(Received for publication, January 28, 1997, and in revised form, May 14, 1997)
From the Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251
To examine the monomer-monomer subunit interface
in the lac repressor, a mutation that generates dimeric
protein (deletion of C-terminal amino acids to disrupt the dimer-dimer
interface) has been combined with amino acid substitutions that alter
the monomer-monomer interface (substitution at Lys84 or
Tyr282). Dimeric proteins with significantly increased
stability to urea denaturation were formed by the introduction of the
apolar amino acids Ala or Leu in lieu of Lys84 in concert
with the deletion of 11 C-terminal amino acids. K84A/11 deletion
protein retained wild-type affinity for operator DNA, while K84L/
11
deletion protein displayed operator affinity similar to its parent
tetramer. To assess further the influence of monomer-monomer interface
stability on assembly and DNA binding, triple mutants were generated
with Y282D, an alteration that disrupts assembly completely in the
wild-type background. The triple mutants were dimeric, but they
exhibited diminished dimer stability to urea denaturation and decreased
operator affinity compared with the double mutations. These results
demonstrate directly the stabilizing influence of apolar substitution
at position 84 on the monomer-monomer interface.
High affinity binding of repressor to operator sequences within the lac operon inhibits transcription of mRNA for the lactose metabolic genes (1). A conformational change in response to binding inducer sugars, including a metabolite of lactose that serves as the in vivo inducer, results in diminished affinity for the operator sites and a consequent increase in lac mRNA transcription (1-3). Each monomer of the homotetrameric lac repressor (4, 5) is composed of 360 amino acids (6-9). The repressor contains two operator binding sites and four inducer binding sites (2, 10-15) and is divided into two domains, one involved primarily in DNA binding (amino acids ~1-59) and the other in assembly and sugar binding (amino acids ~60-360) (14-29). A hinge region, susceptible to protease digestion (19), connects these two domains and not only provides a physical connection for potential communication between the two ligand binding sites but also is involved directly in DNA binding (15). The binding functions of the protein are not only linked through the monomer structure but also are determined by the oligomeric and conformational states of the protein (14, 15, 18, 19, 21, 30-32). Monomeric mutant repressors generated by mutations at Tyr282 do not bind operator DNA (20, 33, 34), while dimeric mutant proteins are unable to form bidentate complexes with DNA (25, 26).
The recently solved x-ray crystallographic structures of the repressor
core domain (amino acids 59-360) and intact protein (14, 15) and
experimental results on a variety of mutant proteins (20, 28, 31, 32,
34, 35) demonstrate that monomer-monomer association to dimers involves
multiple side chain interactions across both N- and C-subdomains within
the core region (Fig. 1). These amino acids are spread
over a significant portion of the primary sequence of the repressor
protein. Lys84 and Tyr282 are each located in
this interface and are positioned to make major energetic contributions
to the association (14, 15, 35). However, the specific effects of side
chain alterations along this interface on the stability of assembly
have not been explored previously.
In contrast to the monomer-monomer interface, the assembly of dimers to tetramer involves a short segment at the C terminus of the protein that forms a four-helix bundle (14, 15). Mutations or deletions in the C-terminal region have demonstrated directly the requirement for the leucine heptad repeat motif found in this region for dimer-dimer assembly (25-27, 29). The importance of oligomerization to operator binding has been studied using the dimeric products of both missense and deletion mutations in the heptad repeat region (25, 27, 29, 36, 37). The observed operator binding affinity of these dimeric proteins was 10-100-fold lower than that for wild-type protein due to a linked equilibrium between monomer association to dimer and dimer-operator binding (36, 38). Although the assembled dimer presumably presents the same face to the operator DNA sequence as the tetramer (14, 15), the dimeric mutants dissociate more readily into monomers in the absence of the C-terminal coiled-coiled region, and the monomer binds with only very low affinity to DNA (20, 33, 34). Thus, the apparent Kd does not reflect the intrinsic DNA binding capacity of dimer species for which the dimer-dimer interface is destabilized (36, 38). In contrast to mutations that eliminate interaction at the C terminus, a construct in which the C terminus of the lac repressor was replaced with the GCN4 sequence that generates a very stable parallel coiled-coil structure (26) exhibited higher stability under denaturing conditions and wild-type DNA binding affinity, as would be anticipated for a stabilized dimer (30, 38).
