©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Mutants Affecting the KRK Sequence in the Carboxyl-terminal Domain of lac Repressor (*)

Likun Li , Kathleen Shive Matthews (§)

From the (1) Department of Biochemistry & Cell Biology, Rice University, Houston, Texas 77251

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
FOOTNOTES
REFERENCES

ABSTRACT

The lac repressor carboxyl-terminal region is required for tetramer assembly and protein stability. To further investigate this region, especially the unusual sequence KRK, four deletion mutants eliminating the carboxyl-terminal 34, 35, 36, and 39 amino acids and five substitution mutants at the position of Arg-326, R326K, R326A, R326E, R326L, and R326W, were constructed using site-specific mutagenesis. The -34-amino-acid (aa) mutant, missing the most carboxyl-proximal lysine from the KRK sequence, exhibited lower affinity for both operator and inducer and lower protein stability than dimeric proteins studied previously. The -35-aa mutant with RK missing, as well as -36 aa and -39 aa, for which the entire KRK sequence was deleted, yielded inactive polypeptides that could be detected only by monoclonal antibody for lac repressor. In the Arg-326 mutant proteins, operator binding affinity was decreased by 6-fold, the shift in inducer binding at elevated pH was diminished, and protein stability was decreased. Dramatic decreases in protein expression and stability occurred with substitution at position 326 by glutamate, leucine, or tryptophan. These results suggest that Arg-326 plays an important role in the formation of the proper tertiary structure necessary for inducer and operator affinity and for protein stability.


INTRODUCTION

lac repressor negatively regulates expression of the lac operon of Escherichia coli by binding to its cognate operator with high affinity and thereby blocking initiation of RNA transcription of the associated structural genes (Jacob and Monod, 1961; Miller and Reznikoff, 1980). Repression is modulated by the interaction of lac repressor with inducer sugars that direct a conformational change to alter affinity for operator sequences (Jobe and Bourgeois, 1972; Barkley et al., 1975; Miller and Reznikoff, 1980). Wild-type lac repressor is a tetrameric protein containing identical subunits of 360 amino acids (Gilbert and Müller-Hill, 1966; Riggs and Bourgeois, 1968) with two operator binding sites and four inducer binding sites (Barkley et al., 1975; O'Gorman et al., 1980a, 1980b; Culard and Maurizot, 1981; Whitson and Matthews, 1986). Each monomer of the repressor protein is composed of two major functional domains with the amino-terminal 59 residues forming a helix-turn-helix DNA binding domain (Adler et al., 1972; Lin and Riggs, 1975; Müller-Hill, 1975) and the residues 60-360 forming a core domain that contains the inducer binding sites and assembly determinants (Platt et al., 1973; Müller-Hill, 1975; Schmitz et al., 1976; Miller, 1979; Miller et al., 1979; Ogata and Gilbert, 1978; Lehming et al., 1988; Alberti et al., 1991; Chakerian et al., 1991; Chen and Matthews, 1992).

Tetramer assembly requires the integrity of the carboxyl-terminal region of the lac repressor protein (Lehming et al., 1988; Brenowitz et al., 1991; Chakerian et al., 1991; Alberti et al., 1991, 1993; Chen and Matthews, 1992; Chen et al., 1994a, 1994b), and evidence demonstrating that the carboxyl-terminal amino acid sequence contributes to protein stability has also been reported (Brenowitz et al., 1991; Chen, 1993; Chen and Matthews, 1994). Alteration or deletion of the leucine heptad repeats at the carboxyl terminus of the repressor yielded dimeric repressors with diminished apparent operator affinity (Chakerian et al., 1991; Alberti et al., 1991; Brenowitz et al., 1991; Chen and Matthews, 1992, 1994). Beyond the leucine heptad repeats, deletion of up to 32 amino acids from the carboxyl terminus yielded dimeric protein with normal inducer binding activity but 150-fold diminution of apparent operator binding affinity (Chen and Matthews, 1992). Similar results were observed with another dimeric mutant repressor lacking 31 amino acids at carboxyl terminus with 15 added missense amino acids (Lehming et al., 1988; Oehler et al., 1990; Brenowitz et al., 1991). A core repressor with the amino-terminal domain and 64 carboxyl-terminal residues removed by chymotrypsin was generated and reported to be a monomer (Müller-Hill, 1975); however, two other laboratories reported that deletion of 43 or 64 amino acids from the carboxyl terminus yielded low levels or no repressor protein in the cell extracts (Betz, 1986; Chen and Matthews, 1992). These observations in the literature raised two questions. Is there a ``boundary'' of dimer assembly between residues 296 and 328 or a ``boundary'' in this region that defines the sequence necessary for the protein to fold properly and thereby to perform its basic function? Genetic studies by phenotypic analysis indicate that arginine at position 326 (Arg-326) is a conserved residue, since significant alterations in phenotype were observed when Arg-326 was substituted by most other amino acids (Kleina and Miller, 1990). On the basis of these investigations and examination of amino acid sequence, we hypothesized that in the region of the unusual KRK sequence, there might be a boundary that defines the sequence necessary for the protein to fold properly and thereby to perform its basic binding function. In order to define this boundary, a series of deletion mutants (-39 aa,() -36 aa, -35 aa, and -34 aa) was generated by site-specific mutagenesis. In addition, amino acids with differing side chain character were selected for substitution at position 326.


