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At least 1,135 records · Page 63Linked to original sources
PubMed1989

Conformational analysis of thioredoxin using organoarsenical reagents as probes. A time-resolved fluorescence anisotropy and size exclusion chromatography study.

Reduced thioredoxin was subjected to chemical modification studies employing organoarsenical reagents specific for "spatially close" thiols. Modification was monitored by the loss in the free thiol content, by the percent incorporation of radiolabelled organoarsenical reagents, and by observing the changes in the amounts of the various thioredoxins by size exclusion chromatography. The rate of modification depends upon the polarity, rigidity, and size of the reagents. Small nonpolar organoarsenical reagents readily modified reduced thioredoxin, whereas polar and large reagents do not. Modifications resulted in the formation of stable 15-membered cyclodithioarsenite ring structures with no apparent changes in the secondary structure of the protein. Modification was reversed by the extrusion of the arsenical moiety by addition of 2,3-dimercaptopropanol. We have further characterized the oxidized, reduced, and modified thioredoxins by size exclusion chromatography and fluorescence anisotropy decay measurements. Both techniques show an increase in the hydrated volume of the protein upon reduction. Upon modification, the hydrodynamic volume of the protein further swells. Fluorescence anisotropy decay reveals that with modification there is loosening of the protein so that a "domain" containing the fluorophores can relax independently of the whole protein structure.

Alkylation↗
PubMed2005

Molecular dynamics simulations of the influenza hemagglutinin fusion peptide in micelles and bilayers: conformational analysis of peptide and lipids.

Molecular dynamics simulations of the influenza hemagglutinin fusion peptide in two differently sized dodecylphosphocholine micelles and a palmitoyl oleoyl phosphatidylcholine bilayer were generated to analyze the influence of the environment. Four independent trajectories (5 ns each for the bilayer, and 2 ns each for the micelles) were generated for each system. The peptide lies at the surface of the micelles, while its N-terminal region inserts deeply in the bilayer. This leads to a substantial increase of the solvation and rigidity of the peptide in micelles as compared to the bilayer. The average structures, nevertheless, are similar in all three systems and agree reasonably with micelle-based NMR structures. When in the bilayer, the peptide increases the chain gauche population and area of adjacent lipids in the same binding leaflet, while it has the opposite effect for the nearby lipids of the other leaflet. These changes, which occur spontaneously to fill voids and defects, cause a decrease in the thickness of the membrane in the neighborhood of the peptide. They would be expected to promote positive curvature, as consistent with the formation of the convex bulge, or "nipple", in the initial stage of membrane fusion. An extension of the classical surfactant theory of Israelachvili based on shapes is proposed to introduce the concept of a "dynamically induced shape" of the membrane lipids by the peptide.

Computer Simulation↗
PubMed1986

Conformational analysis of thymidylate synthase from amino acid sequence and circular dichroism.

Circular dichroism studies were carried out in the vacuum ultraviolet region for thymidylate synthase from Lactobacillus casei and its ligand complexes. The CD spectrum was analyzed for secondary structure by our method and the variable selection method, and both gave similar results. Our method predicts 33% alpha-helix, 25% (23% antiparallel and 2% parallel) beta-sheet, 20% turns, and 16% other structure. The secondary structure of this protein was also predicted from the amino acid sequence by four different methods. Though there is a variation in the prediction among these methods, the prediction of 32% alpha-helix and 23% beta-sheet by combining the four methods is in excellent agreement with our CD results. Further, the location of the predicted regions of alpha-helices and beta-strands along the sequence and the CD characteristics strongly suggest that this protein belongs to an alpha + beta structural class. Binding of the inhibitor FdUMP or the cofactor 5,10-methylenetetrahydrofolate did not change the CD spectrum. However, when both ligands were present, there was a significant change in the CD spectrum and the maximum changes occurred when the concentration of FdUMP was 1 mol/mol of enzyme. The addition of FdUMP and cofactor causes, respectively, a 5% and 6% decrease in beta-sheet and beta-turns and about an 8% increase in "other" structure.

