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PubMed2006

Nonaqueous versus aqueous capillary electrophoresis of alpha-helical polypeptides: effect of secondary structure on separation selectivity.

The CE separation of alpha-helical polypeptides composed of 14-31 amino acid residues has been investigated using aqueous and nonaqueous BGEs. The running buffers were optimized with respect to pH. Generally, higher separation selectivities were observed in nonaqueous electrolytes. This may be explained by a change in the secondary structure when changing from water to organic solvents. Circular dichroism spectra revealed a significant increase in helical structures in methanol-based buffers compared to aqueous buffers. This change in secondary structure of the polypeptides contributed primarily to the different separation selectivity observed in aqueous CE and NACE. For small oligopeptides of two to five amino acid residues no significant effect of the solvent was observed in some cases while in other cases a reversal of the migration order occurred when changing from aqueous to nonaqueous buffers. As these peptides cannot adopt secondary structures the effect may be attributed to a shift of the pKa values in organic solvents compared to water.

Amino Acid Sequence↗
PubMed2001

Length-dependent stability and strand length limits in antiparallel beta -sheet secondary structure.

Designed peptides that fold autonomously to specific conformations in aqueous solution are useful for elucidating protein secondary structural preferences. For example, autonomously folding model systems have been essential for establishing the relationship between alpha-helix length and alpha-helix stability, which would be impossible to probe with alpha-helices embedded in folded proteins. Here, we use designed peptides to examine the effect of strand length on antiparallel beta-sheet stability. alpha-Helices become more stable as they grow longer. Our data show that a two-stranded beta-sheet ("beta-hairpin") becomes more stable when the strands are lengthened from five to seven residues, but that further strand lengthening to nine residues does not lead to further beta-hairpin stabilization for several extension sequences examined. (In one case, all-threonine extension, there may be an additional stabilization on strand lengthening from seven to nine residues.) These results suggest that there may be an intrinsic limit to strand length for most sequences in antiparallel beta-sheet secondary structure.

Nuclear Magnetic Resonance, Biomolecular↗
PubMed1990

Secondary structure of the entomocidal toxin from Bacillus thuringiensis subsp. kurstaki HD-73.

The secondary structure of the toxin from Bacillus thuringiensis subsp. kurstaki (Btk) HD-73 was estimated by Raman, infrared, and circular dichroism spectroscopy, and by predictive methods. Circular dichroism and infrared spectroscopy gave an estimate of 33-40% alpha-helix, whereas Raman and predictive methods gave approximately 20%. Raman and circular dichroism spectra, as well as predictive methods, indicated that the toxin contains 32-40% beta-sheet structure, whereas infrared spectroscopy gave a slightly lower estimate. Thus, all of these approaches are in agreement that the native conformation of Btk HD-73 toxin is highly folded and contains considerable amounts of both alpha-helical and beta-sheet structures. No significant differences were detected in the secondary structure of the toxin either in solution or as a hydrated pellet.

Bacillus thuringiensis↗
PubMed1994

Secondary structure and backbone dynamics of human granulocyte colony-stimulating factor in solution.

The secondary structure and backbone dynamics of the cytokine, human granulocyte colony-stimulating factor (hG-CSF) have been determined by heteronuclear nuclear magnetic resonance (NMR) techniques. Virtually complete NH, C alpha H, C beta H 15N, 13C alpha, and 13C beta assignment of the 175-residue recombinant protein, methionyl-[Cys-17-Ser]-hG-CSF, was achieved by use of three-dimensional (3D) heteronuclear 1H-15N and triple-resonance 1H-15N-13C experiments. Spectra recorded at 750 MHz aided the assignment of severely overlapped regions. The structures of G-CSF from several species have recently been determined by X-ray diffraction [Hill, C. P., Osslund, T. D., & Eisenberg, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5167-5171; Lovejoy, B., Cascio, D., & Eisenberg, D. (1993) J. Mol. Biol. 234, 640-653]. Like several cytokines, hG-CSF has a four-helix topology (A-D) with overhand loop connections, but with an additional helical segment (A') identified in the connection between helix A and helix B. The solution-state determination of the secondary structure is based on short- and medium-range NOEs, backbone J-couplings, and NH exchange data and is corroborated by 13C alpha secondary shifts. The helices are defined as follows: A, 10-38; A',44-53; B, 71-91; C, 102-123; D, 143-172. The dynamics of the amide backbone resonances, investigated using 1H-15N heteronuclear NMR, indicate a rigid protein core with some increased mobility in the AB loop and more pronounced mobility in the CD loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2006

Secondary structure of dengue virus type 4 3' untranslated region: impact of deletion and substitution mutations.

