Eur. J. Biochem. 9Y, 315-322 (1979)
Fluorescently Labelled Histones as Probes of Nucleosome Structure Preparation and General Properties of Methionine-Labelled Histone H4
Peter N. LEWIS Department of Biochemistry, University of Toronto (Received January 17,’June 11, 1979)
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- 102
Electrophoretically 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 pellet contained electrophoretically homogeneous H4 peptide 69-102. The amino acid composition of histone H4 and peptide 69 - 102 agreed with the published sequence [lo, 111. Labelling ofHistone H4 andHistone H4 Peptide69- 102 with N- (Iodoacetylaminoethyl)-8-naphthylamineI-suljonic Acid A typical labelling experiment involved dissolving 50 mg of histone H4 in 5 ml of 8 M urea (SchwartzMann, ultra-pure) adjusted to pH 2.4 with concentrated HCl. To this mixture was added 20mg of N (iodoacetylaminoethyl) - 8 - naphthylamine - 1 - sulfonic acid (Aldrich Chem.Co.) and the sample was incubated in the dark at 37°C for 115 h. The mixture was then desalted on a Sephadex G-25 column (25 x 1.6 cm)
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eluted with 0.01 M HCI. Labelled H4 was recovered from the void volume fractions by precipitation with 7 % perchloric acid. A similar, but scaled-down, procedure was used for peptide 69 - 102. The acid-soluble precipitates were washed once with acidified acetone (0.1 HCI), twice with dry acetone and finally vacuum-dried. The extent of labelling was about 50 % based on the criteria described below. Determination of Amount and Position of Label Incorporated into Histone H4 The extent of labelling was calculated from the absorbance at 345 nm of a labelled H4 sample of known concentration [12] at pH 7 using ~ 3 =~ 5950 5 M c m p l determined from the measured absorption of a solution of known concentration of the free tag under the same conditions. A similar value was reported by Hudson and Weber [I 31 for the 1, 5 isomer after reaction with N-acetylcysteine. The position of the label was determined in two ways, (a) CNBr digestion: samples of the labelled protein were incubated overnight at 20 "C in 70 % formic acid containing 1 "/, CNBr. The resulting solution was then lyophilized to dryness and the lyophilizate examined on 15 % 2.5 M urea/acetic acid gels [14]. (b) Histones H I , H3, H2A and H2B and histone H4 peptides 1 - 23, 25 - 67, 69 - 84 and 86 - 102 prepared as described previously [7,15] were reacted with N-(iodoacetylaminoethy1)-8-naphthylamine-1sulfonic acid under the same conditions given above. After desalting and precipitation, the absorbance of aqueous solutions of known concentration of these compounds at pH 7 were measured at 345 nm and the extent of labelling was computed. Synthetic Chromatin and Nucleosome Preparation An equimolar mixture of histones H2A, H2B, H3 and labelled H4 in 2 M NaCl, 40 mM Tris, 1 mM EDTA, 1 % 2-mercaptoethanol, 1 mM PhMeSOzF pH 8 was mixed with high-molecular-weight calf thymus DNA (Sigma type I) dissolved in the same buffer. A final weight ratio of histone to DNA of 0.75 was chosen with a DNA concentration of 495 pg/ml. The mixture in 2 M NaCl was dialyzed in Spectrapor l tubing (Spectrum Medical Industries) for l h against 1 MNaCl,20mMTris,0.5mM EDTApH8 and then gradient-dialyzedfor 18 M t o 0.14MNaC1,20mM Tris, 0.5 mM EDTA pH 8 with an exponential gradient. The resulting solution was then dialyzed into nuclease digestion buffer (10 mM Tris, 1 mM CaClz pH 7.4) under which conditions the complex precipitated. A portion of the precipitate was suspended in 1 ml of the digest buffer to give a concentration of 40 A260 units/ml, then 80 units of micrococcal nuclease (Sigma grade VI) were added. The enzyme was
Fluorescently Labelled Histone H4
allowed to act for 30 min at 37 "C at which time 5 p1 of 0.5 M EDTA solution was added to terminate the reaction. The sample was spun for 10 min at 10000 x g and the clear supernatant containing 75% of the original Az60 units was loaded onto a Bio-Gel A-5m column (90 x 1.6 cm) containing 10 mM Tris-cacodylate, 0.7 mM EDTA pH 8 as eluant [16]. The 3-ml fractions collected were measured for their fluorescence at 480nm (excitation 370nm) and absorbance at 260 nm. Protein-free 150-base-pair DNA was prepared by deproteinization of the synthetic nucleosome [17]. Electrophoresis The ability of labelled histone H4 to interact with itself and with histone H3 in solutions of moderate ionic strength was measured by electrophoresis on 10 polyacrylamide gels containing 0.05 M sodium phosphate at pH 7, run as described elsewhere [15]. These gels were prerun with 25 pl of 1 M cysteamine hydrochloride to reduce the oxidizing effect of residual ammonium persulfate [9] on histone H3. Synthetic nucleosomes and the constituent DNA were characterized by gel electrophoresis using the 3.5 % Tris/ borate/EDTA gels of Maniatis et al. [IS] as described previously [19]. The histone composition was determined by the discontinuous 18% sodium dodecylsulfate gels of Laemmli [20] as modified by Weintraub et al. [21]. Gels containing fluorescent bands were illuminated by means of a long-wavelength ultraviolet lamp and photographed with an appropriate filter. The acidlurea and phosphate gels were stained with amido black, the 3.5 % Tris/borate/EDTA gels first with ethidium bromide and subsequently with Coomassie blue G in trichloroacetic acid [22], if they contained protein. Scans of stained gels were made with a densitometer attachment for a Pye-Unicam SP1800 spectrophotometer. Spectroscopic Measurements Ultraviolet spectra were recorded by a Bausch and Lomb Spectronic 505 while quantitative absorption measurements were made on a Pye-Unicam SP500 spectrophotometer. Fluorescence emission and excitation spectra were measured with a Baird-Atomic SFI spectrofluorometer. All fluorescence measurements were made at room temperature (z22°C) and are uncorrected for lamp, monochromator and photomultiplier variations. RESULTS AND DISCUSSION Labelling of Histone H4 at Methionine-84 At the beginning of this study it was our intention to label histone H3 in calf whole histone at the two cysteine residues with N-(iodoacetylaminoethyl)-8-
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naphthylamine-I -sulfonic acid [13]. Although the labelling was accomplished, it was found however that the modified histone H3 would no longer complex with histone H4 in free solution in the usual manner. The result was consistent with our previous data on the conformational sensitivity of these H3 sites [15,23]. Due to buried nature of the H3 cysteine residues [24,25], it was necessary to use quite long reaction times to achieve appreciable labelling with this reagent. We observed that during these long reactions even histone H4 became partially labelled. The most likely H4 sites [26] are the two histidine residues at positions 17 and 75 and the single methionine at position 84. Since attaching a probe at a unique position in the more apolar section of this histone seemed very attractive, we attempted to optimize this 'side reaction'. Earlier studies by Vithayathil and Richards [27] indicate that methionine is labelled specifically, albeit slowly, by iodoacetate at low pH values. To further complicate matters, the labelling reagent is fairly insoluble in acidic solutions. We found that by means of a concentrated urea solution, low pH and moderate temperatures, appreciable specific labelling of methionine could be achieved with no detectable labelling at other sites or acid hydrolysis of the histone. Evidence for labelling at only methionine comes from four indirect observations. First, a time course study (shown in Fig.1) of the extent of labelling indicates a limiting value of 1, assuming a pseudofirst-order reaction. Second, of the histone H4 peptides 1-23, 25-67, 69-84 and 86- 102 treated, only the methionine-containing peptide 69 - 84 became fluorescently labelled under the reaction conditions. Third, cyanogen bromide digestion of partially labelled histone H4 yields two peptides, 1- 84 and 85- 102, neither of which are labelled as indicated in Fig.2A. The extent of CNBr cleavage of the histone H4 based on gel scans of the uncleaved H4 and the peptide 1-84 agrees reasonably well with the spectroscopically determined amount of label per H4 molecule, as shown in Table 1. Virtually no label was released during the CNBr cleavage as all the fluorescence was recovered after precipitation with perchloric acid and acetone washing of the reaction lyophilizate. Also, desalting of the lyophilized digest did not reveal any released label. Finally, of the five histones only histones H2B, H3 and H4 became labelled under the acidic reaction conditions. Neither calf histone H1 nor H2A contain methionine. This specificity is shown in gels of Fig. 2B. We conclude from these results that, under the acidic reaction conditions used, this reagent labels only methionine in histone H4. As the sulfonium salt created by the iodoacetate reaction with the methionine sulfur is destroyed in hot acid [26], an amino acid analysis would be of little use in confirming methionine as the labelled residue.
