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INTRODUCTION |
The cold-active and heat-labile
-amylase from the antarctic
bacterium Pseudoalteromonas haloplanktis is an exceptional
case because it is the largest known protein undergoing a reversible unfolding according to a two-state reaction pathway. It has been proposed that this unusual behavior is dictated by the requirement for
an improved flexibility or plasticity of the protein molecule to
perform catalysis at near-zero temperatures (1). Structural investigations have suggested that this is achieved by decreasing the
number and strength of weak interactions stabilizing the native conformation (2-4). Accordingly, one can expect that any mutation designed to improve the stability of this fragile edifice will induce
significant perturbations of the denaturation pattern, thereby
giving evidence for the structural features linked to the unfolding parameters.
Small globular proteins that unfold according to a pure two-state
process, i.e. reversibly and without a stable intermediate between the native and the unfolded states, have allowed researchers to
establish the thermodynamic stability function accounting for the Gibbs
energy change associated with their denaturation over physiological and
nonphysiological temperature ranges. The current availability of this
thermodynamic function, mainly gained by differential scanning
calorimetry, represents much more than the simple physicochemical
characterization of a polymer because it offers a powerful tool for
investigation of the still unexplained "second genetic code,"
i.e. the driving force allowing a polypeptide to fold into a
predetermined native and biologically active conformation (5). The
validity of the bell-shaped stability curve, which predicts protein
unfolding at both high and subzero temperatures, has been established
by the experimental demonstration of cold unfolding (6). Proteins are
said to be marginally stable because the positive Gibbs free energy
change is only a small difference between the large enthalpic and
entropic contributions in the temperature range restrained between the
cold and hot melting temperatures. Basically, denaturation at high
temperature is entropy-driven, because the large and positive thermal
dissipative force exceeds the corresponding enthalpy change. By
contrast, cold unfolding is enthalpy-driven and arises from the
negative contribution of the hydration Gibbs energy of protein groups,
mainly polar, at low temperatures (7).
Four parameters are usually assigned to the unfolding event recorded by
differential scanning calorimetry, (i) the melting point of the
unfolding transition; (ii)
Cp, the difference
in heat capacity between the native and unfolded states arising from the transfer of buried groups in the protein to water, in the random
coil; (iii)
Hcal, the calorimetric
enthalpy corresponding to the total amount of heat absorbed during
unfolding and recorded by the calorimeter; and (iv)
Heff, the effective or van't Hoff enthalpy calculated from the slope of the transition and describing the
cooperativity of the unfolding reaction. In the case of a pure
two-state model,
Hcal and
Heff are nearly identical.
However, large proteins generally unfold irreversibly as a result of
strong heat-induced aggregation, with large deviations from the
two-state model because their multidomain structure induces a stepwise
or overlapping unfolding of domains, behaving as individual
calorimetric units (Fig. 1).
We report here the calorimetric characterization of mutants from the
cold-active
-amylase defined on the basis of its strong homology
with heat-stable mesophilic
-amylases. These mutants reveal the
unsuspected contribution of single amino acid substitutions to the
thermodynamic parameters characteristic of large heat-stable proteins
and provide new insight into the molecular adaptations of enzymes from
psychrophilic organisms.
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EXPERIMENTAL PROCEDURES |
Mutagenesis and Protein Purification--
Mutations in the
P. haloplanktis
-amylase gene were introduced by
polymerase chain reaction as described (8). The nucleotide sequence of
the constructions was checked on an Amersham Pharmacia Biotech ALF DNA
sequencer. The recombinant wild-type
-amylase and the mutant enzymes
were expressed in Escherichia coli at 18 °C and purified
by DEAE-agarose, Sephadex G-100, and Ultrogel AcA54 column
chromatography, as described previously (9). Pig pancreatic
-amylase (PPA)1 was from Roche.
Differential Scanning Calorimetry (DSC)--
Measurements were
performed using a MicroCal MCS-DSC instrument as detailed (1). Samples
(~3 mg/ml) were dialyzed overnight against 30 mM MOPS, 50 mM NaCl, and 1 mM CaCl2, pH 7.2. Thermograms were analyzed according to a non-two-state model in which
Tm,
Hcal, and
Heff of individual transitions are
fitted independently using the MicroCal Origin software (version 2.9).
The magnitude and source of the errors in the Tm and
enthalpy values have been discussed elsewhere (10). Fitting standard
errors on a series of three DSC measurements made under the same
conditions in the present study were found to be ±0.05 K on
Tm and ±1% on both enthalpies.
