Mobile histone tails in nucleosomes. Assignments of mobile segments and investigations of their role in chromatin folding

The 13C NMR spectrum of isolated nucleosome core particles contains many sharp resonances, including resonances of alpha- and beta-carbons, indicating that certain terminal segments of histones rich in basic residues are highly mobile (Hilliard, R. R., Jr., Smith, R. M., and Rill, R. L. (1986) J. Biol. Chem. 261, 5992-5998). Specific histone termini can be removed sequentially from nucleosome core particles by mild treatment with alpha-chymotrypsin or chymotrypsin plus trypsin (Rosenberg, N. L., Smith. R. M., and Rill, R. L. (1986) J. Biol. Chem. 261, 12375-12383). Comparisons of the 13C NMR spectra of native and several partially proteolyzed core particles indicated that a minimum of residues 1-20 of H3 and 1-11 and 118-128 of H2a are contained in mobile segments of native cores. H4 did not appear to contribute to the resonances from mobile histone segments, but a possible contribution of H2b residues 1-16 could not be ruled out. The 13C NMR spectra of oligonucleosomes containing and lacking lysine-rich histones (H1, H5) were similar to each other and to that of native nucleosome cores both when the oligonucleosomes were in an extended conformation at low ionic strength and when they were in a more compact conformation at higher ionic strength. This similarity suggests that histones H1 and H5 must be largely immobilized upon chromatin binding and that the segments of core histones that are mobile in isolated nucleosome cores are not strongly bound to adjacent linker regions in intact chromatin, and are not immobilized by compaction to the degree achieved in 50 mM phosphate buffer.     

The structure and properties of histone F2a comprising the heterologous group F2a1 and F2a2 studied by 13C nuclear magnetic resonance

¹³C n.m.r. spectra have been obtained for aqueous solutions of histones F2a₁ and F2a₂, for the group F2a, for the appropriate amino acid mixturesand for the corresponding hydrolysates. These, when compared with computer simulated spectra give good agreement for secondary structure with that calculated from the known primary structure of the proteins. Evidence based on the spectra obtained at various salt concentrations leads to the conclusion that F2a is not a simple mixture but an interacting heterologous group of histones F2a₁ and F2a₂.     

Dynamic equilibrium in histone assembly: self-assembly of single histones and histone pairs.

The assembly of acid-extracted, purified F2a1, F3, F2a2, and F2b histones and their six possible pairwise combination into organized structures has been studied by: (1) sedimentation velocity, (2) sedimentation equilibrium, (3) electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate after cross-linking the protein solution with dimethyl suberimidate, and (4) electron microscopy. Each of the purified histone fractions can renature and assemble into high molecular weight organized structures. This assembly is dependent on the ionic strength, protein concentration, and temperature of the solutions. The four histones studied assemble into structures of similar dimensions and shape. In each case the first structure observed is a bent rod with a diameter of 22 A. Conditions which favor assembly lead to formation of fibers with diameters of about 44 A. The conditions which lead to assembly into organized structures are similar for the arginine-rich histones, F2a1 and F3. Higher ionic strength is required for the assembly of the lysine-rich histones, F2a2 and F2b. Certain pairs of histones interact. Strong interactions among pairs of histones interfere with the self-assembly of single histones into large structures. Howver, increase in protein concentration or ionic stregth leads to formation of large molecular structures even in solutions of pairs of strongly interacting histones. These structures are similar to those obtained with single histones. The results suggest that aggregation and complexing of histones represent a reversible, ordered process of assembly. The various assembled forms are in a dynamic equilibrium. The final assembled form, which is similar in all cases, is dependent on the environmental conditions to which the histones are exposed. It is suggested that each of the assembled histone structures, regardless whether it is composed of a single histone or a pair of histones, can serve as a core around which the DNA can be wrapped.     

