From the
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
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
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.
[
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
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
The
operator binding activity of each purified repressor protein was
determined by titration using 1
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
K
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.
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, 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.
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.
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`
[ proAB
lac I
lacZ
M15]) was used as the host to prepare single- or
double-stranded DNA for sequencing. E. coli TB1 ( ara,
( lac-pro), StrA, thi,
80d lacZ
M15, 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).
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.
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,
K
is 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
M
P-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, K
is 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.
10
M
P-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
K
SO
, 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.
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.
is 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 K
and 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 K
at 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.
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.
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
-D-galactoside; DTT,
dithiothreitol; KP, potassium phosphate buffer; PBS, phosphate-buffered
saline; PBST, phosphate-buffered saline with Tween 20; ds,
double-stranded.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.