Regulation of the higher-order structure of chromatin by histones H1 and H5

Chicken erythrocyte chromatins containing a single species of linker histone, H1 or H5, have been prepared, using reassembly techniques developed previously. The reconstituted complexes possess the conformation of native chicken erythrocyte chromatin, as judged by chemical and structural criteria; saturation is reached when two molecules of linker histone are bound per nucleosome, as in native erythrocyte chromatin, which the resulting material resembles in its appearance in the electron microscope and quantitatively in its linear condensation factor relative to free DNA. The periodicity of micrococcal nuclease-sensitive sites in the linker regions associated with histone H1 or H5 is 10.4 base pairs, suggesting that the spatial organization of the linker region in the higher-order structure of chromatin is similar to that in isolated nucleosomes. The susceptible sites are cut at differing frequencies, as previously found for the nucleosome cores, leading to a characteristic distribution of intensities in the digests. The scission frequency of sites in the linker DNA depends additionally on the identity of the linker histone, suggesting that the higher-order structure is subject to secondary modulation by the associated histones.     

Investigation of human erythrocyte superoxide dismutase by 1H nuclear-magnetic-resonance spectroscopy.

The 170MHZ 1 H n.m.r. spectra of the Cu(II)/Zn(II), Cu(I)/Zn(II) and apo- forms of human erythrocyte superoxide dismutase (EC 1.15.1.1) are reported. Resonances are assigned to the C-2 and C-4 protons of histidine residues in the active site, and it is suggested that five or six histidine residues serve as ligands to the metal ions in each subunit of the enzyme. The remaining assigned resonances are associated with histidine-41, N-terminal N-acetyl group, histidine- 108 and cysteine- 109. A comparison of the n.m.r. spectra of human and bovine superoxide dismutases suggests significant structural homology.     

Conformation and folding in histones H1 and H5

Denatured histones H1 and H5 can be readily refolded on salt addition. Their digestion by trypsin leads to limit peptides of about 80 residues having the same nmr and CD spectra as those of the intact parent histones. Scanning microcalorimetry shows that (1) the folded structures of H1 and H5 are located entirely in their limit peptides; (2) both have values of the specific denaturation enthalpy typical for small globular proteins; and that (3) both exhibit a classic “2-state” transition (ΔH = ΔH). The heat-denaturation profiles of H5 measured using intrinsic and extrinsic Cotton effect and side-chain nmr peaks do not coincide at all. Only the intrinsic Cotton effects give a T_m and ΔH close to that from microcalorimetry. We conclude that these proteins exhibit large-scale side-chain motions that precede the macroscopic cooperative transition.     

In vivo phosphorylation of histones H1 and H5 in calf thymus and chicken erythrocyte as studied by 31P nuclear magnetic resonance spectroscopy

The 31P NMR method was first applied to characterize in vivo phosphorylation of H1 and H5 in calf thymus and chicken erythrocytes as well as in vitro phosphorylation of H1 and H5 by cAMP-dependent protein kinase. The amino acid residues phosphorylated in vivo in the histones were exclusively serine residues, and the mole fraction of phosphoserine was estimated to be 0.34 and 0.27 per molecule of calf thymus H1 and chicken erythrocyte H5, respectively. Interestingly, chicken erythrocyte H1 was not phosphorylated in vivo. Three H1 subtypes from calf thymus H1 varied in the 31P NMR spectra, and the bisected fragments of calf thymus H1 and chicken erythrocyte H5 exhibited characteristic spectral patterns, indicating that there are considerable diversities of the degree of phosphorylation and phosphorylation sites in very-lysine-rich histones. Furthermore, it was found that the microenvironment of phosphoserine residues phosphorylated in vivo in calf thymus H1 and chicken erythrocyte H5 is quite distinct from that of phosphoserine residues phosphorylated in vitro by bovine heart cAMP-dependent protein kinase.     

Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy

Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities. The secondary structure of this protein in solution has been elucidated by a qualitative interpretation of nuclear Overhauser effects, hydrogen exchange data, and 3JHNH alpha coupling constants. A clear correlation between the 13C alpha chemical shift and secondary structure is found. The secondary structure in the two globular domains of Drosophila CaM in solution is essentially identical with that of the X-ray crystal structure of mammalian CaM [Babu, Y., Bugg, C. E., & Cook, W.J. (1988) J. Mol. Biol. 204, 191-204], which consists of two pairs of a "helix-loop-helix" motif in each globular domain. The existence of a short antiparallel beta-sheet between the two loops in each domain has been confirmed. The eight alpha-helix segments identified from the NMR data are located at Glu-6 to Phe-19, Thr-29 to Ser-38, Glu-45 to Glu-54, Phe-65 to Lys-77, Glu-82 to Asp-93, Ala-102 to Asn-111, Asp-118 to Glu-127, and Tyr-138 to Thr-146. Although the crystal structure has a long "central helix" from Phe-65 to Phe-92 that connects the two globular domains, NMR data indicate that residues Asp-78 to Ser-81 of this central helix adopt a nonhelical conformation with considerable flexibility.     

Quantitative study of secondary structure of histones H1, H2A, and H4 in solution by infrared spectroscopy.

The secondary structure of histones H1, H2A, and H4 (F1, F2a2, and F2a1) has been quantitatively studied in heavy water (2H2O) solutions in a wide range of histone concentration, p2H, and concentration of sodium chloride using an improved infrared spectroscopy method. Under all conditions there are about 5--10% of alpha helix. Conditions favourable for aggregation induce formation of antiparallel pleated sheet structure to an extent of about 15% in H1 and H2A and about 30% in H4. When the p2H and concentration of NaCl are in the physiological range, there is the same content of this structure in H2A and H4 and none in H1.     

Secondary structure of calf-thymus histone H1 by means of 13C-NMR spectroscopy

¹³C-nmr spectroscopy was used to determine the location of the structured segment of the chain of histone H1 in water and NaCl solutions. The segment found (41–89) is contained in the area proposed by others on the basis of ¹H-nmr studies. The number of basic groups left in the mobile ends of the protein matches the number of bases of internucleosomal DNA.     

Separation of nucleosomes containing histones H1 and H5

Hen erythrocyte chromatin was digested with staphylococcal nuclease and fractionated by electrophoresis in polyacrylamide gels. Instead of the three bands described for mouse carcinoma chromatin, four main discrete components (MN1, MN2, MN2E and MN3) were resolved in the mononucleosome fraction of erythrocyte chromatin. MN2 contained all five histones and a DNA fragment of 165–180 base pairs. MN2E comprised four nucleosomal histones plus histone H5 (but not H1) and a DNA fragment of 170–190 base pairs. The relatively nuclease resistant MN3 fraction of erythrocyte nucleosomes contained H1 but no H5 histone. A more accurate analysis of the MN2 fraction in mouse carcinoma nucleosomes revealed some additional microheterogeneity depending on the presence of two different subfractions of H1.     

Alpha-helix in the carboxy-terminal domains of histones H1 and H5.

Although the carboxy-terminal domains of histones H1 and H5 exist as random-coil in aqueous solution, secondary structure prediction suggests that this region has a high potential for alpha-helix formation. We have measured CD spectra in various conditions known to stabilize alpha-helices, to determine whether this potential can be realized in an appropriate environment. Trifluoroethanol increases the helix contents of H1, H5 and their carboxy-terminal fragments, presumably through promotion of axial hydrogen bonding. Sodium perchlorate is also effective and better than sodium chloride, suggesting stabilization by binding of bulky perchlorate ions rather than simple charge screening. Extrapolating from these measurements in solution, and taking into account the occurrence of proline residues throughout the carboxy-terminal domain, we propose that binding to DNA stabilizes helical segments in the carboxy-terminal domains of histones H1 and H5, and that it is this structured form of the domain that is functionally important in chromatin.     