滇黄精中一个三萜皂苷的NMR研究

从滇黄精新鲜根茎中首次分离得到一个三萜皂苷,拟人参皂苷-F11。本文利用1D NMR和2D NMR对其碳、氢信号进行了全归属,并对文献中报道的碳谱数据进行了纠正。     

Natural abundance carbon-13 nuclear magnetic resonance studies of histone and DNA dynamics in nucleosome cores

Natural abundance carbon-13 nuclear magnetic resonance spectra (67.9 MHz) were obtained for native nucleosome cores: cores dissociated in 2 M NaCl and 2 M NaCl, 6 M urea; and cores degraded with DNase I plus proteinase K. Phosphorus-31 NMR spectra of native and dissociated cores and core length DNA were also obtained at 60.7 MHz. The 31P resonance and spin-lattice relaxation time (T1) of DNA were only slightly affected by packaging in nucleosome cores, in agreement with other reports, but 13C resonances of DNA were essentially unobservable. The loss of DNA spectral intensity suggests that rapid internal motions of DNA sugar carbons in protein-free DNA previously demonstrated by 13C NMR methods are partly restricted in nucleosomes. The 13C spectrum of native cores contains many narrow intense resonances assigned to lysine side chain and alpha-carbons, glycine alpha-carbons, alanine alpha- and beta- carbons, and arginine side chain carbons. Several weaker resonances were also assigned. The narrow line widths, short T1 values, and non-minimal nuclear Overhauser enhancements of these resonances, including alpha- and beta-carbons, show that some terminal chain segments of histones in nucleosomes are as mobile as small random coil polypeptides. The mobile segments include about 9% of all histone residues and 25% of all lysines, but only 10% of all arginines. The compositions of these segments indicate that mobile regions are located in amino- or carboxyl-terminal sequences of two or more histones. In addition, high mobility was observed for side chain carbons of 45-50% of all lysines (delta and epsilon carbons) and about 25% of all arginines (zeta carbon) in histones (including those in mobile segments), suggesting that basic residues in terminal histone sequences are not strongly involved in nucleosome structure and may instead help stabilize higher order chromatin structure.     

23Na NMR study of Fibrobacter succinogenes S85: comparison of three chemical shift reagents and calculation of sodium concentration using ionophores

In order to measure intracellular sodium concentrations in resting cells of Fibrobacter succinogenes S85 by (23)Na NMR spectrometry, two methodological aspects were studied. First, three different shift reagents (Dy(PPP(i))(7-)(2), Tm(DOTP)(5-), and Dy(TTHA)(3-)) were tested for their ability to separate internal and external (23)Na NMR resonances. Their toxicity toward F. succinogenes cells was evaluated by in vivo(13)C NMR experiments. Tm(DOTP)(5-) was found to be the most efficient shift reagent while being nontoxic. Second, a new methodology was developed to calculate intracellular sodium concentration in F. succinogenes by using ionophores. This approach avoided the problem of intracellular volume measurement and that of sodium visibility determination.     

Structure of a quinobenzoxazine--G-quadruplex complex by REDOR NMR

Rotational-echo double resonance solid-state (31)P[(19)F] and (13)C[(19)F] NMR spectra have been used to locate the binding of a fluoroquinobenzoxazine to a DNA G-quadruplex labeled by phosphorothioation and [methyl-(13)C]thymidine.     

H-1, C-13, and F-19 nuclear magnetic resonance assignments of 3,3-difluoro-5 alpha-androstane-17 beta-ol acetate.

The molecular structure of 3,3-difluoro-5 alpha-androstane-17 beta-ol acetate was analyzed by 1H, 13C, and 19F nuclear magnetic resonance (NMR) techniques; two-dimensional NMR was used to assigned 1H and 13C resonances. The 1H NMR spectrum in deuterated chloroform shows three sharp singlets (delta = 0.74, 0.79, and 2.00 ppm) integrating for three protons each, an isolated triplet at 4.55 ppm integrating for one proton, and overlapping multiplets between 0.72 and 2.12 ppm integrating for 31 protons. The 13C spectrum shows 18 resonances between 10 and 55 ppm, and three additional resonances at 82.9, 124.0, and 171.5 ppm. The 19F[1H] spectrum shows two sets of doublets (observed 2J = 150 Hz) at 5.00 and -4.80 ppm. Multiplets arising from 19F-13C J-coupling provide the starting assignment for all resonances by means of 1H homonuclear correlation (COSY) and 1H-13C heteronuclear correlation spectroscopy.     

Determination of the number and relative position of tryptophan residues in various albumins.

