Optical anisotropy of chromatin. Flow linear dichroism and electric dichroism studies

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentrations by the use of flow linear dichroism (LD) and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA - 2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA - for chromatins with the linker DNA of 10-30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin. Presented results explain the contradictory data obtained by electrooptical and hydrooptical methods.     

The different binding modes of Hoechst 33258 to DNA studied by electric linear dichroism.

The binding mode of the bisbenzimidazole derivative Hoechst 33258 to a series of DNAs and polynucleotides has been investigated by electric linear dichroism. Positive reduced dichroisms were measured for the poly(dA-dT).poly(dA-dT)- and poly(dA).poly(dT)-Hoechst complexes in agreement with a deep penetration of the drug into the minor groove. Similarly, the drug displays positive reduced dichroism in the presence of the DNAs from calf thymus, Clostridium perfringens and Coliphage T4. Conversely, negative reduced dichroisms were obtained when Hoechst 33258 was bound to poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and poly(dG).poly(dC) as well as with the GC-rich DNA from Micrococcus lysodeikticus indicating that in this case minor groove binding cannot occur. Substitution of guanosines for inosines induces a reversal of the reduced dichroism from negative to positive. Therefore, as anticipated it is the 2-amino group of guanines protruding in this groove which prevents Hoechst 33258 from getting access to the minor groove of GC sequences. The ELD data obtained with the GC-rich biopolymers are consistent with an intercalative binding. Competition experiments performed with the intercalating drug proflavine lend credence to the involvement of an intercalative binding rather than to an external or major groove binding of Hoechst 33258 at GC sequences.     

Anthracycline-DNA interactions studied with linear dichroism and fluorescence spectroscopy

DNA-binding geometry and dynamics of a number of anthracyclines, including adriamycin and 4-demethoxydaunorubicin, interacting with DNA have been studied by means of linear dichroism and fluorescence techniques. The anthracycline chromophore is found to be approximately parallel to the plane of the DNA bases and to have a restricted mobility, as would be expected for an intercalative binding mode, but there are variations between different directions in the chromophore as well as between the drugs. From dichroic spectra of adriamycin in an anisotropic host of poly(vinyl alcohol), absorption components corresponding to transitions with mutually orthogonal polarizations have been resolved. These can be exploited to determine the orientations of the two chromophore axes in the DNA complex relative to the DNA helix axis. In a certain binding regime the long axis of the bound anthracycline chromophores (with the exception of 4-demethoxydaunorubicin) is found to be approximately 10 degrees closer to perpendicular to the helix axis than are the DNA bases. This demonstrates that the average base tilt is at least 10 degrees. By contrast, the short axis of the aglycon moiety is found to be tilted some 20-30 degrees from perpendicular. This may be because it is probing a base direction with a more pronounced, static or dynamic, inclination than the average in DNA. The drug orientation and the DNA orientation (reflecting flexibility) are observed to vary differently and nonmonotonically with binding ratio, suggesting specific binding and varying site geometries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lambda phage cro repressor. DNA sequence-dependent interactions seen by tyrosine fluorescence

The thermodynamics of sickle cell hemoglobin gelation in the presence of oxygen has been investigated by measuring the fractional saturation of the solution and polymer phases, and the solubility. The fractional saturation of the solution phase with oxygen and the solubility were measured by near infrared spectrophotometry after sedimentation of the polymers, while the fractional saturation of the polymer phase was determined from linear dichroism measurements on gels formed by nucleation with an argon ion laser. Using the solution binding data of Gill et al. (1979) to calculate the oxygen pressure corresponding to the solution phase saturation, the initial portion of the polymer binding curve was determined. The self-consistency of the data analysis in terms of the two-phase model for the gel was tested by comparing measured and calculated gel (i.e. solution plus polymer) binding curves, and by comparing the observed solubilities with those calculated from the solution and polymer binding curves using Gibbs-Duhem relations.Oxygen binding to the polymer was found to be non-co-operative up to the maximum measured fractional saturation of 0.14. The binding constant was 0.0059 ± 0.0015 torr^?1 (p₅₀ = 170 ± 40 torr), which is about three times smaller than that of hemoglobin in the low-affinity T quaternary structure. Both the non-co-operative binding and the low affinity could be qualitatively explained in terms of an allosteric model and the current information on the polymer structure.     

pH-dependent forms of the ferryl haem in myoglobin peroxide analysed by variable-temperature magnetic circular dichroism.