MATERIALS AND METHODS

Enzymes and Chemicals T4 polynucleotide kinase was purchased from Promega. T4 DNA ligase and Sequenase version 2.0 kit were obtained from U. S. Biochemical Corp. [C]IPTG was the product of NEN Research Products. ATP, dNTP, IPTG, DNase I, trypsin, and other routine chemicals were from Sigma. Sephadex G-25, Sephadex G-150, and a gel filtration calibration kit (consisting of ribonuclease A, chymotrypsinogen A, ovalbumin, bovine serum albumin, and blue dextran 2000) were from Pharmacia Biotech Inc. Monoclonal antibody was prepared by Sams et al. (1985), and the secondary antibody, anti-mouse IgG (H + L)-peroxidase, was from Boehringer Mannheim Corp. Nitrocellulose membranes were the product of Schleicher & Schuell. Urea (ultrapure) was from Fluka Chemical Corp. Plasmids and Bacterial Strains Plasmid pJC1 (Chen and Matthews, 1992) was used as an expression vector. Uracil-containing single-stranded DNA was prepared from E. coli CJ236 ( dut, ung, thi, rel A, pCJ105(Cm); Kunkel et al. (1987)). E. coli 71/18 ( supE, thi, ( lac-proAB) F` [ proABlac IlacZM15]) was used as the host to prepare single- or double-stranded DNA for sequencing. E. coli TB1 ( ara, ( lac-pro), StrA, thi, 80d lacZM15, r, m) was used as the host to express wild-type and all mutant repressor proteins. Site-specific Mutagenesis Oligonucleotide-directed site-specific mutagenesis was conducted using pJC1 according to the method reported by Kunkel (1985). For all the carboxyl-terminal deletions, the codon at the desired position was converted to a stop codon. For substitutions of Arg-326, the codon for arginine was converted to that for the desired residue. A uracil-containing template was prepared from IR1 or M13KO7 superinfected pJC1-transformed CJ236 cells. The synthetic oligonucleotide was desalted and phosphorylated at the 5` end by T4 polynucleotide kinase followed by hybridization to uracil-containing template (oligo:template 25:1 molar ratio) at 75 °C for 2 min, followed by slow cooling to 35 °C. Sequenase (Version 2.0) and T4 DNA ligase were added, and polymerization and ligation were performed at 37 °C for 2 h. After inactivating the enzymes by heating at 65 °C for 15 min or freezing at -20 °C for 1 h, a portion of the reaction mixture containing newly synthesized and presumably mutated DNA was transformed into E. coli 71/18 cells. Colonies were selected by ampicillin resistance. DNA was prepared using Qiagen plasmid kit (QIAGEN) or Wizard DNA purification system (Promega). Mutations were identified by dideoxy sequencing (Sanger et al., 1980). All mutations at the desired site were further confirmed by sequencing the entire lacI gene to eliminate any possibility of potential mutations at other sites. The confirmed mutant DNA was transformed into E. coli TB1 cells for mutant lac repressor expression. Purification of Repressor Proteins pJC1-transformed E. coli TB1 cells were grown in 2 YT (16 g of tryptone, 10 g of yeast extract, and 5 g NaCl/liter, pH 7.6) in flasks with shaking at 37 °C for 16-20 h. Cells were harvested from 4 to 8 liters of liquid culture by centrifugation and then were frozen and thawed in 3 volumes (v/w) of breaking buffer (0.2 M Tris-HCl, pH 7.6, 0.2 M KCl, 0.01 M MgOAc, 0.3 mM DTT, 5% glucose, 100 µg/ml phenylmethylsulfonyl fluoride, and 250 µg/ml lysozyme).

Wild-type, R326K, and R326A repressors were purified as described previously (O'Gorman et al., 1980a). The -34-aa deletion mutant, R326E, and R326L repressors were purified using a modified procedure. Briefly, after centrifugation of the cell slurry, the supernatant was precipitated with 25% ammonium sulfate. The supernatant from this step was brought to 37% ammonium sulfate and incubated for 45 min, and the mixture was centrifuged at 6,000 g for 40 min. The resulting pellet was dissolved in 0.03 M KP buffer (0.03 M potassium phosphate, pH 7.5, 0.3 mM DTT, 5% glucose) and then desalted. The sample was loaded on a phosphocellulose column pre-equilibrated with 0.05 M KP buffer and eluted with 3 column volumes of 0.05 M KP buffer, followed by elution with a gradient of 0.05-0.15 M KP buffer. The -34-aa repressor was recovered in this step. The R326E and R326L repressors were eluted with a subsequent gradient of 0.15-0.3 M KP buffer. The fractions containing IPTG binding activity were pooled, precipitated with 33% ammonium sulfate, and desalted. The desalted proteins were further purified using a Sephadex G-150 column as described below with loading concentrations of 1 mg/ml. The repressor proteins in each purification step were monitored by IPTG binding assay using the ammonium sulfate precipitation method described by Bourgeois (1971). Protein concentrations were determined using Bio-Rad protein assay (Bradford, 1976). The purity of the proteins was analyzed by 10% SDS-polyacrylamide gel electrophoresis and quantitated by scanning the gel with a computing densitometer. Evaluation of Oligomeric State Sephadex G-150-120 chromatography was carried out to examine the oligomeric state of the repressor proteins and was also used to further purify some of the mutant proteins which could not be purified by phosphocellulose chromatography. The column (1.2 60 cm) was equilibrated with 0.05 M KP buffer, pH 7.5, and calibrated with ribonuclease A, chymotrypsinogen A, ovalbumin, bovine serum albumin, blue dextran 2000, and wild-type lac repressor. For analysis of oligomeric state, purified mutant proteins were loaded at 0.2 mg/ml (1.3 10 M tetramer) in 0.05 M KP buffer. Ammonium sulfate-precipitated proteins were loaded at >5 mg/ml total protein. Column chromatography was performed under constant pressure controlled by a MINIPULS 3 pump. Protein presence was monitored by ultraviolet absorbance detected by a Pharmacia LKB Uvicord SII. Protein fractions corresponding to each peak were assayed by IPTG binding or by monoclonal antibody reaction as described by Sams et al. (1985). The antibody reaction was essential to detect mutants devoid of IPTG binding activity. Concentration of protein at the maximum of the elution peak was 0.5 10 M tetramer.

