(Received for publication, October 6, 1995; and in revised form, December 12, 1995)
From the
The activity of human immunodeficiency virus protease is
markedly increased at elevated salt concentration. The structural basis
of this effect has been explored by several independent methods by
using both the wild-type enzyme and its triple mutant (Q7K/L33I/L63I)
(Mildner, A. M., Rothrock, D. J., Leone, J. W., Bannow, C. A., Lull, J.
M., Reardon, I. M., Sarcich, J. L., Howe, W. J., Tomich, C.-S. C.,
Smith, C. W., Heinrikson, R. L., and Tomasselli, A. G.(1994) Biochemistry 33, 9405-9413), designed to better resist
autolysis. Monitoring the intrinsic fluorescence of the two enzymes
during urea-mediated denaturation has shown that at high NaCl
concentration, both the conformational stability
(G
) and the transition midpoint (D
) between the folded and unfolded states
increase, indicating that the salt stabilizes the enzyme structure.
These equilibrium data are supported by kinetic studies on the
urea-mediated unfolding by measuring fluorescence change, red shifting
in the maximum of the emission spectrum, and far- and near-UV CD. The
salt effects observed in urea-mediated unfolding reactions prevail upon
heat denaturation. All these findings support the existence of a
two-state equilibrium between the folded and unfolded proteins. The pH
dependence of fluorescence intensity indicated that the conformation of
human immunodeficiency virus type 1 protease should change in the
catalytically competent pH region. It is concluded that preferential
hydration stabilizes the protease structure in the presence of salt,
providing entropic contribution to enhance the catalytic activity.
The protease encoded by HIV-1 ()is involved in the
specific processing of large viral polyproteins into individual
structural proteins and enzymes. This prompted extensive investigations
on HIV-1 protease as a potential therapeutic target of AIDS. The enzyme
is a member of the aspartic protease family and exists as homodimer of
identical polypeptide chains, each consisting of 99 amino acid residues (cf.(1) and (2) ). High salt concentration
was found to enhance the catalytic activity, primarily by lowering the
Michaelis constant (K
), and it was
suggested that the salting-out effect of NaCl decreased K
and increased k
/K
, the
specificity rate constant(3, 4) . We have pointed out
that K
values may be markedly dependent
on pH, while the rate enhancement by salt is practically independent of K
and pH(5) . Therefore, other
factors, for example the conformational stability of the protein, may
also be important. Indeed, raising the NaCl concentration frequently
stabilizes the protein structure by preferential hydration (6) . However, the effects of ionic strength on the
conformational stability of HIV-1 protease have not yet been studied.
To reveal whether or not the enhanced catalytic activity is associated
with a more stable structure, we have examined the stability of the
enzyme against urea, pH, and heat denaturation at different salt
concentrations. A mutant enzyme designed to resist autolysis (7) has been used in most experiments.
were carried out in 1.0 ml of 140 nM protein
solutions after incubation for 60-180 min at 25 °C. No change
in fluorescence was observed after that time. For each value of the
measured fluorescence intensity (I
), the
fraction of unfolded protein (f
) was calculated
from ,
where I and I
are the
fluorescence intensities, respectively, of the fully unfolded protein
obtained at high concentrations of urea and the fully folded protein
acquired in the absence of the denaturant. The values of I
and I
were obtained at the
base lines of the transition curves, at which I
became practically invariant at changing urea concentrations. The
fluorescence values were corrected by substraction of the fluorescence
of the corresponding buffer or urea solution in the absence of protein.
For equilibrium unfolding, the change of f with
increasing urea concentration follows (9, 10) ,
where R is the gas constant (8.314 J/degree/mol), T is the absolute temperature, and m is a measure of the
dependence of G on the denaturant concentration.
G
stands for the conformational stability of
the protein at zero concentration of denaturant ([D]
= 0) and indicates how much more stable the native conformation
is compared with the unfolded protein. The urea concentration at the
midpoint of the unfolding curve can be calculated from .