Several studies have generated computer-based predictions of secondary structure of the 3' untranslated region (UTR) of Dengue virus (DEN); however, experimental verification of the formation of these structures in vitro is lacking. This study assessed the congruence of Mfold predictions of secondary structure of the core region of the DEN type 4 3' UTR with nuclease maps of this region. Maps and predictions were largely consistent. Maps supported the existence of previously predicted pseudoknots and identified putative regions of dynamic folding. Additionally, this study investigated previously identified conserved elements in the flavivirus 3' UTR that differ among viruses with different modes of transmission. Specific regions of mosquito-borne DEN type 4 were either deleted or replaced with homologous sequences from tick-borne Langat virus. All of these mutations caused substantial distortion of secondary structure, yet viruses carrying these mutations were viable.

3' Untranslated Regions↗
PubMed2003

Translocation and gross deletion breakpoints in human inherited disease and cancer II: Potential involvement of repetitive sequence elements in secondary structure formation between DNA ends.

Translocations and gross deletions are responsible for a significant proportion of both cancer and inherited disease. Although such gene rearrangements are nonuniformly distributed in the human genome, the underlying mutational mechanisms remain unclear. We have studied the potential involvement of various types of repetitive sequence elements in the formation of secondary structure intermediates between the single-stranded DNA ends that recombine during rearrangements. Complexity analysis was used to assess the potential of these ends to form secondary structures, the maximum decrease in complexity consequent to a gross rearrangement being used as an indicator of the type of repeat and the specific DNA ends involved. A total of 175 pairs of deletion/translocation breakpoint junction sequences available from the Gross Rearrangement Breakpoint Database [GRaBD; www.uwcm.ac.uk/uwcm/mg/grabd/grabd.html] were analyzed. Potential secondary structure was noted between the 5' flanking sequence of the first breakpoint and the 3' flanking sequence of the second breakpoint in 49% of rearrangements and between the 5' flanking sequence of the second breakpoint and the 3' flanking sequence of the first breakpoint in 36% of rearrangements. Inverted repeats, inversions of inverted repeats, and symmetric elements were found in association with gross rearrangements at approximately the same frequency. However, inverted repeats and inversions of inverted repeats accounted for the vast majority (83%) of deletions plus small insertions, symmetric elements for one-half of all antigen receptor-mediated translocations, while direct repeats appear only to be involved in mediating simple deletions. These findings extend our understanding of illegitimate recombination by highlighting the importance of secondary structure formation between single-stranded DNA ends at breakpoint junctions.

3' Flanking Region↗
PubMed2005

JViz.Rna--a Java tool for RNA secondary structure visualization.

Many tools have been developed for visualization of RNA secondary structures using a variety of techniques and output formats. However, each tool is typically limited to one or two of the visualization models discussed in this paper, supports only a single file format, and is tied to a specific platform. In order for structure prediction researchers to better understand the results of their algorithms and to enable life science researchers to interpret RNA structure easily, it is helpful to provide them with a flexible and powerful tool.jViz.Rna is a multiplatform visualization tool capable of displaying RNA secondary structures encoded in a variety of file formats. The same structure can be viewed using any of the models supported, including linked graph, circle graph, dot plot, and classical structure. Also, the output is dynamic and can easily be further manipulated by the user. In addition, any of the drawings produced can be saved in either the EPS or PNG file formats enabling easy usage in publications and presentations.

Computer Graphics↗
PubMed1995

Maintenance of pre-mRNA secondary structure by epistatic selection.

Linkage disequilibrium between polymorphisms in a natural population may result from various evolutionary forces, including random genetic drift due to sampling of gametes during reproduction, restricted migration between subpopulations in a subdivided population, or epistatic selection. In this report, we present evidence that the majority of significant linkage disequilibria observed in introns of the alcohol dehydrogenase locus (Adh) of Drosophila pseudoobscura are due to epistatic selection maintaining secondary structure of precursor mRNA (pre-mRNA). Based on phylogenetic-comparative analysis and a likelihood approach, we propose secondary structure models of Adh pre-mRNA for the regions of the adult intron and intron 2 where clustering of linkage disequilibria has been observed. Furthermore, we applied the likelihood ratio test to the phylogenetically predicted secondary structure in intron 1. In contrast to the other two structures, polymorphisms associated with the more conserved stem-loop structure of intron 1 are in low frequency, and linkage disequilibria have not been observed. These findings are qualitatively consistent with a model of compensatory fitness interactions. This model assumes that mutations disrupting pairing in a secondary structural element are individually deleterious if they destabilize a functionally important structure; a second "compensatory" mutation, however, may restabilize the structure and restore fitness.

