(Received for publication, March 14, 1997, and in revised form, May 5, 1997)
From the Protein Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565, Japan
Escherichia coli ribonuclease HI has a cavity within the hydrophobic core. Two core residues, Ala52 and Val74, resided at both ends of this cavity. We have constructed a series of single mutant proteins at Ala52, and double mutant proteins, in which Ala52 was replaced by Gly, Val, Ile, Leu, or Phe, and Val74 was replaced by Ala or Leu. All of these mutant proteins, except for A52W, A52R, and A52G/V74A, were overproduced and purified. Measurement of the thermal denaturations of the proteins at pH 3.2 by CD suggests that the cavity is large enough to accommodate three methyl or methylene groups without creating serious strains. A correlation was observed between the protein stability and the hydrophobicity of the substituted residue. As a result, a number of the mutant proteins were more stable than the wild-type protein. The stabilities of the mutant proteins with charged or extremely bulky residues at the cavity were lower than those expected from the hydrophobicities of the substituted residues, suggesting that considerable strains are created at the mutation sites in these mutant proteins. However, examination of the far- and near-UV CD spectra and the enzymatic activities suggest that all of the mutant proteins have structures similar to that of the wild-type protein. These results suggest that the cavity in the hydrophobic core of E. coli RNase HI is conformationally fairly stable. This may be the reason why the cavity-filling mutations effectively increase the thermal stability of this protein.
In the interiors of globular proteins, and especially in the protein cores, hydrophobic side chains are generally well packed. Since the formation of a protein core governs the entire folding process of a protein, and because general methods for designing amino acid sequences that fold into the desired core structures have not been established, efforts have been made to evaluate the role of core residues in the stability and function of natural proteins by site-directed mutagenesis (1-21), random mutagenesis (22-24), and theoretical approaches (25-29). The data thus far accumulated suggest that the following phenomena are characteristic to the core mutations. 1) Mutations do not completely abolish the protein functions, unless the hydrophobicity at the core is considerably changed. This means that the hydrophobicity is a sufficient criterion for the formation of a functional core. 2) Mutations to smaller hydrophobic residues usually create cavities, which are either vacant (9, 19, 21) or occupied by a water molecule (15). The protein stability decreases as the volume of such a cavity increases. In this case, the cost for the loss of a single methylene group has been reported to be 1.3 ± 0.5 kcal/mol (30). 3) Mutations to much larger hydrophobic residues or those to polar residues seriously affect the protein structures due to unfavorable interactions in the cores, and thereby almost completely abolish the protein functions. These mutations also dramatically destabilize the proteins.
Although the packing of the hydrophobic side chains is highly efficient in the interiors of globular proteins, it is not perfect, and cavities exist in virtually all large proteins, even within the protein cores (31-34). These cavities may be required to make the proteins highly functional, probably at the cost of reduced stability, because they serve to reduce the strains caused by dense packing of the hydrophobic side chains, and to provide conformational flexibility to proteins. Therefore, it seems likely that the tolerance of proteins to core mutations increases as the volume of the naturally existing cavity at the core increases.
Escherichia coli RNase HI,1 which hydrolyzes only the RNA strand of RNA/DNA hybrids, is a small globular protein with 155 amino acid residues. We have used this protein for studies of protein stability (14, 35-43) and folding (44, 45). This protein is suitable for these studies for the following reasons: (i) highly refined coordinates of this protein are available (46), (ii) an overproduction system for this protein is available (36), and (iii) this protein reversibly unfolds in a single cooperative fashion with thermal and chemical denaturations (38).
In the hydrophobic core of this protein, a cavity exists, which is not
occupied by water molecules. We have previously shown that
cavity-filling mutations, such as Val74 Leu and Ile,
enhanced the protein stability by roughly 1 kcal/mol in
G
(14). We also found by random mutagenesis experiments that the mutation
of Ala52
Val also enhanced the protein stability by 1.7 kcal/mol, probably due to the reduction in the cavity volume around the
mutation site (42). These results allowed us to propose that the
replacement of a hydrophobic residue facing the cavity with a bulkier
and more hydrophobic residue is a general method to increase protein stability. However, proteins are not always stabilized by this method.
