The secondary structure of histone IV and its stability.

The secondary structure within histone IV and its fragments obtained by cyanogen bromide (CNBr) and cleavage at Met 84 has been examined by circular dichroism and spectophotometric pH titration measurements. These studies have confirmed the existence of stable secondary structure within the C-terminal fragment of histone IV (C-peptide which can be perturbed only by 6M urea at pH greater than 8 or 8 M guanidine-HCL. In contrast, the N-terminal fragment (N-peptide) appears to lack significant secondary structure at low ionic strengths but acquires approximately 15% betasheet conformation and 5% alpha-helix upon aggregation at ionic strengths larger than or equal to 0.4. The rates of nitration of the N- and C-peptides by tetranitromethane (TNM) have also been measured as a function of ionic strengths. Under comparable conditions, the rate constant for nitration of the N-peptide was found to be about six times greater than that for the C-peptide, further evidence in support of the presence of stable secondary structure within the C-terminal region of histone IV. After binding these histone IV fragments to DNA, however, the nitration reaction rate constants for the N- and C-peptide in the bound form are found to be 2% and 27% of the corresponding free peptides. Reconstituted nucleohistone IV is about 10% as reactive to TNM as histone IV at comparable ionic strength.     

Conformational changes in histone IV

Conformational change of histone IV, induced by phosphate, have been investigated by observing the intrinsic fluorescence of tyrosine residues and circular dichroism (CD). There is a fast conformational change upon the addition of phosphate, followed by a slow process with time constants in the range of minutes to hours depending upon both the phosphate and histone concentrations. The CD results indicate α-helix formation in the fast process, and β-sheet formation in the slow one, although other secondary and tertiary structural changes also may occur. The histone concentration dependence of the fast process is consistent with dimerization. Divalent phosphate is about ten times more effective than monovalent phosphate in inducing conformational changes. All of the changes are reversible.     

Nuclear magnetic resonance studies of histone IV solution conformation.

The 220-MHz high-resolution proton magnetic resonance (PMR) spectrum of histone IV has been examined as a function of histone concentration, salt concentration, and pD. The hydrophobic C-terminal portion of the histone IV monomer appears to be largely PMR "invisible" indicating that this region of the polypeptide contains rigid secondary structure. Further loss of PMR resonance areas with increased histone IV concentration in neat D2O has been attributed to self-aggregation involving a monomer-dimer equilibrium. An equilibrium between the monomer and large aggregates, on the other hand, appears to dominate at NaCl concentrations above 0.01 M. pD studies reveal an abrupt increase in histone IV aggregation at pD smaller than 0.8 and precipitation of histone IV at pD values in the neighborhood of its isoelectric point, pD similar to 11.     

The conformation of histone f2al (histone IV) in solution at low ionic strength

Hydrodynamic studies of histone f2a1 are performed in dilute salt solution. Protein sedimentation is shown to be dependent upon the partial specific volume and molarity of the supporting electrolyte. To compensate for the secondary chage effect, a solvent of dilute tetramethylammonium chloride, for which (1 ? V?_saltρ) ? 0, is used. To correct for the primary charge effect in sedimentation, a special linear extrapolation to infinte dilution of the protein is employed. Measurements of intrinsic viscosity are interpreted in terms of an increase in molecular dimension with a decrease in ionic strength. The conformation of histone f2a1 in dilute salt solution is interpreted from sedimentation and viscosity data to be that of a highly charged random coil possessing 20–30% of nuclear globular structure.     

Nuclear magnetic resonance studies on the solution conformation of histone IV fragments obtained by cyanogen bromide cleavage.

Two histone IV fragments obtained by cleavage at Met-84 by cyanogen bromide have been examined by proton magnetic resonance (PMR) spectroscopy as a function of temperature, peptide concentration, ionic strength, and pD. Sedimentation and gel electrophoresis studies on these peptides have also been carried out. The 220-MHz PMR spectrum of the N-peptide in both the high- and low-field regions was shown to be almost identical with that calculated for an extended coil N-peptide monomer. The calculated random coil and experimental C-peptide spectra, on the other hand, differ in many respects. Evidence was obtained for the presence of rigid secondary structure in the C-peptide. In addition, the Val, Leu, Ile CH3 resonance displays a prominent high-field satellite band which shifts downfield with increasing temperature. Sedimentation studies on the N-peptide reveal the formation of extremely large, remarkably homogeneous aggregates at ionic strengths larger than or equal to 0.01. The C-peptide, on the other hand, does not appear to form aggregates of sufficient size to be detectable in velocity sedimentation studies of a few hours duration. The relative area changes which have previously been noted in the PMR spectrum of histone IV with increasing ionic strength were also observed for the N-peptide but not the C-peptide. Interpretation of these relative area changes has been made in terms of the amino acid sequence of histone IV, and an effort was made to identify that segment of the polypeptide which undergoes secondary structural change with increasing ionic strength.     

Histone acetylation in Zea mays.I. Activities of histone acetyltransferases and histone deacetylases

DEAE-Sepharose chromatography of extracts from Zea mays meristematic cells revealed multiple histone acetyltransferase and histone deacetylase enzyme forms. An improved method for nuclear isolation allowed us to discriminate nuclear and cytoplasmic enzymes. Two nuclear histone acetyltransferases, A1 and A2, a cytoplasmic B-enzyme and two nuclear histone deacetylases, HD1 and HD2, have been identified. The histone specificity of the different enzyme forms has been studied in an in vitro system, using chicken erythrocyte histones as substrate. The cytoplasmic histone acetyltransferase B is the predominant enzyme, which acetylates mainly histone H4 and to a lesser extent H2A. The nuclear histone acetyltransferase A1 preferentially acetylates H3 and also H4, whereas enzyme A2 is specific for H3. This substrate specificity was confirmed with homologous Z. mays histones. The two histone deacetylases differ from each other with respect to ionic strength dependence, inhibition by acetate and butyrate, and substrate specificity. The strong inhibitory effect of acetate on histone deacetylases was exploited to distinguish different histone acetyltransferase forms.