162 resultados para Induced circular dichroism
Resumo:
The conformation and stability of pearl millet prolamin (pennisetin) were examined by using circular dichroism and C-13 nuclear magnetic resonance spectroscopy. The far UV spectrum of pennisetin in 70% (v/v) aqueous ethanol showed the presence of predominant alpha-helical structure and its occurrence in the alpha + beta class of protein. The far and near UV spectra of pennisetin in ethanol: trifluoroethanol also supported this observation. However pennisetin showed the presence of some helical structure in 8 M urea which is known to be a highly unordered structure forming solvent. A decrease in alpha helical content of native pennisetin was observed with rise in temperature from 5-75-degrees-C and this effect of temperature was found to be reversible. A C-13 NMR spectrum of pennisetin in 70% ethanol suggested a high degree of molecular mobility in ethanol. Comparison of the cross polarization spectrum with the single pulse excitation spectrum suggested pennisetin to be a heterogeneous protein.
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This paper reports a new class of photo-cross-linkable side chain liquid crystalline polymers (PSCLCPs) based on the bis(benzylidene)cyclohexanone unit, which functions as both a mesogen and a photoactive center. Polymers with the bis(benzylidene)cyclohexanone unit and varying spacer length have been synthesized. Copolymers of bis(benzylidene)cyclohexanone containing monomer and cholesterol benzoate containing monomer with different compositions have also been prepared. All these polymers have been structurally characterized by spectroscopic techniques. Thermal transitions were studied by DSC, and mesophases were identified by polarized light optical microscopy (POM). The intermediate compounds OH-x, the monomers SCLCM-x, and the corresponding polymers PSCLCP-x, which are essentially based on bis(benzylidene)cyclohexanone, all show a nematic mesophase. Transition temperatures were observed to decrease with increasing spacer length. The copolymers with varying compositions exhibit a cholesteric mesophase, and the transition temperatures increase with the cholesteric benzoate units in the copolymer. Photolysis of the low molecular weight liquid crystalline bis(benzylidene)-cyclohexanone compound reveals that there are two kinds of photoreactions in these systems: the EZ photoisomerization and 2 pi + 2 pi addition. The EZ photoisomerization in the LC phase disrupts the parallel stacking of the mesogens, resulting in the transition from the LC phase to the isotropic phase. The photoreaction involving the 2 pi + 2 pi addition of the bis(benzylidene)cyclohexanone units in the polymer results in the cross-linking of the chains. The liquid crystalline induced circular dichroism (LCICD) studies of the cholesterol benzoate copolymers revealed that the cholesteric supramolecular order remains even after the photo-cross-linking.
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Polyamides that are structural analogues of the naturally occurring DNA minor groove binding antibiotic distamycin (Dst) are promising candidates as gene modulators. Developing strategies for the large scale screening and monitoring of the cellular distribution of such ligands would aid the faster discovery of molecules, which would have eventual utility in molecular biology and medicine. Attachment of fluorescent tags would be a useful step towards this end. A fundamental question in this connection is whether the tag modifies the DNA binding affinity of the parent compounds. Towards answering this question, we have developed two oligopeptides that bear the dansyl (N, N-dimethylaminonaphthalene sulfonamido fluorophore) coupled directly to the N-terminus of the conjugated N-methylpyrrole carboxamide network, and possess three or four N-methyl pyrrole carboxamide units (abbreviated as Dn3 and Dn4 respectively). DNA binding abilities of these molecules were assessed from fluorescence titration experiments, duplex-DNA T-m analysis (employing both UV and fluorescence spectroscopy), induced circular dichroism measurements (ICD), salt dependence of ICD and apparent binding constant measurements (K-app) employing ethidium bromide (EtBr) displacement assay. Both these molecules 'reported' DNA binding in the form of an enhanced fluorescence emission. As judged from the ICD measurements, salt dependence of ICD, T-m analysis and K-app measurements, the binding affinities of the molecules that possessed dansyl group at their N-termini were lower than the ones with equivalent number of amide units, but possessed N-methylpyrrole carboxamide unit at their N-termini. These results would have implications in the future design of fluorescent polyamides.
