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.     

Linear dichroism characteristics of ethidium-and proflavine-supercoiled DNA complexes

A flow linear dichroism technique is utilized to study the unwinding of supercoiled DNA induced by the binding of ethidium bromide (EB) and proflavine (PF) at different ratios r (drug added/DNA base). In the case of either EB or PF bound to linear calf thymus DNA, the reduced linear dichroism signals LD/A (LD: linear dichroism; A: absorbance, both measured at the same wavelength), determined at 258, and 520 or 462 nm (corresponding to contributions predominantly from the partially oriented DNA bases, intercalated EB, or PF, respectively) are nearly independent of drug concentration. In the case of supercoiled DNA, the magnitude of LD/A at 258 nm first increases to a maximum value near r = 0.04-0.05, and then decreases as r is increased further, mimicking the behavior of the sedimentation coefficients, viscosities, and gel electrophoresis patterns measured by other workers at similar values of r. However, LD/A at 520 nm, which is due to DNA-bound EB molecules, is constant within the range of r values of 0.02-0.06 in which the magnitude of LD/A determined at 258 nm due to the DNA bases exhibits a pronounced maximum. In contrast, in the case of PF, the magnitudes of LD/A determined at 258 or 462 nm are characterized by similar dependencies on r, both exhibiting pronounced maxima at r = 0.05; this parallel behavior is expected according to a simple intercalation model in which the DNA bases and drug molecules are stacked on top of one another, and in which both are oriented to similar extents in the flow gradient. The unexpected differences in the dependencies of (LD/A)258 and (LD/A)520 on r in the case of EB bound to supercoiled DNA, are attributed to differences in the net overall alignment of the EB molecules and DNA bases in the flow gradient. The magnitude of the LD signal at 258 nm reflects the overall degree of orientation of the supercoiled DNA molecules that, in turn, depends on their hydrodynamic shapes and sizes; the LD signals characterizing the bound EB molecules may reflect this orientation also, as well as the partial alignment of individual DNA segments containing bound EB molecules.(ABSTRACT TRUNCATED AT 400 WORDS)     

Orientation and linear dichroism of the reaction centers from Rhodopseudomonas sphaeroides R-26

Linear dicroism of chromatophores and isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides strain R-26 was studied using a novel technique of orientation. The results are discussed in view of the reaction center structure and its position in the membrane. The advantages of the new orientation technique are also outlined.     

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.     

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.     

Orientation and linear dichroism of the reaction centers from Rhodopseudomonas sphaeroides R-26.

Linear dicroism of chromatophores and isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides strain R-26 was studied using a novel technique of orientation. The results are discussed in view of the reaction center structure and its position in the membrane. The advantages of the new orientation technique are also outlined.     

Orientation of the pigments in Photosystem II: A low-temperature linear dichroism and polarized fluorescence emission study of chlorophyll-protein complexes isolated from Chlamydomonas reinhardtii

The orientation of the various absorbing and fluorescing dipoles in Photosystem II have been investigated by linearly polarized light spectroscopy at 5 K, performed on macroscopically oriented PS II complexes derived from Chlamydomonas reinhardtii. Linear dichroism and absorption spectra show that the Q_Y transitions of the chlorophyll molecules are mostly tilted at less than 35° from the plane of largest cross-section of the particle (which in vivo coincides with the plane of the thylakoid membrane). The chlorophyll forms absorbing at 676 and 683 nm are oriented closer to the membrane than the forms absorbing at 665 and 670 nm which are tilted at approximately 35° from the plane. A dip observed around 680 nm in the LD/absorption spectra indicates a component tilted at a larger angle away from the membrane plane than the 676 nm- and 683 nm-absorbing species. A component weakly absorbing around 693 nm and exhibiting a negative LD (tilt larger than 35°) is clearly resolved. The amplitude of the LD at 693 nm relative to that observed at the maximum (676 nm) varies from sample to sample. In the blue spectral region, two populations of carotenoids are observed; one absorbs around 460 and 490 nm, while the other absorbs around 510 nm. They are oriented out of and near to the thylakoid plane, respectively. Comparison of polarized absorption and fluorescence spectra from the same oriented samples allows the assignment of the 695 nm fluorescence emission to the dipoles responsible for the LD signal at 693 nm.     

Infrared linear dichroism of oriented DNA-ligand complexes prepared with the wet-spinning method.

Oriented DNA films prepared by the wet-spinning technique have been complexed with several ligands: the anthracycline antibiotic violamycin BI, the dipeptide L-carnosine, and the oligopeptide antibiotic netropsin. The formation of the DNA-ligand complexes is accompanied by dramatic changes of the conformational flexibility of DNA. The B-A transition which occurs usually between 80% and 70% relative humidity (RH) is more or less suppressed by the ligands. Violamycin BI at a total ligand per DNA base pair ratio, rt, of approximately 0.03 and L-carnosine at rt approximately 1.5 inhibit the B-A transition of approximately 18 and approximately 0.25 base pairs per ligand molecule, respectively. Netropsin at rt = 0.2 induces a very stable B-DNA even at rather low RH (23%). The total hydration of this complex is significantly higher than for a drug-free DNA film. Netropsin-DNA complexes at rt of 0.02 and 0.01 result in an inhibition of approximately 45 base pairs per drug molecule with respect to the B-A transition.     

Orientation of the protonated retinal Schiff base group in bacteriorhodopsin from absorption linear dichroism.

