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Circular Dichroism Measurements (circular + dichroism_measurement)
Selected AbstractsThe stress response protein Gls24 is induced by copper and interacts with the CopZ copper chaperone of Enterococcus hiraeFEMS MICROBIOLOGY LETTERS, Issue 1 2010Jivko V. Stoyanov Abstract Intracellular copper routing in Enterococcus hirae is accomplished by the CopZ copper chaperone. Under copper stress, CopZ donates Cu+ to the CopY repressor, thereby releasing its bound zinc and abolishing repressor,DNA interaction. This in turn induces the expression of the cop operon, which encodes CopY and CopZ, in addition to two copper ATPases, CopA and CopB. To gain further insight into the function of CopZ, the yeast two-hybrid system was used to screen for proteins interacting with the copper chaperone. This led to the identification of Gls24, a member of a family of stress response proteins. Gls24 is part of an operon containing eight genes. The operon was induced by a range of stress conditions, but most notably by copper. Gls24 was overexpressed and purified, and was shown by surface plasmon resonance analysis to also interact with CopZ in vitro. Circular dichroism measurements revealed that Gls24 is partially unstructured. The current findings establish a novel link between Gls24 and copper homeostasis. [source] An Optically Active Polythiophene Exhibiting Electrochemically Driven Light-Interference ModulationADVANCED FUNCTIONAL MATERIALS, Issue 9 2009Hiromasa Goto Abstract Optically active polythiophene (PT*) is successfully prepared by electrochemical polymerization using a cholesteric liquid crystal (CLC) electrolyte solution. Polarizing optical microscopy observations of the polymer reveal a well-resolved fingerprint texture similar to the optical texture of the CLC. Circular dichroism measurements indicate a Cotton effect. The PT* film produced by the asymmetric polymerization in CLC exhibits a variable diffraction function, electrochemically driven refractive index modulation, and electrochromism originating from the periodic dielectric structure, representing a form of structural electrochromism. [source] Overexpression and Characterization of the Rhodobacter sphaeroides PufX Membrane Protein in Escherichia coli,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2007Shiho Onodera Heterologous expression of the PufX membrane protein from purple photosynthetic bacterium Rhodobacter sphaeroides was attempted by using Escherichia (E.) coli cells. The PufX was overexpressed as a recombinant protein with a histidine tag added to the carboxyl terminus, and can be extracted from the cell membrane by various detergents. Circular dichroism measurements showed that the expressed PufX protein had ,-helix contents of 29% in organic solvents and 22,26% in 0.8,2.0% (w/v) n -octyl ,- d -glucopyranoside solutions, suggesting that the PufX contains a substantial ,-helical region composed of 18,22 amino acids. The PufX expressed in E. coli was examined by reconstitution experiments with LH1 ,- and ,-polypeptides and bacteriochlorophyll a. It was shown that the PufX inhibited not only the reconstitution of the LH1 complex, but also the formation of the B820 subunit type complex at high concentrations, indicating that the expressed PufX is biologically active. Large-scale expression of the functional PufX membrane protein provides sufficient quantity for further biophysical and structural analyses of its biological function, and adds another example for producing highly hydrophobic integral membrane proteins using the E. coli expression system. [source] Highly sensitive and quantitative analysis of polyeptides using a new gradient system based on an abrupt change in adsorption of polypeptide to the reversed-phase column packingBIOMEDICAL CHROMATOGRAPHY, Issue 10 2007Ryoya Goda Abstract During a study of 100 µL aliquots of urocortin containing various acetonitrile contents, we hypothesized that a change in the acetonitrile content in the solution across a specific content of acetonitrile (critical threshold) causes an abrupt change in adsorption capacity to the column packing. Circular dichroism measurements suggest that the conformational change induced by acetonitrile in the solution causes the abrupt change in adsorption capacity, and this solvent-induced conformational change is reversible across the critical threshold. This hypothesis can apply to various polypeptides with molecular weights range from 1007 to 6789 and to other organic solvents. A new gradient system utilizing the instant recovery of the adsorption capacity across the critical threshold was designed, and applied to the analysis of a 100 µL aliquot of various polypeptide solutions. The results suggest that use of a solution containing organic solvents more than the critical threshold allows successful dilution of polypeptides up to picomolar concentration range without any loss due to its adsorption during handling procedures and loading onto the LC system, and that a new gradient system enables quantitative analysis of polypeptides at picomolar concentrations in such solutions. Copyright © 2007 John Wiley & Sons, Ltd. [source] Conformations within soluble oligomers and insoluble aggregates revealed by resonance energy transferBIOPOLYMERS, Issue 4 2010Jyothi L. Digambaranath Abstract A fluorescently labeled 20-residue polyglutamic acid (polyE) peptide 20 amino acid length polyglutamic acid (E20) was used to