For studying the effects of salt
concentration on the stability of HIV-1 protease, we have used both the
wild-type enzyme and its mutated form (Q7K/L33I/L63I), which is much
less sensitive to autolysis without significant alteration in the
specific activity(7) . The unfolding of HIV-1 protease and its
mutated form was measured at increasing urea concentration, and the
unfolded fraction, f (), was plotted
against the concentration of denaturant (). Representative
experiments are shown in Fig. 1, and the parameters calculated
from the denaturation curves are given in Table 1. The relative
stabilities of the enzymes are illuminated by the transition midpoints
of denaturation (D
) and by
G
determined at 0.1 and 1.0 M NaCl as well as at pH 5.0
and 7.0. The data show that 1) both enzymes are stabilized by the
higher ionic strength; 2) the effects of salt concentration on both
proteases are more significant at pH 7.0 than at pH 5.0; 3) both
enzymes are more stable at pH 5.0 than at pH 7.0; and 4) the mutant
protease is slightly more stable than the wild-type enzyme.
Figure 1:
Urea denaturation of HIV-1
protease. Unfolding was monitored at 347 nm and 25 °C. a,
0.20 µM wild-type protease at pH 7.0 in the presence of
0.1 M () and 1.0 M (
) NaCl; b,
0.24 µM mutant enzyme (Q7K/L33I/L63I) in the presence of
0.1 M NaCl at pH 5.0 (
) and pH 7.0 (
). In each
case, the data points are taken from two independent experiments. The
curves represent best fits to the experimental points by nonlinear
regression using .
The unfolding of HIV protease has also been examined by measuring the rate of the fluorescence change in 6.3 M urea, assuming that the more stable structure denatures more slowly(13) . The reactions measured at various pH values gave perfect first-order rate constants with both the wild-type and mutant enzymes. It is seen in Fig. 2that the unfolding rate constant is lower at higher ionic strength, i.e. the enzymes are stabilized in the presence of salt. Their greatest conformational stability is found around pH 5. In the vicinity of the minimum of the rate constants, the effects of ionic strength are less pronounced than at the extreme pH values. Furthermore, the difference in the stability of the wild-type and mutant enzymes is smaller in the presence of 1.0 M NaCl than at the lower ionic strength. These kinetic results are consistent with the preceding equilibrium data ( Fig. 1and Table 1).
Figure 2:
pH dependence of the unfolding rate
constant of HIV-1 protease in 6.3 M urea. The concentrations
of native (,
) and mutant (
,
) enzymes were
0.26 and 0.24 µM, respectively.
and
,
reactions in 0.1 M NaCl;
and
, reactions in 1.0 M NaCl.
The spectral change during denaturation in 6.65 M urea has
also been determined. Fig. 3shows that upon unfolding of the
protein, the fluorescence intensity decreases throughout the emission
wavelength range and that the intensity maximum of the spectrum shifts
toward longer wavelengths. The rate constants of unfolding measured at
different wavelengths (330, 340, 347, and 360 nm) are identical within
experimental error (2.93 ± 0.12 10
s
), in accordance with the two-state mechanism (). The rate of the shift in wavelength maximum upon
denaturation (Fig. 4) provided the same first-order rate
constant (2.90 ± 30
10
s
) as obtained from the intensity changes.
Figure 3: Changes in the fluorescence spectrum of the mutant HIV-1 protease upon urea denaturation. The spectra were measured in 6.65 M urea at pH 5.0 in the presence of 0.1 M NaCl at 30, 100, 180, 290, 360, 540, 1200, and 1800 s. The enzyme concentration was 10.6 µM, and the scanning speed was 100 nm/min.
Figure 4: Change with time of the maximum wavelength of the intrinsic fluorescence of the mutant HIV-1 protease upon urea denaturation. The values are taken from the curves in Fig. 3.
Figure 5: Thermal inactivation of the mutant HIV-1 protease. The protein was incubated at 53.0 °C in standard buffer, pH 5.0, containing 1.0 M NaCl. The solid curve represents a single exponential decay.
Figure 6:
pH dependence of the fluorescence
intensity of the mutant HIV-1 protease. The fluorescence intensity of
0.20 µM enzyme is shown in arbitrary units in the presence
of 0.1 M () and 1.0 M (
)
NaCl.
