From the
The chaperonin proteins, GroEL and
GroES
, inhibit protein aggregation and assist in protein
folding in a potassium/ATP-dependent manner. In vitro, assays
for chaperonin activity typically involve adding a denatured substrate
protein to the chaperonins and measuring the appearance of correctly
folded substrate protein. The influence of denaturant is generally
ignored. Low concentrations of guanidinium chloride (<100
mM) had a profound effect on the activity/structure of the
chaperonins. Guanidinium decreased the ATPase activity of GroEL and
attenuated the inhibition of GroEL ATP hydrolysis by GroES. The stable,
asymmetric chaperonin complex which forms in the presence of GroES and
ADP (GroES
ADP
GroEL
-GroEL
) rapidly dissociated upon addition
of 80 mM guanidinium chloride. Dissociation was enhanced at
high ionic strength, but rapid dissociation was guanidinium-specific.
Accelerated release of the GroES from the complex was also
demonstrated. Unfolded proteins alone had no effect on complex
stability. Residual guanidinium depressed the rate of Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase
(Rubisco) folding; an increased aggregation rate also decreased the
yield of folded Rubisco. Chaperonin-assisted folding is therefore best
studied using proteins denatured by means other than guanidinium
chloride.
Molecular chaperones are ubiquitous proteins which assist in the transport, folding, and assembly of other proteins (Lorimer, 1992; Gatenby and Viitanen, 1994). Two Escherichia coli chaperone proteins, GroEL and GroES, exist as double and single heptameric rings, respectively (Hendrix, 1979; Chandrasekhar et al., 1986). These proteins have been termed chaperonins (Ellis, 1992) and assist in protein folding, both in vivo and in vitro (Goloubinoff et al., 1989a, 1989b).
The GroEL protein
stably binds one (Laminet et al., 1990) or two (Viitanen et al., 1991) unfolded protein molecules. In an undefined
mechanism requiring Mg, K
, and
adenine nucleotides, GroEL releases proteins in a form that may
progress to the native state, depending on the folding environment
(Viitanen et al., 1990). Under conditions where the target
protein has been demonstrated not to spontaneously fold (i.e. nonpermissive conditions), the GroES chaperonin is also required
(Schmidt et al., 1994). GroEL hydrolyzes ATP even in the
absence of unfolded polypeptides; this activity can be inhibited by
GroES (Chandrasekhar et al., 1986). The level of GroEL ATPase
inhibition by GroES is dependent on the potassium concentration, with
greater inhibition seen at lower concentrations of potassium (Todd et al., 1993). The GroES co-chaperonin has been proposed to
``coordinate'' GroEL ATP hydrolysis by binding tightly to one
toroid of GroEL, creating an asymmetric complex with ADP trapped at
half of the GroEL ATP-binding sites (GroES
ADP
GroEL
-GroEL
; Todd et
al., 1993). This complex can be isolated by gel filtration and
dissociates with t
of
5 h. If, however, the
isolated complex is allowed to undergo a single round of ATP hydrolysis
(12 s), both GroES
and ADP are completely exchanged (Todd et al., 1994).
Typical in vitro assays for protein
folding involve the addition of denatured target protein to the
chaperonin complex, and the appearance of active target protein is
followed. Initial formation of the denatured protein is achieved either
by using chemical denaturants (urea or guanidinium chloride), heat, or
acid. It is generally assumed that the residual concentration of
denaturant has no effect on chaperonin activity. Although this is
obviously true for dilute concentrations of acid, chemical denaturants
have profound effects both on chaperonin activity and on the folding of
substrate proteins. Concentrations of guanidinium chloride < 0.1 M, typical of those carried over with denatured proteins, are
known to perturb the rate of spontaneous refolding of lactate
dehydrogenase (Zettlemeissl et al., 1979). Similarly, we show
here that guanidinium chloride increases the rate of aggregation of
unfolded conformers of Rubisco, ()thereby decreasing the
yield of folded protein.