The repressor protein has been found to contain discrete regions that contribute to the different properties of the protein, reflected in the structure of the protein (14, 15, 39). Because DNA binding requires the dimeric structure and bidentate loop formation involving two operator sites requires tetramer, stable assembly of the repressor is crucial to its regulatory function. However, the unique influence of alterations in the monomer-monomer interface has been difficult to ascertain in the context of the tetrameric protein. To explore specifically the structural and functional effects of changes in the monomer-monomer interface, we have constructed combinatorial dimeric mutants with specific substitutions in the monomer-monomer interface.
Plasmid pJC-1 was used as the
expression vector (29). Escherichia coli 71-18 (supE thi
(lac-proAB)
F
[proAB+lacIq
lacZ
M15]) was used for DNA isolation, and E. coli
TB-1 (ara,
(lac-pro), StrA,
thi,
80dlacZ
M15, r
,
m+) was used for expression of proteins.
Double-stranded DNA derivatives of pJC-1 encoding repressor mutants either at Lys84 or with the 11-amino acid deletion were isolated using the Promega WizardTM kit. The purified plasmid DNAs were cut with NarI and HindIII restriction enzymes. Appropriate bands were excised from agarose gels, and the DNA was isolated and used in ligation. The circularized derivative plasmid DNA was isolated and used directly in double-stranded sequencing following the protocol of the Sequenase® version 2.0 DNA sequencing kit from U. S. Biochemical Corp. Complete sequencing was carried out to ensure mutation only at selected sites.
Protein Expression and PurificationCells transformed with specific plasmids were grown in 2YT medium (16 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCl, pH 7.6) in a shaking incubator at 37 °C overnight. The cells were pelleted, resuspended in breaking buffer (0.2 M Tris-HCl, pH 7.6, 0.2 M KCl, 0.01 M magnesium acetate, 0.3 mM dithiothreitol, 5% glucose) containing 0.1 mg/ml phenylmethylsulfonyl fluoride (a protease inhibitor) and 250 µg/ml lysozyme at a volume 3 times the cell weight, and frozen. The cells were thawed slowly with the addition of 1-2 ml of 10 mg/ml phenylmethylsulfonyl fluoride and ~7 µl of DNase (10 mg/ml). Following centrifugation, the supernatant was mixed with ammonium sulfate (23.1 g/100-ml volume), and the resuspended precipitate was dialyzed overnight against 0.08 M potassium phosphate buffer (pH 7.5) at 4 °C. The protein solution was centrifuged, and the supernatant was applied to a phosphocellulose column (2.5 × 25 cm) preequilibrated with 0.08 M potassium phosphate (pH 7.5) buffer containing 0.3 mM dithiothreitol and 5% glucose. The column was eluted with the same buffer followed by 0.12 M potassium phosphate buffer. Protein purity (>90% for all proteins) was assessed using SDS-polyacrylamide gels.
Antibody Binding under Denaturing ConditionsProtein (2, 10, and 50 µg) in a total volume of 100 µl was reacted with SDS (0%, 0.01%, and 0.02%) at room temperature for 15 min. The solutions were then filtered onto nitrocellulose paper (presoaked with phosphate-buffered saline), and the proteins were reacted with B-2 antibody as described previously (40). The only difference was that the secondary antibody reaction employed a chemiluminescence kit from Amersham Life Science, with the resulting reactions visualized through exposure to x-ray film.
