A thin quartz cell suitable for vacuum ultraviolet absorption and circular dichroism measurements

The design of a thin quartz cell suitable for absorption and circular dichroism measurements in the vacuum ultraviolet is described. Important features of the cell are (1) that it can be disassembled for cleaning and reproducibly reassembled with path lengths up to 0.3 mm, and (2) that strain in the windows from the compressed sample can be relieved by a sample overflow port. The latter feature allows the cell to be used for circular dichroism as well as absorption measurements.     

On the formation of the double helix between adenine single strands at acidic pH from synchrotron radiation circular dichroism experiments

Here, we present synchrotron radiation circular dichroism spectra for a series of DNA adenine strands, (dA)(n) , n = 2-10, 15, at acidic pH. Reference spectra of a protonated single strand, (dAH(+) )(n) , and a protonated double helix, (dAH(+) )(n) :(dAH(+) )(n) , are provided in the wavelength region from 175 to 330 nm. The largest spectral difference between single and double strands is in the vacuum ultraviolet, where a band changes sign. This new spectral feature that characterizes double helix formation may be useful for analytical purposes but also for shedding light on the underlying complexation mechanism. Furthermore, we find that a minimum of eight or nine bases in a strand is needed for two (dAH(+) )(n) strands to form a duplex. This is a relatively high number as compared with guanine quadruplex and cytosine i-motif formation, which is likely linked to the significant Coulomb repulsion between the all-protonated bases. Finally, spectra recorded as a function of time after sample preparation indicates that the equilibrium is slowly reached, in certain cases taking an hour or more.     

Comprehensive secondary‐structure analysis of disulfide variants of lysozyme by synchrotron‐radiation vacuum‐ultraviolet circular dichroism

To elucidate the effects of specific disulfide bridges (Cys6‐Cys127, Cys30‐Cys115, Cys64‐Cys80, and Cys76‐Cys94) on the secondary structure of hen lysozyme, the vacuum‐ultraviolet circular dichroism (VUVCD) spectra of 13 species of disulfide‐deficient variants in which Cys residues were replaced with Ala or Ser residues were measured down to 170 nm at pH 2.9 and 25°C using a synchrotron‐radiation VUVCD spectrophotometer. Each variant exhibited a VUVCD spectrum characteristic of a considerable amount of residual secondary structures depending on the positions and numbers of deleted disulfide bridges. The contents of α‐helices, β‐strands, turns, and unordered structures were estimated with the SELCON3 program using the VUVCD spectra and PDB data of 31 reference proteins. The numbers of α‐helix and β‐strand segments were also estimated from the VUVCD data. In general, the secondary structures were more effectively stabilized through entropic forces as the number of disulfide bridges increased and as they were formed over larger distances in the primary structure. The structures of three‐disulfide variants were similar to that of the wild type, but other variants exhibited diminished α‐helices with a border between the ordered and disordered structures around the two‐disulfide variants. The sequences of the secondary structures were predicted for all the variants by combining VUVCD data with a neural‐network method. These results revealed the characteristic role of each disulfide bridge in the formation of secondary structures. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Sugar-induced change in near ultraviolet circular dichroism of α-crystallin

In order to study the effect of sugar molecules on the structure of α-crystallin, the protein from bovine lenses was isolated and treated with D-glucose and D-glucose-6-phosphate. Structural studies of the products were carried out using circular dichroism (CD) measurements. In the near-ultraviolet (UV) region, three positive and two negative CD bands are observed for α-crystallin. Upon incubation with the saccharides, the positive ellipticities of these near-UV CD bands increase greatly. The CD spectrum of α-crystallin in the far-UV region, which is typical of the β-conformation of a protein, does not change. The increase does not appear to be caused by glycosylation, or by sugar metabolites such as sorbitol or glycolytic products. When the concentration of sugar is higher than that has been used in this study, the protein tends to aggregate. The results strongly suggest that sugar, upon interaction with α-crystallin, induces a change in the tertiary structure of the protein while its secondary structure remains intact. The interaction of sugar molecules causing such change is weak, noncovalent, and temperature-dependent. The near-UV CD of α-crystallin appears to be a useful probe for the study of such structural changes, which may be significant in the pathogenesis of cataract.     