To confirm the oligomeric state of the wild-type and mutant proteins under DNA binding conditions, a gel retardation experiment was conducted. The binding buffer for gel retardation was modified from that for filter binding (O'Gorman et al., 1980a) by omission of dimethyl sulfoxide and addition of 10% glycerol to the buffer. The binding reactions were performed for 20 min at room temperature with 1 10 M 40-base pair operator DNA (also used in nitrocellulose filter binding, see below) and protein at various concentrations. Gels for retardation assays contained 8% acrylamide (37.5:1), TBE buffer, and 3% glycerol. The gel was pre-electrophoresed to constant current at 100 V with recirculation of the buffer. Reaction mixtures were loaded onto the gel at 300 V. The dyes were loaded in separate lanes; following dye entry into the gel, the voltage was reduced to 100 V, and electrophoresis proceeded for 3 h. The gel was dried and exposed to a FUJI phosphorimaging plate for 1-2 h, and DNA-protein complexes were visualized by scanning the image generated using a FUJI Imaging Analyzer BAS 1000 (FUJI, Photo Film Co., LTD, Japan). Immunological Reactions

Dot Blot

Aliquots of 2 µg and 10 µg of each purified protein, or aliquots of 20 µg and 100 µg of each ammonium sulfate precipitate, were incubated in phosphate-buffered saline (PBS) in the absence or presence of 0.02% SDS and dot-blotted onto a nitrocellulose membrane. The membrane with blotted proteins was blocked with 5% non-fat milk in PBS followed by reaction with monoclonal antibody B-2 as described by Sams et al. (1985).

Western Blot

Proteins were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane using a Semi-dry Transfer Cell (Bio-Rad). Nonspecific sites were blocked by incubation of the membrane in PBS containing 0.05% Tween 20 (PBST) (Batteiger et al., 1982) at room temperature for 1 h, and antibody reaction was also carried out in PBST. Color development was conducted as described for dot-blotting. Fluorescence Measurement of Inducer Binding IPTG binding was determined by fluorescence measurement (O'Gorman et al., 1980b) using an SLM-AMINCO 8000 fluorometer in a buffer containing 0.01 M Tris-HCl (pH 7.5 or pH 9.2), 1 mM EDTA, 0.01 M MgCl, 0.2 M KCl (TMS), with a protein monomer concentration of 1.0 10 M for -34-aa and 1.5 10 M for other mutants, and with varying IPTG concentrations that ranged from 1 10 to 1 10 M. IPTG binding data were subsequently analyzed using the program Igor Version 1.2, to generate fits to the binding equation, R = [IPTG]/(K + [IPTG]), by nonlinear least squares analysis (Chen and Matthews, 1992), where R corresponds to fractional saturation determined by fluorescence, [IPTG] corresponds to the IPTG concentration, Kis the equilibrium dissociation constant, and n is the Hill coefficient. Operator Binding Assay A 40-bp dsDNA containing the lac operator was labeled at 5` ends with [P]ATP by T4 polynucleotide kinase. Binding was carried out in 0.01 M Tris-HCl, pH 7.4, 0.1 mM DTT, 0.1 mM EDTA, 0.15 M KCl, 0.01 M MgOAc, 5% dimethyl sulfoxide, and 100 µg/ml bovine serum albumin, with 1 10 MP-labeled 40-bp operator and a series of concentrations of protein (O'Gorman et al., 1980a). The binding mixtures were transferred onto a nitrocellulose filter by a modified dot-blot apparatus (Wong and Lohman, 1993). The filter was dried and exposed to a FUJI phosphorimaging plate for 30 min, and repressor-operator binding, as measured by operator retention on the filter, was then quantified by scanning and analyzing the image generated using a FUJI Imaging Analyzer BAS1000 (Fuji, Photo Film Co., LTD, Japan). The operator binding data were then analyzed using the program Igor Version 1.2 to generate fits to the binding equation, R = [protein]/( K+ [protein]) (Chen and Matthews, 1992), where R is the fractional saturation, Kis the equilibrium dissociation constant, and [protein] is the protein concentration expression as M dimer. The maximum value of R was not fixed in the fitting process to ensure that saturation was reached. Because the measured stoichiometry of repressor binding to DNA is one operator per dimer unit, it was assumed that each dimer unit could bind to one operator and the protein concentration was expressed in dimer. The binding equation represents the simplest binding process with no linked equilibria and is sufficient to fit the data.

