Circular dichroism and cross-linking studies of bacteriorhodopsin mutants

Summary. Circular dichroism (CD) spectroscopy was employed for native (wild type, WT) bacteriorhodopsin (bR) and several mutant derivatives: R134K, R134H, R82Q, S35C, L66C, and R134C/E194C. Comparative analysis of the CD spectra in visible range shows that only R134C/E194C exhibits biphasic CD, typical for native bR, the other mutants demonstrate CD spectra with significantly smaller or absent negative band. Since the biphasic CD is a feature of hexagonal lattice structure composed by bR trimers in the purple membrane, these mutants and WT were examined by cross-linking studies, which confirmed the same trend towards trimeric organization. Therefore, a single amino acid substitution may lead to drastically different CD spectra without disruption of bR trimeric organization. Thus, although disruption of bR trimeric crystalline lattice structure (e.g., solubilization with detergents) directly results in the disappearance of characteristic bilobe in visible CD, the lack of the bilobe in the CD alone does not predict the absence of trimers.     

Circular dichroism of 1,3-dioxane-type (2'-naphthyl)methylene acetals of glycosides

The CD spectra are reported for a series of 1,3-dioxane-type 4,6-O-(2'-naphthyl)methylene acetals of carbohydrates with and without interacting aromatic protective groups on the C-1, C-2, and C-3 hydroxy groups. In the absence of interacting chromophores, the signs of the (1)B transitions are not sensitive to the configuration of C-4, while the signs of the weak (1)L(a) bands are opposite in the galacto and gluco derivatives. The equatorial parallel conformation is found to be the preferred conformation of the 2-naphthyl group in the solid state by X-ray diffraction. The intense (1)B(a) and (1)B(b) transitions of the naphthalene chromophore allowed a safe configurational assignment by exciton coupled interaction with the aromatic protective groups in para-methoxyphenyl-beta-D-glycosides. The origin of the observed CEs were deduced and the additivity of the interactions was studied. The direction of the hydrogenolytic cleavage of 4,6-O-(2'-naphthyl)methylene acetal of carbohydrates could also be detected by the (1)B(b) transition of the 2-naphthyl chromophore.     

Circular dichroism and absorption spectroscopic data reveal binding of the natural cis-carotenoid bixin to human alpha1-acid glycoprotein

Using circular dichroism (CD) and electronic absorption spectroscopy techniques, interaction of the natural dietary cis-carotenoid bixin with an important human plasma protein in vitro was demonstrated for the first time. The induced CD spectra of bixin obtained under physiological conditions (pH 7.4, 37 degrees C) revealed its binding to the serum acute-phase reactant alpha(1)-acid glycoprotein (AGP), a member of the lipocalin protein family. Spectral features of the extrinsic Cotton effects of bixin suggested the inclusion of a single, chirally distorted ligand molecule into the asymmetric protein environment. Compared with the absorption spectra obtained in ethanol and benzene, the strong red shift of the main absorption peak of AGP-bound bixin indicated that the proposed binding site was rich in aromatic residues, and also suggested that hydrophobic interactions were involved in the binding. Using the data obtained from the CD titration experiments, the association constant (Ka=4.5x10(5)M-1) and stoichiometry of the binding (0.15) were calculated. The low value of the stoichiometry was attributed to the structural polymorphism of AGP. To the authors' knowledge, the current study represents the first human lipocalin protein for which carotenoid binding affinity has been explored in vitro with these techniques.     

Conformational study of the preferred conformations of the peptide sequence VP3(110-121) of HAV by circular dichroism and molecular mechanics

Summary A conformational analysis of the fragment 110–121 of VP3 coating protein of the hepatitis A virus was carried out using circular dichroism spectroscopy and computational studies. The latter studies indicate the tendency of the peptide to adopt hairpin-type structures. Circular dichroism experiments indicate that, in spite of the fact that the isolated peptide exhibits no structure under different experimental conditions, negatively charged liposomes induce a secondary structure that agrees with the results of the computational study.     

