Studies on the Role and Mode of Operation of the Very-Lysine-Rich Histone H1 in Eukaryote Chromatin. The Isolation of the Globular and Non-Globular Regions of the Histone H1 Molecule

Abstract

Digestion of calf thymus H1 histone with thrombin cleaves the molecule at the sequence -(Pro)-Lys-Lys-Ala-, corresponding to a point approximately 122 residues from the N-terminus (about 56% along the molecule). The N-terminal fragment is shown by proton nuclear magnetic resonance (NMR) to possess the globular structure of the intact histone HI molecule, whereas the C-terminal fragment appears to possess little or no structure. The N-terminal fragment separates into two peaks on an ion-exchange column, one of which is shown to originate from a single subfraction of calf thymus histone H1 and the other to originate from the other subfractions, by detailed comparison of the NMR spectra.It thus seems that the structure of the H1 histone in solution under physiological conditions consists of a globular head with a highly basic random coil tail. It is suggested that the globular head has a specific binding site on the subunit structure of the chromosome.The very-lysine-rich histone fraction (Hl) is a class of basic proteins found in eukaryotic chromosomes which are very closely related in amino acid sequence [l]. Interest in this histone fraction has been stimulated by the observation that they are apparently not, as are the other histones, involved in the basic subunit structure of the chromosome [2], but may be involved in its higher order structure. It is now quite clear that the H1 histones are the active agents in salt-induced chromatin condensation [3], and there is good evidence that they are also involved in mitotic chromosomePhysical studies on the H1 molecule in solution have shown that under physiological conditions of pH and ionic strength the molecule folds into a globular structure [S], and there is evidence that this structure does not involve the whole molecule. It would seem reasonable that this globular structure reflects some state of the H1 histone in chromatin, and we have invested some effort in attempts to isolate the globular portion of the molecule by chemical and enzymic cleavage. The object was to produce fragments which contain intact functional parts of the molecule, in isolation from other functional parts, and in this way to study their properties more clearly.

Conformational Studies of Two Non‐histone Chromosomal Proteins and Their Interactions with DNA

“…There are parallels in this respect with thymus HI histones Fig. 8 NMR spectru of recombined complex between high-mohilrtv-group protein 1 ( 3 ntgiml) and DNA (6 mg/ml) in 'H,Ol NaCI, pH 6 9, 20 C, and (above) equivalent spectra of protein alone ( 3 mglml) in 'H,O/NUCI, pH 6 9, 20 C that also forms a globular structure (on increase of pH from 3-6, or by salt addition at acid pH) that involves, at maximum only about one half of the 216 residues in H1 [7,8,13]. In the case of the highmobility-group proteins, lack of sequence data prevents any firm statements on the size of the globular segment, but the general conclusion remains that conformationally the high-mobility-group proteins resemble most closely the lysine-rich histones H1 and H 5, with the exception that the high-mobility-group proteins show aggregation at neutral pH whereas H 1 and H 5 do not.…”

“…An increase in the pH from 3-7 does not by itself induce large structural changes in the conformation of these four histones but this is not found to be the case with H1 which is observed to form both secondary and tertiary structure on pH increase without any concomitant aggregation [7]. At neutral pH (or at pH 3 in high salt) H1 therefore behaves like a single subunit globular protein with the notable difference that only about half of the molecule is included in the fold [8]. The availability of milligram quantities of the high-mobility-group proteins led us to the present structural studies and we have concluded that conformationally they are capable of forming globular structures with specific secondary and tertiary folds.…”

Precise elimination of the N-terminal domain of histone H1

“…Thus they either represent different subfractions cut at the same point in the chain and/or a small amount of C-terminal loss. Although it is often thought (on the basis of partial sequence data) that HI subfractions differ most characteristically at the N-terminus, it is found that the peptide mixture generated by thrombin digestion (Chapman et al, 1976) (which represents residues 122 to the Cterminus from a mixture of fractions) consists of peptides with a wide spread of mobilities. The two peptides of the B IV mixture could both be intact at the C-terminus.…”

“…86 residues in calf thymus HI) and a long unstructured C-terminal domain of very basic composition comprising almost half the molecule (approx. 93 residues in calf thymus HI) (Chapman et al, 1976;Hartman et al, 1977;Puigdomenech et al, 1980;Barbero et al, 1980;Aviles et al, 1978). By reconstituting several HI peptides on to chromatin depleted of its original Hi, it was shown that the central globular domain alone is able to close two full turns of nucleosomal DNA.…”

“…Clearly one or both of the two terminal domains must be involved in the condensation process. To date we have been able to make a peptide consisting of the N-terminal plus globular domains (Chapman et al, 1976), and a test of its ability to condense chromatin Abbreviation used: SDS, sodium dodecyl sulphate.…”