Trace metals were measured by neutron-activation analyses in purified nucleic acids and histone(s) of lymphocytes from patients with acute lymphocytic leukaemia or infectious mononucleosis and from normal donor DNA isolated from lymphocytes of a patient with infectious mononucleosis and a normal donor showed a high a high content of Cr2+, Sb2+, Fe2+, and Zn2+, whereas DNA of lymphoblasts from a patient with acute lymphocytic leukaemia had a lower content of these trace metals, but the Co2+ content was 20-fold higher than in DNA or normal donor lymphocytic cells. Total histones from leukaemic cells had higher contents of most of the trace metals except for Zn2+, which was present in lesser concentration than in histones from normal donor lymphocytic cells. Lysine-rich (F1) histones showed lower contents of Cr2+, Sb2+ and Co2+, whereas arginine-rich (F3) histones had significantly higher contents of these trace metals. These observations may be of interest in that F3 histones more effectively inhibit RNA synthesis in human lymphocytic cells than do other species of histones.     

Kinetic analysis of dynamic 13C NMR spectra: metabolic flux, regulation, and compartmentation in hearts.

Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.     

Kinetics of histone methylation in vivo and its relation to the cell cycle in Ehrlich ascites tumor cells.

The appearance of methylated lysines in newly synthesized histones from Ehrlich ascites tumor cells was measured during one generation time. Newly synthesized histones were pulse-labeled in vivo by L-[3H]lysine, and the time course of the uptake of label into monomethyl, dimethyl and trimethyllysine from gel-electrophoretically isolated histones F2a1 (H4) and F3 (H3) was followed. Methylation starts immediately after histone biosynthesis. It proceeds, however, more slowly than histone synthesis. Both the rate of methylation and the mechanism of methylation in F3 and F2a1 histones differ. F3 methylation can be described by a first-order reaction, i.e. the reaction rate depends only on the concentration of free methylation sites available. Rate constants of approximately 0.21 h-1 were found for all three methylation steps. Methylation in the F2a1 histone proceeds more slowly than in F3. The dimethylation step in this fraction can be described by a zero-order reaction with a rate constant which is the reciprocal of the duration of the DNA synthesis phase. Alternatively this step could be correlated with the transition of the cells from the S phase into the G2 phase. By the end of one generation time all methylation sites in all F2a1 and F3 molecules are occupied by methyl groups at a ratio of about 1:3:1 for monomethyl, dimethyl and trimethyllysine in the F3 histone. In the F2a1 molecule the methyllysines consist mainly of dimethyllysine.     

Effects of hormone and glucose administration on hepatic glucose and glycogen metabolism in vivo. A 13C NMR study

Effects of peripheral venous injection of glucagon and insulin on [1-13C]glucose incorporation into hepatic glycogen of rats were studied by 13C NMR in vivo. Each animal was given a continuous somatostatin infusion and a 100-mg intravenous injection of [1-13C] glucose in NMR experiments or unlabeled glucose in parallel experiments for determination of serum glucose. Insulin administration caused serum glucose to fall below basal levels and accelerated the loss of hepatic [1-13C]glucose; these effects were counteracted by the addition of glucagon. Glucagon administration alone did not affect serum glucose or hepatic [1-13C] glucose but caused the loss of [1-13C]glucose from glycogen and inhibited [1-13C]glucose incorporation into glycogen. Insulin did not alter [1-13C]glucose incorporation into glycogen when given alone or in combination with glucagon. The data are consistent with a model in which liver glycogen synthesis increases linearly with hepatic glucose concentration above a threshold glucose concentration. Insulin did not alter the rate constant or the threshold for synthesis.     

Detergent delipidation and solubilization strategies for high-resolution NMR of the membrane protein bacteriorhodopsin

High-resolution NMR studies of bacteriorhodopsin require the availability of the detergent-solubilized protein with both high concentration and small rotational correlation time. A procedure is described for the optimized preparation of such samples. Bacteriorhodopsin was first delipidated by detergent treatment of purple membrane under nonsolubilizing conditions for the protein. The delipidated aggregated protein could then be solubilized into monomers at concentration close to millimolar by selected detergents. The solubilizing detergent had an important effect on the rotational correlation time of the protein as shown by measuring in each case the temperature-dependent stability of the protein, the size of the detergent-protein complex, and the detergent viscosity. Consistently, a strong influence of the detergent was also found on spectral resolution in 13C NMR spectra of solubilized bacteriorhodopsin labeled with [1-13C]phenylalanine. Best resolution was obtained using n-dodecylmaltoside as detergent, with which relatively narrow well resolved 13C NMR resonances were observed at 50 degrees C. It is suggested that high-resolution NMR studies performed with this detergent may contribute to the structural resolution of bacteriorhodopsin.     

Methylated basic amino acid composition of histones from the various organs from the rat.

