Biochimica et Biophysica Acta, 493 (1977) 24-36
© Elsevier/North-HollandBiomedicalPress BBA 37688 MODIFICATION OF THE CYSTEINE RESIDUES OF THE LACTOSE REPRESSOR PROTEIN USING CHROMOPHORIC PROBES
DIANA S. YANG, ALEX A. BURGUM and KATHLEEN S. MATTHEWS* Department of Biochemistry, Rice University, Houston, Texas 77001 (U.S.A.)
(Received January 3rd, 1977)
SUMMARY Reaction of the lactose repressor protein from Escherichia coli with 2-chloromercuri-4-nitrophenol and 2-bromoacetamido-4-nitrophenol, both environmentally sensitive chromophoric probes, resulted in modification of cysteine residues. Inducer and operator binding activities were not affected by the introduction of either probe into the structure of the protein. The extent of reaction with 2-chloromercuri-4-nitrophenol was not altered by the presence of either inducers or anti-inducers, but reaction with 2-bromoacetamido-4-nitrophenol increased in the presence of inducers. Up to pH 9, increasing the pH yielded an increase in the extent of modification with 2bromoacetamido-4-nitrophenol. Denaturation of the repressor protein exposed all three cysteine residues to modification by both reagents. The reaction of repressor with 2-chloromercuri-4-nitrophenol was prevented by prior modification of repressor using 5,5'-dithiobis-(2-nitrobenzoic acid); also, the 2-mercuri-4-nitrophenol moiety could be dissociated from the protein by the addition of sulfhydryl reagents. The cysteines which reacted with two equivalents of 2-chloromercuri-4-nitrophenol were identified as 268 ( ~ 90 ~), 107 ( ~ 70 ~o) and 140 ( ~ 40 ~). Modification of the protein using one equivalent of this reagent resulted in reaction primarily with cysteine 268 ( >_75 ~). Reaction with 2-bromoacetamido-4-nitrophenol resulted in modification of cysteines 140 (85~o) and 107 (35~). Prior blocking of the cysteine residue 107 by 2-chloromercuri-4-nitrophenol resulted in modification of primarily cysteine 140 by 2-bromoacetamido-4-nitrophenol. The introduction of these two similar chromophoric reporter groups into the repressor protein at known sites provides a probe of repressor structure in regions previously not studied due to the absence of chromophoric groups. In addition, the introduction of the mercury atom of 2-mercuri-4nitrophenol at defined sites in the protein molecule without affecting binding activity provides a heavy atom derivative for X-ray crystallographic studies.
INTRODUCTION The lactose repressor protein obtained from Escheriahia coli is one of the few genetic control proteins which has been isolated. The expression of the lactose operon " To whom reprint requests should be addressed.
25 is regulated by this protein via its interaction with an operator site on the bacterial DNA. This interaction apparently prevents RNA polymerase from transcribing(the region of the DNA proximal to the operator site (for a review of the lactose system, see ref. 1). In the presence of lactose, a metabolite of lactose (1,6-allolactose [2]) called an inducer interacts with the repressor protein, eliciting a conformational change which results in release of the DNA. Transcription of the region of DNA coding for the lac enzymes may then occur. The presence of inducer molecules apparently results in a decreased affinity of repressor for operator DNA (specific interaction) [3], but does not affect the affinity of repressor for non-operator DNA (non-specific interaction) [4]. The difference in Kd for these two types of binding is approximately six orders of magnitude [5]. The presence of an inducer provides a mechanism by which the entire DNA molecule can compete with the operator region (approx. 30 nucleotide pairs) for repressor; in the absence of inducer, the interaction of operator and repressor is sufficiently strong that the remainder of the DNA cannot compete effectively [6]. The isolation of lactose repressor was first carried out by Gilbert and MOilerHill [7]. Availability of strains which overproduce the repressor via promoter mutations and transduction onto phage DNA has allowed the isolation of sufficient quantities of repressor for chemical and physical studies [8, 9]. The lactose repressor protein is a tetrameric protein of molecular weight 150 000 [10], and the amino acid sequence has been determined [11]. Studies on the interaction of the repressor with operator DNA and non-operator DNA have yielded information on dissociation constants, half-life of the complex, and kinetics of association [4, 5, 12, 13]. Ultraviolet and fluorescence spectral techniques have been used to demonstrate that a conformation change occurs in the repressor protein in the presence of inducer molecules, but not anti-inducer molecules (ligands which stabilize the operator-repressor complex) [14, 15]. Platt et al. [16] found that lactose repressor digested with trypsin under native conditions yielded a trypsin-resistant core molecule of approx. 120 000 molecular weight which had lost its amino-terminal region. The core protein was tetrameric and exhibited full inducer binding activity. Operator binding activity, however, was destroyed. Ultraviolet difference spectroscopy utilizing core protein [17] demonstrated spectral changes in response to inducer occurred which were identical to those observed for intact repressor protein, indicating that the spectrally observed conformation change upon binding of inducers involves segments of the protein which are in the core protein and not in the regions removed by trypsin treatment. Chemical modification has been used for proteins to determine differences in reactive residues under various conditions, to detect changes in chromophores introduced into the molecule in response to ligand binding, and to determine the role of specific side chains in the activity/function of the molecules. McMurray and Trentham [18] recently introduced an organomercurial, 2-chloromercuri-4-nitrophenol, which reacts specifically with thiol groups in proteins, and furthermore, acts as an environmentally sensitive chromophoric probe [19]. 2-Chloromercuri-4-nitrophenol has been shown to react with the repressor protein, and the nitrophenol moiety is perturbed by addition of inducers [20]. 2-Bromoacetamido-4-nitrophenol has also been demonstrated to react selectively at low excesses with cysteine residues in proteins and to serve as a chromophoric probe [21]. The absorption characteristics (molar absorptivity and wavelength of maximum absorption) may be affected by the environment of these probes. Environmental changes which alter the pK of the phenol moiety
