Conformational transition of DNA in electroreduction studied by in situ UV and CD thin layer spectroelectrochemistry

Electrochemically induced three conformational transitions of calf thymus DNA from B-10.4 to Z(10.2)-DNA and from B-10.2 to B-10.4 and to C-DNA in 10 mM phosphate buffer solution (pH 7.21) at glassy carbon electrode are found and studied by in situ circular dichroism (CD) thin layer spectroelectrochemistry with singular value decomposition least square (SVDLS) analysis. It indicates that the so-called B-10.2 form and the C-form of DNA may be composed of B-10.4 and left-A DNA and of B-10.4 and right-A DNA, respectively. The irreversible electrochemical reduction of adenine and cytosine groups in the DNA molecule is studied by UV-Vis spectroelectrochemistry. Some electrochemical parameters alphan = 0.17, E-0' = -0.70 V (vs. Ag/AgCl), and the standard heterogeneous electron transfer rate constant, k(0) = 1.8 x 10(-5) cm s(-1) are obtained by double logarithmic analysis and non-linear regression. (C) 2000 Elsevier Science B.V. All rights reserved.

Electric field-induced conformational transition of bovine serum albumin from alpha-helix to beta-sheet

The irreversible conformational transition of bovine serum albumin (BSA) from alpha-helix to beta-sheet, induced by electric field near the electrode surface, was monitored by circular dichroism (CD) with a long optical path thin layer cell (LOPTLC).

Fabrication of layer-by-layer deposited multilayer films containing DNA and its interaction with methyl green

Multilayer films were fabricated by layer-by-layer electrostatic deposition techniques between poly(diallyIdimethylammonium chloride) (PDDA) and calf thymus DNA (CT DNA) on glassy carbon and quartz substrates. Electrochemical impedance spectroscopy (EIS), Fourier transform infrared (FTIR) spectroscopy and UV-vis spectroscopy demonstrated the uniform assembly of PDDA/DNA multilayer films, and X-ray photoelectron spectroscopy confirmed the elemental composition of the films. Moreover, the interaction of DNA in PDDA/DNA films with methyl green was investigated by UV-vis spectroscopy and circular dichroism (CD). (C) 2001 Elsevier Science B.V. All rights reserved.

Electrochemistry and spectroscopy study on the interaction of microperoxidase-11 with lipid membrane

The interaction of microperoxidase-11 (MP11) with cationic lipid vesicles of didodecyldimethylammonium bromide (DDAB) induces an alpha -helical conformation from random coil conformations in solution and this change then makes heme macrocycle more distorted. DDAB-induced MP11 conformations were investigated by cyclic votammetry (CV), circular dichroism (CD) and UV-vis spectrometry. All results indicate that the binding of MP11 in solution to DDAB vesicles and the ordered structure formation are driven by mostly electrostatic interaction between negatively charged residues in the undecapeptide and positively charged lipid headgroups on the membrane surface. Upon binding to DDAB, its half-peak potential was also changed. The mechanism of the interaction between MP11 and DDAB was also discussed. (C) 2001 Elsevier Science B.V. All rights reserved.

Characterization of the structural and functional changes of hemoglobin in dimethyl sulfoxide by spectroscopic techniques

Circular dichroism (CD), fourier transform infrared (FTIR), and fluorescence spectroscopy were used to explore the effect of dimethyl sulfoxide (DMSO) on the structure and function of hemoglobin (Hb). The native tertiary structure was disrupted completely when the concentration of DMSO reached 50% (v/v), which was determined by loss of the characteristic Soret CD spectrum. Loss of the native tertiary structure could be mainly caused by breaking the hydrogen bonds, between the heme propionate groups and nearby surface amino acid residues, and by disorganizing the hydrophobic interior of this protein. Upon exposure of Hb to 52% DMSO for ca. 12 h in a D2O medium no significant change in 1652 cm(-1) band of the FTIR spectrum was produced, which demonstrated that alpha-helical structure predominated. When the concentration of DMSO increased to 57%: (1) the band at 1652 cm(-1) disappeared with the appearance of two new bands located at 1661 and 1648 cm(-1); (2) another new band at 1623 cm(-1) was attributed to the formation of intermolecular beta-sheet or aggregation, which was the direct consequence of breaking of the polypeptide chain by the competition of S=O groups in DMSO with C=O groups in amide bonds. Further increasing the DMSO concentration to 80%, the intensity at 1623 cm(-1) increased, and the bands at 1684, 1661 and 1648 cm(-1) shifted to 1688, 1664 and 1644 cm(-1), respectively. These changes showed that the native secondary structure of Hb was last and led to further aggregation and increase of the content of 'free' amide C=O groups. In pure DMSO solvent, the major band at 1664 cm(-1) indicated that almost all of both the intermolecular beta-sheet and any residual secondary structure were completely disrupted. The red shift of the fluorescence emission maxima showed that the tryptophan residues were exposed to a greater hydrophilic environment as the DMSO content increased. GO-binding experiment suggested that the biological function of Hb was disrupted seriously even if the content of DMSO was 20%. (C) 1998 Elsevier Science B.V. All rights reserved.

