Hyperacetylation of core histones does not cause unfolding of nucleosomes. Neutron scatter data accords with disc shape of the nucleosome

Recent studies report that the frictional resistance of partially acetylated core particles increases when the number of acetyl groups/particle exceeds 10 (Bode, J., Gomez-Lira, M. M. & Schr?ter, H. (1983) Eur. J. Biochem. 130, 437-445). This was attributed to an opening of the core particle though other explanations, e.g. unwinding of the DNA ends were also suggested. Another possible explanation is that release of the core histone N-terminal domains by acetylation increased the frictional resistance of the particle. Neutron scatter studies have been performed on core particles acetylated to different levels up to 2.4 acetates/H4 molecule. Up to this level of acetylation the neutron scatter data show no evidence for unfolding of the core particle. The fundamental scatter functions for the envelope shape and internal structure are identical to those obtained previously for bulk core particles. The structure that gave the best fit to these fundamental scatter functions was a flat disc of diameter 11-11.5 nm and of thickness 5.5-6 nm with 1.7 +/- 0.2 turns of DNA coiled with a pitch of 3.0 nm around a core of the histone octamer. The data analysis emphasizes the changes in pair distance distribution functions at relatively low contrasts, particularly when the protein is contrast matched and DNA dominates the scatter. Under these conditions there is no evidence for the unwinding of long DNA ends in the hyperacetylated core particles. The distance distribution functions go to zero between 11.5 and 12 nm which gives the maximum chord length in a particle of dimension, 11 nm X 5.5 nm. The distance distribution function for the histone octamer contains 85% of the vectors within the 7.0-nm diameter of the histone core. 15% of the histone vectors lie between 7.0 and 12.0 nm, and these are attributed to the N-terminal domains of the core histones which extend out from the central histone core. Histone vectors extending beyond 7.0 nm are necessary to account for the measured radius of gyration of the histone core of 3.3 nm. A similar value of 3.2 nm is calculated for the recent ellipsoidal shape of 11.0 X 6.5 X 6.5 nm from the crystal structure of the octamer. However, the nucleosome model based on this structure is globular, roughly 11 nm in diameter, which does not accord with the flat disc shape core particle obtained from detailed neutron scatter data nor with the cross-section radii of gyration of the histone and DNA found previously for extended chromatin in solution.     

The structure of the chromatin core particle in solution.

The shape and size of the nucleosomal core particle from chromatin has been examined by analysis of neutron and X-ray scattering data from dilute solutions. Calculations of scattering for many different models have been made and only one model was able to account for both the X-ray and neutron profiles. This model is an oblate structure with height about 50A and diameter 110A. The DNA is mainly confined to two annuli located at the top and bottom respectively of the core particle positioned on the outside of a compact protein core which has a height of about 40A and diameter about 73A.     

A low resolution model for the chromatin core particle by neutron scattering

Neutron scattering studies have been applied to chromatin core particles in solution, using the contrast variation technique. On the basis of the contrast dependance of the radius of gyration and the radial distribution function it is shown that the core particle consists of a core containing most of the histone around which is wound the DNA helix,following a path with a mean radius of 4.5 nm,in association with a small proportion of the histones. Separation of the shape from the internal structure, followed by model calculations shows that the overall shape of the particle is that of a flat cylinder with dimensions ca. 11x11x6 nm. Further details of the precise folding of the DNA cannot be deduced from the data, but detailed model calculations support concurrent results from crystallographic studies(25).Images     

Small angle neutron scattering studies of chromatin subunits in solution

Neutron scattering studies have been performed on dilute solutions of the fundamental subunit of chromatin, the nucleosome. The subunits contain approximately 195 base paris (bp) of DNA and histones H2A, H2B, H3, and H4. Measurements of the small angle scattering curves in various H2O/D2O solvents allow the contrast dependence of the radius of gyration of the subunits to be examined and give the mean scattering density of the particle. Further application of contrast variation to the higher angle scatter curves allows the contributions from the shape and internal structure of the subunits to be analyzed separately. From these results, we are able to propose a spherically averaged structure with most of the histones closely packed into a core of radius 3.2 nm surrounded by a loosely packed DNA-rich shell of 2.0 nm thickness resulting in a particle of 5.2 nm average radius. Model calculations for ellipsoids show that the outer shape of the subunit must have an axial ratio between 0.5 and 1.4 but is probably best described by more spherical particle. These results are correlated with the diffraction from chromatin films to provide an explanation for some of the diffraction rings.     

