be broadly consistent with closing of the channel, albeit at distinct regions that may reflect alternate gates of the receptor.

Cheng, Mary Hongying; Cascio, Michael; Coalson, Rob D.

2005-01-01

325

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

PubMed Central

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

Sanchez-Munoz, Orlando L.; Strandberg, Erik; Esteban-Martin, E.; Grage, Stephan L.; Ulrich, Anne S.; Salgado, Jesus

2013-01-01

326

Propofol binding to the resting state of the gloeobacter violaceus ligand-gated ion channel (GLIC) induces structural changes in the inter- and intrasubunit transmembrane domain (TMD) cavities.  

PubMed

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

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

2013-06-14

327

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

PubMed Central

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

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

2002-01-01

328

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

PubMed Central

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

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

2013-01-01

329

A direct comparison of helix propensity in proteins and peptides  

PubMed Central

?-Helical secondary structure occurs widely in globular proteins and its formation is a key step in their folding. As a consequence, understanding the energetics of helix formation is crucial to understanding protein folding and stability. We have measured the helix propensities of the nonpolar amino acids for an ?-helix in an intact protein, ribonuclease T1, and for a 17-residue peptide with a sequence identical to that of the ?-helix in the protein. The helix propensities are in excellent agreement. This shows that when compared in the same sequence context, the helix propensities of the nonpolar amino acids are identical in helical peptides and intact proteins, and that conclusions based on studies of the helix-to-coil transitions of peptides may, in favorable cases, be directly applicable to proteins. Our helix propensities based on ribonuclease T1 are in good agreement with those from similar studies of barnase and T4 lysozyme. In contrast, our helix propensities differ substantially from those derived from studies of alanine-stabilized or salt bridge-stabilized model ?-helical peptides.

Myers, Jeffrey K.; Pace, C. Nick; Scholtz, J. Martin

1997-01-01

330

Conformational dynamics of protein transporter FhaC: large-scale motions of plug helix.  

PubMed

FhaC is an integral outer membrane protein of the whooping cough agent Bordetella pertussis that mediates the transport to the cell surface of a major virulence factor, the filamentous haemagglutinin adhesin FHA. The FHA/FhaC pair is a prototypic TpsA/TpsB system of the widespread 'Two-Partner Secretion' pathway, dedicated to the transport of long extracellular proteins in various pathogenic and environmental Gram-negative bacteria. FhaC belongs to the ubiquitous Omp85 superfamily of protein transporters. The X-ray structure of FhaC shows that the transmembrane ?-barrel channel hypothesized to serve as the FHA-conducting pore is obstructed by two structural elements conserved among TpsB transporters, an N-terminal ? helix and an extracellular loop. Here, we provide evidence for conformational dynamics of FhaC related to the secretion mechanism. Using paramagnetic electron resonance, electrophysiology and in vivo approaches, we showed that FhaC exchanges between open and closed conformations. The interaction with its secretory partner FHA alters this distribution of conformations. The open conformation of FhaC implies a large displacement from the channel of the N-terminal 'plug' helix, which remains in the periplasm during FHA secretion. The membrane environment favours the dynamics of the TpsB transporter. PMID:24646315

Guérin, Jérémy; Baud, Catherine; Touati, Nadia; Saint, Nathalie; Willery, Eve; Locht, Camille; Vezin, Hervé; Jacob-Dubuisson, Françoise

2014-06-01

331

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

332

Left helix of polyproline II type and genesis of ?-structures in spidroins 1 and 2 and their recombinant analogs  

Microsoft Academic Search

The distribution of secondary structure elements along the polypeptide chains of spider silk proteins spidroins 1 and 2 and\\u000a their recombinant analogs has been studied by statistical methods. It was found that these proteins as monomers contain only\\u000a traces of ?-structure, while the Ala-rich and the Gly-rich regions are predicted as ?-helices and as left-handed helices of\\u000a polyproline II type.

N. G. Esipova; L. E. Ragulina; L. I. Davydova; V. M. Lobachev; V. Yu. Makeev; V. G. Bogush; V. G. Tumanyan; V. G. Debabov

2009-01-01

333

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

334

Quadrifilar helix antenna for MIMO system  

Microsoft Academic Search

In this letter, the performance of using a meander line printed quadrifilar helix antenna (MPQHA) structure in a multiple-input-multiple-output (MIMO) system was investigated. It is known that MIMO systems can offer larger capacity than traditional systems, and can exploit transmit or receive diversity. An important parameter is the correlation between the adjacent channels, with a capacity increase for low correlation.