Trypsin DigestionEach protein (0.25 mg/ml in 0.12 M potassium phosphate buffer, pH 7.5) was incubated with 2% (w/w) trypsin (in 0.001 N HCl) for 30 min at room temperature. For control reactions, 0.001 N HCl was added in lieu of trypsin. The reactions were stopped by the addition of 2% (w/v) phenylmethylsulfonyl fluoride (in 100% ethanol). The digested proteins were then run on a 10% SDS-polyacrylamide gel, and protein products were visualized using Coomassie Blue staining.
Analytical Ultracentrifugation of ProteinsDouble sector cells were used for sedimentation velocity studies, and six channel cells were used for equilibrium analysis using a Beckman XL-A analytical ultracentrifuge. In each case, a minimum of three protein concentrations was studied for each mutant. For velocity sedimentation, ~400 µl of protein (0.06-0.6 mg/ml) was loaded, the samples were centrifuged at a maximum speed of 42,000 rpm, and the cells were scanned at 280 nm. The data were analyzed using a software package provided by Beckman, and the sedimentation coefficient was determined at each concentration point using two methods: 1) the second moment algorithm (41, 42) and 2) the time derivative method (43-45). In both cases, the Svedberg values were corrected for solvent effects and viscosity (46).
For equilibrium sedimentation, the cells were loaded with ~100-110 µl of protein at varying concentrations. Samples were centrifuged at 8,000, 10,000, and 16,000 rpm, allowing the sample to reach equilibrium before increasing the speed. Using the data analysis software, plots of ln(Cr) versus r2 were fit using a least squares algorithm, where Cr is the concentration at a specific radial position, r.
Operator Binding AssayA modified procedure of Riggs
et al. (47) and Wong & Lohman (48) was used. Protein at
various dilutions was mixed with 32P-end-labeled 40-base
pair operator DNA (~1012 M). After a short
incubation, reactions were filtered on nitrocellulose using a 96-well
plate, and the nitrocellulose was dried and exposed to a Fuji
bio-imaging for visualization. The plate was read on a Fuji BAS1000
bio-imaging analyzer, and the radioactivity was quantitated using the
program MacBas version 2.0 on a Macintosh computer. Free operator DNA
bound to nitrocellulose was used as background in lieu of
IPTG1 additions due to the very slow
inducer association rate for the mutants in this study. After
subtracting background intensity, the values were expressed as
fractional saturation (R) and plotted against log protein
concentration. The data were fit by nonlinear least squares analysis
using the equation,
![]() |
(Eq. 1) |
From previously reported data, proteins
containing Lys84 substitutions had Kd
values for IPTG binding similar to wild-type (Kd
~106 M) but bound to inducer much more
slowly than wild type (35), precluding use of the standard IPTG binding
assay. A new procedure was developed using individual reactions of 1.5 ml of protein solution at 1 × 10
7 M
concentration (tetramer) with varying concentrations of IPTG. Following
overnight incubation at 4 °C, total fluorescence was measured using
an SLM 8100 spectrofluorometer. The excitation wavelength was 285 nm
directed through a filter (7-54, Corning), and emission fluorescence
was measured using a 350-nm cut-off filter (O-52, Corning).
Fluorescence intensity was converted to fractional saturation
(R), and the data were fit by nonlinear least squares
analysis using the equation,
![]() |
(Eq. 2) |
Stock solutions of either urea (10 or 7 M) or guanidine hydrochloride (6.5 or 4 M) were made fresh daily in 0.1 M potassium sulfate, 0.01 M Tris-HCl, pH 7.5. The concentrations of the solutions were confirmed with a refractometer. Protein (10 µg/ml) was incubated for a minimum of 2 h to reach equilibrium. Using an SLM model AB-2 spectrofluorometer, the solutions were excited at 285 nm, the intensity at 340 nm was utilized for the data analysis, and the intensity was converted to fraction unfolded (Fu) using the equation,
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
Using
restriction enzymes sites that flank the selected regions, the 11
deletion coding sequence was combined with the K84A, K84L, K84A/Y282D,
and K84L/Y282D coding sequences to generate double and triple
mutations. The entire coding region for these combinatorial mutants was
sequenced to verify the desired changes and to eliminate the
possibility of other alterations. Purification of the repressor mutants
utilized the affinity of the DNA binding domain for phosphocellulose.
