Chromatin structure studied by linear dichroism at different salt concentrations

We have studied the linear dichroism (LD) of rat liver chromatin oriented by flow. Soluble chromatin, prepared by brief nuclease digestion, is found to exhibit a positive LD at low ionic strength (1 mM NaCl), with a constant LD/A over the absorption band centered at 260 nm (A, isotropic absorbance). Several previous dichroism studies on soluble chromatin have been performed on sonicated materials and have given negative LD, probably due to the presence of uncoiled DNA. The positive dichroism can be interpreted in terms of a supercoil of DNA in chromatin with a pitch angle larger than 55°, and is, for example, consistent with a model where the cylindrical nucleosome core particles are stacked face to face in the chromatin filament. In contrast to the nuclease-digested chromatin, sonicated chromatin was confirmed to exhibit negative LD. This difference can be attributed to a partial uncoiling of the linker regions between the nucleosomes due to the shearing. The structural transition of chromatin to a compact form can be observed as a reduction of the positive LD of the nuclease-digested chromatin to almost zero in 0.1 M NaCl or in 0.1 mM MgCl₂. This transition is due to a decreased electrostatic repulsion between negative phosphate groups on the DNA chain. In the case of Na⁺, this can be explained as a screening effect due to the bulk concentration of Na⁺. With Mg²⁺ a considerably stronger effect may indicate a more localized binding to the phosphates. At ionic strengths higher than 0.5M NaCl, the dissociation of the histones from DNA leads to uncoiling of chromatin. The change in LD during this process shows that histone H1 contributes only to a small degree to the coiling of the DNA chain, whereas histones H3 and H4 play the major role in the coiling.     

Entropic Enhancement of Protein-DNA Affinity by Oxygen-to-Sulfur Substitution in DNA Phosphate

Dithioation of DNA phosphate is known to enhance binding affinities, at least for some proteins. We mechanistically characterized this phenomenon for the Antennapedia homeodomain-DNA complex by integrated use of fluorescence, isothermal titration calorimetry, NMR spectroscopy, and x-ray crystallography. By fluorescence and isothermal titration calorimetry, we found that this affinity enhancement is entropy driven. By NMR, we investigated the ionic hydrogen bonds and internal motions of lysine side-chain NH₃⁺ groups involved in ion pairs with DNA. By x-ray crystallography, we compared the structures of the complexes with and without dithioation of the phosphate. Our NMR and x-ray data show that the lysine side chain in contact with the DNA phosphate becomes more dynamic upon dithioation. Our thermodynamic, structural, and dynamic investigations collectively suggest that the affinity enhancement by the oxygen-to-sulfur substitution in DNA phosphate is largely due to an entropic gain arising from mobilization of the intermolecular ion pair at the protein-DNA interface.     

Linear dichroism studies of binding site structures in solution: Complexes between DNA and basic arylmethane dyes

The interaction between B-form DNA and twelve cationic triaryl-methane dyes was studied with respect lo optical properties and stabilities, using linear dichroism (LD) and aqueous two-phase partition techniques. Monovalent dyes derived from crystal violet as a rule form a single strong complex (K₁ ca 10⁵ M^?1; site density per nucleotide base n₁ ca 0.1 at 0.1M ionic strength) in which the plane of the dye is at an angle of less than 50° to the local DNA helix axis. The complex with fuchsin is weaker (10⁴M^?1) but can be explained by a similar orientation. For some of the dyes (those with pseudo-C_2v symmetry) XXXre angular orientations of two molecule-fixed axes can be obtained. For the divalent methyl green a second complex appears to be formed at low ionic strength. Methyl green (and to some extent 2-thiophene green and malachite green) show exciton splitting in the LD spectrum and circular dichroism assignable to exciton coupling between transition dipoles roughly parallel to the helical strands, indicating a dye-dye interaction. Tne optical data, supported by fitting experiments with space-filling models, suggests a general structure for the binding site. The dye is not intercalated but is bound to exposed hydrophobic regions in the major groove. The ligand is in part (the charged amino groups) in contact with the phosphoribose chain but its main surface lies against the hydrophobic base-pair stack. For a diphenylmethane dye, Michler's hydrol blue, a perpendicular orientation was observed, possibly due to intercaiation.     

Chromatin model calculations: Arrays of spherical nu bodies.