Shape effects in elastic scattering by orientationally random ensembles of simple model viruses

Recent theoretical work has shown that the complete set of polarized elastic light scattering studies should yield information about particle structure that has so far hardly been utilized. We present calculations of such light-scattering properties for a number of model structures, exploring particularly the size limit at which the new effects should become visible. The particles are assumed to be randomly oriented in aqueous suspension, and all identical to each other. We compare several particle models of differing geometrical shape, but with identical forward scattering power and identical radii of gyration. We find that one of the ten observables shows particularly desirable properties as a general large-particle characterization parameter: it is nonzero for all structures, it approaches zero as particle size decreases, and it shows an angular dependence that distinguishes among models of different shape. Assuming incident light at 350 nm, it differentiates between different shaped particles with radii of gyration as small as 50 nm. Such particles are well below the optical resolution limit and about the size of many types of viruses.     

Higher-order structures of chromatin in solution.

Neutron scatter studies have been made on gently prepared chicken erythrocyte chromatin over a range of ionic strength. At low ionic strength the mass per unit length of the '10 nm nucleofilament corresponds to one nucleosome per 8--12 nm and a DNA packing ratio of between 6 and 9. From the contrast dependence of the cross-section radius of gyration of the nucleofilament the following parameters have been obtained; RgDNA' the cross-section radius of gyration (Rg) when DNA dominates the scatter; RgP, the cross-section Rg when protein dominates the scatter; Rc, the cross-section Rg at infinite contrast and alpha, the constant which describes the dependence of the cross-section Rg on contrast variation. From our understanding of the structure of the core particle, various arrangement of core particles in the nucleofilament have been tested. In models consistent with the above parameters the core particles are arranged edge-to-edge or with the faces of the core particles inclined to within 20 degrees to the axis of the nucleofilament. With increase of ionic strength the transition to the second-order chromatin structure has been followed. This gave the interesting result that above 20 microM NaCL or 0.4 mM MgCL2 the cross-section Rg increases abruptly to about 9 nm with a packing ratio of 0.2 nucleosome/mn and with further increase of ionic strength the Rg increases to 9.5 nm while the packing ratio increases threefold to 0.6 nucleosome/nm. This suggests a family of supercoils of nucleosomes which contract with increasing ionic strength. In its most contracted form the diameter of the hydrated supercoil has been found from the radial distribution function to be 34 nm. Models for the arrangements of core particles in the 34-nm supercoil are discussed.     

X-ray scattering study of the shape of the DNA region in nucleosome core particle with synchrotron radiation.

The structure of the DNA region in rat thymus nucleosome core particle has been studied by synchrotron X-ray scattering analysis and the contrast-variation technique has been applied to determine the contribution of the DNA to the total scatterings. Small-angle contrast-matching measurements show that the entire core particle and isolated histone octamers are contrast-matched by solvents containing 64 and 54% (w/w) sucrose, respectively. At a contrast of 54% sucrose, where the scattering of the DNA dominates, the scattering data extending to higher angle of about 0.05 A-1 have been collected from relatively concentrated solutions (10 mg/ml) of core particles and interpreted on the basis of the regular helical model for the DNA region. The model calculations show that the shape of the DNA around the histone core is approximately by 1.8 turns of regular helix of 42 A radius and 28 A pitch. These values for helical parameters of our model are in good agreement with those of the structure of DNA in crystallized nucleosome cores shown by earlier diffraction studies.     