26 of the chromophores are reflected by concomitant changes in the magnitude of the nitrophenol absorption at wavelengths corresponding to the ionized and unionized forms of the phenol. Therefore, nitrophenol bound to a protein may provide information regarding the protein structure in the immediate vicinity of the chromophoric probe and may reflect environmental perturbations caused by conformational changes in the protein. In this study, 2-chloromercuri-4-nitrophenol and 2-bromoacetamido4-nitrophenol have been used to label the lactose repressor protein in order to explore the effects on the activity of the protein and to characterize the labelled repressor for use in subsequent spectral studies. MATERIALS AND METHODS
Isolation of repressor, lac repressor was isolated from Escherichia coli M96, which carried a temperature-inducible prophage (obtained from David Jackson). The promoter for the repressor protein contains a mutation (is°) which results in overproduction of the repressor protein. The procedure used for isolation was that of Mtiller-Hill et al. [22] using the modification of Platt et al. [23]. Cells were grown in 100-1 batches and stored frozen before use. Yields of 100 mg repressor per 100 g frozen cells were obtained, and the repressor was stored frozen (in 0.24 M phosphate buffer, pH 7.6, 10 -4 M dithiothreitol, 5 ~ glycerol) following elution from the phosphocellulose column. Purified repressor was concentrated with a final (NH4)2SO4 precipitation (33 ~ ) and resuspended in the appropriate buffer. After dialysis to remove the ammonium sulfate, the concentrated protein was stored at 4 °C or frozen. Prior to use for reactions with sulfhydryl reagents, the protein was dialyzed extensively to remove dithiothreitol; the diffusate was saturated with nitrogen before use to remove any traces of oxygen. The purified protein was subjected to sodium dodecyl sulfate gel electrophoresis [24] to check its purity (major band of approx. 95 ~ purity). Routine determinations of protein concentration were made using the Folin-Ciocalteau method [25]. Assay ofrepressor. Isopropyl-fl-D-thiogalactoside binding activity was assayed by the ammonium sulfate precipitation method described by Bourgeois [26] utilizing [14C]isopropyl-fl-D-thiogalactoside (Schwarz-Mann). The D N A binding assay was carried out using nitrocellulose filters as described by Riggs et al. [27]. The DNA, labelled with [3H]thymidine, was obtained from a low-thymine-requiring E. coli strain which carried a temperature-inducible lac prophage (2 plac 5, strain MBC 5, obtained from Mary Barkley). Preparation of phage and purification of DNA were carried out according to Wang et al. [28]. Saturation curves for the repressor samples were obtained. In all assay mixtures, dithiothreitol was eliminated to avoid reversal of the reaction with 2-chloromercuri-4-nitrophenol. Reaction with 2-chloromercuri-4-nitrophenol. 2-Chloromercuri-4-nitrophenol was obtained from F. A. Quiocho and from Eastman. The yellow solid was recrystallized as described by McMurray and Trentham [18]. Melting point was determined to be 237-238 °C (reported, 238 °C). The reagent was dissolved in 0.02 M N a O H and stored frozen, protected from light, at concentrations of 10 mM. For routine use, the solutions were diluted to 1 mM in 0.02 M NaOH. The concentration of the 2-chloromercuri-4-nitrophenol solutions was confirmed by measuring the absorbance at 405 nm of an aliquot diluted into 0.1 M K O H (e = 1.74" 104). Titrations were carried
27 out as described previously [20]. Reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) was carried out by adding 100 #1 of reagent solution (4 mg/ml in 0.1 M sodium phosphate buffer, pH 8) to repressor (0.1 mg/ml) in a 3 ml cuvette. The absorbance at 412 nm was followed until no further change was noted. The extent of reaction was determined using e = 13 600 [29]. Reaction with 2-bromoacetamido-4-nitrophenol. 2-Bromoacetamido-4-nitrophenol was obtained from Sigma. Melting point was determined to be 216-218 °C. The reagent was dissolved in methanol immediately prior to use, with gentle warming, at a concentration of 0.04 M. One-tenth volume of the reagent solution was added to a protein solution ( ~ 0 . 5 mg/ml) in 0.1 M Tris-HC1, 1.0 M NaC1, at the appropriate pH. After the indicated time, the solution was passed through a Sephadex G-25 column equilibrated with the same buffer (at the desired pH) and the protein band was collected. The extent of reaction was determined spectrophotometrically and by amino acid analysis following hydrolysis in 6 M HC1 for 20 h at 1 l0 °C [30]. The hydrolysis product is carboxymethylcysteine. Separation ofpeptides. Repressor (2-5 mg) was reacted with the appropriate reagent and if necessary passed through a Sephadex G-25 column. Indirect determination of the reactive cysteines was carried out by labelling the unreacted residues with iodo [14C]acetamide under denaturing conditions (8 M urea). A 1.5 molar excess of radioactive iodoacetamide (over cysteines) was added followed immediately by the addition of solid urea to 8 M. The mixture was protected from light and allowed to incubate for 30 min at 32 °C. The solution was dialyzed extensively against 0.1 M NH4HCO3 to remove unreacted iodoacetamide. These samples and those used for direct determination of reacted cysteines were treated with trypsin and/or chymotrypsin (I ~ each by weight, 2.5 h each for two additions). The protein was applied directly to a Sephadex G-25 column or lyophilized to dryness for electrophoresis. Peptides were applied to Whatman 3M paper in pH 6.5 electrophoresis buffer (pyridine, 1 0 ~ and acetic acid, 0.3 ~ ) and electrophoresed at 2200 V for 75 min with bromphenol blue as a marker dye. Localization of the peptides was carried out as follows: (a) Nitrophenol-labelled cysteine. The paper was placed in a tank containing ammonia vapor for several minutes. The ionization of the nitrophenol resulted in a visible yellow color. (b) Ninhydrin-positive peptides. The paper was heated to remove traces of ammonia and sprayed with ninhydrin (0.02 ~o in ethanol) to localize the remainder of the peptides. Color development was done in the dark overnight or for 30 min at 80 °C. (c) Tyrosine. A strip along the edge of the peptides was removed and sprayed with ~-nitroso-fl-naphthol solution to identify tyrosine peptides.(d) Radiolabelled peptides. 1-1.5-inch segments of a strip were placed in scintillation fluid and counted to locate the radioactive peptides. Reaction of mercurial labelled protein with 0.01 M iodoacetamide in 8 M urea was also carried out. After removal of the iodoacetamide, the mercurinitrophenol was removed by addition of excess dithiothreitol. The protein was separated from the sulfhydryl reagent, and the previously labelled cysteines were reacted with 2-bromoacetamido-4-nitrophenol in 8 M urea. After dilution into water to precipitate the protein and extensive washing to remove reagent, the protein was treated with trypsin and chymotrypsin and chromatographed on a Sephadex G-50 column in 2 ~ sodium dodecyl sulfate to separate the cysteinecontaining peptides.