Linear dichroism experiments are performed on light-adapted bacteriorhodopsin (BR568) films containing native retinal (A1) and its 3,4-dehydroretinal (A2) analogue to measure the angle between the chromophore transition dipole moment and the membrane normal. QCFF/pi calculations show that the angle between the transition moment and the long axis of the polyene is changed by 3.4 degrees when the C3-C4 bond is unsaturated. The difference vector between the two transition moments points in the same direction as the Schiff base (N----H) bond for the all-trans BR568 chromophore. Because the plane of the chromophore is perpendicular to the membrane plane, a comparison of the transition moment orientations in the A1- and A2-pigments enables us to determine the orientation of the N----H bond with respect to the absolute chromophore (N----C5 vector) orientation. The angles of the transition moments are 70.3 degrees +/- 0.4 degrees and 67.8 degrees +/- 0.4 degrees for the A1- and A2-pigments, respectively. The fact that the change in the transition moment angle (2.5 degrees) is close to the predicted 3.4 degrees supports the idea that the chromophore plane is nearly perpendicular to the membrane plane. The decreased transition moment angle in the A2-analogue requires that the N----H bond and the N----C5 vector point toward the same membrane surface. Available results indicate that the N----C5 vector points toward the exterior in BR568. With this assignment, we conclude that the N----H bond points toward the exterior surface and its most likely counterion Asp-212. This information makes possible the construction of a computer graphics model for the active site in BR568.     

Orientation of the bases of single-stranded DNA and polynucleotides in complexes formed with the gene 32 protein of bacteriophage T4. A linear dichroism study

Linear dichroism measurements were performed in the wavelength region 250 to 350 nm on complexes between the single-stranded DNA binding protein of bacteriophage T4 (gp32) and single-stranded DNA and a variety of homopolynucleotides in compressed polyacrylamide gels. The complexes appeared to orient well, giving rise to linear dichroism spectra that showed contributions from both the protein aromatic residues and the bases of the polynucleotides. In most cases the protein contribution appeared to be very similar, and the linear dichroism of the bases could be explained by similar orientations of the bases for most of the complexes. Assuming a similar, regular structure for most of the polynucleotides in complex, only a limited set of combinations of tilt and twist angles can explain the linear dichroism spectra. These values of tilt and twist are close to (-40 degrees, 30 degrees), (-40 degrees, 150 degrees), (40 degrees, -30 degrees) or (40 degrees, -150 degrees), with an uncertainty in both angles of about 15 degrees. Although the linear dichroism results do not allow a choice between these possible orientations, the latter two combinations are not in agreement with earlier circular dichroism calculations. For the complexes formed with poly(rC) and poly(rA), the linear dichroism spectra could not be explained by the same base orientations. In these two cases also the protein contribution to the linear dichroism appeared to be different, indicating that for some aromatic residues the orientations are not the same as those in the other complexes. The different structures of these complexes are possibly related to the relatively low binding affinity of gp32 to poly(rC), and to a lesser extent to poly(rA).     

Orientation of pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. I. Determination of optimal conditions for linear dichroism measurement

The nature and possible causes of polarized light-scattering artefacts in linear dichroism measurements are investigated. Using criteria described in this article, the available orientation techniques have been critically assessed in order to obtain the linear dichroism spectra of thylakoids and of pigment-protein complexes isolated from pea. It is demonstrated here that the polyacrylamide gel squeezing technique of Abdourakhmanov et al. (Abdourakhmanov, I.A., Ganago, A.O., Erokhim, Yu.E., Solov'ev, A.A. and Chugunov, V.A. (1979) Biochim. Biophys. Acta 546, 183–186) does not lead to pigment degradation and that the linear dichroism spectra obtained in these conditions are essentially free of scattering artefacts. The linear dichroism spectra of light-harvesting complex isolated in different states of aggregation or incorporated into phospholipid vesicles are compared to the spectra of thylakoids. This comparison indicates: (1) that the isolation procedure of Burke et al. (Burke, J.J., Ditto, C.L. and Arntzen, C.J. (1978) Arch. Biochem. Biophys. 187, 252–263) leads to light-harvesting complex in which the in vivo orientation of pigments is preserved; (2) that the antenna chlorophyll a molecules of this complex have a significant degree of orientation with respect to the plane of the thylakoid.     

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.     

Orientation of the pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. III. A quantitative comparison of the low-temperature linear dichroism spectra of thylakoids and isolated pigment-protein complexes

The low-temperature linear dichroism spectrum of thylakoids oriented in polyacrylamide gel can be adequately described by a linear combination of the corresponding spectra of particles of light-harvesting complex, Photosystem I and Photosystem II, isolated by Triton X-100 extraction. The main conclusions which can be derived from this observation are: (1) The in vivo orientation of the pigments within each of the three complexes is not significantly affected by the extraction and purification procedures. (2) The various photosynthetic pigments are oriented roughly to the same extent in each of the three main biochemical constituents of the thylakoid. (3) All the complexes investigated behave like ellipsoids, the largest dimensions of which are lying in the plane of the photosynthetic membrane.     

Induced circular dichroism of DNA-dye complexes

The binding of methylene blue, proflavine, and ethidium bromide with DNA has been studied by spectrophotometric titration. Methylene blue and proflavine or methylene blue and ethidium bromide were simultaneously titrated by DNA. The results indicate that all of these dyes compete for the same bindine sites. The binding properties are discussed in terms of symmetry. The optical properties of the dye–DNA complexes have been studied as a function of DNA/dye ratio. The induced circular dichriosm due to dye–dye interaction was measured at low dye/DNA ratios for cases involving both the same dye and different dyes. A positive Cotton effect for DNA–proflavine complex may be induced at 465 mμ by eithr proflavine or ethidium bromide, whereas a netgative Cotton effect at 465 mμ may be induced by methylene blue. The limiting circular dichroism, with no dye–dye interaction, and the induced circular dichroism spectra are discussed in terms of symmetry rules.