Chromatin fibers consists of globular nucleohistone particles (designated nu bodies) along the length of the chromatin DNA with approximately 6-to7-fold compaction of the DNA within the nu bodies. We have calculated theoretical small-angle x-ray scattering curves and have compared these with experimental data in the literature. Several models predict maxima at the correct angles. The first maximum (approximately 110 degrees A) results from interparticle interference, while both the spatial arrangement and the structure factor the nu bodies can contribute to the additional small-angle maxima. These calculations suggest models which can account for the electron microscopic observation that chromatin is seen as either approximately 100-or approximately 200-to 250 degrees A-diameter fibers, depending on the solvent conditions. They also account for the limited orientability of the x-ray pattern from pulled chromatin fibers.     

Real-time detection of Fe·EDTA/H2O2-induced DNA cleavage by linear dichroism

The conditions for the measurement of linear dichroism (LD) can be adjusted so as to solely reflect the length and the flexibility of DNA. The real-time detection of the EDTA·Fe²⁺-induced oxidative cleavage of double-stranded native and synthetic DNAs was performed using LD. The decrease in the magnitude of the LD at 260 nm, which reflects an increase in the flexibility and a decrease in the length of the DNA, can be described by the sum of two or three exponential curves in relation to the EDTA·Fe²⁺ concentration. The fast component was assigned to the cleavage of one of the double strands, inducing an increase in the flexibility, while the other slower component was assigned to the cleavage of the double strand, resulting in the shortening of DNA. The decrease in the magnitude of the LD of poly[d(A-T)₂] was similar to that of poly[d(I-C)₂], while that of poly[d(G-C)₂] was found to be the slowest, indicating that the resistance of poly[d(G-C)₂] against the Fenton-type reagent was the strongest. This observation suggests that the amine group in the minor groove of the double helix may play an important role in slowing the EDTA·Fe²⁺-induced oxidative cleavage.     

Vibrational CD (VCD) and atomic force microscopy (AFM) study of DNA interaction with Cr3+ ions: VCD and AFM evidence of DNA condensation

The interaction of natural calf thymus DNA with Cr³⁺ ions was studied at room temperature by means of vibrational CD (VCD) and infrared absorption (ir) spectroscopy, and atomic force microscopy (AFM). Cr³⁺ ion binding mainly to N₇ (G) and to phosphate groups was demonstrated. ψ‐Type VCD spectra resembling electronic CD (ECD) spectra, which appear during ψ‐type DNA condensation, were observed. These spectra are characterized mainly by an anomalous, severalfold increase of VCD intensity. Such anomalous VCD spectra were assigned to DNA condensation with formation of large and dense particles of a size comparable to the wavelength of the probing ir beam and possessing large‐scale helicity. Atomic force microscopy confirmed DNA condensation by Cr³⁺ ions and the formation of tight DNA particles responsible for the ψ‐type VCD spectra. Upon increasing the Cr³⁺ ion concentration the shape of the condensates changed from loose flower‐like structures to highly packed dense spheres. No DNA denaturation was seen even at the highest concentration of Cr³⁺ ions studied. The secondary structure of DNA remained in a B‐form before and after the condensation. VCD and ir as well as AFM proved to be an effective combination for investigating DNA condensation. In addition to the ability of VCD to determine DNA condensation, VCD and ir can in the same experiment provide unambiguous information about the secondary structure of DNA contained in the condensed particles. © 2002 Wiley Periodicals, Inc. Biopolymers 61: 243–260, 2002     

Non-intercalative binding mode of bridged binuclear chiral Ru(II) complexes to native duplex DNA

A pair of chiral binuclear ruthenium(II) complexes were prepared and their binding affinities towards double stranded native DNA were assessed by observing isotropic absorption, polarized light spectra - circular and linear dichroism (CD and LD), fluorescence quenching and DNA thermal denaturation. Upon binding to DNA, the complexes produced LD signals consisting of positive and negative signals in the absorption region, although they exhibited red shift and hypochromism in the absorption spectrum. These contrasting observations indicated that the binding modes of the complexes are largely deviated from classical intercalative binding. Groove binding of the complexes to DNA was found to be more likely than intercalative binding. The small increase of DNA melting temperature in the presence of the complexes indicated a predominance of DNA groove binding. The absence of “molecular light switch effect” further supported non-intercalative binding. The groove binding propensity of complexes was also supported by comparison of the resulting data with the [Ru(phen)₂(dppz)]²⁺.     