28 RESULTS
Reaction of repressor with 2-chloromercuri-4-nitrophenol The lactose repressor protein has been reacted with 2-chloromercuri-4-nitrophenol [20]; the equivalence point of the titration curve at all pH values corresponded to 2 tool mercurinitrophenol per mol repressor monomer, indicating that one cysteine was not available for reaction [20]. By titrating at pH 8.1, it was possible to observe two equivalence points for the repressor protein at 1 and 2 mol mercurinitrophenol per mol repressor monomer [20]. The presence of two equivalence points implies that the molar absorptivities for the mercurinitrophenol groups attached to the two cysteines are slightly different at this pH. Since two equivalence points were observed, the two cysteines must also react at different rates; if the rates of reaction were identical, each point on the titration curve would be an equal mixture of the decrease in absorbance observed for each individual cysteine residue and would yield a single slope.
Reaction of repressor with 2-bromoacetamido-4-nitrophenol Repressor protein was reacted with a 200-fold excess of reagent followed by separation of the modified protein from reagent and cyclized reagent on Sephadex G-25. The isolated modified protein was then used in subsequent studies. A time course of modification indicated that reaction was essentially complete within 60 min at pH 9.0 (Fig. 1). This rate appears to be slow compared to that expected for free sulthydryl groups, i.e. on the order of minutes [21]. At lower pH values, the extent of reaction was decreased significantly (Table I). Amino acid analysis indicated that 1.2 cysteine residues were modified at pH 9.0 and no other residues were affected. In addition, correlations between absorbance at 425 nm and numbers of modified cysteine residues indicated no other nitrophenol was introduced into the molecule.
Effects of modification on the binding activities Assays were carried out to determine the effects of repressor reaction with chromophoric labels on the inducer and operator DNA binding activities of the protein. Inducer binding activity of the repressor was determined after modification with increasing concentrations of 2-chloromercuri-4-nitrophenol at both pH 7 and pH 9; binding activity remained at > 9 5 ~ of unmodified repressor [20]. In addition, a time course was carried out to determine if any effects could be found over 30 min after reaction. No loss in inducer binding activity was observed. Repressor modified with 2-bromoacetamido-4-nitrophenol was also assayed for inducer binding activity (Fig. 1). Activity remained at >90~o of unmodified repressor over the entire time course of reaction. Binding of these nitrophenol chromophores to the cysteine residues does not affect inducer binding, implying that these residues are not an essential part of the inducer binding site. Anti-inducer competes with inducer for binding to modified repressor; therefore, this interaction is not affected by reaction. Operator binding activity of the most reacted species was measured for both modified repressors and found to be identical to protein which had not been exposed to reagent (Fig. 1). The cysteine residues which react with these reagents are either not a part of the operator binding site or do not interfere with operator binding even if modified.
29 i 100
LAA • (3 -
75 %) when only one equivalent of mercurial was present. Using the excess iodoacetamide method described for two equivalents, it was possible to show that cysteine 268 contained 70% of the chromophore label (Fig. 3). Therefore, it appears that cysteine 268 can be selectively modified. (c) 2-Bromoacetamido-4-nitrophenol. Repressor modified with 2-bromoacetamido-4-nitrophenol was treated with a mixture of trypsin and chymotrypsin and
33 the resultant peptides electrophoresed at pH 6.5. Nitrophenol absorbance was noted at regions corresponding to cysteines 107 and 140 (Fig. 4). Chromography of peptides from tryptic/chymotryptic digestion of modified repressor (Fig. 4) allowed quantitation of the extent of reaction of 107 (35 ~) and 140 (85 ~o). By reacting the protein with two equivalents of 2-chloromercuri-4-nitrophenol prior to reaction with 2-bromoacetamido-4-nitrophenol, it was possible to limit reaction with the latter primarily to cysteine 140. The mercurial can be easily removed after reaction by addition of a sulfhydryl reagent (e.g. dithiothreitol) prior to passage through Sephadex G-25. ELECTROPHORESIS, pH. G.5
FRE~t
f
.Pc4sO G,N
~
# f
c s,4o
.DYE .CK,N0
IO7
~--
CYS 140
.G
,,~,
CYS 107
I
~.4
12 • 08 cu
IX
o .2
)4
uo cO •
O u'~
_75 ~o with one equivalent of 2-chloromercuri-4-nitrophenol, providing the possibility for selective modification of this residue. The cysteines in the repressor protein appear to be removed from solvent, since reaction of the protein with sulfhydryl reagents which normally react with exposed residues (iodoacetamide, iodoacetate, dyecatalyzed oxidation) did not result in modification, and the rates of reaction with the nitrophenols are slow compared to other proteins [35]. It is interesting to note that the two reagents have different patterns of modification, overlapping at cysteine 107. Cysteine 268 appears unreactive to 2-bromoacetamido-4-nitrophenol, while cysteine 140 is either unreactive or only partially reactive to 2-chloromercuri-4-nitrophenol. Cysteine 140 occurs in a region of the sequence which is quite apolar (there are no charged residues from amino acids 