Additional information concerning the fluorescence change may be acquired from the position of the maximum of the fluorescence spectrum, which exhibits a significant red shift when the tryptophan residues become more exposed to water. Slight red shifting with decreasing salt concentration is indeed observed in the spectra of HIV-1 protease measured at pH 7.0 (344 nm at 1 M NaCl and 345 nm without the addition of salt). No shift in the wavelength maximum is found at pH 3.0 and 5.0.
The activity of HIV-1 protease markedly increases with increasing ionic strength(5, 14, 15, 16) . The activation has been explained by assuming that the substrates are ``salting in'' to the active site of the enzyme(3, 4) . Ionic strength may also modify the conformational stability of the protein. In particular, the substrate-binding flaps (17) may change their flexibility, thereby affecting the catalytic activity. However, the dependence of protein structure on salt concentration has not yet been measured.
Unfolding studies using urea denaturation have clearly indicated
that the stability of both the wild-type and mutant HIV-1 proteases is
significantly enhanced at higher ionic strength ( Fig. 1and Table 1). This is apparent from both the G
and D
values. The
G
values obtained in this study are
4 times less than that
found previously for the wild-type HIV-1 protease(11) . The
latter value of 14.2 kcal/mol (the conversion factor between joules and
calories is 4.18) calculated with a more complicated equation, which
also involved protein concentration, was determined at pH 6.0 in the
presence of 0.2 M NaCl and 2% glycerol, not too different from
the conditions employed here. The value of 14.2 kcal/mol is rather high
since the conformational stability of a globular protein is generally
between 5 and 15 kcal/mol (21-63 kJ/mol), with one of the most
stable proteins, bovine pancreatic trypsin inhibitor, having 14.3
kcal/mol energy(18) . Our results indicate that the HIV-1
protease belongs to a class of much less stable proteins. The
transition midpoint of urea denaturation (D
= 2.6 M) of the previous study(11) ,
however, is in accordance with our findings.
The preferable autolysis sites of the wild-type HIV-1 protease have been changed in the mutant (Q7K/L33I/L63I), so that the specificity for cleaving the sensitive peptide bonds has been lost(7, 19) . It is, therefore, interesting that the mutant enzyme is stabilized not only against proteolysis, but also against urea denaturation. Since the leucine-isoleucine exchange is a fairly conservative modification, the enhanced conformational stability may primarily be attributed to Lys-7. In fact, creation of specific cation- or anion-binding sites on the surface of a protein through genetic engineering may increase the conformational stability by strengthening the water shell around the protein(18) .
It cannot be ruled out that some autolysis occurs during unfolding, and this promotes denaturation, in particular with the wild-type enzyme. However, autolysis may not be significant because the unfolded enzyme can be renatured to at least 70% in the case of the wild-type enzyme (11) , which we have confirmed, and we have obtained even higher activity with the mutant enzyme (data not shown). Furthermore, the recovery of activity is somewhat better in the presence of 1.0 M NaCl relative to 0.1 M NaCl. Since the enzyme is more active at high salt concentration, greater autolysis and lower recovery of activity would be expected in 1.0 M NaCl if autolysis were a crucial factor. Hence, the stabilization by NaCl appears to overbalance the effect of autolysis.
The generally used equilibrium method (Fig. 1) requires relatively long incubation of the enzyme at different denaturant concentrations. In the transition region, where both folded and unfolded proteases are present, the danger of proteolysis is imminent. Therefore, we have developed a simple kinetic method that markedly diminishes the residence of the protein in the transition region. Specifically, the enzyme is incubated at high urea concentration, while the rate of unfolding is monitored fluorometrically. Under this condition, the reaction rapidly proceeds through the transition region, thereby considerably reducing the time allowed for autolysis. This simple method has shown that the high salt concentration lowers the unfolding rate constants and enabled us to readily encompass a wider pH range (Fig. 2), where a minimum rate constant representing the maximum stability of the protease was revealed near pH 5.0. In all cases, the rate constants were perfectly first-order, consistent with a two-state denaturation mechanism ().