Amino Acid Sequence↗
PubMed2006

Glycosyldisulfides from dynamic combinatorial libraries as O-glycoside mimetics for plant and endogenous lectins: their reactivities in solid-phase and cell assays and conformational analysis by molecular dynamics simulations.

Dynamic combinatorial library design exploiting the thiol-disulfide exchange readily affords access to glycosyldisulfides. In order to reveal lectin-binding properties of this type of non-hydrolyzable sugar derivative, libraries originating from a mixture of common building blocks of natural glycans and thiocompounds were tested against three plant agglutinins with specificity to galactose, fucose or N-acetylgalactosamine, respectively, in a solid-phase assay. Extent of lectin binding to matrix-immobilized neoglycoprotein presenting the cognate sugar could be reduced, and evidence for dependence on type of carbohydrate was provided by dynamic deconvolution. Glycosyldisulfides also maintained activity in assays of increased physiological relevance, that is, using native tumor cells and also adding to the test panel an endogenous lectin (galectin-3) involved in tumor spread and cardiac dysfunction. N-Acetylgalactosamine was pinpointed as the most important building block of libraries for the human lectin and the digalactoside as most potent compound acting on the toxic mistletoe agglutinin which is closely related to the biohazard ricin. Because this glycosyldisulfide, which even surpasses lactose in inhibitory capacity, rivals thiodigalactoside as inhibitor, their degrees of intramolecular flexibility were comparatively analyzed by computational calculations. Molecular dynamics runs with explicit consideration of water molecules revealed a conspicuously high degree of potential for shape alterations by the disulfide's three-bond system at the interglycosidic linkage. The presented evidence defines glycosyldisulfides as biologically active ligands for lectins.

Binding Sites↗
PubMed2002

2-Ethyl and 2-ethylidene analogues of 1alpha,25-dihydroxy-19-norvitamin D(3): synthesis, conformational analysis, biological activities, and docking to the modeled rVDR ligand binding domain.

Novel 19-nor analogues of 1alpha,25-dihydroxyvitamin D(3) were prepared and substituted at C-2 with an ethylidene group. The synthetic pathway was via Wittig-Horner coupling of the corresponding A-ring phosphine oxides with the protected 25-hydroxy Grundmann's ketones. Selective catalytic hydrogenation of 2-ethylidene analogues provided the 2alpha- and 2beta-ethyl compounds. The 2-ethylidene-19-nor compounds with a methyl group from the ethylidene moiety in a trans relationship to the C(6)-C(7) bond (E-isomers) were more potent than the corresponding Z-isomers and the natural hormone in binding to the vitamin D receptor. Both geometrical isomers (E and Z) of (20S)-2-ethylidene-19-norvitamin D(3) and both 2alpha-ethyl-19-norvitamins (in the 20R- and 20S-series) have much higher HL-60 differentiation activity than does 1alpha,25-(OH)(2)D(3). Both E-isomers (20R and 20S) of 2-ethylidene vitamins are characterized by very high calcemic activity in rats. The three-dimensional structure model of the rat vitamin D receptor and the computational docking of four synthesized (20R)-19-norvitamin D(3) analogues into its binding pocket are also reported.

Animals↗
PubMed1999

Conformational analysis of the hydrophobic peptide alphas1-casein(136-196).