Thermal Inactivation--
Thermograms generated by starch
hydrolysis were recorded on a MCS isothermal titration calorimeter. The
technical background for such an experiment has been described
elsewhere (11). Starch concentration was 2% in 50 mM
Hepes, 50 mM NaCl, 1 mM CaCl2, pH 7.2, and was saturating during the time course of the experiment. Reactions were initiated by injecting 10-50 µl of a 30 µg/ml
enzyme solution into the same buffer. The activity decay at
45 °C was recorded continuously for 30 min, and data were fitted on
a monoexponential function to determine the first order constant rate
of enzyme inactivation at 45 °C, S.E. <1%.
Enzyme Assay and Kinetics--
The kcat
values were determined at 25 °C using 3.5 mM
4-nitrophenyl-
-D-maltoheptaoside-4,6-O-ethylidene
as substrate (8). The kcat/Km
values for substrate hydrolysis were determined from initial rates
using the following relation.
kcat/Km =
v0/S0E0, which is
valid at S0
Km for systems that obey Michaelis-Menten kinetics. The validity of this equation was
ascertained by performing determinations at three substrate concentrations.
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RESULTS |
Selection of Potentially Stabilizing Mutations--
The
psychrophilic
-amylase from P. haloplanktis (AHA) belongs
to chloride-dependent
-amylases. This group includes all
known animal
-amylases and the enzymes from some Gram-negative
bacteria, covering a large range of living temperatures (12). The high degree of amino acid sequence identity between
chloride-dependent
-amylases allowed us to construct a
multiple sequence alignment of the available primary structures (12)
and to detect about 20% of 453 residues that are specific to the
cold-adapted enzyme. The involvement of these residues in the weak
stability of the psychrophilic
-amylase was assessed by comparison
of its crystal structure (3, 4) with that of PPA, the closest
structural homolog, as well as with the structure of
-amylases from
human salivary and pancreatic glands and from the insect Tenebrio
molitor. Molecular modeling was then used to check the consistency
of mutations aimed to introduce weak interactions found in mesophilic
-amylases in the heat-labile AHA. The selected mutations are listed
in Table I along with the restored
interactions. Following production in E. coli and
purification, the stability and kinetic parameters of 14 mutant enzymes
were determined (Table II). Mutants are
listed according to their main properties, as discussed below.
Fig. 1 depicts the microcalorimetric
behavior of the recombinant wild-type AHA and of heat-stable
-amylases, including PPA taken as reference for mutation
selection.

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Fig. 1.
Thermal unfolding of
-amylases recorded by DSC. The heat-stable PPA
and BAA are characterized by higher Tm (top of
the transition) and Hcal
(area under the transition) values, by a flattening of the
transition and by the occurrence of calorimetric domains
(deconvolutions are in thin lines). All thermograms
are base line-subtracted and normalized for protein
concentrations.
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Enthalpic Stabilization by Electrostatic
Interactions--
Enthalpic stabilization involves an increase of the
denaturation enthalpy and of the melting point. Such stabilization of the heat-labile
-amylase is obtained when a salt bridge (N150D), polar aromatic interactions (V196F), or an additional H-bond (K300R) is
introduced in the protein. Thermal unfolding of some of these mutants
is illustrated in Fig. 2. These
additional electrostatic interactions induce a slightly higher
Tm value (Table II) associated, however, with a
large increase of the calorimetric enthalpy
(
Hcal). Characterization of the
double mutant N150D/V196F revealed several interesting features. The
effect of the mutations on the melting point is additive (Table II,
Fig. 2). Moreover, both mutations affect
Hcal and
Heff differently, whereas their cumulative effect in the double mutant leads to a
Hcal/
Heff ratio at unity, indicative of a perfect two-state unfolding
pattern.

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Fig. 2.
Enthalpic stabilization. Thermal
unfolding of AHA and of single and double mutants (NV,
N150D/V196F) characterized by increased Tm and
Hcal values.