Karpusas et al. (1) found two large cavities in T4 lysozyme
and designed two mutations (Leu133
Phe and
Ala129
Val) to fill them. Eijsink et al. (8)
introduced various mutations to fill the cavities in the neutral
protease from Bacillus stearothermophilus. None of these
mutations resulted in a significant increase in the protein stability.
It was shown for the T4 lysozyme mutants that the hydrophobic effects
gained by the mutation were canceled by the strains and van der Waals
contacts. Determination of the crystal structures of the RNase HI
mutants revealed that the conformation of the cavity was not seriously
altered by the mutation, except for the mutation site, and the cavity
volume around the mutation site was remarkably reduced (14). Therefore, it seems likely that the conformation of the cavity in the hydrophobic core of E. coli RNase HI is rather rigid and stable. The
difference in the conformational stability of the cavity may affect the
dependence of the protein stability on the cavity-filling mutation. To
examine whether E. coli RNase HI is tolerant to mutations in
which polar or extremely bulky side chains are introduced into the
cavity, we have constructed a series of single and double mutant
proteins at Ala52 and Val74, and have analyzed
their conformational stabilities and enzymatic activities.
Here we report that the size of the cavity in the hydrophobic core of
E. coli RNase HI is large enough to introduce three methyl
or methylene groups without causing significant steric hindrance. The
thermal stabilities of a series of the mutant proteins suggest that
this cavity is more effectively filled by the double mutations
(Ala52 Val and Val74
Leu) than by the
single mutation (Ala52
Ile). We also report that this
cavity seems to be conformationally fairly stable and the protein is
tolerant to mutations that seriously affect the hydrophobicity and the
packing of the cavity residues. Thus, the structure of this protein was
not severely damaged when the cavity residues were replaced by either
aromatic or charged residues, although these mutations greatly reduced
both the protein stability and the enzymatic activity.
The wild-type E. coli RNase HI protein (36) and the mutant protein A52V (42), in which Ala52 was replaced by Val, were prepared previously. Restriction enzymes and modifying enzymes for recombinant DNA technology were from Takara Shuzo Co., Ltd. Other chemicals were of reagent grade.
Cells and PlasmidsThe plasmids pJAL600 (36), pJAL52V (42),
and pJAL74L and pJAL74A (14) for the overproduction of the wild-type
protein, and the mutant proteins A52V, V74L, and V74A, respectively,
were constructed previously. These plasmids bear the wild-type or the mutant rnhA gene under the control of the bacteriophage
promoters PR and PL, the
cIts857 gene, and the bacteriophage fd transcription
terminator. Competent cells of E. coli
HB101(F
, hsd S20, recA13, ara-14, proA2, lac Y1,
gal K2, rps L20(str), xyl-5, mtl-1, sup E44, leuB6, thi-1) were
from Takara Shuzo Co., Ltd. E. coli HB101 transformants with
pJAL600 derivatives were grown in Luria-Bertani (LB) medium (47)
containing 100 mg/liter ampicillin at 30 °C.
Alteration of the rnhA gene was
carried out by site-directed mutagenesis using polymerase chain
reaction, as described previously (36). The DNA oligomers used as the
5- and 3
-mutagenic primers (30-40 bases long) were synthesized by
Sawady Technology Co., Ltd. These DNA oligomers were designed such that
the codon for Ala52 was changed from GCT to ATC for Ile,
CTG for Leu, TGT for Cys, ATG for Met, TTC for Phe, ACT for Thr, CAG
for Gln, GAA for Glu, CCG for Pro, TCT for Ser, AAC for Asn, GAT for
Asp, TAC for Tyr, GGT for Gly, CAT for His, AAA for Lys, TGG for Trp,
and CGT for Arg. For the construction of the single mutant proteins at
position 52, the wild-type rnhA gene in the plasmid pJAL600
was used as a template. For the construction of the double mutant
proteins at positions 52 and 74, the mutant rnhA gene in
either plasmid pJAL74A or pJAL74L was used as a template. The resultant
plasmids for the overproduction of the mutant proteins were designated as either pJAL52X or pJAL52X74X
, in which X and X
represent the amino
acid residues (one-letter notation) substituted for Ala52
and Val74, respectively. Likewise, the mutant proteins were
designated as either A52X or A52X/V74X
. All of the nucleotide
sequences of the mutant rnhA genes were confirmed by the
dideoxy chain termination method (48).