Table 1, Time course of. histone H 4 lahelling with N-(iodoaceiylaminoethyl)~-naphtliylamine-I -sulfonic acid as assayed hq' ahsorhance and CNBr digestion Labelling was calculated from the absorbance at 345 nm by comparison with that of a solution of labelled histone of known 4 5950 ~ M - ' cm-' for the free tag. Labelling concentration and ~ 3 = was measured by CNBr digestion by calculating the relative areas of peaks from scanned gels due to histone H4 and peptide 1 - 84. Corrections were made for the smaller size of peptide 1-84 by the multiplicative factor 1.21 and for the efficiency of the cleavage of unlabelled H4 (8673, due presumably to the formation of methionine oxides Time
Labelling measured by CNBr digestion
A345
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0 12 24 31 46 62
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Fig. 1. Time course of histone H4 labelling h j N-(iodoucet~~lumincieth~l)-~-naphthq'lamine-l-sulfonic acid in 8 M urea p H 2.4. The histone H4 and tag concentrations were 10 and 2 mg/ml respectively. The ratio of covalently bound label to H4 was calculated from the absorbance at 345 nm and the concentration of a pH-7 solution of the histone H4 as a function of time of reaction at 37 -C
Interacting Properties of the Labelled Histone H4 Unlabelled histone H4 aggregates by itself in solutions of moderate ionic strength and forms a tetrameric complex with histone H3 (for a review see [28,29]). One of the major requirements of our study was that the labelled H4 should still participate in these processes in the same manner as the unlabelled protein. As mentioned earlier, -SH-labelled H3 does not satisfy this condition. In most of our preparations of labelled histone H4 the extent of labelling is about 50 %. We have attempted to subfractionate the labelled histone directly from the unlabelled histone with no success. Even CNBr digestion of the unlabelled H4
Fluorescently Labelled Histone H 4
318
and separation of the peptide 1 - 84 from the labelled H4 by the self-aggregation method of Ziccardi and Schumaker [30] did not work as a significant fraction of the peptide 1-84 also aggregates. Small amounts of pure labelled H4 were obtained by preparative electrophoresis of the CNBr digest on acidlurea gels and quantitative fluorescent measurements confirm the visual observation that peptide 1 - 84 is unlabelled. That methionine-labelled histone H4 behaves as its unlabelled parent can be seen from Fig. 3 which shows H4 self-interaction and histone H3/H4 complex formation on phosphate gels [15,23]. Competition experiments involving the addition of unlabelled histone H4 to an equimolar mixture of labelled histone H4
A
H4 1-84 -
b
a
d
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Fig. 3. Ability of luhelled histone H4 io self-associute and compler with histone H3. Gel pair 1 : an equimolar mixture of 50 labelled histone H4 with histone H3 run on a non-denaturing 10% 0.05 M phosphate pH 7 gel; left-hand gel was illuminated with 330-nm light, the right-hand one was stained with amido black. Gel pair 2 : the same H3, labelled H4 mixture run on a 157; urea/acetic acid gels. Gel pair 3 ; 50% labelled histone H4 run on the 10% 0.05 M phosphate pH 7 gel; all of the H4 histone aggregates and remains at the gel origin, as does a sample of unlabelled protein
and histone H3, followed by gel analysis, showed qualitatively that the labelled H4 had the same affinity for H3 as the unlabelled histone H4. Quantitative measurements confirming this were also made by eluting the protein from a macerated gel with 0.1 % sodium dodecylsulfate and measuring the fluorescence and by eluting the dye from a duplicate stained gel [15]. Fluorescent Properties on Labelled Histone H4 in Solution
H -1 - 2B,3
- 2A -4 e
f
Fig. 2. Clrea,'clcetic cicitl gels u j ( A ) C'NBr-digested lahrlled H 4 und ( B ) acid-extracted whole calfthymus labelled hi.stonr. (A) Gels a, b and c show the N-(iodoacetylaminoethy1)-8-naphthylamine-1sulfonic acid reaction after (a) 0, (b) 70 and (c) 140 h, stained with amino black. Gel d shows the 140-h reaction, photographed while illuminated with 330-nm light; similar, but less intense, fluorescent bands were obtained for shorter reaction times. Fragment 85- 102 ran too fast on these eels relative to H4 and ueutide . . 1-84 to be conveniently displayed; no fluorescence was detected in this fast-moving band. (B) Acid-extracted whole calf thymus histone reacted for 70 h with the above fluorescent label in 8 M urea at pH 2.4. Gel d was photographed while illuminated with 330-nm light, gel e was stained with amido black. The horizontal lines on the left indicate the visible fluorescent bands L
The absorption spectra of partially labelled histone H4 as well as unlabelled H4 are shown in Fig. 4. The covalently bound label has two main absorption bands centered at 260 and 345 nm, in good agreement with the results of Hudson and Weber [13] for the label alone. The fluorescence emission spectra of labelled H4 in solutions of increasing ionic strength at pH 8 are shown in Fig. 5A. About a fivefold increase in the fluorescent emission intensity and a wavelength maximum shift from 505 nm in water to 480 nm in 2 M NaCl is observed. It is well known that under these conditions H4 changes from a relatively disordered conformation to a highly ordered structure including aggregate formation [7]. This large increase in quantum yield was also observed by Hudson and Weber [13] for the free label when it was transferred from an aqueous solution to one containing 60% ethanol. As an indication of the relative apolar environment that the label is experiencing, an emission spectrum in 0.2% sodium dodecylsulfate is also shown in Fig. 5 A . Each Of the ionic strength spectra shown in Fig. 5 A change to the dodecylsulfate spec-
319