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Calorimetric Domain Acquirement by Nonpolar Groups--
Aliphatic
side chains were introduced in AHA in order to reconstruct hydrophobic
core clusters found in mesophilic
-amylases (Q164I, T232V, N288V,
and M379V). Interestingly, each substitution results in a non-two-state
unfolding of the mutant enzymes and in the appearance of two
calorimetric units or domains (Fig. 3). As also shown in Fig. 3, the separation between both calorimetric units
in the mutants can be improved by adjusting the experimental conditions, providing evidence for the coexistence of two protein domains of distinct stability. Deconvolution of the heat capacity function revealed that one domain always has an increased
Tm compared with that of AHA (Table II), underlining
the stabilization induced by the reconstituted hydrophobic cluster. By
contrast, the lower Tm of the first domain seems to
be the consequence of strain imposed in one domain that is not
compensated in other protein regions. This is supported by the
combination of mutations Q164I and V196F resulting in a mutant enzyme
displaying two stabilized domains, as far as Tm is
concerned. The mutant T232V deserves special comments. Indeed, the
shape of its heat capacity function is similar to that of PPA (Fig. 3)
as well as the relative contribution of each domain to
Hcal and
Heff (Table II). Therefore, this
mutant consistently reproduces the microcalorimetric unfolding pattern
of the heat-stable PPA but at lower temperatures.

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Fig. 3.
Calorimetric domains induced by additional
nonpolar groups in hydrophobic core clusters (inset,
T232V, pH 8.0).
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Cooperativity of Unfolding--
The comparison of
Hcal and
Heff values allows us to analyze
the unfolding cooperativity by microcalorimetry. Several mutations listed in Table II affect the unfolding cooperativity without noticeable change of
Hcal. Thermal
unfolding of mutants Del6, L219R and R64E (Fig.
4) clearly illustrates the relation
between
Heff and the steepness of
the transition. Furthermore, the thermogram slope of mutants L219R and
R64E is considerably decreased and approaches the profile of large and
stable proteins such as Bacillus amyloliquefaciens
-amylase (BAA) (Fig. 1).

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Fig. 4.
Alteration of unfolding cooperativity.
Decreasing the steepness of the transition maintains a higher level of
native structures at elevated temperatures.
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Folding and Unfolding Reversibility--
In these
experiments, the mutant enzymes were 100% unfolded, then allowed to
refold during a cooling period of 15 min at 15 °C. Reversibility was
calculated by the recovery of
Hcal
during a second scan (Table II). Substitution of a single amino acid side chain is sufficient to drastically alter this critical parameter of protein folding, as demonstrated by mutant N12R that displays only
25% renaturation. Mutants Q164I, V196F, and Q164I/V196F that are
stabilized by hydrophobic groups also decrease the unfolding reversibility significantly. In the same conditions, unfolding of the
heat-stable PPA and BAA is completely irreversible.
Thermal Inactivation Recorded by Isothermal Titration
Calorimetry--
Determination of inactivation rate constants usually
requires the recording of the residual enzyme activity after incubation at high temperature. In the case of AHA and of its mutants, the model
of Lumry and Eyring (13) is valid:
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(Eq. 1)
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where k2 is the first order rate
constant of thermal inactivation. However, the various degrees of
reversibility noted for the mutants (kinetically characterized by
k
1) strongly impair the validity of the
results obtained by conventional methods. We have therefore devised a
new method using isothermal titration calorimetry and recording
activity at 45 °C as the heat released by the hydrolysis of starch
glycosidic bonds. This method provides a direct and continuous
monitoring of activity at the denaturing temperature (Fig.
5). As a prerequisite, the same
experiment was followed by fluorescence (without substrate) showing
that the structural modifications accompanying a temperature shift from 15 to 45 °C are fast with a k1 value of about
0.04 s
1. Therefore, the transition N
U is one order
of magnitude faster than the transition U
I, and the disappearance
of the active state N can be taken as a measure of
k2, as reported in Table II. It is significant
that 10 of 14 mutants are protected against thermal inactivation and
that mutants carrying newly introduced electrostatic interactions
display the lowest inactivation rate constants (Fig. 5). A plot of
unfolding reversibility versus thermal inactivation (Fig.
6A) shows that these
parameters for the mutant enzymes are clustered between the respective
values for AHA and PPA.

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Fig. 5.
Thermal inactivation recorded by isothermal
titration calorimetry. Upper panel, following enzyme
injection (arrow), activity at 45 °C is recorded as the
heat flow of the reaction. Note the stability of PPA and the activity
decay of AHA. Lower panel, semilogarithmic plot of residual
activity versus time illustrating the lower inactivation
rate of some mutants. NV, N150D/V196F.
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Fig. 6.
Correlation of stability (A)
and kinetic (B) parameters for wild-type
( ) and mutant ( )
-amylases.