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The homogeneous serine hydroxymethyltransferase from monkey liver was optimally activate at 60°C and the Arrhenius plot for the enzyme was nonlinear with a break at 15°C. The monkey liver enzyme showed high thermal stability of 62°C, as monitored by circular dichroism at 222 nm, absorbance at 280 nm and enzyme activity. The enzyme exhibited a sharp co-operative thermal transition in the range of 50°-70° (Tm= 65°C), as monitored by circular dichroism. L-Serine protected the enzyme against both thermal inactivation and thermal disruption of the secondary structure. The homotropic interactions of tetrahydrofolate with the enzyme was abolished at high temperatures (at 70°C, the Hill coefficient value was 1.0). A plot of h values vs. assay temperature of tetrahydrofolate saturation experiments, showed the presence of an intermediate conformer with an h value of 1.7 in the temperature range of 45°-60°C. Inclusion of a heat denaturation step in the scheme employed for the purification of serine hydroxymethyltransferase resulted in the loss of cooperative interactions with tetrahydrofolate. The temperature effects on the serine hydroxylmethyltransferase, reported for the first time, lead to a better understanding of the heat induced alterations in conformation and activity for this oligomeric protein.
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ALUMINIUM exposure has been shown to result in aggregation of microtubule-associated protein tau in vitro. In the light of recent observations that the native random structure of tau protein is maintained in its monomeric and dimeric states as well as in the paired helical filaments characteristic of Alzheimer's disease, it is likely that factors playing a causative role in neurofibrillary pathology would not drastically alter the native conformation of tau protein. We have studied the interaction of tau protein with aluminium using circular dichroism (CD) and 27(Al) NMR spectroscopy. The CD studies revealed a five-fold increase in the observed ellipticity of the tau-aluminium assembly. The increase in elipticity was not associated with a change in the general conformation of the protein and was most likely due to an aggregation of the tau protein induced by aluminium. Al-27 NMR spectroscopy confirmed the binding of aluminium to tau protein. Hyperphosphorylation of tau in Alzheimer's disease is known to be associated with defective microtubule assembly in this condition. Abnormally phosphorylated tau exists in a polymerized form in the paired helical filaments (PHF) which constitute the neurofibrillary tangles found in Alzheimer's disease. While it is hypothesized that its altered biophysical characteristics render abnormally phosphorylated tau resistant to proteolysis, causing the formation of stable deposits,the sequence of events resulting in the polymerization of tau are little understood, as are the additional factors or modifications required for tills process. Based on the results of our spectroscopic studies, a model for the sequence of events occurring in neurofibrillary pathology is proposed.
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We have used circular dichroism as a probe to characterize the solution conformational changes in RecA protein upon binding to DNA. This approach revealed that RecA protein acquires significant amounts of alpha-helix upon interaction with DNA. These observations, consistent with the data from crystal structure (Story, R. M., Weber, I., and Steitz, T. (1992) Nature 355, 318-325), support the notion that some basic domains including the DNA binding motifs of RecA protein are unstructured and might contribute to the formation of alpha-helix. A comparison of nucleoprotein filaments comprised of RecA protein and a variety of DNA substrates revealed important structural heterogeneity. The most significant difference was observed with poly(dG). poly(dC) and related polymers, rich in GC sequences, which induced minimal amounts of alpha-helix in RecA protein. The magnitude of induction of alpha-helix in RecA protein, which occurred concomitant with the production of ternary complexes, was 2-fold higher with homologous than heterologous duplex DNA. Most importantly, the stimulation of ATP hydrolysis by high salt coincided with that of the induction of alpha-helix in RecA protein. These conformational differences provide a basis for thinking about the biochemical and structural transitions that RecA protein experiences during the formal steps of presynapsis, recognition, and alignment of homologous sequences.