Marked changes in the GroEL tertiary structure (decreased ellipticity, fluorescence, decreased molecular weight) have been observed at guanidinium concentrations in excess of 1.0 M (Price et al., 1993). The ATPase activity was more sensitive, with a 50% reduction in the ATP hydrolysis rate with 300 mM guanidinium chloride (Price et al., 1993). Here we demonstrate that lower concentrations of guanidinium (<150 mM) also inhibit the ATPase of GroEL and destabilize the asymmetric chaperonin complex, thus relieving the inhibition of GroEL ATPase by GroES.
Spontaneous folding was examined by rapidly
diluting 20 µl of 5.0 µM acid-denatured Rubisco (in 10
mM HCl) at 25 °C to 1.0 ml with buffer, resulting in a
folding reaction containing 100 nM Rubisco folding
intermediate, 300 mM NaCl, 100 mM HEPES, 10 mM DTT, 5 mM Mg(CHCO
)
, 5
mM KCH
CO
, 0.1 mM EDTA, 5
µM bovine serum albumin, and various concentrations of
guanidinium or urea. At the indicated times, 5 µl of 2.86
µM GroEL
was added to 100 µl of the
reaction, effectively quenching further folding by binding unfolded
Rubisco species (U + U
in Fig. SII). Samples
were then assayed for Rubisco activity.
Scheme II: Scheme II
Aggregation of Rubisco was
determined by rapidly diluting Rubisco (5-10 µM in
10 mM HCl at 25 °C) 50 times, to 100 nM in
100 mM HEPES (pH 7.8), 10 mM DTT, 0.1 mM EDTA, 5 µM bovine serum albumin, and various
concentrations of guanidinium chloride or urea. At the indicated times,
the amount of Rubisco folding intermediate which could productively
bind GroEL was determined by adding 5 µl of 2.86 µM GroEL
to 100 µl of the aggregation reaction.
GroEL-bound Rubisco was supplemented with 10 µl of 50 mM Mg(CH
CO
)
, 50 mM KCH
CO
, 10 mM ATP, 2.5 µM GroES
to allow productive folding to occur. Controls
lacking this addition confirmed no spontaneous folding occurred in the
absence of chloride ion (Schmidt et al., 1994). The residual
amount of Rubisco folding intermediate present as a function of time
was fit to a pseudo-first order decay function, 1 -
exp
(-k
1
time), using Kaleidagraph.
Light scattering measurements were done on a Perkin-Elmer LS-5B luminescence Spectrometer at 25 °C. Excitation was at 500 nm (10 nm slit), and scattered light was measured by monitoring emission at 500 nm (10 nm slit) for the indicated time.
Figure 1:
Effect of salts on rate of GroEL ATP
hydrolysis. The rate of ATP hydrolysis by GroEL was determined at 25
°C in buffer 1 (100 mM Tris-HCl (pH 7.8), 10 mM MgCl, 1 mM DTT, 0.1 mM EDTA) plus
the indicated concentrations of freshly prepared guanidinium chloride (circles) or NaCl (squares). Assays were begun by
adding [
-
P]ATP (final concentration
= 100 µM, 52 Ci/mol) to reactions containing 0.50
µM GroEL
plus 0 (filled
symbols) or 1.0 µM GroES
(open
symbols) and 100 mM (A) or 1 mM (B) KCl. The concentration of liberated P
was
determined at
4 times within 15 min. Linear regression analysis (r
0.995) gave initial rates of ATP hydrolysis. Note the
design of these assays ignores the burst of GroEL ATP hydrolysis seen
in the presence of GroES (Todd et al., 1993). Less than 15% of
the total amount of ATP present was hydrolyzed under these conditions.
Assays at 1 mM KCl included 1 mM creatine phosphate
and 1 unit/ml creatine phosphokinase to prevent accumulation of ADP,
which is a potent ATPase inhibitor of GroEL
GroES at low
potassium concentrations (Todd et al., 1993). At 1 mM potassium, less than 10% of the total
[
P]P
present was liberated, thus
dilution of specific activity by the ATP-regenerating system was
negligible. Rates of asymmetric complex dissociation (asterisk) were calculated from Fig. 2A.
