(Received for publication, December 12, 1994; and in revised form, January 27, 1995)
From the
Hydrophobic exposure on the chaperonin GroEL is increased
6-10-fold after the protein is treated with the His-reactive
reagent diethyl pyrocarbonate (DEP), or the solution pH is lowered to
5.5. The induced hydrophobic surfaces have the same
1,1`-bis(4-anilino)naphthalene-5,5`-disulfonic acid (bis-ANS) binding
characteristics as unperturbed GroEL: a K
3.5 µM, a maximum intensity at
500
nm, and an average fluorescence lifetime of
8.0 ns. The
pK
for the pH-induced transition is 6.6,
most likely attributable to the only histidine in GroEL, His-401,
located in the intermediate domain. The modification of one histidine
residue per monomer upon DEP treatment is supported by the correlation
between the change in the absorbance at 242 nm for the N-carbethoxyhistidyl derivative and the increase in bis-ANS
fluorescence. GroEL at pH 5.5 is tetradecameric and can capture
urea-denatured rhodanese and release it as active enzyme. The
GroEL-rhodanese complex is more stable to dissociation by 2.25 M urea than the complex formed at pH 7.8. We propose that His-401 is
in a conformationally sensitive region such that protonation or
modification can lead to increased exposure of hydrophobic surfaces
capable of binding folding intermediates.
The proper folding and transport of nascent polypeptide chains in vivo can be facilitated by the group of proteins termed
molecular chaperones (1, 2). One class of chaperones, the chaperonins,
has been found in all prokaryotes, mitochondria, and chloroplasts (3).
The extensively studied chaperonin from Escherichia coli, GroEL, the homologue of the mitochondrial matrix protein hsp60
(heat shock protein with a M = 60,000), has
been shown to facilitate in vitro refolding of several
chemically denatured proteins, including rhodanese (4, 5),
ribulose-bisphosphate carboxylase/oxygenase (Rubisco) (6, 7), and
citrate synthase (8). GroEL is a tetradecamer (14-mer) of 57.2-kDa
subunits arranged as two stacked seven-membered rings (3, 9, 10). It
has been shown that the range of possible substrates for this
chaperonin is rather broad, with GroEL capable of binding about half of
the soluble proteins from E. coli cell lysate with relatively
high affinity (11). Such promiscuity brings into question the specific
recognition motif, which now appears to involve in some cases, but is
not necessarily restricted to, an amphipathic helix that may be
stabilized or induced by binding GroEL (12-16). Despite studies
supporting a model of GroEL interacting with substrate through multiple
binding sites (17, 18), the mechanism of binding and functional release
is still obscure, especially since the differing requirements for
release indicate various degrees of interaction between chaperonin and
substrate in the binary complexes(16) . Some proteins require
only K
and MgATP for release of functional enzyme from
the binary complex, whereas others also require the co-chaperonin GroES
(4-6, 19).
It has been proposed that the chaperonin binds the
``molten globule'' or ``compact intermediate'' form
of substrate proteins, a fairly compact state displaying significant
secondary structure, increased exposure of hydrophobic surfaces
compared with the native protein, and lacking a rigid tertiary
structure (4, 20). These hydrophobic surfaces have been suggested to
mediate the binding between chaperonin and substrate(2) .
However, according to quantitative bis-ANS ()binding
studies(5) , there are very few available sites on unperturbed
GroEL for such binding. Even in the papers discussing molten globule
intermediates as substrates, it is assumed that increased binding of a
hydrophobic probe by the complex is due entirely to exposure of
surfaces on the substrate protein(4) , without any concomitant
increase in the exposure of hydrophobic surfaces on GroEL. It has not
been shown by direct biochemical evidence that GroEL has the necessary
complementary hydrophobic surfaces for interaction with a folding
intermediate of substrate.