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Fig. 5. Uncorrected,fluo,.escence emission spectra of ( A ) labelled histone H4, ( B ) labelled H4 peptide 69- 102 and ( C ) labelled hisronr H4 with 150-base-pair calf thymus DNA. In (A) the histone concentration was 25 pg/ml and the buffer was 1 m M Tris p H 8 ; NaCl concentration as indicated. The dashed line corresponds t o that spectrum obtained when any of the samples which gave the solid-line spectra were adjusted to 0.2% sodium dodecylsulfate. The peptide concentration in (B) was 4 pg/ml and the buffer was 10 mM Tris p H 8. Solid line corresponds to no added salt, the dashed line to the addition of NaCl to 2 M. In (C) labelled histone H4 was mixed directly with the DNA to give final concentrations of 12 and 44 pg/ml respectively. The solution conditions were: curve 1, 10 mM Tris p H 8; curve 2, 2 M NaCI/10 m M Tris pH 8 ; curve 3, 8 M urea/lO mM Tris pH 8 ; curve 4, 10 m M Tris p H 8 but no D N A ; curve 5, 8 M urea/l0 mM Tris pH 8 but no DNA. The excitation wavelength in all spectra was 370 nm
trum when the detergent is added. This suggests that the origin of the increased emission intensity for the H4-bound label is a structural one, rather than one that is due to the effect of salt on the label itself. To reinforce this, the emission spectra of similarly labelled H4 peptide 69 - 102 in low and high ionic strength buffers is shown in Fig. 5B. Virtually no salt-induced spectral changes occur. These results for labelled H4 and peptide 69- 102 agree well with our previous studies [7,31] on histone H4 which showed that while
the region 69 - 102 of intact H4 participates in the salt-induced structure formation, the free peptide remains in the random coil state. A quantitative analysis of these fluorescence changes will be reported in a future publication. Fluorescent Properties of Labelled Histone H4 Bound to D N A The fluorescence emission spectrum of labelled histone H4 mixed directly with 150-base-pair DNA
320
in various solvents is shown in Fig. 5 C. The magnitude and wavelength of the shift on binding to DNA are similar but larger than those found for this histone in high salt (see Fig. 5 A). This suggests that histone H4 aggregates on binding to DNA in a manner similar to that which occurs in free solution in the presence of salt. Addition of solid urea reduces the emission intensity to nearly that of the histone in a low ionic strength buffer in the absence of DNA. Since H4 is still bound to the DNA in the presence of urea [32,33], the increase in fluorescence is unlikely to be due to a direct interaction with the DNA. This is supported by the observation that labelled peptide 69- 102 undergoes only a comparatively small (75 %) increase in fluorescence emission on binding to the same DNA. Rather the emission changes probably result from the formation of an apolar environment near methionine-84 due to histone H4 folding and self-association when binding to DNA. These results are consistent with previous studies [30] which have shown that histone H4, when alone, binds to DNA in a cooperative fashion producing local regions of high protein concentration along the DNA molecule. Preparation and Properties of Synthetic Chromatin and Nucleosomes Containing Fluorescently Labelled Histone H4 In chromatin and isolated nucleosomes histone H4 is found to be associated with histones H2A, H2B and H3 in an octameric complex [34]. A synthetic chromatin and nucleosome were prepared using the methionine-labelled H4 as described in Materials and Methods. The elution profile of micrococcal-nucleasedigested synthetic chromatin is shown in Fig.6. The four fractions that contained appreciable fluorescent
Fluorescently Labelled Histone H4
intensity and 260-nm absorbance were pooled and characterized. The DNA size of this synthetic nucleosome was found to be 151 k 26 base pairs and the protein/DNA ratio was 1.08. All the four input histones were present in approximately equal proportions as judged from 18 % sodium dodecylsulfate gels. The mobility of this synthetic nucleosome on 3.5% Tris/borate/EDTA gels was 0.61 k 0.04 relative to a bromophenol blue marker and agrees exactly with that found by us for an 11-S native calf thymus 140base-pair core nucleosome on the same gel system [19]. The thermal denaturation derivative profile at 260 nm for this synthetic nucleosome was quite similar to that obtained for a native particle (not shown). The fluorescence emission spectra of the synthetic nucleosome in various solvents are shown in Fig.7. Essentially the same spectral changes were also obtained from the synthetic chromatin, indicating that no changes in fluorescence were caused by the nuclease digestion or subsequent purification steps. Two main points can be made from these results. First, in the synthetic chromatin and nucleosome the histone H4 methionine-84 is exposed to an apolar environment resulting in a high quantum yield and a blue-shifted emission maximum at 472 nm. The emission intensity increase relative to labelled H4 in 10 mM Tris pH 8 is not as great (3.3) as for labelled H4 in a highly aggregated state (4.4) or for labelled H4 when bound to DNA alone (5.3). Second, addition of solid salt to 2 M results in a large decrease in fluorescence as well as a red shift to 480 nm. A similar, but larger decrease and wavelength shift was found when the synthetic chromatin or nucleosome was made 8 M in urea. The large increases in fluorescence emission and wavelength maxima when H4 undergoes either self-
321
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Ionic strength (mM) Fig. 8. Ionic-strength-induced changes in ,fluorescence emission of labelled histone H4 incorporated into a synthetic nucleosome. The DNA concentration was 26 pg/ml and the protein/DNA ratio was determined as 1.08 (w/w). The sample was initially in 0.25 mM EDTA pH 8, aliquots of 0.1 M NaCl were added and the fluorescence measured at 475 nm. Excitation was at 370 nm. The relative fluorescence is normalized to the initial value with no added salt 400
500 Wavelength (nm)
600
Fig. 7. Uncorrected .fluorescence emission spectra of histone-H4labelled mononucleosome in various solutions. The histone H 4 concentration was 12 pg/ml. Curve 1, 10 mM Tris pH 8 ; curve 2, 0.2% sodium dodecylsulfate, 30 mM Tris pH 8 ; curve 3, 2 M NaCI, 10 mM Tris p H 8. A curve similar to curve 3 was obtained with 8 M urea. Excitation was at 370 nm