Figure 2:
Stability of GroES
ADP
GroEL
-GroEL
complex:
retention of [
-
P]ADP. A, percent
of initially isolated [
-
P]ADP which remains
associated with the chaperonin complex after 2 min incubation in buffer
2 [10 mM Tris-HCl (pH 7.4), 5 mM Mg(CH
CO
)
] plus:
guanidinium chloride (
), NaCl (
), Tris-HCl (pH 7.8)
(
), or Mg(CH
CO
)
(
).
Complex dissociation was also measured in 10 mM Tris-HCl (pH
7.4), 50 mM Mg (CH
CO
)
with
increasing guanidinium chloride (
), or NaCl (
). B, percent of initially isolated
[
-
P]ADP which remains associated with the
chaperonin complex after incubation in buffer 2 with 0 (
) or 80
(
) mM guanidinium chloride.
At low potassium concentration, the
affinity of GroEL for ATP is much reduced (Todd et al., 1993),
and the effect of guanidinium is more pronounced, with an 80%
decrease in activity upon addition of 150 mM (Fig. 1B). A portion of this inhibition could be
reproduced by increasing ionic strength alone, as evidenced by a 50%
decrease in ATPase activity in the presence of NaCl. Fig. 1B also emphasizes the specificity of GroEL for K
in
the presence of several hundredfold excess of Na
. The
inhibition of GroEL by GroES at low potassium concentrations is nearly
complete and occurs after one GroEL toroid has turned over (Todd et
al., 1993). Guanidinium chloride (
60 mM) relieved
this inhibition, and ATP hydrolysis became independent of GroES. The
similarity in rates of ATP hydrolysis regardless of GroES indicated
that guanidinium-induced dissociation of the complex became faster than
the rate of ATP turnover by GroEL alone. Elevated concentration of NaCl
alone, however, could also relieve GroES inhibition, suggesting that
the GroEL
GroES
ADP complex is unstable at higher ionic
strengths.
The rate of
nucleotide loss (Fig. 2B) in 80 mM guanidinium
was 3.1 min, a 100-fold increase in the dissociation
rate. At any given guanidinium concentration, the dissociation rate was
decreased by the presence of additional ADP, (
)consistent
with ADP binding on the second ring of GroEL stabilizing the chaperonin
complex. Similar effects of ADP on complex stability have been
demonstrated at 0 °C (Todd et al., 1994).
Figure 3: Dissociation of GroES from asymmetric complex as measured by gel filtration. Isolated asymmetric chaperonin complex was injected onto a TSK 4000 after: (i) 15-min, (ii) 75-min, and (iii) 5-min treatment with 80 mM guanidinium chloride or (iv) 5-min incubation with 20 mM EDTA. The GroEL and chaperonin complex elutes at 6.9 ml (Peak A), and GroES elutes at 9.3 ml (Peak B). Absorbance at 212 nm was monitored and is shown offset by 70, 60, and 30 multiabsorbance units for runs i, ii, and iii, respectively.
Figure 4:
Effect of guanidinium chloride on Rubisco
folding. A, chaperonin-assisted Rubisco folding in the
presence of 0 (), 50 (
), 100 (asterisk), 150
(
), or 200 (+) mM guanidinium chloride. B,
spontaneous folding of acid-denatured Rubisco was measured in the
presence of 0 (
), 30 (
), 60 (
), or 90 (asterisk) mM guanidinium chloride or 200 mM urea (
).
