Trinitrobenzenesulfonate modification of the lysine residues in lactose repressor protein

Modification of the lysine residues in the lactose repressor protein has been carried out with trinitrobenzenesulfonate. Reaction of lysine residues at positions 33, 37, 108, 290, and 327 was observed. Inducer binding was increased by modification with this reagent, while both nonspecific DNA binding and operator DNA binding were diminished, although to differing degrees. The loss in operator DNA binding capacity was complete with modification of approximately 2 equiv of lysine per monomer. The extent of reaction was affected by the presence of both sugar and DNA ligands; binding activities of the modified protein and reaction pattern of the lysines were perturbed by these ligands. The presence of operator or nonspecific DNA during the reaction protected against specific and nonspecific DNA binding activity loss. This protection presumably occurs by steric restriction of reagent access to lysine residues which are essential for both nonspecific and operator binding interactions. Lysines-33 and -108 were protected from modification in the presence of DNA. These experiments suggest that the charge on the lysine residues is important for protein interaction with DNA and that steric constraints for operator DNA interaction with the protein are more restrictive than for nonspecific DNA binding. In contrast, inducer (isopropyl beta-D-thiogalactoside) presence partially protected lysine-290 from modification while significantly enhancing reaction at lysine-327. Conformational alterations consequent to inducer binding are apparently reflected in these altered lysine reactivities.     

Chemical modification of buried lysine residues of bovine serum albumin and its influence on protein conformation and bilirubin binding.

Using a double modification technique about 20% of the lysine residues of bovine serum albumin (BSA) which are not easily accessible in the native protein have been modified. The technique involved approximately 80% modification of lysine residues of BSA with citraconic anhydride followed by chemical modification of the remaining lysine residues with acetic anhydride, succinic anhydride, potassium cyanate, or O-methylisourea. Finally, these preparations were decitraconylated under mild acidic conditions to yield acetylated, succinylated, carbomylated or guanidinated BSA. All of these preparations were found to be homogeneous with respect to charge and size. The spectral, hydrodynamic and bilirubin binding properties of these preparations are described. In contrast to most of the highly modified proteins these preparations with the exception of succinylated BSA are very similar to native BSA in their spectral and hydrodynamic properties. However, the equilibrium association constant (Ka) with bilirubin measured by fluorescence quenching was decreased by about 100-fold in acetylated, carbamylated and succinylated BSA, but only 3-fold in guanidinated BSA. Since conformationally acetylated and carbamylated BSAs are identical to guanidinated BSA we conclude that the decrease in Ka in these preparations is solely due to loss of positive charge on 'critical' lysine residues. The results support a binding model for BSA in which bilirubin binding site is buried and the protein undergoes a series of relaxational changes in conformation upon interaction with bilirubin.     

Kinetics of chemical modification of arginine and lysine residues in calf thymus histone H1

The arginine and lysine residues of calf thymus histone H1 were modified with large molar excesses of 2,3-butanedione and O-methylisourea, respectively. Kinetic study of the modification reaction of the arginine residue revealed that the reaction is divided into the two pseudo-first-order processes. About a third (1 Arg) of the total arginine residues of the H1 molecule was rapidly modified without causing any detectable structural change of the molecule, and the slow modification of the remaining arginine residues (2 Arg) led to a loss of the folded structure of H1. In the case of lysine residue modification, 93% (56 Lys) of the total lysine residues of the H1 was modified with the same rate constant, while 7% (4 Lys) of lysine residue remained unmodified. When the reaction was performed in the presence of 6M guanidine-HCl, all of lysine residues were modified. It is concluded that the 2 arginine and 4 lysine residues resistant to modification are buried in interior regions of the H1 molecule and play an important role in the formation of the H1 globular structure, while the other 1 arginine and 56 lysine residues are exposed to solvent.     

Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding

Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding. Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue. The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function. The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity. In contrast, inducer binding was not recovered upon reversal of the histidine modification. However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity. Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine. The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss. Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29. Results from the modification of core domain paralleled observations with intact repressor.     

Modification of arginine and lysine in proteins with 2,4-pentanedione.

