Interaction of dipalmitoyl-phosphatidylcholine with calf thymus histone H1

The interaction between dipalmitoyl-phosphatidylcholine and calf thymus histone H1 has been studied. A protein-phospholipid complex, resulting from this interaction, has been isolated by centrifugation in a sucrose gradient. The phospholipid-histone interaction causes an increase in the alpha-helix content of the protein; the corresponding conformational transition is observed by CD studies in the far-u.v. region. The only tyrosine residue of the protein can be advantageously used as an intrinsic fluorescent probe; thus, fluorescence spectra indicate that protein folding induced by phospholipids is concomitant with the tyrosine transfer into a more hydrophobic environment. The trypsin-resistant core of the histone is also folded in the presence of the phospholipid but the conformational transition occurs at lower lipid concentration than for the intact protein. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene indicates that the protein shifts the transition temperature of the phospholipid from 41.5 to 44.0 degrees. Secondary structure prediction of the trypsin-resistant core of the histone indicates the existence of an amphipathic helix that could be responsible for the lipid-protein interaction.     

Conformational changes in subfractions of calf thymus histone H1.

This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.     

Phosphorylation of calf thymus H1 histone by muscle glycogen phosphorylase kinase

Muscle glycogen phosphorylase kinase [EC 2.7.1.38] has the ability to phosphorylate five fractions of calf thymus histone. H1 histone is the most preferable substrate, and maximally about 1.3 mol of phosphate is incorporated into every mole of this histone. This reaction absolutely depends on CA2+, and the molecular activity is about one third of that of cyclic AMP-dependent protein kinase (protein kinase A). The affinity of phosphorylase kinase for H1 histone is higher than that of protein kinase A. Calmodulin stimulates this histone phosphorylation. Analysis of the N-bromosuccinimide-bisected fragments of fully phosphorylated H1 histone has revealed that the enzyme phosphorylates mostly seryl residues in both amino- and carboxyl-terminal portions, although phosphorylation of the carboxyl-terminal portion is twice as much as that of the amino-terminal portion. Fingerprint analysis indicates that the phosphorylation sites in H1 histone for this enzyme are different from the sites phosphorylated by protein kinase A. This catalytic activity also differs from that of a newly found multifunctional protein kinase which may be activated by the simultaneous presence of Ca2+ and phospholipid.     

Effects of chemical modification of lysine residues in trypsin

Chemical modifications are a simple method to identify and modify functional determinants of enzymes. In the case of serine proteases, it is possible to induce characteristics which are advantageous for peptide synthesis. In this work, we investigated the influence of guanylation and succinylation of lysine residues on the S′-subsite specificity, the catalytic behavior and stability of trypsin. We have found, that succinylation leads to an about 10-fold better acceptance of basic residues in P1′, whereas guanylation shows no remarkable effects. Furthermore, guanylation enhances, succinylation reduces the general enzyme–substrate interactions in P2′. The structural fundamentals of these specificity changes are discussed. The catalytic behavior of trypsin was not influenced by guanylation and succinylation but an enhancement of the stability against autolytic processes by introducing additional negative charges into the protein was observed.     

Characteristics of the tertiary structure of histone H1 from the calf thymus

By optical methods it has been previously shown that the globular "head" of histone H1 forms a hydrophobic cavity containing Tyr72. The latter is screened from the polar water surrounding and its intramolecular mobility is drastically hindered. As a consequence of the alteration in the micromilieu are a long wave shift (lambda max = 279,5 nm) and a more pronounced longwave absorption spectra, higher anisotropy (A = 0,11), augmented quantum yield of fluorescence (approximately 0,2) and a decrease of the Stern-Volmer constant for Hl at fluorescence quenching by acrylamide. It was found that changes in fluorescence intensity of histones are connected with alterations in the quantum yield of fluorescence at lambda exc = = 265 nm, but not at lambda exc = 280 nm. The changes in fluorescence intensity at light excitation 280 nm (F280) and 265 nm (F265) are in good accordance with shift delta E286 in differential absorption spectra. Introduction of parameter Cf = F280/F265 allows to study shifts of excitation spectra instead of shifts in absorption spectra of histones. This method has certain advantages, since it permits investigations with lower protein concentrations and in turbid solutions. The data obtained allow to draw out Tyr72 of histone Hl into a special class of fluorescent-tyrosyls, that differ in properties from those of other tryptophandevoided proteins: RNAse, insulin and core-histones H2A, H2B, H3 and H4.     