The mutant proteins were overproduced in E. coli HB101 cells harboring the pJAL600 plasmid derivatives by raising the cultivation temperature from 30 to 42 °C, and were purified to homogeneity, as described previously (36). The protein concentration was determined from the UV absorption, assuming that the mutant proteins obtained in this experiment have the same A2800.1% value of 2.0 as that of the wild-type protein (49). The production levels of the mutant proteins in the cells were estimated by subjecting whole cell lysates to SDS-polyacrylamide gel electrophoresis on a 15% polyacrylamide gel (50), followed by staining with Coomassie Brilliant Blue.
RNase H ActivityThe enzymatic activity was determined at 30 °C and pH 8.0 in the presence of 10 mM MgCl2, 50 mM NaCl, 1 mM 2-mercaptoethanol, and 100 µg/ml bovine serum albumin, by measuring the radioactivity of the acid-soluble digestion product from a 3H-labeled M13 RNA/DNA hybrid, as described previously (35). One unit of enzymatic activity is defined as the amount of enzyme producing 1 µmol of acid-soluble material/min. For the kinetic analyses, the substrate concentration was varied from 0.1 to 1.6 µM (nucleotide phosphate concentration). The hydrolysis of the substrate with the enzyme follows Michaelis-Menten kinetics, and the kinetic parameters, Km and Vmax, were determined from the Lineweaver-Burk plot.
Circular Dichroism SpectraThe CD spectra were measured on
a J-720 automatic spectropolarimeter (Japan Spectroscopic Co., Ltd.) at
25 °C in 10 mM glycine-HCl (pH 3.2). For the measurement
of the far-ultraviolet (UV) CD spectra (200-260 nm), the protein
concentration was approximately 0.2 mg/ml, and a cell with an optical
path length of 2 mm was used. For the measurement of the near-UV CD
spectra (250-320 nm), the protein concentration was 0.5-1.0 mg/ml and
a cell with an optical path length of 10 mm was used. The mean residue
ellipticity (, deg·cm2·dmol
1) was
calculated by using an average amino acid molecular weight of 110.
Thermal denaturation curves and the
temperature of the midpoint of the transition (Tm)
were determined at pH 3.2 by monitoring the change in the CD value at
220 nm, as described previously (38). Proteins were dissolved in 10 mM glycine-HCl (pH 3.2). The enthalpy change of unfolding
at the Tm (Hm) was calculated
by van't Hoff analysis. The difference in the free energy change of
unfolding between the mutant and wild-type proteins at the
Tm of the wild-type protein (
Gm) was estimated by the relationship given
by Becktel and Schellman (51),
Gm =
Tm·
Sm.
Tm is the change in Tm of a
mutant protein relative to that of the wild-type protein.
Sm is the entropy change of the wild-type protein
at the Tm. We used the
Sm
value of 0.304 kcal/(mol·K), which was previously determined at pH
3.0 (38), for the calculation of the
Gm values
at pH 3.2.
Ala52 and Val74,
which face the hydrophobic cavity of E. coli RNase HI, are
located in the I and
II helices, respectively (Fig. 1). In addition to these residues, Thr9,
Leu49, Leu56, Leu67,
Leu107, and Trp118 form the cavity. Of them,
Thr9 is located in the
A strand, Leu49 and
Leu56 are located in the
I helix, Leu67 is
located in the
D strand, Leu107 is located in the
IV
helix, and Trp118 is located in the
E strand. All of
these residues are almost fully buried inside the protein molecule. The
hydroxyl group of Thr9 does not face the cavity but forms
the hydrogen bond with the hydroxyl group of Thr69 (46).