study structural changes which occur in E20 as it co-aggregates with other unlabeled polyE peptides. Resonance energy transfer (RET) was performed using an o -aminobenzamide donor at the N-terminus and 3-nitrotyrosine acceptor at the C-terminus of E20. PolyE aggregates were not defined as amyloid, as they were nonfibrillar and did not bind congo red. Circular dichroism measurements indicate that polyE aggregation involves a transition from ,-helical monomers to aggregated ,-sheets. Soluble oligomers are also produced along with aggregates in the reaction, as determined through size exclusion chromatography. Time-resolved and steady-state RET measurements reveal four dominant E20 conformations: (1) a partially collapsed conformation (24 Å donor,acceptor distance) in monomers, (2) an extended conformation in soluble oligomers (>29 Å donor,acceptor distance), (3) a minor partially collapsed conformation (22 Å donor-acceptor distance) in aggregates, and (4) a major highly collapsed conformation (13 Å donor,acceptor distance) in aggregates. These findings demonstrate the use of RET as a means of determining angstrom-level structural details of soluble oligomer and aggregated states of proteins. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 299,317, 2010. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Comparison of the aggregation properties, secondary structure and apoptotic effects of wild-type, Flemish and Dutch N-terminally truncated amyloid , peptidesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2001N. Demeester Abstract The Dutch (E22Q) and Flemish (A21G) mutations in the ,APP region of the amyloid precursor protein (APP) are associated with familial forms of Alzheimer dementia. However, patients with these mutations express substantially different clinical phenotypes. Therefore, secondary structure and cytotoxic effects of the three A,(12,42) variants [wild-type (WT), Dutch and Flemish] were tested. At a concentration of 5 µm the aggregation of these peptides followed the order: A,(1,42) WT > A,(12,42) WT > A,(12,42) Flemish >,A,(12,42) Dutch. The stability of the secondary structure of these peptides upon decreasing the trifluoroethanol (TFE) concentration in the buffer was followed by circular dichroism measurements. WT peptides progressively lost their ,-helical structure; this change occurred faster for both the Flemish and Dutch peptides, and at higher percentages of TFE in the buffer, and was accompanied by an increase in ,-sheet and random coil content. Apoptosis was induced in neuronal cells by the A,(12,42) WT and Flemish peptides at concentrations as low as 1,5 µm, as evidenced by propidium iodide (PI) staining, DNA laddering and caspase-3 activity measurements. Even when longer incubation times and higher peptide concentrations were applied the N-truncated Dutch peptide did not induce apoptosis. Apoptosis induced by the full length A,(1,42) peptide was weaker than that induced by its N-truncated variant. These data suggest that N-truncation enhanced the cytotoxic effects of A, WT and Flemish peptides, which may play a role in the accelerated progression of dementia. [source] The conformational stability of the Streptomyces coelicolor histidine-phosphocarrier proteinFEBS JOURNAL, Issue 11 2004Characterization of cold denaturation, protein interactions Thermodynamic parameters describing the conformational stability of the histidine-containing phosphocarrier protein from Streptomyces coelicolor, scHPr, have been determined by steady-state fluorescence measurements of isothermal urea-denaturations, differential scanning calorimetry at different guanidinium hydrochloride concentrations and, independently, by far-UV circular dichroism measurements of isothermal urea-denaturations, and thermal denaturations at fixed urea concentrations. The equilibrium unfolding transitions are described adequately by the two-state model and they validate the linear free-energy extrapolation model, over the large temperature range explored, and the urea concentrations used. At moderate urea concentrations (from 2 to 3 m), scHPr undergoes both high- and low-temperature unfolding. The free-energy stability curves have been obtained for the whole temperature range and values of the thermodynamic parameters governing the heat- and cold-denaturation processes have been obtained. Cold-denaturation of the protein is the result of the combination of an unusually high heat capacity change (1.4 ± 0.3 kcal·mol,1·K,1, at 0 m urea, being the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements) and a fairly low enthalpy change upon unfolding at the midpoint temperature of heat-denaturation (59 ± 4 kcal·mol,1, the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements). The changes in enthalpy (m,Hi), entropy (m,Si) and heat capacity (m,Cpi), which occur upon preferential urea binding to the unfolded state vs. the folded state of the protein, have also been determined. The m,Hi and the m,Si are negative at low temperatures, but as the temperature is increased, m,Hi makes a less favourable contribution than m,Si to the change in free energy upon urea binding. The m,Cpi is larger than those observed for other proteins; however, its contribution to the global heat capacity change upon unfolding is small. [source] X-ray magnetic circular dichroism measurements using an X-ray phase retarder on the BM25 A-SpLine beamline at the ESRFJOURNAL OF SYNCHROTRON RADIATION, Issue 3 2010Roberto Boada Circularly