The Conformation of Histone H5

Abstract

Treatment of chicken erythrocyte histone H5 with trypsin in a high-ionic-strength medium results in very rapid initial digestion and the formation of a 'limiting' resistant product peptide. Under these solution conditions the H5 molecule is maximally folded by spectroscopic criteria and it is concluded that the resistant peptide, GH5, represents a globular folded region of the molecule whilst the rapidly digested parts are disordered. The peptide GH5 is shown to comprise the sequence 22-100. In support of this conclusion it is shown that whilst intact histone H5 is hydrodynamically far from being a compact globular shape, peptide GH5 is approximately spherical by hydrodynamic and scattering criteria. Further more, peptide GH5 retains all the whelical structure of intact H5 (circular dichroism) and appears to also maintain all the tertiary structure (nuclear magnetic resonance). It follows that in solution at high ionic strength, histone H5 consists of three domains: an N-terminal disordered region 1-21, a compact globular central domain 22-100 and a long disordered C-terminal chain 101 -185. Structural parallels are drawn with the three-domain structure of the histone H1 moleculeThe nucleated erythrocytes of birds, reptiles, amphibians and fish contain a basic histone H5 that partially, but not completely replaces histone HI in the mature erythrocyte 11 -51. These two histones have a similar molecular weight (HI : 217 residues [6], H5: 185 residues [7]) and amino acid composition. The main distinction is that histone H5 contains substantially more arginine and serine than histone H I . Studies in this laboratory have demonstrated that histone H1 contains three distinct conformational domains : the N-terminal sequence of 35 residues and the C-terminal 95 residues are disordered in solution, whilst the central region of about 88 residues is compact and approximately spherical [8]. In two similar studies on histone H5 [7,9] the conclusion was reached that in free solution there is a compact folded conformation in the N-terminal half of the molecule which does not extend far beyond residue 99 and inay extend right up to the N-terminus. In the present study histone H5 has been digested with trypsin to give a limiting product peptide, and this peptide has been isolated and identified. It is then shown that although the intact H5 molecule is far from fully compact, the tryptic peptide appears to be fully compact and approximately spherical by hydrodynamic and scattering criteria. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy are Ahbrrviatiom. NM R, nuclear magnctic resonance; CD, circularused to demonstrate that the peptide retains all the helicity and the features of tertiary folding that are observed in intact H5. MATERIALS AND METHODS Preprution of Histone H5Chicken erythrocyte histone H5 was prepared by the acid extraction method of Murray et al. [lo] and purified by chromatography on a Biogel P-60 column, eluting with 20 mM HCI, 50 mM NaCI. Gel ElectrophoresisA modified Laemmli sodium ...

In vitro phosphorylation of histones H5, H2A, H2B and of the dimer H2A‐H2B by a cyclic AMP‐dependent protein kinase from rat pancreas

“…September 1980 and Serz9 in H5) which are located inside the structured apolar regions of the proteins [28,29], are brought to the surface of the protein by a &turn structure and therefore are accessible to the protein kinase [ 131. The major sites of phosphorylation are placed outside the organised apolar regions of the proteins [29].…”

“…The remaining 32P-label is found in the slightly basic N-terminal part of the molecule at the edge of the globular region of the protein (residues 23-99) [28].…”

Phosphorylated sites of chicken erythrocyte histone H5 by a cyclic AMP‐independent protein kinase from mouse plasmocytoma cells

“…The specificity of these two enzymes contrasts sharply with that of cAMP-dependent protein kinase from pig brain which preferentially phosphorylates serine residues located in the globular domain (residues 22-100) of histone H5 [7,9]. Four of the 5 serine residues phosphorylated in vitro in H5 by the H1-specific cAMP-independent kinase are identical to those found phosphorylated in vivo [5].…”

“…Sites of phosphorylation of histone H5 have been investigated in vivo [4], and in vitro using the catalytic subunit of cyclic AMPdependent protein kinase from pig brain [7] and from rat pancreas [8]. Even if the specificity is different from one kinase to another, the sites of phosphorylation in histone H5 are distributed in vitro as in vivo in two distinct regions of the protein: the amino-terminal region (residues 1-21) and the carboxy-terminal region (residues 101-189); in all cases, no site has been detected in the globular part of the molecule (residues 22-100) [9]. This fact has been already mentioned for histone H 1 which, like histone H5, conrains 3 different domains: a short apolar amino-terrninal part in random coil (residues 1-34); a globular central part (residues 35-120); and a highly basic carboxy-terminal part in random coil (residues 121-213) [10].…”

Isolation of a 167 basepair chromatosome containing a partially digested histone H5

“…The time-course of digestion was followed both by extraction with 5% perchloric acid (which extracts only histones Hl and H5 and their peptides) and by precipitation with trichloroacetic acid (which yields all chromosomal proteins). Fig.lB shows the rate of digestion of the lysine-rich histones set against a sample of pure histone H5 and a purified sample of the globular peptide of H5 obtained by a digestion in solution [9] and designated GHS: by 45 min the lysine-rich histones have been degraded to their limit peptides of which that from H5 presumably dominates. Detailed analysis of these limit products has shown that principally N-terminal residues are lost from core histones and that the resulting core particle is not dramatically changed in structure WI.…”