For a comprehensive analysis of the effect of the mutation at the
cavity on the protein stability, we have constructed a series of single
mutant proteins, in which Ala52 is replaced by the 19 other
amino acid residues. Position 52 was chosen as the site for the
introduction of the series of mutations, because Ala52 is
the smallest residue among those forming the cavity and therefore it is
possible to introduce into the cavity a variety of side chains, that
greatly differ in size and hydrophobicity. In addition, we have
constructed 10 double mutant proteins, in which Ala52 is
replaced by Gly, Val, Leu, Ile, or Phe and Val74 is
replaced by Ala or Leu. These mutant proteins were constructed to
estimate the number of methylene groups that could be introduced into
the cavity without creating serious strains, and to examine whether
effects other than hydrophobicity, such as packing, at the cavity
contribute to the protein stability.
Overproduction and Purification
All of the single mutant
proteins, except for A52W and A52R, were overproduced and purified in
an amount sufficient for biochemical characterizations. The mutant
proteins A52W and A52R could not be purified, because of the extremely
low production levels in cells (data not shown). The mutations of
Ala52 Trp and Arg may dramatically destabilize the
protein and thereby increase the susceptibility to proteolytic
degradation, probably because these residues are too large to fill the
cavity without causing significant steric hindrance. The ionized group
in the Arg side chain may also contribute to altering the protein
conformation, because it is unlikely that an ionized group remains
alone without a hydrogen-bonding or ion-pair partner within the
hydrophobic core of a protein. In fact, the cellular production levels
of the mutant proteins A52D, A52E, A52K, A52Y, and A52F, in which Ala52 is replaced by ionic or aromatic residues, were
considerably lower than that of the wild-type protein (data not shown).
In contrast, the cellular production levels of the other mutant
proteins were similar to that of the wild-type protein. Consequently,
the yields of the mutant proteins A52D, A52E, A52K, A52Y, and A52F from
1-liter cultures were 3-10 mg, and those of the other mutant proteins
were 27-62 mg (Table I). Likewise, of the 10 double mutant proteins, only the mutant protein A52G/V74A could not be purified, because of the extremely low production level in cells (data
not shown). The simultaneous introduction of the two cavity-creating mutations probably dramatically destabilizes the protein.
|
The
far-UV CD spectra of all of the single mutant proteins are basically
the same as that of the wild-type protein (Fig.
2a). In contrast, the near-UV CD spectra of
these mutant proteins differed from one another. They were roughly
classified into three groups (types A-C), based on the CD values at
260-280 nm (Fig. 2b). The wild-type protein and the mutant
proteins A52C, A52P, and A52G gave the type A spectrum. In the type B
and type C spectra, the CD values at 260-280 nm increased as compared
with those in the type A spectrum. The extent of this increase is
relatively small for the type B spectrum and is relatively large for
the type C spectrum. The mutant proteins A52I, A52L, A52V, A52N, A52D,
A52Q, A52E, A52S, and A52T gave the type B spectrum. The mutant
proteins A52F, A52Y, A52M, A52H, and A52K gave the type C spectrum.
Thus, the extent of the increase in the CD values at 260-280 nm seems to be correlated with the volume of the side chain of the replaced residue. Nevertheless, the shape of the near-UV CD spectrum of the
protein, especially that around 290 nm, is not markedly changed by the
mutations. These results suggest that the mutations of Ala52 cause a local conformational change, but only to a
small extent, even when an aromatic or ionized group is introduced into
the cavity by the mutation.