The recently released crystal structure
of a GroEL tetradecamer shows each monomer with an equatorial,
intermediate, and apical domain (21). The authors attribute the lack of
resolution for residues 222-248 in the apical domain to a region
of probable flexibility. As shown by the companion mutagenesis study,
single mutations of hydrophobic residues in this region and flanking it
(Y199E, Y203E, F204E, L234E, L237E, L259S, V263S, and V264S) abolish
peptide binding (22). Interestingly, the putative hydrophobic binding
pocket defined by the crystal structure and mutagenesis work is not
buried in the structure or apparently blocked by it but rather lines
the entrance to the inner channel and should be readily accessible to
binding by hydrophobic probes in solution. In fact, several studies
visualize via electron microscopy or suggest based on biochemical
evidence that GroEL binds substrate proteins in this channel (23-26).
The intermediate domain is composed of two stretches of amino acids
(134-190 and 377-408) which form three -helices and
three
-strands that not only connect the equatorial and apical
domains, but also make inter-monomer contacts between two highly
conserved stretches of residues(21) . Mutagenesis of residues
at several places in the intermediate domain (I150E, S151V, A152E,
A383E, A405E, and A406E) shows significant effects on one or more
properties of the chaperonin: folding of substrate, ATPase activity,
substrate binding, or GroES binding. Thus, the intermediate and apical
domains appear to have dynamic properties, critical to the interaction
between GroEL and substrate, that are not revealed by the static
crystal structure.
In this study, we show that GroEL displays a
pH-dependent exposure of hydrophobic surfaces, as measured by the
fluorescence of the hydrophobic probe bis-ANS. At pH 5.5, under
conditions where the protein retains its tetradecameric structure,
exposure of hydrophobic surfaces on GroEL can be induced to a level of
6-10-fold over that observed at pH 7.6. The pH dependence of the
hydrophobic increase shows a pK
6.6. This value, in addition to experiments with the His-reactive
reagent diethylpyrocarbonate, leads us to suggest that the single
histidine in GroEL monomers, residue 401 in the intermediate domain, is
in a region of the protein that has flexibility or is sensitive to
structural perturbations that can account for the observed pH-triggered
exposure of hydrophobic surfaces. Furthermore, binding of non-native
rhodanese to GroEL at pH 5.5 occurs to produce a complex that is more
stable to urea dissociation than a comparable complex at pH 7.8
described previously(17) .
Rhodanese was prepared as described previously (27) and stored at
-70 °C as a crystalline suspension in 1.8 M ammonium
sulfate. Rhodanese concentrations were determined using A = 1.75 (28) and a molecular mass of 33
kDa (29). Rhodanese activity was assayed using a colorimetric method
based on the absorbance at 460 nm of the complex formed between the
reaction product, thiocyanate, and ferric ion(28) . The
chaperonin, GroEL, was purified from lysates of E. coli cells
bearing the multicopy plasmid pGroESL which were grown at 37 °C
(30). The purification was a modified version of previously published
protocols (31), except that the Mono Q and hydroxylapatite columns were
excluded, but including the batch treatment with Affi-Gel blue as
described in(19) . After purification, GroEL was dialyzed
against 50 mM Tris-HCl, pH 7.5, and 1 mM dithiothreitol, then made 10% (v/v) in glycerol, rapidly frozen in
liquid nitrogen, and stored at -70 °C. The protomer
concentration of GroEL was measured using the bicinchoninic acid
protein assay (Pierce) according to the procedure recommended by the
manufacturer.
Reaction mixtures with diethylpyrocarbonate,
displaying maximal absorbance change at 242 nm, were treated with
hydroxylamine adjusted to pH 7 (0.5 M final concentration).
The extent and rate of the reaction with NHOH were followed
spectrophotometrically, either by taking spectra between 220 and 300 nm
or by following the absorbance change at 242 nm.
Figure 1:
Bis-ANS fluorescence of DEP-treated and
untreated GroEL at different pH values. A solution with 1 µM GroEL was treated with 0.25 mM DEP at room temperature
for 1 h (filled diamonds) or with 0.50 mM DEP at room
temperature for 50 min (open circles). bis-ANS (final
concentration, 10 µM) was added to the treated protein,
and the fluorescence at 500 nm was measured with excitation at 360 nm.
