The structure of nucleohistones and nucleoprotamines

Abstract

Unoriented gels and solutions of DNA, nucleohistone (DNH) and nuoleoprotamine (DNP) have been studied systematically as a function of water content and ionic strength, by small-angle X-ray diffraction techniques. This experimental approach, which is formally equivalent to a phase diagram analysis, permits the investigation of the structure of biological macromolecules under conditions similar to those that exist in the living cell; furthermore, intact biological samples can sometimes be studied by the same technique. Dilute isotropic solutions, in which the solute is randomly dispersed, give a diffuse halo around the incident beam. When the solute particles are rod-like in shape the scattered intensity has a typical angular distribution from which the radius of gyration of the rods around their axis can be determined; if the experiments are carried out on an absolute scale, the mass per unit length of the rods can be determined as well. At higher concentration organized liquid-crystalline structures are encountered, the X-ray diffraction patterns of which are formed by bands or sharp lines. A systematic study, as a function of concentration, of systems containing DNA, DNH and DNP reveals the existence of various types of structure, each characterizing one phase; some of the parameters of each structure can be determined. The results obtained with DNA-water systems, containing variable amounts of electrolyte, show that at all concentrations from 1 to 50% the DNA molecules are rod-like in shape and that the mass per unit length of the rods is in excellent agreement with the Watson-Crick model. Furthermore, at high concentrations the DNA rods are aligned with their axes parallel and organized in hexagonal arrays, each molecule being separated from the others by the solvent. The molecules are effectively independent, the distance between them depending only on the concentration. The structures observed in DNH are more complex. In the dilute solutions rod-like particles are found, the mass per unit length and the axial radius of gyration of which are consistent with a bundle of four DNA molecules surrounded by histone. At higher concentrations, several liquid crystalline phases are observed, some of which give fairly sharp reflections at high spacings (50 Å). One phase, which exists at concentrations close to 38%, is formed by parallel associations of pairs of DNA molecules accompanied by histone. Another phase, present in the concentration range 55 to 65%, is a hexagonal array of parallel DNA rods with water and histones filling the gap between the DNA molecules. At still higher concentration (circa >70%) another phase is found, the structure of which is not discussed here. The various phases are observed in a variety of DNH samples; the salient features of the different structures are shown to be typical of the nucleohistones. In the DNP-water system two phases are found: the isotropic dilute solution, which exists at very low DNP concentration and cannot be studied by the X-ray techniques, and a hexagonal organization of DNA molecules, with water and protamine in the interstices. The amount of water that can be taken up by this structure is much smaller than in the case of DNA and DNH. The structure of DNP is more ionic-strength dependent than either DNA or DNH. These structures are highly specific for DNH and DNP; no intermediate structure is found in systems containing a mixture of DNH and DNP. Furthermore, it is shown that the X-ray pictures obtained with intact fowl erythrocyte nuclei and with trout sperm heads are very similar to those either of DNH-water or of DNP-water systems, taken at appropriate concentrations. The results of this small-angle X-ray scattering study are compared with the previous X-ray scattering investigations of oriented nucleoprotein fibres and with the information provided by hydrodynamic and electron microscope techniques.