Conformational states and reversibility of histone complexes

Abstract

The core histone complex (H3:H4:H2A:H2B)2 and products of dissociation, the H2A:H2B: dimer and the H3:H4 tetramer, were isolated from chicken erythrocyte chromatin by several literature methods as well as gel filtration on Bio-Gel A15m at various salt concentrations. The conformational and oligomeric characteristic of these histone complexes were compared to analogous histone complexes prepared by renaturation of individually acid-extracted histones by circular dichroism (CD) and analytical gel filtration chromatography. The salt-extracted core histone complex (independent of method of preparation), the purified dissociation products, the H2A:H2B dimer, and the H3:H4 tetramer in 2 M NaCl, 10 mM sodium phosphate, 0.25 mM EDTA and 0.1 mM DTT, pH 7.0, have conformation which are identical, by the criteria of similar CD spectra, with complexes prepared from acid-extracted histones, Likewise, the salt-extracted complexes may be cycled through solvents of low ionic strength (10 mM sodium phosphate, pH 7.0 or 50 mM NaOAc, pH 5.0) or 1 mM HCl and returned to 2.0 M NaCl, 10 mM sodium phosphate, 0.25 mM EDTA, and 0.1 mM DTT, pH 7.0, in a completely reversible manner. Thus it would appear that acid-denatured histones are capable of being fully renatured to yield native-like complexes.

Crystallographic Structure of the Octamer Histone Core of the Nucleosome

“…1 and figure 2 in (2)] and the constraints imposed by the crystallographic symmetry elements (2, p. 547). Five helices, not two, were mentioned in our paper (2), and these plus several smaller helices make up about 50 percent of the protein mass, consistent with circular dichroism and Raman spectroscopy (16). Furthermore, we have reported that the chains of H2A and H2B have been traced nearly from end to end, and sufficient segments of characteristic amino acid sequences have been identified in the map to allow the assignment of the polypeptides.…”

Expression of a Zinc-Binding Domain of Boar Spermatidal Transition Protein 2 in Escherichia coli

“…The fractions containing TP2Z were lyophilized, and the lyophilized proteins were dissolved in 0.71 M 2-mercaptoethanol/7 M GuHCl/50 mM sodium acetate-HCl buffer, pH 2.0, and further purified by gel filtration HPLC on Diol-120. The purified TP2Z was refolded in the same manner as intact TP2 (15) by a slight modification of the method of Beaudette et al (20). TP2Z was dissolved in 32 mM DTT/7 M GuHCl/10 mM Tris-HCl, pH 8.8, and incubated for 1 h at 40°C.…”

Isolation of intact transition protein 2 with three zinc finger motifs from boar late spermatid nuclei

“…TP2 was refolded by a slight modification of the method of Beaudette et al (16). TP2 was dissolved in 32 mM dithiothreitol/8 M urea/10mM Tris-HCl, pH 8.8, and incubated for 1 h at 40~ After the pH was adjusted to 2.0 with i0 % trifluoroacetic acid, the solution was dialyzed against 50 ~M ZnCl2/O.l mMdithiothreitol/lO mMTricine-NaOH, pH 7.4 with or without 0.15MKF, or 10mMEDTA/0.1 mM dithiothreitol/10 mM Tricine-NaOH, pH 7.4, for 24 h, and then dialyzed against the same buffer without ZnCl 2 or EDTA for 48 h. Gel electrophoresis SDS-PAGE (17) and AU-PAGE (18) were performed on 15 % gel.…”