Synthesis and characterization of modified nucleotides in the 970 hairpin loop of Escherichia coli 16S ribosomal RNA

The synthesis of the 6-O-DPC-2-N-methylguanosine (m²G) nucleoside and the corresponding 5′-O-DMT-2′-O-TOM-protected 6-O-DPC-2-N-methylguanosine phosphoramidite is reported [DPC, diphenyl carbamoyl; DMT, 4,4′-dimethoxytrityl; TOM, [(triisopropylsilyl)oxy]methyl]. The availability of the phosphoramidite allows for syntheses of hairpin RNAs with site-selective incorporation of 2-N-methylguanosine modification. Four 18-nt hairpin RNA analogues representing the 970-loop region (helix 31 or h31; U960–A975) of Escherichia coli 16S rRNA were synthesized with and without modifications in the loop region. Subsequently, stabilities and conformations of the singly and doubly modified RNAs were examined and compared with the corresponding unmodified RNA. Thermodynamic parameters and circular dichroism spectra are presented for the four helix 31 RNA analogues. Surprisingly, methylations in the loop region of helix 31 slightly destabilize the hairpin, which may have subtle effects on ribosome function. The hairpin construct is suitable for future ligand-binding experiments.     

Steric analysis of glycerophospholipids by circular dichroism. Stereospecifity of phospholipase D catalyzed transesterification.

The circular dichroism spectra of natural glycerophospholipids and synthetic 1-$\text{sn}$-phosphatidic acid were recorded. 3-$\text{sn}$-phosphatidic acid derivatives were found to show a positive Cotton effect, while 1-$\text{sn}$-phosphatidic acid revealed a negative Cotton effect. The results are interpreted in terms of the carboxyl sector rule. By this method phospholipase D was shown to produce stereospecifically 3-$\text{sn}$-phosphatidyl-1-$\text{sn}$-glycerol when incubated with egg yolk lecithin and exess of glycerol.     

Inhibitory effect of 8-substituted adenosine derivatives on Ca++ and modulator protein-dependent phosphodiesterase activity.

8-Substituted adenosine and cyclic AMP derivatives exhibited some negative Cotton effects in circular Dichroism at B_2u band in pH 7.5 solution, suggesting that these derivatives take syn conformation. The adenosine derivatives, as well as cyclic AMP derivatives, competitively inhibited the cyclic AMP hydrolyzing activity in Ca⁺⁺ and modulator protein-dependent phosphodiesterase preparation from hog brain cortex. The inhibitory potential of an adenosine derivative was lower than that of the cyclic AMP derivative having the same substituent by the lack of the phosphate moiety for which affinity was 0.5 kcal / mol. These results may suggest that the cyclic AMP hydrolyzing site on the enzyme requires the syn conformation of purine riboside.     

Conformational study of calf brain tubulin

The conformation of calf brain tubulin has been monitored by circular dichroism, optical rotatory dispersion, and spectrophotometric titration as a function of pH, temperature, ligand concentrations, and denaturants. At pH 7, calf brain tubulin maintains its structural integrity between 5 and 37 °C as determined by circular dichroism. Furthermore, the presence of MgCl ₂ up to 1.6 × 10^?2m does not induce any observable changes in the circular dichroism spectra, nor does 10^?4m CaCl₂. With increasing pH, the spectral data can best be described as a gradual loosening of the secondary structure between pH 7 and 9. Both spectral and titrimetric data suggest a major unfolding of tubulin between pH 9 and 10. The apparent pK of tyrosine shifts from 10.85 to 9.98 upon transferring from buffer to 6 m guanidine hydrochloride, indicating that at least 14 of the 15 tyrosine groups are not fully accessible to protons in the native protein. The single disulfide bridge in calf brain tubulin helps to maintain a domain which is highly resistant to unfolding by denaturants.     

