Tyrosine residues in histones. Kinetics of histones F1 and F2A1 nitration by tetranitromethane]

The kinetics of nitration of tyrosine residues in histones F1 and F2a1 by tetranitromethane has been investigated. At low ionic strength and 30-fold molar excess of nitrating agent the nitration reaction results in fast modification of all tyrosine residues in both histones. At the same time the rates of modification of different tyrosine residues in histone F2a1 are not identical and markedly exceed the rate of N-Ac-OEt-Tyr nitration in a model system. The increase of reaction mixture ionic strength causes an increase of modification rates. The differential UV-absorption spectra of histone F1 obtained by temperature perturbation show an abnormal positive characteristic maximum at 286.8 nm. Analysis of the dependence of nitration rates of tyrosine residues in histones in saline solutions upon the ionic strength and of difference UV-absorption spectra of histones leads to a conclusion that there are specific interactions of definite parts of histone polypeptide chains. These interactions may arise from aggregation of histone molecules.     

Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives

The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49-64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118-123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the pKa of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (pKa = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine pKa shift in the colipase micelle complex may explain why nitrated colipase can reactivate lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5.     

Comparison of the role of tyrosine residues in human IgG and rabbit IgG in binding of complement subcomponent C1q.

Treatment of covalently cross-linked or heat-aggregated oligomers of human IgG with 4 mM-tetranitromethane abrogated their C1q-binding activity. In contrast, tetranitromethane modification of rabbit IgG oligomers, under identical conditions, had no effect upon their C1q-binding activity. The tetranitromethane treatment led to nitration of about ten tyrosine residues per IgG molecule in both species, and the modification was specific for tyrosine residues. Reduction of the nitrated protein with Na2S2O4 did not lead to recovery of C1q-binding activity in human IgG oligomers or to loss of activity in rabbit IgG oligomers. Tryptic peptides from the nitrated proteins were isolated and a peptide containing nitrotyrosine-319 was recovered from human IgG, as well as peptides from both species corresponding to the region around nitrotyrosine-278. These data are consistent with the inactivation of C1q-binding activity in human IgG being the result of nitration of tyrosine-319; the rabbit IgG is unaffected by nitration because position 319 is phenylalanine. The evidence supports the C1q-receptor site proposed by Burton, Boyd, Brampton, Easterbrook-Smith, Emanuel, Novotny, Rademacher, van Schravendijk, Sternberg & Dwek [(1980) Nature (London) 288, 338-344]: residues 316-338.     

Probing structural differences in prion protein isoforms by tyrosine nitration

Two conformational isomers of recombinant hamster prion protein (residues 90-232) have been probed by reaction with two tyrosine nitration reagents, peroxynitrite and tetranitromethane. Two conserved tyrosine residues (tyrosines 149 and 150) are not labeled by either reagent in the normal cellular form of the prion protein. These residues become reactive after the protein has been converted to the beta-oligomeric isoform, which is used as a model of the fibrillar form that causes disease. After conversion, a decrease in reactivity is noted for two other conserved residues, tyrosine 225 and tyrosine 226, whereas little to no effect was observed for other tyrosines. Thus, tyrosine nitration has identified two specific regions of the normal prion protein isoform that undergo a change in chemical environment upon conversion to a structure that is enriched in beta-sheet.     

Exposed tyrosine residues of lambda cro repressor protein evidenced by nitration and photo CIDNP experiments

The tyrosine residues of lambda cro repressor were partially nitrated with tetranitromethane under mild conditions. After digestion by Achromobacter protease I, the extent of nitration was determined by HPLC and amino acid analysis. Tyr 26 was most easily nitrated and Tyr 51 followed it. Tyr 10 was resistant to nitration. By comparison of the proton magnetic resonance spectrum of the partially nitrated cro protein with the above result, the aromatic proton resonances of the tyrosine side chains could be assigned to individual tyrosine residues. The extent of nitration is parallel to the accessibility to a flavin dye as measured by photo CIDNP (chemically induced dynamic nuclear polarization).     