Structure of the nucleosome core particle at 8 A resolution

The x-ray crystallographic structure of the nucleosome core particle has been determined using 8 A resolution diffraction data. The particle has a mean diameter of 106 A and a maximum thickness of 65 A in the superhelical axis direction. The longest chord through the histone core measures 85 A and is in a non-axial direction. The 1.87 turn superhelix consists of B-DNA with about 78 base pairs or 7.6 helical repeats per superhelical turn. The mean DNA helical repeat contains 10.2 +/- 0.05 base pairs and spans 35 A, slightly more than standard B-DNA. The superhelix varies several Angstroms in radius and pitch, and has three distinct domains of curvature (with radii of curvature of 60, 45 and 51 A). These regions are separated by localized sharper bends +/- 10 and +/- 40 base pairs from the center of the particle, resulting in an overall radius of curvature about 43 A. Compression of superhelical DNA grooves on the inner surface and expansion on the outer surface can be seen throughout the DNA electron density. This density has been fit with a double helical ribbon model providing groove width estimates of 12 +/- 1 A inside vs. 19 +/- 1 A outside for the major groove, and 8 +/- 1 A inside vs. 13 +/- 1 A outside for the minor groove. The histone core is primarily contained within the bounds defined by the superhelical DNA, contacting the DNA where the phosphate backbone faces in toward the core. Possible extensions of density between the gyres have been located, but these are below the significance level of the electron density map. In cross-section, a tripartite organization of the histone octamer is apparent, with the tetramer occupying the central region and the dimers at the extremes. Several extensions of histone density are present which form contacts between nucleosomes in the crystal, perhaps representing flexible or "tail" histone regions. The radius of gyration of the histone portion of the electron density is calculated to be 30.4 A (in reasonable agreement with solution scattering values), and the histone core volume in the map is 93% of its theoretical volume.     

Low resolution models of self-assembled histone fibers from X-ray diffraction studies.

X-ray diffraction data from self-assembled histone fibers are presented for three systems: H4, H3-H4, and the four core histones H2A, H2B, H3 and H4. These data have been obtained under conditions of high ionic strength and high protein concentration which are thought to promote histone conformation similar to that found in intact chromatin. The low angle equatorial scattering (R less than .05 A-1) is analysed, and, with additional constraints imposed by electron microscopy data, four low resolution fibrillar models are derived. Two features common to all the possible models are a maximum outer diameter of approximately 60 A and a subfibril diameter of approximately 25 A. It is the interference of the protein subfibrils across a central region of low electron density - a 10 A "hole" - which gives rise to the characteristic diffraction peak at 36 A. Possible relationships of the models of the histone fibers to the structure of the histone component of chromatin are suggested.     

Neutron scatter and diffraction techniques applied to nucleosome and chromatin structure

Neutron scatter and diffraction techniques have made substantial contributions to our understanding of the structure of the nucleosome, the structure of the 10-nm filament, the "10-nm----30-nm" filament transition, and the structure of the "34-nm" supercoil or solenoid of nucleosomes. Neutron techniques are unique in their properties, which allows for the separation of the spatial arrangements of histones and DNA in nucleosomes and chromatin. They have equally powerful applications in structural studies of any complex two-component biological system. A major success for the application of neutron techniques was the first clear proof that DNA was located on the outside of the histone octamer in the core particle. A full analysis of the neutron-scatter data gave the parameters of Table 3 and the low-resolution structure of the core particle in solution shown in Fig. 6. Initial low-resolution X-ray diffraction studies of core particle crystals gave a model with a lower DNA pitch of 2.7 nm. Higher-resolution X-ray diffraction studies now give a structure with a DNA pitch of 3.0 nm and a hole of 0.8 nm along the axis of the DNA supercoil. The neutron-scatter solution structure and the X-ray crystal structure of the core particle are thus in full agreement within the resolution of the neutron-scatter techniques. The model for the chromatosome is largely based on the structural parameters of the DNA supercoil in the core particle, nuclease digestion results showing protection of a 168-bp DNA length by histone H1 and H1 peptide, and the conformational properties of H1. The path of the DNA outside the chromatosome is not known, and this information is crucial for our understanding of higher chromatin structure. The interactions of the flexible basic and N- and C-terminal regions of H1 within chromatin and how these interactions are modulated by H1 phosphorylation are not known. The N- and C-terminal regions of H1 represent a new type of protein behavior, i.e., extensive protein domains that are designed not to fold up into secondary and tertiary protein structures. This behavior is increasingly observed in DNA and chromatin binding proteins, and in the case of the high-mobility group proteins HMG 14 and 17, the entire polypeptide chain is a flexible random coil over a wide range of solution, ionic, and pH conditions. It follows that the native conformations are probably imposed on these flexible domains and molecules by their binding sites in chromatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