Animals↗
PubMed1993

Mechanism of adenylate kinase. 1H, 13C, and 15N NMR assignments, secondary structures, and substrate binding sites.

Backbone 1H, 13C, and 15N NMR assignments were obtained for the complex of chicken muscle adenylate kinase (AK) with its bisubstrate analog, MgAP5A [magnesium P1,P5-bis(5'-adenosyl)-pentaphosphate]. The assignments were used to elucidate the secondary structures and the enzyme-MgAP5A interactions. The work involves two unusual features: the molecular weight of AK (21.6 kDa) is one of the largest, on a monomeric basis, for which nearly complete assignment has been reported to date, and the assignment was performed at pH 7.1 instead of the acidic pH used for most other proteins. The results are summarized as follows. Firstly, unambiguous sequential assignments of backbone resonances have been achieved effectively by the combined use of two sequential assignment methods: NOE-directed assignments and the recently developed 1J-coupling-directed assignments. The starting points of the assignments were provided by several specifically labeled enzyme samples. Over 90% of the backbone 1H, 13C, and 15N resonances have been assigned. Secondly, spin system information was obtained from the HCCH-TOCSY and HCCH-COSY experiments as well as from 2D homonuclear NMR data. Overall, the side-chain resonances of ca. 40% of the residues, including most of the those displaying NOEs with the adenosine moieties of MgAP5A, have been assigned. Thirdly, secondary structural elements in the AK-MgAP5A complex were identified by extensive analyses of 1H-15N 2D HMQC-NOESY and 3D NOESY-HMQC spectra. Overall, the enzyme consists of ca. 60% alpha-helices and a five-stranded parallel beta-sheet. The results are compared with the secondary structure of the free AK from porcine muscle in crystals [Dreusicke, D., Karplus, P. A., & Schulz, G. E. (1988) J. Mol. Biol. 199, 359-371]. Lastly, most of the intermolecular NOEs between AK and the adenosine moieties of MgAP5A have been identified: Thr39, Leu43, Gly64, Leu66, Val67, Val72, and Gln101 are in proximity to the adenosine moiety of the adenosine 5'-monophosphate site, whereas Thr23 is in proximity to that of the adenosine 5'-triphosphate site. These data are discussed in relation to previous results from site-directed mutagenesis, NMR, and X-ray studies and in relation to the mechanism of catalysis.

Adenylate Kinase↗
PubMed1996

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers.

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.

Amino Acid Sequence↗
PubMed1984

Evidence for the transcript secondary structures predicted to regulate transcription attenuation in the trp operon.

We have examined the leader transcript of the Serratia marcescens trp operon to determine if the alternate secondary structures postulated to control transcription attenuation actually form in purified trp leader RNA. RNA secondary structures were analyzed by partial digestion of 32P end-labeled leader transcripts with single and double strand-specific nucleases. We found that the 176 nucleotide wild-type transcript formed the predicted hairpin structures designated 1:2 and 3:4; the latter structure is believed to signal transcription termination. We constructed a deletion plasmid, pSm delta 1,4, to determine whether the postulated RNA antiterminator structure 2:3 could also form. This plasmid lacked the DNA regions corresponding to RNA segments 1 and 4. We found that the leader transcript from pSm delta 1,4 formed structure 2:3. This provides the first direct evidence establishing the formation of a predicted antitermination secondary structure.

Base Sequence↗
PubMed1985

Prediction of the secondary structure of Bowman-Birk soybean protease inhibitor from LTS sequence.

The secondary structure of Bowman-Birk soybean inhibitor (BBI) has been predicted from its amino acid sequence (71 residues) using the statistical method of Chou and Fasman (1974) as well as the informational method of Garnier et al. (1978). According to both methods, no alpha-helical region is predicted in BBI. Short beta-strands at 11-15, 39-43, 48-52 and 57-59 are predicted by Chou and Fasman's method, and at 11-14, 39-43, 48-52 and 57-59 by the method of Garnier et al. Predicted beta-turn tetrapeptides are residues at 1-4, 6-9, 14-17, 18-21, 24-27, 30-33, 36-39, 60-63, 63-66 and 68-71, according to the method of Chou and Fasman. These predictive results indicate that BBI consists essentially of beta-turn, beta-strand, and unordered structures, which represent about 56, 25 and 19% (by difference) of its secondary structure, respectively. The predicted reverse turn amount in BBI is very high, in comparison with that found usually in proteins. No fitting of the experimental circular dichroism spectrum (in the 195-240 nm region), which was reported by Kay (1976), has proved satisfactory when using the parameters for helical, beta-sheet, beta-turn and unordered conformations proposed by several authors.

