THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 1, Issue of January 10, pp. 110-121, 1973

Printed in U.S.A.

Lac Repressor

SPECIFIC PROTEOLYTIC DESTRUCTION OF THE NH,-TERMINAL REGION AND LOSS OF THE DEOXYRIBONUCLEIC ACID-BINDING ACTIVITY*

(Received for publication, July 11, 1972)

TERRY PLATT,~ JAMES G. FILES, AND KLAUS WEBER

From the Biological Laboratories, Harvard Unitiersity, 16 Divinity Avenue, Cambridge, Massachusetts 0,838

SUMMARY

Lac repressor is characterized by the amino acid sequence of its first 59 amino acid residues and the sequence of addi- tional peptides, accounting for a total of 104 residues, or about 30% of the molecule.

Trypsin and chymotrypsin readily inactivate the DNA bind- ing of repressor without a loss in binding properties for the inducer isopropyl-P-D-thiogalactoside. Characterization of the trypsinized repressor shows that digestion under native conditions yields a trypsin-resistant core molecule, which is still a tetramer and exhibits full inducer binding. The in- ducer binding activity as well as the tetrameric structure can be recovered after denaturation in a random coil solvent. The monomer molecular weight of the tryptic core is approxi- mately 28,000, compared to 38,000 for the intact repressor polypeptide chain indicating the release of about 80 to 90 amino acid residues in the form of tryptic peptides. Charac- terization of these peptides indicates that they include the NHQ-terminal59 residues of the Zac repressor and that they all probably come from the NHZ-terminal region of the mole- cule. Thus, trypsin or chymotrypsin is able to attack the NHz-terminal regions of the repressor tetramer without in- ducing any further internal nicks in the polypeptide chain.

These findings provide substantial support for the hypothe- sis that the NH&erminal region of lac repressor is required for binding to the Iac operator, but not for the binding of inducer or for the folding of the subunit and its association into a tetrameric structure.

The expression of the lactose operon in Escherichia coli is regu- Iated by the protein product of the i-gene (the Zac repressor) at a site on the bacterial chromosome called the lac operator (1). The operator is located between the Zac promoter, where transcription of the operon is initiated (2-4), and the structural genes of the operon. The repressor binds to the operator (5)? and this binding

* This work was supported by Grants GM-17662 and GM-69541 from the National Institutes of Health.

1 National Science Foundation Predoctoral Fellow. Present address, Department of Biological Sciences, Stanford University, Stanford, California 94305.

apparently bIocks the progress of RNA polymerase along the DNA (6), thus preventing the transcription of Zac messenger RNA. Derepression occurs when an “inducer” of the operon binds to the repressor, releasing it from the DNA (1, 5). Upon derepres- sion, Zac operon mRNA is transcribed and the enzymes necessary for the utilization of lactose are synthesized at up to 1000 times their basal (repressed) levels (1).

Assays have been developed to follow the inducer binding ac- tivity (7-10) and the operator binding activity (5, 11) of the repressor. The possibility that these two activities may be at least partially independent of each other is suggested by the existence of certain classes of mutations in the repressor. One class exhibits a weakened affinity for inducer with little change in DNA binding activity (10, 12). These are called “super-repres- sor” (is) mutations since is repressors are not easily induced in viva. Conversely, there is a class of nonrepressing, transdominant (imd) mutations which do not affect the inducer binding prop- erties of the repressor, but virtually eliminate the affinity for the lac operator (13, 14). Genetic mapping results have indicated that i-d mutations occur at the left hand end of the i-gene, while the is mutations tend to cluster in more central positions (15). Since the direction of reading of the i-gene is from left to right (16, 17), these results suggest that regions critical to operator binding may be located in the NHS-terminal region of the Zac repressor, whereas inducer binding depends primarily on a region occurring nearer the middle of the polypeptide chain.

Biochemical support for this hypothesis has come from the recent demonstration that a mutant repressor molecule lacking the first 42 amino acids is unable to repress, but retains normal affinity for the inducer IPTGI and is able to maintain a stable tetrameric structure (18). These results raised the possibility that limited proteolysis of the wild type molecule might be used as a probe to define the functional regions of the Zac repressor more precisely. Some proteins may be split into smaller stable components by mild proteolytic digestion (see Hill (19) for a review). In certain cases, proteolysis proceeds very specifically and to a limited extent, but with a wide spectrum of effects on both the activity and the tertiary or quaternary structure of the treated enzyme. Excellent examples are provided by studies on ribonuclease (20), y-globulin (21, 22), ,&galactosidase (23), DNA polymerase (24, 25), and diphtheria toxin (26, 27). The results

1 The abbreviations used are: IPTG, isopropyl-b-n-thiogalac- toside: SDS, sodium dodecyl sulfate; PMSF, phenyl-methane- sulfonyl-fluoride; dansyl,5-dimethylaminonaphthalene-l-sulfonyl.

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of mild proteolytic digestion on Zac repressor under nnbive con- ditions are presented below, along with amino acid sequence results which include a continuous stretch from residues 1 to 59.

MATl3RIALS AND METHODS

Repressor PuriJication-A strain constructed by J. H. XIiller (28), which carries the isQ mutation on a temperature-inducible defective lac prophage, was used to obtain 1000 times the normal amount of repressor by temperature induction of the prophage in a loo-liter fermenter. The purification varies somewhat from the procedure of Miiller-Hill et al. (29). Frozen cells (500 g) are broken into pieces and thawed in 2 liters of Buffer B (0.2 M KCl, 0.2 M Tris-HCl (pH 7.6 at 4”), 0.01 M MgAc, 3 X low4 nf dithio- threitol, and 5% glycerol) which is 10 lg per ml in DNase (Worth- ington Biochemical, crude). The sample is homogenized briefly (15 s) in a chilled Waring Blendor, then stirred gently at 4” until the viscosity is greatly reduced. Cell debris is removed by centrif- ugation and the clear supernatant (about 1500 ml) is poured off. The remaining viscous layer is extracted with 3 volumes of Buffer B, centrifuged again, and the second clear supernatant is pooled with the first. This combined sample is made up to 2500 ml, and 277 g of crystalline ammonium sulfate (231 mg per ml) is added slowly while stirring at 4”. After 1 hour, the precipitate is col- lected by centrifugation, and resuspended in 150 to 200 ml of Buffer B. This sample is dialyzed against 0.12 M Buffer KP (0.10 M K2HP04, 0.02 M KHzPOd, 3 X 1O-4 M dithiothreitol, 5% glycerol), pH 7.4, with at least three changes of buffer. The insoluble material is then removed from the sample by centrifu- gation and the clear supernatant (about 2 mg per ml in Zac repres- sor) is applied to a 500-ml phosphocellulose column (Whatman P-11) equilibrated with 0.12 M Buffer KP (pH 7.4). The sample is washed through at 80 to 100 ml per hour until the effluent &SO is less than 0.1. A linear gradient (500 ml each side) is begun from 0.12 M KP to 0.24 M KP (0.20 M K2HPOI, 0.04 RI KHsPO4, 3 x 1O-.4 Y dithiothreitol, 5% glycerol) at the same flow rate, and &ml fractions are collected. The repressor act,ivity elutes at 0.17 to 0.19 M KP and is located using the filter assay for IPTG binding (8) on every third tube with aliquots of 25 to 50 ~1. The pooled peak is precipitated with ammonium sulfate at 231 mg per ml as above and gives a clean white pellet upon centrifugation. This pellet is redissolved in 1 M Tris-HCl (pH 7.6 at 4’), 3 X 10m4 M dithiothreitol. Lac repressor is soluble to nearly 40 mg per ml under these conditions; the addition of glycerol to 307, (v/v) permits storage at -20” and prevents aggregation of the repressor. The purified molecule possesses 50% of its theoretical operator binding activity and is stable in this respect for several months. From 500 g of frozen cells we obtain 600 to 700 mg of repressor (a yield of 90%); the sample is better than 98% pure (as judged by electrophoresis on SDS-polyacrylamidc gels) and has an A280/260 ratio of 1.9.

AZlcyLation-Repressor (10 mg per ml in 6 M guauidine hydro- chloride, 0.25 M Tris-HCl pH 7.8) was reduced at 37” for 3 hours with a 50.fold molar excess of 2-mercaptoethanol. Alkylation of the cysteine residues occurred upon the addition of iodoacetic acid (in 6 M guanidine hydrochloride, 0.25 M Tris-HCl pH 7.8) in 1.5 molar excess over the 2-mercaptoethanol. The reaction was carried out for 20 min in the dark at 37” and stopped with an excess of 2-mercaptoethanol. The pH of the solution did not fall below 7.4. The protein solution was dialyzed for 15 hours at 4” in the dark, against several changes of distilled water, and the flocculent white precipitate was collected by centrifugation.