Hydrophobic interactions are important in the self-association of milk proteins, including alphas1-casein. The extent to which casein interaction sites are influenced by local secondary structure is not widely known. Both primary amino acid sequence and local secondary structure are shown to affect the self-association of the hydrophobic peptide alphas1-casein(136-196). The peptide is aggregated at low concentrations (7 microM and above), as determined by 1H nuclear magnetic resonance (NMR) measurements at pH 6.0 in phosphate buffer. Increase in temperature is shown to induce side chain mobility (melting) as indicated by both 1H NMR and near-UV circular dichroism (CD) measurements. As determined by far-UV CD, there is also a loss in the global amount of extended structure with increasing temperature, while beta-turn structures and some aromatic dichroism are conserved at temperatures as high as 70 degrees C. Similar retention of structure occurs at pH 2 and in 6 M guanidine HCl. The observed stability of beta-turns and some side chains in alphas1-casein(136-196) supports previous assumptions that hydrophobic, proline-based turns are important interaction sites in the self-association of alphas1-casein, and possibly in the formation of the calcium transport complexes, the casein micelles. It may be speculated that these areas of the peptide represent a 'molten globule-like', heat stable, core structure for alphas1-casein.

Amino Acid Sequence↗
PubMed2002

Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia.

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278-300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic alpha helix ranging from Phe280 to Tyr291, followed by a type I beta turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278-292 and YopD271-292, lacked helical structure, implicating the beta turn in helix stability. An interaction between YopD278-300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide-LcrH complex. These effects were not observed for the unstructured peptide, YopD278-292, which confirms that the alpha helical structure of the YopD amphipathic domain is a critical binding region of LcrH.

Amino Acid Sequence↗
PubMed1994

Production, inhibitory activity, folding and conformational analysis of an N-terminal and an internal deletion variant of chicken cystatin.

Two deletion variants of chicken cystatin were produced after cassette mutagenesis of the recombinant Arg-Glu-Phe-[Met1, Ile29, Leu89]-chicken egg white cystatin gene in Escherichia coli. The variant des-Ser1-Pro11-[Ala12, Glu13, Phe14, Met15, Ile29, Leu89]-chicken cystatin (N-del 2) and the variant Arg-Glu-Phe-[Met1, Ile29]-des-Cys71-Met89-chicken cystatin (del-helix II) were purified and characterized by inhibition kinetics, far-ultraviolet-CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied. The del-helix II variant, shortened by 19 amino acids, is a basic, stefin-like mini-cystatin with one disulfide bridge. Its inhibitory properties are identical to chicken cystatin and its stability against Gdn/HCl is similar. The folding of the del-helix II variant corresponds best to a single step process. In contrast to this, the reversible folding of natural and recombinant chicken cystatin is more complex when recorded by either tryptophan fluorescence or far-ultraviolet-CD. With increasing Gdn/HCl concentration, a stabilization of secondary-structural elements is initially observed, followed by unfolding with minor but distinct intermediate states. The N-del 2 variant has a neutral pI and shows folding behaviour very similar to natural and recombinant chicken cystatin. However its inhibition constants with papain, actinidin and cathepsin B and L are 1000-100,000-fold higher than those obtained with natural and recombinant chicken cystatin.

Amino Acid Sequence↗
PubMed2000

An NMR conformational analysis of cyclic bradykinin mimics. Evidence for a beta-turn.

A detailed NMR study is carried out in acetonitrile/water solutions on three novel cyclic bradykinin antagonist analogues, BKM-824, BKM-870, and BKM-872, to examine their solution structures, and to correlate the structures with bradykinin antagonist and anti-cancer activities. The solution structures of the cyclic peptides are correlated with the structural data for known linear bradykinin antagonists. The sequences are: BKM-824 c[Ava-Ig1-Ser-DF5F-Oic-Arg] where Ava is 5-aminovaleric acid, Ig1 is alpha-(2-indanyl)glycine, F5F is pentafluorophenylalanine, and Oic is (2S,3aS,7aS)-octahydroindole-2-carboxylic acid; BKM-870; c[DArg-Arg-Add-DF5F-Oic-Arg] where Add is 12-aminododecanoic acid; and BKM-872; c[DArg-Arg-Eac-Ser-DF5F-Oic-Arg] where Eac is 6-aminocaproic acid. BKM-824 was the only peptide within this series that possessed a discernable solution structure. The NMR data indicate the presence of a type I beta-turn between residues F5F4 and Ava1, a C-terminal-like end. Molecular dynamics calculations show that a type I beta-turn from DF5F4 to Ava1 does exist although the turn was somewhat distorted. This result differs from the structures seen in linear bradykinin antagonists, which usually possess a type II'beta-turn at the C-terminal end and the presence of a defined turn is correlated with bradykinin antagonist activity. There is no solution structure for BKM-870 and BKM-872 but a correlation between the primary sequence Arg(terminal)-DArg1-Arg2-long chain aliphatic amino acid and anti-cancer activity is evident.