The reaction product of myoglobin and H2O2 exists in two different forms according to the external pH. Varied-temperature magnetic-circular dichroism (m.c.d.) spectroscopy demonstrates that both contain the oxyferryl ion Fe(IV) = O. Alkaline myoglobin peroxide has often been used as a model for oxidized intermediates in the catalytic cycles of haem-containing peroxidases, but absorption and m.c.d. spectra show that the acid form is much more closely related to species such as horeradish peroxidase Compound II. The differences are tentatively ascribed to ionization of the proximal histidine ligand in alkaline myoglobin peroxide. It is also shown that the m.c.d. method allows an estimate of the zero-field splitting parameter of both forms, values of D = 28.0 +/- 3 cm-1 and 35.0 +/- 5 cm-1 being obtained for the alkaline and acid forms respectively.     

Membrane-phorbol ester interactions monitored by circular dichroism

The interaction of phorbol 12,13-dibutyrate (PDBu), 12-O-retinoylphorbol 13-acetate (RPA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) with L-alpha-phosphatidylserine-containing small unilamellar vesicles or erythrocyte ghosts was monitored by circular dichroism (CD). No change in the CD spectra of PDBu was observed upon binding, while RPA and TPA spectra were slowly affected by the interaction. The changes in RPA and TPA spectra were assigned to the embedding of these molecules in the membrane bilayers. In the presence of 10(8) cells/ml, after one minute incubation, about 2 to 5% of the amount of phorbol ester added is embedded in the membrane. It is suggested that either phorbol esters entering the membrane is not a prerequisite for protein kinase C activation or the amount of phorbol esters necessary to activate protein kinase C is very small.     

Analysing DNA complexes by circular and linear dichroism

The application of linear and circular dichroism (LD and CD) in nucleic acid research id illustrated by recent results aimed at answering specific structural problem in the interaction of DNA with molecules of biological importance. We first consider the circumstances under which ligands, such as DAPI (4′, 6-diamidino-2-phenylindole), change their preferred binding mode in the minor groove to major groove binding or intercalation. As an extension of this problem we refer to the switch between groove binding and intercalation of structurally similar ligands such as ellipticines and trigonal ruthenium complexes. We also explore the use of LD and CD in the determination of the structure of the complex formed between the polynucleotide poly(dA) and the novel ‘peptide nucleic acid’, consisting of nucleic acid bases joined by a polyamide homomorphous with the deoxyribose-phosphate backbone of DNA. Finally, the structure and interaction of the recombination enzyme RecA with DNA is discussed, in particular the influence of the presence of the intercalators, groove binders or covalent DNA adducts.     

Drug-DNA interactions: spectroscopic and footprinting studies of site and sequence specificity of elliptinium.

The binding of the antitumoral ellipticine derivative 2-methyl-9-hydroxyellipticinium acetate (elliptinium; NMHE) to DNA was analyzed by the combined use of DNase I footprinting and spectroscopic methods. Using two fragments of pBR322 DNA, five discrete NMHE binding sites of 5-7 protected base pairs (bp) were detected by footprinting at 4 degrees C on the analyzed regions. These corresponded to alternating pyrimidines and purines. The inactive derivative 2-methyl ellipticinium acetate L(NME) lacking a hydroxy group failed to demonstrate DNA protection even at low temperature. Ultraviolet-absorption and 1H-nmr analysis was performed using two autocomplementary octanucleotides d(TGACGTCA) (I) and d(ACTGCAGT) (II). The uv-absorption titrations resulted in an intercalative binding mode for NMHE in the oligomers. Analysis of the derived biphasic Scatchard plots yielded two binding sites corresponding to approximately 6-bp and 2-bp sizes and characterized by apparent association constants K1 approximately 10(8) M-1 and K2 approximately 10(6) M-1, respectively. The 1H-nmr analysis of exchangeable (imino) protons and nonexchangeable protons performed in the one- and two-dimensional modes confirmed the intercalation of NMHE, and further revealed the existence of multiple sites on DNA. Assuming that imino resonance line width concerned the sole kinetic effects, 10-ms order lifetimes were estimated for the drug-oligonucleotide complexes at 7 degrees C, pH 7, and 0.1 ionic strength. Finally, examination of every drug-DNA spectra in the light of the footprinting results indicated that there was a preference for binding of NMHE to the CpG (octamer I) and TpG (octamers I and II) steps.     