Langaside,a novel secoiridolactone glycoside derivative from Tachiadenus longiflorus Griseb. (Gentianaceae) formed by a [2+2] cycloaddition reaction

Langaside (1), a secoiridoid lactone glucoside possessing a novel skeleton formed by a [2 + 2] cycloaddition reaction between the secoiridolactone glucoside, 1,9-trans-9,5-cis-sweroside, and p-coumaric acid was isolated from the fruits and flowers of the Malagasy Tachiadenus longiflorus Griseb. (Gentianaceae), alongside another seven known compounds. The structure of langaside was established using HRESIMS, IR and NMR spectroscopy and comparison of experimental and calculated electronic circular dichroism (ECD) spectra. Langaside was screened for its neuritogenic activity against SHSY-5Y cells and anticancer activity against the NCI59 human tumour cell panel but not found to be active.     

Circular dichroism of metallothioneins. A structural approach.

A comprehensive study on circular dichroism of metallothioneins containing Zn, Cd and Cu was carried out. The contributions of the metals, the sulphur and the polypeptide chain to the observed Cotton effects was shown. From the pH dependency of the extrinsic Cotton effects which are due to the metal-thiolate chromophore the stability of the metal clusters was found to decrease in the order Cu greater than Cd greater than Zn. The pH values corresponding to the dissociation of half of the bound metal ions are 0.44 for Cu-thionein, 3.05 for Cd-thionein and 4.6 for Zn-thionein. The extrinsic Cotton effects of Cd, Zn-thioneins of varying Cd to Zn ratio could be simulated using the difference circular dichroic spectra of Cd-thionein (bands at 227, 242.5 and 262 nm), Zn-thionein (bands at 225 and 244 nm) and the circular dichroic spectrum of cysteine-thionein (band at 200 nm, shoulder at 225 nm). Since during the dissociation of the metals the circular dichroic spectra exhibited changes only in amplitude and not in shape we can conclude that the dissociation of the metal ions involves the complete sequential degradation of metal clusters. In the near-ultraviolet region the metal-free proteins show only Cotton effects attributable to a disulphide chromophore. Thus Cotton bands are observed for cystine-thionein at 282.5 and 260 nm. From the intrinsic circular dichroism of Cd- and Zn-thionein (negative Cotton effect at 200 nm, shoulder at 225 nm) it follows that the protein conformation consists of less than 5% helical or pleated sheet structure and therefore has to be classified as unordered structure or "fixed" random coil     

Absolute Configurations of Four Resorcylic Acid Lactones,Paecilomycins J − M,by CD/TDDFT Calculations

The absolute configurations of four resorcylic acid lactones (RALs), paecilomycins J ? M ( 1 ? 3 and 5 ), were assigned by Time‐Dependent Density‐Functional Theory (TDDFT) calculations of their electronic circular dichroism (CD) spectra. The previously reported structure 4 for paecilomycin M was found to be incorrect and should be changed to structure 5 . Analysis of structure‐spectrum relationship for this group of RALs suggested that V′‐shape conformations give type I CD spectra (two negative Cotton effects around 300 and 260 nm, a positive Cotton effect around 220 nm) while V‐shape conformations yield type II spectra (signs of three Cotton effects were opposite to those in type I). Chirality 26:44–50, 2013. © 2013 Wiley Periodicals, Inc.     

Importance of DNA conformation in the reaction with cis-dichlorodiammine platinum (II)

Cis-dichlorodiammine platinum (II) has been reacted with synthetic polynucleotides either in B or in Z conformation. The binding of cis-dichlorodiammine platinum (II) stabilizes the Z conformation when reacted with poly (dG-m⁵dC) ·poly (dG-m⁵dC) in the Z conformation as shown by circular dichroism and by the antibodies to Z-DNA. On the other hand, the binding of cis-dichlorodiammine platinum (II) stabilizes a new conformation when reacted with poly(dG-dC)·poly(dG-dC) or poly (dG-m⁵dC)·poly(dG-m5dC) in the B conformation. The antibodies to Z-DNA bind to these platinated polynucleotides. In rabbits, the injection of platinated poly (dG-dC) poly (dG-dC) induces the synthesis of antibodies which recognize Z-DNA. In low salt conditions, the circular dichroism spectra of these platinated polynucleotides differ from those of B-DNA or Z-DNA. The characteristic³¹P nuclear magnetic resonance spectrum of Z-DNA is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

Rotatory dispersion and circular dichroism of low-molecular-weight poly- -benzyl-L-glutamate