Amino Acid Sequence↗
PubMed2002

Correlation of MFOLD-predicted DNA secondary structures with separation patterns obtained by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis.

We have studied 57 different mutations within three beta-globin gene promoter fragments with sizes 52 bp, 77 bp, and 193 bp by fluorescent capillary electrophoresis CE-SSCP analysis. For each mutation and wild type, energetically most-favorable predicted secondary structures were calculated for sense and antisense strands using the MFOLD DNA-folding algorithm in order to investigate if any correlation exists between predicted DNA structures and actual CE migration time shifts. The overall CE-SSCP detection rate was 100% for all mutations in three studied DNA fragments. For shorter 52 bp and 77 bp DNA fragments we obtained a positive correlation between the migration time shifts and difference in free energy values of predicted secondary structures at all temperatures. For longer 193 bp beta-globin gene fragments with 46 mutations MFOLD predicted different secondary structures for 89% of mutated strands at 25 degrees C and 40 degrees C. However, the magnitude of the mobility shifts did not necessarily correlate with their secondary structures and free energy values except for the sense strand at 40 degrees C where this correlation was statistically significant (r = 0.312, p = 0.033). Results of this study provided more direct insight into the mechanism of CE-SSCP and showed that MFOLD prediction could be helpful in making decisions about the running temperatures and in prediction of CE-SSCP data patterns, especially for shorter (50-100 bp) DNA fragments.

Algorithms↗
PubMed1994

Effect of template secondary structure on the inhibition of HIV-1 reverse transcriptase by a pyridinone non-nucleoside inhibitor.

The importance of RNA secondary structure on HIV-1 reverse transcriptase catalyzed polymerization and on the potency of the pyridin-2-one inhibitor 3-(4,7-dichlorobenzoxazol-2-ylmethylamino)-5-ethyl-6-meth ylpyridin-2(1H)-one, L-697,661, were investigated by employing heteromeric primer-template systems. Our data revealed that a stem-loop hairpin secondary structure in the RNA template could lead to strong hindrance of reverse transcription in the reaction catalyzed by HIV-1 reverse transcriptase resulting in the build up of intermediate-length (pause) polymerization products. The presence of L-697,661 greatly enhanced the accumulation of the pause products suggesting that the rate of enzyme translocation from the pause product might be more potently inhibited than polymerization up to the pause site. Model experiments using a synthetic RNA template containing a stem-loop hairpin revealed that the inhibitory potency of L-697, 661 increased 2-fold upon polymerization to within four bases of the secondary structure. Inhibitor potency was enhanced over 6-fold when primer-extension proceeded through the duplex region of the stem-loop.

Animals↗
PubMed2004

Protein secondary structure assignment through Voronoï tessellation.

We present a new automatic algorithm, named VoTAP (Voronoï Tessellation Assignment Procedure), which assigns secondary structures of a polypeptide chain using the list of alpha-carbon coordinates. This program uses three-dimensional Voronoï tessellation. This geometrical tool associates with each amino acid a Voronoï polyhedron, the faces of which unambiguously define contacts between residues. Thanks to the face area, for the contacts close together along the primary structure (low-order contacts) a distinction is made between strong and normal ones. This new definition yields new contact matrices, which are analyzed and used to assign secondary structures. This assignment is performed in two stages. The first one uses contacts between residues close together along the primary structure and is based on data collected on a bank of 282 well-refined nonredundant structures. In this bank, associations were made between the prints defined by these low-order contacts and the assignments performed by different automatic methods. The second step focuses on the strand assignment and uses contacts between distant residues. Comparison with several other automatic assignment methods are presented, and the influence of resolution on the assignment is investigated.

Algorithms↗
PubMed1999

Intrinsic helical propensities and stable secondary structure in a membrane-bound fragment (S4) of the shaker potassium channel.