Newborn rats received 5 muCi each of [3H]lysine and [methyl-14C]methionine/g body weight. They were killed 10 days later and the nuclei prepared from the kidneys, liver, cerebrum, cerebellum, and thymus. The five major histones were extracted from these nuclei by the method of Johns and further purified on Bio-Gel P-10. The histones were hydrolyzed and the basic amino acids fractionated on Beckman PA-35 resin. Only the F3 and Fia1 histones contained any significant amounts of methylated amino acid residues as measured by chemical or radiological assay. The product of methylation of F2a1 was predominantly dimethyllysine with trace quantities of monomethyllysine detectable in rapidly proliferating tissue. The products of methylation of F3 were mono-, di-, and trimethyllysine in an approximate molar ratio of 0.55:1.0:0.35. This ratio did not vary significantly in the F3 histones prepared from the different organs. No methylarginine or methylhistidine was detected in any of the histones prepared from the five organs. The total amount of dimethyllysine in F2a1 from the different organs of adult rats was approximately 2.0 mol/mol of polypeptide. It appears that the distribution of methyl groups on the lysyl residues in the F3 and F2a1 histones from the different organs is similar and does not contribute to tissue heterogeneity.     

Structural features of an exocyclic adduct positioned opposite an abasic site in a DNA duplex

Structural studies have been extended to dual lesions where an exocyclic adduct is positioned opposite an abasic site in the center of a DNA oligomer duplex. NMR and energy minimization studies were performed on the 1,N2-propanodeoxyguanosine exocyclic adduct (X) positioned opposite a tetrahydrofuran abasic site (F) with the dual lesions located in the center of the (C1-A2-T3-G4-X5-G6-T7-A8-C9).(G10-T11-A12-C-13-F14-C15 -A16-T17-G-18) X.F 9-mer duplex. Two-dimensional NMR experiments establish that the X.F 9-mer helix is right-handed with Watson-Crick A.T and G.C base pairing on either side of the lesion site. NOEs are detected from the methylene protons of the exocyclic ring of X5 to the imino protons of G4.C15 and G6.C13 which flank the lesion site, as well as to the H1' and H1" protons of the cross strand F14 tetrahydrofuran moiety. These NMR results establish that the exocyclic adduct X5 is positioned between flanking G4.C15 and G6.C13 base pairs and directed toward the abasic lesion F14 on the partner strand. These studies establish that the exocyclic ring of the 1,N2-propanodeoxyguanosine adduct fits into the cavity generated by the abasic site.     

Vancomycin derivative with damaged D-Ala-D-Ala binding cleft binds to cross-linked peptidoglycan in the cell wall of Staphylococcus aureus

Des-N-methylleucyl-4-(4-fluorophenyl)benzyl-vancomycin (DFPBV) retains activity against vancomycin-resistant pathogens despite its damaged d-Ala-d-Ala binding cleft. Using solid-state nuclear magnetic resonance (NMR), a DFPBV binding site in the cell walls of whole cells of Staphylococcus aureus has been identified. The cell walls were labeled with d-[1-(13)C]alanine, [1-(13)C]glycine, and l-[epsilon-(15)N]lysine. Internuclear distances from (19)F of the DFPBV to the (13)C and (15)N labels of the cell-wall peptidoglycan were determined by rotational-echo double-resonance (REDOR) NMR. The (13)C{(19)F} and (15)N{(19)F} REDOR spectra show that, in situ, DFPBV binds to the peptidoglycan as a monomer with its vancosamine hydrophobic side chain positioned near a pentaglycyl bridge. This result suggests that the antimicrobial activity of other vancosamine-modified glycopeptides depends upon both d-Ala-d-Ala stem-terminus recognition (primary binding site) and stem-bridge recognition (secondary binding site).     

GLUTARALDEHYDE FIXATION OF ISOLATED EUCARYOTIC NUCLEI : Evidence for Histone-Histone Proximity

Isolated chicken erythrocyte nuclei have been incubated with dilute concentrations of the bifunctional cross-linking agent glutaraldehyde (0–20 mM) in order to stabilize histone-histone interactions within the native nucleus. The kinetics of the disappearance of acid-soluble histones, free amino groups, and of individual histones have been observed to be pseudo first-order. Apparent first-order rate constants for the disappearance of individual histones correlate with the lysine mole percent of that fraction and follow the ranking, k_app: F1 > F2C > F2B ≥ F2A2, F2A1, F3. Histone polymers were observed to form very rapidly during the fixation reaction. Partial fractionation and amino acid analyses of these polymers support the view that they are composed principally of cross-linked (F2C)_n molecules (where n = 2 to ~8). The rate of glutaraldehyde reaction with free amino groups in histones is drastically reduced in solvents that promote chromatin decondensation (i.e., low ionic strengths in the absence of divalent cations) whereas the formation of cross-linked F2C polymers is less severely reduced. It is proposed that some F2C histones exist in close proximity within the isolated erythrocyte nucleus.     