Daniel K. C. Chew; I. Morfis; Dimitrios Mavrakis; Stavros Stavrou

2004-01-01

335

Dramatic Structural Changes Resulting from the Loss of a Crucial Hydrogen Bond in the Hinge Region Involved in C-Terminal Helix Swapping in SurE: A Survival Protein from Salmonella typhimurium  

PubMed Central

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

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

2013-01-01

336

Crystallization and preliminary X-ray diffraction analysis of post-fusion six-helix bundle core structure from Newcastle disease virus F protein.  

PubMed

Fusion of virus members from the Paramyxoviridae family involves two glycoproteins. They are termed attachment glycoprotein (HN, H or G) and fusion protein (F). The F protein contains two highly conserved heptad-repeat (HR) regions, HR1 and HR2. Through conformational changes in the F protein, HR1 and HR2 are believed to form a stable six-helix coiled-coil bundle during the membrane-fusion process. However, no crystal structure has yet been documented for this state in the Newcastle disease virus (NDV, a member of the Paramyxoviridae family) F protein, despite the recent success on its F(0) crystal structure (Chen et al., 2001), which was thought to represent the pre-fusion conformation of F glycoprotein. In this study, a single-chain polypeptide constructed by linking two truncated HR regions of the NDV F protein has been expressed, purified and crystallized by means of the hanging- or sitting-drop vapour-diffusion method. Crystals in hexagonal and trapezoid forms with a resolution limit of 2.6 A were obtained. These crystals belonged to space group C2, with unit-cell parameters a = 66.4, b = 38.2, c = 102.0 A, beta = 100.2 degrees. Crystals in the rhombic form with a resolution limit of 2.5 A were also obtained. These crystals belonged to space group P2(1), with unit-cell parameters a = 59.0, b = 31.9, c = 62.3 A, beta = 117.0 degrees. This will add to the repertoire of viral fusion protein post-fusion state structures and help further the understanding of the molecular mechanism of enveloped virus fusion. PMID:12832792

Li, Peng-Yun; Zhu, Jie-Qing; Wu, Bei Li; Gao, Feng; Tien, Po; Rao, Zihe; Gao, George F

2003-07-01

337

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

PubMed

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

Khadria, Ambalika S; Senes, Alessandro

2013-10-29

338

A novel approach for simulation of a coaxial coupler for helix TWTs using HFSS  

Microsoft Academic Search

This paper describes a novel approach to model and analyze a helix to coaxial transition using three-dimensional electromagnetic computer code, the High Frequency Structure Simulator (HFSS) based on the Finite Element Method (FEM). For a K-Ka band helix TWT, RF Coaxial Coupler consisting of a helix to coaxial transition and a RF coaxial window is designed. The novelty of this

A. K. Agrawal; S. Raina; L. Kumar

2003-01-01

339

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

340

iBioSeminar: Seven Transmembrane Receptors  

NSDL National Science Digital Library

Highlights include overcoming initial skepticism that the receptors even existed; isolating the receptors as discrete biochemical entities and demonstrating their ligand binding and functional activating properties; discovering their seven transmembrane spanning arrangement and homology with the visual light receptor rhodopsin, thereby leading to the discovery of the wider seven transmembrane receptor superfamily; determination of the structure â function relationships of the receptors by mutagenesis and chimeric receptor construction; discovery of constitutively active mutant receptors; discovery of the phosphorylation of the receptors by G protein coupled receptor kinases, and of the Beta-arrestins and of their universal mechanism for desensitizing the receptors.