138-150 and only two polar residues in this region) [11]. By contrast, cysteines 107 and 268 are both surrounded by regions containing polar and charged amino acid side chains. Despite the polar character of the amino acids in the vicinity of the two mercurinitrophenol-reactive cysteines at least in the primary structure, it appears that these residues are not readily available to solvent, since reaction with iodoacetate and iodoacetamide is not observed. Ultraviolet difference spectroscopy and fluorescence studies have indicated that the repressor protein undergoes a structural change in response to binding inducer molecules [14, 15]. The absorption and fluorescence properties of tryptophan and tyrosine in the repressor molecule are altered by inducer binding and the solvent exposure of these aromatic residues is also decreased. Changes in the sedimentation properties have also been observed (Barkley, M., personal communication). The introduction of visible chromophores into the repressor protein provides an opportunity to observe changes in other regions of the repressor protein; since the sites of modification are known, it should be possible to correlate the results of studies involving aromatic residues with those utilizing probes at the cysteine sites. The demonstration that the chromophores can be perturbed by binding inducer molecules
35 opens the possibility to study in detail the characteristics of the various cysteines using spectral techniques [36]. Furthermore, it is of some importance to note that reaction of 2-chloromercuri-4-nitrophenol with repressor introduces heavy metal atoms into the structure of the protein at a defined site (Cys-268). Since the reaction does not result in alteration of the activity of the repressor, this modification would be useful for X-ray crystallographic studies. ACKNOWLEDGEMENTS
The suggestion and gift of 2-chloromercuri-4-nitrophenol from Florante Quiocho; many helpful discussions with Florante Quiocho and David Miller; and technical assistance by Clarence Sams and Robert Hord are gratefully remembered. D.S.Y. was a Robert A. Welch Foundation Pre-doctoral Fellow. This work, was supported by the Robert A. Welch Foundation (C-576) and the National Science Foundation (GB-39936). REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Reznikoff, W. (1972) Ann. Rev. Gen. 6, 133-167 Jobe, A. and Bourgeois, S. (1972) J. Mol. Biol. 69, 397-404 Riggs, A. D., Newby, R. F. and Bourgeois, S. (1970) J. Mol. Biol. 51, 303-314 Lin, S.-Y. and Riggs, A. D. (1970) Nature 228, 1184--1186 Lin, S.-Y. and Riggs, A. D. (1972) J. Mol. Biol. 72, 671-681 Von Hippel, P. H., Revzin, A., Gross, C. A. and Wang, A. C. (1974) Proc. Natl. Acad. Sci. U.S. 71,4808-4812 Gilbert, W. and Mtiller-Hill, B. (1966) Proc. Natl. Acad. Sci. U.S. 56, 1891-1898 Mfiller-Hill, B., Crapo, L. and Gilbert, W. (1968) Proc. Natl. Acad. Sci. U.S. 59, 1259-1264 Mfiller-Hill, B. (1971) Angew. Chem. Int. Edn. 3, 160-172 Riggs, A. D. and Bourgeois, S. (1968) J. Mol. Biol. 34, 361-364 Beyreuther, K., Adler, K., Geisler, N. and Klemm, A. (1973) Proc. Natl. Acad. Sci. U.S. 70, 35763580 Riggs, A. D., Suzuki, H. and Bourgeois, S. (1970) J. Mol. Biol. 48, 67-83 Riggs, A. D., Bourgeois, S. and Cohn, M (1970) J. Mol. Biol. 53, 401-417 Laiken, S. L., Gross, C. A. and Von Hippel, P. H. (1972) J. Mol. Biol. 66, 143-155 Matthews, K. S., Matthews, H. R., Thielmann, H. W. and Jardetzky, O. (1973) Biochim. Biophys. Acta 295, 159-165 Platt, T., Files, J. G. and Weber, K. (1973) J. Biol. Chem, 248, 110-121 Matthews, K. S. (1974) Biochim. Biophys. Acta 359, 334-340 McMurray, C. H. and Trentham, D. R. (1969) Biochem. J. 115, 913-921 Quiocho, F. A. and Thomson, J. W. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2858-2862 Yang, D. S. and Matthews, K. S. (1976) J. Mol. Biol. 103, 433-437 Horton, H. R. and Koshland, D. E. (1967) Methods Enzymol. XI, 866-870 M(iller-Hill, B., Beyreuther, K. and Gilbert, W° (1971) Methods Enzymol. XXI D, 483-487 Platt, T., Weber, K., Ganem, D. and Miller, J. H. (1972) Proc. Natl. Acad. Sci. U.S. 69, 897-901 Weber, K., Pringle, J. R. and Osborn, M. (1972) Methods Enzymol. XXVI C, 3-27 Chou, S.-C. and Goldstein, A. (1960) Biochem. J. 75, 109-115 Bourgeois, S. (1971) Methods Enzymol. XXI D, 491-500 Riggs, A. D., Bourgeois, S., Newby, R. F. and Cohn, M. (1968) J. Mol. Biol. 34, 365-368 Wang, J. C., Barkley, M. D. and Bourgeois, S. (1974) Nature 251,247-249 Habeeb, A. F. S. A. (1972) Methods Enzymol. XXV, 457-463 Spackman, D., Stein, W. and Moore, S. (1958) Anal. Chem. 30, 1190-1197 Pfahl, M. (1972) Genetics 72, 393-409 Pfahl, M., Stockter, C. and Gronenborn, B. (1974) Genetics 76, 669-679
36 33 Miller, J. H., Coulondre, C., Schmeissner, U., Schmitz, A. and Lu, P. (1975) in Symposium on Protein Ligand Interactions (Sund, H. and Blauer, G., eds.), pp. 238-252, Konstanz, de Gruyter, Berlin 34 Adler, K., Beyreuther, K., Fanning, E., Geisler, N., Gronenborn, B., Klemm, A., Mfiller-Hill, B., Pfahl, M. and Schmitz, A. (1972) Nature 237, 322-327 35 Friedman, B. E., Olson, J. S. and Matthews, K. S. (1976) J. Biol. Chem. 251, 1171-1174 36 Sams, C. F., Friedman, B. E., Burgum, A. A. and Matthews, K. S. (1977) J. Biol. Chem., in the press
© Elsevier/North-HollandBiomedicalPress BBA 37688 MODIFICATION OF THE CYSTEINE RESIDUES OF THE LACTOSE REPRESSOR PROTEIN USING CHROMOPHORIC PROBES
DIANA S. YANG, ALEX A. BURGUM and KATHLEEN S. MATTHEWS* Department of Biochemistry, Rice University, Houston, Texas 77001 (U.S.A.)