Electropolymeric generation of optically active polypyrrole

The facile synthesis of optically active polypyrrole has been achieved via the enantioselective electropolymerization of pyrrole on indium-tin-oxide-coated glass electrodes in aqueous solution containing D-(+) or L-(-) tartaric acid, (1S)-(+)-10-camphorsulfonic acid and L-lactic acid. The dark films of conducting polypyrroles salt formed under electrostatic conditions (+0.65V vs. Ag/AgCl) exhibited strong circular dichroism (CD) spectra typical of polymers possessing helical chirality. The quantitative reversal of the CD spectrum of the salt grown in (+)-tartaric acid as opposed to (-)-tartaric acid suggests that electropolymerization is highly enantioselective, with one helical screw of the polymer chain being preferentially produced depending on the hand of the tartaric anion incorporated. (C) 1997 Elsevier Science Ltd.

A NEW SIMPLE LONG-PATH-LENGTH THIN-LAYER SPECTROELECTROCHEMICAL CELL IN VARIOUS USES

This article describes the design of a new type of long-path-length thin-layer cell (LPTLC), which is constructed by inserting two Teflon cell bodies into a standard cuvette. This cuvette holder enables the LPTLC to combine with photometric instrumentation conveniently. Gold, platinum, glassy carbon, and other materials can be used for the working electrode. Since no soluble adhesive material is used, the cell can meet various needs (in situ conventional and derivative W-vis, in situ circular dichroism, etc.) in both aqueous and nonaqueous systems.

HELIX-INDUCED ASYMMETRIC POLYMERIZATION

Asymmetric polymerization could be induced by an already formed optically active living prepolymer with one-handed screw sense helix conformation. The usually formed anionic active centre on the prepolymer could be changed to cationic, radical and even of Ziegler-Natta type. These living prepolymers with various kinds of active centre were all effective to induce a consequent asymmetric polymerization of a monomer which may be other than that in the prepolymer, to afford an optically active helical chain with the same screw sense as that of the prepolymer. Eight monomers have been used in the work. Optical rotation, circular dichroism and gelpermeation chromatography have been taken to prove the helix-induced asymmetric polymerization.

Characterization of the main light-harvesting chlorophyll a/b-protein complex of green alga, Bryopsis corticulans

The main light-harvesting chlorophyll a/b -protein complex (LHC II) has been isolated directly from thylakoid membranes of shiphonous green alga, Bryopsis corticulans Setch. by using two consecutive runs of anion exchange and gel-filtration chromatography. Monomeric and trimeric subcomplexes of LHC 11 were obtained by using sucrose gradient ultracentrifugation. Pigment analysis by reversed-phase high performance liquid chromatography showed that chlorophyll a (Chl a), chlorophyll b (Chl b), neoxanthin, violaxanthin and siphonaxanthin were involved in LHC 11 from B. corticulans. The properties of electronic transition of monomeric LHC II showed similarities to those of trimeric LHC II. Circular dichroism spectroscopy showed that strong intramolecular interaction of excitonic dipoles between Chl a and between Chl b exist in one LHC II apoprotein, while the intermolecular interaction of these dipoles can be intensified in the trimeric structure. The monomer has high efficient energy transfer from Chl b and siphonaxanthin to Chl a similarly to that of the trimer. Our results suggest that in B. corticulans, LHC II monomer has high ordered pigment organization that play effective physiological function as the trimer, and thus it might be also a functional organization existing in thylakoid membrane of B. corticulans.

Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand.

Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.

De novo design and molecular assembly of a transmembrane diporphyrin-binding protein complex.

The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPP's) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.

Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.