X-ray diffraction study of a new crystal form of the nucleosome core showing higher resolution

Crystals have been grown of intact (unproteolysed) nucleosome cores from a variety of sources. The unit cells are all very similar, with one core particle per asymmetric unit. The X-ray diffraction patterns extend to about 5 Å in the direction perpendicular to the plane of the flat particle, and to somewhat less than this in other directions. The arrangement of particles in the unit cell has been deduced from Patterson projection maps, which also indicate the presence of a particle dyad. The data are consistent with the earlier proposed model for the core particle in which the 146 base-pairs of DNA are wound in about $\text{1}\text{3}\text{4}$ turns of superhelix about a histone octamer core.High angle diffuse X-ray scattering from the crystals shows that the DNA of the core particle is in the B form. The anisotropy of the diffuse scattering shows that the DNA is not firmly fixed to the histone core all along the superhelix path, but only over limited regions whose location correlates well with those in which the DNA is differentially protected against nuclease digestion.     

Small-angle X-ray scattering on malate synthase from baker's yeast. The native substrate-free enzyme and enzyme-substrate complexes.

Malate synthase from baker's yeast has been investigated in solution by the small-angle X-ray scattering technique. Size, shape and structure of the native substrate-free enzyme and of various enzyme-substrate complexes have been determined. As the enzyme was found to be rather unstable against X-rays, several precautions as well as sophisticated evaluation procedures had to be adopted to make sure that the results were not influenced by radiation damage. These included use of low primary intensity, short time of measurement, the presence of high concentrations of dithiothreitol, combined use of the conventional slit-collimation system and the new cone-collimation system. 1. For the native substrate-free enzyme the following molecular parameters could be established: radius of gyration R = 3.96 +/- 0.02 nm, maximum particle diameter D = 11.2 +/- 0.6 nm, radius of gyration of the thickness Rt = 1.04 +/- 0.04 nm, molecular weight Mr = 187000 +/- 3000, correlation volume Vc = 338 +/- 5 nm3, hydration x = 0.35 +/- 0.02 g/g, mean intersection length - l = 5.0 +/- 0.2 nm. Comparison of the experimental scattering curve with theoretical curves for various models showed that the enzyme is equivalent in scattering to an oblate ellipsoid of revolution rather than to a circular cylinder. The semiaxes of this ellipsoid are a = b = 6.06 nm and c = 2.21 nm. Thus with an axial ratio of about 1:0.36 the enzyme is of very anisometric shape. 2. Binding of the substrates (acetyl-CoA, glyoxylate) or the substrate analogue pyruvate causes slight structural changes of the enzyme. These changes are reflected mainly by a slight decrease of the radius of gyration (0.3--1.3%, as established both with the slit-smeared and the desmeared curves). Concomitantly there occurs a decrease of the maximum particle diameter and an increase of the radius of gyration of the thickness. These changes imply an increase of the axial ratio by 2.2--6.9%, i.e. substrate binding induces a decrease of anisometry. While the particle volume appears to be unchanged on binding glyoxylate or its analogue pyruvate, binding of acetyl-CoA causes slight changes of this parameter. In a similar manner the binding of acetyl-CoA leads to a slight enhancement of the molecular weight; this increase corresponds to the binding of 2.7 +/- 1 molecules of acetyl-CoA.     