Microliter aliquots of 1 mM HCl were used to adjust the pH of
the solution between 7.8 and
4.5. At each pH value the
fluorescence intensity was read and the pH, as shown, was measured. Filled circles, a solution with 1 µM GroEL
(untreated) and 10 µM bis-ANS was titrated with HCl
between pH
7.8 and
4.5. As above, the pH was measured at each
point where the fluorescence was measured. Open squares, a
solution at pH 5.5, containing the same concentrations of GroEL and
bis-ANS used above, was titrated with NaOH to pH
7.6.
To further test the possibility that the observed pH-dependent exposure of hydrophobic surfaces was due to the protonation of the His imidazole group, a His-reactive reagent was used to pretreat GroEL before acid titration. DEP reacts with His side chains to produce an N-carbethoxyhistidyl derivative (35-37). The top curve in Fig. 1shows the titration of GroEL that had been pretreated with 0.25 mM DEP at room temperature for 1 h (filled diamonds). The approximate maximum intensity of bis-ANS fluorescence achievable upon addition of acid (e.g. the intensity at pH 5.5) was not affected, but the modified protein displayed considerably more hydrophobic surfaces at neutral pH. In fact, when GroEL was pretreated with 0.5 mM DEP at room temperature for 50 min, prior to the acid titration analysis, 97.3% of the maximum fluorescence seen at pH 5.5 was seen at neutral pH (Fig. 1, open circles). Thus, modification with DEP abolished the observed pH-dependent change in bis-ANS fluorescence described above.
Reaction of DEP with residues other than histidine,
especially lysine or tyrosine, has been demonstrated (35, 38). However,
since the reaction requires an unprotonated nucleophile, modification
of His is the most likely explanation given the solution conditions
used here. To support this, we carried out several experiments to
verify that the modification was occurring at His-401. First, the
absorbance of the reaction was monitored. As shown in Fig. 2A, the result was an increase in absorbance at
242 nm as has been previously demonstrated for DEP modification of
histidine in other
proteins(35, 36, 37, 38) , whereas
there was no significant change at any other wavelength, including 278
nm (the wavelength at which modified tyrosines absorb). Second,
hydroxylamine was used to test for the reversibility of DEP
modification. Melchior and Fahrney (39) found that 0.5 M NHOH, adjusted to pH 7, removes N-carbethoxy
groups from imidazole. In the present case, absorbance spectroscopy
showed that hydroxylamine completely reversed the modification by DEP
(data not shown). Since reversal has been shown to be possible with
only histidyl and tyrosyl derivatives, but not with other modified
residues(35, 38) , and since tyrosine does not appear
to account for the modification observed here, we take this as evidence
of a His modification. It is also evidence that the His residue is only
singly modified and has not formed a derivative that is modified at
both imidazole nitrogens, which is not reversible by treatment with
NH
OH(35) .
Figure 2:
A,
absorbance spectra of DEP-modified GroEL. A solution of 16.6 µM GroEL in 100 mM NaHPO
, pH 7, was
treated with 0.5 mM DEP at room temperature and the difference
absorbance spectra were taken at various times. Representative curves
are shown at 40 s, 105 s, 5 min, and 7 min 40 s (starting from bottom). B, relationship between fluorescence of DEP-modified GroEL and
the number of histidine residues modified. Filled circles, the
absorbance and fluorescence of solutions containing 16.6 or 1
µM GroEL, respectively, 100 mM NaH
PO
, pH 7, and 0.5 mM DEP were
followed for 30 min. For various time points, the fluorescence is
plotted versus number of His residues modified (based on
absorbance, using
= 3200 M
cm
). The solid line is a best fit to
the first seven data points, extrapolated to 100% fluorescence. The
extrapolated value for the number of His residues modified/monomer
GroEL at 100% fluorescence is 1.19.
Since DEP modification produced an
increase in bis-ANS fluorescence and in absorbance at 242 nm, the
kinetics for the reaction were followed by both absorbance and
fluorescence spectroscopy. In each case the data were best fit by
exponential curves, displaying pseudo-first order kinetics with a
theoretical A = 0.10 and k = 0.004 s
; a F
= 5.0 and k = 0.009 s
. Fig. 2B shows the results after the data was treated as
described previously (35, 36, 37) . The curve
relating fluorescence to the number of His residues modified (based on
absorbance) extrapolates at 100% fluorescence to
1.19 His residues
modified per GroEL monomer, which is consistent with the interpretation
that one histidine per monomer was modified.