Primary amines react with 2,4-pentanedione at pH 6-9 to form enamines, N-alkyl-4-amino-3-penten-2-ones. The latter compounds readily regenerate the primary amine at low pH or on treatment with hydroxylamine. Guanidine and substituted guanidines react with 2,4-pentanedione to form N-substituted 2-amino-4,6-dimethylpyrimidines at a rate which is lower by at least a factor of 20 than the rate of reaction of 2,4-pentanedione with primary amines. Selective modification of lysine and arginine side chains in proteins can readily be achieved with 2,4-pentanedione. Modification of lysine is favored by reaction at pH 7 or for short reaction times at pH 9. Selective modification of arginine is achieved by reaction with 2,4-pentanedione for long times at pH 9, followed by treatment of the protein with hydroxylamine. The extent of modification of lysine and arginine side chains can readily be measured spectrophotometrically. Modification of lysozyme with 2,4-pentanedione at pH 7 results in modification of 3.8 lysine residues and less than 0.4 arginine residue in 24 hr. Modification of lysozyme with 2,4-pentanedione at pH 9 results in modification of 4 lysine residues and 4.5 arginine residues in 100 hr. Treatment of this modified protein with hydroxylamine regenerated the modified lysine residues but caused no change in the modified arginine residues. One arginine residue seems to be essential for the catalytic activity of the enzyme.     

Sequence-specific endonuclease Bgl I. Modification of lysine and arginine residues of the homogeneous enzyme.

The sequence-specific endonuclease Bgl I from Bacillus globigii (RUB561) has been purified to homogeneity as determined by denaturing polyacrylamide gel analysis. The active form of the enzyme is a single polypeptide with a molecular weight of 32,000. The enzyme requires Mg2+ in the reaction mixture and displays a broad pH and monovalent cation requirement. Bgl I is not sensitive to sulfhydryl reagents but was affected by reagents that modify lysine and arginine residues. When lysine residues were modified by pyridoxal 5'-phosphate, both binding and catalysis were diminished while modification of arginine residues by 2,3-butanedione inhibited the enzyme activity but had no effect on its binding properties.     

Reductive alkylation of lysine residues in subtilisin DY

Only lysine epsilon-amino groups (and the N-terminal alpha-amino group) in native subtilisin DY were reductively alkylated by glyceraldehyde in the presence of sodium cyanoborohydride. The modified protein molecule was cleaved by TosPheCH2Cl-trypsin or cyanogen bromide and the two sets of peptides obtained were fractionated and purified by gel filtration and HPLC. For determination of the degree of modification of each lysine residue, selected peptides were subjected to sequence analysis combined with quantitative estimation of the containing PTH-Lys and PTH-epsilon-DHP-Lys. The data obtained showed that the lysine residues in positions 12, 15, 27, 43, 136, 141, 265 were entirely modified, those in positions 170, 184, 237 were partially modified, and Lys22 and Lys94 were unaccessible for the reagent. The caseinolytic activity decreased by 23% when the maximum number of lysine residues (8.6 of the total 12 residues) in subtilisin DY were modified. The CD-spectra of native and modified enzyme showed only slight differences. Both these experiments suggest that the lysine residues do not take part directly in the catalytic reaction but are responsible for maintaining the native three-dimensional enzyme structure. The data obtained for the accessibility of the different lysine residues in subtilisin DY correlated very well with the positions of these residues in a video model of the structure of subtilisin Carlsberg, thus suggesting that the spatial structures of these two enzymes are very similar.     

Conformational stability of citraconylated ovalbumin.

The lysine residues were modified to varying degrees (50-91%) with citraconic anhydride to determine the extent of conformational change in ovalbumin. Major findings included: 1. Sixteen of the 20 lysine residues are located on the protein surface, while the remaining four are buried. 2. The tertiary structure changed progressively with the degree of modification. 3. However, the secondary structure was disrupted only after one or more of the four buried lysines had been citraconylated. 4. Although the secondary structure was unaltered, the alpha-helix was nevertheless progressively destabilized as the surface 16 lysine residues were modified. This destabilization was due to electrostatic repulsions introduced by the entering citraconyl groups.     