interactions or becomes incorporated into a synthetic nucleosome indicate that position 84 on H4 is clearly a site for histone-histone interactions. Based on the properties of the label itself in solutions of decreasing polarity [13], one might argue that this region of H4 experiences a range of apolar environments depending upon the particular interaction considered. This is particularly evident when the nucleosome is exposed to 2 M NaC1. Under these conditions the site 84 on H4 is probably much more exposed to the solvent that it is in the low ionic strength situation. Thomas et al. [35] have shown that in 2 M NaCl, even though the histone core is dissociated from the DNA, the secondary structure of the protein is largely preserved. Our result demonstrates that while this may be true on a gross scale, locally major structural changes are probably occurring on binding of the octamer core to the DNA. The sizeable changes in fluorescence emission shown in Fig.7 represent the extremes of complete protein unfolding or core histone octamer dissociation of the synthetic nucleosome. Preliminary experiments indicate that the fluorescence emission intensity and fluorescence polarization values can be made to vary with only small changes in parameters such as ionic strength and histone integrity. While the results of such experiments will form the basis of future reports, as an illustration of the utility of this approach the relative fluorescence intensity of the labelled histone H4 synthetic nucleosome as a function of ionic
strength is shown in Fig.8. From this it can be seen that in the ionic strength range 0-40 mM there is a sharp transition centered at 1.3 mM, and possibly a second beyond 5 mM, which is quite broad. These fluorescent changes can be compared to the sedimentation coefficient changes reported by Gordon et al. [36]in native core nucleosomes at 1 mM and 7.5 mM ionic strength. The increase in emission intensity with ionic strength shown in Fig. 8 is consistent with their folding proposal for the nucleosome [36]. In a more compact nucleosome it is plausible that the environment around methionine-84 would become less polar. Correlations of this type indicate that certain specific chemical modifications can be made to the histones without drastically interfering with their structural function. For this reason we are very encouraged by these results which demonstrate that extrinsic labels will probably yield considerable information about the conformational flexibility of this fundamental particle. Approaches of this type might ultimately reveal how a single entity can function in both transcriptionally active and inactive regions of the genome [37]. Fluorescent Properties of Labelled Histone H4 in the Presence of Other Histones The section of histone H4 containing methionine84 has been implicated [23,38,39] in the interacting surface of the tetrameric histone H3 . H4 complex. It might be expected therefore that the fluorescent label at this locus would experience an altered environment in the presence of histone H3 under complexing conditions [23,40]. The relative values of the fluorescence emission intensities at 470 nm for mixtures of labelled histone H4 with the three other core
P. N. Lewis: Fluorescently Labelled Histone H4
322 Table 2. Changes in the fluorescence emission intensity of labelled histone H4 in the presence of other histones Equimolar amounts of labelled histone H4 and other histones in 20 p1 10 mM HC1 were added to 1.5 ml of 10 mM sodium phosphate p H 7.4. The fluorescence emission at 470 nm (excited at’ 360 nm) was measured immediately. The fluorescence value given is normalized to the value for labelled H4 alone in the same buffer. Histone H4 concentration was 1 pM Sample
Fluorescence at 470 nm
~~~~
~~
Labelled Labelled Labelled Labelled Labelled
H4 H4 and H4 and H4 and H4 and
histone H2A histone H2B histone H 3 DNA
1 .oo 1.04 102 1.45 2.50
histones confirms this as shown in Table 2. Only histone H3 gives rise to a significantly altered value and this indicates that under the conditions of measurement (0.01 M phosphate pH 7.4, 1 pM histone H4, the label is in a more apolar environment that it is alone or with histones H2A and H2B. This is almost certainly due to the high stability ofthe histone H3 . H4 complex compared to the others [40]. The fluorescence emission intensity of the labelled histone H4/H3 mixture is considerably lower than the value obtained for the binding of labelled H4 to DNA or for the value obtained when this histone is incorporated in a synthetic nucleosome. It is, however, quite similar to the value obtained for the synthetic nucleosome containing labelled H4 when exposed to 2 M NaCl as shown in Fig.7. While this result does not prove that the labelled H4 is involved in the same structure in both the H3 . H4 tetramer and high-saltdissociated histone octamer, it is at least consistent with this notion. This research was supported by a grant from the Medical Research Council of Canada.
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P. N. Lewis, Department of Biochemistry, University of Toronto, Medical Sciencies Building, Toronto, Ontario, Canada, M5S 1A8
7. Lewis, P. N., Bradbury, E. M. Sr Crane-Robinson, C. (1975) Biochemistry, 14, 3391 - 3400. 8. Hooper, J. A. Sr Smith, E. L. (1973) J . Biol. Chem. 248, 32553260. 9. Alfageme, C. R., Zweidler, A., Mahowald, A. Sr Cohen, L. (1974) J . Bid. Chem. 249,3729-3736. 10. Ogawa, Y., Quagliarotti, G., Jordan, J., Taylor, C. W., Starbuck, W. C. Sr Busch, H. (1969) J . B i d . Chem. 244, 43874392. 11. DeLange, R . J., Fambrough, D. M., Smith, E. L. Sr Bonner, J. (1969) J . Bid. Chem. 244, 319-334. 12. Lowry, 0. H., Rosebrough, N . J., Farr, A. L. Sr Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. 13. Hudson, E. N . Sr Weber, G. (1973) Biochemistry, 12, 41544161. 14. Panyim, S. Sr Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337-346. 15. Lewis, P. N. (1976) Can. J . Biochem. 54, 641 -649. 16. Shaw,B. R., Corden, J. L., Sahasrabuddhe,C.G. Sr Van Holde, K. E. (1974) Biochem. Biophys. Res. Commun. 61, 11931198. 17. Britten, R. J., Graham, D. E. Sr Neufeld, 8 . R. (1974) Methods Enzymol. 29, 363-418. 18. Maniatis, T., Jeffrey, A. Sr Van dc Sande, H. (1975) Biochrmistry, 14, 3787- 3194. 19. Lewis, P. N. (1977) Can. J . Biochem. 55, 736-746. 20. Laemmli, U . K. (1971) Nature (Lond.) 227, 1. 21. Weintraub, H., Palter, K. Sr Van Lente, F. (1975) Cell, 6, 85110.