Mononuclear and binuclear Co(III)-dioxygen adducts of bleomycin. Circular dichroism and electron paramagnetic resonance studies

Co(II) interacts with bleomycin in aqueous solution, in the presence of air, to give a short lived mononuclear superoxo-Co(III) complex (I) identified previously, by Sugiura, by electron paramagnetic resonance measurements. This complex rapidly releases O₂ to yield the dinuclear μ-peroxo-Co(III) complex (II), but is stabilized by the presence of DNA yielding a new superoxo long lived species (I′). The absorption and circular dichroism spectra of the three species (I,I′,II) have been characterized.     

Conformational study of the preferred conformations of the peptide sequence VP3(110-121) of HAV by circular dichroism and molecular mechanics

A conformational analysis of the fragment 110–121 of VP3 coating protein of the hepatitis A virus was carried out using circular dichroism spectroscopy and computational studies. The latter studies indicate the tendency of the peptide to adopt hairpin-type structures. Circular dichroism experiments indicate that, in spite of the fact that the isolated peptide exhibits no structure under different experimental conditions, negatively charged liposomes induce a secondary structure that agrees with the results of the computational study.     

湖南尖吻蝮蛇毒出血毒素的圆二色性和化学修饰

用远紫外ＣＤ谱研究了湖南产尖吻蝮蛇毒的两个出血毒素（ＤａＨＴ－１、ＤａＨＴ－２）的溶液构象,计算得ＤａＨＴ－１的α螺旋、β折叠、无规卷曲的含量分别为３６．９％、３５．５％、２７．６％;ＤａＨＴ－２的α螺旋、β折叠、无规卷曲分别为２３．４％、３１．３％、４５．３％。随ｐＨ的增大或减小,峰位蓝移,酸性条件下的变化比碱性条件下的变化大。构象单元含量计算表明：α螺旋减少,无规卷曲增多,β折叠基本未变。温度和ｐＨ对ＣＤ谱的影响相似,５０℃时峰位蓝移,α螺旋减少,无规卷曲增多．ＥＤＴＡ对ＣＤ谱影响显著,０．０２ｍｏｌ／ＬＥＤＴＡ便导致两个出血毒素呈极度的无序状态。ＥＤＴＡ完全抑制,半胱氨酸部分抑制,胰蛋白酶不影响它们的出血活性。     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls in nematic liquid crystals. I. Electric and weak magnetic field effects on the dichroism spectra

Dichroism spectra of chlorophyll a, chlorophyll b and bacteriochlorophyll a in various nematic liquid crystals are reported. The initial orientation of chlorophylls in such a sample is determined by the interaction of the aggregate formed from the pigment and the liquid crystal molecules with the electrode surface on the cell windows. Reorientation is carried out by either an electric or magnetic field. The analysis of the circular dichroism spectra obtained from these samples on the basis of the Mueller matrix shows that the intensity is predominantly related to the texture of the sample. Chlorophyll molecules can be aggregated with liquid crystals in two ways: (1) through the chlorin magnesium atom, which results in the liquid crystal chain being almost perpendicular to the porphyrin ring, or (2) attached parallel to the line connecting the first and third pyrrole rings of the chlorin, the chlorin now lying in the plane of the liquid crystal chains. By comparing the dichroism spectra of various chlorophylls in the same liquid crystal we can draw conclusions concerning the preferred type of aggregation, not only with liquid crystals, but also with biological molecules. These liquid crystal systems are models of the orientation effects found for chlorophyll in lamellae. The model studied in this work is much simpler than the lamellar system but it does exhibit several common properties with the latter. Both systems are anisotropic and show much more intense dichroism signals, often of opposite sign, compared with those observed for photosynthetic pigments in isotropic solutions. Dichroism signals of organism fragments are much more complex than those of our model, which can either be related to the occurrence in the organism of several types of pigments or, for a given type of pigment, could be the result of exciton splitting. On the basis of our model it is shown that small changes in the anisotropy of the pigment in the surroundings have a strong influence on the sign and amplitude of the observed circular dichroism signal. Such effects may be responsible for the structure of the dichroism spectra observed for biological samples. Such structures can be partially related to the superposition of the dichroism signal from various ‘domains’ of chromophore which are different in both pigment arrangement and in the anisotropy of the surroundings of the pigment molecules themselves.     