Chemical modification of human alpha 1-proteinase inhibitor by tetranitromethane. Structure-function relationship.

Nitration of tyrosine residues of alpha 1-proteinase inhibitor (alpha 1-PI) by tetranitromethane yielded a product that maintained its inhibitory activity against trypsin but lost most of its inhibitory activity against elastase. Chemical analysis of the product showed that four out of the six tyrosine residues in alpha 1-PI had been nitrated to various degrees: Tyr-38 and Tyr-297 were not nitrated, whereas Tyr-138, Tyr-160, Tyr-187 and Tyr-244 were nitrated to extents in the range 40-80%. We interpreted these data to mean that modification of these tyrosine residues decreased the association constant between alpha 1-PI and the proteinases and that the decrease differs from one proteinase to the other. When either alpha 1-PI-trypsin or alpha 1-PI-elastase complex was nitrated, nitration took place only to a very slight extent at these latter four tyrosine residues. On the other hand, Tyr-38 and Tyr-297 underwent nitration to about 20%. We concluded that Tyr-138, Tyr-160, Tyr-187 and Tyr-244 were located on the surface of alpha 1-PI that interacts with either trypsin or elastase in the formation of complexes, and were therefore protected from nitration.     

Optical measurement of a solvent-induced isomerization in a proline-containing hexapeptide.

The hexapeptide Gly-Gly-Pro-Tyr-Gly-Gly has been synthesized and its tyrosine residue converted to nitrotyrosine by reaction with tetranitromethane. When diluted from dimethylsulfoxide into aqueous solution, the nitrated hexapeptide undergoes a slow conformational change characterized by a change in the ionization state of the nitrotyrosine group. This slow reaction is not observed with peptides containing nitrotyrosine and no proline. Also, the rate and activation enthalpy of this slow conformational change suggest that it could be due to proline cis-trans isomerization. The possibility of measuring the rate of cis-trans isomerization of proline residues in a polypeptide chain is discussed.     

The status of tyrosyl residues in a Formosan cobra cardiotoxin.

Spectrophotometric titration of Formosan cobra cardiotoxin showed that two of the three tyrosyl residues were titrated freely with a normal apparent pKa of 9.6 whereas the remaining one ionized at pH above 11.0. Nitration of cardiotoxin in Tris . HCl buffer with tetranitromethane resulted in the selective nitration of tyrosine 11 and tyrosine 22. It also revealed that tyrosine 51 was the abnormal one in the spectrophotometric titration. Complete nitration occurred in the presence of 6.0 M guanidine hydrochloride. Compared with the conformation of native cardiotoxin, the peptide conformation of the partially nitrated cardiotoxin did not change significantly but the conformation of the completely nitrated cardiotoxin changed remarkably. The biological activity of cardiotoxin was indeed affected by nitration, but the immunological activity was nearly intact even when all the tyrosine residues were nitrated.     

Identification of an essential tyrosine residue in nitroalkane oxidase by modification with tetranitromethane

The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with production of nitrite and hydrogen peroxide. The enzyme is irreversibly inactivated by incubation with tetranitromethane, a tyrosine-directed reagent, at pH 7.3. The inactivation is time-dependent and shows first-order kinetics for two half-lives of inactivation. Further inactivation can be achieved upon a second addition of tetranitromethane. A saturation kinetic pattern is observed when the rate of inactivation is determined versus the concentration of tetranitromethane, indicating that a reversible enzyme-inhibitor complex is formed before irreversible inactivation occurs. Values of 0.096 +/- 0.013 min(-1) and 12.9 +/- 3.8 mM were determined for the first-order rate constant for inactivation and the dissociation constant for the reversibly formed complex, respectively. The competitive inhibitor valerate protects the enzyme from inactivation by tetranitromethane, suggesting an active-site-directed inactivation. The UV-visible absorbance spectrum of the inactivated enzyme is perturbed with respect to that of the native enzyme, suggesting that treatment with tetranitromethane resulted in nitration of the enzyme. Comparison of tryptic maps of nitroalkane oxidase treated with tetranitromethane in the presence and absence of valerate shows a single peptide differentially labeled in the inactivated enzyme. The spectral properties of the modified peptide are consistent with nitration of a tyrosine residue. The amino acid sequence of the nitrated peptide is L-L-N-E-V-M-C-(NO(2)-Y)-P-L-F-D-G-G-N-I-G-L-R. The possible role of this tyrosine in substrate binding is discussed.     