Amino Acid Sequence↗
PubMed1985

Nuclease activity of 1,10-phenanthroline-copper ion. Conformational analysis and footprinting of the lac operon.

The nuclease activity of 1,10-phenanthroline-copper [(OP)2Cu+] preferentially nicks the wild-type, Ps, and L8-UV-5 lac promoters in the conserved promoter specific sequence (Pribnow box). The preferred sites of attack of the wild-type fragment within this region are at positions -13 and -12 on the template strand. When the comparable fragment from the Ps promoter, which differs from the wild type at position -9 (T instead of C), is cleaved with (OP)2Cu+, a new strong band at position -10 in the gel patterns is clear. An apparent increase in cutting at position -11 can also be observed. The conversion of the Ps promoter to the L8-UV-5 promoter (a change from an A to a T at position -8 and a change from a C to a T at position -66) results in alteration of the relative intensities of the four prominent bands at positions -13 to -10. Most notably, the intensity at position -10 is attenuated in L8-UV-5. The hypersensitivity of the Pribnow box region to the coordination complex is also apparent if the cutting of the missense strand is analyzed. The region of strong nicking in this case ranges from positions -11 to -3, and the relative intensities of the bands depend on the primary sequence of the promoters. These data suggest that a single base change induces local variation in the DNA structure. This new structure may be responsible for the notable difference in the efficiency of the promoters. Pancreatic deoxyribonuclease I (DNase I) does not preferentially cleave the Pribnow box relative to other regions of the sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1990

Conformational analysis of the immunodominant epitopes of the circumsporozoite protein of Plasmodium falciparum and knowlesi.

All of the coat proteins of the sporozoite and merozoite stages of Plasmodium, determined to date, contain tandem repeats and most of these contain at least one proline residue. These tandemly repeated segments of the circumsporozite (CS) proteins of P. falciparum and P. knowlesi have been shown to constitute an immunodominant epitope. Antibodies to these peptide segments have been shown to be protective and cause the shedding of the CS protein, known as the CSP reaction. In this study, four synthetic peptides were prepared by solid-phase peptide synthesis. The first peptide corresponds to the tetrapeptide tandem repeat in the CS protein of P. falciparum, repeated eight times, (NANP)8. The second peptide is an analogue of the first in which glycine is substituted for proline, (NANG)8. The third peptide corresponds to the tandem repeat of P. knowlesi, PK(1-24), which is repeated twice (QAQGDGANAGQP)2. The fourth peptide is a tetrapeptide repeat, corresponding to the C-terminal tetrapeptide of PK(1-24) and is repeated eight times, (AGQP)8. It is shown by CD measurements that the presence of proline in these repeats induces an increase in beta-sheet (beta-turn) content in the (NANP)8 peptide relative to the repeat of (NANG)8 and PK(1-24) peptide in aqueous media. The (AGQP)8 peptide has the highest beta-sheet (beta-turn) content of all peptides studied. The Chou-Fasman predictive algorithm indicates a high beta-turn content in the synthetic peptides. It is concluded that this increase in defined structure correlates well with and hence may contribute to the increased antigenicity in these repeats.

Amino Acids↗
PubMed2006

Conformational analysis of gamma' peptide (410-427) interactions with thrombin anion binding exosite II.