Intensity of cooperative vibronic transitions in the Eu(III), Gd(III) and Tb(III) formates spectra

The luminescence and absorption spectra of the lanthanide ions in solids and coordination compounds are characterized by sharp pure electronic lines, which are accompanied by much weaker lines of vibronic transitions. The vibronic spectroscopy is a good probing tool for investigations of the properties of surrounding ion ligands. The lanthanides formates are efficient luminescent crystals and can be viewed as the elementary type in the whole class of the oxygen-containing lanthanide coordination compounds. The intensity of vibronic transitions in spectra of luminescence and excitation europium (⁵D₀→⁷F₂, ⁷F₀→⁵D₂), terbium (⁷F₆→⁵D₄), gadolinium (⁶P_7/2→⁸S_7/2) in anhydrous formates of the type Ln(HCOO)₃ (Ln = Eu, Tb, Gd) and Y(HCOO)₃.2H₂O doped with Eu³⁺ and Tb³⁺ (C ~1 mol%) are reported. Also, the infrared and Raman spectra were obtained for the same compounds. Related integral intensity vibronic sidebands depend on the type of electronic transition of the same ion and varies for the same electronic transitions in different crystals. The obtained experimental data referring to the rate constants of vibronic transitions and intensity distribution in vibronic spectra on normal vibrations of the formate groups are in agreement with the predictions based on the Stavola–Dexter theory of cooperative vibronic transitions.     

Circular dichroism and aggregation studies of amyloid beta (11-8) fragment and its variants

Aggregation of Abeta peptides is a seminal event in Alzheimer's disease. Detailed understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Here comparative conformational and aggregation studies using CD spectroscopy and thioflavine T fluorescence assay are presented. As a model peptide, the 11-28 fragment of Abeta was used. This model peptide is known to contain the core region responsible for Abeta aggregation. The structural and aggregational behaviour of the peptide was compared with the properties of its variants corresponding to natural, clinically relevant mutants at positions 21-23 (A21G, E22K, E22G, E22Q and D23N). In HFIP (hexafluoro-2-propanol), a strong alpha-helix inducer, the CD spectra revealed an unexpectedly high amount of beta-sheet conformation. The aggregation process of Abeta(11-28) variants provoked by water addition to HFIP was found to be consistent with a model of an alpha-helix-containing intermediate. The aggregation propensity of all Abeta(11-28) variants was also compared and discussed.     

Exciton coupling effects and conformational change of perhexyloligosilanes with optically active methyl(1-naphthyl)phenylsilyl terminals

Perhexyloligosilanes (R,R)-(+)-MeNpPhSi*(Hex(2)Si)(n)Si*PhNpMe (n = 2; (R,R)-(+)-4a, n = 4; (R,R)-(+)-6a, n = 6; (R,R)-(+)-8a) with chiral methyl(1-naphthyl)phenylsilyl terminals were synthesized and characterized. The absorption wavelengths lambda(max) by (1)L(a,Ph) transition of phenyl chromophore conjugated with oligosilane units in (R,R)-(+)-4a - (R,R)-(+)-8a show bathochromic shift of about 3-4 nm compared with those of the alpha,omega-phenyl substituted perhexyloligosilanes Ph(Hex(2)Si)(m)Ph (m = 4; 4b, m = 6; 6b, m = 8; 8b) having the same silicon chain length. Longer chain length induces the separated lambda(max) of (1)L(a,Ph) from (1)B(b,Np) of naphthyl chromophore with positive exciton chiralities. In (R,R)-(+)-8a, although the extremum wavelengths lambda(ext) of exciton coupling between (1)B(b,Np) and (1)L(a,Ph) are separated by about 80 nm, the compound retains the positive exciton chirality, which provides definite information on the absolute configuration of terminal chiral silicon atoms. Bulky terminal substituents and lowering the temperature affect the conformation of the main chain, inducing extended silicon backbone structure.     