Improved sequence-based prediction of protein secondary structures by combining vacuum-ultraviolet circular dichroism spectroscopy with neural network

Synchrotron-radiation vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy can significantly improve the predictive accuracy of the contents and segment numbers of protein secondary structures by extending the short-wavelength limit of the spectra. In the present study, we combined VUVCD spectra down to 160 nm with neural-network (NN) method to improve the sequence-based prediction of protein secondary structures. The secondary structures of 30 target proteins (test set) were assigned into alpha-helices, beta-strands, and others by the DSSP program based on their X-ray crystal structures. Combining the alpha-helix and beta-strand contents estimated from the VUVCD spectra of the target proteins improved the overall sequence-based predictive accuracy Q(3) for three secondary-structure components from 59.5 to 60.7%. Incorporating the position-specific scoring matrix in the NN method improved the predictive accuracy from 70.9 to 72.1% when combining the secondary-structure contents, to 72.5% when combining the numbers of segments, and finally to 74.9% when filtering the VUVCD data. Improvement in the sequence-based prediction of secondary structures was also apparent in two other indices of the overall performance: the correlation coefficient (C) and the segment overlap value (SOV). These results suggest that VUVCD data could enhance the predictive accuracy to over 80% when combined with the currently best sequence-prediction algorithms, greatly expanding the applicability of VUVCD spectroscopy to protein structural biology.     

Absorption, circular dichroism, magnetic circular dichroism and emission study of rat kidney Cd,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra of rat liver and rat kidney cadmium-, zinc- and copper-containing metallothioneins (MT) are reported. The absorption, CD and MCD data of native rat kidney Cd,Cu-MT protein closely resemble data recorded for the rat liver Cd,Zn-MT. This suggests that the major features in all three spectra of the native Cd,Cu-MT are dominated by cadmium-related bands. The CD spectrum of the Cd,Cu-MT recorded at pH 2.7 has the same band envelope that is observed for a Cd,Cu-MT formed in vitro by titration of Cd,Zn-MT with Cu(I), suggesting that the copper occupies the zinc sites in Cd,Cu-MT formed both in vivo and, at low molar ratios, in vitro. Remetallalion of the metallothionein from low pH in the presence of both copper and cadmium results in considerably less cadmium bound to the protein than was present in the native sample. It is suggested that this is due to the effect of the distribution of the copper amongst all available binding sites, thus inhibiting cluster formation by the cadmium. Emission spectra are reported for the first time for a cadmium- and copper-containing metallothionein. An emission band at 610 nm is shown to be a sensitive indicator of Cu(I) binding to metallothionein. Both the native Cd,Cu-MT and a Cd,Cu-MT formed in vitro exhibit an excitation spectrum with a band in the copper-thiolate charge-transfer region.     

蛋白质二级结构的真空紫外圆二色性研究

利用同步辐射真空紫外圆二色谱仪和特制的样品池,测定溶液中蛋白质的真空紫外圆二色谱,测定波长低至175nm,并应用一种新的计算法分析计算了蛋白质5种二级结构的含量,所得结果与用X射线衍射法测定的结果一致.讨论了获得好的真空紫外圆二色谱的几个重要因素.结果表明,真空紫外圆二色法是目前测定溶液中蛋白质二级结构的较好方法之一.     

Absolute configuration of (−)-myrtenal by vibrational circular dichroism

The VCD spectrum of the monoterpene (−)-myrtenal (1) was compared with theoretical spectra using ab initio density functional theory (DFT) calculations at the B3LYP/6-31G(d,p), B3LYP/6-31G+(d,p), B3LYP/6-311G+(d,p), B3LYP/DGDZVP, and B3PW91/DGTZVP levels of theory. Conformational analysis of 1 indicated that the lowest energy conformer was s-trans-C2-C10, which contributes more than 98.5% to the total conformational population regardless of the employed level of theory. The use of a recently developed confidence level algorithm demonstrated that VCD spectra calculated for the main conformer, using the indicated hybrid functionals and basis set, gave no significant changes, from where it follows that B3LYP/DGDZVP calculations provide a superior balance between computer cost and VCD spectral accuracy. The DGDZVP basis set demanded around a quarter the time than the 6-311G+(d,p) basis set while providing similar results. The spectral comparison also provided evidence that the levorotatory enantiomer of myrtenal has the 1R absolute configuration.     