Copper(I)-incorporation into spinach apoplastocyanin

Spinach plastocyanin was converted into the apoprotein. CuSO₄ and oxidized Cu(II)- thionein reacted with the apoprotein to Cu(II) plastocyanin. Cu(I) transfer from Cu(I)0-thionein was only 15%. The structural analogue of the copper thiolate chromophore [Cu(I)(thiourea)₃]Cl as well as [Cu(CH₃CN)₄]ClO₄ successfully formed the Cu(I)- holoprotein. Characteristic circular dichroism bands at θ₂₈₄ (?5300 deg·cm²·dmol^?1 and θ₃₁₀ (+3300 deg·cm²·dmol^?1) were seen. Upon oxidation with ferricyanide and dialysis against phosphate buffer the correct Cu(II) binding into the active centre of Cu(II) plastocyanin was confirmed by EPR-measurements. The use of [Cu(I)(thiourea)₃] Cl as a convenient Cu(I) source for reconstitution studies on copper proteins is highly recommended.     

Molecular arrangement of DNA in bacteriophage T4

The degree of preferred orientation and the coiling of the deoxyribonucleic acid within phage T₄ was studied by two independent techniques, namely, polarization of fluorescence and uv linear dichroism. A correlation between the two kinds of data was obtained, which indicated that a significant proportion (about 30%) of total phage DNA is aligned preferentially along the long axis of phage heads. Analyses of the data suggest that all of the phage DNA cannot be in a highly supercoiled helical configuration. A few models of the DNA arrangement in T₄ have been discussed in which linear sidewise packings of DNA would be predominant and may explain the observed longitudinal orientation of intraphage DNA.     

Light microscope study of mixed helices in reconstituted Salmonella flagella

In this morphological study of bacterial flagella, single flagellar filaments in solutions were photographed by dark-field light microscopy to determine parameters to describe their intact shapes. First, I measured pitches of helices I, II and III assumed by copolymers of flagellins from Salmonella strains SJ25 and SJ814 (Asakura & Iino, 1972), and combined the data with electron microscopic data of the contour length per period of each filament to calculate the pitch angles of the three helices. (The pitch angle is the angle between a tangent to the filament and the helical axis.)Secondly, filaments which consisted of two blocks assuming different helices were prepared by two-step copolymerization of SJ25 and SJ814 flagellins and the configurations of these mixed-type filaments were examined. In filaments of any mixed type, the axes of the constituent blocks were oriented at the same angle, called the “block angle” Ψ. This angle was found to be approximated by Ψ = 180 ° ? ¦θ₁ ? θ₂¦, where θ₁ and θ₂ are the pitch angles of the mixed helices. On the basis of this finding, the morphology of mixed-type filaments is discussed.     

The nonplanar peptide unit. III. Quantum chemical calculations for related compounds and experimental X-ray diffraction data.

The possible nonplanar distortions of the amide group in formamide, acetamide, N-methylacetamide, and N-ethylacetamide have been examined using CNDO/2 and INDO methods. The predictions from these methods are compared with the results obtained from X-ray and neutron diffraction studies on crystals of small open peptides, cyclic peptides, and amides. It is shown that the INDO results are in good agreement with observations, and that the dihedral angles θ_N and Δω defining the nonplanarity of the amide unit are correlated approximately by the relation θ_N = ?2Δω, while θ_C is small and uncorrelated with Δω. The present study indicates that the nonplanar distortions at the nitrogen atom of the peptide unit may have to be taken into consideration, in addition to the variation in the dihedral angles (?,ψ), in working out polypeptide and protein structures.     

Conformational studies on polynucleotide chains. IV. Backbone structure of ribonucleic acids.

In preceding papers the energies associated with the internal rotations in the sugar–phosphate–sugar complex were described with an analytical potential consisting of a Lennard-Jones 6–12 term and an intrinsic torsional term and representing the best fit to a large number of energies computed with a quantum mechanical ab initio technique. The complex considered there (of 37 atoms and with the chemical formula C₁₀H₁₈O₈P) is repesentative of deoxyribonucleic acids. In this paper we apply our potential to evaluating the intramolecular energies of the 39-atom complex C₁₀H₁₈O₁₀P, representative of the ribonucleic acids. The potential energies for the internal rotations (considered independent from one another) and the energy maps for rotations about consecutive bonds of the backbone chain are critically compared, both with those obtained for the deoxy system and with those obtained from different theoretical approaches as available from literature. It is shown that, at least for certain combinations of the internal rotation angles, the choice of the starting geometry for the sugarphosphate–sugar molecule (bond lengths and valence angles) strongly affects the value of the computed energy. If a proper geometry is used, very low energies are predicted by our potential in correspondence of the sets of torsional angles found in various RNAs by x-ray crystallography.     