Neutron scattering studies of chromatosomes

Neutron scattering data establish that the radius of gyration of the DNA in chicken erythrocyte chromatosome particles is significantly higher, by about 0.3 nm, than the radius of gyration of the DNA in the core particle. Corresponding information of the radius of gyration of the protein component in the chromatosomes (3.75 nm) indicated an enlargement, compared to the radius of gyration of the octamer of histone proteins both in core particles and in the histone octamer stabilised in 2 M NaCl (3.25 nm). From the latter data, we could calculate the distance in the chromatosome between the centre of mass of the linker histone and the histone octamer as 5.5 nm. These results impose severe limitations for the organisation of the 22 bp extra DNA and the possible location of H1/H5 in the chromatosome, implying that the H1/H5 is close to the centre turn of the core particle DNA.     

Reconstruction of protein form with X-ray solution scattering and a genetic algorithm

We have reconstructed, from experimental approximately 2 nm resolution X-ray solution scattering profiles, the corresponding shapes and sizes of myoglobin, troponin C, spermadhesin PSP-I/PSP-II, chymotrypsinogen A, superoxide dismutase, ovalbumin, tubulin, nitrite reductase, catalase, the structural change of troponin C upon dissociation of the two high affinity Ca(2+), and the solution model structure of a tandem pair of fibronectin type III cytoplasmic domains of integrin alpha6beta4 before determination of its crystal structure. To this purpose we have designed a new genetic algorithm which gradually explores a discrete search space and evolves convergent models made of several hundred beads (down to 0.3 nm radius) best fitting the scattering profile upon Debye calculation, without geometrical constraints or penalty for loose beads. This is a procedure of effective numerical transformation of the one-dimensional scattering profiles into three-dimensional model structures. The number of beads in models is correlated with the protein molecular mass (with one exception). The shape and approximate dimensions of each protein have been retrieved by a set of ten solution models, essentially superimposable with the available crystal structures.     

Chromatin fiber dimensions and nucleosome orientation: a neutron scattering investigation.

Chromatin fibers were studied in solutions of mM monovalent salt by small angle neutron scattering. The variation of the cross section radius of gyration with H2O/D2O contrast shows that DNA is at much larger average radial distances from the fiber axis than histone. Consequently, the coils of DNA in a core particle must be approximately parallel to the fiber direction. The radii of gyration suggest that the maximum diameter of chromatin and nucleosomes is approximately 14 nm and that the DNA id distributed in two radial layers. The concentration dependence of the scattering maxima near 14 nm spacings furnishes independent support for a 14 nm external diameter and can be interpreted by a double DNA layer configuration.     

Scatter analysis of discrete-sized chromatin fragments favours a cylindrical organization

Fragments of chromatin containing 23 +/- 2.5 nucleosomes have been fractionated after light nuclease treatment of chicken erythrocyte nuclei. Low-angle scattering measures the total z-average radius of gyration of the already well-defined particles and the shape of scatter curves can be compared with three-dimensional analysis as opposed to cross-section analysis of long chromatin fragments. The data show that the particles are not spherical, have no detectable hole in the center of the structure and are best represented by a solid rod-like shape such as that generated by a coil of nucleosomes with the centre perhaps filled with linker DNA and histone H1/H5. 23 nucleosome fragments, where the DNA is partially fragmented, have near-identical scatter curves to the above-defined intact particles, indicating the primary importance of histone proteins in maintaining the integrity of the chromatin higher-order structure. Neutron scattering shows the radii of gyration to be contrast-independent, which fits in with the model calculations for solenoids. Particles with fragmented DNA and the intact particles, therefore, behave as sections of a solenoidal higher-order structure and possibly are observed as "superbeads' only during the folding and unfolding pathways of nucleosome multimers.     

