Structural transitions of chromatin at low salt concentrations: a flow linear dichroism study

We have studied the structure of nuclease-solubilized chromatin from Ehrlich ascites cells by flow linear dichroism (LD) using the anisotropic absorption of the DNA bases and of two intercalated dyes, ethidium bromide and methylene blue. It is confirmed that intercalation occurs preferentially in the linker part of the chromatin fiber, at binding ratios (dye/base) below 0.020. Using this information, we determined the orientation of the linker in relation to the average DNA organization in chromatin. The LD measurements indicate that the conformation of chromatin is considerably changed in the ionic strength interval 0.1-10 mM NaCl: with increasing salt concentration, the LD of the intrinsic DNA base absorption changes signs, from negative to positive, at approximately 2.5 mM NaCl. The LD of the intercalated dyes also changes signs, however, at a somewhat higher salt concentration. The results are analyzed in terms of possible allowed combinations of tilt angles of nucleosomes and pitch or tilt angles of linker DNA sections relative to the fiber axis, at different salt concentrations in the interval 0.1-10 mM NaCl. Two models for the salt-induced structural change of chromatin are discussed.     

Stepwise unfolding of chromatin by urea. A flow linear dichroism and photoaffinity labeling study

The unfolding of chromatin by urea (0-7 M) was studied by means of flow linear dichroism, photoaffinity labeling and nuclease digestion. The linear dichroism results indicate that the unfolding of the DNA is accomplished through two distinct transitions at 1-2 M urea and 6-8 M urea, respectively. The photoaffinity labeling studies indicate that an opening of the nucleosome histone core occurs above 2 M urea, accompanied by general loosening of the structure. Based on the results a model for the unfolding of chromatin fibers by urea is proposed, which includes a stretching of the linker DNA (0-2 M urea) followed by a "loosening" of the nucleosome core, possibly to a one-loop DNA conformation (2-6 M urea), and finally resulting in an almost total stretching of the DNA (greater than 6 M urea).     

Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10^?3M Na⁺. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle.     

Structure of hyperacetylated chromatin: light scattering and flow linear dichroism study

Cation-induced folding of 10 nm chromatin filament to 30 nm fiber was studied with hyperacetylated chromatin using light scattering at 90 degrees and flow linear dichroism. Acetylated chromatin folded in a way indistinguishable from that of the control chromatin: both the compactness of chromatin and the orientation of nucleosomes relative to the fiber axis were identical at a given salt concentration.     

Effector-induced conformational transitions in Ascaris suum phosphofructokinase. A fluorescence and circular dichroism study.

Results of activity and spectral studies using fluorescence and circular dichroism show that AMP and fructose 2,6-bisphosphate (F-2,6-P2) activate Ascaris suum phosphofructokinase through specific and similar conformational changes. Inorganic compounds like (NH4)2SO4 and KH2PO4 also induce structural alterations in the enzyme in a manner different from those caused by AMP and F-2,6-P2. The enzyme is activated by both AMP and F-2,6-P2, in 20 mM phosphate buffer, pH 6.6, with 0.2 mM ATP and 1 mM F-6-P. The Kact values for AMP and F-2,6-P2 are 25 +/- 3 microM and 1.5 +/- 0.2 microM, respectively. Both effectors quench enzyme tryptophan fluorescence in phosphate, pH 6.6, in a concentration-dependent manner. The Kd values determined from the decrease in emission intensity at 342 nm as a function of effector concentration are 24 +/- 3 microM for AMP and 1.00 +/- 0.15 microM for F-2,6-P2, in excellent agreement with the values of Kact. Both effectors also produce dramatic changes in the CD spectrum of the enzyme, in the region from 240 to 190 nm representing the peptide backbone. Secondary structure calculations suggest an increase in the alpha-helical content of the enzyme in the presence of either effector. The Kd values obtained from the concentration dependence of the decrease in ellipticity at 210 nm are 22.8 +/- 5.3 microM and 1.3 +/- 0.2 microM, respectively, for AMP and F-2,6-P2, once again in close agreement with the Kact values for these effectors. The data imply that activation of phosphofructokinase by these effectors is concomitant with structural changes in the enzyme. Further, comparison of the difference CD spectra for the effects of AMP and F-2,6-P2 show that both of them produce similar conformational changes and probably stabilize a similar final activated state of the enzyme. Other hexose phosphate analogues such as fructose 6-phosphate, glucose 1,6-bisphosphate, and fructose 1,6-bisphosphate do not affect the CD spectrum of the enzyme. Ammonium sulfate has no effect on the CD spectrum of the enzyme in phosphate buffer but does cause a significant alteration in the spectrum obtained in Mes. Gel filtration high performance liquid chromatography using a Borosil TSK 400 column shows that the tetrameric state of the native enzyme is not affected by the presence of the effectors.     