Alkylation of the methionine residues was carried out using a modification of the procedure of Wilkinson (30). The CM-

cysteine repressor was redissolved at a concentration of I5 mg per ml in 6 M guanidine hydrochloride, 0.2 M sodium formate which had been adjusted to pH 3.5 with formic acid. Iodo[l-14C]acetic acid (3.2 &i per pmole, New England Nuclear) was added to this sample at 0.5 mole per mole of methionine (30) and incubated in the dark at 37” for 24 hours. After adding an excess of unlabeled iodoacetic acid to insure complete reaction (10 moles per mole of methionine), the sample was incubated for an additional 24 hours in the dark at 37”. The reaction was stopped with a large molar excess of 2-mercaptoethanol and the sample was dialyzed ex- tensively against 0.1 M ammonium bicarbonate.

Cyanogen Bromide Cleavage-Cyanogen bromide was used in lOO-fold molar excess over the methionyl residues, in 70% formic acid at room temperature for 10 to 15 hours. The sample was then diluted 20-fold and lyophilized to remove excess reagent and solvent.

Carboxymethyi-celkdose Chromatography of Cyancgen Brow&k Fragments-A cyanogen bromide digest of 1 to 2 pmoles (40 to 80 mg) of carboxymethylated Zac repressor was taken up in 3 to 5 ml of 6 M urea, 0.01 M sodium acetate buffer (pH 5.7) and applied to a carboxymethylcellulose column (1.2 X 35 cm) (Whatman CM-52) equilibrated with the same buffer at 4”. A linear gradient was run at a flow rate of 20 ml per hour from 0.01 to 0.20 M sodjum acetate in 6 M urea (pH 5.7), 140 ml on each side.

Digestion of Repressor under Native Conditions-A sample of purified Zac repressor was dialyzed into 0.1 it1 ammonium bicar- bonate at 4”. The concentration after dialysis was generally about 10 mg per ml; the slight precipitate which formed did not affect the resulm of the subsequent digestion. To a 4-mg (0.1 pmole) aliquot of dialyzed repressor was added l”/ by weight (40 kg) of trypsin or chymotrypsin for 20 to 30 min at 37”. Di- gestion was stopped by the addition of phenyl-methane-sulfonyl fluoride. No insoluble material was present, and the sample was either passed immediately through a Sephades G-50 column or lyophilized.

Enzymatic Digestions-Tryptic and chymotryptic digests were generally carried out at protein concentrations of 10 to 20 mg per ml in 0.05 M ammonium bicarbonate at 37”. The enzyme was used at 1: 100 (w/w) for 2 hours, then an additional aliquot was added for 2 hours more. Digestion was stopped with PMSF. Subtilisin digestion was carried out at room temperature in 0.05 M ammonium bicarbonate for 1 hour at a 1:lOO (w/w) ratio. Leucine aminopeptidase was dialyzed before use against 0.2 M

sodium bicarbonate, 0.01 M MgCl2. The peptide was taken up in 30 ~1 of the same buffer and the enzyme was added in a I :20 molar ratio. After incubation at 37” for 15 hours, the reaction was stopped by the addition of 0.2 M acetic acid, and a portion run directly on a Beckman amino acid analyzer. Carboxypep- tidase A and B were purified by acetone precipitation immediately before use, and redissolved in 0.2 M sodium bicarbonate. Di- gestions u-ere carried out at 1: 20 molar ratios in 0.2 M sodium bicarbonate for 1 to 4 hours at 37”. The sample was acidified, and a portion of the soluble fraction run directly on a Beckman analyzer.

SuccinyZaAon-Lyophilized material (0.2 to 0.3 pmole) was taken up in 1.0 ml of 6 M urea, 0.2 M sodium borate, adjusted to pH 9.0 with HCI. [14C]Succinic anhydride (9 PCi per pmole, New England Nuclear) was dissolved in 50 ~1 of acetone (dried just before use by passing through a small column of basic alu- minum oxide). Radioactive succinic anhydride wa.s added to the peptide in a a-fold molar excess over free amino groups, and incu- bated at room temperature for 3 min. Unlabeled succinic anhy- dride was then added in IO-fold molar excess under the same

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conditions. The buffer strength appeared adequate to compen- sate for pH changes without further adjustment. After excess reagent was removed by passing the sample through a Sephadex G-25 column in 0.05 M ammonium bicarbonate, the radioactively labeled peptide was lyophilizcd.

Electrophoresis and Chromatography-Lyophilized peptides were taken up in 50 to 200 ~1 of pH 3.5 electrophoresis buffer (5.0% acetic acid, 0.5y0 pyridine), and applied to Whatman No. 3ililM paper (46 X 57 cm) in a 5-cm band. The paper was thor- oughly wet with buffer (to concentrate the sample at the same time) and run at 2200 volts, 180 ma, for 55 to 85 min in a Savant high voltage electrophoresis tank. Basic fuchsin was used as a dye marker and ran 12 to 20 cm under these conditions, with a mobility of about 0.4 relative to free lysine. After an initial drying, the sample was concentrated to a narrow band with elec- trophoresis buffer and dried again. The paper was run in a second dimension perpendicular to the first in ascending chro- matography for 20 hours at room temperature in a closed tank. The chromatographic solvent used was (by volume) 7.6% acetic acid, 37.8% butanol, 24.4% pyridine, and 30.2% water. Fol- lowing chromatography, the paper was removed and dried in a hood.

Peptide Location and Elution-14C-Labeled peptides were located by autoradiography using Kodak NS-2T X-ray film. Other peptides were located by spraying the chromatogram with 0.02% ninhydrin in 95% ethanol. After a 12-hour incubation in the dark, it was possible to see less than 10 nmoles of a peptide. No significant destruction of the NHz-terminal residues was detectable by amino acid composition, and no difficulty was encountered in sequencing these peptides. The peptides were eluted with 2.5 ml of 0.2 N acetic acid for 12 hours at room tem- perature and lyophilized.

Amino Acid Analysis-Peptide samples were hydrolyzed in sealed, evacuated tubes containing 2 ml of 6 N HCl and 20 ~1 of phenol for 18 to 24 hours at 108”. After hydrolysis, the sample was dried down and redissolved in 0.5 to 1.0 ml of sodium citrate buffer (pH 2.2) for application onto a Beckman analyzer. Under these conditions it was possible to detect the presence of trypto- phan residues, although the recovery was low. The major hy- drolysis products of carboxymethylmethionine eluted at two positions from the long column, as described by Gundlach et al. (31). One derivative was methionine itself, and the other was carboxymethylhomocysteine, which eluted 1 min after proline. The yields under our conditions were 30 to 40% for methionme and 40 to 50% for carboxymethylhomocysteine. Succinyl-lysine was recovered intact from leucine aminopeptidase digestions and eluted between proline and glycine on the long column.

Determination of cysteine residues was carried out using 5,5’- dithiobis(2-nitrobenzoic acid) as described by Sedlak and Lindsay (32). Aliquots of repressor were mixed with a solution 8 M in guanidine-hydrochloride and 3 x 10M3 M in 5,5’-dithiobis(k nitrobenzoic acid), using 0.2 M TrisHCl pH 8.2 as a buffer. Ab- sorbance at 412 nm was read immediately against a blank without protein. The estinct.ion coefficient used was &? = 1.36 X 104, the value given by Ellman (33). Protein concentration was determined by amino acid analysis. Analysis of cysteic acid after performic acid oxidation yielded 3.0 residues of cysteic acid per polypeptide chain. This result is in good agreement with the value of 2.7 obtained by the optical method.

Sequence Determination-NH*-terminal sequence analysis of intact repressor was performed according to the SDS-dansyl- Edman procedure of Weiner et al. (34). Sequence analysis of neot,ides was carried out in tubes (6 x 50 mm) using 2 to 10

nmoles of sample. The only significant departures from the method of Hartley (35) were to shorten incubation times such that each complete cycle takes about 2 hours. The coupling reaction (100 ~1 of 507; pyridine, 5 ~1 phenylisothiocyanate) was incubated at 50” for 30 min; cyclization and cleavage was carried out with 100 ~1 of trifluoroacetic acid at 50” for 5 min. The tri- fluoroacetic acid was blown off with nitrogen and the sample dried for 10 min in a vacuum desiccator at 60”. After the n-butyl- acetate extraction, the sample was finally taken up in 25 ~1 of water, and a portion was removed for dansylation to identify the NH%-terminal residue before beginning another cycle of Edman degradation.