Figure 3:
Binding of bis-ANS to GroEL at pH 5.5 and
pH 7.8. Separate samples of 1 µM GroEL at pH 5.5 (as
adjusted by HCl), with increasing amounts of bis-ANS, were excited at
360 nm, and the fluorescence intensity was read at 500 nm (upper
curve, squares). The curves were generated using a nonlinear least
squares procedure in the software program, PS Plot (Poly Software
International, Salt Lake City, UT). The K and I
, as taken from the fitted
curve, are 3.9 and 4.9, respectively. Separate samples were prepared
and fluorescence measured as above, except that a 50 mM Tris-HCl, pH 7.8, buffer was used (lower curve, circles).
The K
and I
for
this curve (as taken from the fitted curve) are 3.5 and 0.47,
respectively.
Consistent with this is the
fact that the wavelength of maximum fluorescence intensity did not
change significantly between pH 7.8 and pH 5.5 (data not shown). The
wavelength of bis-ANS fluorescence shifted 60 nm upon binding to
GroEL from aqueous solution, whereas the maximum shift upon pH change
was <5 nm. This suggests that the hydrophobic nature of the binding
sites did not change significantly, since it has been shown that the
wavelength maximum of bis-ANS fluorescence is sensitive to the
hydrophobic character of the binding site (40, 41).
To confirm that
these observed changes in fluorescence intensity were in fact due to an
increase in the number of bis-ANS molecules bound, and not due to
different fluorescence quantum yields at the different pH values,
lifetime measurements were made at the two extremes (pH 7.8 and 5.5).
The decay at each pH was best fit by three components. At pH 7.8:
1 = 1.78 ns,
1 = 0.188;
2 = 6.57
ns,
2 = 0.794; and
3 = 130.24 ns,
3
= 0.0185. At pH 5.5:
1 = 1.51 ns,
1 =
0.230;
2 = 6.93 ns,
2 = 0.751; and
3
= 128.45 ns,
3 = 0.0191. The reduced
values for the data at pH 7.8 and 5.5 were 2.94 and 0.904,
respectively, consistent with the fact that the sampling error
decreases as the signal increases, and the reduced
approaches 1. Thus, the average lifetimes were 7.96 ns and 8.00
ns at pH 5.5 and 7.8, respectively. The average lifetimes and the
fractional contributions of the individual components indicate that the
fluorescence lifetime did not vary with pH. Therefore, there was no
significant change in the quantum yield of the bis-ANS as a function of
pH so that an increase in the area of hydrophobic surface best accounts
for the observed increase in fluorescence.
As a control to verify that this apparent difference in urea
stability was due to the stability of the complex and not due to the
difference in urea effectiveness at different pH values, possibly
because of differential cyanylation, the stability of the GroEL
tetradecamer at the two pH values against urea dissociation was
examined as described previously(50) . The results were
consistent with the fact that the low pH form shows higher
fluorescence, even in 0 M or low concentrations of urea (not
shown), and were also consistent with an unfolding transition and
decreased fluorescence between approximately 2 and 4 M urea.
Based on these results, there was no significant difference between the
effectiveness of urea at pH 5.5 and pH 7.8, meaning that the greater
stability of the GroEL-Rho complex was not due to a
decreased effectiveness of the chaotrope.
The mechanism for the capture of folding intermediates by
GroEL and the release of functional proteins is not well understood,
although pieces of the scheme are coming together from different lines
of investigation. There is now good evidence that hydrophobicity is a
necessary characteristic for the substrate (42-44) and that in some
cases it must be present in an amphiphilic
context(12, 13) . Additionally, based on the crystal
structure and mutant studies (21, 22) , there is now a
better idea of where this complementary hydrophobic binding site might
exist on the chaperonin. The challenge is to incorporate this
structural information with current models based on the dynamics and
biochemistry of the system. To bind substrate proteins in non-native,
molten globule states, GroEL must be able to display significantly more
hydrophobic surfaces than has previously been demonstrated(5) .