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.     

Quasi-elastic light scattering study of salt induced conformational transitions of chromatin subunit

The translational diffusion coefficient D_T of monodisperse solutions of 146 base pairs (bp) core particles was studied by the quasi-elastic light scattering technique. When the salinity was raised a change of D_T from 1.9 × 10^?7 cm² s^?1 to 3.2 × 10^?7 cm² s^?1 was detected at about 2 mM NaCl, followed by a smooth decrease of D_T beyond 0.6 M NaCl. The measurements of particle concentration and scattering vector effects on the D_T showed that the influence of interactions between particles can be disregarded. The interaction between particles and counterions is also discussed and does not appear to be the origin of the actual changes in D_T. These transitions of D_T are hence related to changes of shape and size of the particles. It is shown that the single transition at low salinity corresponds to a conformational change while the variation of D_T at high salinity can be interpreted by a destabilization of the edifice. In different regions of salinities, the observed values of D_T can lead to reasonable hydrodynamic models.     

Circular dichroism and saccharide-induced conformational transitions of lectins from Ricinus communis.

Conformational studies on lectins from castor beans (Ricinus communis), RCA_I and RCA_II, were performed by using circular dichroism (CD). The CD spectra were similar showing several negative bands at 270–320 nm, a positive region at 230–250 nm, several negative bands at 205–225 nm, and a positive peak at about 195 nm. However, significant differences were observed in the band strength between RCA_I and RCA_II. Lactose, melibiose, and d-fucose induced marked Conformational alterations in RCA_I, whereas weaker effects were produced by d-galactose and l-rhamnose. Saccharide-induced conformational alterations were weaker in RCA_II than in RCA_I, with only lactose and melibiose inducing significant alterations. d-Glucose and 2-acetamido-2-deoxy-d-glucose, which do not inhibit hemagglutination by RCA_I or RCA_II, did not influence lectin conformation. Acetylation of tyrosyl groups with N-acetylimidazole produced changes in the CD bands in the near uv indicating involvement of tyrosine residues. The saccharide effect was most pronounced at 285 nm, a band that was assigned to a tyrosine chromophore. Analysis of the CD bands in the far-uv zone indicated the presence of approximately 50% pleated sheet (β) structure, and 13–15% α-helix in both RCA_I and RCA_II. According to the CD results, the polypeptide chain backbone in the lectins was not affected by the saccharides, whereas significant disorganization occurred in 7 m guanidine-HCl.     

Electric and flow linear dichroism of unfolded and condensed chromatin: a comparative study at low and intermediate ionic strength

Identical samples containing polynucleosomal chains of chicken erythrocyte (CE) and Ehrlich ascites tumour (EA) chromatin were studied under various ionic conditions with regard to electric linear dichroism (ELD) and flow linear dichroism (FLD). Both orientation techniques consistently confirmed that, in the limit of very low ionic strength and in the absence of multivalent cations, the reduced linear dichroism of chromatin is negative in the DNA-base absorption band, as expected for an extended zig-zag polynucleosomal conformation. With increasing electrolyte content, both ELD and FLD decreased drastically in amplitude, but in contrast to the ELD which remains negative in an intermediate range of low ionic strength (0.1-0.5 mM Mg2+) the FLD changes sign and becomes positive. The ELD and FLD amplitudes decrease with higher Mg2+ concentrations and FLD even vanishes in the region of 0.2-0.4 mM; both signals are positive above 0.4-0.5 mM Mg2+. The origin of the dissimilarities between ELD and FLD observations is still not fully understood. Several possibilities are considered: ELD signals are more influenced than FLD by the presence of short chromatin chains, nucleosomes and small pieces of naked DNA, while FLD is more susceptible to the presence of large, easily orientable, scattering aggregates. Different preferred orientation directions of the chromatin fibre with respect to electric and hydrodynamic fields may also be involved. Finally, FLD and ELD probably "see" different features of the chromatin structure.     

The temperature and pH dependence of conformational transitions of the chromatin subunit.