Robert Lefkowitz (ASCB;)

2011-06-06

341

The Gramicidin A Transmembrane Channel: Characteristics of Head-to-Head Dimerized ?(L,D) Helices  

PubMed Central

A series of helical structures for gramicidin A, with alternating L and D residues, are characterized as to number of residues per turn, atoms in hydrogenbonded rings, and dihedral angles. Because of alternating peptide C-O directions, these helices are capable of forming head-to-head hydrogen-bonded dimers with the capacity of functioning as transmembrane channels. The dimers are characterized as to channel length, pore size, and expected ion selectivity. In a test of the proposed head-to-head association for channel formation, the malonyl dimer [N,N?-(dideformyl gramicidin A)-malonamide] was synthesized. The chemical and conformational integrity of the product was verified by nuclear magnetic resonance; in lipid bilayer studies, the dimer was found to be a potent mediator of ion conductance with the predicted concentration dependence. Thus, the results on malonyl gramicidin A prove head-to-head association in formation of the transmembrane channel, and the results are consistent with the specific geometrical configuration involved in head-to-head dimerization of ?(L,D) helices. At this stage, the action of gramicidin A on membranes with lipid-layer thicknesses of 30 Å or less can best be understood in terms of the ?(L,D) helix with 6.3 residues per turn.

Urry, D. W.; Goodall, M. C.; Glickson, J. D.; Mayers, D. F.

1971-01-01

342

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

PubMed

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

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

2013-01-15

343

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

PubMed Central

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

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

2012-01-01

344

Transmembrane Topology of a CLC Chloride Channel  

Microsoft Academic Search

CLC chloride channels form a large and conserved gene family unrelated to other channel proteins. Knowledge of the transmembrane topology of these channels is important for understanding the effects of mutations found in human myotonia and inherited hypercalciuric kidney stone diseases and for the interpretation of structure--function studies. We now systematically study the topology of human CIC-1, a prototype CLC

Thomas Schmidt-Rose; Thomas J. Jentsch

1997-01-01

345

Different secondary structure elements as scaffolds for protein folding transition states of two homologous four-helix bundles.  

PubMed

Comparison of the folding processes for homologue proteins can provide valuable information about details in the interactions leading to the formation of the folding transition state. Here the folding kinetics of 18 variants of yACBP and 3 variants of bACBP have been studied by Phi-value analysis. In combination with Phi-values from previous work, detailed insight into the transition states for folding of both yACBP and bACBP has been obtained. Of the 16 sequence positions that have been studied in both yACBP and bACBP, 5 (V12, I/L27, Y73, V77, and L80) have high Phi-values and appear to be important for the transition state formation in both homologues. Y31, A34, and A69 have high Phi-values only in yACBP, while F5, A9, and I74 have high Phi-values only in bACBP. Thus, additional interactions between helices A2 and A4 appear to be important for the transition state of yACBP, whereas additional interactions between helices A1 and A4 appear to be important for the transition state of bACBP. To examine whether these differences could be assigned to different packing of the residues in the native state, a solution structure of yACBP was determined by NMR. Small changes in the packing of the hydrophobic side-chains, which strengthen the interactions between helices A2 and A4, are observed in yACBP relative to bACBP. It is suggested that different structure elements serve as scaffolds for the folding of the 2 ACBP homologues. PMID:15690348

Teilum, Kaare; Thormann, Thorsten; Caterer, Nigel R; Poulsen, Heidi I; Jensen, Peter H; Knudsen, Jens; Kragelund, Birthe B; Poulsen, Flemming M

2005-04-01

346

Fluoroalcohols induced unfolding of Succinylated Con A: native like beta-structure in partially folded intermediate and alpha-helix in molten globule like state.  

PubMed

Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20-60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2-40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0-90% v/v TFE and HFIP) and resulted in stable alpha-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both beta-sheet and alpha-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-anilinonaphthalene-1-sulfonic acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from beta-sheet to alpha-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A. PMID:16970906

Fatima, Sadaf; Ahmad, Basir; Khan, Rizwan Hasan

2006-10-15

347

The substrates of the gamma-aminobutyric acid transporter GAT-1 induce structural rearrangements around the interface of transmembrane domains 1 and 6.  