Modification of tyrosine residues of the lactose repressor protein.

Reaction of the lactose repressor protein from Escherichia coli with high molar excesses (up to 800 fold) of tetranitromethane resulted in modification of tyrosine residues in the amino-terminal and core regions of the molecule. Tyrosines 7 and 17 exhibit significant reactivity at low levels (5-10 fold molar excess) of tetranitromethane. The loss of operator binding activity upon nitration at these low concentrations of reagent indicates involvement of these two tyrosines in the binding process. Inducer binding activity was maintained at approx. 90% of unreacted repressor for all excesses of reagent studied. Addition of inducer to the repressor prior to reaction resulted in decreased modification of tyrosines in the core region, but anti-inducers did not affect the reaction significantly. The effect of inducers on the pattern of reaction apparently reflects the conformational change which occurs upon binding of these ligands. Acetylation of the repressor protein with N-acetylimidazole modified lysines and tyrosines with complete loss of operator binding activity and retention of 75-80% of inducer binding activity.     

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.     

Tyrosine modification of glucose dehydrogenase from Bacillus megaterium. Effect of tetranitromethane on the enzyme in the tetrameric and monomeric state

The active tetrameric glucose dehydrogenase from Bacillus megaterium is rapidly inactivated upon reaction with tetranitromethane. The inactivation is correlated with the nitration of a single tyrosine residue/subunit. The nitration does not influence the dissociation-reassociation process of the enzyme. The inactivation is prevented by the presence of NAD, AMP, ATP. The sequence around the nitrated tyrosine residue was determined and the residue was identified as Tyr-254 in the covalent structure of the enzyme. After dissociation of the enzyme into its monomers two tyrosine residues become susceptible to nitration. The nitrated subunits are unable to reassociate to the tetramer. Isolation and sequence analysis of the peptides containing nitrotyrosine indicated that two different tyrosine residues are predominantly modified. One residue is Tyr-254 which is essential for the catalytic activity and the other one is Tyr-160 which seems to be located in the subunit binding area.     

Chemical modification of the F0 part of the ATP synthase (F1F0) from Escherichia coli. Effects on proton conduction and F1 binding

The purified F0 part of the ATP synthase complex from Escherichia coli was incorporated into liposomes and chemically modified by various reagents. The modified F0-liposomes were assayed for H+ uptake and, after reconstitution with F1, for total and dicyclohexylcarbodiimide-sensitive ATPase activity. The water-soluble carbodiimide, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide methiodide, (1.2 mM), inhibited H+ uptake to a great extent. Binding of F1 was almost unaffected, but the hydrolysis of ATP was uncoupled from H+ transport. This is reflected by the inhibition of dicyclohexylcarbodiimide-sensitive ATPase activity. Woodward's reagent K, N-ethyl-5-phenylisoxazolium-3'-sulfonate, inhibited both H+ uptake and total ATPase activity. Modification of arginine residues by phenylglyoxal (20 mM) was followed by inhibition of the F1 binding activity by 80% of the control. H+ translocation was reduced to 70%. Diethylpyrocarbonate (3 mM) exhibited a strong inhibiting effect on H+ uptake but not on F1 binding. Modification of tyrosine (by tetranitromethane) as well as lysine residues (by succinic anhydride) did not affect F0 functions. From the data presented we conclude that carboxyl-groups, different from the dicyclohexylcarbodiimide-binding site, are involved in H+ translocation through F0 and, in part, in the functional binding of F1. Furthermore, for the latter function, also arginine residues seem to be important. The role of histidine residues remains unclear at present.     