Interaction of lac repressor with alternating poly d (A-T) and poly d (G-C). Circular dichroism studies

The binding of lac repressor to poly d(A-T) and poly d(G-C) has been studied using circular dichroism. The results indicate that the binding induces the same conformational change of both polynucleotides and perturbs the same number of nucleic acid bases (28 bases). It is shown that in 0.1 M phosphate buffer the CD measurement can be used to determine the binding constant of lac repressor to poly d(A-T). Competition experiments performed at various salt concentrations show that the stronger interaction of lac repressor for poly d(A-T) than for poly d(G-C) is based on difference in the dissociation rate of the complexes whereas the association rate for both polymers are similar.     

Conformational studies of a benzodiazepine-like peptide in SDS micelles by circular dichroism, 1H NMR and molecular dynamics simulation

Summary The conformation of a benzodiazepine-like decapeptide corresponding to the YLGYLEQLLR fragment of a casein has been examined in a sodium dodecyl sulfate micellar medium using circular dichroism, two-dimensional¹H NMR spectroscopy and restrained molecular dynamics simulation. The decapeptide adopts an amphipathic 3₁₀-helicoid structure in which the E⁶...R¹⁰ ionic bridge stabilizes the C-terminus.     

DNA-binding and cleavage studies of novel binuclear copper(II) complex with 1,1'-dimethyl-2,2'-biimidazole ligand

DNA-binding properties of novel binulear copper(II) complex [Cu(2)(Dmbiim)(4)(H(2)O)(2)](ClO(4))(4).6H(2)O, where Dmbiim = 1,1'-Dimethyl-2,2'-biimidazole are investigated using electronic absorption spectroscopy, fluorescence spectroscopy, viscosity measurement and voltammetry. The results show that the copper(II) complex interacts with DNA through minor groove binding. The interaction between the complex and DNA has also been investigated by gel electrophoresis, interestingly, we found that the copper(II) complex can cleave circular plasmid pBR322 DNA efficiently in the presence of AH(2) (ascorbic acid) at pH 8.0 and 37 degrees C.     

Toxicity of Pyroglutaminated Amyloid β-Peptides 3(pE)-40 and -42 Is Similar to That of Aβ1-40 and -42

An N-terminal truncated isoform of the amyloid beta-peptide (A beta) that begins with a pyroglutamate (pE) residue at position 3 [A beta3(pE)-42] is the predominant isoform found in senile plaques. Based upon previous in vitro studies regarding A beta N-terminal truncated isoforms, it has been hypothesized that A beta3(pE)-x isoforms may aggregate more rapidly and become more toxic than corresponding Abeta1-x peptides. However, the toxicity and aggregation properties of A beta3(pE)-42 and A beta3(pE)-40 have not previously been examined. After initial solubilization and 1-week preaggregation of each peptide at 37 degrees C and pH 7.4, the toxicity of 5-50 microM A beta3(pE)-42 was similar to that of A beta1-42. Moreover, the toxicity of A beta3(pE)-40 paralleled that induced by A beta1-40 in both 1 day in vitro (DIV) cortical and 7 DIV hippocampal cells. Circular dichroism spectra did not reveal major differences in secondary structure between aged A beta1-42, A beta3(pE)-42, A beta3(pE)-40, and A beta1-40 or freshly solubilized forms of these peptides. Overall, the data indicate that the loss of the two N-terminal amino acids and the cyclization of glutamate at position 3 do not alter the extracellular toxicity of A beta.     