Rotatory dispersion and circular dichroism of low-molecular-weight poly-γ-benzyl-L -glutamate, which was prepared from the N-carboxyanhydride by n-hexylamine initiation at [A]/[I] 3, 4, and 8, have been measured in ethylene dichloride and dioxane at different concentrations. The rotatory properties of the polypeptides are all characterized by a trough at 233 mμ of a negative Cotton effect or by a negative circular dichroic band at 223 mμ. With increasing A/I value or concentration, dextrorotation increases and the negative dichroic band becomes deeper. Both the trough magnitude and the negative ellipticity are linearly dependent on the content of β-structure, and the rotatory parameters for the pure β-structure are estimated by extrapolation of the linear relations. Circular dichroism and infrared spectra of the polypeptides have also been measured in trifluoroethanol, and the effect of solvents on the polypeptide conformation is discussed.     

Conformational structure of bombesin as studied by vibrational and circular dichroism spectroscopy

Raman and Fourier transform infrared (FTIR) spectroscopies and circular dichroism (CD) have been applied to investigate the secondary structure of bombesin in the solid state and in phosphate buffer solution (pH 3.8). At concentrations around 10⁻⁵ M, circular dichroism reveals that bombesin exists as an irregular or disordered conformation. However, the secondary structure of the peptide appears to be a mixture of disordered structure and intermolecular β-sheets in 0.01 M sodium phosphate buffer when the peptide concentrations are higher than around 6.5 mM. The tendency of bombesin to form aggregated β-sheet species seems to be originated mainly in the sequence of the residues 7–14, as supported by the Raman spectra and β-sheet propensities (P_β) of the amino-acid residues. It is the hydrophobic force of this amino-acid sequence, and not a salt bridge effect, that is the factor responsible for the formation of peptide aggregates.     

Proline-glutamate chimera’s side chain conformation directs the type of β-hairpin structure

Our aim was to study the impact of two proline chimeras, containing a glutamic acid side chain in cis- or trans-configuration, on secondary structure formation. We further investigated to what extent the configuration of the side chain contributes to the overall peptide conformation. We used a 10 residue peptide (IYSNPDGTWT) that forms a β-hairpin in water. The turn-forming proline was substituted with either a cis- or trans-proline-glutamic acid chimera, resulting in the peptides IYSNP ^{cis -E} DGTWT (P1_P ^cis-E ) and IYSNP ^{trans -E} DGTWT (P1_P ^trans-E ). We studied the conformation of the modified peptides by circular dichroism (CD) and NMR-spectroscopy, and SEC/static light scattering (SLS) analysis. NMR analysis reveals that the modified peptides maintain the β-hairpin conformation in aqueous solution. At 5 °C and pH 4.3, the peptide (P1_P ^cis-E ) was found to adopt two coexisting β-hairpin conformations (2:2 β-hairpin, and 3:5 β-hairpin). In contrast to that, the peptide (P1_P ^trans-E ) adopts a 2:2 β-hairpin that exists in equilibrium with a 4:4 β-hairpin conformation. The adoption of ordered β-hairpin structures for both modified peptides could be confirmed by CD spectroscopy, while SEC/SLS analysis showed a monomeric oligomerization state for all three investigated peptides. With the combination of several NMR methods, we were able to elucidate that even small alterations in the side chain conformation of the proline-glutamate chimera (cis or trans) can significantly influence the conformation of the adopted β-hairpin.     

Synthesis of Morin–Zinc(II) Complex and its Interaction with Serum Albumin

A morin–zinc(II) complex (MZ) was synthesized and its interaction with bovine serum albumin (BSA) were studied by molecular spectroscopy including fluorescence emission spectra, UV-visible spectra, circular dichroism (CD) spectra, three-dimensional fluorescence spectra, and synchronous fluorescence spectra. The interaction mechanism of BSA and MZ was discussed by fluorescence quenching method and Förster non-radiation energy transfer theory. The thermodynamic parameters ΔH ^θ, ΔG ^θ, ΔS ^θ at different temperatures were calculated and the results indicate the interaction is an exothermic as well as entropy-driven process. Hydrogen bond forces played the most important role in the reaction. The fluorescence probe experiment showed that the binding site of MZ is in subdomain IIA of BSA and the distance between BSA and MZ is 3.17 nm at normal body temperature. The conformation changes of BSA in presence of MZ were investigated by CD spectra and three-dimensional fluorescence spectra.     