The 1H nuclear-magnetic-resonance spectroscopy of cobalt(II)-beta-lactamase II.

The 1H n.m.r. spectra of beta-lactamase II in the presence of Co(II) were studied. Analysis of the spectra suggests that Co(II) binds at the same two metal-binding sites as does Zn(II). The binding of Co(II) at the first site is much weaker than the binding of Zn(II) at this site, whereas the binding of Co(II) at the second site is tighter than the binding of Zn(II). The binding of Co(II) to the mono-zinc(II)-enzyme caused only one marked change in the spectrum, namely a decrease in the intensity of the resonances assigned to the C-2 and C-4 protons of one histidine residue (residue E). However, when the spectra of the apoenzyme and the Co(II)-enzyme were compared, there were many differences. A significant fraction of the protons in the whole molecule are affected by the binding of Co(II) at the first metal-ion-binding site (where the ligands are the enzyme's sole thiol group and three histidine residues). This may be because the first site is internal, or because of a difference in conformation between the apoenzyme and the mono-Co(II)-enzyme. The second site may be located on the surface of the molecule.     

Carbon-13 nuclear-magnetic-resonance spectroscopy of whole cells and of cytochrome C from Neurospora crass grown with (S-Me-13C)methionine.

Neurospora crassa cytochrome C biosynthetically labelled with [S-Me-13C]methionine was prepared and analysed by 13C nuclear-magnetic-resonance spectroscopy. The methyl group of methionine is extensively incorporated into an N-trimethyl-lysine-72 residue arise from S-adenosylmethionine transmethylation, and that the methyl carbons of methionine residues are sufficiently close to the haem centre to experience chemical shifts from the ring currents of the tetrapyrrole pi electrons and broadening due to binding of methionine-80 with the haem, as well as interaction of the S-E113C]methyl groups with the paramagnetic iron centre. Although whole cells of the labelled Neurospora produced a 13C resonance at the expected position for the methionyl methyl group most of the methyl label was diverted into N-tetra-alkyl ammonium compounds. After an active state of growth these labelled N-methyl compounds appear, in the main, to be low-molecular-weight derivatives of choline which, if associated with membrane, are in a sufficiently fluid environment to have short rotational correlation times. During a subsequent dormant growth period these compounds become associated to some extent with relatively more immobile phases as a result of membrane binding or an increase in membrane rigidity.     

Effect of ions on phospholipid layer structure as indicated by Raman spectroscopy.

Various anions and cations are found to induce changes in the layered structure of phosphatidylcholine-water systems as indicated by Raman Spectroscopy. From the ratio of Raman intensities, I1064/I1089, it is inferred that dipositive ions decrease the proportion of gauche character in the hydrocarbon chains, with the relative influence being: Ba2+ less than Mg2+ less than Ca2+ similar to Cd2+. Unipositive ions (Li+, K+ and Na+) produce no observed changes in the Raman spectrum of the lecithin dispersion. The proportion of gauche character of the hydrocarbon chains is found to be nearly independent of the anion for: Br-, Cl-, acetate-, I-, ClO4-, CNS- and SO42-. Dispersions prepared with a solution of KI+I2 produced Raman spectra in which the 1089cm-1 peak, which is characteristic of random lipid chains, was greatly intensified, presumably because of the presence of I3- which is known to penetrate the lipid lamellae. The observed trends are discussed.     

Footprinting of linker histones H5 and H1 on the nucleosome.