Hydrodynamic, spectroscopic, and chemical crosslinking studies on monomer chromatin subnits are reported as a function of ionic strength, pH, and temperature. In earlier studies, two salt-dependent conformational transitions were described (Gordon et al., Proceedings of the National Academy of Science, 75, 660, 1978). Transition one occurred between 0.7 and 2.0 mM ionic strength and transition two occurred between 5.0 and 11.0 mM ionic strength. Crosslinking at 11 mM ionic strength with formaldehyde suppressed both transitions. In this communication we report that the second transition was characterized by changes in the circular dichroism spectra in the 260--320 nm region as well as by changes in the hydrodynamic properties. As the ionic strength was increased from 5.0 to 11.0 mM, [theta]282 decreased from 2000 TO 1500 DEG CM2/DMOLE AND [THETA]295 decreased from 0 to -400 deg cm2/dmole. Both transitions occurred in the pH range from pH 6.0 to 9.2. At pH 5.0, the two ionic strength-dependent transitions were no longer observed and the characteristic changes in the circular dichroism spectra were suppressed. The spectra of the monomer subunits at pH 5.0 showed only small changes with ionic strength and resembled the spectra of the subunits at 11 mM ionic strength above pH 6.0. In order to characterize the transitions in thermodynamic terms an ionic strength near the midpoint of each transition was selected. Then, changes in s20,w and D20,w were measured as a function of temperature. These data allow an estimation to be made of the enthalpies and entropies of the transitions.     

Interaction of benz[a]pyrene diol epoxide with chromatin studied by flow linear dichroism

Chromatin isolated from Ehrlich ascites cells was incubated with the tumourigenic compound (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenz[a]pyrene [(+)-anti-BPDE] at low ionic strength and the modified chromatin was analysed using flow linear dichroism (LD). The results confirm that (+)-anti-BPDE preferentially binds to the DNA in the linker regions, and furthermore show that the long axis of the bound pyrenyl chromophore is oriented parallel or close to parallel to the average orientation of the chromatin fiber axis. The data indicate that the binding geometry of (+)-anti-BPDE in chromatin is similar to that in pure DNA and deoxyguanosine-containing double-helical oligonucleotides.     

The conformation of proteins in chromatin. A circular dichroism study below 250 nm.

This paper is an investigation of the circular dichroism (CD) spectra of DNA and protein in chromatin. The circular dichroism (CD) of chromatin below 250 nm is due to DNA and protein peptide chromophores. The spectrum in this region is resolved into contributions from salt-extractable proteins (histone and non-histone proteins extractable with sodium chloride), residual non-histone proteins (not extractable with 3 M sodium chloride), and DNA. Below 250 nm, DNA in chromatin has the same CD spectrum as DNA free in solution, in contrast to the CD of DNA above 250 nm (Hjelm, R. and Huang, R. C., (1974), Biochemistry 13, 5275). Histones and salt-extractable non-histone proteins in chromatin are seen to have an average CD like those observed for globular proteins. The average CD of the residual non-histone proteins is consistent with a population of proteins with more extended conformation. The CD of each of these components is found to be the same in chromatins isolated from tissues having different nuclear synthetic activities: chick embryo brain, pig cerebellum, myeloma K41, calf thymus, and chicken erythrocyte.     

pH-induced conformational changes in chromatin subunits measured by circular dichroism and immunochemical reactivity

Changes in the conformational state of chromatin core particles from chicken erythrocytes were studied by both immunochemical and biophysical methods as a function of pH and ionic strength. When the pH of core particles in a solution of ionic strength 3, 60 or 220 mM was lowered from pH 7.5, a sharp transition in the circular dichroism spectrum of DNA monitored between 320 and 260 nm was observed at pH 6.65. This change in DNA ellipticity was totally reversible. Binding to core particles of antibodies specific for histones H2B, H2A, H3 and for the IRGERA (synthetic C-terminal) peptide of H3 was used to follow changes in histone antigenicity. Binding was studied in the pH range 7.5-5.35, and at ionic strength of 60 and 220 mM. A change in reactivity of some histone epitopes was observed around pH 6.2–6.5. However, the changes observed by circular dichroism and antibody binding pertain to different components of chromatin subunits and they probably reflect independent phenomena. The alteration in accessibility of these determinants at the surface of core particles was completely reversible and was dependent on ionic strength. The conformation changes in core particles occurring near physiological ionic strength and pH may reflect dynamic changes in chromatin structure that possess functional significance.     