The effect of manganese(II) on the structure of DNA/HMGB1/H1 complexes: electronic and vibrational circular dichroism studies

The interactions were studied of DNA with the nonhistone chromatin protein HMGB1 and histone H1 in the presence of manganese(II) ions at different protein to DNA and manganese to DNA phosphate ratios by using absorption and optical activity spectroscopy in the electronic [ultraviolet (UV) and electronic circular dichroism ECD)] and vibrational [infrared (IR) and vibrational circular dichroism (VCD)] regions. In the presence of Mn2+, the protein-DNA interactions differ from those without the ions and cause prominent DNA compaction and formation of large intermolecular complexes. At the same time, the presence of HMGB1 and H1 also changed the mode of interaction of Mn2+ with DNA, which now takes place mostly in the major groove of DNA involving N7(G), whereas interactions between Mn2+ and DNA phosphate groups are weakened by histone molecules. Considerable interactions were also detected of Mn2+ ions with aspartic and glutamic amino acid residues of the proteins.     

Circular dichroism studies of copper(II)- hyaluronic acid complexes in relation to conformation of the polymer

The study of the Cu(II)-hyaluronate complexes by absorption and CD spectra, as well as by acid–base titration and viscosity, provides information about the nature of ligands and the conformation of the polymer. Three different complexes have been identified. The first (complex I), which is formed between pH 3 and 6, involves mainly the carboxyl groups of the polymer as ligands and is characterized by a strong absorption band at 238 nm. In this complex formation, the CD properties of hyaluronate do not charge appreciably. The second (complex II) forms between pH 6 and 8 bad shows a major change in CD properties. The changes include (1) a new positive CD band at 250 nm and a strong negative on in the π → π* amide transition region and (2) the disappearance of the negative n → π* amide CD band near 210 nm. A sharp increase in absorbance at 238 nm from complex I to II has been attributed to a conformational transition which is also manifested in the CD features of hyaluronate. Complex II involves, in addition to the carboxyl group, the nitrogen atom of the deprotonated acetamido group coordinated to Cu(II). The absorption at 230–280 nm is associated with the optically active charge-transfer transitions involving ligands to metal ion. At higher concentrations of the polymer or at higher pH, complex II aggregates to a gel, complex III. Chondroitin, differing from hyaluronic acid in the C-4 hydroxyl group configuration of the glucosamine moiety, does not show any CD change in the presence of Cu(II).The results provide further support to our fourfold helical structure of Cu(II)–hyaluronate complex at pH between 6 and 8. Intrinsic viscosities of hyaluronate in the presence of the cupric ion is lower than in the presence of other monovalent or bivalent cations, indicating a compact conformation of the polymer when it is complexed with Cu(II).     

Mapping the Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) using modeling techniques,site directed mutagenesis,circular dichroism and ligand binding assays

The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics.     

Studies on the dehydrative cyclization of epimeric 4-(L-xylo and L-lyxo-tetritol-1-yl)-2-phenyl-2H-1,2,3-triazoles. Circular dichroism and NMR assignment of the formed anomeric C-nucleoside L-threofuranosyl triazole analogs

Dehydrative cyclization of epimeric 4-(L-xylo- and L-lyxo-tetritol-1-yl)-2-phenyl-2H-1,2,3-triazoles afforded the anomeric alpha and beta-L-threofuranosyl analogs. The anomeric configuration of the formed anomeric C-nucleoside analogs was determined by circular dichroism and NMR spectroscopy.     

Studies on the interaction of copper complexes of (-)-epicatechin gallate and (-)-epigallocatechin gallate with calf thymus DNA

The interaction of copper complexes of (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) with calf thymus DNA (ct-DNA) was investigated by UV-visible (UV-Vis), fluorescence and circular dichroism along with melting studies. It was observed that both copper complexes quench the fluorescence intensity of ethidium bromide bound ct-DNA upon binding, resulting in a ground state complex formation by a static quenching process. The binding constants evaluated from fluorescence data were supported by the UV-Vis study. The values ranged from 0.84 to 1.07 × 10⁵ M⁻¹ and 1.14 to 1.04 × 10⁵ M⁻¹ for Cu(II)-ECG and Cu(II)-EGCG, respectively for the temperature range 21-42 °C with two binding sites. Thermodynamic parameters obtained are suggestive of the involvement of different modes of interaction during binding for each complex although both were found to be intercalating in nature. Circular dichroism studies and variations in the melting temperature reveal unwinding of the ct-DNA helix with conformational changes due to binding.