Conformational study on poly [gamma-(alpha-phenethyl)-L-glutamate] using vibrational circular dichroism spectroscopy

Vibrational circular dichroism (VCD) and IR absorption spectra are obtained in a chloroform solution for poly[gamma-((R)-alpha-phenethyl)-L-glutamate] (PRPLG) and poly[gamma-((S)-alpha-phenethyl)-L-glutamate] (PSPLG), whose only structural difference is an opposite chiral center in the side chain. Their characteristic amide A, I, and II bands show VCD patterns quite similar to those of poly[gamma-benzyl-L-glutamate] (PBLG), indicating that the secondary structure of these polypeptides is a right-handed alpha-helix. The VCD spectra in the CH stretching region exhibit different patterns for PRPLG and PSPLG, reflecting the chirality difference in the side chains. This difference is interpreted on the basis of the additivity of optical activity contributions from the main chain conformation and the chirality difference in the side chains. The results indicate that a VCD difference spectrum of the CH stretching region is a useful diagnostic tool for elucidating local chirality differences.     

Vibrational and electronic circular dichroism monitoring of copper(II) coordination with a chiral ligand

Novel copper(II) coordination compounds with chiral macrocyclic imine ligands derived from R-/S-camphor were asymmetrically synthesized and characterized with the aid of chiroptical spectroscopies. Crystal structures of both enantiomers were determined by single crystal X-ray diffraction analysis. Circular dichroism (CD) spectra were analyzed using a simplified exciton model as well as quantum chemical computations. The absolute configuration of the copper(II) coordination compounds determined from CD was found consistent with the crystal data. The copper(II) complexes were further investigated by vibrational CD (VCD) measurement combined with density functional theory calculation. The complex formation was evidenced by spectral shifts of the characteristic bands in the CD and VCD spectra.     

Phase equilibrium in poly(rA).poly(rU) complexes with Cd2+ and Mg2+ ions, studied by ultraviolet, infrared, and vibrational circular dichroism spectroscopy

Ultraviolet (UV) and infrared (IR) absorption and vibrational circular dichroism (VCD) spectroscopy were used to study conformational transitions in the double-stranded poly(rA). poly(rU) and its components-single-stranded poly(rA) and poly(rU) in buffer solution (pH 6.5) with 0.1M Na+ and different Mg2+ and Cd2+ (10(-6) to 10(-2) M) concentrations. Transitions were induced by elevated temperature that changed from 10 up to 96 degrees C. IR absorption and VCD spectra in the base-stretching region were obtained for duplex, triplex, and single-stranded forms of poly(rA) . poly(rU) at [Mg2+],[Cd2+]/[P] = 0.3. For single-stranded polynucleotides, the kind of conformational transition (ordering --> disordering --> compaction, aggregation) is conditioned by the dominating type of Me2+-polymer complex that in turn depends on the ion concentration range. The phase diagram obtained for poly(rA) . poly(rU) has a triple point ([Cd2+] approximately 10(-4)M) at which the helix-coil (2 --> 1) transition is replaced with a disproportion transition 2AU --> A2U + poly(rA) (2 --> 3) and the subsequent destruction of the triple helix (3 --> 1). The 2 --> 1 transitions occur in the narrow temperature interval of 2 degrees -5 degrees . Unlike 2 --> 1 and 3 --> 1 melting, the disproportion 2 --> 3 transition is a slightly cooperative one and observed over a wide temperature range. At [Me2+] approximately 10(-3) M, the temperature interval of A2U stability is not less than 20 degrees C. In the case of Cd2+, it increases with the rise of ion concentration due to the decrease of T(m) (2-->3). The T(m) (3-->1) value is practically unchanged up to [Cd2+] approximately 10(-3)M. Differences between diagrams for Mg(2+) and Cd2+ result from the various kinds of ion binding to poly(rA).poly-(rU) and poly(rA).     