Chemical relaxation of co-operative conformational transitions of linear biopolymers

The kinetics and chemical relaxation of co-operative conformational changes of linear (bio)polymers (e.g. helix-coil transitions of polypeptides) are discussed on the basis of the linear ISING model. Chemical relaxation is in general shown to be described by 4N−5 relaxation times if the polymer chain consists of N elementary reaction sites. It is pointed out that nevertheless substantial simplifications of the theory can be achieved in many special cases of practical interest. Sufficiently short chains exhibit first-order kinetics resulting in a single relaxation time whereas for certain medium chain lengths zero-order kinetics plays a principal role in the relaxation process. For the particularly interesting case of very long chains a set of four relaxation equations is derived. The corresponding relaxation times are calculated assuming strong co-operativity and slow nucleation rates. However, it is almost exclusively the largest one of these relaxation times which actually controls the conformational change as turns out by means of a new approach to compute amplitude factors.     

Conformational transitions in phosvitin with pH variation. Vibrational circular dichroism study

The vibrational circular dichroism (VCD) spectra of metal-free phosvitin are presented as a function of pH and analyzed both qualitatively and by using a factor analysis approach referenced to a protein data set. The qualitative pattern of both the IR and VCD changes is consistent with a coil-to-sheet transition occurring as pH is progressively decreased to values lower than 3. A similar transition was seen in commercial preparation of phosvitin which still contained metal ions, but there the transition was more gradual and occurred at somewhat different pH values. Such a gradual change is also evident in the solution phase absorption band profile but is made clearer using Fourier deconvolution. Based on VCD results, the low pH transition appears to occur with two distinct manifestations of the beta-sheet form. However, at the lowest pH values the sample may precipitate. These two forms are not distinguishable with Fourier transform infrared alone and may be due to a twist of the beta-sheet form or to aggregation.     

Circular dichroism of histone-bound regions in chromatin.

Native, NaCl-treated, trypsin-treated, and polylysine-bound nucleohistones were studied in 2.5 × 10^?4 M EDTA, pH 8.0, using circular dichroism (CD) and thermal denaturation. Removal of histone I by 0.6 M NaCl has a much smaller effect on both Δε₂₂₀ and Δε₂₇₈ than the removal of other histones. This indicates that histone I has less helical content and less conformational effect on the DNA in nucleohistone. By extrapolating to 100% binding by histones other than I, the positive CD band near 275 nm is close to zero. Comparison is also made between the effects of binding by the more basic and the less basic halves of histones by trypsin-digestion and polylysine-binding experiments. Trypsin digestion of nucleohistone reduces melting band IV at 82°C much more than melting band III at 72°C. However, the CD changes of Δε₂₇₈ and Δε₂₂₀ induced by trypsin digestion are small, unless melting band III is also reduced by the use of a higher trypsin level. This implies that the less basic halves of histones, which stabilize DNA to 72°C (melting band III), have more helical structure and are more responsible for conformational change in DNA than are the more basic halves, which stabilize DNA to 82°C (melting band IV). Polylysine binding to nucleohistone diminishes melting band III but has no effect on melting band IV. This binding affects only slightly the Δε₂₂₀ of nucleohistone, indicating that polylysine interferes very little with the structure of the less basic halves of bound histones. The implications of these studies with respect to chromatin structure are discussed.     

Higher order structure of chromatin: evidence from photochemically detected linear dichroism

We have used photochemically detected linear dichroism to measure the separate average angular orientations of nucleosomes and linker DNA in 30-nm chromatin fibers of varying linker size (20-80 base pairs). Our results indicate that the average tilt angles vary with linker size, but not in a monotonic manner, suggesting that the constancy of geometry of the 30-nm fiber is maintained by compensatory changes of nucleosomal tilt which accommodate packing of variable lengths of linker DNA. We discuss the compatibility of our results with the various classes of models that have been proposed for the 30-nm fiber, including the continuous solenoid model and models built from the basic unit of the zig-zag ribbon. Many models can be eliminated, and all have to be modified to fit our results for chromatins with very long linkers.     

A method for the experimental study of DNA conformational transitions in fibers

The method proposed for the study of DNA conformational transitions is based on the proportionality, experimentally observed, between the length of a DNA fiber and the axial rise per nucleotide characterizing the molecular helix. Precise curves for the A-B and B-C transitions as a function of the relative humidity are obtained by using X-ray fiber data and measurements of fiber dimensions. It is thus shown that the A-B transition is a cooperative process between two different states, whereas the B-C transition can be considered as a progressive change of conformation. The present method is applied on two natural DNAs differing in base composition so that the effect of the nucleotide content on the conformational changes can be estimated.