The operator binding activity of each purified repressor protein was determined by titration using 1 10 MP-labeled operator (for -34 aa, the operator concentration was 2 10 M). The data from activity assay were analyzed using an CA-Cricket Graph III program assuming a stoichiometry of one operator per dimer. Urea Denaturation Urea denaturation experiments were performed in 0.1 M KSO, 0.01 M Tris-HCl, pH 7.5 (Chen and Matthews, 1994). Urea stock solution (10 M) was prepared daily using ultrapure urea (Fluka Chemical Corp.) and filtered before use. The denaturing reactions were completed by incubation of each protein (10 µg/ml) with varying concentrations of urea at room temperature for 2-3 h. The denatured samples were examined by fluorescence intensity measurements using an SLM AMINCO 8000 fluorometer with excitation at 285 nm and emission at 354 nm. The resulting data were plotted using a CA-Cricket Graph III program. Trypsin Digestion Reactions were performed at a protein concentration of 0.25 mg/ml in 0.12 M potassium phosphate (KP) buffer containing 0.3 mM DTT, 5% glucose, and 2% (w/w) trypsin (stock made in 0.001 N HCl, Chang et al. (1994)) at room temperature for 30 min. The reaction was stopped by the addition of 2% (w/v) phenylmethylsulfonyl fluoride (stock made in 100% ethanol, Matthews (1979)). The digested proteins were analyzed by electrophoresis on a 10% SDS-polyacrylamide gel followed by silver staining.


RESULTS AND DISCUSSION

Selection of Mutant Sites

To examine the carboxyl-terminal region encompassing the unusual KRK sequence, we produced a series of deletion mutants extending beyond the previously characterized mutant with a 32-amino acid deletion (-32 aa): -34 aa, with one lysine deleted from the KRK sequence, -35 aa with RK deleted, -36 aa and -39 aa that deleted the entire KRK sequence (Fig. 1). In addition, we generated five substitutions for arginine at position 326 using amino acids with side chains differing in character: lysine (polar, positive charge), alanine (apolar, short chain), glutamate (polar, negative charge), leucine (apolar, long chain), and tryptophan (bulky indole ring). Each of the designed mutants was generated by site-directed mutagenesis, and the entire lacI gene was sequenced to ensure that no other alterations were present.


Figure 1: Sequence of lac repressor carboxyl-terminal 43 amino acids. The sequence KRK, which is hypothesized to be important to the structure and function of lac repressor, is underlined. For the carboxyl-terminal deletions, the codon 3` to the terminal amino acid (at the end sites indicated by ***) was converted to a stop codon. For the single substitutions at position 326, the selected residues, alanine, glutamic acid, lysine, leucine, and tryptophan, replace arginine at position 326.



Expression Levels of Mutant Repressors

All nine mutant repressor proteins were present in the cell extracts based on reaction with monoclonal antibody to the repressor protein (see also Fig. 7 ); however, the yields of some mutants differed greatly from wild-type. Protein profiles by denaturing gel electrophoresis of the 37% ammonium sulfate precipitation fractions (Fig. 2 A) and Western blot of these same fractions (Fig. 2 B), as well as by IPTG binding assays of crude cell extracts (Fig. 2 C), indicated that R326K and R326A had expression levels close to that of wild-type repressor protein. However, -34 aa had a significantly lower steady-state expression level than wild-type, and R326E and R326L were expressed at even lower levels. The mutants -35 aa, -36 aa, -39 aa, and R326W were undetectable by staining, activity assays, or Western blot using comparable amounts of cell extract, although use of higher amounts of protein allowed detection of these polypeptides in ammonium sulfate precipitates (see below). The extremely low steady-state expression levels of the latter seven mutants suggests the possibility that the structures of these polypeptide chains are altered from wild-type, and, therefore, the proteins become more susceptible to proteolytic degradation in the cell.


Figure 7: Immunoblotting of mutant repressors with B2 monoclonal antibody. The proteins shown are as follows: A, wild-type; B, -34 aa; C, -35 aa; D, -36 aa; E, -39 aa; F, R326K; G, R326A; H, R326E; I, R326L; J, R326W. The reactions were carried out in the absence ( a) and presence ( b) of 0.02% SDS. For wild-type, -34 aa, R326K, R326A, R326E, and R326L, 2 µg of purified protein was used for duplicate samples labeled 1 and 2 and 10 µg for duplicate samples labeled 3 and 4. For -35 aa, -36 aa, -39 aa, and R326W, 20 µg of total protein from a 37% ammonium sulfate precipitate of the cell extract was used for samples labeled 1 and 2 and 100 µg for samples labeled 3 and 4.




Figure 2: Protein expression levels of mutant repressors. A, protein profiles generated by SDS-polyacrylamide gel electrophoresis of 37% ammonium sulfate precipitate from cell extracts. Lane 1, purified wild-type protein as marker; lane 2, purified -34-aa protein as marker; lane 3, wild-type; lane 4, -34 aa; lane 5, -35 aa; lane 6, -36 aa; lane 7, -39 aa; lane 8, R326K; lane 9, R326A; lane 10, R326E; lane 11, R326L; and lane 12, R326W. One µg of purified protein was used for the markers in lanes 1 and 2, and 25 µg of total protein for lanes 3-12. B, Western blot of the same samples as A except for omission of purified wild-type and -34-aa proteins. The reactions with B2 monoclonal antibody were carried out as described under ``Materials and Methods.'' C, IPTG binding activity in cell extracts. IPTG binding assay was performed as described under ``Materials and Methods.'' IPTG binding activity was expressed as cpm/mg of protein.