SDS-polyacrylamide gels and Western blotting indicate high purity of
the purified mutant proteins (Fig. 2). The yield for
triple mutant proteins was lower than that for double mutant proteins,
the first suggestion that the triple mutant proteins were less
stable.
Antibody Binding
Antibody binding under denaturing conditions
allows an indirect measurement of conformation and stability of a
mutant protein as compared with wild-type repressor (40). Previous
experiments have shown that native wild-type protein will react with
B-2 monoclonal antibody only in the presence of low concentrations of
SDS, while monomeric repressor protein is reactive with this monoclonal
antibody in the absence of SDS (33, 40). The results for dimeric
proteins examined previously tend to fall somewhere in between these
two extremes (29, 30, 37). The double mutants in this study reacted
more strongly in the absence of SDS and demonstrated only partial
reactivity in the presence of SDS (Fig. 3). These
results indicate that the mutations at residue 84 impart conformational changes that expose the antibody epitope located in the C terminus in
the dimer and make the epitope less available in the presence of SDS.
The differences seen between the K84A/Y282D/11 deletion and the
K84L/Y282D/
11 deletion suggest that the character of the side chain
at position 84 plays an important role in the conformation of the dimer
that determines epitope availability. The K84A/Y282D/
11 deletion is
more reactive to antibody, regardless of SDS, as compared with the
K84L/Y282D/
11 deletion. This effect may be a consequence of the
differential structural rigidity imparted by leucine versus alanine substitution at position 84.
Trypsin Digestion
Mild proteolysis results in the cleavage of
the DNA binding domain (amino acids 1-59) from the core domain of the
lac repressor (19, 52). The double and triple mutants, as
well as wild type, were reacted with 2% (w/w) trypsin (Fig.
4). The results for the double and triple mutants
clearly indicate their higher susceptibility to trypsin digestion
compared with wild-type protein, which is digested to a single species
under identical conditions. The digestion patterns for the double
mutants and the K84L/Y282D/11 deletion are similar, while the pattern
of protein fragments for the triple mutant K84A/Y282D/
11 deletion
appears to have a larger fraction of a faster migrating species
(lane 8). This difference may be a consequence of lower
oligomer stability in this particular mutant compared with the
corresponding Leu substitution. The generally increased susceptibility
of the double and triple mutants to protease digestion may derive from
distortions in the hinge region and N-subdomain; these distortions may
be generated by more rigid N-subdomain association that also results in
increased monomer-monomer stability (see below).
Analytical Ultracentrifugation of Wild-type and Mutant Proteins
Sedimentation velocity experiments were performed to
determine the Svedberg value, s, for the mutant and
wild-type proteins. For comparison, the value of s was
determined and compared using two separate methods: 1) the second
moment or boundary spreading method (41), which calculates s
(42) based on the movement of the boundary position; and 2) the time
derivative method, which uses a time derivative of the concentration at
each radial position to calculate an apparent sedimentation coefficient
distribution pattern (g(s*)) (43-45). In both
cases, only those scans that had generated a depleted area between the
meniscus and the boundary were used. In each case, values for
s were corrected for solvent density and specific volume
effects. The resulting s20,w values are
shown in Table I. The corrected s values for
the wild-type lac repressor range from 6.6 × 1013 (second moment) to 7.6 × 10
13
(time derivative), in agreement with previously reported values of
~7.0 × 10
13 (53). The values for the mutants,
which are all smaller than wild-type values, indicate their dimeric
state. The slight differences in the s values among the
mutants probably arise from either their intrinsic association
properties or differences in packing of the structure.
|
While the sedimentation velocity results established that the mutants differ significantly in shape and size from the wild-type protein, we wished to establish the molecular weights directly by equilibrium sedimentation. The derived molecular weight values for the double and triple mutant proteins as well as wild type can be seen in Table I. These molecular weight determinations were obtained from a global fit of the data taken at varying sedimentation speeds and/or varying protein concentrations. These data provide direct evidence that the double and triple mutants exist as dimeric species.