Influence of divalent cations on the conformation of phosphorothioate oligodeoxynucleotides: a circular dichroism study

Phosphorothioate oligodeoxynucleotides (ODNs) have been extensively investigated in vivo and in vitro for antisense control of gene expression. It has been shown that cellular uptake of phosphorothioate ODNs in some in vitro cell systems increases in the presence of divalent cations. In this work, we analyze the conformation of phosphorothioate ODNs and specific changes induced in it by various divalent cations using circular dichroism (CD) spectroscopy. CD data were obtained with several phosphorothioate ODNs in the absence and presence of the divalent cations Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺ and Mn²⁺. All CD spectra indicated stable conformations of the ODNs in solution. The spectra were strongly dependent on ODN sequence and composition. Some ODNs such as T₂₃ and another with ‘random’ distribution of bases showed CD spectra characteristic of B-form DNA. Other ODNs which had at least three consecutive guanines in their sequences exhibited spectra characteristic of parallel G-tetraplexes. CD spectra of antisense ODNs exhibited specific responses to divalent cations. Changes in the conformation were not simply due to ionic strength effects. Mn²⁺ diminished secondary structure in some ODNs. Group II divalent ions stabilized the parallel G-tetraplexes, and Mg²⁺ generally had the weakest stabilizing efficiency. Each sequence/ion combination had a specific response so these effects cannot be generalized. These sequence-dependent, divalent ion-sensitive, and structurally unique solution conformations may be related to ion-mediated ODN uptake.     

Synthesis of 6-N-(benzothiazol-2-yl)deoxyadenosine and its exciton-coupled circular dichroism

6-N-(Benzothiazol-2-yl)deoxyadenosine (A^BT) was synthesized and incorporated into DNAs. Although, the multipoint benzothiazole (BT) modification of oligodeoxynucleotides reduced the stability of duplexes with their complementary strands, it induced the strong exciton coupling between BT moieties. The circular dichroism (CD) spectra of this exciton coupling interaction were observed at wavelengths above 300 nm and overlapping with the UV absorption bands of nucleotides could be avoided. The shapes of the CD spectra due to this interaction were strongly influenced by the helicity of two BT groups.     

Influence of hemin on the conformation of cyanogen bromide-cleaved peptides of apomyoglobin

The complexes of the three BrCN-cleaved fragments of sperm whale apomyoglobin with hemin were studied by circular dichroism (CD). In native myoglobin, the heme is located in the middle fragment; the isolated peptide (residues 56–131), however, produces little extrinsic Cotton effects by the addition of hemin, although about four molecules of hemin are bound to this peptide. In marked contrast, the COOH-terminal peptide (residues 132–153), which binds three hemin molecules, shows strong Cotton effects in the Soret bands and drastically changes its conformation from unordered to highly helical. The Arg-modified or Lys-deaminated peptide no longer undergoes conformational changes by the addition of hemin, suggesting that the two propionic acid groups of one hemin molecule interact with the Arg residue and one of the Lys residues, which stabilizes the induced helical conformation. The NH₂-terminal peptide (residues 1–55) binds one hemin molecules, and the helicity of this fragment is slightly enhanced by the addition of hemin. Both the CD and difference absorption spectra indicate that the mode of interaction between the peptides and hemin are different for the three apomyoglobin fragments.     

Circular dichroism studies on α- and β-ketosides of 5-acetamido-3,5-dideoxy- -glycero- -galacto-nonulopyranosonic acid (N-acetylneuraminic acid) and of some of its derivatives