Tyrosine nitration in prostaglandin H(2) synthase

In this study, we investigated the effects of various nitrogen oxide (NO(x)) species on the extent of prostaglandin H(2) synthase-1 (PGHS-1) nitration in purified protein and in vascular smooth muscle cells. We also examined PGHS-1 activity under these conditions and found the degree of nitration to correlate inversely with enzyme activity. In addition, since NO(x) species are thought to invoke damage during the pathogenesis of atherosclerosis, we examined human atheromatous tissue for PGHS-1 nitration. Both peroxynitrite and tetranitromethane induced Tyr nitration of purified PGHS-1, whereas 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7; a nitric oxide-releasing compound) did not. Smooth muscle cells treated with peroxynitrite showed PGHS-1 nitration. The extent of nitration by specific NO(x) species was determined by electrospray ionization mass spectrometry. Tetranitromethane was more effective than peroxynitrite, NOC-7, and nitrogen dioxide at nitrating a tyrosine-containing peptide (12%, 5%, 1%, and <1% nitration, respectively). Nitrogen dioxide and, to a lesser extent, peroxynitrite, induced dityrosine formation. Using UV/Vis spectroscopy, it was estimated that the reaction of PGHS-1 with excess peroxynitrite yielded two nitrated tyrosines/PGHS-1 subunit. Finally, atherosclerotic tissue obtained from endarterectomy patients was shown to contain nitrated PGHS-1. Thus, prolonged exposure to elevated levels of peroxynitrite may cause oxidative damage through tyrosine nitration.     

F0 portion of Escherichia coli ATP synthase: orientation of subunit c in the membrane

Incubation of right-side-out oriented membrane vesicles of Escherichia coli with tetranitromethane resulted in the nitration of tyrosine residues (Tyr-10 and Tyr-73) of subunit c from the ATP synthase. Cleavage of the protein with cyanogen bromide and separation of the resulting fragments, especially of the tyrosine-containing peptides, clearly demonstrated that the distribution of the nitro groups is similar at any time and at any pH value chosen for the analysis. Furthermore, the percentage of 3-nitrotyrosine present in the two peptide fragments was in good agreement with that obtained for the intact polypeptide chain. While the modification of the tyrosine residues in subunit c with the lipophilic tetranitromethane is independent of the orientation of the membrane vesicles, the subsequent partial conversion of the 3-nitrotyrosine to the amino form only occurred when membrane vesicles with right-side-out orientation were treated with the ionic, water-soluble sodium dithionite, which at certain concentrations cannot penetrate biological membranes. Cleavage of subunit c isolated from nitrated and subsequently reduced membrane vesicles and separation of the resulting fragments by high-pressure liquid chromatography showed that the 3-nitrotyrosine in the Tyr-73-containing peptides has been completely reduced, while the nitro group in peptides containing Tyr-10 remained nearly unaffected.     

Iodination of nucleosomes at low ionic strength: conformational changes in H4 and stabilization by H1.

Radioactive iodine has been used to probe the relative reactivities of nucleosomal H4 tyrosine residues under various conditions of subphysiological ionic strength. We observe that tyrosine 72 of H4, which is not reactive over the range 20-150 mM NaCl, becomes the predominant site of iodination within H4 when nucleosomes are subjected to conditions of very low ionic strength. Conversely, the other H4 tyrosine residues, which are reactive within nucleosomes in solutions of moderate ionic strength (20-150 mM NaCl), become nonreactive when the ionic strength is reduced. This "flip-flop" in the H4 iodination pattern is the manifestation of a reversible nucleosomal conformational change. A method is presented which enables the conformational status of H4 in nucleosomes to be determined by simply electrophoresing the histones on a Triton gel after probing nucleosomes with labeled iodine. Using this technique, we demonstrate that the presence of H1 on one side of the nucleosome stabilizes a histone core domain on the other side so that all four tyrosines of H4 are maintained in their physiological ionic strength conformation even under conditions of no added salt.     