PubMed

The sodium- and chloride-coupled gamma-aminobutyric acid (GABA) transporter GAT-1 is essential for efficient synaptic transmission by this neurotransmitter. GAT-1 is the first cloned member of the neurotransmitter-sodium-symporter family. Here we address the idea that during transport the extracellular halves of transmembrane domains (TM) 1 and 6, TM 1b/TM 6a, move relative to the binding pocket. Therefore, we have probed the aqueous accessibility of TM 6a and its proximity to TM 1b in the presence and absence of its substrates. Cysteines were introduced, one by one, at all TM 6a positions. In several mutants, transport activity was inhibited by the impermeant sulfhydryl reagent (2-trimethylammonium)methanethiosulfonate, whereas wild type GAT-1 was basically insensitive. This inhibition was potentiated by sodium, whereas GABA was protective. Moreover, we used paired cysteine mutagenesis in conjunction with treatments with copper(II)(1,10-phenanthroline)(3) (CuPh). CuPh did not affect the activity of wild type GAT-1 but potently inhibited transport by the TM 6a mutant D287C. Such inhibition was not observed with D287C/C74A, indicating that Asp-287 is close to Cys-74 of TM 1b. Inhibition of transport of D287C by CuPh, but not by (2-trimethylammonium)methanethiosulfonate, was potentiated when sodium and GABA were both removed. Thus, the degree of inhibition by CuPh is not a simple function of the accessibility of the individual cysteines but also involves structural rearrangements around the TM 1b/TM 6a interface. PMID:18381286

Rosenberg, Alex; Kanner, Baruch I

2008-05-23

348

Synthesis and structural characterisation as 12-helix of the hexamer of a beta-amino acid tethered to a pyrrolidin-2-one ring.  

PubMed

Starting from (3S,4R,1'S)-3-amino-2-oxo-1-[1'-(4-methoxyphenylethyl)]pyrrolidine carboxylic acid (2), the first synthesis of a beta-foldamer containing pyrrolidin-2-one rings is described, whose 12-helix conformation is assigned by NMR analysis and confirmed by molecular dynamics (MD) simulations. PMID:17136245

Menegazzo, Ileana; Fries, Alexander; Mammi, Stefano; Galeazzi, Roberta; Martelli, Gianluca; Orena, Mario; Rinaldi, Samuele

2006-12-21

349

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

PubMed Central

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

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

2002-01-01

350

A statistically derived parameterization for the collagen triple-helix  

Microsoft Academic Search

The triple-helix is a unique secondary structural motif found primarily within the collagens. In collagen, it is a homo- or hetero-tripeptide with a repeating primary sequence of (Gly-X-Y)n, displaying characteristic peptide backbone dihedral angles. Studies of bulk collagen fibrils indicate that the triple-helix must be a highly repetitive secondary structure, with very specific constraints. Primary sequence analysis shows that most

Jan K. Rainey; M. Cynthia Goh

2002-01-01

351

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

352

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

PubMed Central

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

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

2014-01-01

353

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

Microsoft Academic Search

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

C. D. Bahl; C. Morisseau; J. M. Bomberger; B. A. Stanton; B. D. Hammock; G OToole; D. R. Madden

2010-01-01

354

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

PubMed

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

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

2013-04-01

355

Modelling Sodium Channel Kinetics using the Sliding Helix Concept  

NASA Astrophysics Data System (ADS)

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

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

1998-03-01

356

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch  

Microsoft Academic Search

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface

Philip L. Yeagle; Michael Bennett; Vincent Lemaître; Anthony Watts

2007-01-01

357

Coil-helix transition of biopolymer confined in finite cylinder  

NASA Astrophysics Data System (ADS)

An off-lattice dynamic Monte Carlo method is used to study the conformations of biopolymers confined in finite cylinders. The coil-helix transition is observed with the increase of bending energy b for the polymer chain confined in small cylinder, and helical structure becomes more perfect for larger b. Periodicity of tangent-tangent correlation function C(s) begins to disappear when the cylinder height H increases, and the influence of R and b on the ability of helix formation of polymer chain weakens significantly. Therefore, the semiflexible and stiff polymer chains can fold into helix in small cylinder.

Sun, Tingting; Yang, Zhiyong

2014-07-01

358

Alternative C-Terminal Helix Orientation Alters Chemokine Function  

PubMed Central

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

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

2013-01-01

359

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

Microsoft Academic Search

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

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

2001-01-01

360

The double helix and the 'wronged heroine'.  