Although a significant decrease in expression level was observed with removal of the carboxyl-proximal lysine from the KRK sequence (-34 aa), this mutant still possessed the basic functions characteristic of lac repressor. It is important to note that further deletion of one residue to remove RK (-35 aa) resulted in inactive protein with an extremely low expression level. The significant difference between -34-aa and -35-aa mutants suggested that the guanidino side chain at position 326 may be important to the function of the lac repressor, consistent with in vivo phenotypic analysis of a number of substitutions at this site generated by Kleina and Miller (1990). This hypothesis was further confirmed by single substitutions for R326. Small differences in the expression levels compared with wild-type were observed when Arg-326 was replaced by amino acids containing a positively charged side chain (lysine) or a small apolar side chain (alanine); however, dramatic changes in protein expression occurred when amino acids with a negatively charged side chain (glutamate) or a long chain apolar side chain (leucine) were selected for substitution. Moreover, a remarkably low protein expression was observed when an amino acid containing a bulky aromatic side chain (tryptophan) was introduced at position 326. These results suggested that Arg-326 may play an important role in the formation of proper tertiary structure necessary for expression of repressor protein and are consistent with the conservation of Arg-326 indicated by in vivo phenotypic analysis (Kleina and Miller, 1990).

Purification of the Mutant Repressor Proteins

Phosphocellulose chromatography normally employed for the purification of lac repressor was effective for R326K and R326A using the procedure described previously (O'Gorman et al., 1980a). However, due to the low expression levels of the altered proteins, purification of other mutant repressor proteins by phosphocellulose chromatography was not successful. To overcome this problem, several modified steps were employed (see ``Materials and Methods''), including additional ammonium sulfate precipitation steps, altered conditions for elution from phosphocellulose, and Sephadex G-150 chromatography. The purities of the repressor proteins were examined by both density scanning of the stained protein species separated by SDS gel electrophoresis and stoichiometric assay of operator binding. The electrophoretic analysis of the purified mutant repressors is shown in Fig. 3, and stoichiometric operator binding binding curves are shown in the insets to Fig. 5 . The quantitative data are summarized in . For most mutants, the results from both methods were concordant; however, the purity of the -34-aa protein obtained from analysis of gel electrophoresis profiles was 90%, while the purity obtained from stoichiometric operator binding assay was only 50%. This difference most likely arises from the thermodynamic linkage of dimer assembly and operator binding (Brenowitz et al., 1991; Chen and Matthews, 1994). Even at elevated operator concentration (2 10 M), the binding curve measured for the -34-aa mutant does not appear to be stoichiometric (note the first three data points in the inset to Fig. 5B); thus, the subunit affinity in this deletion mutant is diminished significantly, and the apparent operator affinity measured (see below) is presumed to be significantly weaker than the intrinsic operator affinity for this protein.


Figure 3: Purity of mutant repressor proteins. Protein purity was examined by electrophoresis on a 10% SDS-polyacrylamide gel subsequently stained by Coomassie Blue. Two µg of protein were loaded to each lane. Lane 1, wild-type; lane 2, -34 aa; lane 3, R326K; lane 4, R326A; lane 5, R326E; lane 6, R326L.




Figure 5: Operator binding of mutant repressors. The fractional saturation ( R) for operator DNA binding was measured as the ratio of DNA retained on a nitrocellulose membrane at a specific protein concentration (expressed in M dimer) to DNA retained at saturating concentrations. The titrations were performed as described under ``Materials and Methods.'' The inset to each panel shows operator binding activity assayed under stoichiometric conditions using 10 M operator for wild-type and R326 substitution mutants and 2 10 M operator for -34 aa. The values shown for each data point are the average of three determinations; standard deviation at each point is shown as an error bar. A, wild-type; B, -34 aa; C, R326K; D, R326A; E, R326E; F, R326L.



Oligomeric State of Mutant Repressor Proteins

The oligomeric form of each protein in this series of mutant repressors was determined by Sephadex G-150 filtration at a concentration >0.5 10 M tetramer (Fig. 4 A). All mutant repressors with substitutions at Arg-326 were tetramer at this concentration, migrating at the same position as wild-type repressor; these data suggest that Arg-326 does not contribute significantly to tetramer assembly, despite the effects of substitution at this position on protein expression and stability (see below). Since dissociation of a modified repressor protein was found to occur in the nanomolar concentration range (Royer et al., 1990), we have employed gel retardation experiments to ascertain the oligomeric state of repressor at the low concentrations where DNA binding occurs. R3 protein, in which the carboxyl-terminal leucine heptad repeat region was replaced by the oligomerization domain of GCN4 to form a stable dimer (Alberti et al., 1993; Chen et al., 1994b), was selected as a marker for the dimeric form of the protein. As shown in Fig. 4 B, a complex of the same mobility is formed by the wild-type lac repressor at protein concentrations both above and below the previously measured range of dissociation (1 10 M and 5.0 10 M). The mobility of this complex is distinct from that for the presumably dimeric R3-operator species, and the complex is deduced to consist of tetramer-operator. All of the proteins with substitutions at Arg-326 form a complex with mobility identical with wild-type and are therefore identified as tetramer-operator complexes, while the -34-aa protein yields a complex with DNA in retardation analysis corresponding to a dimeric species (Fig. 4 B).