Inducer BindingThe Kd for wild-type
protein at pH 7.5 is ~2 × 106 M,
while at pH 9.2 the affinity for IPTG decreases ~8-fold (54, 55). For
the dimeric C-terminal deletion mutants, the IPTG binding properties
were very similar to wild-type protein (29). For the parent tetrameric
mutant proteins (K84A, K84L, K84A/Y282D, and K84L/Y282D), IPTG binding
affinity was similar to that of wild-type protein at neutral pH, but
with no significant decrease in affinity at elevated pH levels.
However, because the rate of inducer binding for the Lys84
mutants was diminished more than 100-fold, a new procedure was developed to examine IPTG binding by fluorescence for the double and
triple mutants in this study. Individual reaction mixtures at a protein
concentration (tetramer) of 1 × 10
7 M
containing differing amounts of IPTG were incubated overnight at
4 °C. To ensure the viability of this procedure, wild-type protein
was analyzed alongside the other mutant proteins. Table II summarizes the binding affinity for this set of
proteins, and the individual binding curves can be seen in Fig.
5. Although affinity measurements for the wild type are
consistent with previous data, the Hill coefficients for wild-type
protein did not correspond to previously determined values, perhaps due
to the long incubation times; these values are not reported. The
similarity of the derived binding affinities of the double and triple
mutants at neutral pH to wild type indicates that disruption of either
or both subunit interfaces does not markedly affect sugar binding
affinity, consistent with previous studies (29, 34). In the double
mutants examined, affinity for the inducer is decreased ~2-fold at pH
9.2; the added stability provided by apolar substitution in the
N-subdomain subunit interface (see Fig. 1) may preclude the
conformational changes with pH values that generate diminished affinity
for the inducer, as observed for the parent tetrameric protein. In
contrast, at elevated pH the triple mutants exhibit lower affinity for
IPTG, and the pH-associated shift is greater than wild-type or the
parent tetramer for the K84A/Y282D/-11 deletion protein. Disruption of the C-subdomain interface coupled with apolar substitution at position
84 may provide the requisite flexibility for pH-induced structural
shifts. The effect of pH on the inducer binding behavior of the double
and triple mutants correlates loosely with the stability of the
monomer-monomer interface (see below), with lower stability corresponding to greater pH response.
|
Operator Binding
The binding of the wild-type lac
repressor to a 40-base pair operator DNA sequence in 0.1 M
K2SO4 buffer is ~2 × 1010
M (38). The operator binding affinities for dimeric
proteins range from equivalent to 10-100-fold lower than wild-type
protein (Table II) (29, 38). The individual binding curves for the proteins with double and triple mutations can be seen in Fig. 6. The double mutant K84A/
11 deletion binds operator
comparably with the wild-type protein, as did its tetrameric parent
K84A (35); these data indicate that this dimer does not dissociate to
monomer under conditions used for DNA binding. The wild-type affinity
for the operator occurs because interactions of the apolar amino acid
alanine presumably provide energetic compensation for the loss of
monomer-monomer stability normally provided by the C terminus.
Given the change to an apolar amino acid at position 84 for the
K84L/11 deletion, this protein might also be expected to bind DNA
with wild-type affinity. However, this mutant binds operator DNA with
~10-fold lower affinity than wild type but with an affinity similar
to that of its tetrameric parent, K84L (35). The lower affinity for
both tetrameric and dimeric mutant proteins with this substitution may
derive from the effect of the bulky, apolar leucine side chain at the
subunit interface, resulting in alteration of the orientation and/or
alignment of the N-terminal DNA-binding domains crucial for optimal DNA
binding (15). However, dissociation to monomer does not appear to
contribute substantially to the diminished DNA affinity for this
mutant, since the K84L tetramer and K84L/
11 deletion dimer affinities
for the operator are similar.