DNA binding of a spermine derivative: Spectroscopic study of anthracene-9-carbonyl-n1-spermine with poly[d(G-C)·(d(G-C))] and poly[d(A-T) · d(A-T)]

The binding of polyamines, including spermidine ( 1 ) and spermine ( 2 ), to poly[d(G-C) · d(G-C) ] was probed using spectroscopic studies of anthracene-9-carbonyl-N¹-spermine ( 3 ); data from normal absorption, linear dichroism (LD), and circular dichroism (CD) are reported. Ligand LD and CD for transitions located in the DNA region of the spectrum were used. The data show that 3 binds to DNA in a manner characteristic of both its amine and polycyclic aromatic parts. With poly [(dG-dC) · (dG-dC)], binding modes are occupied sequentially and different modes correspond to different structural perturbations of the DNA. The most stable binding mode for 3 with poly[d(G-C) · d(G-C)] has a site size of 6 ± 1 bases, and an equilibrium binding constant of (2.2 ± 1.1) × 10⁷ M^?1 with the anthracene moiety intercalated. It dominates the spectra from mixing ratios of approximately 133:1 until 6:1 DNA phosphate: 3 is reached. The analogous data for poly [d(A-T) · d(A-T)] between mixing ratios 36:1 and 7:1 indicates a site size of 8.3 ± 1.1 bases and an equilibrium binding constant of (6.6 ± 3.3) × 10⁵ M^?1. Thus, 3 binds preferentially to poly [d(G-C) · d(G-C)] at these concentrations. © 1994 John Wiley & Sons, Inc.     

Interactions of metallic ions with DNA. V. DNA renaturation mechanism in the presence of Cu 2+

New experimental data were obtained by means of circular dichroism, melting, renaturation, and kinetic experiments, upon Cu²⁺ binding to DNA, poly dAT, and poly dGdC. They enable us to propose a model of binding giving a satisfactory explanation to all of the data found in the literature. Two types of binding sites are proposed: (a) a “sandwich” of Cu²⁺ between two adjacent G-C pairs giving a charge-transfer complex, and (b) a chelate between a phosphate group and a nitrogen atom of the bases (N₇ of guanine and N₃ of cytosine at room temperature, N₃ of adenine after thermal opening of A-T pair). Type (a) stabilizes the helix and keeps the two strands linked. Type (b) destabilizes the helix and explains why the kinetic rate of renaturation is the same as that of copper release.     

Stereochemistry of nucleic acids and their constituents. IV. Allowed and preferred conformations of nucleosides,nucleoside mono-, di-, tri-, tetraphosphates,nucleic acids and polynucleotides

A uniform notation and convention is suggested to describe the torsional angles in nucleic acids and their derivatives. The torsional angle χ, relating the stereochemistry of the base with respect to the sugar, shows more variation for the β-purine glycosides than for the β-pyrimidine glycosides. This variation is attributed to the fact that the β-purine derivatives may form intramolecular O(5′)-H…N(3) hydrogen bonding. The χ values for the α-purine and α-pyrimidine glycosides show preference for the –syn-clinal (or anti) conformation. The mode of puckering of the sugar also influences the χ value. The various possible conformations for the furanose ring are described by the torsional angles τ₀ τ₁, τ₂, τ₃, τ₄, about the five ring bonds. From an analysis of the torsional angles (ω, ?, ψ, ψ′, ?′, ω′) about the sugar phosphate bonds in the x-ray structures of the known nucleosides, nucleotides, phosphodiesters, nucleic acids, and related compounds, and from a consideration of molecular models, it is found that the possible conformations for the backbone of helical nucleic acids is strikingly limited. Most importantly, the preferred conformation of the nucleotide unit in poly nucleotides and nucleic acids turns out to be the same as that found for the nucleotide in the crystal structure. It is observed that base “stacking” is a consequence of the restricted backbone conformation. The torsional angles are illustrated in the form of conformational “wheels”. Interrelation between the torsion angles about successive pairs of sugar-phosphate bonds are presented in the form of conformational maps: ω,?; ?,ψ; ψ.ψ′; ψ′,?′; ?′,ω′; ω′,ω. The ω′,ω map shows the perferred conformations about the inter-nucleotide bonds of right- and left-handed helices and the possible conformations of phosphodiesters. The preferred conformation of the pyrophosphate and triphosphate is that in which the phosphate oxygens display a staggered arrangement when viewed along the P–P axis. A plausible structure and conformation for the ATPM^2? backbound complex is presented. This structure differs from that proposed by SzentGyorgi in that the metal (only transition metals are considered here) is not bound to the NH₂ nitrogen of adenine, but rather is simultaneously bound to N(7) of the ring and three phosphates (α, β, γ), or N(7) of the ring and two phosphates (β, γ). The remaining metal coordination may be satisfied by solvent–metal or enzyme–metal bonds.     