Figure 4: Evaluation of oligomeric state of mutant repressors. A, the molecular mass of the mutant repressors was evaluated by Sephadex G-150 column chromatography of purified proteins and ammonium sulfate precipitates where purification was not possible. The concentration of the purified proteins applied to the column was 0.2 mg/ml (1.3 10 M tetramer). The concentration of ammonium sulfate-precipitated proteins applied to the column was >5 mg/ml. Closed circles correspond to K of standard proteins and wild-type repressor with the following molecular masses (in daltons): ribonuclease A, 13,700; chymotrypsinogen A, 25,000; ovalbumin, 43,000; bovine serum albumin, 67,000; wild-type repressor, 150,000. Proteins evaluated were: A, -34 aa; B, -35 aa; C, -36 aa; D, -39 aa; E, R326K; F, R326A; G, R326E; H, R326L; I, R326W. Proteins in the eluate were identified by IPTG binding for purified proteins ( A, E, F, G, and H) or B2 monoclonal antibody reaction in ammonium sulfate precipitates for proteins that could not be purified and were undetectable by IPTG binding ( B, C, D, and I). B, gel retardation assays for wild-type and mutant proteins. The binding reactions were performed as described under ``Materials and Methods.'' The binding mixtures containing P-labeled 40-base pair operator (1 10 M) and proteins at the indicated concentrations were incubated and then subjected to electrophoresis on an 8% polyacrylamide gel; the radiolabeled bands were visualized by scanning the image generated using a phosphorimaging device. The concentrations of proteins employed were as follows: lane 1, DNA alone; lane 2, 1 10 M wild-type repressor; lane 3, 5 10 M wild-type repressor; lane 4, 1 10 M R3 protein; lane 5, 5 10 M R3 protein; lane 6, 2.5 10 M R326K; lane 7, 2.5 10 M R326A; lane 8, 2.5 10 M R326E; lane 9, 2.5 10 M R326L; lane 10, 1.4 10 M -34 aa.



The -34-aa, -35-aa, -36-aa, and -39-aa deletion mutant repressors were detected in gel filtration experiments at the position expected for dimer, near the elution volume of bovine serum albumin (67 kDa) (Fig. 4 A and ). The presence of protein at the dimer position for the -35-aa, -36-aa, and -39-aa deletion mutants was indicated by antibody analysis; whether these polypeptides are unfolded (and hence elute with a larger volume than anticipated for the monomer, i.e. close to dimer) or are actually folded and assembled was not possible to determine. However, the former interpretation is tempting based on the absence of detectable inducer binding capacity in these mutants. An effective boundary for activity, if not folding and assembly, is indicated by the dramatic change from functional dimer for the -34-aa mutant to no activity for the -35-aa mutant, corresponding to the deletion of Arg-326.

Operator Binding Properties

Operator binding capacity is a sensitive probe of structural perturbations in mutant lac repressor proteins, since this activity requires both proper tertiary folding and quaternary assembly at least to dimer in order to generate the optimal orientation of a pair of helix-turn-helix motifs for operator contact (Lehming et al., 1990; Kisters-Woike et al., 1991; Chang et al., 1993, 1994). The results of operator binding analyses are shown in Fig. 5 and summarized in . Compared with wild-type, mutant repressors substituted at Arg-326 showed a lower operator binding affinity (6-7-fold), while the -34-aa protein showed a dramatically decreased apparent operator binding affinity (400-fold). These results indicate that the structure required for operator binding was only slightly perturbed by substitution for Arg-326, even with a large apolar side chain (R326L), although protein expression levels of both R326E and R326L were significantly decreased compared to R326K and R326A. The oligomeric state of the repressors at the concentrations employed was identified by gel retardation as tetramer for the substitution mutants and dimer for -34 aa (Fig. 4 B). In addition, the binding isotherms are noncooperative (except for -34 aa, see below), a stoichiometry of 2 operators per tetramer is found, and the data can be fit by a simple equilibrium of dimer-operator binding. Although it cannot be discerned unequivocally whether operator influences a dimer-tetramer equilibrium ( i.e. promotes tetramer formation), there is no evidence for coupled equilibria in the Arg-326 substitution mutants. It should also be noted that neither of the conditions employed in these experiments (either for monitoring equilibrium constant or stoichiometry) would allow facile detection of any cooperativity in operator DNA binding.

In contrast to the Arg-326 substitution mutants, the -34-aa deletion mutant displays much lower apparent affinity, and the binding is not a simple equilibrium, presumably due to linkage of monomer-monomer assembly to dimer and operator binding equilibria (Brenowitz et al., 1991; Chen and Matthews, 1994). The larger shift in apparent operator affinity for -34 aa compared to other deletion dimers indicates that the dimer-monomer Kis significantly greater than the corresponding value for the well-characterized -11-aa dimer (8 10 M, Chen and Matthews (1994)). This lowered monomer-monomer affinity correlates to deletion of a single lysine from the KRK sequence; it should be noted again that deletion that includes RK in this unusual sequence results in inactive protein detected only by B2 monoclonal antibody reaction.

Inducer Binding Properties

Wild-type repressor binds IPTG noncooperatively at neutral pH with a Kof 1.1 10 M and binds cooperatively at pH 9.2 with 7-fold increased apparent Kand a Hill coefficient of 1.6 ( Fig. 6and ). This allosteric behavior indicates the importance of subunit communication in repressor function (Daly and Matthews, 1986; Matthews, 1987; Chakerian and Matthews, 1991; Chen and Matthews, 1992). R326K has the same inducer binding affinity as wild-type repressor at neutral pH, but exhibits a decreased shift in Kat pH 9.2 and elevated cooperativity. R326A and R326E have only slightly lower affinity for inducer at neutral pH and exhibit diminished pH shift and slightly lower cooperativity at pH 9.2 compared to wild-type protein. R326L has 4-fold diminished inducer affinity at neutral pH with no pH shift and diminished cooperativity at both pH 7.5 and 9.2; the apparent negative cooperativity in this mutant suggests that the presence of a bulky apolar group influences the allosteric change that accompanies inducer binding in this mutant. In contrast to other dimeric repressors examined (Lehming et al., 1988; Chen and Matthews, 1992), the -34 aa exhibits not only greatly diminished apparent operator affinity, but also about 4-fold decreased inducer affinity at neutral pH with almost no cooperative binding at elevated pH (Hill coefficient = 1.1). These effects on inducer binding are observed even at increased protein concentration (1 10 M, Fig. 6). These differences apparently do not derive from the presence of substantial monomeric protein, as gel filtration indicates the presence of dimer at the corresponding protein concentrations. This loss in function and decrease in communication between subunits compared with the -32-aa dimer (Chen and Matthews, 1992) may derive from disruption of KRK sequence, either by deletion of the carboxyl-terminal lysine residue or the proximity of the carboxyl terminus, since similar effects on pH shift and cooperativity were observed for some Arg-326 substitution mutants.