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Kinetic Parameters--
The kinetic parameters
kcat and Km were recorded
using a chromogenic maltoheptaose oligosaccharide (Table II). From these results, four main aspects should be pointed out. (i) As reported
previously, AHA possesses a higher activity
(kcat) than PPA in order to compensate for the
slow chemical reaction rates at low temperature. (ii) The general trend
of the mutations is to decrease both kcat and
Km. Fig. 6B shows that both kinetic
parameters for mutants all fall within a narrow region of the plot,
between the values of AHA and PPA. (iii) As a result, the mutations
tend to raise the specificity constant or catalytic efficiency
kcat/Km to values close to
that of PPA (Table II). (iv) Most mutations protecting the enzymes
against thermal inactivation also concomitantly decrease the
kcat value.
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DISCUSSION |
Stability of Large Proteins--
The magnitude of the
contribution, as recorded by microcalorimetry, of a single amino acid
side chain to the stability parameters of a large protein is one of the
most significant features revealed by engineering the heat-labile
-amylase. Such individual contributions are generally masked in
stable proteins by the complex network of interactions involved in the
native state conformation and by the fact that frequently, heat-induced
unfolding of large proteins is kinetically driven as a result of
pronounced irreversibility. Our results show that the selected
electrostatic interactions provide the largest contribution to
stability per se, by increasing both Tm
and
Hcal and by protecting mutants
against thermal inactivation (Table II). Thermal unfolding of the
double mutant N150D/V196F follows the two-state transition model and is
essentially reversible. Accordingly, its thermodynamic stability can be
analyzed using the modified Gibbs-Helmholtz equation:
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(Eq. 2)
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This function corresponds to the energy required to disrupt the
native state (14) and is illustrated in Fig.
7. One can note that despite a modest
increase of the melting point, the conformational energy of the double
mutant is twice that of the wild-type AHA around 10 °C. Similarly,
the contribution at 20 °C of the ion pair in N150D can be estimated
to 4.4 kcal/mol, of the H-bond in K300R to 0.7 kcal/mol, and of a polar
aromatic interaction in V196F (assuming two interplanar interactions)
to 0.8 kcal/mol. These values are in good agreement with data obtained by ab initio calculations (15) or by experimental studies
(16, 17). These free energy gains originate, however, from very large enthalpic-entropic compensation effects. The denaturation enthalpy and
entropy can be calculated according to the relations (18):
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(Eq. 3)
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(Eq. 4)
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For instance, the denaturation enthalpy at 20 °C for AHA is
10.5 kcal/mol, and the entropic term (T
S)
amounts to 2.2 kcal/mol. The corresponding values for mutant N150D are
60.9 and 48.2 kcal/mol, respectively. Therefore, the gain of 4.4 kcal/mol in conformational energy provided by the ion pair arises from
a 6-fold increase of the denaturation enthalpy balanced by a 20-fold
increase of the entropic cost. It should be added that mutant N150D
restores a surface salt bridge. Although the net contribution of
solvent-exposed ion pairs has been frequently questioned (19), our
results demonstrate that such interactions can provide a substantial
increment of conformational stability. This is especially relevant for
thermophilic and hyperthermophilic proteins that are characterized by
an abundance of surface ion pairs (20), in some instances organized in
interconnected networks (21, 22). On the other hand, about 8 ion pairs,
15 arginine residues and 10 aromatic interactions are lacking in the
crystal structure of AHA when compared with the heat-stable PPA (3).
The disappearance of these electrostatic based features is obviously a
main determinant for both the low Tm and melting
enthalpy of the psychrophilic
-amylase.

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Fig. 7.
Stability curves of AHA and of the double
mutant N150D/V196F (NV). Dashed line,
theoretical curve for a heat-stable -amylase, using data from Ref.
41.
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It is generally agreed that the hydrophobic effect is the main factor
in stabilizing folded conformations (23). As shown in Fig. 3 and Table
II, the insertion of a nonpolar group into a hydrophobic core cluster
creates a stabilization center in the protein and in the peculiar case
of AHA promotes the appearance of calorimetric domains. Unfortunately,
large deviations from the two-state model preclude any reliable
calculation for these mutants. It should be noted, however, that the
increases of Tm for the stabilized transition are of
the same magnitude or even higher (M379V) than that provided by
electrostatic interactions. Analysis of the hydrophobic clusters in AHA
shows that 20 substitutions decrease the hydrophobicity index of the
involved residues relative to PPA. As indicated by the calorimetric
behavior of the mutations selected for this work (Fig. 3), the overall
low core hydrophobicity of the heat-labile AHA accounts to a large
extent for the lack of discrete unfolding intermediates. It is usually
accepted that extreme cooperativity is only achieved in small molecules
with tight packing of groups (14). This obviously is not the case for
the large multidomain heat-labile
-amylase. As suggested by the
mutation effects, its unfolding cooperativity arises from the very low
number of interactions maintaining the native state, which therefore
results in the simultaneous disruption of all structural elements and
in the unusual two-state unfolding of this large protein. Considering
protein adaptation to temperature, a practical implication of the
flattening of the unfolding transition in thermostable
-amylases
such as BAA (Fig. 1) is to maintain a higher level of native
conformation at elevated temperatures. Fig. 4 shows that the same holds
true between 45 and 55 °C for mutants of the heat-labile
-amylase with altered cooperativity. The relation between unfolding
reversibility and thermal inactivation (Fig. 6A) is another
unsuspected result gained from the
-amylase mutant analysis.