Flow linear dichroism spectra of four filamentous bacteriophages: DNA and coat protein contributions

In this study, we have separated the contributions of DNA and protein to the absorption and linear dichroism (LD) of each of four phages: fd, IKe, Pf1, and Pf3. We have found that the DNA packaged in each of the phages is hypochromic relative to the purified single stranded DNA, suggesting that bases are stacked in all of the phages. We have oriented the phages by flow and for the first time report the intrinsic LD from 320 to 190 nm for each of these phages. From the intrinsic LD of the phages and the isotropic absorption of the individual components, we have determined the reduced dichroism of the DNA within the phages and, subsequently, the maximum angle of inclination of the DNA bases (from the helix axis) for the packaged DNA. The maximum angles were 63° and 64° for the DNAs of class I phages fd and IKe, respectively. The angles were significantly less, 51° and 49°, for the DNAs of the class II phages Pf1 and Pf3, respectively. Thus, the two classes of phage differ in the structures of their packaged DNA, the DNA bases of the class II phages being more parallel to the long axis of the phage than are the DNA bases of the class I phages.     

Comparison of base inclination in ribo-GC and deoxybribo-GC polymers,and synthesis of poly(rGrC)-poly(rGrC)

The inclination angle between the base normal and the helix axis, and the axes around which the bases incline, are measured for ribo-GC polymers in buffer by using flow linear dichroism (LD), and compared to measurements for deoxyribo-GC polymers in buffer and under dehydrating conditions. A new method is designed to synthesize poly(rGrC) -poly(rGrC), which is not available commercially, in large quantities. The LD of this RNA reveals inclination angles that are similar to the B-form DNA in buffer, although the axes are different. The CD of poly(dGdC)-poly(dGdC) under the dehydrating conditions is similar to poly(rGrC)-poly(rGrC), indicating it is in the A form, and the LD gives larger inclination angles than either the B form or the corresponding RNA. Poly(dG)-poly(dC) is in the A form in buffer. Comparison among poly(rG)-poly(rC) in buffer, and poly (dG)-poly(dC) in buffer and under dehydrating conditions, reveals similar inclination angles and axes, although the LD shows that the DNA has the largest inclination angles. Except for poly(rGrC)-poly(rGrC), which has a unique reduced dichroism, all the axes for G are similar, as are the axes for C. © 1995 John Wiley & Sons, Inc.     

Serine and β-turns

Serine is one of the most frequent residues included within β-turns in proteins. Conformational investigations have been carried out on the LL and LD model dipeptides Bu^tCO-Pro-Ser-NHMe in the solute state by ir and nmr spectroscopies and in the solid state by x-ray diffraction. LL and LD sequences are folded in the βI- and βII-types, respectively, and in both cases a weak intramolecular interaction takes place between the NH and O^γ sites of the serine residue.     

Impact of wastewater fouling on contact angle

Capillary dependent systems are highly influenced by surface fouling and may degrade as material surface properties change. In anticipation of a spacecraft microgravity fluids management system exposed to highly variable wetting conditions, the impact of urine wastewater fouling on capillary contact angle was examined. The results indicate that, in general, surface fouling can decrease the contact angle when crystalline structures or biofilms form. Small crystalline growth on the order of 10 μm can lower advancing contact angles θ_adv by approximately 30°, while biofilm growth can lower it by approximately 15°. Vacuum drying of fouled surfaces increased θ_adv by about 8°, and defects greater in height than 5% of the capillary length increased θ_adv by approximately 30°. These trends may indicate that promotion of wastewater fouling may improve the performance of capillary dependent fluids management systems. These results may also influence terrestrial technologies, including medical catheters and sustainable wastewater treatment systems exposed to wastewater fouling.