We further examined the effect of denaturants on the spontaneous folding reaction at 25 °C (Fig. 4B, Table 2). Spontaneous folding of R. rubrum Rubisco at 25 °C is dependent on chloride ions (Lorimer et al., 1993). Addition of guanidinium chloride had no effect on the rate of spontaneous folding, yet the yield of folded protein decreased considerably (Table 2), even though additional chloride ion was being added. Urea, on the other hand, slightly reduced both the rate and the yield of Rubisco folding. A reduced yield, with no effect on the rate-limiting step of folding, suggested that the partitioning between productive and unproductive folding pathways had changed; possibly through an increase in the formation of species which do not fold to the native state. This hypothesis was first tested by measuring the rate at which Rubisco conformer(s) became unrecognizable by GroEL. Unfolded Rubisco conformer(s) were created by diluting acid-denatured protein into buffer-containing denaturants. In the absence of chloride ions, these Rubisco conformers do not spontaneously fold (Lorimer et al., 1993; Schmidt et al., 1994), and light scattering demonstrated that these species do not form large aggregates (see below). Excess GroEL was added at various times to bind these Rubisco conformers, and the amount of trapped Rubisco was measured. Guanidinium (but not urea) increased the rate at which these conformer(s) became unrecognizable to GroEL in a concentration-dependent manner, almost tripling the rate (Table 2). Urea slightly slowed this rate.
The simplest interpretation for the disappearance of unfolded conformers (other than progressing to the native state) is that these species aggregate. Chaperonins are known to suppress formation of high molecular weight aggregates which can easily be detected using light-scattering techniques (Buchner et al., 1991). Therefore we tested guanidinium for its effect on the rate of Rubisco aggregation. In the absence of guanidinium, neutralization of acid-denatured Rubisco resulted in a species which did not scatter light for at least 15 min (Fig. 5B). Upon addition of guanidinium HCl (but not urea), the rate and extent of light scattering increased. The increased scattering was not simply due to increased ionic strength, since addition of up to 250 mM potassium acetate failed to demonstrate a corresponding increase in aggregation. Rather, guanidinium allowed growth of Rubisco species, no longer recognizable by GroEL into particles large enough to scatter light.
Figure 5:
Effect of guanidinium chloride on the rate
of Rubisco aggregation. A, acid-denatured Rubisco was added to
buffers containing 0 (), 30 (
), 60 (
), or 90 (asterisk) mM guanidinium chloride or 200 mM urea (
). GroEL was added at the indicated time to trap any
Rubisco folding intermediate which had not progressed irreversibly to
unproductively folded states. The trapped Rubisco was subsequently
released and allowed to fold. B, light scattering of unfolded
Rubisco (added at 30 s, first arrow) was monitored for 15 min (trace a). After 300 s (second arrow), 60 (trace
b), 90 (trace c), 120 (trace d) mM guanidinium chloride, or 200 (trace e) mM urea
was added.
Fig. SIsummarizes the ATPase cycle of the GroEL
double-toroid (as viewed from the side, species A;
GroEL-GroEL
). In the absence of GroES,
hydrolysis proceeds through Cycle I, with the rate of ATP hydrolysis
being slower than either ATP binding or P
release (Jackson et al., 1993; Todd et al., 1993). In the presence of
GroES, the reaction cycle involves the formation of an asymmetric
complex (GroES
ADP
GroEL
-GroEL
), which subsequently breaks down,
then reforms (either through species B
D
F
A, or
through species B
D
E
C). At low potassium
concentrations, the affinity of the unliganded toroid of GroEL for ATP
is greatly reduced (Todd et al., 1993), and species B is
stable and can be isolated. The affinity for ATP can be subsequently
increased by raising the concentration of K
and ATP
hydrolysis restored. With each turnover on the free face of GroEL, the
tightly bound ADP on the opposite toroid is completely exchanged (Todd et al., 1994). If all seven nucleotide binding sites on the
unliganded toroid must be simultaneously occupied for quantized
turnover to occur (Todd et al., 1994), a reduced affinity for
ATP at each binding site decreases the probability of complete
occupancy and hence the frequency of ATP hydrolysis by that ring. Thus
at low potassium concentrations the affinity for ATP is reduced to the
extent that species D is never completely occupied, and the asymmetric
complex B remains completely inhibited. However, increases in the ionic
strength in general, and increases in the concentration of guanidinium
HCl in particular restored ATP hydrolysis (Fig. 1B).