Restriction enzyme kinetics monitored by UV linear dichroism

The use of linear dichroism (LD) spectroscopy for biological applications has been brought to the forefront recently by our development of thermostated microvolume Couette cells. We present a method for following the digestion of DNA by restriction endonucleases in real time without the use of any extrinsic dyes or labels. This is accomplished using linear dichroism spectroscopy (the differential absorbance of light polarized parallel and perpendicular to the sample orientation axis). The differential absorbance signal depends on the degree of alignment of the molecules. In this case the DNA is aligned by Couette flow (flowing the solution in the annular gap between two concentric cylinders), and we monitor the increase in alignment upon linearization of a circular DNA molecule. In addition, we observe a decrease in alignment upon further digestion and subsequent shortening of the DNA. Ten enzymes were investigated: seven enzymes with a single cut site (EcoRI, KpnI, NdeI, NotI, NruI, SmaI, XbaI), two enzymes with two cut sites (BstZ17I, EagI), and one enzyme with no cut site (ClaI). LD, as implemented in this new assay, is broadly applicable across a wide range of DNA-modifying enzymes and compounds and, as such, is a useful addition to the toolbox of biological characterization.     

Orientation and linear dichroism of chloroplasts and sub-chloroplast fragments oriented in an electric field

Whole or broken spinach chloroplasts, bacterial chromatosphores and CPI chlorophyll · protein complexes in aqueous suspensions at room temperature can be oriented in externally applied electric fields. The orientation is observed by monitoring the electric field induced linear dichroism (LD). With whole chloroplasts a detectable LD signal is observed using voltages as low as 2–3 V (50 Hz alternating voltage) across an 0.3 cm electrode gap, and nearly complete orientation is observed at fields of 30 V · cm^?1. The wavelength dependence of the LD signals using either orienting electric fields ($\text{E}$) alone, or magnetic fields ($\text{B}$) alone, are similar but opposite in sign with $\text{E}$ and $\text{B}$ pointing in the same direction. The chloroplasts tend to orient in such a way that the membrane planes are parallel to $\text{E}$. The CPI complexes and bacterial chromatophores require much higher electric fields for orientation than whole chloroplasts (for CPI complexes E > 2000 V · cm^?1); rectangular, millisecond duration, voltage pulses are utilized for the observation of electric field induced LD spectra in these cases. Oriented CPI complexes exhibit LD maxima of the same sign at 685 and at 440 nm. The oriented chromatophores exhibit an LD spectrum of either positive or negative sign, depending on the wavelength. The mechanisms of the orientation are discussed.     

Flow orientation of brain microtubules studies by linear dichroism

Flow orientation of bovine brain microtubules has been studies using phase-modulation detected linear dichroism, LD, in a Couette cell with radial light propagation. LD could be sensitively measured in a wide flow gradient interval: 10^-3–10³ s^-1, without any apparent degradation of the microtubule structure. An extremely small flow gradient, 10^-3 s^-1 is sufficient to give significant orientation, and 10 s^-1 rapidly produced a very high degree of orientation. It is also shown that thermal convection effectively orients microtubules in vitro.The apparent linear dichroism is dominated by an anisotropic scattering from the aligned microtubules, superimposed on a weaker absorption dichroism due to intrinsic chromophores. The linear dichroism due to anisotropic turbidity, LD, is found to be an excellent tool for monitoring the formation of microtubules and in contrast to ordinary turbidity measurements, non-specific aggregates contribute to a far less extent.Time resolved LD was used to study the orientational relaxation of microtubules upon stopped shear. The relaxation towards random orientation can be described by a slow, multi-exponential decay. With increasing protein concentration the relaxation becomes slower and above approximately 1 mg/ml a fraction with a semipermanent orientation is formed. Finally, the development of orientation with time upon applying a small, constant gradient has been measured and the results are considered in terms of a model for flow orientation of rigid rods.     

Flow-induced alignment of amyloid protofilaments revealed by linear dichroism

Amyloid fibrils underlying various serious amyloidoses including Alzheimer and prion diseases form characteristic deposits in which linear fibrils with an unbranched and rigid morphology associate laterally or radially, e.g. radial senile amyloid plaques of amyloid beta. To clarify the formation of these high order amyloid deposits, studying the rheology is important. A 22-residue K3 peptide fragment of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis, forms long and homogeneous protofilament-like fibrils in 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2). Here, using circular dichroism and linear dichroism, we observed the flow-induced alignment of fibrils. Analysis of far- and near-UV linear dichroism spectra suggested that both the net pi-pi* transition moment of the backbone carbonyl group and L(b) transition moment of the Tyr(26) side chain are oriented in parallel to the fibril axis, revealing the structural details of amyloid protofilaments. Moreover, the intensities of flow-induced circular dichroism or linear dichroism signals depended critically on the length and type of fibrils, suggesting that they are useful for detecting and characterizing amyloid fibrils.     