Heparin-induced circular dichroism of chloroquine

Circular dichroism (CD) and UV/Visible absorption (UV/Vis) spectroscopy techniques were used to investigate the interaction between heparin and chloroquine, an antimalarial drug that has shown potential as an anti-prion agent. CD spectra of rac-chloroquine upon addition of heparin provide evidence of glycosaminoglycan (GAG) binding, support recent findings suggesting that interactions between heparin and antimalarial drugs are largely due to electrostatic interactions, and represent the first reported GAG-induced CD signal of a bicyclic, aromatic compound. The association constant (∼10³ M⁻¹) between chloroquine and heparin was calculated from a UV titration curve and provided additional insight into the nature of the association between these two compounds.     

Vacuum-ultraviolet circular dichroism study of saccharides by synchrotron radiation spectrophotometry

Vacuum-ultraviolet circular dichroism (VUVCD) spectra of five monosaccharides (D-glucose, D-mannose, D-galactose, D-xylose, and D-lyxose) and five disaccharides (maltose, isomaltose, cellobiose, gentiobiose, and lactose) were measured to 160 nm using a synchrotron-radiation VUVCD spectrophotometer in aqueous solution under high vacuum at 25 degrees C. Most of the saccharides show a positive peak with some shoulders at around 170 nm, except for D-galactose and lactose, which show two distinct negative peaks at around 165 and 177 nm. These spectra are influenced by such structural factors as alpha and beta anomers at C-1, axial and equatorial hydroxyl groups at C-2 and C-4, trans (T) and gauche (G) conformations of the hydroxymethyl group at C-5, and the type of glycosidic linkage. Deconvolution of the VUVCD spectra of D-glucose, D-mannose, and D-galactose into six independent Gaussian components for alpha-GG, alpha-GT, alpha-TG, beta-GG, beta-GT, and beta-TG conformations suggests that the alpha anomer has red-shifted spectra relative to the beta anomer, and that GG and GT conformations have positive and negative circular dichroism signs, respectively, while the sign for TG conformation is anomer dependent. These speculations from the deconvolution analyses are also supported by the VUVCD spectra of disaccharides. These results give new insight into the equilibrium conformations of saccharides, demonstrating the usefulness of synchrotron-radiation VUVCD spectroscopy.     

Effect of conformation and configuration of arabinose residues on the circular dichroism of C-glycosylflavones

Examination of a variety of arabinose containing C-glycosylflavones has shown that the sign and intensity of the CD band at 250–275 nm (charge-transfer band) reflect not only the point of attachment of the sugar to the flavone but also depend upon the absolute and anomeric configuration, ring-size and ring-conformation in addition to the preferred rotameric conformation of the sugar about the C-aryl, C-l″ bond. A change in stereochemistry of arabinose from the α to β anomer resulted in sign inversion of the 250–275 nm CD band for 6-C-l-arabinosylflavones. Furthermore, a 6-C-arabinosylflavone containing α-l-arabinose exhibited an oppositely signed charge-transfer CD band in comparison to one which contained α-d-arabinose. 6,8-Di-C-glycosylflavones containing arabinose and glucose exhibited CD bands resulting from contributions due to both sugars, if the arabinose was not present as the β-pyranose form (¹C₄, conformation).     

Determination of the absolute configuration and solution conformation of a novel disubstituted pyrrolidine acid A by vibrational circular dichroism

Compound A, a novel disubstituted pyrrolidine acid, is a member of a new class of agents that are potentially useful for the treatment of diabetes and dyslipidemia. The absolute configuration of this compound was determined by using vibrational circular dichroism (VCD). The results are in agreement with the assignments based on both X-ray analysis and the stereo-selective chemical synthesis. During VCD analysis, the solution conformation for a portion of compound A in CDCl(3) was also established. The compound is found to associate as an H-bonded carboxylic acid "dimer" in CDCl(3) solution, and VCD calculations on a model dimer fragment were required to establish the absolute configuration.     

Time-resolved circular dichroism in carbonmonoxy-myoglobin: the central role of the proximal histidine

A calculation of the circular dichroism (CD) spectra of carbonmonoxy- and deoxy-myoglobin is carried out in relation to a time-resolved CD experiment. This calculation allows us to assign a dominant role to the proximal histidine in the definition of the electronic normal modes and to interpret the transient CD structure observed in a strain of the proximal histidine. This strain builds up in 10 ps and relaxes in 50 ps as the protein evolves towards its deoxy form.     