PubMed

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

Maddox, Brenda

2003-01-23

361

Theoretical and experimental TWT helix loss determination  

Microsoft Academic Search

As efficiency and power requirements for broad-band traveling-wave tubes (TWT) increase, the necessity to reduce losses becomes more and more important. It is particularly important to reduce circuit losses because, when efficient multistage depressed collectors are used, each 2-percent increase in circuit efficiency yields a 1-percent increase in overall efficiency! Attempts to predict losses in helix structures have produced answers

M. R. Gillette; Jenn-Tsung Chen

1979-01-01

362

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

Microsoft Academic Search

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

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

2010-01-01

363

A Structurally Altered d , l Amino Acid TCR? Transmembrane Peptide Interacts with the TCR? and Inhibits T-Cell Activation in Vitro and in an Animal Model †  

Microsoft Academic Search

Protein-protein interactions in the membrane are pivotal for the cellular response to receptor- sensed stimuli. Recently, it has been demonstrated that an all- D-amino acids analogue of the TCRR transmembrane peptide (CP) is recruited to the TCR complex and inhibits T-cell activation in Vitro and in ViVo, similarly to the wild-type CP peptide. Here we investigated the relative contributions of

Francisco J. Quintana; Doron Gerber; Itai Bloch; Irun R. Cohen; Yechiel Shai

2007-01-01

364

Spitzer\\/IRAC observations of the Helix Planetary Nebula  

Microsoft Academic Search

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 PN, and therefore provides an opportunity to resolve the small-scale structure in the nebula. The emission from this PN in the 5.8 and 8.0 micron IRAC bands is dominated by the pure rotational

J. L. Hora; W. B. Latter; M. Marengo

2005-01-01

365

GEOTROPIC EXCITATION IN HELIX  

PubMed Central

Rotation of an inclined surface on which Helix is creeping straight upward, such that the axis of the animal is turned at a right angle to its previous position, but in the same plane, leads to negatively geotropic orientation after a measurable latent period or reaction time. The duration of the latent period is a function of the slope of the surface. The magnitude of the standard deviation of the mean latent period is directly proportional to the mean latent period itself, so that the relative variability of response is constant. The dependence of reaction time upon extent of displacement from symmetrical orientation in the gravitational field is found also by tilting the supporting surface, without rotation in the animal's own plane. On slopes up to 55°, the relation between latent period and the sine of the slope is hyperbolic; above this inclination, the latent period sharply declines. This change in the curve is not affected by the attachment of moderate loads to the snail's shell (up to 1/3 of its own mass), and is probably a consequence of loss of passive stable equilibrium when rotated. When added loads do not too greatly extend the snail's anterior musculature, the latent period for the geotropic reaction is decreased, and, proportionately, its ?. These facts are discussed from the standpoint that geotropic excitation in these gasteropods is due to impressed muscle-tensions.

Hoagland, H.; Crozier, W. J.

1931-01-01

366

Bifilar Helix for Backward-Wave Oscillators  

Microsoft Academic Search

A bifilar helix used as the slow wave circuit for the backward-wave oscillator is investigated in this paper. Calculations show that the bifilar helix has a higher impedance than that of the ordinary single-wire helix. The propagating characteristics of the bifilar helix are analyzed and some experimental results of cold measurements are presented. It is also shown that with a

Ping Tien

1954-01-01

367

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

368

The infrared dichroism of transmembrane helical polypeptides.  

PubMed Central

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

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

1995-01-01

369

Role of alpha-helix seven of Bacillus thuringiensis Cry1Ab delta-endotoxin in membrane insertion, structural stability, and ion channel activity.  

PubMed

Domain I of the Cry1Ab insecticidal toxic protein has seven alpha-helices and is considered to be involved in the ion channel activity. While other alpha-helices, particularly alpha-4 and alpha-5, have been extensively explored, the remaining alpha-helices have been slightly studied. Site-directed mutagenesis was used to generate mutations throughout sequences encoding the alpha-helix 7 to test its role in ion channel function. Every amino acid residue in alpha-helix 7 was mutated to alanine. Most resultant proteins, e.g., D225A, W226A, Y229A, N230A, R233A, R234A, D242A, and F247A yielded no protoxin or were sensitive to degradation by trypsin or Manduca sexta midgut juice. Other mutant proteins, R224A, R228A, and E235A, were resistant to degradation to the above proteases but were 8, 30, and 12 times less toxic to M. sexta, respectively, than the wild-type Cry1Ab. Circular dichroism spectroscopy indicated a very small change in the R228A spectrum, while R224A and E235A display the same spectrum as the wild-type protein. These three mutant proteins showed little differences from Cry1Ab when analyzed by saturation binding and competition binding kinetics with (125)I-labeled toxin or by surface plasmon resonance to M. sexta brush border membrane vesicles. More conservative amino acid substitutions were introduced into alpha-helix 7 residues: R228K, F232Y, E235Q, and F247Y. In comparison with wild-type Cry1Ab, mutant proteins R228K, F232Y, E235A, and E235Q selectively discriminate between K+ and Rb+, while R224A and R228A had reduced inhibition of short-circuit current for both ions, when analyzed by voltage clamping of M. sexta midguts. PMID:11327876