The restoration of ATP hydrolysis by species B requires either (i) that
the affinity of the unliganded GroEL ring for ATP be increased so as to
populate species D or (ii) that GroES dissociates from species B, thus
returning the system to the uninhibited Cycle I. Since we have
demonstrated that the rate of GroES dissociation is increased
>100-fold in the presence of 80 mM guanidinium HCl ( Fig. 2and Fig. 3), we favor the latter explanation. At
low potassium concentrations, an increase in ionic strength or in the
concentration of guanidinium HCl causes the chaperonin complex to
dissociate and return to cycle I (i.e. species B
F
A). The rate of the dissociation step in various concentrations
of guanidinium was calculated from the data in Fig. 2and is
shown in Fig. 1B (asterisk). At concentrations
in excess of 100 mM, the complex dissociated faster than it
could be formed, thus ATP hydrolysis became independent of GroES. At
higher potassium concentrations, the asymmetric complex dissociation
may remain rate-limiting, or the affinity for ATP may be sufficiently
high to permit formation of species D. We cannot presently distinguish
these two alternatives.
Scheme I: Scheme I
The addition of guanidinium chloride to a
fully inhibited chaperonin complex temporarily relieves GroES
inhibition, allowing hydrolysis of additional ATP. Under such
circumstances GroEL continues hydrolyzing ATP until the ADP
concentration again rises to inhibitory levels (Todd et al.,
1993). The addition of guanidinium chloride thus induces a
``burst'' of ATP hydrolysis. The magnitude of this burst will
depend on the the ionic strength and on the concentrations of
K, guanidinium chloride, ATP, and ADP. Martin et
al.(1991) observed just such a burst upon adding rhodanese,
unfolded in guanidinium chloride, to a fully inhibited chaperonin
complex. They attributed this phenomenon to a specific stimulation of
the chaperonin ATPase activity. In view of the inability of unfolded
proteins, denatured by other methods, to induce the dissociation of the
chaperonin complex (Table 1), plus the ability of even modest
concentrations of guanidinium chloride alone to induce bursts of ATP
hydrolysis, we question the interpretation of Martin et al. (1991).
Spontaneous folding
experiments demonstrated that guanidinium decreased the yield, but had
no effect on the rate of folding. Instead, guanidinium increased the
rate of aggregate formation (k) (Fig. 5),
decreasing the amount of protein available for folding (U + U
). The chaperonin-assisted folding
reaction was less susceptible to aggregation, since GroEL could re-bind
the misfolded conformers and allow re-partitioning to occur.
GroEL
has now been observed to increase the rate of folding for two proteins:
Rubisco (4-fold in Table 2; Lorimer et al., 1993) and
malate dehydrogenase (Staniforth et al., 1994). This rate
increase could occur simply by increasing the concentration of the
species which has the potential to fold (Fig. SII, species U).
An increase in the rate constant, however, indicates that
there is a change in the rate-determining step, perhaps from k to k
. In agreement
with this hypothesis, Sosnick et al.(1994) demonstrated that
the spontaneous folding rate for the majority of cytochrome c conformers is limited by the reversal of misfolded structures (k
). For many proteins GroEL decreases the
rate of folding, perhaps by lowering the concentration of U by binding U
. A net decrease in protein available for
folding results when k
< k
, an increase occurs when k
> k
.
While many folding characteristics are specific to the protein being studied, the difficulties encountered by using guanidinium could be more universal. In comparing the folding yields of several proteins (Table 3), higher yields of spontaneous folding occurred when proteins were denatured in urea than when guanidinium chloride was used. Given the relatively slow mixing which occurs upon dilution of these viscous solutions (seconds), and thus the high local concentration of both denaturant and unfolded polypeptide, it is possible that enhanced intermolecular hydrophobic associations result in increased aggregation. Many in vitro protein folding reactions are performed by diluting a small volume of highly concentrated denatured protein by a large factor resulting in some variability in the final denaturant concentration. Given the sensitivity of the chaperonin interactions to low concentrations of guanidinium chloride and its possible effects on unfolded proteins, we suggest that denaturants other than guanidinium chloride be used when assessing chaperone-assisted folding activity.