Dansylation of the peptide samples (0.1 to 0.2 nmoles provided ample material for identification) was carried out in 10 pl of 0.1 M sodium bicarbonate with the addition of 5 ~1 of dansyl chloride (5 mg per ml in acetone) for 30 min at 37’. The reaction mixture was dried down, 100 ~1 of 6 N HCI was added, and the 6 x 50 mm tube was sealed under a water aspirator vacuum to hydrolyze for 4 to 6 hours at 108”. After hydrolysis, the dansyl-amino acids were identified on 5 x 5 cm polyamide plates in four suc- cessive solvent systems as described in Weiner et al. (34). This procedure does not distinguish between acids and amides, nor is it possible to identify tryptophan and carboxymethylmethionine, presumably because multiple derivatives are present. Thus, when one of these residues was encountered in the sequence, no positive dansyl identification was possible. However, subsequent Edman degradation following a “blank” at one of these positions generally continued to yield clear results, and the missing residue could be assigned by difference.

RESULTS

Table I shows the amino acid composition of lac repressor, calculated for a polypeptide chain of molecular weight 38,000. SDS-dansyl-Edman degradation (34) was used on intact repressor

TABLE I

Amino acid composition of lac repressor

The composition is calculated for a molecular weight of 38,000, which has been obtained for the lac repressor monomer by electro- phoresis on SDS-polyacrylamide gels according to the methods of Weber and Osborn (36,37) using a number of different polypeptide chains as standards. The residue values given were obtained by averaging three duplicate sets of 22-hour hydrolysates of repres- sor. The threonine and serine values given are extrapolations from 24- to 72-hour hydrolysates. The valine and isoleucine values are taken from 72-hour hydrolysates. The half-cystine content was determined by performic acid oxidation and titration with 5,5’-dithiobis(2-nitrobenzoic acid). Tryptophan was deter- mined according to Liu and Chang (38). The amino acid com- position is in good agreement with the one reported previously by Miiller-Hill et al. (29), except for the phenylalanine content which has been reported to be 8 rather than 5 residues per polypeptide chain. Presently we have no explanation for this discrepancy.

Residue Value Residue Value

Lysine . . . . 11.9 Alanine.............. 40.7 Histidine . . . . . . 7.0 Cysteine............. 2.9 Arginine, 19.8 Valine. 32.6 Aspartic acid. . . 28.8 Methionine . . 9.0 Threonine. . . . . 18.1 Isoleucine . . 16.6 Serine . . . . . 29.8 Leucine . . . 39.6 Glutamic acid.. . . 43.7 Tyrosine . . . . . . . . . 7.6 Proline _ . . . , . . . . 11.5 Phenylalanine. . . . 4.9 Glycine............. 21.0 Tryptophan . . . . 2.0

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to determine the NHt-terminal sequence. The result obtained by this procedure for residues 1 to 16 is Met-Lys-Pro-Val-Thr- Leu-Tyr-Asx-Val-,4la-Glx-Tyr-Ala-Gly-Val-Ser-. Amide resi- dues lvere assigned by further studies on isolated peptides and the full KHz-terminal sequence for residues 1 to 59 is shown in Fig. 1. The methods used for this sequence determination are described below.

Cyanogen Bromide Cleavage Fragments A cyanogen bromide digest of carboxymethyl-lated Zac repressor

was chromatographed on CM-cellulose in 6 RI urea, 0.01 M sodium

acetate, pH 5.7, with a linear gradient to 0.20 RI sodium acetate. The elution profile from this column is shown in Fig. 2. The fractions corresponding to Peak E were pooled, and t,he urea was removed by dialysis against distilled water in acetylated mem- branes. The peptide formed a light precipitate under these conditions. SDS-dansyl-Edman degradation (34) of this sample yielded an NIlz-terminal sequence Lys-Pro-Val-Thr-. Com- parison with the first 16 residues of intact repressor demonstrates that this sequence corresponds to residues 2 to 5 in the polypeptide chain. Since the NHz-terminal methionine will be removed due to the specificity of cyanogen bromide cleavage, this peptide

CN-L CN-II . -

-:

Met-Lys-Pro-Val-Thr-Leu-Tyr Asp-Val-Ala-Glu-Tyr Ala-Gly-Val-Ser-Tyr Gln-Thr-Val-Ser-Arg

.

T-1; r T;; ‘i ‘.:’ 1 ,:’ 1

C b 4 0

m-1 2 ,Q m-2 CH-3 I- s

CN-II

T-2

T-2a

T-3

al-Val-Ai;-Gln-Ala-Ser-His-Vyl-Ser-Ala-Lys 50

E F-2a I- I- .

F-2 4

G

CN-III

FIG. 1. NH*-t.erminal sequence of Zac repressor, residues 1 to 59. sequence of residues 1 to 16 has been determined on intact re- The figure summarizes sequence data given in the text. All of pressor by SDS-dansyl-Edman degradation (34). These results the peptides indicated above the sequence are derivatives of cyan- are in agreement with the sequence obtained using a Beckman ogen bromide fragments, except for CH-4 and CH-5 which were automated Sequencer through residue 42. Adler et al. (39) have found in a chymotryptic digest of repressor under native condi- independently det,ermined the NHz-terminal sequence through tions. All of the peptides below the sequence were obtained from residue 50; however they have not assigned all the amide substi- the digestion of repressor under native conditions, except for F-2 tutions. Their assignment of residue 25 as aspartic acid is in (and its chymotryptic derivatives F-&z and F-Zb) which was iso- disagreement with our finding of asparagine (based on digestion lated as a radioactively labeled methionine-containing peptide. of peptide T-Za with Ieucine aminopeptidase). At, present we In addition to the data indicated in the figllre, the NHz-terminal have no explanation for this discrepancy.

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CM-CELLULOSE COLUMN pH 5.7

0

0.60 -

A280

0.40 -

0.20 c

A

h

Fraction Number

FIG. 2. CM-cellulose chromatography of cyanogen bromide fragments. A cyanogen bromide digest of 1 pmole (40 mg) of carboxymethylatcd lac repressor was taken up in 3 ml of 6 M urea, 0.01 M sodium acetate buffer pH 5.7 and applied to a CM-cellulose (Whatman CM-52) column (1.2 X 35 cm) equilibrated with the same buffer at 4”. Fractions of 3 ml were collected, at a flow rate of 20 ml per hour. At Fraction 35, a linear gradient was begun from 0.01 to 0.20 M sodium acetate (pH 5.7) in 6 M urea (140 ml on each side of the gradient maker). The absorbance at 280 nm was

plotted as shown; no material absorbing at either 280 nm or 230 nm was found between Fract.ions 35 and 75. Peaks A, B, 6, and D were mixtures and have not been further analyzed at present; Peak E contains only the first internal cyanogen bromide frag- ment (CN-II) and was analyzed as described in the text.

must be the first internal cyanogen bromide fragment (CN-II) of the Zac repressor. The amino acid composition of this peptide is given in Table II, and we shall show that it accounts for residues 2 to 42 of the Zac repressor.

Treatment of CN-II with a mixture of carboxgpeptidase A and

B resulted in the release of 1.6 residues of alanine per 1.0 residue of homoserine. This suggests a COOH-terminal sequence of (Ala)-Ala-Hsr for CN-II.

CN-II contained 3 residues of lysine and 2 residues of arginine. The Iysine residues were labeled with [K$uccinic anhydride and CN-II was then digested with trypsin (see “Materials and Methods”), which would be expected to yield only three peptides, corresponding to cleavage at each of the two arginines. The digested peptide was lyophilized and extracted with 0.2 M acetic acid, which yielded both a soluble and an insoluble fraction. Amino acid composition of the acid-insoluble portion showed a

high tyrosine content and suggested that this fraction contained the NHz-terminal peptidc of CN-II, since tyrosine residues occur at positions 7 and 12 in the sequence determined on the intact molecule. This acid-insoluble peptide, T-l, was further digested with chymotrypsin and fingerprinted. A single radioactively IabeIed peptide was observed upon autoradiography (T-la), and three more were found after staining with ninhydrin (T-lb, T-lc, and T-Jd). Each of these was analyzed as described below. The radioactive succinyl-lysine residue is indicated as “Lys*.”