The circular dichroism spectra of a number of N-acetylneuraminic acid derivatives in aqueous solution were studied. For all compounds, the Cotton effects were found to be in the spectral range of the acetamido and carboxyl chromophores. The c.d. curves of the methyl, ethyl, and allyl α- -ketosides are characterized by a broad, positive band centered at λ ≈ 195 nm with a slight skew towards the higher wavelengths and weak bands between λ 225 and 255 nm, whereas the methyl β- -ketoside and the corresponding methyl ester show only an intense positive band with a broad shoulder in the same spectral range. 5-Acetamido-3,5-dideoxy- -glycero-β- -galacto-nonulopyranose, its methyl β- -ketoside, and 5-acetamido-3,5-dideoxy- -glycero- -galacto-nonulopyranosonamide containing only the acetamido chromophore showed one single positive Cotton effect centered at λ ≈ 192 nm. The c.d. spectrum of 5-acetamido-3,5-dideoxy- -glycero- -galacto-nonulopyranosonic acid confirms the β- configuration of the free acid in aqueous solution, whereas the shape of the c.d. curve of O-(N-acetyl-α- -neuraminopyranosyl)-(2→3)-O-β- -galactopyranosyl-(1→4)- -glucopyranose resembles that of the methyl, ethyl, and allyl α- -ketosides 2-4.     

Partial analysis of the bands seen in circular dichroism spectra of green and blue-green algal cells and thylakoids

A comparison was made of the circular dichroism (C.D.) spectra of Chlorella, Euglena, and Anacystis cells and thylakoids. Analyses of the spectra reveal that these C.D. bands are similar to those observed previously in whole spinach choloroplasts and subchloroplast particles. C.D. spectra of Euglena chloroplasts show bands at longer wavelengths than previously reported. From comparisons of circular dichroism spectra and fine structure, it was concluded that: (a) bands seen in circular dichroism spectra were not the result of light scattering from thylakoid membranes; and (b) bands seen in the C.D. spectra of nonmembranous systems (previously reported) could account for circular dichroism of algae. We also concluded that comparisons would have to be made with model systems in order to correct for effects of absorption flattening, concentration obscuring, and differential light scattering of membranous systems.     

Purification, characterization and reassembly of the bacteriophage T4D tail sheath protein P18.

P18, the sole component of T4 tail sheath, has been isolated in a monomeric active form from extended sheaths of intact tails which were dissociated at low ionic strength. The molecular weight of P18 is determined to be 65,000 from sedimentation equilibrium and 73,000 from sodium dodecyl sulphate/gel electrophoresis. Combining the diffusion constant (D_20,w = 5·5× 10^?7cm²s^?1)and the sedimentation constant (s⁰_20,w = 4·2 S) a value of 67,000 is obtained. The circular dichroism spectra reveal a striking similarity of the structure of P18 in the monomeric state and in the extended sheath conformation.The purified P18 is found to reassemble into extended sheaths if the core-baseplate complex is present, forming normal length tails. Structures similar to polysheath are formed in the absence of core-baseplates.     

Structure and absolute configuration of a visamminol derivative using IR and vibrational circular dichroism

The aerial parts of Arracacia tolucensis (Kunth) Hemsl. (Apiaceae) provided the new visamminol derivative (S)-(+)-4′-O-angeloylvisamminol (1), along with the known angular pyranocoumarin 2. Analysis of HMBC NMR correlations did not allow distinction of the linear dihydrofurochromone 1 from pyranochromone 5. The structure and S absolute configuration of (+)-1 was therefore established by comparison of the IR and vibrational circular dichroism spectra of the natural product with the DFT B3LYP/DGDZVP calculated spectra for (S)-1 and (S)-5. Structural verification followed by single crystal X-ray diffraction of (+)-1 and chemical correlation.     

Linear and circular dichroism of membranes from Rhodopseudomonas capsulata

Absorption, linear dichroism and circular dichroism spectra of Rhodopseudomonas capsulata (wild-type-St. Louis strain, mutant Y5 and mutant Ala⁺) are particularly sensitive to the nature of the light-harvesting bacteriochlorophyll-carotenoid-protein complexes. Evidence for exciton-type interactions is seen near 855 nm in the membranes from the wild-type and from mutant Y5, as well as in an isolated B-800 + 850 light-harvesting complex from mutant Y5. The strong circular dichroism that reflects these interactions is attenuated more than 10-fold in membranes from the Ala⁺ mutant, which lacks both B-800 + 850 and colored carotenoids and contains only the B-875 light-harvesting complex. These results lead to the conclusion that these two light-harvesting complexes have significantly different chromophore arrangements or local environments.