Phosphorylation of calf thymus H1 histone by calcium-activated,phospholipid-dependent protein kinase

Ca²⁺-activated, phospholipid-dependent protein kinase recently found in mammalian tissues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) $\text{J.}$$\text{Biol.}$$\text{Chem.}$$\text{254}$, 3692–3695) is able to phosphorylate five fractions of calf thymus histone. H1 histone serves as a preferential substrate, and approximately two moles of phosphate are incorporated into every mole of this histone. Analysis on the N-bromosuccinimide-bisected fragments of this radioactive histone has revealed that the enzyme phosphorylates preferentially seryl and threonyl residues located in the carboxyl-terminal half of this histone molecule.     

Selective chemical modification of arginine residues in mitochondrial malate dehydrogenase

Mitochondrial malate dehydrogenase (L-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) from porcine heart exhibits a time dependent loss in enzymatic activity in the presence of the reagent butanedione. The inhibition occurs concomitant with the modification of 2.4 residues of arginine per molecular weight of 70,000. The presence of the reduced coenzyme, NADH, protects the enzyme from inhibition by butanedione and from modification of arginine residues, suggesting that the residues modified are located near the coenzyme binding site and hence at or near the enzymatic active center of this enzyme.     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as ε-N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of ε-N-methyllysines (1.40, 1.66, and 5.62 mol% for ε-N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of ε-N-methyllysines in histone H1.     

Adenosine diphosphate ribosylation of histone H1 by purified calf thymus polyadenosine diphosphate ribose polymerase

The mechanism of poly ADPR synthesis and the transfer of poly ADPR to histone H1 molecule by electrophoretically homogenous calf thymus poly ADPR polymerase containing DNA was examined. 1) An acid insoluble radioactive complex (I) was obtained after incubation of purified enzyme with [3H] NAD. The stability of (I) was examined by SDS-polyacrylamide gel electrophoresis. The complex (I) was stable against acid, SDS, urea, DNase and RNase, but labile against pronase, trypsin, alkali and snake venom phosphodiesterase treatment. The molecular weight of (I) was about 130 000 daltons estimated by SDS-gel electrophoresis. The radioactive products of successive alkali, venom phosphodiesterase and Pronase hydrolysis of (I) were PR-AMP and AMP. The mean chain length of poly ADPR of (I) was 20--30. These results suggest that the complex (I) is poly ADP-ribosylated poly ADPR polymerase. 2) Besides (I), a second radioactive peak (II) was observed when acid insoluble products obtained from an incubation mixture containing purified poly ADPR polymerase, [3H] NAD and purified histone H1 were analyzed on SDS-polyacrylamide gel electrophoresis. The molecular weight of (II) was estimated to be about 23 000 daltons. The complex (II) is eluted like histone H1 on CM-cellulose columns and hydrolyzed by alkali, trypsin and snake venom phosphodiesterase but not by DNase, or RNase. The comples (II) was extracted selectively by 5 per cent perchloric acid or 5 per cent trichloroacetic acid from mixture of (I) and (II). The mean chain length of poly ADPR of complex (II) and 5--20; these results suggest that the complex (II) is poly ADP-ribosylated histone H1. 3) Results 1) and 2) indicate that purified DNA containing, thus DNA independent, poly ADPR polymerase catalyzes two different reactions, the ADPR transfer onto the enzyme itself and onto histone H1 and the elongation of ADPR chains. Dimeric forms of ADP-ribosylated histone H1 was not observed. Free poly ADPR was observed only when very small quantities of enzyme were used for incubation.     