Additionally, the generality of this kind of interaction suggests that
exposure of these ``sticky'' surfaces would somehow be
regulated, with exposure predicated on induced fit of the non-native
substrate or a triggered response, such that nonproductive interactions
do not interfere with the chaperonin. Such an induced response has been
proposed for another chaperone, SecB (45-47), is consistent with
previous reports for folding of -lactamase by GroEL (48) and is
reasonable considering the structural transition in GroEL recently
shown by Hansen and Gafni (10, 49).
That GroEL displays the same
significant enhancement of bis-ANS fluorescence when the pH is lowered
or when treated with DEP suggests very similar structural changes in
the tetradecamer under the two conditions. It is important to realize
that this large conformational change in the oligomer occurs simply
with the protonation or N-carbethoxylation of His-401. The His
residue in question falls in the middle of a highly conserved, extended
-helix in the intermediate domain which, at its N-terminal end,
makes contacts to a highly conserved region of the adjacent
monomer(363-378) in the tetradecamer(21) . Based on the
amino acid sequence, this
-helix has considerable hydrophobic
character in the region around 401 (3, 50), and although we cannot say
where the side chain lies, until the three-dimensional coordinates are
released, it is reasonable that it is buried in a hydrophobic region of
the protein such that protonation perturbs the native structure.
Introducing a charge into a hydrophobic environment might be expected
to have dramatic conformational consequences, explaining the increased
exposure of hydrophobic surfaces. Alternatively, the arrangement of
side chains may be such that N-carbethoxylation causes a
steric clash that distorts the structure of the intermediate domain. We
are not suggesting a mechanistically important role for His-401, since
published sequences show that the His is not a highly conserved residue
(51), and since not all site-directed mutations at this position are
deleterious (see below). Rather, our contention is that His-401 is in a
structurally sensitive region where small perturbations, especially due
to the introduction of charge, can produce large increases in exposure
of hydrophobic surfaces. This would be consistent with the idea that
the intermediate domain serves as a hinge region within a monomer
and/or as a connecting region that allows ``communication''
between adjacent monomers in the tetradecamer(21) . In fact,
the mutations made by Fenton et al.(22) in the
intermediate domain show results that support a similar conclusion.
It is interesting that neither the His Phe mutant made by
Fenton et al.(22) nor a similarly uncharged
substitution we have made (His
Leu) showed any non-native
characteristics, when evaluated according to urea stability,
ATP-dependent chaperonin activity, and ability to arrest rhodanese
refolding. (
)This is in contrast to another mutant, where a
constitutive negative charge was introduced (His
Asp), which
yielded little soluble, recoverable GroEL in the supernatant after
centrifugation of the E. coli cell lysates (where GroEL is
normally found during purification). The protein was instead found
aggregated in inclusion bodies.
These seemingly disparate
results make sense within the confines of the present model. If
exposure of hydrophobic surfaces on GroEL is affected by flexibility in
the intermediate domain, then mutation to a constitutive negative
charge at residue 401 could significantly change the structure in that
region. The result would be a protein that aggregates in vivo due to the exposure of large amounts of hydrophobic surface. It is
interesting to note here that DEP-modified GroEL, which constitutively
displays significant hydrophobic surface, can arrest the spontaneous
refolding of denatured rhodanese, but will not release the active
enzyme, even upon addition of MgATP. (
)Similarly, some of
the single mutations of residues in the intermediate domain (I150E,
S151V, A383E, and A405E) result in a protein that lacks the ability to
release substrate or has a decrease in release(22) .
Other
recent work (50, 52) shows that various salts and polycations can
induce exposure of hydrophobic surfaces on GroEL without dissociation
of the tetradecamer or can increase the affinity between GroEL and
substrate. Based on that work and the present results, it is tempting
to speculate that exposure of functional hydrophobic surfaces on GroEL
can be triggered by ionic perturbations. This would be in keeping with
the earlier results demonstrating that GroEL preferentially binds
amphiphilic -helices that are positively charged (12, 13, 53) and
makes sense, given the recent results suggesting that divalent cations
cause structural changes in GroEL that increase the rates of
cross-linking between monomers (54).