The thermal denaturation curves of the wild-type and mutant proteins
are shown in Fig. 3. All of the mutant proteins
reversibly unfolded in a single cooperative manner with thermal
denaturation. The parameters characterizing the thermal denaturation of
the mutant proteins, which were determined based on the assumption that
these proteins unfold in a two-state mechanism, are summarized in Table
I. Of the 17 mutant proteins, those in which Ala52 is
replaced by more hydrophobic aliphatic or sulfur-containing residues
were more stable than the wild-type protein by 0.5-1.9 kcal/mol in
G. In contrast, the other mutant proteins were less stable than the wild-type protein by 0.5-5.9 kcal/mol. We have analyzed the thermal stabilities of these mutant proteins at pH 5.5 in
the presence of 1 M guanidine hydrochloride, conditions under which the wild-type protein reversibly unfolds (38). However, except for the apparent Tm values, the thermodynamic parameters could not be determined, because the thermal denaturations of all mutant proteins were not reversible under these conditions. The
Tm values of the mutant proteins relative to that of
the wild-type protein (
Tm) at pH 5.5 were
comparable with those determined at pH 3.2, except for those of the
mutant proteins A52D and A52E (data not shown). The stabilities of
these mutant proteins relative to that of the wild-type protein at pH 5.5 were much lower than those at pH 3.2, probably because Asp and
Glu are not ionized at pH 3.2, but are at least partially ionized
at pH 5.5. Because the mutant proteins unfold irreversibly at pH 5.5, only the thermodynamic values at pH 3.2, at which all the mutant
proteins unfold reversibly, are discussed in this report.
The enzymatic activities of the mutant proteins varied from 0.1 to 112% of that of the wild-type protein (Table I). To determine whether the mutation affects the catalytic efficiency or substrate binding, the kinetic parameters were determined for some of the mutant proteins with poor enzymatic activities. The results are summarized in Table II. The Km values of these mutant proteins increased only by at most 2.5-fold, whereas their Vmax values decreased by 12-44-fold, as compared with those of the wild-type protein, suggesting that the mutation does not seriously affect substrate binding, but affects the catalytic efficiency. The conformations of the active-site residues may be altered by the mutations, so that the catalytic efficiencies of the enzymes are considerably decreased.
|
The cavity volumes of
the mutant proteins V74A and V74L are larger and smaller than that of
the wild-type protein, respectively (14). Accordingly, the mutant
protein V74A must be much more tolerant of mutations at
Ala52 to bulkier hydrophobic residues than the V74L
protein. The conformational changes of the proteins caused by the
mutations at Ala52 were analyzed by CD. Comparison of the
near-UV CD spectra of the double mutant proteins with that of the
parent single mutant protein (V74A or V74L) revealed that the changes
in the CD spectra by the mutations at position 52 are much smaller for
V74A than for V74L (Fig. 4). These results suggest that
the tolerance of the protein conformation to the cavity mutations
increases as the volume of the cavity increases. The enzymatic
activities are summarized in Table III. The enzymatic
activities of the double mutant proteins A52X/V74L were always lower
than those of the corresponding mutants A52X/V74A. In addition, some of
the double mutant proteins A52X/V74A, such as A52I/V74A and A52F/V74A,
were more active than the corresponding single mutant proteins at
position 52. Therefore, the increase in the cavity volume apparently
contributes to reducing the strains caused by the cavity filling
mutations that are unfavorable for the enzymatic activity.
|
The parameters characterizing the thermal denaturation of the double
mutant proteins are also summarized in Table III. Among these
parameters, Gm
, instead of
Gm, reflects the effect of the mutation at
Ala52 on the stability of the mutant protein V74X
, and
Gm, which is calculated as
Gm
(A52X/V74X
)
Gm(A52X), reflects the difference between the
effect of the mutation at Ala52 on the stability of the
wild-type protein (
Gm(A52X)) and that on the
stability of the mutant protein V74X
(
Gm
(A52X/V74X
)). The double mutant proteins
A52X/V74X
should give positive
Gm values,
if the mutation at Ala52 creates strains at the cavity of
the wild-type protein, that are unfavorable for the protein stability,
and if the mutation at Val74 contributes to eliminating
these strains. In contrast, they should give negative
Gm values, if the mutation at
Val74 contributes to creating additional strains. As shown
in Table III, all of the double mutant proteins A52X/V74A gave positive
Gm values. Whereas, all of the double
mutant proteins A52X/V74L, in which Ala52 is replaced by
bulkier hydrophobic residues, gave negative
Gm values. These results suggest that the
mutation of Ala52 to Val, Leu, Ile, or Phe creates strains
within the cavity of the wild-type protein due to a collision between
the substituted residue and the surrounding residues. These strains
must be at least partially eliminated when Val74 is
replaced by Ala, because of the increase in the cavity volume. In
contrast, additional strains must be created when Val74 is
replaced by Leu, because of the decrease in the cavity volume. It
should be noted that the A52V/V74A mutant gave a positive
Gm value, but of only
0.1 kcal/mol.