(Received January 3rd, 1977)
SUMMARY Reaction of the lactose repressor protein from Escherichia coli with 2-chloromercuri-4-nitrophenol and 2-bromoacetamido-4-nitrophenol, both environmentally sensitive chromophoric probes, resulted in modification of cysteine residues. Inducer and operator binding activities were not affected by the introduction of either probe into the structure of the protein. The extent of reaction with 2-chloromercuri-4-nitrophenol was not altered by the presence of either inducers or anti-inducers, but reaction with 2-bromoacetamido-4-nitrophenol increased in the presence of inducers. Up to pH 9, increasing the pH yielded an increase in the extent of modification with 2bromoacetamido-4-nitrophenol. Denaturation of the repressor protein exposed all three cysteine residues to modification by both reagents. The reaction of repressor with 2-chloromercuri-4-nitrophenol was prevented by prior modification of repressor using 5,5'-dithiobis-(2-nitrobenzoic acid); also, the 2-mercuri-4-nitrophenol moiety could be dissociated from the protein by the addition of sulfhydryl reagents. The cysteines which reacted with two equivalents of 2-chloromercuri-4-nitrophenol were identified as 268 ( ~ 90 ~), 107 ( ~ 70 ~o) and 140 ( ~ 40 ~). Modification of the protein using one equivalent of this reagent resulted in reaction primarily with cysteine 268 ( >_75 ~). Reaction with 2-bromoacetamido-4-nitrophenol resulted in modification of cysteines 140 (85~o) and 107 (35~). Prior blocking of the cysteine residue 107 by 2-chloromercuri-4-nitrophenol resulted in modification of primarily cysteine 140 by 2-bromoacetamido-4-nitrophenol. The introduction of these two similar chromophoric reporter groups into the repressor protein at known sites provides a probe of repressor structure in regions previously not studied due to the absence of chromophoric groups. In addition, the introduction of the mercury atom of 2-mercuri-4nitrophenol at defined sites in the protein molecule without affecting binding activity provides a heavy atom derivative for X-ray crystallographic studies.
INTRODUCTION The lactose repressor protein obtained from Escheriahia coli is one of the few genetic control proteins which has been isolated. The expression of the lactose operon " To whom reprint requests should be addressed.
25 is regulated by this protein via its interaction with an operator site on the bacterial DNA. This interaction apparently prevents RNA polymerase from transcribing(the region of the DNA proximal to the operator site (for a review of the lactose system, see ref. 1). In the presence of lactose, a metabolite of lactose (1,6-allolactose [2]) called an inducer interacts with the repressor protein, eliciting a conformational change which results in release of the DNA. Transcription of the region of DNA coding for the lac enzymes may then occur. The presence of inducer molecules apparently results in a decreased affinity of repressor for operator DNA (specific interaction) [3], but does not affect the affinity of repressor for non-operator DNA (non-specific interaction) [4]. The difference in Kd for these two types of binding is approximately six orders of magnitude [5]. The presence of an inducer provides a mechanism by which the entire DNA molecule can compete with the operator region (approx. 30 nucleotide pairs) for repressor; in the absence of inducer, the interaction of operator and repressor is sufficiently strong that the remainder of the DNA cannot compete effectively [6]. The isolation of lactose repressor was first carried out by Gilbert and MOilerHill [7]. Availability of strains which overproduce the repressor via promoter mutations and transduction onto phage DNA has allowed the isolation of sufficient quantities of repressor for chemical and physical studies [8, 9]. The lactose repressor protein is a tetrameric protein of molecular weight 150 000 [10], and the amino acid sequence has been determined [11]. Studies on the interaction of the repressor with operator DNA and non-operator DNA have yielded information on dissociation constants, half-life of the complex, and kinetics of association [4, 5, 12, 13]. Ultraviolet and fluorescence spectral techniques have been used to demonstrate that a conformation change occurs in the repressor protein in the presence of inducer molecules, but not anti-inducer molecules (ligands which stabilize the operator-repressor complex) [14, 15]. Platt et al. [16] found that lactose repressor digested with trypsin under native conditions yielded a trypsin-resistant core molecule of approx. 120 000 molecular weight which had lost its amino-terminal region. The core protein was tetrameric and exhibited full inducer binding activity. Operator binding activity, however, was destroyed. Ultraviolet difference spectroscopy utilizing core protein [17] demonstrated spectral changes in response to inducer occurred which were identical to those observed for intact repressor protein, indicating that the spectrally observed conformation change upon binding of inducers involves segments of the protein which are in the core protein and not in the regions removed by trypsin treatment. Chemical modification has been used for proteins to determine differences in reactive residues under various conditions, to detect changes in chromophores introduced into the molecule in response to ligand binding, and to determine the role of specific side chains in the activity/function of the molecules. McMurray and Trentham [18] recently introduced an organomercurial, 2-chloromercuri-4-nitrophenol, which reacts specifically with thiol groups in proteins, and furthermore, acts as an environmentally sensitive chromophoric probe [19]. 2-Chloromercuri-4-nitrophenol has been shown to react with the repressor protein, and the nitrophenol moiety is perturbed by addition of inducers [20]. 2-Bromoacetamido-4-nitrophenol has also been demonstrated to react selectively at low excesses with cysteine residues in proteins and to serve as a chromophoric probe [21]. The absorption characteristics (molar absorptivity and wavelength of maximum absorption) may be affected by the environment of these probes. Environmental changes which alter the pK of the phenol moiety
26 of the chromophores are reflected by concomitant changes in the magnitude of the nitrophenol absorption at wavelengths corresponding to the ionized and unionized forms of the phenol. Therefore, nitrophenol bound to a protein may provide information regarding the protein structure in the immediate vicinity of the chromophoric probe and may reflect environmental perturbations caused by conformational changes in the protein. In this study, 2-chloromercuri-4-nitrophenol and 2-bromoacetamido4-nitrophenol have been used to label the lactose repressor protein in order to explore the effects on the activity of the protein and to characterize the labelled repressor for use in subsequent spectral studies. MATERIALS AND METHODS