The triple mutant proteins, K84A/Y282D/11 deletion and
K84L/Y282D/
11 deletion, had an observed Kd
~30-60-fold lower than either wild-type protein or K84A/
11
deletion mutant protein. While the apolar K84A or K84L substitution can
compensate for the
11 deletion and maintain a stable dimeric state in
the double mutants, further introduction of the Y282D mutation in the
C-terminal subdomain disrupts the monomer-monomer interface
significantly, generating diminished operator binding. Although these
proteins form dimer in the concentration range employed for
sedimentation experiments, the diminished stability of the
monomer-monomer interface may result in dissociation to monomer at the
protein concentrations necessary to measure operator affinity.
The equilibrium constant, and
consequently the free energy change, between the assembled/folded and
unfolded states can be obtained through the use of denaturants such as
urea, guanidine hydrochloride, or temperature. Since the denaturation
experimental data define an equilibrium constant, for analytical
purposes the reaction must reach equilibrium, and further the reaction
must be reversible (51). Using methods previously employed for
lac repressor mutant proteins (37, 38), denaturation
reactions in urea were allowed to reach equilibrium at room temperature for 2-24 h before fluorescence intensity measurements were taken, and
it was found that the shorter time allowed equilibrium to be achieved.
Reversibility was established for urea denaturation of the triple
mutants K84A/Y282D/11 deletion and K84L/Y282D/
11 deletion and the
11 deletion protein (38). However, denaturation of the mutant
proteins K84A/
11 deletion and K84L/
11 deletion was incomplete even
at the maximum urea concentrations obtainable (Fig. 7).
To explore this phenomenon further, the stronger denaturant guanidine
hydrochloride was employed, despite the fact that unfolding of the
triple mutants and
11 deletion mutant occurred at very low
concentrations in this denaturant (Fig. 7). Unfortunately, the
unfolding and refolding of the more stable mutants did not follow the
same pathway in guanidine hydrochloride, precluding assessment of the
equilibrium constant and therefore the free energy of denaturation.
Lowering the pH to 6.5 in an attempt to destabilize the double mutant
proteins did not alter their behavior, and using lower pH values
resulted in the precipitation of wild-type protein.
The calculated values for G at zero denaturant
concentration (
G0) and m, which is
a measure of the dependence of
G on denaturant concentration (51), can be found in Table III for the
urea data. The data for the K84A/
11 deletion in urea were derived
from the partial unfolding observed for this protein; no value could be estimated for the K84L/
11 deletion, indicating a stability of >21
kcal/mol, presuming a similar m value for this protein. The individual fits for fractional unfolding versus denaturant
can be seen in Fig. 8. From the calculated
G0 values, the pattern of stability of the
mutant proteins, ranked from the most to the least stable, is as
follows: K84L/
11 deletion > K84A/
11 deletion >
11
deletion > K84L/Y282D/
11 deletion > K84A/Y282D/
11
deletion. Not only does the apolar substitution at residue 84 strengthen the dimeric structure, but these changes generate sufficient
stability to compensate for Y282D mutation, an alteration that normally
disrupts the monomer-monomer interface completely to generate monomer
in the wild-type background. The denaturation data provide convincing
evidence for the stabilizing effect of apolar substitution at residue
84 and underscore the key contribution of the monomer-monomer interface
to overall protein stability. Attempts to fit the DNA binding data to a
linked equilibrium for dimer association and dimer binding to the
operator resulted in intrinsic affinities for operator DNA greater than
the wild-type value. These analyses assume that the free energy for
monomer unfolding corresponds with that of the Y282D protein, an
assumption that may not apply for monomers with apolar substitutions at
position 84.