Likewise, the A52V/V74L mutant gave a negative
Gm value, but of only
0.4 kcal/mol. Therefore, the mutation of Ala52
Val probably does not
create a significant strain at the cavity of the wild-type protein,
even when its volume is reduced by the mutation of Val74
Leu. Consequently, the double mutant protein A52V/V74L was the most
stable mutant protein (
Gm of 2.43 kcal/mol) among those constructed in this experiment, and it was more stable than
the most stable single mutant protein, A52I, by 0.6 kcal/mol.
As previously reported for a series of tryptophan synthase subunit mutants (52) and T4 lysozyme mutants (53) with multiple amino
acid substitutions at a unique position in the protein interior, a
correlation was observed between the changes in the thermal stability
(
Gm) and the hydrophobicities of the
substituted residues for the RNase HI variants with a series of
mutations at Ala52 (Fig. 5). However, the
data for the RNase HI variants substituted with Tyr, Phe, His, Lys,
Pro, and Gly do not fall on the straight line that was obtained from a
least-square fit of the data for 12 proteins with Ile, Leu, Cys, Val,
Met, Ala, Thr, Ser, Gln, Asn, Glu, and Asp at position 52. The
stabilities of the mutant proteins A52Y, A52F, A52H, A52K, A52P, and
A52G are much lower than those expected from the hydrophobicities of
the substituted residues.
The mutant proteins A52Y, A52F, A52H, and A52K are unexpectedly
unstable, probably because the introduction of an aromatic or ionized
group into the cavity alters the protein conformation. In fact, the
near-UV CD spectra of these mutant proteins are significantly different
from that of the wild-type protein, whereas those of the other mutant
proteins are similar to or only slightly different from that of the
wild-type protein (Fig. 2). Of the six Trp residues in the protein
molecule, Trp118 faces the cavity. Computer modeling
suggests that this Trp residue is forced to change its orientation to
avoid a collision with the bulky residues substituted for
Ala52.2 It is therefore likely
that the changes in the near-UV CD spectra mainly reflect a
conformational change of Trp118. This Trp residue, as well
as other residues that are located around the cavity, may change their
conformations, so that the strains caused by the introduction of an
aromatic or ionized side chain into the cavity can be eliminated.
However, these conformational changes must create additional
interactions that are unfavorable for protein stability. Nevertheless,
the mutant proteins A52Y, A52F, A52H, and A52K accumulated in the cells
in an amount sufficient for purification. In contrast, the mutant
proteins A52W and A52R did not accumulate in the cells. Since Trp and
Arg are the largest aromatic and ionized residues, respectively, the
mutations to these residues may create a serious conformational change
that makes the protein extremely unstable. Considerable destabilization due to the introduction of aromatic residues or Arg into a space in the
interior of the protein molecule has also been reported for the
tryptophan synthase subunit mutants (52).
The stabilities of the RNase HI variants substituted with Glu and Asp were unexpectedly low, only when these residues were ionized. The poor cellular production levels of the mutant proteins A52E and A52D, and the normal cellular production level of A52H (data not shown), also suggest that these mutant proteins are considerably unstable only when the substituted residues are ionized. At around pH 7.0, at which the cells grow, Asp and Glu may be ionized, whereas His may not. Since the introduction of a polar group into the cavity neither unexpectedly destabilizes the protein nor seriously affects the near-UV CD spectrum of the protein, unless it is ionized, it seems likely that a polar group can be introduced into the cavity without creating significant strain. In addition to the hydrophobic residues that surround the cavity (Leu49, Ala52, Leu56, Leu67, Val74, Leu107, and Trp118), many hydrophobic residues, such as Val5, Ile7, Tyr22, Leu26, Tyr28, Phe35, Ile53, Leu59, Val65, Ile78, Leu103, Leu111, Ile116, and Trp120, form the hydrophobic core of the protein. The extensive hydrophobic interactions among these residues probably make the conformation of the cavity fairly stable and thereby make it tolerant of the mutations. In the wild-type protein, the cavity is vacant and is not occupied by water molecules. However, it remains to be determined whether the introduction of a polar group into the cavity is accompanied by the introduction of a water molecule.
