Proton magnetic resonance, circular dichroism and other studies of whole and cleaved calf thymus histone H1 (formerly F1) reveal the presence of specific folded structures in the region approximately from residue 40-115. Ionic, hydrogen-bond and hydrophobic interactions all appear to contribute to the stability of the structure, which is predicted to contain a-helices in regions 42-55 and 58-75. No evidence was found for 8-structures, either inter or intramolecular, or for any structure formation outside the region 40-115. At 18 "C and a protein concentration of 2 mM the first-order exchange rate between random-coil and structured forms is slower than 80 s-'; at 40 "C the exchange rate is faster than 330 s-The very-lysine-rich histone H 1 (formerly called F1 or I; the nomenclature used in this paper is taken from [I]), exhibits several features which distinguish it from the other histone fractions found in eukaryote chromatin. It has the highest molecular weight (approx. 23000) and has a very high lysine : arginine ratio of over 15 : 1, varying up to 21 : 1 for some subfractions.Over 25 % of the molecule consists of lysine residues, but despite this very basic nature histone H1 is the fraction most easily removed from chromatin on increasing the ionic strength of the solution. Histone H1 has been implicated in the condensation of chromatin in two ways : increase in ionic strength of a chromatin gel in the region below that required to remove histone H1 (0.1-0.3 M NaC1) causes a ten-fold physical contraction of the gel which is dependent on the Abbreviations. NMR, nuclear magnetic resonance; CD, circular dichroism. presence of histone H1 [2]; and in the true slime mould P. polycephalum the very-lysine-rich histone H1 undergoes a peak of phosphorylation late in G2 phase at a point in the cell cycle which corresponds to chromosome condensation [3]. This latter observation has led to the proposal that phosphorylation of histone H1 may be part of a mitotic trigger mechanism [4,5]. Furthermore, a histone Hl/DNA reconstituted complex also exhibits physical condensation at the same ionic strengths as those required for chromatin. This effect and that of histone H1 in chromatin gel will be described in the succeeding paper of this series.Mammalian histone H1 has been subjected to several sequence determinations, which reveal some sequence microheterogeneity which is both species and tissue specific [7,8]. The experiments described in this paper were carried out using unfractionated calf thyEur. J. Biochem. 52 (1975)

Spectroscopic studies (nuclear magnetic resonance, circular dichroism and infrared) have been carried out on chicken erythrocyte histone H 5 and on three peptides cleaved therefrom: 1-31, 32-197 and 58-197. It is shown that at ionic strengths above 0.1M part of the H 5 molecule takes up a globular conformation containing 14% a helix but no p sheet structure. Several details of the circular dichroism and nuclear magnetic resonance spectra indicate that the globular region is located in the N-terminal half of the molecule and this proposal is supported by the observation that the peptide 32-197 is largely incapable of folding and the peptide 59-197 is completely incapable of folding. Structural similarities and differences between histone H 5 and histone H 1 are discussed.The nucleated erythrocytes of birds, reptiles, amphibians and fish contain a very basic histone, H5, that has not been found in other tissues [l -51. H 5 largely, but not completely, replaces H 1 and the two histones are not dissimilar in amino acid composition and molecular weight [4,6 -81. A significant difference between the two histones is that H 5 contains 11 % arginine whilst H 1 contains only 2%. In this respect H 5 resembles the 4 1 histones of marine invertebrate sperm (in particular sea urchins) which totally replace the H I and also contain about 11% arginine in addition to roughly 25% lysine 191. Early observations of the occurrence and composition of H 5 resulted in the suggestion that its function is the total suppression of the genome in the fully mature erythrocyte [lo]. Detailed studies on anemic chickens have subsequently shown, however, that H 5 can be detected even in erythroblasts (when a wide range of protein synthesis is taking place), and does not appear suddenly at the mature erythrocyte stage when protein synthesis is finally terminated [I 1 -151. The function of H 5 in relation to genome suppression is, therefore, not as simple as originally thought. H 5 nevertheless is closely related to H I [I61 and a number of experiments have implicated H 1 in the condensation of diffuse chromatin [17 -191. In particular the state of chromatin is thought to depend on the degree of phosphorylation of H 1 [20,21] and recently it has been shown that H 5 is phosphorylated and dephosphorylated in viuo [22]. There is at present no evidence to Abbreviations. NMR, nuclear magnetic resonance; CD, circular dichroism.suggest why H 1 should be replaced in large part by H 5 and the present structural investigation of H 5 was carried out to point out the similarities and differences between H 5 and H 3 . Related studies on H 1 have already been published [23,24]. Hydrodynamic studies have shown that H 5 does not aggregate at high ionic strength [8,28] and thereby resembles H 1 and not the remaining four histone fractions. The monomeric state of H 1 has led to proposals that its function is quite separate from that of the other histones and this may also be true for H5. In chicken erythrocytes the stoichiometry of histones H 5 and H 1 together, wi...

The nuclear magnetic resonance (NMR) spectrum of chromatin at ionic strengths below about 0.5 M may be attributed solely to its histone H1 component. The effect of various ions and urea on the complex has been investigated using NMR and confirm that the contraction of the complex on increase of ionic strength is largely due to electrostatic interactions. A detailed study of the H1 . DNA complex has also been undertaken. The behaviour of H1 in the two cases is virtually identical, implying that in chromatin the HI is complexed with the DNA rather than with the other histones. Microcalorimetric measurements reveal that the binding of H I to DNA is athermic or involves a heat of reaction which is very small indeed. This is thc second in a series of papers in which the role of the very-lysine-rich histone H1 in Chromatin structure is investigated by N M R and other methods. The first of the series [ l ] was devoted to the structure of the isolated histone.Chromatin can easily be extracted from calf thymus by isolation of clean nuclei, followed by gentle breakage of nuclear membrane to release the chromatin. The standard product of this process is a uniform gel having a DNA concentration in the region of 6 mgjg chromatin, together with the five histone fractions and a few percent of non-histone proteins. As has been reported previously [2] the gel may be caused to contract to about 15 ?< of its original volume by dialysing against sodium chloride solutions in the region of 0.05 -0.45 molar, while higher concentrations of sodium chloride remove the H1 and cause the contracted gel to relax to its original volume. The contraction of chromatin is dependent on the presence of H I , and the nuclear magnetic resonance (NMR) spectra of the gel may be attributed entirely to the HI component of the complex, the other histones apparently being bound much more tightly, so that their spectra are broadened and are unobservable under the conditions used. A more detailed study of the effects of various ions and of urea on chromatin gel Ahhreviurions. NMK, nuclear magnetic resonance; HI, hislone H1 (FI).has now been undertaken, and the results are presented in this communication. In addition studies have been made of the interaction between H1 and DNA alone, with the interesting result that both the NMR spectra and the macroscopic behaviour of the complex exactly parallel those in chromatin. This may be taken as an indication that the operation of HI in chromatin is largely or completely independent of the other histones. The formation of the H1 .

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