Low-angle X-ray scattering analysis of the Thermoplasma acidophilum nucleoprotein subunit

A DNA-protein complex isolated from Thermoplasma acidophilum has been examined using low-angle X-ray scattering measurements. In agreement with the results of electron-microscopic studies a diamter of 5.5 nm is deduced. Finally, a simplified model of the DNA-protein particles is discussed postulating a kinked DNA.     

Low-angle neutron scattering analysis of Na/K-ATPase in detergent solution

Purified Na/K-ATPase from guinea pig renal outer medulla has been delipidated and solubilized in Brij 58 (polyoxyethylene ether; C-16, E-20). At a concentration of 2 mg of Brij 58/mg of protein, about one-half the enzyme complement was solubilized and almost 50% of Na/K-ATPase activity was retained by the enzyme-micelle complex. Guinier plots of the neutron scattering profiles yielded no evidence of heterogeneity with respect to subunit composition or the state of aggregation in the solubilized oligomers. Contrast matching with D2O used to obtain estimates of the molecular weight of the micellar form of Na/K-ATPase gave a mean value of 310,000 +/- 42,700, which corresponds to an alpha 2 beta 2 tetramer. A Stuhrmann plot of the neutron scattering data yielded an estimated radius of gyration of 67 A. The Stuhrmann plot also indicated an asymmetrical distribution of neutron scattering density. On the basis of the Stuhrmann plot parameters, the estimated molecular weight, and the radius of gyration, a low-resolution model was formulated of the oligomeric unit of Na/K-ATPase.     

Electron microscopy of chromatin subunit particles

Electron microscope studies of PS-particles obtained by partial nuclease digestion of calf thymus chromatin shows them to be very similar in size and shape to particles which have been detected in native chromatin. Furthermore, measurement of the spacing between pairs of such particles, together with the known length of the DNA coiled in the particles gives an estimate of about 200 base pairs for the chromatin “subunit”.     

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.     

The subunit structure of chromatin from Physarum polycephalum.

Nucleosome DNA repeat lengths in Physarum chromatin, determined by nuclease digestion experiments, are shorter than those observed in most mammalian chromatin and longer than those reported for chromatin of certain other lower eukaryotes. After digestion with staphylococcal nuclease for short periods of time an average repeat length of 190 base pairs is measured. After more extensive digestion an average repeat length of 172 base pairs is measured. Upon prolonged digestion DNA is degraded to an average monomer subunit length of 160 base pairs, with only a small amount of DNA found in lengths of 130 base pairs or smaller. Mathematical analysis of the data suggests that the Physarum nucleosome DNA repeat comprises a protected DNA segment of about 159 base pairs with a nuclease-accessible interconnecting segment which ranges from 13 to 31 base pairs. The spacing data are compatible with measurements from electron micrographs of Physarum chromatin.     

Nucleoprotein chromatin subunit from Physarum polycephalum

The nucleoproteins resulting from digestion of the nuclei of the true slime mold Pysarum polycephalum with micrococcal nuclease have been resolved according to the size classes in linear sucrose gradients containg 0.5 M NaCl, and analysed for DNA, RNA and protein content. The basic nucleoprotein subunit has been found to contain a DNA fragment of about 150--170 base pairs complexed with an approximately equal amount, on a weight basis, of basic proteins and a relatively small amount of non-histone proteins (about 35% of the amount of DNA). Higher nucleoprotein oligomers were shown to contain spacer DNA fragments between adjacent subunits and a considerably higher ratio of non-histone proteins to DNA than the basic subunit. Both the basic subunit and higher nucleoprotein oligomers of Physarum chromatin contain some amount of tightly bound RNA. However, in contrast to the distribution of the non-histone proteins, the ratio of RNA to RNA is similar in both fractions.     

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     

Picosecond molecular motions in bacteriorhodopsin from neutron scattering.

The characteristics of internal molecular motions of bacteriorhodopsin in the purple membrane have been studied by quasielastic incoherent neutron scattering. Because of the quasihomogeneous distribution of hydrogen atoms in biological molecules, this technique enables one to study a wide variety of intramolecular motions, especially those occurring in the picosecond to nanosecond time scale. We performed measurements at different energy resolutions with samples at various hydration levels within a temperature range of 10-300 K. The analysis of the data revealed a dynamical transition at temperatures Td between 180 K and 220 K for all motions resolved at time scales ranging from 0.1 to a few hundred picoseconds. Whereas below Td the motions are purely vibrational, they are predominantly diffusive above Td, characterized by an enormously broad distribution of correlation times. The variation of the hydration level, on the other hand, mainly affects motions slower than a few picoseconds.     

Shape and subunit organisation of the DNA methyltransferase M.AhdI by small-angle neutron scattering

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects DNA from cleavage by the endonuclease. The DNA methyltransferase M.AhdI is a 170 kDa tetramer with the stoichiometry M(2)S(2) and has properties typical of a type I MTase. The M.AhdI enzyme has been prepared with deuterated S subunits, to allow contrast variation using small-angle neutron scattering (SANS) methods. The SANS data were collected in a number of (1)H:(2)H solvent contrasts to allow matching of one or other of the subunits in the multisubunit enzyme. The radius of gyration (R(g)) and maximum dimensions (D(max)) of the M subunits in situ in the multisubunit enzyme (50 A and 190 A, respectively) are close of those of the entire MTase (51 A and 190 A). In contrast, the S subunits in situ have experimentally determined values of R(g)=35 A and D(max)=110 A, indicating their more central location in the enzyme. Ab initio reconstruction methods yield a low-resolution structural model of the shape and subunit organization of M.AhdI, in which the Z-shaped structure of the S subunit dimer can be discerned. In contrast, the M subunits form a much more elongated and extended structure. The core of the MTase comprises the two S subunits and the globular regions of the two M subunits, with the extended portion of the M subunits most probably forming highly mobile regions at the outer extremities, which collapse around the DNA when the MTase binds.     