Figure 6: Fluorescence titration of IPTG binding to mutant repressors. The fractional saturation ( R) was measured as ratio of the change in fluorescence at a specific IPTG concentration to the total change in fluorescence at saturating IPTG concentrations. The titrations were carried out at pH 7.4 ( closed circle) and at pH 9.2 ( open circle). Protein concentrations were 1.5 10 M monomer for wild-type and R326 substitution mutants and 1.0 10 M monomer for -34 aa. The values shown for each data point are the average of three determinations; standard deviation at each point is shown as an error bar. A, wild-type; B, -34 aa; C, R326K; D, R326A; E, R326E; F, R326L.



Immunoblotting Assay

In the native wild-type repressor, the epitope recognized by B2 monoclonal antibody is buried in the tetramer so that reactivity can be detected only in the presence of low concentrations of detergents ( e.g. 0.02% SDS), which dissociate the tetrameric protein but maintain the monomer in a relatively native form (Hamada et al., 1973; Sams et al., 1985). At high concentrations of detergent, reaction of B2 monoclonal antibody with wild-type repressor is diminished, presumably due to unfolding of the monomer. Most dimeric repressors react with B2 monoclonal antibody partially in their native form and exhibit full reaction in the presence of 0.02% SDS (Chen and Matthews, 1992). In the absence of detergent, monomeric or dimeric protein with a large carboxyl-terminal deletion ( e.g. -32 aa) displays full reactivity with the antibody (Daly and Matthews, 1986; Chen and Matthews, 1992). Thus, the extent of exposure of the epitope between amino acids 280 and 328 (Sams et al., 1985) in the mutant repressors may reflect quaternary subunit association pattern and/or tertiary folding of the mutant proteins.

As shown in Fig. 7 and summarized in I, the wild-type repressor reacts with the antibody only in its dissociated form. R326K reacts with the antibody partially in its native form, indicating that some conformation change occurs when arginine is replaced by lysine at position 326. R326A reacts with the antibody in its native form more strongly than R326K, suggesting that substitution by alanine causes more serious conformational perturbation in this region than lysine. R326E, R326L, R326W, and all deletion mutants react fully with the antibody in their native forms, indicating that the epitope in these mutant repressors is fully exposed in their native forms. The reaction of these mutant repressors with B2 antibody is significantly diminished in the presence of 0.02% SDS, a result consistent with the possibility that monomers of these mutant proteins may be more unstable than wild-type monomer and unfold even at low concentrations of detergent. As demonstrated by Sephadex G-150 filtration (Fig. 4, ), the Arg-326 substitution mutants are tetrameric proteins at the concentrations employed for these experiments and when bound to DNA at low concentrations; however, their antibody reaction patterns differ from wild-type repressor. These differences indicate the importance of the guanidino side chain at position 326 in the formation of tertiary structure of the lac repressor that shields the B2 target epitope from reaction. Among the substitution mutants, R326E, R326L, and R326W have antibody reaction patterns similar to monomeric or dimeric proteins with large carboxyl-terminal deletions; thus, the substitution of Arg-326 with glutamate, leucine, or tryptophan results in conformational alterations in these tetrameric proteins that allow B2 monoclonal antibody to access fully its epitope even in their native forms.

Urea Denaturation Properties

Urea denaturation of dimeric mutants has indicated that the process monitored by fluorescence emission spectra is two-state and corresponds to dimer to unfolded monomer (Chen and Matthews, 1994). While detailed thermodynamic analysis of tetramer dissociation/unfolding is beyond the scope of this paper, we have examined the five purified mutant repressors, R326K, R326A, R326E, R326L, and -34 aa to compare their behavior to the wild-type protein as a monitor of relative stability. As shown in Fig. 8and summarized in I, wild-type repressor began unfolding at 2 M urea, with a midpoint of 2.5 M and a steep transition. R326K and R326A displayed similar behavior with unfolding beginning near 1.5 M urea, a midpoint near 1.8 M urea, and a transition less steep than the wild-type protein. R326E began unfolding below 1 M urea with a midpoint near 1.8 M and an even shallower transition, while R326L appeared to unfold at very low urea concentrations, midpoint 1.3 M, with a transition similar in shape to R326E. The -34-aa protein underwent unfolding beginning below 1 M urea and had a midpoint of 1.8 M with a transition slope similar to R326E and R326L. Although it is not possible to assign differences in stability to a specific transition, the relative order of stability can be deduced from these data. Presuming that intermediate states are largely unpopulated and that the transitions observed are reversible, the urea denaturation results suggest the following order of stability: wild-type > R326K, R326A > 34 aa, R326E > R326L. It is noteworthy that this order follows precisely the relative expression levels of these mutant lac repressors (Fig. 2). The lower stability of these proteins is also consistent with the results of antibody reaction that suggested unfolding (lower reactivity) of the monomeric structure at low detergent concentrations.