Regional ligand domain is involved in scavenger receptor-mediated recognition of maleyl-albumin by rat sinusoidal liver cells

Scavenger receptor-mediated endocytosis of maleyl-albumin was studied with rat sinusoidal liver cells. Upon maleylation of greater than 28 mol lysine residues per protein, bovine serum albumin became an active ligand. Further modification of up to 37 mol lysine residues per protein resulted in a sharp increase in the ligand activity, reaching a maximum level thereafter. Removal of maleyl moieties from maleyl-albumin (demaleylation) from 53 mol to 14 mol lysine residues per protein did not affect the ligand activity. However, further demaleylation to less than 5 mol lysine residues per protein led to complete loss of the ligand activity. Thus, the covalently incorporated maleyl moieties are needed for the ligand activity. The ligand activity was also generated when two peptides (Frag N and Frag C) from cyanogen bromide-cleaved albumin were maleylated, indicating that the formation of an active ligand would not require a whole albumin molecule. Maleyl Frag C was further separated into three peptides; maleyl Frag C-1 (261 amino acid residues), maleyl Frag C-2 (102 residues) and maleyl Frag C-3 (36 residues). The cellular binding and endocytic degradation of maleyl-albumin or acetylated low density lipoprotein were effectively competed for by maleyl Frag C-1 and maleyl Frag C-2 but not by maleyl Frag C-3. Thus, regional domains might be involved in the ligand recognition by the scavenger receptor.     

Study of heat denaturation of human serum albumin in water-alcohol and water-salt solutions in the presence of organic ligands

The method of differential scanning microcalorimetry was used to show a decrease in heat stability of serum albumin in the presence of aliphatic alcohols. In aqueous-alcohol media, the melting temperature, denaturation transition enthalpy were decreased, and the protein intermolecular aggregation enhanced. When the alcohol concentration in aqueous solution was elevated, the number of epsilon-amino groups of lysine residues in human serum albumin exposed to the solvent rose from 6-7 in aqueous solution to maximum 20 groups in the aqueous-alcohol solution, respectively. The elevation of ionic strength also induced an increase in the number of exposed lysine residues and was accompanied by an enhancement of protein aggregation. The modification of six amino groups by pyridoxal phosphate or three by glucose in the initial albumin stabilized the protein incubated at 65 degrees-70 degrees C both in the aqueous-alcohol media. At the given concentration and temperature the native protein was denatured and fully aggregated. Aliphatic alcohols displaced fatty acids from the binding sites on the molecule of serum albumin, which resulted in a change in the number of peaks of the melting curve.     

Chemical modification of proteinsby “smart” polymers

The modification of duck ovomucoid, a proteinaceous proteinase inhibitor from egg white, by poly-N,N-diethylacrylamide possessing a low critical solution temperature (LCST) has been investigated. The free amino groups of the lysine residues and the N-terminal residue of the ovomucoid molecule were modified; as a result, the inhibitor activity towards trypsin decreased significantly and that towards chymotrypsin decreased slightly. The transformation of ovomucoid antitryptic centers into antichymotryptic centers was observed upon the heating of the solutions of the modified protein above the LCST. The hydrophobization of the lysine residues situated in the reactive centers of the inhibitor was shown to cause this phenomenon. The structure of the binding loop was not distorted and the modified lysine residues could be recognized by chymotrypsin molecules, similarly to the hydrophobic amino acid residues of the antichymotryptic center.     

Chemical reactivity of brome mosaic virus capsid protein

Viral particles are biological machines that have evolved to package, protect, and deliver the viral genome into the host via regulated conformational changes of virions. We have developed a procedure to modify lysine residues with S-methylthioacetimidate across the pH range from 5.5 to 8.5. Lysine residues that are not completely modified are involved in tertiary or quaternary structural interactions, and their extent of modification can be quantified as a function of pH. This procedure was applied to the pH-dependent structural transitions of brome mosaic virus (BMV). As the reaction pH increases from 5.5 to 8.5, the average number of modified lysine residues in the BMV capsid protein increases from 6 to 12, correlating well with the known pH-dependent swelling behavior of BMV virions. The extent of reaction of each of the capsid protein's lysine residues has been quantified at eight pH values using coupled liquid chromatography-tandem mass spectrometry. Each lysine can be assigned to one of three structural classes identified by inspection of the BMV virion crystal structure. Several lysine residues display reactivity that indicates their involvement in dynamic interactions that are not obvious in the crystal structure. The influence of several capsid protein mutants on the pH-dependent structural transition of BMV has also been investigated. Mutant H75Q exhibits an altered swelling transition accompanying solution pH increases. The H75Q capsids show increased reactivity at lysine residues 64 and 130, residues distal from the dimer interface occupied by H75, across the entire pH range.     