|
Conclusion
The goal of this effort was to combine mutations
that affect monomer-monomer interactions (residues 84 and 282) with
those that affect dimer-dimer interactions (C-terminal deletions) to examine independently the influence of substitutions in the
monomer-monomer interface on protein structure and function. All mutant
proteins generated were dimeric, as expected due to the requirement of the leucine heptad repeat region for tetramerization (14, 15, 24-27,
29, 56, 57). The ability of the double mutants to resist unfolding in
high concentrations of urea and their high affinity operator binding
provide evidence for an extremely stable dimer of lac
repressor. Significantly increased stability associated with the
replacement of polar amino acids involved in a salt bridge network by
hydrophobic residues has also been observed in the Arc repressor
protein (58). These results combine to suggest that properly positioned
apolar residues may provide a significant stabilizing force for
assembly and consequently for functional properties associated with the
oligomeric structure. However, the specific binding characteristics
generated may depend on whether the altered monomer can fold into a
wild-type conformation, as illustrated by the differences observed
between the K84A/11 deletion and K84L/
11 deletion proteins.
The operator binding affinities for the double mutants were similar to
those reported for the parent Lys84 lac
repressor mutants. The replacement of Lys84 with the apolar
amino acid Ala imparts to the structure both stability and preservation
of wild-type DNA binding affinity in the 11 deletion background; the
dissociation of dimer to monomer appears to be prevented by this apolar
substitution, and the intrinsic Kd for DNA binding
is observed. Complete disruption of the C-subdomain subunit interface
of K84A/
11 deletion protein by substitution at Tyr282 is
required to diminish the high affinity operator binding and dimer
stability of this protein, although even this disruption is
insufficient to preclude assembly completely. Although monomer-monomer stability appears to be increased when Lys84 is replaced
with the more bulky and apolar Leu, this substitution may alter the
orientation of DNA binding domains, resulting in lower affinity for
operator DNA in the parent tetramer and in the dimer.
Both triple mutants are dimeric at protein concentrations in the micromolar range but apparently dissociate at lower concentrations due to the effects of Asp at position 282, a substitution that generates monomeric protein in the wild-type background (20, 28, 31, 34). This dissociation contributes at least partially to diminished affinity of the triple mutants for operator DNA. Nonetheless, the assembly of the triple mutants attests to the exceptional stabilizing influence of apolar residues at position 84. The amino acid changes in these combinatorial mutants result in relatively little influence on the inducer binding affinities at neutral pH. However, binding characteristics are altered at elevated pH, an effect that may be linked to decreased rates of inducer binding associated with substitution at position 84 (35), to disrupted communication between monomers, or to altered flexibility in the structure. Further study will be required to address the origin of these effects.
Construction of these mutations has provided proteins in which the
monomer-monomer interface is either more stable than wild type or 11
deletion (K84A/
11 deletion; K84L/
11 deletion) or less stable than
wild type or
11 deletion (K84A/Y282D/
11 deletion; K84L/Y282D/
11
deletion). The differences between these two groups highlight the
importance of the monomer-monomer interface in the overall function of
this protein. These data underscore the pivotal role of the region
surrounding Lys84 in the core N-subdomain (Fig. 1) in
forming the monomer-monomer interface, consistent with the location of
this residue in the x-ray crystallographic structures (14, 15). Apolar
residues in this position provide compensatory energy for contacts lost from the monomer-monomer interface in the distant C-subdomain (Y282D
mutation). Results from the combination of these specific mutations not
only provide insight into the role of the monomer-monomer interface in
protein assembly but also highlight the generation of different
conformational states of the protein that have consequences for both
DNA binding and IPTG binding. This combinatorial approach allows
examination of the influence of specific interactions on the structure,
stability, and function of a protein and explores the ways in which
compensatory interactions may generate comparable functional
outcome.
We thank Dr. Jie Chen for assistance and advice on the protein denaturation studies and Dr. Mark Hargrove for help in data analysis and curve fitting.