22. Blakesley, R. W. 91 Boezi, J. A. (1977) Anal. Biochem. 82, 580- 582. 23. Lewis, P. N . (1976) Can. J . Biochem. 54, 963-970. 24. Hyde, J. E. 91 Walker, I. 0. (1974) Nucleic Acids Res. I , 203 -215. 25. Garrad, W. T., Nobis, P. s( Hancock, R. (1977) J . Biol. Chem. 252, 4962-4967. 26. Curd, F. R. N. (1967) Methods Enzymol. 11, 532-541. 27. Vithayathil, P. J. Sr Richards, F. M. (1960) J . Bid. Chem. 235, 2343 - 2351. 28. Van Holde, K. E. Sr Isenberg, I. (1975) Acc. Chem. Res. 8, 327-335. 29. Kornberg, R. D. (1977) Annu. Rev. Biochem. 46, 931 -954. 30. Ziccardi, R. Sr Schumaker, V. (1973) Biochemistry, 12, 3231 3235. 31. Crane-Robinson, C., Hayashi, H., Cary, P. D., Briand, G., Sautiere, P., Krieger, D., Vidali, G., Lewis, P. N. Sr TomKun, J. (1977) Eur. J . Biochem. 79, 535-548. 32. Chalkley, R. Sr Jensen, R. H. (1968) Biochemistry, 7, 43804387. 33. Olins, D . E., Bryan, P. N., Harrington, R. E., Hill, W. E. s( Olins, A. (1977) Nucleic Acids Res. 4, 1911 -1931. 34. Thomas, J. 0. Sr Kornberg, R. D. (1975) Proc. Narl Acad. Sci. U.S.A. 72, 2626-2630. 35. Thomas, G. J., Prescott, B. Sr Olins, D . E. (1977) Science (Wash. D.C.) 197, 385-388. 36. Gordon, V. C., Knobler, C. M., Olins, D. E. h Schumaker, V. N. (1978) Proc. Natl Acad. Sci. U.S.A. 75, 660-663. 37. Weintraub, H. Sr Groudine, M. (1976) Science (Wash. D.C.) 193, 848 - 856. 38. Moss, T., Cary, P. D., Crane-Robinson, C. Sr Bradbury, E. M . (1976) Biochemistry, 15, 2261 -2267. 39. Bohm, L., Hayashi, H., Cary, P. D., Moss, T., Crane-Robinson, C. Sr Bradbury, E. M. (1977) Eur. J . Biochem. 77, 487 -493. 40. D’Anna, J. A. Sr Isenberg, 1. (1974) Biochemistry, 13, 49924997.
Fluorescently Labelled Histones as Probes of Nucleosome Structure Preparation and General Properties of Methionine-Labelled Histone H4
Peter N. LEWIS Department of Biochemistry, University of Toronto (Received January 17,’June 11, 1979)
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- 102
Electrophoretically 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 pellet contained electrophoretically homogeneous H4 peptide 69-102. The amino acid composition of histone H4 and peptide 69 - 102 agreed with the published sequence [lo, 111. Labelling ofHistone H4 andHistone H4 Peptide69- 102 with N- (Iodoacetylaminoethyl)-8-naphthylamineI-suljonic Acid A typical labelling experiment involved dissolving 50 mg of histone H4 in 5 ml of 8 M urea (SchwartzMann, ultra-pure) adjusted to pH 2.4 with concentrated HCl. To this mixture was added 20mg of N (iodoacetylaminoethyl) - 8 - naphthylamine - 1 - sulfonic acid (Aldrich Chem.Co.) and the sample was incubated in the dark at 37°C for 115 h. The mixture was then desalted on a Sephadex G-25 column (25 x 1.6 cm)
316
eluted with 0.01 M HCI. Labelled H4 was recovered from the void volume fractions by precipitation with 7 % perchloric acid. A similar, but scaled-down, procedure was used for peptide 69 - 102. The acid-soluble precipitates were washed once with acidified acetone (0.1 HCI), twice with dry acetone and finally vacuum-dried. The extent of labelling was about 50 % based on the criteria described below. Determination of Amount and Position of Label Incorporated into Histone H4 The extent of labelling was calculated from the absorbance at 345 nm of a labelled H4 sample of known concentration [12] at pH 7 using ~ 3 =~ 5950 5 M c m p l determined from the measured absorption of a solution of known concentration of the free tag under the same conditions. A similar value was reported by Hudson and Weber [I 31 for the 1, 5 isomer after reaction with N-acetylcysteine. The position of the label was determined in two ways, (a) CNBr digestion: samples of the labelled protein were incubated overnight at 20 "C in 70 % formic acid containing 1 "/, CNBr. The resulting solution was then lyophilized to dryness and the lyophilizate examined on 15 % 2.5 M urea/acetic acid gels [14]. (b) Histones H I , H3, H2A and H2B and histone H4 peptides 1 - 23, 25 - 67, 69 - 84 and 86 - 102 prepared as described previously [7,15] were reacted with N-(iodoacetylaminoethy1)-8-naphthylamine-1sulfonic acid under the same conditions given above. After desalting and precipitation, the absorbance of aqueous solutions of known concentration of these compounds at pH 7 were measured at 345 nm and the extent of labelling was computed. Synthetic Chromatin and Nucleosome Preparation An equimolar mixture of histones H2A, H2B, H3 and labelled H4 in 2 M NaCl, 40 mM Tris, 1 mM EDTA, 1 % 2-mercaptoethanol, 1 mM PhMeSOzF pH 8 was mixed with high-molecular-weight calf thymus DNA (Sigma type I) dissolved in the same buffer. A final weight ratio of histone to DNA of 0.75 was chosen with a DNA concentration of 495 pg/ml. The mixture in 2 M NaCl was dialyzed in Spectrapor l tubing (Spectrum Medical Industries) for l h against 1 MNaCl,20mMTris,0.5mM EDTApH8 and then gradient-dialyzedfor 18 M t o 0.14MNaC1,20mM Tris, 0.5 mM EDTA pH 8 with an exponential gradient. The resulting solution was then dialyzed into nuclease digestion buffer (10 mM Tris, 1 mM CaClz pH 7.4) under which conditions the complex precipitated. A portion of the precipitate was suspended in 1 ml of the digest buffer to give a concentration of 40 A260 units/ml, then 80 units of micrococcal nuclease (Sigma grade VI) were added. The enzyme was
Fluorescently Labelled Histone H4
allowed to act for 30 min at 37 "C at which time 5 p1 of 0.5 M EDTA solution was added to terminate the reaction. The sample was spun for 10 min at 10000 x g and the clear supernatant containing 75% of the original Az60 units was loaded onto a Bio-Gel A-5m column (90 x 1.6 cm) containing 10 mM Tris-cacodylate, 0.7 mM EDTA pH 8 as eluant [16]. The 3-ml fractions collected were measured for their fluorescence at 480nm (excitation 370nm) and absorbance at 260 nm. Protein-free 150-base-pair DNA was prepared by deproteinization of the synthetic nucleosome [17]. Electrophoresis The ability of labelled histone H4 to interact with itself and with histone H3 in solutions of moderate ionic strength was measured by electrophoresis on 10 polyacrylamide gels containing 0.05 M sodium phosphate at pH 7, run as described elsewhere [15]. These gels were prerun with 25 pl of 1 M cysteamine hydrochloride to reduce the oxidizing effect of residual ammonium persulfate [9] on histone H3. Synthetic nucleosomes and the constituent DNA were characterized by gel electrophoresis using the 3.5 % Tris/ borate/EDTA gels of Maniatis et al. [IS] as described previously [19]. The histone composition was determined by the discontinuous 18% sodium dodecylsulfate gels of Laemmli [20] as modified by Weintraub et al. [21]. Gels containing fluorescent bands were illuminated by means of a long-wavelength ultraviolet lamp and photographed with an appropriate filter. The acidlurea and phosphate gels were stained with amido black, the 3.5 % Tris/borate/EDTA gels first with ethidium bromide and subsequently with Coomassie blue G in trichloroacetic acid [22], if they contained protein. Scans of stained gels were made with a densitometer attachment for a Pye-Unicam SP1800 spectrophotometer. Spectroscopic Measurements Ultraviolet spectra were recorded by a Bausch and Lomb Spectronic 505 while quantitative absorption measurements were made on a Pye-Unicam SP500 spectrophotometer. Fluorescence emission and excitation spectra were measured with a Baird-Atomic SFI spectrofluorometer. All fluorescence measurements were made at room temperature (z22°C) and are uncorrected for lamp, monochromator and photomultiplier variations. RESULTS AND DISCUSSION Labelling of Histone H4 at Methionine-84 At the beginning of this study it was our intention to label histone H3 in calf whole histone at the two cysteine residues with N-(iodoacetylaminoethyl)-8-