In contrast to the mutations to Tyr, Phe, His, and Lys, the mutations
to Pro and Gly did not seriously affect the protein conformation,
suggesting that these mutations do not create unfavorable van der Waals
contacts within the cavity. Ala52 is located in the I
helix. Therefore, the instabilities of the mutant proteins A52P and
A52G must reflect the intrinsic helix-destabilization associated with
the mutation of Ala52
Pro or Gly. Statistical analyses
in natural proteins suggest that Pro is an
helix breaker, and the
introduction of a Pro residue into an
helix kinks it by an average
of 26 ± 5° (54). However, the similarities in the near-UV CD
spectra and the enzymatic activity between the mutant protein A52P and
the wild-type protein suggest that the conformation of the
I helix
is not seriously affected by the mutation of Ala52
Pro.
Energy minimization was carried out to see the effect of the mutation
of Ala52
Pro on the protein structure by the molecular
mechanics program PRESTO (55). The result supports the hypothesis that
the
I helix is not appreciably kinked at the position where Pro is
introduced (data not shown). The crystallographic (56) and computer
(57) analyses for the structures of the mutant proteins of T4 lysozyme, in which Pro is introduced into
helices, also support this
hypothesis. The long interdomain
helix in T4 lysozyme, which is
originally kinked by 8.5°, was shown to be additionally kinked by
only 5.5° by the mutation of Asp72
Pro (56). All of
the mutant proteins of T4 lysozyme with Pro in the helix are
dramatically less stable than the wild-type protein, by 2.7-8.2
kcal/mol. In contrast, the RNase HI mutant A52P is less stable than the
wild-type protein by only 1.6 kcal/mol. In addition, this mutant
protein is enzymatically fully active. The
I helix of E. coli RNase HI seems to be more tolerant to Pro substitutions than
the
helices of T4 lysozyme, probably because the
I helix of
E. coli RNase HI is located in the interior of the protein
molecule and is highly stabilized through extensive hydrophobic
interactions with the rest of the protein. In fact, the mutation of
either Ala51 or Ala55
Pro within the
I
helix neither seriously destabilizes the protein nor dramatically
affects the enzymatic activity.3
The cavity-filling mutations effectively increase the stability of E. coli RNase HI, probably because the conformation of the cavity is fairly stable. Some of the stabilized mutant proteins, such as A52C and A52M, are enzymatically as active as the wild-type protein. Therefore, this cavity may not provide flexibility to the protein, which is required to make it enzymatically active. However, the enzymatic activity of the protein is rather sensitive to mutations within the cavity, probably because the cavity is located close to the active site. The determination of the kinetic parameters of the mutant proteins with poor RNase H activities suggests that the mutations within the cavity affect the catalytic efficiency, rather than the substrate binding (Table II). Some of the catalytic residues, such as Asp10 and Asp70, may change their conformations when the conformation of the cavity is altered. Comparison of the effects of the mutations on the enzymatic activity, the protein stability, and the near-UV CD spectrum suggests that the changes in the enzymatic activity roughly correlate with the changes in the CD spectrum. However, some of the mutant proteins with small changes in the CD spectrum, such as A52L and A52Q, are considerably less active than the wild-type protein. These results indicate that conformational changes around the cavity that affect the enzymatic activity are not always detected by measuring the near-UV CD spectrum. The CD spectra of mutant proteins A52L and A52Q were measured at pH 3.2, instead of pH 8.0, at which the enzymatic activity was determined. However, it is unlikely that the CD spectra of these mutant proteins are considerably changed at pH 8.0. In contrast to the correlation between the changes in the enzymatic activity and the near-UV CD spectrum, the changes in the enzymatic activity do not correlate with the changes in the protein stability.