Further evidence for the two-state mechanism has been obtained by analyzing the change of the emission spectrum during urea denaturation. The two tryptophan residues of HIV-1 protease are not uniformly exposed to water; Trp-6 is more buried than Trp-42, as can be judged from the three-dimensional structure of the enzyme(20) . If the unfolding reactions of two tryptophan residues are not simultaneous, the kinetics measured at various emission wavelengths may be different(21) . In contrast, the rate of unfolding is independent of the wavelength (Fig. 3), and the rate constants are identical within experimental error.
The intrinsic fluorescence of HIV-1 protease is
approximately constant between pH 4.5 and 7.5 (Fig. 6). The
pH-rate profiles of the enzyme reactions conform to bell-shaped curves,
with pK values of
3 and 4.8-6.2 with
different substrates(5, 22, 23) . These
pK
values are attributed to the ionization of the
catalytically competent aspartic acids. It is clear, however, from Fig. 6that at pH
3, there is a considerable conformational
change, which may arise from the ionization of one of the catalytic
aspartic acids or some other acidic group involved in the stabilization
of the protein structure, for example by ion pair
formation(11) .
From the pH dependence of fluorescence intensity, it appears that the tryptophans are buried to the greatest extent between pH 4.5 and 7.5 (Fig. 6), where the enzyme is most compact. The compact form of a protein is expected to be more stable than a loose form. However, the urea-mediated denaturation ( Fig. 1and Fig. 2) indicates that the HIV-1 protease is most stable at pH 5 and is significantly less stable at pH 7. This apparent contradiction may be resolved by considering that the fluorescence intensity at a given pH reflects the static form of the enzyme, while the urea-mediated denaturation is a dynamic probe, which offers information about the unfolding reaction.
The stabilization of proteins by NaCl can be attributed to preferential hydration of the surface of the molecule(6) . A substantial portion of the protective water shell is apparently released at high temperature, and this may, in part, explain why the salt effects are less important at 53 °C.
This study has shown that the enzyme is most stable around pH 5 and less stable under more acidic and more alkaline conditions. Furthermore, the less stable the enzyme is, the more effectively it is stabilized. Proteins are generally most stable near their isoelectric points, where the net charge of the protein is low(18, 24) . In contrast, HIV-1 protease is most stable at pH 5, which is far from pI 8.66 and 9.35, the calculated isoelectric points of the wild-type and mutant enzymes, respectively. In the case of HIV-1 protease, the sum of the charges rather than the equality of the positive and negative species controls the stability. Specifically, the total number of charges is practically unchanged between pH 5 and the isoelectric point. Thus, the number of charges of the mutant enzyme is 13.87, 21.03, 21.35, and 21.70 at pH 3.00, 5.00, 7.00, and 9.35, respectively. The reduced stability at pH 3.0 is explicable in terms of diminished hydration. It may also be inferred from the calculated data that the stabilization by a water shell could be similar at pH 5.00 and 9.35. However, the stability is substantially decreased with increasing pH above 5, and this may indicate that some ion pairs of conformational importance decompose, such as Asp-29-Arg-8`, Asp-29`-Arg-8, and/or the C- and N-terminal electrostatic interaction between Pro-1 and Phe-99` and between Pro-1` and Phe-99.
The most intriguing question concerns the effects of
salt on the catalytic activity. One factor of the promotion of
catalysis may be the salting in of substrate to the enzyme surface,
which is supported by the lower K at higher ionic
strength(3, 4) . We propose that an additional factor
to be considered is the conformational stability of the enzyme. In the
light of the present data, it is clear that the enhanced ionic strength
increases not only the catalytic activity, as has been shown
previously(5, 14, 15, 16) , but also
the stability of the enzyme structure. The contribution of the increase
in stability to catalysis may be surprising because of the implication
in the catalysis of the flexible flaps that cover the substrate in the
binding cleft. This requires flexibility rather than stability. On the
other hand, better binding occurs at a rigid active site because of
entropic reasons. In the case of HIV-1 protease, the latter factor
appears to overrule the former one. Some flexibility in the active site
of HIV-1 protease is indispensable in order that the enzyme be able to
accommodate to the unlike sites of its natural polyprotein substrates.
A proper balance between flexibility and rigidity should hold when a
sufficiently wide specificity and catalytic efficiency are
simultaneously required.