Chemical modification of lysine and arginine residues in the myosin regulatory light chain inhibits phosphorylation

The contribution of lysine and arginine residues to the substrate specificity of the myosin light-chain kinase has been studied using chemically modified myosin light chains. Succinylation or maleylation of the myosin light chains caused complete inhibition of their phosphorylation. Modification of 50% of the lysine residues resulted in 90% inhibition of phosphorylation and this was accompanied by a 25-fold increase in the apparent Km. In contrast, phosphorylation of the myosin light chains by the cAMP-dependent protein kinase was relatively insensitive to lysine modification, with only a 15% reduction in phosphorylation following succinylation of 50% of the lysine residues. Treatment with either cyclohexane-1,2-dione or camphorquinone-10-sulfonic acid resulted in between 90 and 98% inhibition of myosin light-chain phosphorylation. These reagents caused modification of both lysine and arginine residues, and accordingly only part of the inhibition can be attributed to arginine modification. Modification of all of the cysteine and methionine residues caused only a 40% inhibition of phosphorylation. The results of this study support the concept that lysine and arginine residues act as essential specificity determinants for the myosin light-chain kinase in protein substrates.     

Intrinsic fluorescence, difference spectrophotometry and theoretical studies on tertiary structure of calf thymus histone H1

Tyr-72 is included in the hydrophobic cleft which is formed in the histone H1 globular head. Tyr-72 is screened against polar aqueous environment and its intramolecular mobility is sharply retarded. This microenvironment causes a red shift (lambda max = 279 nm) and a sharpening of the longer wavelength shoulder of absorption spectra, a high fluoresence anisotropy value (A = 0,11), high quantum yield of fluoresence (approximately 0.2) and a decrease of the Stern-Volmer Constant during quenching of histone H1 fluorescence by acrylamide. It has been found that the change in the intensity of histone fluorescence at lambda excit = 265 nm, but not at lambda excit = 280 nm, is due to the changes in the quantum yield of fluorescence. The increase of fluorescence intensity at lambda excit = 280 nm depends on the changes in the quantum yield and molar extinction coefficient of histone H1 tyrosyl chromophore. The change in the ratio of fluorescence intensity exited at 280 nm (F280) to the fluorescence intensity excited at 265 nm (F265) corresponds to the change of delta epsilon 286 in difference absorption spectra. The introduction of the parameter Cf = F280/F265 allows one to go over to studying excitation spectrum shifts instead of histone absorption spectrum shifts, which is much more convenient methodologically since in this case it is possible to carry out research using lower protein concentrations and turbid solutions. The results make it possible to designate Tyr-72 of histone H1 as a special class of fluorescent tyrosyls whose properties differ from those of tyrosyls of other tryptophane-free proteins: RNAase, insulin, core histones--H2A, H2B, H3, H4 and some others.     

Distribution of histone F1 on calf thymus nucleohistone DNA.

A method of large-scale preparation of the histone F1-DNA complex by removing all other proteins from calf thymus nucleohistone was established. This involved gel filtration of nucleohistone through a column containing a band of sodium dodecyl sulfate. The F1-DNA complex obtained had the original amount of F1 and no other. The F1-DNA complex exhibited distinct two-step melting on thermal denaturation. The first step was apparently attributable to naked DNA regions and the second step, about 30 deg. C higher than the first step, to the regions covered with F1. Buoyant density experiments with the complex after fixation with formalin revealed that F1 was distributed fairly evenly over DNA fragments of an average molecular weight of about 4 × 10⁶. Electron microscopic examination of the complex after various degrees of denaturation with formalin indicated that the longest stretch of unbound DNA was about 0·3 μm.     

Structure, chemical modification and interaction of histone H1 with chromatin components

Structure, chemical modification, and interaction of histone H1 and its individual fragments with DNA and structural elements of chromatin are considered. Special attention is paid to phosphorylation of histone H1 molecules. Recent data concerning localization and mobility of histone H1 in chromatin as well as mechanisms of nucleosomal chain condensation are reviewed.