Surface characterization of human serum albumin and sodium perfluorooctanoate mixed solutions by pendant drop tensiometry and circular dichroism

The interfacial behavior of mixed human serum albumin (HSA)/sodium perfluorooctanoate (C8FONa) solutions is examined by using two experimental techniques, pendant drop tensiometry and circular dichroism spectroscopy. Through the analysis of the surface tension of the mixed solutions, surface competitive adsorption at the air-water interface between C8FONa and HSA is detected. The dynamic adsorption curves exhibit the distinct regimes in their time-dependent surface tension. The nature of these regimes is further analyzed in terms of the variation of the molecules surface areas. As a consequence, a compact and dense structure was formed where protein molecules were interconnected and overlapped. Thus, a reduction of the area occupied per molecule from 100 to 0.2 nm(2) is interpreted as a gel-like structure at the surface. The presence of the surfactant seems to favor the formation of this interfacial structure. Finally, measurements of circular dichroism suggests a compaction of the protein due to the association with the surfactant given by an increase of alpha-helix structure in the complexes as compared to that of pure protein.     

Vibrational circular dichroism of gramicidin D in vesicles and micelles

The vibrational circular dichroism (VCD) and absorption spectra of gramicidin D in three model membranes (dioctadecyldimethylammonium chloride vesicles, dimyristoyl-phosphatidylcholine vesicles, and sodium dodecyl sulfate micelles) are presented. The absorption and VCD spectra suggest that the stable gramicidin D conformation in the model membranes is different from those in organic solvents. The presence of cations does not change the membrane-bound conformation of gramicidin D.     

Synchrotron radiation circular dichroism of various G‐quadruplex structures

Here we report synchrotron radiation circular dichroism spectra of various G‐quadruplexes from 179 to 350 nm, and a number of bands in the vacuum ultraviolet (VUV) are reported for the first time. For a tetramolecular parallel structure, the strongest band in the spectrum is a negative band in the VUV at 182 nm; for a bimolecular antiparallel structure with diagonal loops, a new strong positive band is found at 190 nm; for a bimolecular parallel structure with edgewise loops, a strong positive band at 189 nm is observed; and for a self‐folded chair‐type structure, the strongest band in the spectrum is a positive band at 187 nm. For the tetramolecular parallel structure, the CD signals at all wavelengths are dominated by contributions from quartets of G bases, and the signal strength is approximately proportional to the number of quartets. Our experiments on well‐characterized G‐quadruplex structures lead us to question past attributions of CD signals to helix handedness and G quartet polarity. Although differences can be observed in the VUV region for the various quadruplex types, there do not appear to be clear‐cut spectral features that can be used to identify specific topological features. It is suggested that this is because a dominant positive band in the VUV seen near 190 nm in all quadruplex structures is due to intrastrand guanine–guanine base stacking. However, our spectra can serve as reference spectra for the G‐quadruplex structures investigated and, not least, to benchmark theoretical calculations and empirical models. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 429–433, 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     

Ultraviolet-circular dichroism spectroscopy and potentiometric study of the interaction between human serum albumin and sodium perfluorooctanoate

The interaction of a fluorinated surfactant, sodium perfluorooctanoate, with human serum albumin (HSA) has been investigated by a combination of ultraviolet-circular dichroism (UV-CD) spectroscopy and potentiometry (by a home-built ion-selective electrode) techniques to detect and characterize the conformational transitions of HSA. By using difference spectroscopy, the transition was followed as a function of temperature, and the data were analyzed to obtain the parameters characterizing the thermodynamics of unfolding. The results indicate that the presence of surfactant drastically changes the melting unfolding, acting as a structure stabilizer and delaying the unfolding process. Potentiometric measurements were used to determine the binding isotherms and binding capacity for this system. The isotherm shows a high affinity of surfactant molecules for HSA. The average number of surfactant molecules absorbed per protein molecule (at 28 mM of surfactant concentration) was found to be approximately 900, about 6 g of surfactant per gram of protein. The shape of the binding capacity curve and the relation between binding capacity and extend of cooperativity were examined. From these analysis, the values of g (number of ligand-binding sites), KH (Hill binding constant), and nH (Hill coefficient) were determined.