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Experimental studies have observed significant changes in both structure and function of lysozyme (and other proteins) on addition of a small amount of dimethyl sulfoxide (DMSO) in aqueous solution. Our atomistic molecular dynamic simulations of lysozyme in water-DMSO reveal the following sequence of changes on increasing DMSO concentration. (i) At the initial stage (around 5% DMSO concentration) protein's conformational flexibility gets markedly suppressed. From study of radial distribution functions, we attribute this to the preferential solvation of exposed protein hydrophobic residues by the methyl groups of DMSO. (ii) In the next stage (10-15% DMSO concentration range), lysozome partially unfolds accompanied by an increase both in fluctuation and in exposed protein surface area. (iii) Between 15-20% concentration ranges, both conformational fluctuation and solvent accessible protein surface area suddenly decrease again indicating the formation of an intermediate collapse state. These results are in good agreement with near-UV circular dichroism (CD) and fluorescence studies. We explain this apparently surprising behavior in terms of a structural transformation which involves clustering among the methyl groups of DMSO. (iv) Beyond 20% concentration of DMSO, the protein starts its final sojourn towards the unfolding state with further increase in conformational fluctuation and loss in native contacts. Most importantly, analysis of contact map and fluctuation near the active site reveal that both partial unfolding and conformational fluctuations are centered mostly on the hydrophobic core of active site of lysozyme. Our results could offer a general explanation and universal picture of the anomalous behavior of protein structure-function observed in the presence of cosolvents (DMSO, ethanol, tertiary butyl alcohol, dioxane) at their low concentrations. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.3694268]
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We report a pH-dependent conformational transition in short, defined homopolymeric deoxyadenosines (dA(15)) from a single helical structure with stacked nucleobases at neutral pH to a double-helical, parallel-stranded duplex held together by AH-HA base pairs at acidic pH. Using native PAGE, 2D NMR, circular dichroism (CD) and fluorescence spectroscopy, we have characterized the two different pH dependent forms of dA(15). The pH-triggered transition between the two defined helical forms of dA(15) is characterized by CD and fluorescence. The kinetics of this conformational switch is found to occur on a millisecond time scale. This robust, highly reversible, pH-induced transition between the two well-defined structured states of dA(15)represents a new molecular building block for the construction of quick-response, pH-switchable architectures in structural DNA nanotechnology.
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Achieving stabilization of telomeric DNA in G-quadruplex conformation by Various organic compounds has been an important goal for the medicinal chemists seeking to develop new anticancer agents. Several compounds are known to stabilize G-quadruplexes. However, relatively few are known to induce their formation and/or alter the topology, of the preformed quadruplex DNA. Herein, four compounds having the 1,3-phenylene-bis(piperazinyl benzimidazole) unit as a basic skeleton have been synthesized, and their interactions with the 24-mer telomeric DNA sequences from Tetrahymena thermophilia d(T(2)G(4))(4) have been investigated using high-resolution techniques Such as circular dichroism (CD) spectropolarimetry, CD melting, emission spectroscopy, and polyacrylamide gel electrophoresis. The data obtained, in the presence of one of three ions (Li+, Na+, or K+), indicate that all the new compounds have a high affinity for G-quadruplex DNA, and the strength of the binding with G-quadruplex depends on (1) phenyl ring substitution, (ii) the piperazinyl side chain, and (iii) the type of monovalent cation present in the buffer. Results further Suggest that these compounds are able to abet the conversion of the Intramolecular quadruplex into parallel stranded intermolecular G-quadruplex DNA. Notably, these compounds are also capable of inducing and stabilizing the parallel stranded quadruplex from randomly structured DNA in the absence of any stabilizing cation. The kinetics of the structural changes Induced by these compounds could be followed by recording the changes in the CD signal as a function of time. The implications of the findings mentioned above are discussed in this paper.