317
P. N. Lewis
naphthylamine-I -sulfonic acid [13]. Although the labelling was accomplished, it was found however that the modified histone H3 would no longer complex with histone H4 in free solution in the usual manner. The result was consistent with our previous data on the conformational sensitivity of these H3 sites [15,23]. Due to buried nature of the H3 cysteine residues [24,25], it was necessary to use quite long reaction times to achieve appreciable labelling with this reagent. We observed that during these long reactions even histone H4 became partially labelled. The most likely H4 sites [26] are the two histidine residues at positions 17 and 75 and the single methionine at position 84. Since attaching a probe at a unique position in the more apolar section of this histone seemed very attractive, we attempted to optimize this 'side reaction'. Earlier studies by Vithayathil and Richards [27] indicate that methionine is labelled specifically, albeit slowly, by iodoacetate at low pH values. To further complicate matters, the labelling reagent is fairly insoluble in acidic solutions. We found that by means of a concentrated urea solution, low pH and moderate temperatures, appreciable specific labelling of methionine could be achieved with no detectable labelling at other sites or acid hydrolysis of the histone. Evidence for labelling at only methionine comes from four indirect observations. First, a time course study (shown in Fig.1) of the extent of labelling indicates a limiting value of 1, assuming a pseudofirst-order reaction. Second, of the histone H4 peptides 1-23, 25-67, 69-84 and 86- 102 treated, only the methionine-containing peptide 69 - 84 became fluorescently labelled under the reaction conditions. Third, cyanogen bromide digestion of partially labelled histone H4 yields two peptides, 1- 84 and 85- 102, neither of which are labelled as indicated in Fig.2A. The extent of CNBr cleavage of the histone H4 based on gel scans of the uncleaved H4 and the peptide 1-84 agrees reasonably well with the spectroscopically determined amount of label per H4 molecule, as shown in Table 1. Virtually no label was released during the CNBr cleavage as all the fluorescence was recovered after precipitation with perchloric acid and acetone washing of the reaction lyophilizate. Also, desalting of the lyophilized digest did not reveal any released label. Finally, of the five histones only histones H2B, H3 and H4 became labelled under the acidic reaction conditions. Neither calf histone H1 nor H2A contain methionine. This specificity is shown in gels of Fig. 2B. We conclude from these results that, under the acidic reaction conditions used, this reagent labels only methionine in histone H4. As the sulfonium salt created by the iodoacetate reaction with the methionine sulfur is destroyed in hot acid [26], an amino acid analysis would be of little use in confirming methionine as the labelled residue.
Table 1, Time course of. histone H 4 lahelling with N-(iodoaceiylaminoethyl)~-naphtliylamine-I -sulfonic acid as assayed hq' ahsorhance and CNBr digestion Labelling was calculated from the absorbance at 345 nm by comparison with that of a solution of labelled histone of known 4 5950 ~ M - ' cm-' for the free tag. Labelling concentration and ~ 3 = was measured by CNBr digestion by calculating the relative areas of peaks from scanned gels due to histone H4 and peptide 1 - 84. Corrections were made for the smaller size of peptide 1-84 by the multiplicative factor 1.21 and for the efficiency of the cleavage of unlabelled H4 (8673, due presumably to the formation of methionine oxides Time
Labelling measured by CNBr digestion
A345
"0° - 0.75
h
(7)
0 22 45 70 114 160
0 12 24 31 46 62
0 8 17 34 39 46
3
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0.25
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/
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150
Time (h)
Fig. 1. Time course of histone H4 labelling h j N-(iodoucet~~lumincieth~l)-~-naphthq'lamine-l-sulfonic acid in 8 M urea p H 2.4. The histone H4 and tag concentrations were 10 and 2 mg/ml respectively. The ratio of covalently bound label to H4 was calculated from the absorbance at 345 nm and the concentration of a pH-7 solution of the histone H4 as a function of time of reaction at 37 -C
Interacting Properties of the Labelled Histone H4 Unlabelled histone H4 aggregates by itself in solutions of moderate ionic strength and forms a tetrameric complex with histone H3 (for a review see [28,29]). One of the major requirements of our study was that the labelled H4 should still participate in these processes in the same manner as the unlabelled protein. As mentioned earlier, -SH-labelled H3 does not satisfy this condition. In most of our preparations of labelled histone H4 the extent of labelling is about 50 %. We have attempted to subfractionate the labelled histone directly from the unlabelled histone with no success. Even CNBr digestion of the unlabelled H4
Fluorescently Labelled Histone H 4
318
and separation of the peptide 1 - 84 from the labelled H4 by the self-aggregation method of Ziccardi and Schumaker [30] did not work as a significant fraction of the peptide 1-84 also aggregates. Small amounts of pure labelled H4 were obtained by preparative electrophoresis of the CNBr digest on acidlurea gels and quantitative fluorescent measurements confirm the visual observation that peptide 1 - 84 is unlabelled. That methionine-labelled histone H4 behaves as its unlabelled parent can be seen from Fig. 3 which shows H4 self-interaction and histone H3/H4 complex formation on phosphate gels [15,23]. Competition experiments involving the addition of unlabelled histone H4 to an equimolar mixture of labelled histone H4
A
H4 1-84 -
b
a
d
C
B
Fig. 3. Ability of luhelled histone H4 io self-associute and compler with histone H3. Gel pair 1 : an equimolar mixture of 50 labelled histone H4 with histone H3 run on a non-denaturing 10% 0.05 M phosphate pH 7 gel; left-hand gel was illuminated with 330-nm light, the right-hand one was stained with amido black. Gel pair 2 : the same H3, labelled H4 mixture run on a 157; urea/acetic acid gels. Gel pair 3 ; 50% labelled histone H4 run on the 10% 0.05 M phosphate pH 7 gel; all of the H4 histone aggregates and remains at the gel origin, as does a sample of unlabelled protein