T-l a (Residues W to ‘?‘): Lys*-Pro-Val-Thr-Leu-Tyr-The amino acid composition of this peptide is identical with that expected for a peptide spanning residues 2 to 7 in the sequence. No positive dansyl identification could be made for the NH2 terminus of T-la. This result is consistent with the proposed position of T-la as the

TABLE II

Amino acid composition of CN-II and its tryptic peptides

CN-II elutes pure from the CM-cellulose column (Peak E in Fig. 2). The values for each residue were rounded off t.o the nearest integer for presentation in t.he t,able (maximum deviation less than 10%). The tryptic peptides T-l, T-2, and T-3 were isolated as described in the text; it can be seen that their com- bined composition is in agreement with the composition of CN-II.

Residue T-l T-2 T-3 CN.11

Lysine............... Histidine Arginine. . Aspartic acid.. Asparagine Threonine. . Serine................ Glutamine.. Glutamic acid.. Proline. Glycine. Alanine.............. Valine. Leucine Tyrosine . Homoserine..........

Total................

1

1 1

2

2

1 1 1 1 2

4

1

3

21

1 1 1

1 1 2

1

2

3

13

3

1

2

1

1 3

4

2

3

1 1 6

8

1 3

1

41

NHz-terminal peptide of CN-II, since the NH?-terminal Iysine of W-11 was converted into di-succinyl-lysinc by succinylation. The blocked a-amino group prevents analysis by dansylation or Edman degradation. T-la contains proline and leucine, and CN-II itself contains a sole residue each of proline and leucine. Direct Edman degradation on the intact repressor molecule has placed these residues unambiguously at positions 3 and 6, respec- tively; thus, T-la can only compromise residues 2 to 7.

T-lb (Residues 8 to 12): Asp-Val-Ala-Glu-Tyr-Dansylation indicated an NH&erminal residue of Ass and the amino acid composition was consistent with a peptide spanning the residues 8 to 12 determined by direct Edman degradat,ion. Digestion by leucine aminopeptidase yielded aspartic and glutamic acid resi- dues rather than asparagine and glutamine.

T-lc (Residues 13 to 17): Ala-Gly-Val-Ser-Tyr-The NHZ- terminal alanine was identified by dansylation. The composition suggests that this peptide overlaps with residues 13 to 16 in the sequence obtained by direct Edman degradation; the tyrosine is placed at residue 17 by difference.

T-id (Residues 18 to 22): Gin-Thr-Val-Ser-Arg-Edmnn degra- dation provided the full sequence of this peptide. The presence of the arginine residue placed peptide T-ld as the COOH-terminal chymotryptic peptide of T-l. Leucinc aminopeptidase digestion indicated an NH&erminal glutamine rather than glutamic acid residue.

The amino acid composition of T-l (TabIe II) is completely accounted for by the combined compositions of its chymotryptic digestion products T-la, b, c, and d. The first three of these were ordered by the sequence of residues 1 to 16 as determined directly by SDS-dansyl-Edman degradation on intact repressor; by dif- ference, T-ld provides the COOH-terminal region. The full sequence of T-l (residues 1 to 22) is shown in Fig. 1.

The remaining tryptic peptides of CN-II were acid-soluble and were fingerprinted directly. Two spots (T-2 and T-3) were

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visible upon autoradiography of the chromatogram. After staining, no other ninhydrin-positive peptides were found. Since tryptic digestion of succinylated CN-II was expected to yield three peptides, it appeared likely that T-l, T-2, and T-3 would account for the enbire sequence of CN-II. Table II shows that this is indeed the case. T-3 is the COOH-terminal peptide of CN-II as judged by the presence of homoserine and its COOH- terminal sequence. Peptide T-2 may be placed by difference between T-l and T-3.

T-2 (Residues 23 to 35): Val-Val-Am-Gin-Ala-Ser-His-Vol-Ser- Ala-Llls*-Thr-,4rg-The NHz-terminal portion of this sequence was obtained by Edman degradation through the 10th residue (alanine). By amino acid composition, only 1 residue each of lysine, threonine, and arginine remained to be placed in order. To determine the sequence in this region, it was necessary to perform a tryptic digest of nonsuccinylated CN-II. This proce- dure yielded the two smaller peptides (T-2a and T-2b) that, per- mitted an unambiguous determination for the COOH-terminal sequence of T-2.

T-2a (Residues 23 to 33): Val-Val-Asn-Gln-Ala-Ser-His-Val- Ser-Ala-Lys-The first 4 residues of this peptide were obtained by Edman degradation and shown to agree with the NHz-terminal region of peptide T-2, Val-Val-Asx-Glx. Leucine aminopep- tidase demonstrated the presence of asparagine and glutamine rather than aspartic and glutamic acid. The sequence of the first 10 residues of T-2a may be inferred by comparison with T-2, and the lysine residue may be placed COOH-terminal by dif- ference.

T-2b (Residues SJ$ to 35): Thr-Arg-Dansylation assigned threonine as the SHs-terminal residue. The presence of this peptide along wit)h T-2a in a tryptic digest of nonsuccinylated CN-II verifies the (‘OOH-terminal sequence indicated above for peptide T-2.

T-3 (Residues 36 to 42): Glu-Lys*-Val-Glu-Ala-Ala-Hsr- Edman degradation provided the first 4 residues in this sequence. The homoserine was placed COOH-terminal by the specificity of cyanogen bromide cleavage, and the Ala-Ala sequence was placed by difference. Leucine aminopcptidase digestion indicated 2 residues of glutamic acid rather than glutamine; succinyl-lysine did not block the progress of the enzyme, and eluted between proline and glycinc on the amino acid analyzer.

Xethionine-containing Peptides

Since cyanogen bromide cleavage occurs specifically COOH- terminal to methionine residues, the isolation of peptides con- taining internal methionines can provide the overlaps between the cyanogen bromide fragments. We modified the procedure of Wilkinson (30) to carbosymethylate methionyl residues with iodo[%]acetic acid following alkylation of the cysteinyl residues with unlabeled iodoacetic acid (see “Materials and Methods”). The radioactively labeled repressor was digested with trypsin and this sample was passed through a Sephadex G-25 column; the radioactivity profile is shown in Fig. 3.

The fractions corresponding to Peaks II and III were separately

pooled and lyophilized. Each of these was run on paper in two dimensions (see “Materials and Methods”). Only one radio-

actively labeled peptide (F-l) was found upon autoradiography of the chromatogram run on the sample from Peak III. Two

labeled peptides were isolated from Peak II. One of these (F-2)

was sequenced entirely; further digestion of the other was neces-

sary to obtain a pure fragment F-3. With the exception of F-2, these peptides have not been placed within the sequence of the

wild type repressor. In the sequence data presented below, the

--- G-25 COLUMN 2000 I- iT I ‘t

‘UUJ ,f-%% i L& - 40 Fraction number

100 Fraction Number TOP

FIG. 3 (left). Gel filtration of methionine-containing tryptic peptides. dne micromole (40 mg) of [‘4C]carboxyme&yl~e~hi- onine-labeled Zac repressor (see “Materials and Methods”) was digested with tryps& for 4 hours at 37”. The insoluble material was removed by centrifugation, and the supernatant applied to a Sephadex G-25 column (2.5 X 95 cm) in 0.05 M ammonium bicar- bonate. The flow rate was 25 ml per hour, and each fraction was 4 ml; lOO+l aliquots were counted, and the radioactivity profile is shown in the figure. The ratios of the neaks are consistent with the number of methionine-containing *peptides in each. One peptide (F-l) was isolated from Peak III, two (F-2 and F-3) from Peak II, and four out of six (F-4, F-5. F-6. and F-7) were recovered from Peak I. Sequence analysis of’ea& of these is presented in the text.