Lumbricus terrestris hemoglobin: A comparison of small-angle X-ray scattering and cryoelectron microscopy data

The quaternary structure of Lumbricus terrestris hemoglobin was investigated by small-angle x-ray scattering (SAXS). Based on the SAXS data from several independent experiments, a three-dimensional (3D) consensus model was established to simulate the solution structure of this complex protein at low resolution (about 3 nm) and to yield the particle dimensions. The model is built up from a large number of small spheres of different weights, a result of the two-step procedure used to calculate the SAXS model. It accounts for the arrangement of 12 subunits in a hexagonal bilayer structure and for an additional central unit of cylinder-like shape. This model provides an excellent fit of the experimental scattering curve of the protein up to h = 1 nm⁻¹ and a nearly perfect fit of the experimental distance distribution function p(r) in the whole range. Scattering curves and p(r) functions were also calculated for low-resolution models based on 3D reconstructions obtained by cryoelectron microscopy (EM). The calculated functions of these models also provide a very good fit of the experimental scattering curve (even at h > 1 nm⁻¹) and p(r) function, if hydration is taken into account and the original model coordinates are slightly rescaled. The comparison of models reveals that both the SAXS-based and the EM-based model lead to a similar simulation of the protein structure and to similar particle dimensions. The essential differences between the models concern the hexagonal bilayer arrangement (eclipsed in the SAXS model, one layer slightly rotated in the EM model), and the mass distribution, mainly on the surface and in the central part of the protein complex. © John Wiley & Sons, Inc. Biopoly 45: 289–298, 1998     

Determination of domain structure of proteins from X-ray solution scattering

An ab initio method for building structural models of proteins from x-ray solution scattering data is presented. Simulated annealing is employed to find a chain-compatible spatial distribution of dummy residues which fits the experimental scattering pattern up to a resolution of 0.5 nm. The efficiency of the method is illustrated by the ab initio reconstruction of models of several proteins, with known and unknown crystal structure, from experimental scattering data. The new method substantially improves the resolution and reliability of models derived from scattering data and makes solution scattering a useful technique in large-scale structural characterization of proteins.     

Solution structure of a short DNA fragment studied by neutron scattering

The solution structure of a DNA fragment of 130 base pairs and known sequence has been investigated by neutron small-angle scattering. In 0.1 M NaCl, the overall structure of the DNA fragment which contains the strong promoter A1 of the Escherichia coli phage T7 agrees with that expected for B-DNA. The neutron scattering curve is well fitted by that of a rigid rod with a length of 44 nm and a diameter of 2 nm. The results were confirmed by quasi-elastic light scattering and analytical centrifugation. The neutron measurements in H2O and D2O buffer reveal a cross-sectional inhomogeneity not detected by X-ray small-angle scattering. This inhomogeneity is caused by the hydration layer around the DNA core and not by the helical structure. The primary solvent shell has a density increased by at least 4-9% compared to bulk water.     

Structure of bacteriophage T7. Small-angle X-ray and neutron scattering study.

Small-angle x-ray and neutron scattering techniques were applied to bacteriophage T7 solutions at different scattering densities. Scattering curves determined under a variety of experimental conditions were used to derive a set of parameters characterizing the shape, size, and weight of the whole phage particle and of its DNA and protein components. The T7 head has an icosahedral shape with an edge of 37.7 +/- 0.5 nm, a volume of (12.0 +/- 1.0) x 10(4) nm3, and a small tail amounting to 6--7% of the head volume. The intraphage DNA region is most probably a hollow sphere. The best fit to the data was obtained with a model in which the hollow sphere filled with a protein core with a diameter of 24 nm. The average degree of swelling (i.e., the ratio of the hydrated to the dry volume) of the particle is 2.3; the degree of swelling of the DNA component is higher, 3.2, and that of the protein part is lower, 1.2.