and histone H3, followed by gel analysis, showed qualitatively that the labelled H4 had the same affinity for H3 as the unlabelled histone H4. Quantitative measurements confirming this were also made by eluting the protein from a macerated gel with 0.1 % sodium dodecylsulfate and measuring the fluorescence and by eluting the dye from a duplicate stained gel [15]. Fluorescent Properties on Labelled Histone H4 in Solution
H -1 - 2B,3
- 2A -4 e
f
Fig. 2. Clrea,'clcetic cicitl gels u j ( A ) C'NBr-digested lahrlled H 4 und ( B ) acid-extracted whole calfthymus labelled hi.stonr. (A) Gels a, b and c show the N-(iodoacetylaminoethy1)-8-naphthylamine-1sulfonic acid reaction after (a) 0, (b) 70 and (c) 140 h, stained with amino black. Gel d shows the 140-h reaction, photographed while illuminated with 330-nm light; similar, but less intense, fluorescent bands were obtained for shorter reaction times. Fragment 85- 102 ran too fast on these eels relative to H4 and ueutide . . 1-84 to be conveniently displayed; no fluorescence was detected in this fast-moving band. (B) Acid-extracted whole calf thymus histone reacted for 70 h with the above fluorescent label in 8 M urea at pH 2.4. Gel d was photographed while illuminated with 330-nm light, gel e was stained with amido black. The horizontal lines on the left indicate the visible fluorescent bands L
The absorption spectra of partially labelled histone H4 as well as unlabelled H4 are shown in Fig. 4. The covalently bound label has two main absorption bands centered at 260 and 345 nm, in good agreement with the results of Hudson and Weber [13] for the label alone. The fluorescence emission spectra of labelled H4 in solutions of increasing ionic strength at pH 8 are shown in Fig. 5A. About a fivefold increase in the fluorescent emission intensity and a wavelength maximum shift from 505 nm in water to 480 nm in 2 M NaCl is observed. It is well known that under these conditions H4 changes from a relatively disordered conformation to a highly ordered structure including aggregate formation [7]. This large increase in quantum yield was also observed by Hudson and Weber [13] for the free label when it was transferred from an aqueous solution to one containing 60% ethanol. As an indication of the relative apolar environment that the label is experiencing, an emission spectrum in 0.2% sodium dodecylsulfate is also shown in Fig. 5 A . Each Of the ionic strength spectra shown in Fig. 5 A change to the dodecylsulfate spec-
319
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\\
I
I
1 250
1
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-
\ \
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300 Wavelength (nrn)
Fig. 4. Absorption spectrum of H4 and fluorescently labelled H4 in 10 m M Tris p H 8. (----)
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.
.
.
.
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.
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Fig. 5. Uncorrected,fluo,.escence emission spectra of ( A ) labelled histone H4, ( B ) labelled H4 peptide 69- 102 and ( C ) labelled hisronr H4 with 150-base-pair calf thymus DNA. In (A) the histone concentration was 25 pg/ml and the buffer was 1 m M Tris p H 8 ; NaCl concentration as indicated. The dashed line corresponds t o that spectrum obtained when any of the samples which gave the solid-line spectra were adjusted to 0.2% sodium dodecylsulfate. The peptide concentration in (B) was 4 pg/ml and the buffer was 10 mM Tris p H 8. Solid line corresponds to no added salt, the dashed line to the addition of NaCl to 2 M. In (C) labelled histone H4 was mixed directly with the DNA to give final concentrations of 12 and 44 pg/ml respectively. The solution conditions were: curve 1, 10 mM Tris p H 8; curve 2, 2 M NaCI/10 m M Tris pH 8 ; curve 3, 8 M urea/lO mM Tris pH 8 ; curve 4, 10 m M Tris p H 8 but no D N A ; curve 5, 8 M urea/l0 mM Tris pH 8 but no DNA. The excitation wavelength in all spectra was 370 nm
trum when the detergent is added. This suggests that the origin of the increased emission intensity for the H4-bound label is a structural one, rather than one that is due to the effect of salt on the label itself. To reinforce this, the emission spectra of similarly labelled H4 peptide 69 - 102 in low and high ionic strength buffers is shown in Fig. 5B. Virtually no salt-induced spectral changes occur. These results for labelled H4 and peptide 69- 102 agree well with our previous studies [7,31] on histone H4 which showed that while
the region 69 - 102 of intact H4 participates in the salt-induced structure formation, the free peptide remains in the random coil state. A quantitative analysis of these fluorescence changes will be reported in a future publication. Fluorescent Properties of Labelled Histone H4 Bound to D N A The fluorescence emission spectrum of labelled histone H4 mixed directly with 150-base-pair DNA
320
in various solvents is shown in Fig. 5 C. The magnitude and wavelength of the shift on binding to DNA are similar but larger than those found for this histone in high salt (see Fig. 5 A). This suggests that histone H4 aggregates on binding to DNA in a manner similar to that which occurs in free solution in the presence of salt. Addition of solid urea reduces the emission intensity to nearly that of the histone in a low ionic strength buffer in the absence of DNA. Since H4 is still bound to the DNA in the presence of urea [32,33], the increase in fluorescence is unlikely to be due to a direct interaction with the DNA. This is supported by the observation that labelled peptide 69- 102 undergoes only a comparatively small (75 %) increase in fluorescence emission on binding to the same DNA. Rather the emission changes probably result from the formation of an apolar environment near methionine-84 due to histone H4 folding and self-association when binding to DNA. These results are consistent with previous studies [30] which have shown that histone H4, when alone, binds to DNA in a cooperative fashion producing local regions of high protein concentration along the DNA molecule. Preparation and Properties of Synthetic Chromatin and Nucleosomes Containing Fluorescently Labelled Histone H4 In chromatin and isolated nucleosomes histone H4 is found to be associated with histones H2A, H2B and H3 in an octameric complex [34]. A synthetic chromatin and nucleosome were prepared using the methionine-labelled H4 as described in Materials and Methods. The elution profile of micrococcal-nucleasedigested synthetic chromatin is shown in Fig.6. The four fractions that contained appreciable fluorescent
Fluorescently Labelled Histone H4