polarized X-rays produced by a diamond X-ray phase retarder of thickness 0.5,mm in the Laue transmission configuration have been used for recording X-ray magnetic circular dichroism (XMCD) on the bending-magnet beamline BM25A (SpLine) at the ESRF. Field reversal and helicity reversal techniques have been used to carry out the measurements. The performance of the experimental set-up has been demonstrated by recording XMCD in the energy range from 7 to 11,keV. [source] Characterization of the G-quadruplex structure of a catalytic DNA with peroxidase activityBIOPOLYMERS, Issue 5 2009De-Ming Kong Abstract It has been reported that the complexes formed by hemin and some G-quadruplexes can be developed as a new class of DNAzyme with peroxidase activity. This kind of DNAzyme has received a great deal of attention. But to date, the actual G-quadruplex structure that can provide hemin with enhanced peroxidase activity is in doubt. Herein, the G-quadruplex structure of CatG4, a 21-nucleotide DNA oligomer which was previously reported to bind hemin and the resulting complex exhibiting enhanced peroxidase activity, was characterized by fluorescence and circular dichroism measurements. The results suggest that the catalytically active form of CatG4 may be a unimolecular parallel quadruplex rather than a unimolecular chair-type antiparallel quadruplex or a multistranded parallel quadruplex. In addition, the fluorescence analysis of labeled oligonucleotides may be developed as a supplementary tool for the study of DNA conformations. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 331,339, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Syntheses, Phase Behavior, Supramolecular Chirality, and Field-Effect Carrier Mobility of Asymmetrically End-Capped Mesogenic OligothiophenesCHEMISTRY - A EUROPEAN JOURNAL, Issue 14 2009Qingwei Meng Dr. Abstract Supramolecular chirality and liquid crystalline OFET: Achiral end-capped oligothiophenes can be tuned to exhibit supramolecular chirality with unique striped textures showing distinct circular dichroism signals as well as a highly ordered SmE phase that leads to high hole carrier mobility. A novel series of asymmetrically end-capped mesogenic oligothiophenes, with various oligothiophene core lengths, alkoxy tail lengths, and molecular polarities through introducing alkylsulfanyl or alkylsulfonyl functionalities as the terminal group, have been synthesized by palladium-catalyzed Suzuki cross-coupling and Kumada cross-coupling reactions as key steps. For the single end-capped oligothiophenes, CmO-Ar-OT(4)-H in which m=10, 12, 14, 16, and 18, all of these oligomers exhibited a broad temperature range of highly ordered smectic E and enantiotropic nematic phases, apart from the one with the longest octadecyloxy tail. For the double end-capped series C10O-Ar-OT(n)-R, R=Ph-SC6 or Ph-SO2C6 in which n=1, 2, 3, and 4, oligomers with more than one thiophene ring exhibited smectic A and smectic C phases, various crystal polymorphs and/or unusual low-temperature condensed phases. In the nonpolar, alkylsulfanylphenyl-substituted oligothiophene series, both the crystal/solid melting point and mesogenic clear point increased significantly with an increasing oligothiophene conjugation length. In the polar, alkylsulfonylphenyl-substituted oligothiophene series, all the oligomers showed increased melting points, but decreased mesogenic temperature intervals than those of their corresponding alkylsulfanyl counterparts. Remarkably, two different helical structures showing distinct striated textures or striped patterns were observed with a pitch of several to tens of micrometers under a polarized optical microscope upon cooling from their preceding fluidic smectic phases. The unusual twisted smectic layer structures in the thin solid films exhibiting distinct supramolecular chirality of both handednesses, revealed by circular dichroism measurements, were further confirmed by XRD analyses characterized by a sharp layer reflection together with its higher orders and diffuse wide-angle scatterings. In addition, initial studies showed that the highly ordered smectic phase of the single end-capped oligothiophenes can be utilized to improve field-effect charge mobility. C10O-Ar-OT(4)-H showed a hole mobility of 0.07,cm2,V,1,s,1 when deposited on octyltrichlorosilane-treated substrates at 140,°C and the on/off current ratios reached 5×105; on the other hand, its mobility was only 8×10,3,cm2,V,1,s,1 on the same substrate when deposited at room temperature. [source] Polarization control of ultrashort mid-IR laser pulses for transient vibrational circular dichroism measurements,CHIRALITY, Issue 1E 2009Mathias Bonmarin Abstract Linear dichroism and birefringence artifacts are a major source of concern in transient circular dichroism measurements. They mainly arise from interaction of an imperfectly circular polarized probe beam with a nonisotropic sample. We present in this article a procedure to generate mid-IR pulses of highly symmetric left and right handed circular or elliptical polarization states for transient VCD measurements. An infrared femtosecond laser source is synchronized to the natural frequency of a photo elastic modulator. Residual static birefringence of the modulator and the sample cell can be largely compensated by carefully controlling the arrival time of the ultrashort probe pulses at the modulator. Chirality 21:E298,E306, 2009. © 2009 Wiley-Liss, Inc. [source] | |||||||||||||