FIG. 4 (right). Sucrose gradients on “core” repressor species. Following digestion of samples of repressor under native condi- tions, as described under “Materials and Methods,” with either trypsin or chymotrypsin, aliquots of the digested samples were run on 5 ml gradients, 5 to 20% sucrose in 0.01 M Tris (pH 7.6), 0.05 M KCI, 3 X 10-h M dithiothreitol, ST0 glycerol. The gradients were centrifuged for 15 hours at 58,000 rpm, 4”, in a Beckman SW- 65 rotor. Each gradient was dripped in 19 to 20 fractions of 0.25 ml. The IPTGrbinding activity-was located using 100.~1 ali- quots of each fraction in the Millinore filter assav (8). which meas- &es the binding of [l%]IPTG to the filter by Zac repressor. Four marker proteins were used with each set of gradients: aldolase (I), wild type Zac repressor (Z), bovine serum albumin (.s), and myoglobin (4). The tryptic core gradient profile is indicated by solid circles (a), and the chymotryptic core gradient profile by crosses (X). Both repressor derivatives have the majority of the IPTG-binding activity sedimenting at 6.4 S, which corre- sponds to a species of about 120,000 in molecular weight.

radioactively labeled carboxgmethylmethionine residue is indi- cated as “Met*.” Table III summarizes the sequence results for the mcthionine-containing peptides.

F-l: Ala-Leu-Ala-Asp-Xer-Leu-l~~et~-Gln-Leu-Ala-Arg-The complete sequence of this peptide was obtained by Edman deg-

radation, and is in agreement with the amino acid composition. A positive dansyl identification was not possible for the methionine

residue (see “Materials and Methods”) ; thus the carboxymethyl- methionine residue is placed by difference. F-l is identical with

peptide “D” (see below). In order to obtain further proof for this sequence, an aliquot of F-l was digested with chymotryp-

sin and the new labeled peptide (F-la) was purified by electro-

phoresis in one dimension at pH 3.5. F-la: Ala-Asp-Ser-Leu-Alet*-Gln-Leu-Edman degradation

provided this sequence, with the methionine placed by difference. The result is in agreement with the sequence of F-l from residue 3 through residue 9.

F-2 (Residues 36 to 51): Glu-Lys-Val-Glu-Ala-Ala-Met*-Ala- Glu-Leu-Asn-Tyr-Ile-Pro-Asn-Arg-The first 11 residues (through asparagine) were determined by direct Edman degradation. A

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TABLZ III Methionille-containing peptides of lac repressor

The sequences of seven out of nine methionine-containing peptides have been determined as described in the text. F-2 and F-4 are the only peptides whose position in the primary struc- ture are known (residues 36 to 51 and 1 to 7). F-2 and F-l are both released upon tryptic digestion of Zac repressor under native conditions.

I Ala-Leu-Ala-Asp-Ser-Leu-Met-Gln-Leu-Ala-Arg

Glu-Lys-Val-Glu-Ala-Ala-~-Ala-Glu-Leu-Asn-

-Tyr-Ile-Pro-Asn-Arg

Val-Ser-Met-Val-Glx-Arg

Met-Lys-Pro-Val-Thr-Leu-Tyr

Glu-Gly-Asp-Trp-Ser-Ala-Met-Ser-Gly-Phe

Gly-Ala-Met-Arg

Asx-Glx-Met-Ala-Leu -

positive dansyl result was not obtained at position 7; however, the sequence prior to this is identical with the first 6 residues of peptide T-3. This permits the assignment of carboxymethyl- methionine to position 7 (corresponding to the homoserine residue in T-3). From the amino acid composition of F-Z, 5 residues beyond the asparagine at position I1 are unaccounted for (tyrosine, isoleucine, proline, asparagine or aspartic acid, and arginine). To determine this region of the sequence, F-2 was digested with chymotrypsin and fingerprinted. One major radioactively la- beled spot was found (F-2a), and one major unlabeled ninhydrin- positive spot (F-2b).

F-$&a (Residues 36 to 45): Glu-Lys-Val-Glu-Ala-Ala-Net*- Ala-Glu-Leu-Edman degradation provided the first 9 residues, and the COOH-terminal leucine residue was placed by difference.

F-db (Residues .Q+ to 51): Tyr-Zle-Pro-Asn-Arg-This peptide was fully sequenced by Edman degradation, and provides the order of the missing residues in F-2. Since the asparagine is COOH-terminal to proline, it is not available to either carboxy- peptidase or leucine aminopeptidase. The assignment of as- paragine rather than aspartic acid is based on the electrophoretic mobility of the peptide.

The amino acid compositions of F-2a and F-2b account for the sequence of F-2 except for the asparagine residue at position 46. Since F-Z is identical with peptide “G” in Fig. 7b, which yielded 1 asparagine residue rather than aspartic acid upon leucine aminopeptidase digestion, the same assignment may be used for the corresponding residue in F-2.

The second radioactive peptide isolated from Peak II proved to be too long for direct sequence determination. It was there- fore digested with chymotrypsin and again fingerprinted. We observed a single spot (F-3) from this digest after autoradiog- raphy.

F-S: Val-Xer-Met*-Val-Glx-Arg-The amino acid composition agrees with the complete sequence as determined by Edman degradation. The carboxymethylmethionine was placed by difference. The Clx assignment has not yet been made.

Peptides F-l, F-2, and F-3 are the only methionine-containing peptides occurring in Peaks II and III of the G-25 column profile shown in Fig. 3. Since the composition of Zac repressor indicates a total of nine methionine residues, the remaining six methionines must be accounted for by Peak I, and the peptides containing them must be significantly larger than the 16 residues found for peptide F-2 (Peak 11). The fractions corresponding to Peak I were

pooled, lyophilieed, and digested with chymotrypsin. A finger- print of this digest yielded four strong spots upon autoradiography, one of intermediate intensity, and several weaker spots. The intermediate spot (F-4) and two of the strong spots (F-5 and F-6) were sufficiently pure for sequence analysis.

F-4 (Residues 1 to 7): Met*-Lys-Pro-Val-Thr-Leu-Tyr-The NHz-terminus could not be identified positively by dansylation, but Edman degradation yielded lysine at position 2. Amino acid composition confirmed that this peptide corresponds to the NHZ- terminal region of Zac repressor and is identical to peptide “c” in Fig. 7a.

F-5: Glu-Gly-Asp-TrpSer-Ala-Met*-Ser-Gly-Phe-This peptide was fully sequenced by Edman degradation. Leucine amino- peptidase digestion indicated residues of aspartic and glutamic acid rather than asparagine and glutamine. No positive dansyl result was obtained at either position 4 (tryptophan) or position 7 (carboxymethylmethionine). However, a minor radioactively- labeled spot was found on the chromatogram (F-5a) whose se- quence provides an unambiguous assignment for the methionine- containing region.

F-5~: Xer-Ala-Met*-Ser-Gly-Phe-Edman degradation provided the full sequence, and the carboxymethylmethionine has been placed by difference.

F-6: Gly-Ala-Met*-Arg-The sequence was determined by Edman degradation, and the carboxymethylmethionine was placed by difference.

The remaining two radioact.ively labeled spots on the chymo- tryptic fingerprint of Peak I moved only very slowly in either dimension. NHz-terminal analysis by dansylation showed that neither was sufficiently pure for sequence determination. Further digestion, however, has allowed us to sequence a portion of one of these peptides (F-7).

F-7: Asx-Glx-Net*-Ala-Leu-The sequence was determined by Edman degradation. Composition verified the placement of the carboxymethylmethionine by difference. This peptide was ob- tained after subtilisin digestion of one of the large chymotryptic methionine-containing peptides from Peak I and was purified by electrophoresis in one dimension.

The radioactively labeled peptides F-l through F-7 account for 7 of the 9 methionine residues in the Zac repressor. Future work along this line should permit sequence determination of the regions surrounding the remaining 2 residues of methionine. The results presented here allow the ordering of three cyanogen bromide fragments (CN-I, CN-II, and CN-III). When the remaining seven are isolated and purified, the methionine-containing peptides are expected to provide an unambiguous order for all of them.

Digestion of lac Repressor under Native Conditions

Native repressor was digested with t.rypsin for 30 min as de- scribed under “Materials and Methods.” At various times after the addition of trypsin, aliquots of the sample were removed and the enzymatic digestion was st,opped by the addition of PMSF. SDS-polyacrylamide gels were run on the samples for each time point. Intact repressor polypeptide chains (molecular weight 38,000) disappeared within the first 10 min, but three main bands were evident on the gels in the 28,000 to 33,000 molecular weight range. No digestion products could be seen in the 10,000 to 25,000 molecular weight range. These results suggest that diges- tion yields a “core” of the Zac repressor with a molecular weight of about 30,000 per polypeptide chain which is resistant to further tryptic digestion. The experiment was also performed with chymotrypsin, and similar results were obtained. The only dif- ference between these two digestions was that the core, after the

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addition of chymotrypsin, appeared slightly smaller in molecular weight than the tryptic core. We shall refer to the core material obtained by trypsin as the “T-core” and that obtained by chymo- trypsin as the “C-core”.