DNase I has been used to footprint the linker histones H5 and H1 on the nucleosome of chicken erythrocyte chromatin. Rate constants have been derived for digestion at the principal sites of attack on chromatosome length DNA (168 bp), located about 10 bp apart, and compared with those observed for linker histone-depleted chromatosomes. Complete protection was found for site S7 on the dyad axis and decreasing partial protection seen at symmetrically positioned sites on each side of S7. Strong, but not complete protection was noted at S14, the site corresponding to the end of the core particle, situated less than 1/4 of a turn away from the dyad. Uniform partial protection was observed for sites S2, S3, S4 and S10, S12 on the far side of the chromatosome. The simplest interpretation of these results is that the globular domain of H5/H1 is responsible for the protection at S7, whilst extended N- and C-domains give rise to the partial protection at sites away from the dyad axis.     

Amino acid sequence homologies between H1 and H5 histones

Ehrlich ascites cell microsomes catalyze the exchange of the acyl group of acyl dihydroxyacetone phosphate with free fatty acids. The reaction does not require ATP and CoA.     

Time-resolved 1H and 13C NMR spectroscopy for detailed analyses of the Azotobacter vinelandii mannuronan C-5 epimerase reaction

AlgE2, AlgE4, and AlgE6 are members of a family of mannuronan C-5 epimerases encoded by Azotobacter vinelandii, and are active in the biosynthesis of alginate, where they catalyze the post-polymerization conversion of beta-D-mannuronic acid residues into alpha-L-guluronic acid residues. To study the kinetics and mode of action of these enzymes, homopolymeric mannuronan and other alginate samples with various composition were epimerized by letting the enzymatic reaction take place in an NMR tube. Series of 1H NMR spectra were recorded to obtain a time-resolved picture of the epimerization progress and the formation of specific monomer sequences. Starting from mannuronan, guluronic acid contents of up to 82% were introduced by the enzymes, and the product specificity, substrate selectivity, and reaction rates have been investigated. To obtain direct information of the GulA-block formation, similar experiments were performed using a 13C-1-enriched mannuronan as substrate. The NMR results were found to be in good agreement with data obtained by a radioisotope assay based on 3H-5-labeled substrates.     

Aspects of the chemical structure of soil organic materials as revealed by solid-state13C NMR spectroscopy

Solid-state cross-polarisation/magic-angle-spinning³C nuclear magnetic resonance (CP/MAS¹³C NMR) spectroscopy was used to characterise semi-quantitatively the organic materials contained in particle size and density fractions isolated from five different mineral soils: two Mollisols, two Oxisols and an Andosol. The acquired spectra were analysed to determine the relative proportion of carboxyl, aromatic, O-alkyl and alkyl carbon contained in each fraction. Although similar types of carbon were present in all of the fractions analysed, an influence of both soil type and particle size was evident.The chemical structure of the organic materials contained in the particle size fractions isolated from the Andosol was similar; however, for the Mollisols and Oxisols, the content of O-alkyl, aromatic and alkyl carbon was greatest in the coarse, intermediate and fine fractions, respectively. The compositional differences noted in progressing from the coarser to finer particle size fractions in the Mollisols and Oxisols were consistent with the changes noted in other studies where CP/MAS¹³C NMR was used to monitor the decomposition of natural organic materials. Changes in the C:N ratio of the particle size fractions supported the proposal that the extent of decomposition of the organic materials contained in the fine fractions was greater than that contained in the coarse fractions. The increased content of aromatic and alkyl carbon in the intermediate size fractions could be explained completely by a selective preservation mechanism; however, the further accumulation of alkyl carbon in the clay fractions appeared to result from both a selective preservation and anin situ synthesis.The largest compositional differences noted for the entire organic fraction of the five soils were observed between soil orders. The differences within orders were smaller. The Mollisols and the Andosol were both dominated by O-alkyl carbon but the Andosol had a lower alkyl carbon content. The Oxisols were dominated by both O-alkyl and alkyl carbon.A model describing the oxidative decomposition of plant materials in mineral soils is proposed and used to explain the influence of soil order and particle size on the chemical composition of soil organic matter in terms of its extent of decomposition and bioavailability.