Probing the ligand-induced conformational change in HLA-DR1 by selective chemical modification and mass spectrometric mapping

Peptide binding induces conformational changes in class II MHC proteins that have been characterized using a variety of hydrodynamic and spectroscopic approaches, but these changes have not been clearly localized within the overall class II MHC structure. In this study, empty and peptide-loaded complexes of HLA-DR1, a common class II MHC variant, were chemically modified using the side chain-specific chemical modifiers p-hydroxyphenylglyoxal (arginine), tetranitromethane (tyrosine), N-bromosuccinimide (tryptpophan), and NHS-biotin (lysine). Modified proteins were subjected to in-gel digestion with trypsin and subsequent analysis by MALDI/MS. Three arginine residues and two lysine residues were differentially reactive, modified in the empty form but not the peptide-loaded form of the protein, indicating that the chemical reactivity of these regions differs in the two conformations. Three of the differential modifications were located on a single lateral face of the protein, indicating that this region is involved in the conformational change. Additionally, a number of lysine and tyrosine modification sites were present in both protein conformations. Overall, the pattern of reactivity is inconsistent with the idea that empty MHC molecules exist as molten globules or other partially unfolded intermediates, and suggests that the peptide-induced conformational change is localized to only a few regions of the protein.     

The secondary structure of histone IV and its stability.

The secondary structure within histone IV and its fragments obtained by cyanogen bromide (CNBr) and cleavage at Met 84 has been examined by circular dichroism and spectophotometric pH titration measurements. These studies have confirmed the existence of stable secondary structure within the C-terminal fragment of histone IV (C-peptide which can be perturbed only by 6M urea at pH greater than 8 or 8 M guanidine-HCL. In contrast, the N-terminal fragment (N-peptide) appears to lack significant secondary structure at low ionic strengths but acquires approximately 15% betasheet conformation and 5% alpha-helix upon aggregation at ionic strengths larger than or equal to 0.4. The rates of nitration of the N- and C-peptides by tetranitromethane (TNM) have also been measured as a function of ionic strengths. Under comparable conditions, the rate constant for nitration of the N-peptide was found to be about six times greater than that for the C-peptide, further evidence in support of the presence of stable secondary structure within the C-terminal region of histone IV. After binding these histone IV fragments to DNA, however, the nitration reaction rate constants for the N- and C-peptide in the bound form are found to be 2% and 27% of the corresponding free peptides. Reconstituted nucleohistone IV is about 10% as reactive to TNM as histone IV at comparable ionic strength.     

Ca2+ and Zn2+-binding properties of nitrated S-100b protein from bovine brain.

The single tyrosine residue in S-100b protein was nitrated by treatment with tetranitromethane in 0.1 M-Tris/HCl buffer, pH 8.0, containing 2 mM-EDTA. The nitrated protein did not differ significantly in secondary structure from its native unmodified counterpart, as revealed by far-u.v. c.d. measurements. The effect of Ca2+ on the modified protein was different from that on the native protein, e.g. addition of Ca2+ resulted in a loss of helical content from 55 to 47% with the native protein whereas Ca2+ had no significant effect on the gross conformation of the nitrated derivative. Near-u.v. c.d. studies also indicated a very minimal effect on the tyrosine residue and this was also reflected in the u.v.-absorption difference spectrum. Polyacrylamide-gel electrophoresis in the absence of SDS showed the nitrated S-100b to move faster in the presence of EDTA compared with the calcium-bound state, suggesting that the modified protein does bind Ca2+ although it does not undergo a major conformational change in response to Ca2+ addition. In contradistinction, Zn2+ binding was not influenced by nitration, as demonstrated by aromatic c.d. and u.v.-difference spectroscopy. It is clear from this study that the single tyrosine residue in S-100b is critical to sense the Ca2+-induced conformational changes in the protein.