Inducer Binding and Tetrameric Structure Are Retained

The ability to bind the inducer IPTG is not affected by this partial digestion. After a 20-min incubation with trypsin under native conditions, the digestion was stopped by adding PMSF, and the sample was diluted to a concentration of 0.2 mg per ml in bovine serum albumin at 20 mg per ml. Aliquots of the digested sample (200 ~1 each) were assayed in parallel by equilibrium dialysis (7) against [14C]IPTG at four different con- centrations (0.1, 0.3, 1.1, and 3.1 x 10m6 M) for 3 hours at 4”. The presence of bovine serum albumin in loo-fold excess over repressor minimizes any residual effects of trypsin that may occur, and permits more accurate determinations of the protein con- centration after dialysis. A Scatchard plot of the results from this assay yielded a value for the IPTG binding constant K, = 0.8 (hO.2) x 10e6 M. This result is considered to agree within experimental error with the value reported for the normal repressor of 1.3 X lO-‘j M (7). Thus, digestion with trypsin under native conditions has no significant effect on the ability of the repressor to bind inducer.

The core samples obtained after tryptic or chymotryptic diges- tion were run on 5 to 20% sucrose gradients and the results of this experiment are shown in Fig. 4. Under these conditions, native repressor has a molecular weight of 150,000 and sediments as a tetrameric species with an S value of 7.2. This was used as one of the markers (No. 2) in this experiment. The figure indicates that most of the IPTG binding activity of both the tryptic and chymotryptic cores sediments at about 6.4 S. This S value cor- responds to a molecular weight (for a globular protein) of about 120,000. An aliquot of one of the peak fractions from the T-core gradient was run on SDS-polyacrylamide gels, and a comparison of this sample with the intact repressor polypeptide chain is shown in Fig. 5. For the T-core sample, a single band predominates in the 28,000 to 30,000 molecular weight range, which is significantly smaller than the undigested repressor species of molecular weight 38,000. From the size of the T-core monomer, we may infer that the tryptic and chymotryptic core species which sediment at 6.4 S are tetrameric. End-group determination of both the T-core and C-core tetramers by dansylation (34) yielded multiple resi- dues, demonstrating that neither of these species has a homoge- neous NH2 terminus.

Operator Binding Is Destroyed

Native repressor binds to the Zac operator with an affinity of about 2 X lo-I3 M (40). Two minutes after the addition of trypsin to a sample of active native repressor, no binding to Zac operator DNA could be detected, whereas the undigested con- trol was still completely active, when assayed according to Riggs et al. (11). There was no detectable protective effect when the digestion was carried out in the presence of (a) lo+ M IPTG, (b) saturating concentrations of calf thymus DNA (under conditions where repressor binds nonspecifically), or (c) 10e3 M o-nitrophenyl- fucoside (a competitive inhibitor of IPTG that does not relieve repression). In all three cases the gel patterns after 20 min looked identical with those from a digestion carried out in the absence of added substrates.

Pure repressor tends to aggregate when it is stored at high con- centrations in the cold over long periods of time (this may be avoided by storage in 30% glycerol at -20”). When such a

FIG. 5. SDS-polyacrylamide gels of T-core and wild type ZUC repressor. An aliquot from the peak (Fraction 8) of the tryptic core sucrose gradient shown in Fig. 4 was run on a 12-cm SDS- polyacrylamide gel in parallel aith an aliquot of undigested re- pressor, according to the method of Weber and Osborn (3G). The T-core sample (left-hand gel) is compared to intact Zac repressor separately (right-hand gel) and as a mixture of the two samples (midcUe gel). The molecular weight of the tryptic core polgpep- tide is approximately 28,000, compared to the wild type polypep- tide chain of 38,000. No intact repressor can be seen on the T-core gel, nor can any material of smaller molecular weight than the main band. This results suggests a molecular weight loss of about 10,000 as the result of digestion, in the absence of internal nicks in the polypeptide chain. The molecular weight of the T-core polypeptide indicates that the main species shown in the sucrose gradient profile (Fig. 4) is tetrameric.

sample was run on a sucrose gradient comparable to those shown in Fig, 4, all of the IPTG-binding activity pelleted at the bottom of the tube. Thus, the predominant species of repressor is an aggregate of larger molecular weight than the normal tetramer. We attempted to compare the effect of trypsin on an aggregated sample of repressor with its effect on the tetrameric species and on a sample which had pTecipitated upon dialysis into low salt (0.05 M ammonium bicarbonate). Roth the aggregated and precipitated samples were initially turbid. Shortly after the addi- tion of trypsin, the solutions became clear. The sucrose gradient profiles of all three digested samples were identical to those shown in Fig. 4. Thus, it appears that trppsinization effects disaggre- gation of insoluble samples of repressor without causing dissocia- tion of the normal tetrameric state or destroying the inducer- binding activity.

_ -

Partial Renaturation of Activity

The inducer-binding activity of wild type repressor can be renatured with high efficiency (41), although conditions have not

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yet been found to recover DNA-binding activity. Under the conditions described below, the proteolytic cores could also be reconstituted after denaturation in a random coil solvent.

,4 sample of native repressor was digested with trypsin for 20 min, the reaction was stopped with PMSF, and an aliquot was incubated in 6 M guanidine hydrochloride, 0.1 M 2-mercaptoetha- nol, 0.2 M Tris HCl (pH 7.4) at 37” for 1 hour. This sample was then diluted dropwise in the cold (4”) into 10 volumes of 0.2 M

Tris-HCl (pH 7.6), 0.1 M 2-mercaptoethanol. To remove the excess guanidine hydrochloride, the renatured sample was dia- lyzed against a buffer containing 0.1 M Tris-HCl, 0.1 M KCI, 5 loll MgAc, 2 x lo+ M dithiothreitol, and 5% glycerol. As a control, an undigested aliquot of repressor was subjected to the same treatment omitt’ing the guanidine hydrochloride step. The IPTG-binding a&iv&y was measured by the Millipore filter assay (8), and both sample and control had identical activities (within 10%). This result indicates a complete recovery of IPTG- binding activity upon renaturation of the tryptic core.

The sedimentation coefficient of the renatured species was de- termined by sucrose gradient centrifugation as described above and shown in Fig. 4. For the renatured sample, 50% of the IPTG-binding activity was found to sediment with an S value of 6.4. About 25tz of the activity formed a broad slower moving peak, and the other 25yc sedimented considerably faster than 6.4 S. This experiment demonstrates that the polypeptide chain of the tryptic core may renature into a tetrameric species with sig- nificant recovery. Since the T- core tetramer contains no indi- vidual polypeptides of molecular weight greater than 30,000 (see gels in Fig. 5), we may conclude that folding of the polypeptide chain into the proper inducer-binding conformation and the ability to aggregate into oligomers does not depend on the missing region of the polypeptide chain.

Separation of Peptides Released by Digestion

Gel filtration of a tryptic digest of native repressor yields two peaks (as illustrated in Fig. 6). Peak A represents the trypsin resistant core, eluting at the void volume of the column. Peak B must represent one or more peptides released by the digestion; its elution position corresponds to that of a peptide of 15 to 30 amino acid residues. This sample (Peak B) was pooled, lyophi- lized, digested wit,h chymotrypsin, and fingerprinted as described under “Materials and Methods.” The four predominant spots are shown in Fig. 7n. Following elution, the four peptides were characterized by amino acid composition, NH*-terminal residue (dansylation) and leucine aminopeptidase digestion when neces- sary. Comparison of these peptides with the chymotryptic peptides obtained from T-l suggest that they comprise the Nl&- terminal tryptic peptide and may be ordered as “c” (residues I to 7), “a” (residues 8 to 12). “b” (residues 13 to 17), and “d” (residues 18 to 22).

:1n alternative technique was used to analyze the remaining peptides released by the digestion of native repressor with trypsin. After stopping the digestion with PMSF, the sampIe is lyophi- lized, and taken up in “electrophoresis buffer” at pH 3.5 (see “Materials and Met.hods”). The T-core and the NHz-terminal tryptic peptide are insoluble in this buffer. When the soluble peptides are fingerprinted (see “Materials and Methods”), the pattern shown in Fig. 7b is obtained. There are seven major ninhydrin-positive spots, and four of these (“E,” “B,” “G,” and “C”) account for residues 23 to 59 in the repressor sequence.