Lactose repressor protein modified with dansyl chloride: activity effects and fluorescence properties

Chemical modification using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) has been used to explore the importance of lysine residues involved in the binding activities of the lactose repressor and to introduce a fluorescent probe into the protein. Dansyl chloride modification of lac repressor resulted in loss of operator DNA binding at low molar ratios of reagent/monomer. Loss of nonspecific DNA binding was observed only at higher molar ratios, while isopropyl beta-D-thiogalactoside binding was not affected at any of the reagent levels studied. Lysine residues were the only modified amino acids detected. Protection of lysines-33 and -37 from modification by the presence of nonspecific DNA correlated with maintenance of operator DNA binding activity, and reaction of lysine-37 paralleled operator binding activity loss. Energy transfer between dansyl incorporated in the core region of the repressor protein and tryptophan-201 was observed, with an approximate distance of 23 A calculated between these two moieties.     

Inhibition of milk xanthine oxidase by fluorodinitrobenzene

Milk xanthine oxidase reacted with fluorodinitrobenzene resulting in the modification of two lysine residues with a 6-fold decrease in catalytic activity. Continued reaction with fluorodinitrobenzene up to a total of 11 dinitrophenyl residues/equivalent of enzyme-bound FAD resulted in no further decrease in activity. Stopped flow studies revealed that the modification perturbed the reduction of the enzyme by xanthine; this was 6-fold lower with modified than with native enzyme. The reaction of the reduced modified enzyme with oxygen was qualitatively and quantitatively the same as with native enzyme. One nitro group of each dinitrophenyl lysine residue is slowly reduced by xanthine; reduction of both nitro groups is achieved by dithionite. The two dinitrophenyl lysine reduces can be distinguished on the basis of their kinetics of reduction. One appears to be located on the protein surface and is reduced in an intermolecular reaction, while the other appears to be located in a pocket of the enzyme and is reduced in a slow intramolecular reaction.     

Modification of proteins by 3-hydroxyanthranilic acid: the role of lysine residues

The mechanism of reaction of proteins with 3-hydroxyanthranilic acid (3OHA) under oxidizing conditions has been examined. A range of proteins were found to tan when exposed to oxidized 3OHA. One exception was lysozyme which tanned only after being denatured by reduction and carboxymethylation. Chemical modification experiments using bovine serum albumin (BSA) suggested that lysine was the primary site of reaction in 3OHA-mediated protein tanning. This reactivity of 3OHA toward lysine was confirmed by autoxidizing 3OHA in the presence of amino acid homopolymers. The rate of modification of both BSA and polylysine was pH dependent. At neutral pH, a component of the coloration of the protein was found to be due to the formation of a lysyl-p-quinone adduct. Other products appear to arise through addition to the 3OHA quinone imine. Poly-(Glu,Lys) was tanned by 3OHA at a greatly reduced rate, suggesting that electrostatic interactions may influence the reaction with lysine residues and may provide an explanation for the lack of tanning of lysozyme. Despite the reaction between 3OHA and lysine, amino acid analysis revealed little quantitative change in the lysine content of proteins even after exposure to 3OHA for a period of 24 h. These results support the proposal that reaction with lysine residues is the major route of protein tanning by 3-hydroxyanthranilic acid.     