Alcantara, E P; Alzate, O; Lee, M K; Curtiss, A; Dean, D H

2001-02-27

370

Interactions between relay helix and Src homology 1 domain helix (SH1) drive the converter domain rotation during the recovery stroke of myosin II  

PubMed Central

Myosin motor protein exists in two alternative conformations, pre-recovery state M* and post-recovery state M**, upon ATP binding. The details of the M*-to-M** transition, known as the recovery stroke to reflect its role as the functional opposite of the force-generating power stroke, remain elusive. The defining feature of the post-recovery state is a kink in the relay helix, a key part of the protein involved in force generation. In this paper we determine the interactions that are responsible for the appearance of the kink. We design a series of computational models that contain three other segments, relay loop, converter domain and Src homology 1 domain helix (SH1), with which relay helix interacts, and determine their structure in accurate replica exchange molecular dynamics simulations in explicit solvent. By conducting an exhaustive combinatorial search among different models we find that: 1) the converter domain must be attached to the relay helix during the transition, so it does not interfere with other parts of the protein, 2) the structure of the relay helix is controlled by SH1 helix. The kink is strongly coupled to the position of SH1 helix. It arises as a result of direct interactions between SH1 and the relay helix and leads to a rotation of the C-terminal part of the relay helix which is subsequently transmitted to the converter domain.

Baumketner, Andrij

2012-01-01

371

The determinants of hydrophobic mismatch response for transmembrane helices.  

PubMed

Hydrophobic mismatch arises from a difference in the hydrophobic thickness of a lipid membrane and a transmembrane protein segment, and is thought to play an important role in the folding, stability and function of membrane proteins. We have investigated the possible adaptations that lipid bilayers and transmembrane ?-helices undergo in response to mismatch, using fully-atomistic molecular dynamics simulations totaling 1.4 ?s. We have created 25 different tryptophan-alanine-leucine transmembrane ?-helical peptide systems, each composed of a hydrophobic alanine-leucine stretch, flanked by 1-4 tryptophan side chains, as well as the ?-helical peptide dimer, gramicidin A. Membrane responses to mismatch include changes in local bilayer thickness and lipid order, varying systematically with peptide length. Adding more flanking tryptophan side chains led to an increase in bilayer thinning for negatively mismatched peptides, though it was also associated with a spreading of the bilayer interface. Peptide tilting, bending and stretching were systematic, with tilting dominating the responses, with values of up to ~45° for the most positively mismatched peptides. Peptide responses were modulated by the number of tryptophan side chains due to their anchoring roles and distributions around the helices. Potential of mean force calculations for local membrane thickness changes, helix tilting, bending and stretching revealed that membrane deformation is the least energetically costly of all mismatch responses, except for positively mismatched peptides where helix tilting also contributes substantially. This comparison of energetic driving forces of mismatch responses allows for deeper insight into protein stability and conformational changes in lipid membranes. PMID:22995244

de Jesus, Armando J; Allen, Toby W

2013-02-01

372

TRAMPLE: the transmembrane protein labelling environment.  