Isolation of repressor, lac repressor was isolated from Escherichia coli M96, which carried a temperature-inducible prophage (obtained from David Jackson). The promoter for the repressor protein contains a mutation (is°) which results in overproduction of the repressor protein. The procedure used for isolation was that of Mtiller-Hill et al. [22] using the modification of Platt et al. [23]. Cells were grown in 100-1 batches and stored frozen before use. Yields of 100 mg repressor per 100 g frozen cells were obtained, and the repressor was stored frozen (in 0.24 M phosphate buffer, pH 7.6, 10 -4 M dithiothreitol, 5 ~ glycerol) following elution from the phosphocellulose column. Purified repressor was concentrated with a final (NH4)2SO4 precipitation (33 ~ ) and resuspended in the appropriate buffer. After dialysis to remove the ammonium sulfate, the concentrated protein was stored at 4 °C or frozen. Prior to use for reactions with sulfhydryl reagents, the protein was dialyzed extensively to remove dithiothreitol; the diffusate was saturated with nitrogen before use to remove any traces of oxygen. The purified protein was subjected to sodium dodecyl sulfate gel electrophoresis [24] to check its purity (major band of approx. 95 ~ purity). Routine determinations of protein concentration were made using the Folin-Ciocalteau method [25]. Assay ofrepressor. Isopropyl-fl-D-thiogalactoside binding activity was assayed by the ammonium sulfate precipitation method described by Bourgeois [26] utilizing [14C]isopropyl-fl-D-thiogalactoside (Schwarz-Mann). The D N A binding assay was carried out using nitrocellulose filters as described by Riggs et al. [27]. The DNA, labelled with [3H]thymidine, was obtained from a low-thymine-requiring E. coli strain which carried a temperature-inducible lac prophage (2 plac 5, strain MBC 5, obtained from Mary Barkley). Preparation of phage and purification of DNA were carried out according to Wang et al. [28]. Saturation curves for the repressor samples were obtained. In all assay mixtures, dithiothreitol was eliminated to avoid reversal of the reaction with 2-chloromercuri-4-nitrophenol. Reaction with 2-chloromercuri-4-nitrophenol. 2-Chloromercuri-4-nitrophenol was obtained from F. A. Quiocho and from Eastman. The yellow solid was recrystallized as described by McMurray and Trentham [18]. Melting point was determined to be 237-238 °C (reported, 238 °C). The reagent was dissolved in 0.02 M N a O H and stored frozen, protected from light, at concentrations of 10 mM. For routine use, the solutions were diluted to 1 mM in 0.02 M NaOH. The concentration of the 2-chloromercuri-4-nitrophenol solutions was confirmed by measuring the absorbance at 405 nm of an aliquot diluted into 0.1 M K O H (e = 1.74" 104). Titrations were carried
27 out as described previously [20]. Reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) was carried out by adding 100 #1 of reagent solution (4 mg/ml in 0.1 M sodium phosphate buffer, pH 8) to repressor (0.1 mg/ml) in a 3 ml cuvette. The absorbance at 412 nm was followed until no further change was noted. The extent of reaction was determined using e = 13 600 [29]. Reaction with 2-bromoacetamido-4-nitrophenol. 2-Bromoacetamido-4-nitrophenol was obtained from Sigma. Melting point was determined to be 216-218 °C. The reagent was dissolved in methanol immediately prior to use, with gentle warming, at a concentration of 0.04 M. One-tenth volume of the reagent solution was added to a protein solution ( ~ 0 . 5 mg/ml) in 0.1 M Tris-HC1, 1.0 M NaC1, at the appropriate pH. After the indicated time, the solution was passed through a Sephadex G-25 column equilibrated with the same buffer (at the desired pH) and the protein band was collected. The extent of reaction was determined spectrophotometrically and by amino acid analysis following hydrolysis in 6 M HC1 for 20 h at 1 l0 °C [30]. The hydrolysis product is carboxymethylcysteine. Separation ofpeptides. Repressor (2-5 mg) was reacted with the appropriate reagent and if necessary passed through a Sephadex G-25 column. Indirect determination of the reactive cysteines was carried out by labelling the unreacted residues with iodo [14C]acetamide under denaturing conditions (8 M urea). A 1.5 molar excess of radioactive iodoacetamide (over cysteines) was added followed immediately by the addition of solid urea to 8 M. The mixture was protected from light and allowed to incubate for 30 min at 32 °C. The solution was dialyzed extensively against 0.1 M NH4HCO3 to remove unreacted iodoacetamide. These samples and those used for direct determination of reacted cysteines were treated with trypsin and/or chymotrypsin (I ~ each by weight, 2.5 h each for two additions). The protein was applied directly to a Sephadex G-25 column or lyophilized to dryness for electrophoresis. Peptides were applied to Whatman 3M paper in pH 6.5 electrophoresis buffer (pyridine, 1 0 ~ and acetic acid, 0.3 ~ ) and electrophoresed at 2200 V for 75 min with bromphenol blue as a marker dye. Localization of the peptides was carried out as follows: (a) Nitrophenol-labelled cysteine. The paper was placed in a tank containing ammonia vapor for several minutes. The ionization of the nitrophenol resulted in a visible yellow color. (b) Ninhydrin-positive peptides. The paper was heated to remove traces of ammonia and sprayed with ninhydrin (0.02 ~o in ethanol) to localize the remainder of the peptides. Color development was done in the dark overnight or for 30 min at 80 °C. (c) Tyrosine. A strip along the edge of the peptides was removed and sprayed with ~-nitroso-fl-naphthol solution to identify tyrosine peptides.(d) Radiolabelled peptides. 1-1.5-inch segments of a strip were placed in scintillation fluid and counted to locate the radioactive peptides. Reaction of mercurial labelled protein with 0.01 M iodoacetamide in 8 M urea was also carried out. After removal of the iodoacetamide, the mercurinitrophenol was removed by addition of excess dithiothreitol. The protein was separated from the sulfhydryl reagent, and the previously labelled cysteines were reacted with 2-bromoacetamido-4-nitrophenol in 8 M urea. After dilution into water to precipitate the protein and extensive washing to remove reagent, the protein was treated with trypsin and chymotrypsin and chromatographed on a Sephadex G-50 column in 2 ~ sodium dodecyl sulfate to separate the cysteinecontaining peptides.