Figure 8: Urea denaturation of mutant repressors. Denaturing reactions were performed by incubation of each protein at a concentration of 10 µg/ml (2.7 10 M monomer) monomer with the indicated concentrations of urea at room temperature for 2-3 h. The denatured samples were examined by fluorescence intensity measurements with excitation at 285 nm and emission at 354 nm. Different symbols in each panel indicate independent triplicate measurements for each protein. A, wild-type protein; B, -34 aa; C, R326K; D, R326A; E, R326E; F, R326L.



Trypsin Digestion

Under mild conditions, the amino-terminal 59 amino acids of wild-type lac repressor can be removed by trypsin digestion, and the resulting core protein remains relatively trypsin-resistant (Platt et al., 1973; Matthews, 1979). The sensitivity of mutant repressors to trypsin may provide information regarding alterations in protein conformation (Chang et al., 1994). As shown in Fig. 9, with mild treatment by trypsin, wild-type repressor was digested primarily into core protein and amino terminus (which is not visible on the gel) with a small amount of intact protein remaining. The digestion of the Arg-326 substitution mutants and -34-aa deletion mutant yielded similar behavior overall, with primarily digestion to the core domain, although densitometric analysis of gels indicated that the amount of protein recovered after digestion was less for these proteins. Thus, the protein susceptibility to digestion appeared to increase for these mutants. These results are consistent with other data presented that indicate that the guanidino side chain at position 326 plays a role in maintaining the tertiary structure of wild-type lac repressor and that alteration at this site renders the protein less stable.


Figure 9: Trypsin digestion of mutant repressors. The reactions were performed at a protein concentration of 0.25 mg/ml (6.7 10 M monomer) in 0.12 M KP buffer containing 0.3 mM DTT, 5% glucose, and 2% trypsin at room temperature for 30 min. The digested proteins were examined by electrophoresis on a 10% SDS-polyacrylamide gel subsequently stained by silver staining reagent. One µg of protein was loaded to each well. Odd lanes are protein without trypsin exposure, and even lanes are proteins digested with trypsin as described. Lanes 1 and 2, wild-type repressor; lanes 3 and 4, -34 aa; lanes 5 and 6, R326K; lanes 7 and 8, R326A; lanes 9 and 10, R326E; lanes 11 and 12, R326L.




CONCLUSION

Characterization of mutant proteins produced by C-terminal deletion or substitution for Arg-326 suggests that the unique KRK sequence in the carboxyl terminus may play an important role in the structure and function of lac repressor protein. The -34-aa mutant with the carboxyl-proximal lysine missing from the KRK sequence exhibits lower affinity for both operator and inducer and lower protein stability than other dimeric proteins studied previously (Brenowitz et al., 1991; Chen and Matthews, 1992, 1994). The -35-aa mutant missing the RK sequence, as well as -36 aa and -39 aa, for which the entire KRK was deleted, yielded inactive polypeptides detected only by monoclonal antibody for lac repressor. All deletion mutant repressors reacted fully with B2 monoclonal antibody in the absence of detergent, indicating the B2 epitope in these proteins is fully exposed. The significant difference between -34 aa and -35 aa coupled with results of in vivo phenotypic analysis (Kleina and Miller, 1990) suggested initially that Arg-326 might be important to the structure and function of lac repressor. This observation was further confirmed by characterization of proteins with single amino acid substitutions for Arg-326. Dramatic decreases in steady-state protein expression level occurred when a negatively charged moiety (glutamate) or a long chain apolar group (leucine) was employed to substitute for the guanidino side chain at position 326. Moreover, remarkably low protein expression and complete loss of function were observed when a bulky apolar side chain (tryptophan) was introduced at position 326. Immunological reaction with B2 monoclonal antibody and trypsin digestion provide different assessments of the conformation of the mutant repressors. Substitution at Arg-326 causes the exposure of the B2 antibody target epitope, which is buried within the tetrameric structure of wild-type repressor and leads to partial (R326K) or full (R326A, R326E, R326L, and R326W) reaction with the antibody in the native form. Urea denaturation data also indicate that the substitution and deletion mutants have decreased stability compared to the wild-type protein. Moreover, all substitutions at Arg-326 result in a 6-fold decrease in operator binding affinity, and most of these substitutions also diminish allostery associated with inducer binding. In the cases of large apolar chains, substitution at Arg-326 leads to obviously lower affinity and cooperativity for inducer binding (R326L) or even complete loss of function (R326W). Consistent with these data, in the recently reported structure of the purine repressor (Schumacher et al., 1994), which has significant amino acid homology to the lac repressor protein (Schumacher et al., 1993), the residue homologous in the primary structure to Arg-326 forms multiple hydrogen bonds that would contribute to the stability of the tertiary fold of this protein. In addition, this residue is positioned so that it might exert influence on subunit interactions and affect the conformational change in the monomer. In composite, the results reported here suggest that the side chain of arginine at position 326 plays an important role in the formation of the proper tertiary structure necessary to generate wild-type parameters for inducer and operator binding and for protein stability.

  
Table: Summary of protein purity and oligomeric state of mutant repressors


  
Table: Binding affinities of mutant repressors to operator and inducer


  
Table: Immunological reaction and urea denaturation analysis of mutant repressors



FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM22441 and Robert A. Welch Foundation Grant C-576 and employed the facilities of the Keck Center for Computational Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 713-527-4015; Fax: 713-285-5154.

The abbreviations used are: aa, amino acid; IPTG, isopropyl-1-thio--D-galactoside; DTT, dithiothreitol; KP, potassium phosphate buffer; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with Tween 20; ds, double-stranded.


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