Functional studies on transfected cell microarray analysed by linear regression modelling

Transfected cell microarray is a promising method for accelerating the functional exploration of the genome, giving information about protein function in the living cell. The microarrays consist of clusters of cells (spots) overexpressing or silencing a particular gene product. The subsequent analysis of the phenotypic consequences of such perturbations can then be detected using cell-based assays. The focus in the present study was to establish an experimental design and a robust analysis approach for fluorescence intensity data, and to address the use of replicates for studying regulation of gene expression with varying complexity and effect size. Our analysis pipeline includes measurement of fluorescence intensities, normalization strategies using negative control spots and internal control plasmids, and linear regression (ANOVA) modelling for estimating biological effects and calculating P-values for comparisons of interests. Our results show the potential of transfected cell microarrays in studying complex regulation of gene expression by enabling measurement of biological responses in cells with overexpression and downregulation of specific gene products, combined with the possibility of assaying the effects of external stimuli. Simulation experiments show that transfected cell microarrays can be used to reliably detect even quantitatively minor biological effects by including several technical and experimental replicates.     

Attenuation of DNA-protein interactions associated with intrinsic, sequence-dependent DNA curvature.

Inherently curved DNA segments, associated with short runs of adenines, have been identified in many gene regulatory regions, yet their physiological significance remains unknown. The observations reported in this study indicate that intrinsically bent nucleic acid fragments are characterized by substantially attenuated affinities toward DNA-binding proteins involved in structural functions, such as H1 histone and protamine, as well as toward various DNA-modifying enzymes including ligases and exo- and endonucleases. Two mechanisms might be responsible for the altered binding properties. According to the first mechanism, the attenuated binding affinities and the bending represent two independent consequences of the unique structural parameters exhibited by A-tracts. Indeed, analysis of the degradation products obtained upon exposure of the curved sequences to various chemical nucleases points toward the narrowing of the DNA minor groove, a conformational modulation known to characterize A-tracts and to run along the axially-bent motifs, as a potential determinant of the observed binding attenuation. Alternatively, the conformational constraints which result from the stable bending might act to modulate the strength of DNA-protein interactions. Although the factor directly responsible for the altered binding affinities revealed by the bent sequences cannot as yet be conclusively resolved, it is proposed that a reiteration of this specific factor, being either an A-tract or a bend, in phase with the DNA helical repeat acts to amplify the modulation of the binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-induced linear dichroism in photoreversibly photochromic sensor pigments. – III. Chromophore rotation estimated by polarized light reversal of dichroism

Phytochrome from oats ( Avena sativa L. cv. Sol II), partially purified on brushite, was immobilized on Sepharose beads to which antiphytochrome immunoglobulin had been covalently linked. The immobilized phytochrome was first brought to the Pr form with unpolarized far-red light. The change in linear dichroism at 660 nm induced by plane polarized red light, and its reversal by plane polarized far-red light were then studied using a dual-wavelength spectrophotometer equipped with polarizing filters. The far-red light was most effective in reversing red-induced dichroism when the angle between the planes of polarization of red and far-red light was approximately 23°. From this it was computed that the long-wavelength transition moment of phytochrome rotates about 29° (or 180°–29°) with respect to the protein during conversion from Pr to Pfr. The reverse experiment, using unpolarized red light followed first by polarized far-red light and then polarized red light, with dichroism monitored at 730 nm, also gives most effective reversal for an angle of about 23° between polarization planes, but this corresponds to a transition moment rotation of about 36° (or 180°–36°). The present method is more straightforward but less accurate and confirms our earlier conclusion that the rotation angle is close to 32° (or 180°–32°) in contrast to the "in vivo" value of 90° found by several workers.     