PubMed

TRAMPLE (http://gpcr.biocomp.unibo.it/biodec/) is a web application server dedicated to the detection and the annotation of transmembrane protein sequences. TRAMPLE includes different state-of-the-art algorithms for the prediction of signal peptides, transmembrane segments (both beta-strands and alpha-helices), secondary structure and fast fold recognition. TRAMPLE also includes a complete content management system to manage the results of the predictions. Each user of the server has his/her own workplace, where the data can be stored, organized, accessed and annotated with documents through a simple web-based interface. In this manner, TRAMPLE significantly improves usability with respect to other more traditional web servers. PMID:15980454

Fariselli, Piero; Finelli, Michele; Rossi, Ivan; Amico, Mauro; Zauli, Andrea; Martelli, Pier Luigi; Casadio, Rita

2005-07-01

373

Molecular Content of the Helix Nebula  

NASA Astrophysics Data System (ADS)

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

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

2012-06-01

374

Designed di-heme binding helical transmembrane protein.  

PubMed

De novo designing of functional membrane proteins is fundamental in terms of understanding the structure, folding, and stability of membrane proteins. In this work, we report the design and characterization of a transmembrane protein, termed HETPRO (HEme-binding Transmembrane PROtein), that binds two molecules of heme in a membrane and catalyzes oxidation/reduction reactions. The primary structure of HETPRO has been optimized in a guided fashion, from an antimicrobial peptide, for transmembrane orientation, defined 3D structure, and functions. HETPRO assembles into a tetrameric form, from an apo dimeric helical structure, in complex with cofactor in detergent micelles. The NMR structure of the apo HETPRO in micelles reveals an antiparallel helical dimer that inserts into the nonpolar core of detergent micelles. The well-defined structure of HETPRO and its ability to bind to heme moieties could be utilized to develop a functional membrane protein mimic for electron transport and photosystems. PMID:24829076

Mahajan, Mukesh; Bhattacharjya, Surajit

2014-06-16

375

Structural properties of a peptide derived from H +-V-ATPase subunit a  

Microsoft Academic Search

The 3D structure of a peptide derived from the putative transmembrane segment 7 (TM7) of subunit a from H+-V-ATPase from Saccharomyces cerevisiae has been determined by solution state NMR in SDS. A stable helix is formed from L736 up to and including Q745, the lumenal half of the putative TM7. The helical region extends well beyond A738, as was previously

Louic S. Vermeer; Valérie Réat; Marcus A. Hemminga; Alain Milon

2009-01-01

376

Unusually stable helix formation in short alanine-based peptides.  

PubMed Central

Short, 16-residue, alanine-based peptides show stable alpha-helix formation in H2O. This result is surprising when contrasted with the classical view that regards the alpha-helix as a marginally stable structure in H2O and considers short helices unstable. The alanine-based peptides are solubilized by insertion of three or more residues of a single charge type, lysine (+) or glutamic acid (-). The results cannot be explained by helix stabilization resulting from concentration-dependent association or by the interaction of charged residues with the helix dipole. Our results are not predicted by the parameters for alanine and lysine that have been determined by the "host-guest" method: these parameters predict that a 16-residue peptide should not show measurable alpha-helix formation. Analysis of the role of the hydrophobic interaction in alpha-helix formation [Richards, F.M. & Richmond, T. (1978) in Molecular Interactions and Activity in Proteins, Ciba Foundation Symposium 60, ed. Wolstenholme, G.E. (Excepta Medica Amsterdam), pp. 23-25] does not show an unusually strong hydrophobic interaction in a helical block of alanine residues. The likely explanation for our results is, therefore, that individual alanine residues have a high helical potential. It is not yet known whether any other amino acids show this property, and the origin of this property is also unknown.

Marqusee, S; Robbins, V H; Baldwin, R L

1989-01-01

377

Expression of the helix-loop-helix protein, Id, during branching morphogenesis in the kidney  

Microsoft Academic Search

Expression of the helix-loop-helix protein, Id, during branching morphogenesis in the kidney. Id, a member of the helix-loop-helix protein family, is an inhibitor of transcriptional activation by basic-helix-loop-helix proteins. In the developing mouse kidney, Id mRNA was observed as early as 12.5 days post-coitum (dpc) specifically in the condensed mesenchyme surrounding the ureteric buds by in situ hybridization. At 14.5

Melinda K Duncan; Tetsuo Shimamura; Kiran Chada

1994-01-01

378

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

PubMed

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

Beisswenger, Michael; Cabrele, Chiara

2014-09-01

379

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

380

Helix/coil nucleation: a local response to global demands.  

PubMed

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

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

2009-12-01