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)

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A simple method for the computer simulation of high-resolution nuclear magnetic resonance spectra is described and applied to the analyses of salt-induced changes in the spectra of histones. The method is more objective than the earlier approach of visual examination of spectral changes.It allows the information contained in the spectral envelope to be used and has led to some modifications of the earlier proposals of the segments of histones involved in salt-induced conformational changes, interhistone interactions and interactions with DNA. A feature of these newer proposals is their self-consistency, particularly of the results obtained for F2B, the cleaved amino and carboxyl halves of F2B and of F2B * DNA.The direct relationship between the amino-acid sequence of a protein, its three-dimensional structure and its function is now an accepted part of the dogma of molecular biology. I n the case of most proteins, the tertiary structure is determined within quite close limits by the intramolecular interactions between the various parts of its constituent polypeptide chain, and any changes in the shape of the molecule are restricted to small effects resulting from, for example, the binding of substrates, or to the drastic and frequently irreversible process of denaturation. I n the light of this general observation, the group of basic nuclear proteins known as histones stand out as being unusual in that they undergo large and reversible changes in conformation with relatively small changes in solvent conditions. They are further distinguished from most other proteins by the highly asymmetrical way in which residues of differing characters, for example apolar or basic residues, are grouped along the molecular chain, as demonstrated by the sequence for histones F2A1 [I] and F2B [2] and the partial sequence of F1 [3]. Thus the character of one section of a given histone molecule, as expressed in its readinness to form helical conformations or in its ability to interact with other molecules, differs greatly from that of other sections of the same molecule [I-71.These characteristics of the histones lead naturally to the proposal that different parts of the histone Abbreviation. NMR, nuclear magnetic resonance.molecule perform different functions in the structure of chromatin and the complex changes which chromatin undergoes during mitosis. It is therefore of considerable interest to study isolated histone fractions in order to elucidate the properties of individual sections of the molecules and the intramolecular and intermolecular changes which may be induced in them, with a view to the eventual, though as yet remote, solution of the problem of their precise role in the structural changes of the chromosomes.Of the techniques available for studying proteins in solution, nuclear magnetic resonance appears one of the most promising in that it is capable, in t,heory at least, of providing data on each individual residue in the molecule. I n practice, however, even a t the highest frequencies available or projected for t...

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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