Studies on the Role and Mode of Operation of the Very-Lysine-Rich Histones in Eukaryote Chromatin. The Conformation of phi 1 Histones from Marine Invertebrate Sperm

Abstract

Proton magnetic resonance, circular dichroism and infrared spectroscopy are used to investigate the secondary and tertiary structures of three very lysine-rich histones from marine invertebrate sperm. At high ionic strength both Arhacia lisula and Holothuria tuhulosa histone cpl are observed to contain 25-30",, r-helix, no /&structure and to form specific folded structures. Both cpl proton magnetic resonance spectra have perturbed methyl resonances at chemical shifts close to those observcd for calf thymus H 1, suggesting analogies in tertiary structure. Mytilus edulis histone cpl however, shows no spectroscopic evidence of secondary and tertiary structure on salt addition.The formation of specific folded structures in calf thymus histone H1 induced by increase of ionic strength or of pH has been demonstrated in the first paper of this series [l]. It appears that in histone H1 a highly ordered structure exists. which involves at least the central and less hydrophilic rcgion of the molecule. This conclusion follows from the appearance of signals in the high-field part of the nuclear magnetic resonance (NMR) spectra of the protein, which are shifted by the ring-current effects of aromatic side chains. Furthermore the circular dichroism (CD) spectrum of H l demonstrated the presence of some cc-helix in the folded state.In the present work we have extended our conformational studies to a group of sperm histones from marine invertebrates (coded as cpl histones) which have been extracted from the species Arhacia li.~rda (sea urchin). Holothiuia tiihulosa (sea cucumber) and Mjstilrrs ethrlis (sea mussel). These basic proteins can be considered analogous to histone H1 from somatic tissues of vertebrates in the sense that they have been prcpared in the same way as histone H1 by the methods of Johns [2], they are electrophoretically homogeneous [3,4] and, with the exception of M . edulis histone 471, they resemble calf thymus histone H1 in amino acid composition (see Table 1). The most striking features of the composition of these histones are the great Ahhreviatiotis. N M R . nuclear magnetic resonance: CD. circular dichroism.variability in arginine content, the small number of amino acids present in histone cpl of M . edulis and the considerable variation in aromatic residues and in histidine. Despite these variations they are clearly closely related to calf thymus H1 histone.As earlicr stated [3] the high basicity of these histones could be related to the synthetic inactivity of the sperm nucleus as a whole. This also seems to be the case in difl'ercnt types of sperm [6] and in avian erythrocytes [7,8] which possess the additional histone H5. rich in lysine and in arginine.Our aim in this paper is to investigate structural homologies and differences within the cpl histones and to compare them with calf thymus histone H1. Such data are important to define which aspects of the secondary and tertiary structure of these histones are relevant for the preservation of any specific (though at prcsent unknown) functionality ...

The Structure of Sea‐Urchin‐Sperm Histone φ1 (H1) in Chromatin and in Free Solution

“…The aromatic protons are shown in Fig.5. At pH 3, 1 M NaCl, the two histidine C2H protons of G41 and LG41 are at their characteristic shifts of 8.7 and 8.8 ppm as found in intact $1 [13]. Likewise the C-33 protons of the two tyrosine residues are found at characteristic shifts of 6.7 and 6.8 ppm [13].…”

“…cm2 dmol-l as characteristic of 100% helix [17]. It has previously been shown that at high ionic strength, $1 does not contain p structures [13]. The measured ellipticity of intact $1 at 222 nm was -4700 deg.…”

“…At pH 3, 1 M NaCl, the two histidine C2H protons of G41 and LG41 are at their characteristic shifts of 8.7 and 8.8 ppm as found in intact $1 [13]. Likewise the C-33 protons of the two tyrosine residues are found at characteristic shifts of 6.7 and 6.8 ppm [13]. From both the upfield and the aromatic spectrum it is concluded that the tertiary structure of intact histone 41 is essentially preserved in both peptides G41 and LG$l.…”

“…It has previously been demonstrated that at high ionic strength ( % 1 M NaCl), histone 41 folds to form secondary and tertiary structure [13]. The progress of digestion was followed by turbidity and by gel electrophoresis (Fig.1).…”

“…5(C) was the spectrum after 2 h. By comparing the area of the resonance at 8.48 ppm in Fig. 5 with that of single histidine C-2 protons The composition of & I is taken from our previous work [13]. The number of residues have been calculated in all cases supposing two tyrosines.…”