Accessibility and mobility of lysine residues in beta-lactoglobulin

N epsilon-[2H6]Isopropyllysyl-beta-lactoglobulin was prepared by reductive alkylation of beta-lactoglobulin with [2H6]acetone and NaBH4 to provide a 2H (NMR) probe for the study of lysine involvement in lipid-protein interactions. Amino acid analysis showed 80% of the protein's 15 lysine residues to be labeled. Unmodified lysine residues were located through peptide maps produced from CNBr, tryptic, and chymotryptic digests of the labeled protein. Lys47 was not modified; Lys135,138,141, located along an amphipathic helical rod, were each partially unmodified. All other lysine residues were at least 90% modified. Average correlation times calculated from 2H NMR spectra were 20 and 320 ps for 8.7 and 3.3 residues, respectively, in 6 M guanidine hydrochloride; in nondenaturing solution, values of 70 and 320 ps were obtained for 6.5 and 3.2 residues, respectively, with the remaining 2.3 modified residues not observed, suggesting that side chains of lysine residues in unordered or flexible regions were more mobile than those in stable periodic structures. 2H NMR spectra of the protein complexed with dipalmitoylphosphatidylcholine confirmed the extrinsic membrane protein type behavior of beta-lactoglobulin previously reported from 31P NMR studies of the phospholipids complexed with beta-lactoglobulin. Although no physiological function has yet been identified, comparison of these results with the X-ray structure [Papiz et al. (1986) Nature (London) 324, 383-385] supports the hypothesis that residues not accessible for modification may help to stabilize the cone-shaped beta-barrel thought to contain binding sites for small lipid-soluble molecules.     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

Mass spectrometric characterization of circulating and functional antigens derived from piperacillin in patients with cystic fibrosis

A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag to stimulate T cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time and concentration dependent, with selective modification of Lys(541) observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys(190), Lys(195), Lys(432), and Lys(541)) were detected in patients' plasma. Piperacillin-specific T lymphocyte responses (proliferation, cytokines, and granzyme B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T cells with characterized synthetic conjugates. Analysis of minimally modified T cell-stimulatory albumin conjugates revealed peptide sequences incorporating Lys(190), Lys(432), and Lys(541) as principal functional epitopes for T cells. This study has characterized the multiple haptenic structures on albumin in patients and showed that they constitute functional antigenic determinants for T cells.     

Application of the reaction of dithioesters with epsilon-amino groups in lysine to the chemical modification of proteins

The reaction of lysine with dithioesters was applied to horseradish peroxidase donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) using carboxymethyl dithiotridecanoate: three to four lysine residues were modified. The modified enzyme was soluble and active in diethyl ether. Papain (EC 3.4.22.2) was modified with carboxymethyl dithiobenzoate: two lysine residues were modified. The modified enzyme was soluble and active in dimethylsulfoxide. From these results it is concluded that dithioesters are efficient reagents for the modification of peripheral lysine residues of proteins. Aromatic dithioesters, less reactive but more selective, should be recommended for thiol-dependent enzymes such as papain.     

Biotinylation of reactive amino groups in native recombinant human interleukin-1 beta

Recombinant human interleukin-1 beta (rIL-1 beta) was chemically modified by a 10-fold molar excess (reagent:protein) of sulfosuccinimidyl 6-(biotinamido) hexanoate (sulfo-NHS-LC-biotin) or sulfosuccinimidobiotin (sulfo-NHS-biotin) under mild conditions. The primary product was purified in each case by cation exchange high performance liquid chromatography (HPLC) and digested with endoproteinase Lys C. Peptide mapping by C18 reverse phase HPLC permitted identification of three sites of biotinylation using both reagents; N-terminal alanine, lysine 93, and lysine 94. Few additional singly modified rIL-1 beta products were obtained under these conditions, despite the presence of 15 lysine residues in this protein. These data support the view that the N terminus as well as the trilysine sequence (residues 92-94) are readily susceptible to chemical modification and are exposed on the surface of the protein. Chromatography of intact biotinylated rIL-1 beta by C4 reverse phase HPLC resolved a protein modified exclusively at the N-terminal alanine from two proteins modified singly at either lysine 93 or lysine 94. In addition, a protein product modified at lysine 103 was also obtained when rIL-1 beta was similarly modified with sulfo-NHS-biotin. Since the only difference between the two biotinylation reagents relates to spacer length and its associated hydrophobicity, these data suggest that lysine 103 is not as accessible to surface modification reagents as are lysine 93, lysine 94, or alanine 1. Initial experiments indicate that none of the modifications described above decrease thymocyte proliferation by more than one order of magnitude. Therefore, these amino acid residues are not crucial for bioactivity, and we anticipate the use of these monobiotinylated proteins in structure/function analysis of IL-1 beta.