28 RESULTS
Reaction of repressor with 2-chloromercuri-4-nitrophenol The lactose repressor protein has been reacted with 2-chloromercuri-4-nitrophenol [20]; the equivalence point of the titration curve at all pH values corresponded to 2 tool mercurinitrophenol per mol repressor monomer, indicating that one cysteine was not available for reaction [20]. By titrating at pH 8.1, it was possible to observe two equivalence points for the repressor protein at 1 and 2 mol mercurinitrophenol per mol repressor monomer [20]. The presence of two equivalence points implies that the molar absorptivities for the mercurinitrophenol groups attached to the two cysteines are slightly different at this pH. Since two equivalence points were observed, the two cysteines must also react at different rates; if the rates of reaction were identical, each point on the titration curve would be an equal mixture of the decrease in absorbance observed for each individual cysteine residue and would yield a single slope.
Reaction of repressor with 2-bromoacetamido-4-nitrophenol Repressor protein was reacted with a 200-fold excess of reagent followed by separation of the modified protein from reagent and cyclized reagent on Sephadex G-25. The isolated modified protein was then used in subsequent studies. A time course of modification indicated that reaction was essentially complete within 60 min at pH 9.0 (Fig. 1). This rate appears to be slow compared to that expected for free sulthydryl groups, i.e. on the order of minutes [21]. At lower pH values, the extent of reaction was decreased significantly (Table I). Amino acid analysis indicated that 1.2 cysteine residues were modified at pH 9.0 and no other residues were affected. In addition, correlations between absorbance at 425 nm and numbers of modified cysteine residues indicated no other nitrophenol was introduced into the molecule.
Effects of modification on the binding activities Assays were carried out to determine the effects of repressor reaction with chromophoric labels on the inducer and operator DNA binding activities of the protein. Inducer binding activity of the repressor was determined after modification with increasing concentrations of 2-chloromercuri-4-nitrophenol at both pH 7 and pH 9; binding activity remained at > 9 5 ~ of unmodified repressor [20]. In addition, a time course was carried out to determine if any effects could be found over 30 min after reaction. No loss in inducer binding activity was observed. Repressor modified with 2-bromoacetamido-4-nitrophenol was also assayed for inducer binding activity (Fig. 1). Activity remained at >90~o of unmodified repressor over the entire time course of reaction. Binding of these nitrophenol chromophores to the cysteine residues does not affect inducer binding, implying that these residues are not an essential part of the inducer binding site. Anti-inducer competes with inducer for binding to modified repressor; therefore, this interaction is not affected by reaction. Operator binding activity of the most reacted species was measured for both modified repressors and found to be identical to protein which had not been exposed to reagent (Fig. 1). The cysteine residues which react with these reagents are either not a part of the operator binding site or do not interfere with operator binding even if modified.
29 i 100
LAA • (3 -
75 %) when only one equivalent of mercurial was present. Using the excess iodoacetamide method described for two equivalents, it was possible to show that cysteine 268 contained 70% of the chromophore label (Fig. 3). Therefore, it appears that cysteine 268 can be selectively modified. (c) 2-Bromoacetamido-4-nitrophenol. Repressor modified with 2-bromoacetamido-4-nitrophenol was treated with a mixture of trypsin and chymotrypsin and
33 the resultant peptides electrophoresed at pH 6.5. Nitrophenol absorbance was noted at regions corresponding to cysteines 107 and 140 (Fig. 4). Chromography of peptides from tryptic/chymotryptic digestion of modified repressor (Fig. 4) allowed quantitation of the extent of reaction of 107 (35 ~) and 140 (85 ~o). By reacting the protein with two equivalents of 2-chloromercuri-4-nitrophenol prior to reaction with 2-bromoacetamido-4-nitrophenol, it was possible to limit reaction with the latter primarily to cysteine 140. The mercurial can be easily removed after reaction by addition of a sulfhydryl reagent (e.g. dithiothreitol) prior to passage through Sephadex G-25. ELECTROPHORESIS, pH. G.5
FRE~t
f
.Pc4sO G,N
~
# f
c s,4o
.DYE .CK,N0
IO7
~--
CYS 140
.G
,,~,
CYS 107
I
~.4
12 • 08 cu
IX
o .2
)4
uo cO •
O u'~