Referring to Equation 1, mutants protected against thermal inactivation
are also those that refold less efficiently, mimicking the behavior of
thermostable
-amylases. This could be explained by the large number
of all types of weak interactions required for proper folding and
stability in mesophilic and thermophilic enzymes. As a consequence,
there will be more possibilities of side reactions during refolding,
either by intramolecular mismatch or by aggregation due to replacement
of the regular intramolecular pairing by intermolecular interactions
(17).
Cold Adaptation--
It was shown that all 24 side chains in the
active site performing H-bonding to a large pseudosaccharide inhibitor
in the transition state conformation are strictly conserved in both AHA and PPA crystal structures (4, 24). As a consequence, changes in
primary structure that occur far from the active site should be
responsible for the high specific activity of AHA. As a matter of fact,
all mutations in this study designed to restore a mesophilic character
are located far from the catalytic center and tend to decrease both
kcat and Km (Fig.
6B). It has been convincingly argued that increases in
flexibility of cold-adapted enzymes lead to a broader distribution of
conformational states, translated into higher Km
values for enzymes devoid of adaptive changes within the active site
(25, 26). In this context, rigidifying the molecule or part of the
molecule by the newly engineered weak interactions in the psychrophilic
-amylase should contribute to decreased Km values
by reducing the conformational entropy of the binding-competent
species. The kinetic parameters of the mutants (Fig. 6B)
give experimental support to the previous hypothesis that
temperature-adaptive increases in kcat occur
concomitantly with increases in Km (26).
It has been proposed that stability and activity are not physically
linked (27). Laboratory evolution can indeed yield nonnatural catalysts
that are thermostable and active either at high (28) or low (29, 30)
temperatures. Nevertheless, when the selection is only applied on low
temperature activity (the relevant parameter for psychrophilic
enzymes), especially by in vivo functional selection systems
(allowing cell growth, for instance), the evolved enzymes generally
display a concomitant reduced thermostability (31-34). It was assumed
that the selective advantage for these optimized catalysts depends on a
high kcat and not on maintaining stability (27).
However, our structure-based approach demonstrates that natural
mutations occurring in the cold-active
-amylase associate high
activity and low stability. The convergence of the results from both
random and rational studies stresses that the lack of selective
pressure for stable proteins at low temperatures does not entirely
account for the instability of psychrophilic proteins. On the contrary,
heat lability or conformational plasticity seems to be intimately
interlaced with sustained activity in cold environments. This is
possibly the easiest route taken by natural selection on the
evolutionary time scale or because large fluctuations around the native
state are required by the energetics of substrate binding (35)
and catalysis at low temperatures, an aspect that is not documented for
such enzymes to date. The mutant N288V represents an extreme case. This
mutant possesses a heat-labile domain with the lowest
Tm (Table II) but is marginally stable even at
4 °C, with a half-life of 2 h at 25 °C and requiring fast
purification procedures. This behavior is consistent with the proposal
that AHA has reached a state close to the lowest accessible stability of its native state and cannot be further destabilized (1). If indeed
stability is linked to activity in cold-adapted enzymes, this
limitation explains the imperfect adaptation of psychrophilic enzymes,
i.e. their activity at low temperatures remains lower than
that of mesophiles at 37 °C.
Concluding Remarks--
This mutational analysis of a large
protein demonstrates that the psychrophilic
-amylase has lost
numerous weak interactions during evolution to reach the proper
conformational flexibility at low temperatures. These adaptive
adjustments contribute to improve kcat without
alteration of the catalytic mechanism, because the active site
architecture is not modified, but at the expense of a weaker substrate
binding affinity. On the other hand, thermophilic enzymes strengthen
the same type of interaction to gain in structural stability at high
temperatures but at the expense of poor activity at room temperature
(36-38). These aspects underline the continuum in the strategy of
temperature adaptation in proteins and reinforce the concepts of
"compromise" (39) between activity and stability leading to
"corresponding states" (40) of enzymes adapted to different thermal environments.