Probing the influence of sequence-dependent interactions upon α-helix stability in alanine-based linear peptides

The observation that short, linear alanine-based polypeptides form stable α-helices in aqueous solution has allowed the development of well-defined experimental systems with which to study the influence of amino acid sequence upon the stability of secondary structure. We have performed detailed conformational searches upon six alanine-based peptides in order to rationalize the observed variation in the α-helical stability in terms of side-chain-backbone and side-chain-side-chain interactions. Although a simple, gas-phase, potential model was used to obtain the conformational energies for these peptides, good agreement was obtained with experiment regarding their relative α-helical stabilities. Our calculations clearly indicate that valine, isoleucine, and phenylalanine residues should destabilize the α-helical conformation when included within alanine-based peptides because of energetically unfavorable side-chain-backbone interactions, which tend to result in the formation of regions of 3₁₀-helix. In the case of valine, the destabilization most probably arises from entropic effects as the isopropyl side chain can assume more orientations in the 3₁₀-helical form of the peptide. A detailed examination of very short-range interactions in these peptides has also indicated that an interaction, involving fewer than five consecutive residues, whose stabilizing effect reinforces that of the (i, i + 4) hydrogen bond may be the basis of the requirement for increased nucleation (σ) and propagation parameters (s) required by Zimm–Bragg theory to predict the α-helical content for compounds in this class of short peptides. Our calculations complement recent work using modified Zimm–Bragg and Lifson–Roig theories of the helix–coil transition, and are consistent with molecular dynamics simulations upon linear peptides in aqueous solution. © 1993 John Wiley & Sons, Inc.     

Orientation of photosystem-I pigments. Investigation by low-temperature linear dichroism and polarized fluorescence emission

Using absorption and fluorescence experiments at low temperature with polarized light on oriented samples, the orientation of PS-I-related pigments, both in green plants and in Chlamydomonas reinhardtii, has been investigated on isolated pigment-protein complexes and intact thylakoids. The following observations have been made. (i) The isolation procedure of PS I₁₁₀, PS I₆₅, LHC I and CP0) particles from pea and C. reinhardtii do not alter significantly the intrinsic orientation of the pigments inside the complexes; (ii) Chl b is a structural component of PS I, linked to the peripheral antenna, with an orientation with respect to the thylakoid plane different from that observed in the main light-harvesting complex (iii) PS I₆₅ (i.e., ‘core’ PS I) of pea and C. reinhardtii contains identical chromophores having the same orientation with respect to the geometrical longest axis (axes) of the complexes. (iv) LHC I and CP0 (i.e., PS I ‘peripheral antenna’) of pea and C. reinhardtii have identical oriented chromophores, except that a long-wavelength component with a high anisotropy is only present in green plants. This set of pigments, which absorbs at 705–725 nm, has the same orientation as the dipoles emitting F₇₃₅ and also as the Q_Y transition of P-700. (v) All the long-wavelength fluorescence properties of the various studied membranes are explained by these data on isolated PS I complexes: wild-type C. reinhardtii and Chl-b-less barely fluoresce from the core pigments, while a CP1 deficient mutant of C. reinhardtii and wild-type barley fluoresce from the antenna pigments.     

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.     

Heme-heme interactions in circular dichroism spectra

Using the theory of optical activity by Kirkwood and Tinoco, the CD spectra of the heme peptides in mono- and dimeric forms have been calculated according to the simple spectral model of heme of Simpson.     

Nucleic acid vibrational circular dichroism, absorption, and linear dichroism spectra. II. A DeVoe theory approach.

The DeVoe polarizability theory is used to calculate vibrational circular dichroism (VCD) and infrared (IR) absorption spectra of four polyribonucleotides: poly(rA) x poly(rU), poly(rU) x poly(rA) x poly(rU), poly(rG) x poly(rC), and poly(rC+) x poly(rI) x poly(rC). This is the first report on the use of the DeVoe theory to calculate VCD, oriented VCD, IR absorption, and IR linear dichroism (LD) spectra of double- and triple-stranded polyribonucleotides. Results are reported for DeVoe theory calculations--within the base-stretching 1750-1550 cm(-1) spectral region--on several proposed multistranded polyribonucleotide geometries. The calculated spectra obtained from these proposed geometries are compared with previously reported measured and calculated VCD and IR spectral results. Base-base hydrogen-bonding effects on the frequencies and magnitudes of the base carbonyl stretching modes are explicitly considered. The good agreements found between calculated and measured spectra are proposed to be further evidence of the usefulness of the DeVoe theory in drawing three-dimensional structural conclusions from measured polyribonucleotide VCD and IR spectra.