_75 ~o with one equivalent of 2-chloromercuri-4-nitrophenol, providing the possibility for selective modification of this residue. The cysteines in the repressor protein appear to be removed from solvent, since reaction of the protein with sulfhydryl reagents which normally react with exposed residues (iodoacetamide, iodoacetate, dyecatalyzed oxidation) did not result in modification, and the rates of reaction with the nitrophenols are slow compared to other proteins [35]. It is interesting to note that the two reagents have different patterns of modification, overlapping at cysteine 107. Cysteine 268 appears unreactive to 2-bromoacetamido-4-nitrophenol, while cysteine 140 is either unreactive or only partially reactive to 2-chloromercuri-4-nitrophenol. Cysteine 140 occurs in a region of the sequence which is quite apolar (there are no charged residues from amino acids 138-150 and only two polar residues in this region) [11]. By contrast, cysteines 107 and 268 are both surrounded by regions containing polar and charged amino acid side chains. Despite the polar character of the amino acids in the vicinity of the two mercurinitrophenol-reactive cysteines at least in the primary structure, it appears that these residues are not readily available to solvent, since reaction with iodoacetate and iodoacetamide is not observed. Ultraviolet difference spectroscopy and fluorescence studies have indicated that the repressor protein undergoes a structural change in response to binding inducer molecules [14, 15]. The absorption and fluorescence properties of tryptophan and tyrosine in the repressor molecule are altered by inducer binding and the solvent exposure of these aromatic residues is also decreased. Changes in the sedimentation properties have also been observed (Barkley, M., personal communication). The introduction of visible chromophores into the repressor protein provides an opportunity to observe changes in other regions of the repressor protein; since the sites of modification are known, it should be possible to correlate the results of studies involving aromatic residues with those utilizing probes at the cysteine sites. The demonstration that the chromophores can be perturbed by binding inducer molecules
35 opens the possibility to study in detail the characteristics of the various cysteines using spectral techniques [36]. Furthermore, it is of some importance to note that reaction of 2-chloromercuri-4-nitrophenol with repressor introduces heavy metal atoms into the structure of the protein at a defined site (Cys-268). Since the reaction does not result in alteration of the activity of the repressor, this modification would be useful for X-ray crystallographic studies. ACKNOWLEDGEMENTS
The suggestion and gift of 2-chloromercuri-4-nitrophenol from Florante Quiocho; many helpful discussions with Florante Quiocho and David Miller; and technical assistance by Clarence Sams and Robert Hord are gratefully remembered. D.S.Y. was a Robert A. Welch Foundation Pre-doctoral Fellow. This work, was supported by the Robert A. Welch Foundation (C-576) and the National Science Foundation (GB-39936). REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Reznikoff, W. (1972) Ann. Rev. Gen. 6, 133-167 Jobe, A. and Bourgeois, S. (1972) J. Mol. Biol. 69, 397-404 Riggs, A. D., Newby, R. F. and Bourgeois, S. (1970) J. Mol. Biol. 51, 303-314 Lin, S.-Y. and Riggs, A. D. (1970) Nature 228, 1184--1186 Lin, S.-Y. and Riggs, A. D. (1972) J. Mol. Biol. 72, 671-681 Von Hippel, P. H., Revzin, A., Gross, C. A. and Wang, A. C. (1974) Proc. Natl. Acad. Sci. U.S. 71,4808-4812 Gilbert, W. and Mtiller-Hill, B. (1966) Proc. Natl. Acad. Sci. U.S. 56, 1891-1898 Mfiller-Hill, B., Crapo, L. and Gilbert, W. (1968) Proc. Natl. Acad. Sci. U.S. 59, 1259-1264 Mfiller-Hill, B. (1971) Angew. Chem. Int. Edn. 3, 160-172 Riggs, A. D. and Bourgeois, S. (1968) J. Mol. Biol. 34, 361-364 Beyreuther, K., Adler, K., Geisler, N. and Klemm, A. (1973) Proc. Natl. Acad. Sci. U.S. 70, 35763580 Riggs, A. D., Suzuki, H. and Bourgeois, S. (1970) J. Mol. Biol. 48, 67-83 Riggs, A. D., Bourgeois, S. and Cohn, M (1970) J. Mol. Biol. 53, 401-417 Laiken, S. L., Gross, C. A. and Von Hippel, P. H. (1972) J. Mol. Biol. 66, 143-155 Matthews, K. S., Matthews, H. R., Thielmann, H. W. and Jardetzky, O. (1973) Biochim. Biophys. Acta 295, 159-165 Platt, T., Files, J. G. and Weber, K. (1973) J. Biol. Chem, 248, 110-121 Matthews, K. S. (1974) Biochim. Biophys. Acta 359, 334-340 McMurray, C. H. and Trentham, D. R. (1969) Biochem. J. 115, 913-921 Quiocho, F. A. and Thomson, J. W. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2858-2862 Yang, D. S. and Matthews, K. S. (1976) J. Mol. Biol. 103, 433-437 Horton, H. R. and Koshland, D. E. (1967) Methods Enzymol. XI, 866-870 M(iller-Hill, B., Beyreuther, K. and Gilbert, W° (1971) Methods Enzymol. XXI D, 483-487 Platt, T., Weber, K., Ganem, D. and Miller, J. H. (1972) Proc. Natl. Acad. Sci. U.S. 69, 897-901 Weber, K., Pringle, J. R. and Osborn, M. (1972) Methods Enzymol. XXVI C, 3-27 Chou, S.-C. and Goldstein, A. (1960) Biochem. J. 75, 109-115 Bourgeois, S. (1971) Methods Enzymol. XXI D, 491-500 Riggs, A. D., Bourgeois, S., Newby, R. F. and Cohn, M. (1968) J. Mol. Biol. 34, 365-368 Wang, J. C., Barkley, M. D. and Bourgeois, S. (1974) Nature 251,247-249 Habeeb, A. F. S. A. (1972) Methods Enzymol. XXV, 457-463 Spackman, D., Stein, W. and Moore, S. (1958) Anal. Chem. 30, 1190-1197 Pfahl, M. (1972) Genetics 72, 393-409 Pfahl, M., Stockter, C. and Gronenborn, B. (1974) Genetics 76, 669-679
36 33 Miller, J. H., Coulondre, C., Schmeissner, U., Schmitz, A. and Lu, P. (1975) in Symposium on Protein Ligand Interactions (Sund, H. and Blauer, G., eds.), pp. 238-252, Konstanz, de Gruyter, Berlin 34 Adler, K., Beyreuther, K., Fanning, E., Geisler, N., Gronenborn, B., Klemm, A., Mfiller-Hill, B., Pfahl, M. and Schmitz, A. (1972) Nature 237, 322-327 35 Friedman, B. E., Olson, J. S. and Matthews, K. S. (1976) J. Biol. Chem. 251, 1171-1174 36 Sams, C. F., Friedman, B. E., Burgum, A. A. and Matthews, K. S. (1977) J. Biol. Chem., in the press