Peptide E (Residues 23 to 33): Val-Val-Asn-Gln-Ala-Ser-His- Val-Ser-Ala-LysDansylation provided the NH2-terminal va-

line; composition and digestion with leucine aminopeptidase

G-50 P COLUMN

A I.OL

A280

0.5-

B

I I / I 20

Froc+ion4Lnber 60

After FIG. 6. Gel filtration of repressor digest.ed with trypsin. digesting 4 mg (0.1 pmole) of repressor with trypsin under native conditions in 0.1 M ammonium bicarbonate (see “Materials and Methods”) the sample was completely soluble. This sample (0.4 to 0.6 ml) was passed through a Sephadex G-50 column (1.2 X 20 cm) equilibrated with 0.1 M ammonium bicarbonate, at about 15 ml per hour, with a fraction size of 0.4 ml. The column profile at Azso is shown in the figure. Peak A represents the void volume of the column. Peak B contains the NHz-terminal tryptic peptide of the repressor (residues 1 to 22) in which there are 3 tyrosine residues.

verified that this peptide is identical with peptide T-2a described above.

Peptide B (Residues $4 to 36): Thr-Arg-The NHz-terminal threonine was identified by dansylation, the arginine placed by difference. Peptide B is identical to T-2b.

Peptide G (Residues 36 to 51): Glu-Lys-Val-Glu-Ala-Ala-Met- Ala-Glu-Leu-Asn-Tyr-Ile-Pro-Asn-Arg-Characterization of this peptide by amino acid composition, dansylation, and leucine aminopeptidase digestion demonstrated that it was identical to the methionine-containing peptide (F-2) described above.

Peptide C (Residues 52 to 59): Val-Ala-Gln-Gln-Leu-Ala-Gly- Lys-Edman degradation provided the sequence through the glycine at position 7; the lysine was placed COOH-terminal by difference. Leucine aminopeptidase digestion indicated 2 resi- dues of gluta.mine rather than glutamic acid. The arguments which allow the ordering of this peptide in the overall sequence are presented below.

The following three peptides (“D,” “F,” and “A”) have not yet been placed in the NHz-terminal sequence of the repressor.2

Peptide D: Ala-Leu-Ala-Asp-Xer-Leu-Met-Gln-Leu-Ala-Arg- Dansylation yielded an NHz-terminal alanine. This result and the amino acid composition suggest that D is identical with pep- tide F-l whose sequence determination has been presented above. Leucine aminopeptidase digestion of D indicated 1 residue each of aspartic acid and glutamine rather than asparagine and glu- tamic acid.

Peptide F: Gin-Val-Ser-Arg-Edman degradation provided the full sequence of this peptide. The presence of a glutamine residue was indicated by leucine aminopeptidase digestion.

Peptide A: Leu-Glx-Ser-Gly-Glx-The peptide was fully se- quenced by Edman degradation. Digestion with leucine amino-

2 Preliminary sequence data on a cvanogen bromide fragment starting in position 43(CN-III) supports-the assumption that the three peptides (D, F, and A) are located in CN-III and there- fore can be assigned to the NHz-terminal part of the molecule.

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c 6 ---__ -- ‘Z 0 -

0 Dye Lys 0 Gin-Gin-Leu-The NHz-terminal Asn-Tyr-Ile-Pro- sequence was provided by Edman degradation, and identifies peptide CH-4 as beginning at residue 46 in the wild type sequence. The aspara- gine and glutamine residues have been assigned by homology

OC Ob

with those in peptides G and C. The COOH-terminal sequence of CH-4 was provided by another chymotryptic peptide, CH-5.

Od

CH-5 (Residues 51 to 56): Arg-Val-Ala-Gin-Gin-Leu--The full sequence was provided by Edman degradation. The two gluta- mine residues have been assigned by homology with peptide C, since positions 2 to 6 correspond to the NH?-terminal sequence of peptide C as shown above. These resuks have been used to overlap the NH2 terminus of C with the COOH terminus of CH-4. The NH2 terminus of CH-4 in turn overlaps with the COOH terminus of G.

-~__-----------_--___ Three other chymotryptic peptides were obtained in high

- electrophoresis yield from the digestion under native conditions. These are indicated in Fig. 1 and correspond to the peptides c, a, and b mentioned above which comprise residues 1 to 17 in the NHZ-

0 Dye LYSO terminal sequence. These chymotryptic peptides are CH-1 (residues 1 to 7), CH-2 (residues 8 to 12) and CH-3 (residues 13 to 17).

nD

The additional peptides released by chymotrypsin under na- tive conditions appear to be large and ha.ve not yet been ob- tained in sufficient yield for sequence determination.

VerQication and Extension of NHz-terminal Sequence

0 1 OA OG nC I

4 .- t5 I - electrophoresis

FIG. 7. Fingerprints of peptides released from intact Zac re- pressor by digestion under native conditions. A, fingerprint of a chymotryptic digest of the NHz-terminal tryptic peptide of re- pressor (residues 1 to 22). As described in the text, peptide c spans residues 1 to 7, peptide a spans residues 8 to 12, peptide 6 spans residues 13 to 17, and peptide d spans residues 18 to 22. The location of these peptides in the primary st.ructure is indicated in Fig. 1. B, fingerprint of the soluble peptides obtained from a trip& digest of repressor under n&&e condit,ions as described under “Materials and Methods.” The seauence of these uentides is given in the text; four of them (E, B, G, and C) have been or- dered in the primary structure of the NHz-terminal region of the repressor as shown in Fig. 1. The location of the remaining three (D, F, and A) in the sequence is not yet known.2

peptidase demonstrated the presence of 1 residue each of gluta- mine and glutamic acid, although the positions have not been assigned. It appears more likely that the glutamine residue is COOH-terminal and the peptide arises by a chymotryptic-like cleavage, since peptide A contains no basic residues but is ob- tained reproducibly and in high yield after tryptic digestion of native repressor.

We attempted an analogous digest with chymotrypsin, to analyze the peptides released from native repressor by this en- zyme. After lyophilization of the digest, the soluble chymo- tryptic peptides were fingerprinted on paper as before. Two of these peptides (CH-4 and CH-5) have provided the primary evidence for the ordering of peptide C (residues 52 to 59) as immediately following peptide G (residues 36 to 51) shown above.

CH-4 (Residues 46 to 56): Asn-Tyr-Ile-Pro-Asn-Arg-Val-Ala-

The sequence presented above for residues 1 to 51 was ob- tained by overlapping the sequences of the first internal cyano- gen bromide fragment (CN-II) with a methionine-containing tryptic peptide (F-2). After determining this sequence, we were able to confirm the data shown in Fig. 1 through residue 42 by the use of a Beckman automated sequencer. The results from enzymatic digestion of Zac repressor under native conditions have permitted us to extend the NHz-terminal sequence through residue 59, as described above and illustrated in Fig. 1. The overlap between the “native” tryptic peptides G (residues 36 to 51) and C (residues 52 to 59) was provided by the native chymotryptic peptides CH-4 (residues 46 to 56) and CH-5 (residues 51 to 56). The sequence data of the additional methi- onine-containing peptides account for a total of 104 residues, or approximately 30% of the polypeptide chain.

The majority of the tryptic peptides released upon digestion of native repressor comprise the NHt-terminal region of the molecule. Three peptides (D, F, and A), however, have not yet been localized in the amino acid sequence of the repressor. Since SDS gel electrophoresis of tryptic or chymotryptic core molecule indicates the absence of internal nicks in the polypep- tide chains, the remaining three peptides should therefore come from either the NH2 terminus or the COOH terminus of the molecule. Adler el al. (41) have shown that carboxypeptidase digestion of Zac repressor dissociates the tetrameric molecule. Since the core molecules are tetramers we prefer the interpreta- tion that the COOH terminus is still intact in the tryptic core. Final proof for the hypothesis, that the native tryptic peptides D, F, and A will permit extension of the NHz-terminal sequence, can only come from further studies2 It should also be noted that the possibility of the release of a few additional undetected peptides upon tryptic digestion cannot be eliminated.

DISCUSSION

We have previously shown that an early nonsense mutation in the i-gene gives rise to a reinitiated fragment of Zac repressor (18). The NHz-terminal sequence of the reinitiated protein was de-

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TABLE IV

Ph,ysical properties of lac repvessor, the tryptic coTe of lac repres- SOT, ad the iloo reinitiation fragment

Data are taken from Platt et al. (18).