The location and stability of helical secondary structure in a fragment comprising an extended sequence of the S4 transmembrane segment of the Shaker potassium channel was determined in methanol, and when bound to vesicles composed of egg phosphatidylcholine: egg phosphatidylglycerol (4:1; mol:mol) in water. The N-acetylated, C-amidated peptide corresponds to the sequence comprising residues A355-I384 in the Shaker potassium channel. Although NOEs characteristic of helical structure encompass essentially the full peptide sequence in methanol, analysis of amide and CH(alpha) chemical shifts, and amide exchange protection factors establish that stable helical structure comprises only around the first 22 amino acids of the 30 residue peptide. This sequence corresponds to that predicted to have the highest helical stability in water, indicating that while helical structure is considerably stabilised in methanol, the relative helical propensities of amino acids in methanol may be similar to those in water. In the presence of vesicles containing negatively charged lipids, helical structure corresponding to a maximum of around 40 % of the extended S4 peptide is induced; no helical structure is induced in the presence of vesicles composed only of neutral lipids. The location of stable helical structure in the membrane-bound peptide was determined by amide hydrogen-deuterium exchange trapping, and was shown to encompass the sequence between residues near M2 and I18. This sequence is similar to that having high helix propensity in water and methanol, supporting the idea that intrinsic helical propensities are important in defining the location of stable helical structure in polypeptides bound in the interfacial region of lipid bilayers. The study defines an approach to determining the location of, and contributions to, the stability of helical secondary structure in membrane-reconstituted polypeptides.

Amino Acid Sequence↗
PubMed2005

Secondary structure, conformational stability and glycosylation of a recombinant Candida rugosa lipase studied by Fourier-transform infrared spectroscopy.

The secondary structure of lipase 1 from Candida rugosa, a model system for large monomeric enzymes, has been studied by FTIR (Fourier-transform infrared) spectroscopy in water and 2H2O. The secondary structure content, determined by the analysis of the amide I band absorption through second derivative and curve fitting procedures, is in agreement with that estimated by X-ray data and predicts, in addition, the existence of two classes of alpha-helices. We have also investigated the enzyme stability and aggregation at high temperature by following the protein unfolding. The thermal stability determined by FTIR is in excellent agreement with the temperature dependence of the lipase activity. Furthermore, new insights on the glycosylation of the recombinant protein produced in Pichia pastoris and on its heterogeneity related to different fermentation batches were obtained by the analysis of the IR absorption in the 1200-900 cm(-1) carbohydrate region. A drastic reduction of the intensity of this band was found after enzymic deglycosylation of the protein. To confirm that the FTIR absorption in the 1200-900 cm(-1) region depends on the carbohydrate content and glycoform distribution, we performed an MS analysis of the protein sugar moieties. Glycosidic structures of the high mannose type were found, with mannoses ranging from 8 to 25 residues.

Amides↗
PubMed1988

Secondary structure of the Neurospora crassa plasma membrane H+-ATPase as estimated by circular dichroism.

In a previous communication, a water-soluble, hexameric form of the Neurospora crassa plasma membrane H+-ATPase was described (Chadwick, C. C., Goormaghtigh, E., and Scarborough, G. A. (1987) Arch. Biochem. Biophys. 252, 348-356). To facilitate physical studies of the hexamers, the H+-ATPase isolation procedure has been improved, resulting in a structurally and functionally stable hexamer preparation that contains only 5 to 10% non-ATPase protein, approximately 12 mol of enzyme-bound lysophosphatidylcholine/mol of H+-ATPase monomer, and little or no residual plasma membrane phospholipid. Importantly, when activated by lysophosphatidylglycerol, which satisfies the acidic phospholipid requirement of the enzyme, the hexameric quaternary structure of the enzyme is retained, indicating that the functional properties of the water-soluble hexamers are relevant to those of the native, membrane-bound enzyme. The circular dichroism (CD) spectrum of this H+-ATPase preparation has been measured from 184 to 260 nm and used to estimate the secondary structure of the enzyme. The H+-ATPase is estimated to consist of approximately 36% helix, 12% antiparallel beta-sheet, 8% parallel beta-sheet, 11% beta-turn, and 26% other (irregular) structure. There is no change in the CD spectrum when known enzyme ligands are added to the H+-ATPase solution, suggesting that any changes in secondary structure that might occur during ligand binding and/or catalytic cycling are either minor or result in compensatory changes in secondary structure. The CD spectrum of the H+-ATPase is also compared to published spectra of the animal cell Na+/K+- and Ca2+-ATPases and is shown to be quite similar in shape and intensity, suggesting that all of these ATPases, which have significant sequence homology and are mechanistically similar, may have similar secondary structure composition as well.

Adenosine Triphosphate↗