Interparticle effects in low-angle x-ray and neutron diffraction from chromatin.

Published diffraction data are critically reviewed, and replotted in a new way to show the variation with concentration of the 8- to 25- nm diffraction maximum. Most of the early data are found to be consistent with a single model for a liquid-type array of mutually repulsive particles, whose molecular weight is calculated to be that of a nucleosome or possibly a dimer. The data for all but the highest concentrations, where distortion due to dehydration is possible, support no particular model for the higher-order coiling of chains of nucleosomes, and cannot be used to support models for "native" chromatin. Only in the presence of excess salts or after isolation with polyamines is there aggregation in solution of nucleosomes, which then give peaks at 11 and 5.5 nm that do not change much with concentration. Recent work by the authors confirms that under some conditions nucleosome undergo a transition to a state whose diffraction is consistent with hexagonal packing of extended DNA to which histones are still attached. This state is probably responsible for much of the strong 2.7-nm peak previously obtained from certain samples, which was in some cases assigned to nucleosome structure. Only the peak at 3.7 nm is clearly attributable to the form factor of the isolated native nucleosome.     

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.     

Subunit associations among chromatin particles.

The self-association of oligonucleosomal chromatin particles in solution has been studied by light scattering and sedimentation. In the absence of magnesium ions no association is observed. In the presence of 70mM sodium or 2mM magnesium ions mono, di, tri and tetranucleosomes self-associate only if they contain bound histone 1. This association leads to the formation of compact aggregates and is continuous and non-cooperative. The relevance to higher order arrangements of nucleosomes is discussed.     

Physical characteristics of human transferrin from small angle neutron scattering.

The technique of small angle neutron scattering has been used to determine the molecular shape, the volume, and the molecular weight of pooled human transferrin in an aqueous solution isotonic with blood. Analysis of the measurements assuming a spheroidal molecular shape indicates that an oblate spheroid with semi-axes of length 46.6 +/- 1.4, 46.6 +/- 1.4 and 15.8 +/- 3.8 A, and a molecular volume of (144 +/- 45) X 10(3) A3 is the best simple approximation to the shape of the transferrin molecule. The radius of gyration, Rg, determined from a Guinier plot is 30.25 +/- 0.49 A, in agreement with Rg calculated for the oblate spheroidal shape. The molecular weight is determined to be (75 +/- 5) X 10(3). The shape-independent molecular volume is found to be (98 +/- 10) X 10(3) A3. The difference in the two volumes suggests that transferrin is not a uniform spheroid but may have a more complex shape.     

The subunit structure of mouse satellite chromatin.

Atomic coordinates are presented for the 3740 atoms other than hydrogen in the dimeric molecule of chicken muscle triose phosphate isomerase. They are derived from an electron-density map at 2.5Åresolution, interpreted in terms of the known amino-acid sequence, and they have been adjusted systematically to give stereochemically appropriate bond lengths and angles.     

Chromatin subunit small angle neutron scattering: A DNA rich coil surrounds a protein-DNA core

The small angle neutron scattering radii of gyration of 185 base pair subunits have been determined in H₂O and D₂O These values suggest that the outer diameter is 120 to 150Å. The results are not consistent with models in which all of the DNA is in an external shell. The neutron scattering profiles are in good agreement with a model based upon freeze etching electron microscopy (4) having two concentric coils of DNA with 80Åand 150Åexternal diameters.     

Kinetic asymmetry of subunit exchange of homooligomeric protein as revealed by deuteration-assisted small-angle neutron scattering

We developed a novel, to our knowledge, technique for real-time monitoring of subunit exchange in homooligomeric proteins, using deuteration-assisted small-angle neutron scattering (SANS), and applied it to the tetradecamer of the proteasome α7 subunit. Isotopically normal and deuterated tetradecamers exhibited identical SANS profiles in 81% D₂O solution. After mixing these solutions, the isotope sensitive SANS intensity in the low-q region gradually decreased, indicating subunit exchange, whereas the small-angle x-ray scattering profile remained unchanged confirming the structural integrity of the tetradecamer particles during the exchange. Kinetic analysis of zero-angle scattering intensity indicated that 1), only two of the 14 subunits were exchanged in each tetradecamer and 2), the exchange process involves at least two steps. This study underscores the usefulness of deuteration-assisted SANS, which can provide quantitative information not only on the molecular sizes and shapes of homooligomeric proteins, but also on their kinetic properties.     

Hydration of NaDNA by neutron quasi-elastic scattering.

Preliminary results of neutron quasi-elastic scattering experiments are reported for hydrated paracrystals of sodium deoxyribonucleic acid (NaDNA). The samples were investigated at two water contents: 3.5 +/- 1.0 and 9.5 +/- 1.5 mol H2O per mole nucleotide. The results of the scattering experiments were almost independent of whether the NaDNA fibers were oriented parallel or perpendicular to the momentum transfer. The data indicate that at the lower hydration the water molecules do not diffuse appreciably on the time scale of the neutron measurements (approximately 3 X 10(-10) s). At the higher hydration the water molecules diffuse isotropically in a sphere of 9 A in diameter with a diffusion coefficient of (5 +/- 2) X 10(-6) cm2 s-1.