Norclerodane diterpenoids from rhizomes of Dioscorea bulbifera

Three norclerodane diterpenoids, diosbulbins K-M, and one analogous enolglycoside, diosbulbinoside G, together with four norclerodane diterpenoids, diosbulbins B, E, F and G, were isolated from rhizomes of Dioscorea bulbifera. Their structures were established by spectroscopic and chemical methods, including ¹H and ¹³C NMR, NOESY, HSQC, HMBC, and HRMS analyses. The relative configurations of diosbulbins K and L, and diosbulbin F were confirmed by X-ray crystallographic diffraction analysis, and the absolute stereochemistry of diosbulbin K was determined by a modified Mosher’s method. The ¹³C NMR spectroscopic data for diosbulbins E, F and G were also measured for the first time. The compounds did not show significant cytotoxic and anti-bacterial activities.     

Backbone structure confirmation and side chain conformation refinement of a bradykinin mimic BKM-824 by comparing calculated (1)H, (13)C and (19)F chemical shifts with experiment

Calculated and experimental (1)H, (13)C and (19)F chemical shifts were compared in BKM-824, a cyclic bradykinin antagonist mimic, c[Ava(1)-Igl(2)-Ser(3)-DF5F(4)-Oic(5)-Arg(6)] (Ava=5-aminovaleric acid, Igl=alpha-(2-indanyl)glycine, DF5F=pentafluorophenylalanine, Oic=(2S,3aS,7aS)-octahydroindole-2-carboxylic acid). The conformation of BKM-824 has been studied earlier by NMR spectroscopy (M. Miskolzie et al., J. Biomolec. Struct. Dyn. 17, 947-955 (2000)). All NMR structures have qualitatively the same backbone structure but there is considerable variation in the side chain conformations. We have carried out quantum mechanical optimization for three representative NMR structures at the B3LYP/6-31G* level, constraining the backbone dihedral angles at their NMR structure values, followed by NMR chemical shift calculations at the optimized structures with the 6-311G** basis set. There is an intramolecular hydrogen bond at Ser(3) in the optimized structures. The experimental (13)C chemical shifts at five C(alpha) positions as well as at the Cbeta, Cgamma and Cdelta position of Ava(1), which forms part of the backbone, are well reproduced by the calculations, confirming the NMR backbone structure. A comparison between the calculated and experimental H(beta) chemical shifts in Igl(2) shows that the dominant conformation at this residue is gauche. Changes of proton chemical shifts with the scan of the chi(1) angle in DF5F(4) suggest that chi(1)180 degrees. The calculated (1)H and (13)C chemical shifts are in good agreement with experiment at the rigid residue Oic(5). None of the models gives accurate results for Arg(6), presumably because of its positive charge. Our study indicates that calculated NMR shifts can be used as additional constraints in conjunction with NMR data to determine protein conformations. However, to be computationally effective, a database of chemical shifts in small peptide fragments should be precalculated.     

Electrophoretic analysis of liver and testis histones of the frog Rana pipiens

Histones were extracted from frog livers and testes and analyzed by electrophoresis on long polyacrylamide gels and on sodium dodecyl sulfate (SDS)-containing polyacrylamide gels. Frog histones were found to be similar to those of calf thymus except that frog histone fraction F2A2 showed a marked dependence on the temperature at which the long gels were run, and frog histone fraction F3 could be separated from frog F2B on SDS-containing gels. Comparisons between frog liver and frog testis histones indicated that the testis contains as its major F1 component a fast migrating species not found in liver. Testis histones also showed less microheterogeneity of fractions F3 and F2A1 than liver histones. These were the only differences observed between liver and testis histones, even when testis histones were prepared from sperm suspensions that were rich in cells in the late stages of spermiogenesis. Thus it seems that, in Rana, the electrophoretic properties of the basic proteins of sperm differ from those of somatic cells only in the nature of histone F1 and in the degree of microheterogeneity of fractions F2A1 and F3.