intensity and 260-nm absorbance were pooled and characterized. The DNA size of this synthetic nucleosome was found to be 151 k 26 base pairs and the protein/DNA ratio was 1.08. All the four input histones were present in approximately equal proportions as judged from 18 % sodium dodecylsulfate gels. The mobility of this synthetic nucleosome on 3.5% Tris/borate/EDTA gels was 0.61 k 0.04 relative to a bromophenol blue marker and agrees exactly with that found by us for an 11-S native calf thymus 140base-pair core nucleosome on the same gel system [19]. The thermal denaturation derivative profile at 260 nm for this synthetic nucleosome was quite similar to that obtained for a native particle (not shown). The fluorescence emission spectra of the synthetic nucleosome in various solvents are shown in Fig.7. Essentially the same spectral changes were also obtained from the synthetic chromatin, indicating that no changes in fluorescence were caused by the nuclease digestion or subsequent purification steps. Two main points can be made from these results. First, in the synthetic chromatin and nucleosome the histone H4 methionine-84 is exposed to an apolar environment resulting in a high quantum yield and a blue-shifted emission maximum at 472 nm. The emission intensity increase relative to labelled H4 in 10 mM Tris pH 8 is not as great (3.3) as for labelled H4 in a highly aggregated state (4.4) or for labelled H4 when bound to DNA alone (5.3). Second, addition of solid salt to 2 M results in a large decrease in fluorescence as well as a red shift to 480 nm. A similar, but larger decrease and wavelength shift was found when the synthetic chromatin or nucleosome was made 8 M in urea. The large increases in fluorescence emission and wavelength maxima when H4 undergoes either self-
321
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Ionic strength (mM) Fig. 8. Ionic-strength-induced changes in ,fluorescence emission of labelled histone H4 incorporated into a synthetic nucleosome. The DNA concentration was 26 pg/ml and the protein/DNA ratio was determined as 1.08 (w/w). The sample was initially in 0.25 mM EDTA pH 8, aliquots of 0.1 M NaCl were added and the fluorescence measured at 475 nm. Excitation was at 370 nm. The relative fluorescence is normalized to the initial value with no added salt 400
500 Wavelength (nm)
600
Fig. 7. Uncorrected .fluorescence emission spectra of histone-H4labelled mononucleosome in various solutions. The histone H 4 concentration was 12 pg/ml. Curve 1, 10 mM Tris pH 8 ; curve 2, 0.2% sodium dodecylsulfate, 30 mM Tris pH 8 ; curve 3, 2 M NaCI, 10 mM Tris p H 8. A curve similar to curve 3 was obtained with 8 M urea. Excitation was at 370 nm
interactions or becomes incorporated into a synthetic nucleosome indicate that position 84 on H4 is clearly a site for histone-histone interactions. Based on the properties of the label itself in solutions of decreasing polarity [13], one might argue that this region of H4 experiences a range of apolar environments depending upon the particular interaction considered. This is particularly evident when the nucleosome is exposed to 2 M NaC1. Under these conditions the site 84 on H4 is probably much more exposed to the solvent that it is in the low ionic strength situation. Thomas et al. [35] have shown that in 2 M NaCl, even though the histone core is dissociated from the DNA, the secondary structure of the protein is largely preserved. Our result demonstrates that while this may be true on a gross scale, locally major structural changes are probably occurring on binding of the octamer core to the DNA. The sizeable changes in fluorescence emission shown in Fig.7 represent the extremes of complete protein unfolding or core histone octamer dissociation of the synthetic nucleosome. Preliminary experiments indicate that the fluorescence emission intensity and fluorescence polarization values can be made to vary with only small changes in parameters such as ionic strength and histone integrity. While the results of such experiments will form the basis of future reports, as an illustration of the utility of this approach the relative fluorescence intensity of the labelled histone H4 synthetic nucleosome as a function of ionic
strength is shown in Fig.8. From this it can be seen that in the ionic strength range 0-40 mM there is a sharp transition centered at 1.3 mM, and possibly a second beyond 5 mM, which is quite broad. These fluorescent changes can be compared to the sedimentation coefficient changes reported by Gordon et al. [36]in native core nucleosomes at 1 mM and 7.5 mM ionic strength. The increase in emission intensity with ionic strength shown in Fig. 8 is consistent with their folding proposal for the nucleosome [36]. In a more compact nucleosome it is plausible that the environment around methionine-84 would become less polar. Correlations of this type indicate that certain specific chemical modifications can be made to the histones without drastically interfering with their structural function. For this reason we are very encouraged by these results which demonstrate that extrinsic labels will probably yield considerable information about the conformational flexibility of this fundamental particle. Approaches of this type might ultimately reveal how a single entity can function in both transcriptionally active and inactive regions of the genome [37]. Fluorescent Properties of Labelled Histone H4 in the Presence of Other Histones The section of histone H4 containing methionine84 has been implicated [23,38,39] in the interacting surface of the tetrameric histone H3 . H4 complex. It might be expected therefore that the fluorescent label at this locus would experience an altered environment in the presence of histone H3 under complexing conditions [23,40]. The relative values of the fluorescence emission intensities at 470 nm for mixtures of labelled histone H4 with the three other core
P. N. Lewis: Fluorescently Labelled Histone H4
322 Table 2. Changes in the fluorescence emission intensity of labelled histone H4 in the presence of other histones Equimolar amounts of labelled histone H4 and other histones in 20 p1 10 mM HC1 were added to 1.5 ml of 10 mM sodium phosphate p H 7.4. The fluorescence emission at 470 nm (excited at’ 360 nm) was measured immediately. The fluorescence value given is normalized to the value for labelled H4 alone in the same buffer. Histone H4 concentration was 1 pM Sample
Fluorescence at 470 nm
~~~~
~~
Labelled Labelled Labelled Labelled Labelled
H4 H4 and H4 and H4 and H4 and
histone H2A histone H2B histone H 3 DNA
1 .oo 1.04 102 1.45 2.50
histones confirms this as shown in Table 2. Only histone H3 gives rise to a significantly altered value and this indicates that under the conditions of measurement (0.01 M phosphate pH 7.4, 1 pM histone H4, the label is in a more apolar environment that it is alone or with histones H2A and H2B. This is almost certainly due to the high stability ofthe histone H3 . H4 complex compared to the others [40]. The fluorescence emission intensity of the labelled histone H4/H3 mixture is considerably lower than the value obtained for the binding of labelled H4 to DNA or for the value obtained when this histone is incorporated in a synthetic nucleosome. It is, however, quite similar to the value obtained for the synthetic nucleosome containing labelled H4 when exposed to 2 M NaCl as shown in Fig.7. While this result does not prove that the labelled H4 is involved in the same structure in both the H3 . H4 tetramer and high-saltdissociated histone octamer, it is at least consistent with this notion. This research was supported by a grant from the Medical Research Council of Canada.
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