Thrombin utilizes two anion binding exosites to supplement binding of fibrinogen to this serine protease. Approximately 7-15% of the fibrinogen gamma chain exists as the highly anionic gamma' variant (408VRPEHPAETEY(S)DSLY(S)PEDDL427). This segment has been demonstrated to target thrombin ABE-II and can accommodate sites of phosphorylation in place of sulfonation without sacrificing binding affinity. The present work employed 1D and 2D solution NMR to characterize the structural features of the bound gamma' peptide (410-427) and to evaluate the requirement of sulfonation for effective thrombin interaction. The results indicate the gamma' residues 414-427 make significant contact with the enzyme, a beta-turn exists between residues 422-425 in the presence of thrombin, and there is a large cluster of through-space interactions involving residues 418-422. Effective contact with ABE-II requires the presence of at least one phosphotyrosine residue with Y(P)422 being the more important player. Hydrogen-deuterium exchange (HDX) coupled with MALDI-TOF MS was implemented to examine the location of the gamma' peptide-thrombin interface and to screen for changes in solvent exposure at distant sites. The HDX results demonstrate that the gamma' peptide interacts with or is in close proximity to thrombin residues R93, R97, R173, and R175. The binding of the gamma' peptide also protects other regions of thrombin from deuterium exchange. Affected regions include segments of ABE-I, the autolysis loop, the edge of the active site region, and the A-chain. Finally, thrombin forms a ternary complex with the gamma' peptide and PPACK, generating an enzyme whose solvent-exposed regions are even further stabilized from HDX.

Animals↗
PubMed1999

Conformational analysis of peptide fragments derived from the peripheral subunit-binding domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: evidence for nonrandom structure in the unfolded state.

There is currently a great deal of interest in the early events in protein folding. Two issues that have generated particular interest are the nature of the unfolded state under native conditions and the role of local interactions in folding. Here, we report the results of a study of a set of peptides derived from a small two-helix protein, the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex. Five peptides of overlapping sequence were prepared, including sequences corresponding to each of the helices and to the region connecting them. The peptides were characterized by CD and, where possible, nmr. A peptide corresponding to the second helix is between 12 and 17% helical at neutral pH. CD also indicates a lower percentage of helical structure in the peptide corresponding to the first alpha-helix, although the values of the alpha-proton chemical shifts suggest some preference for nonrandom structure. Peptides corresponding to the interhelical loop, which in the full domain contains two overlapping beta-turns and a 5-residue 3(10)-helix, are less structured. There is no significant change in the helicity of any of these peptides with pH. To test for fragment complementation, CD spectra of the two peptides derived from each helix and the long connecting peptide were compared to the spectra of each possible pair, as well as to a mixture containing all three. No increase in structure was observed. We complement our peptide studies by characterizing a point mutant, D34V, which disrupts a critical hydrogen bonding network. This mutant is unable to fold and provides a useful model of the denatured state. The mutant is between 9 and 16% helical as judged by CD. The modest amount of helical structure formed in some of the peptide fragments and in the point mutant suggests that the denatured state of the peripheral subunit binding domain is not completely unstructured. This may contribute to the very rapid folding observed for the intact protein.

Amino Acid Sequence↗
PubMed1998

Double missense mutation in exon 41 of the human dystrophin gene detected by double strand conformation analysis.

Development of late-onset Becker muscular dystrophy is reported in a patient whose two healthy brothers showed high serum creatine kinase level. No cases of neuromuscular disorders had been previously reported in this family. The analysis of the dystrophin gene showed that the three brothers had A-->C transversion at nucleotide 6092 in exon 41, a missense mutation which converts lysine into glutamine. The symptomatic patient showed an additional mutation in the same exon, a T-->C transition at nucleotide 6119, converting a phenylalanine to leucine. The possible pathogenic role of this mutation is discussed.

Dystrophin↗