Resumo:
The extremities of chromosomes end in a G-rich single-stranded overhang that has been implicated in the onset of the replicate senescence. The repeated sequence forming a G-overhang is able to adopt a four-stranded DNA structure called G-quadruplex, which is a poor substrate for the enzyme telomerase. Small molecule based ligands that selectively stabilize the telomeric G-quadruplex DNA, induce telomere shortening eventually leading to cell death. Herein, we have investigated the G-quadruplex DNA interaction with two isomeric bisbenzimidazole-based compounds that differ in terms of shape (V-shaped angular vs linear).While the linear isomer induced some stabilization of the intramolecular G-quadruplex structure generated in the presence of Na+ the other, having V-shaped central planar core, caused a dramatic structural alteration of the latter, above a threshold concentration. This transition was evident from the pronounced changes observed in the circular dichroism spectra and from the get mobility shift assa involving the G-quadruples DNA. Notably, this angular isomer could also induce the G-quadruplex formation in the absence of any added cation. The ligand-quadruples complexes were investigated by computational molecular modeling, providing further information on structure-activity relationships. Finally, TRAP (telomerase repeat amplification protocol) experiments demonstrated that the angular isomer is selective toward the inhibition of telomerase activity.
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Hyoscyamine 6 beta-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6 beta-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. mete! (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. mete! (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6 beta-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K-m values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50 mu M each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of alpha-helicity in the secondary structure. From the fluorescence studies, Stern-Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14 M-1 and 0.56 M-1, respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes. (C) 2010 Elsevier Masson SAS. All rights reserved.
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An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAACCCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.
Resumo:
An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAACCCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.
Resumo:
An analysis of the base pair doublet geometries in available crystal structures indicates that the often reported intrinsic curvature of DNA containing oligo-(d(A).d(T)) tracts may also depend on the nature of the flanking sequences. The presence of CA/TG doublet in particular at the 5' end of these tracts is expected to enhance their intrinsic bending property. To test this proposition, three oligonucleotides, d(GAAAAACCCCCC), d(CCCCCCAAAAAG), d(GAAAAATTTTTC), and their complementary sequences were synthesized to study the effect of various flanking sequences, at the 5' and 3' ends of the A-tracts, on the curvature of DNA in solution. An analysis of the polyacrylamide gel electrophoretic mobilities of these sequences under different conditions of salts and temperatures (below their melting points) clearly showed that the oligomer with CA/TG sequence in the center was always more retarded than the oligomer with AC/GT sequence, as well as the oligomer with AT/AT sequence. Hydroxyl radical probing of the sequences with AC/GT and CA/TG doublet junctions gives a similar cutting pattern in the A-tracts, which is quite different from that in the C-tracts, indicating that the oligo(A)-tracts have similar structures in the two oligomers. KMnO4 probing shows that the oligomer with a CA/TG doublet junction forms a kink that is responsible for its inherent curvature and unusual electrophoretic mobility. UV melting shows a reduced thermal stability of the duplex with CA/TG doublet junction, and circular dichroism (CD) studies indicate that a premelting transition occurs in the oligomer with CA/TG doublet step before global melting but not in the oligomer with AC/GT doublet step, which may correspond to thermally induced unbending of the oligomer. These observations indicate that the CA/TG doublet junction at the 5' end of the oligo(A)-tract has a crucial role in modulating the overall curvature in DNA.
Resumo:
The antiparallel intramolecular G quartet structure for the 3.5 copy Oxytricha telomeric sequence d(G(4)T(4))(3)G4 has been established using a combination of spectroscopic and chemical probing methods. In the presence of Naf ions, this sequence exhibits a circular dichroism spectrum with a positive band at 295 nm and a negative band around 265 nm, characteristic of an antiparallel G quartet structure. Further, we show that d(G(4)T(4))(3)G(4) adopts an antiparallel intramolecular G quartet structure even in K+ unlike d(G(4)T(4)G(4)). KMnO4 probing experiments indicated the existence of intra and interloop interactions in the Na+ induced structure. We have found that K+ not only increases the thermal stability of,G quartet structure but also binds to the loop region and disrupts stacking and interloop interactions. Biological consequences of such cation-dependent conformational micro-heterogeneity in the loop region of G quartet structures is also discussed.