Analyses of the stabilities of a series of double mutant proteins, in
which Ala52 and Val74 are simultaneously
replaced by other hydrophobic residues, allow us to discuss the effects
of the mutations within the cavity on the protein stability in more
detail. The changes in stability and conformation induced by the single
and double mutations at positions 52 (Ala52 Ile) and 74 (Val74
Ala and Leu) are schematically illustrated in
Fig. 6. The mutation of Val74
Ala
increases the cavity volume, whereas the mutation of Val74
Leu decreases it. The former destabilizes the protein by 2.3 kcal/mol and the latter stabilizes it by 1.1 kcal/mol. The mutation of
Ala52
Ile increases the stability of the V74A mutant by
3.5 kcal/mol. These results indicate that a single methyl or methylene
group contributes to increase the protein stability by 1.1 kcal/mol. This value is comparable to the empirically derived value of 1.3 ± 0.5 kcal/mol reported by Pace (30). The Ile side chain within the
cavity of the V74A mutant probably does not create a strain. In
contrast, the mutation of Ala52
Ile increases the
stabilities of the wild-type protein and the V74L mutant by only 1.9 and 0.1 kcal/mol, respectively. Therefore, it seems likely that the
stabilizing effect caused by the burial of the additional methyl or
methylene groups is partially or almost fully canceled by the
destabilizing effect caused by the unfavorable van der Waals contacts
in these proteins.
Dependence of the protein stability on the number of the methyl or
methylene group(s) that are introduced into or removed from the cavity
is shown in Fig. 7. It suggests that the volume of the
cavity in the wild-type protein is large enough to allow the
introduction of three methyl or methylene groups without creating considerable strain. However, the Gm value
varied from 1.3 to 2.4 kcal/mol for the mutant proteins A52V/V74L,
A52I, and A52L, in which three methyl or methylene groups are
introduced into the cavity. This suggests that the shape of the
substituted residue is important for the cavity-filling mutations. The
A52I and A52L mutants are less stable than the A52V/V74L mutant by 0.6 and 1.1 kcal/mol, respectively, probably due to unfavorable packing
effects. Similar effects have been observed for the core mutations in
T4 lysozyme (6) and N-terminal domain of
repressor (7). Thus, the
cavity must be most effectively filled in the A52V/V74L mutant.
However, the strains still contribute to destabilizing this mutant
protein. If the introduction of the double mutation of
Ala52
Val and Val74
Leu into the
wild-type protein did not create strain, it would increase the protein
stability by ~3.3 kcal/mol. However, the A52V/V74L mutant is more
stable than the wild-type protein by only 2.4 kcal/mol.
It has been reported that "swapped" mutant proteins, such as V35I/I47V of the gene V protein (3, 21) and L121A/A129L of T4 lysozyme (17), in which the core residues are reversed, are considerably less stable than the parent wild-type protein, by 2.9 and 1.1 kcal/mol, respectively. These results indicate that the packing effects are the major determinants of the stabilities of the protein variants with core mutations. However, the swapped RNase HI mutant, A52V/V74A, is less stable than the wild-type protein by only 0.5 kcal/mol. The packing effects may not be major determinants of the stabilities of the proteins with mutations at the cavity. A space in the cavity may serve to reduce the unfavorable contacts caused by a swapped mutation.
In this report, the Gm values estimated from
the equation of Becktel and Schellman (51) were used to evaluate the
effects of the mutations on the protein stability. These values are
almost identical with those calculated by using the enthalpy change of
unfolding (
Hm) and the change in the heat capacity (
Cp) as reported previously (58), except for that of the mutant protein A52K (data not shown). The estimated
Gm value of the mutant protein A52K (
5.93
kcal/mol) is different from the calculated one (
5.35 kcal/mol), but
only by 10%. These results indicate that the equation of Becktel and Schellman (51) is valid for the almost entire range of
Tm values obtained.
We thank R. Tanimura for computer modeling of the structures of the single mutant proteins and S. Kawakita for providing the backbone structure of E. coli RNase HI.