Fluorescently Labelled Histones as Probes of Nucleosome Structure

Abstract

A fluorescent derivative of calf thymus histone H4 has been prepared by the reaction of methionine-84 with N-(iodoacetylaminoethyl)8-naphthylamine-l -sulfonic acid at pH 2.4 in 8 M urea. The preparation and characterization of this labelled histone is described. Fluorescence emission measurements indicate that the label on H4 undergoes a 3 -5-fold increase in emission intensity when H4 self-interacts or binds to DNA alone or is incorporated in a synthetic nucleosome. The changes observed are consistent with the formation of varied apolar environments around methionine-84, due most likely to histone-histone rather than histone-DNA interactions. Preliminary experiments indicate that the precise emission intensity of labelled H4 in the nucleosome is quite sensitive to conditions of ionic strength and histone integrity.There is now considerable interest in the structure of the fundamental chromosomal unit, the nucleosome (for a review see [l]). While X-ray and neutron diffraction studies [2,3] reveal details of the folding of this entity what is also needed is the ability to examine directly the possibility of conformational isomerism. It may be that the basic nucleosome folding scheme can adopt one of a number of discrete structures in response to variables such as ionic strength, degree of histone modification, and non-histone composition.One approach to studying this possibility involves the attachment of environment-sensitive probes to unique sites in the nucleosome [4-61. These probes could then be used to monitor structural changes, both equilibrium and dynamic, in response to variations in the parameters mentioned above. In this connection we wish to report the preparation and general properties of histone H4 labelled with N-(iodoacetylarninoethy1)-8-naphthylamine-l-sulfonic acid at the single methionine-84. This paper will deal mainly with the qualitative behaviour of this covalently bound fluorescent label in free solution and when complexed with DNA and the other histones in a nucleosome. Subsequent communications will be concerned with the quantitative aspects of these interactions and the use of this and other probes covalently attached to the other histones in the nucleosome to elucidate conformational transitions.Abbreviution. PhMeS02F, phenylmethylsulphonyl fluoride. Enzyme. Micrococcal nuclease (EC 3.1.4.7). MATERIALS AND METHODS Preparation of Histone H4 and H4 Peptide 69-102Electrophoretically homogeneous H4 was isolated by gel filtration on Bio-Gel P-10 [7] from a roughly equimolar mixture of histones H2A and H4 prepared by the method of Hooper and Smith [8]. To ensure the absence of methionine oxidation, the H4 samples were treated overnight with 50 % thioglycolic acid [91 and were recovered by acetone precipitation. A crude fraction of H4 peptide 69-102 was prepared by gel filtration on Sephadex G-25 of a partial digest (0.25 M acetic acid) of histone H4 [7]. Contaminating peptide 1-23 was removed by bringing the peptide mixture to 12% perchloric acid and spinning down the precipitate. The pelle...

Intermolecular histone H4 interactions in core nucleosomes

“…We report here the results of experiments with histone H4 labeled with pyrene at which indicate the existence of a histone H4-H4 contact in the vicinity of the bound fluor when this histone is incorporated by reconstitution into a nucleosome-like particle. Further we show that the pyrene excimer fluorescence which results from the close proximity of two pyrenes in the same nucleosome depends on the ionic strength of the solution and agrees well with other studies on the conformation of the nucleosome in its native form (Ausio et al, 1984;Uberbacher et al, 1983) and when containing dansyl-labeled histone H4 (Lewis, 1979; Chung & Lewis, 1985).…”

“…show that this procedure results in labeled nucleosomes that are virtually indistinguishable from native particles by a variety of criteria (Lewis, 1979; Lewis & Chiu, 1980; Chung & Lewis, 1985). The reconstituted nucleosomes, after purification on 5-20% sucrose gradients, were routinely checked for their homogeneity, mobility on 5% TCE (10 mM Tris-cacodylate and 0.7 mM EDTA, pH 7.4) polyacrylamide gels (Maniatis et al, 1975), histone integrity, and stoichiometry.…”

Nucleosome Structure

“…The DNA in these terminal regions of the core particle may have a different twist than does DNA in the core particle interior (see above). salt range have also been observed in the core particle diffusion coefficient (168), circular dichroism (169), (170), electric dichroism (171), and in the increased solvent accessibility of fluorescent probes (127,129). Low ionic strength also prevents the fo rmation of a cross-link between histones H2B and H4 but not between H2A and H2b (172).…”

Internal architecture of the core nucleosome: fluorescence energy transfer studies at methionine-84 of histone H4

“…Fluorophore Met-84 H4 Labeled Particles Are Similar to Native Nucleosomes. As we have shown earlier (Lewis, 1979; Chung & Lewis, , 1986, Met-84 fluorophore modified histone H4 together with unmodified histones H2A, H2B, and H3 and core length DNA is easily incorporated into nucleosome like particles which can be isolated in homogeneous form from 5-20% sucrose gradients. Analysis of these labeled particles reveals that they are virtually indistinguishable from native nucleosomes on the basis of their histone composition, sedimentation velocity, gel mobility, thermal denaturation characteristics, and DNase I digestion products as shown in Figure 1.…”

“…Chicken core length DNA and electrophoretically homogeneous core histones were isolated as described previously . Histone H4 was specifically modified at its single Met-84 residue by the method of Lewis (1979) using N-[ [ (iodoacetyl) amino] ethyl] -5-naphthylamine-1 -sulfonic acid (1,5-IAEDANS)* 1 **(tritiated as well as cold) and 5-(iodoacetamido)fluorescein (5-IAF). Protein concentrations were determined by the method of Lowry et al (1951).…”