Comparison of the thermodynamics and base-pair dynamics of a full LNA:DNA duplex and of the isosequential DNA:DNA duplex

Locked nucleic acids (LNA), conformationally restricted nucleotide analogues, are known to enhance pairing stability and selectivity toward complementary strands. With the aim to contribute to a better understanding of the origin of these effects, the structure, thermal stability, hybridization thermodynamics, and base-pair dynamics of a full-LNA:DNA heteroduplex and of its isosequential DNA:DNA homoduplex were monitored and compared. CD measurements highlight differences in the duplex structures: the homoduplex and heteroduplex present B-type and A-type helical conformations, respectively. The pairing of the hybrid duplex is characterized, at all temperatures monitored (between 15 and 37 degrees C), by a larger stability constant but a less favorable enthalpic term. A major contribution to this thermodynamic profile emanates from the presence of a hairpin structure in the LNA single strand which contributes favorably to the entropy of interaction but leads to an enthalpy penalty upon duplex formation. The base-pair opening dynamics of both systems was monitored by NMR spectroscopy via imino protons exchange measurements. The measurements highlight that hybrid G-C base-pairs present a longer base-pair lifetime and higher stability than natural G-C base-pairs, but that an LNA substitution in an A-T base-pair does not have a favorable effect on the stability. The thermodynamic and dynamic data confirm a more favorable stacking of the bases in the hybrid duplex. This study emphasizes the complementarities between dynamic and thermodynamical studies for the elucidation of the relevant factors in binding events.

Structure and denaturation of 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1

The secondary structure of the trimeric protein 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1, the second of three enzymes involved in the dechlorination of 4-chlorobenzoate to form 4-hydroxybenzoate, has been examined. E(mM) for the enzyme was 12.59. Analysis by circular dichroism spectrometry in the far uv indicated that 4-chlorobenzoyl coenzyme A dehalogenase was composed mostly of alpha-helix (56%) with lesser amounts of random coil (21%), beta-turn (13%) and beta-sheet (9%). These data are in close agreement with a computational prediction of secondary structure from the primary amino acid sequence, which indicated 55.8% alpha-helix, 33.7% random coil and 10.5% beta-sheet; the enzyme is, therefore, similar to the 4-chlorobenzoyl coenzyme A dehalogenase from Pseudomonas sp. CBS-3. The three-dimensional structure, including that of the presumed active site, predicted by computational analysis, is also closely similar to that of the Pseudomonas dehalogenase. Study of the stability and physicochemical properties revealed that at room temperature, the enzyme was stable for 24 h but was completely inactivated by heating to 60 degrees C for 5 min; thereafter by cooling at 1 degrees C min(-1) to 45 degrees C, 20.6% of the activity could be recovered. Mildly acidic (pH 5.2) or alkaline (pH 10.1) conditions caused complete inactivation, but activity was fully recovered on returning the enzyme to pH 7.4. Circular dichroism studies also indicated that secondary structure was little altered by heating to 60 degrees C, or by changing the pH from 7.4 to 6.0 or 9.2. Complete, irreversible destruction of, and maximal decrease in the fluorescence yield of the protein at 330-350 nm were brought about by 4.5 M urea or 1.1 M guanidinium chloride. Evidence was obtained to support the hypothetical three-dimensional model, that residues W140 and W167 are buried in a non-polar environment, whereas W182 appears at or close to the surface of the protein. At least one of the enzymes of the dehalogenase system (the combined 4-chlorobenzoate:CoA ligase, the dehalogenase and 4-hydroxybenzoyl coenzyme A thioesterase) appears to be capable of association with the cell membrane.

Identification of the region of non-A beta component (NAC) of alzheimer's disease amyloid responsible for its aggregation and toxicity

The non-beta-amyloid (Aß) component of Alzheimer's disease amyloid (NAC) and its precursor a-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and a-synuclein both form ß-sheet structures upon ageing, aggregate to form fibrils, and are neurotoxic. We recently established that a peptide comprising residues 3±18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY-5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8±18) and NAC(8±16) are toxic, whereas NAC(12±18), NAC(9±16) and NAC(8±15) are not. Circular dichroism indicates that none of the peptides displays ß-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce ß sheet, fragments NAC(8±18) and NAC(8±16) both form ß-sheet structure. Only NAC(8±18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8±16 of NAC, equivalent to residues 68±76 in a-synuclein, comprise the region crucial for toxicity.