Zac repressor Tryptic core ilo reinitiated

fragment

7-

epres- ion in YiVO

Yes

No

lr.w

1.3 7.2 0.8 6.4 1.0 G.G

Subunit state of a size aggregation i

38,000 Tetramer 0

28,000 Tetramer -90

34,000 Tetramer 42

No. of amino

cids miss- ng at the NH2 ter-

minus

termined by SDS-dansyl-Edman degradation (18, 34), and the information presented here proves that this sequence corresponds uniquely to a region within the wild type sequence beginning at residue 43 (alanine). The repressor fragment resulting from this abnormal translational start lacks 42 amino acid residues and does not repress, although it retains the normal tetrameric structure and inducer-binding properties of Dhe wild type mole- cule. These results suggested the possibility of obtaining similar fragments from the wild type molecule by mild proteolytic treat- ment in vitro.

The experiments presented here demonstrate that digestion of the native molecule with either trypsin or chymotrypsin pro- ceeds to a limited extent, leaving a resistant core. The physical properties of the tryptic core, the iloo repressor protein, and the normal Zac repressor are compared in Table IV. The core poly- peptide chains, as judged by SDS gel electrophoresis, have a molecular weight of about 28,000, whereas the intact polypep- tide chaiu has a molecular weight of 38,000. Therefore, a total of about 90 amino acid residues must be released in the form of tryptic or chymotryptic peptides. Five of the eight major tryptic peptides account completely for residues 1 to 59 of the NHz-terminal sequence of the repressor, and the remaining three probably also come from the NHS-terminal region2

The tryptic and chymotryptic cores, which lack more than 80 residues, cannot bind to DNA in vitro. The capacity to bind inducer is not affected by the digestion, and sucrose gradient centrifugation of the cores shows that the digested species is still primarily a tetramer. Thus, some amino acid residues critical for the DNA binding ability must be located in the portion of the molecule which is removed from the NH, terminus by di- gestion (more than 207; of the polypeptide chain). By con- trast, the NHe-termiual region of the repressor does not contain residues that are necessary for maintaining a t,etrameric struc- ture or for the bindiilg of iuducer. These results argue for a spatial separation of the functional sites in the repressor. Sup- port for this conclusion is provided by the esistencc of the re- init,iat.cd iloo reprc~sor, \vhich proved that a region necessary for binding to operator actually occurs before residue 43 in the wild type repressor sequence (18). It has not yet been possible to determine whether the critical residues in this region are (a) directly involved in the nlechanism of repression; or (5) only required for the repressor to assume the correct operator-binding conformation.

not affected by this altered inducer binding (10). Conversely, the iYd (nonrepressing, transdominant) repressors lack only operator-binding activity; they are still tetrameric and bind inducer normally (13, 14, 39). Pfahl (15) has placed many i-” (operator-binding) mutations at the NHz-terminal end of the gene, but found that the is (inducer-binding) mutations tend to cluster near the center of the gene. These results further sup- port the conclusion that the DNA-binding and IPTG-binding sites are separated in the repressor polypeptide.

Another feature of the core species is that the missing region is not required for the correct three-dimensional folding of the residual portion of the repressor molecule. The IPTG-binding activity can be completely recovered by renaturation of the tryptic core from a random-coil solvent (guanidine hydrochlo- ride), and sucrose gradient centrifugation demonstrates that 50% of this IPTG-binding activity sediments at about 6.4 S, which corresponds to the initial tetrameric species.

A recent proposal that all 4 subunits are necessary for repres- sion and bind t.o repeating sequences in the lac operator in a linear fashion has been put forward by Adler et al. (39). The NHz-terminal region of the repressor is hypothesized to form a “protuberance” or “loop” necessary for binding to base pairs within the groove of the DNA double helix, and particular resi- dues in the NH%-terminal sequence of Zac repressor are predicted to be directly involved in binding to the operator site. Al- though we are not yet able to test the particular predictions of this model, the experiments presented here support the idea that the NHs-terminal region of the repressor is indeed exposed to the external environment. It may form either a protuberance or be a “globule” connected to the main body of the protein by a thin polypeptide bridge (as is seen in the y-globulins (21, 22)). Mild proteolytic digestion of the repressor specifically destroys the ability of the molecule to recognize and bind to the Zuc operator site on the DKA, concomitant with the cleavage of peptides from the NH2 terminus of the polypeptide. This region is therefore not only critical to DNA binding, it is also readily accessible to attack by trypsin or chymotrypsin. A protuberance able to bind within the DNA groove might be expected to be similarly exposed to proteolytic degradation. Support for this idea also comes from the observation that removal of the NH2 terminus by mild digestion enhances the solubility of the tetramer. A protruding NH2 terminus could conceivably interact with nearby molecules to cause aggregation.

The interpretations of t,hese data are in agreement with the genetm location and properties of functional mutations in the i-gene, The is (“super-repressor”) molecules have weaker . . _. .

The IPTG-binding activity and oligomeric structure of the tryptic core (modified by proteolytic attack in vitro) as well as the mutant iloo repressor (synthesized in vivo in a modified form) are very stable, although large portions of the polypeptide chain are missing. This is in marked contrast to molecules which have alterations at the COOH terminus. For example, a small deletion at the COOH terminus of the mutant Ll repressor results in a physiologically altered molecule. In &JO, the Ll molecule is an extremely poor repressor (2, 28), either because it is unable to form tetramers or because of an alteration at the DNA-binding site itself. In vitro, the Ll repressor sediments as a much broader, slower moving peak than the wild type, in- dicating the loss of a smble tetrameric structure, though it re- tains the normal affinity for IPTG (28). Although the subunit polypeptide chain is approximately the same size as that of the wild type repressor, the COOH terminal alteration confers a striking instability upon the mut,ant molecule: the Ll repressor appears to be actually degraded in the cell, whereas the wild type repressor is stable (37). Thus, the oligomeric structure is

affinities for inducer than normal, but their other properties are significantly affected by the Ll deletion, and in addition the sub-

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121

units themselves must form a less compact structure that results in high susceptibility to proteolytic attack. This is confirmed by the observation that the IPTG-binding activity of the Ll repressor which itself is stable in vitro, is very sensitive to the action of trypsin (37). By contrast, digestion of the wild type repressor in vitro under native conditions does not affect the IPTG-binding activity (see “Results”). Thus, the presence of a slight alteration at the COOH terminus of the repressor has a profound effect on the stability of the tertiary and quaternary structure of the molecule. To cite additional support for this interpretation, Adler et al. (39) have reported that high concen- trations of carboxypeptidase 13 alter the aggregation properties of t,he wild type tetramer. This effect is remarkable when it is compared to the stability of the tryptic or chymotg-ptic cores. The latter have lost more than 80 residues from the NH2 termi- nus, yet remain free of internal nicks and maintain a tetrameric structure with normal inducer binding. These findings are all consistent with the possibility that the COOH-terminal region of the repressor is intimately involved in the formation of oligo- mers and in maintaining the over-all stability of the tertiary structure.

For the purpose of future work, a significant practical advan- tage of the proteolytic digestion of the native Zac repressor is that the tryptic or chymotryptic cores can be separated from the released peptides by gel filtration. It should be noted that direct evidence for “release” has been presented only for the NHz-terminal tryptic peptide; the possibility remains that some peptides produced by trypsin may still be noncovalently bound to the repressor core. In any case, the core may be treated essentially as a smaller protein of only 28,000 molecular weight per subunit, or about 260 amino acid residues. This should prove advantageous for both functional studies and sequence

work.

The cleavage of Ohc peptides themselves from the polypeptide chain has provided a simple technique to confirm and extend the primary structure of the repressor directly from the NH* terminus. The simplicity of the peptide map obtained after trypsinization provides an excellent technique for localizing ea.rly point mutations in the amino acid sequence of the repres- sor, which may be used as a further tool for the determination of structure-function relationships in the wild type molecule. In experiments to be report,ed elsewhere, we have located four dif- ferent mutations wit.hin the first 59 residues, working on a scale of lOA7 moles (4 mg) for each of the purified mutant repressors (42).

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Terry Platt, James G. Files and Klaus WeberACID-BINDING ACTIVITY

NH2-TERMINAL REGION AND LOSS OF THE DEOXYRIBONUCLEIC Repressor : SPECIFIC PROTEOLYTIC DESTRUCTION OF THELac

1973, 248:110-121.J. Biol. Chem. 

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