From the Division of Biochemical Sciences, National Chemical
Laboratory, Pune 411008, India
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INTRODUCTION |
The folding and assembly of a protein into its biologically active
conformation is a complex succession of reactions involving the
formation of secondary and tertiary structures and domains, the pairing
of domains, and the oligomerization of folded monomers (1, 2).
Elucidation of the various processes that govern protein folding has
been the focus of intense research for the past decades. Numerous
in vitro protein folding experiments have demonstrated that
many proteins successfully achieve their correct native structures in
the complete absence of other cellular factors and without input of
energy. This led to the expectation that protein folding is determined
by the information encoded by the amino acid sequence and proceeds
in vivo by the same spontaneous mechanism (1, 2). However,
in vivo folding and assembly of proteins occur in a highly
complex heterogeneous environment, in which high concentrations of
proteins in various stages of folding and with potentially interactive
surfaces coexist that may change the folding potentials inherent in the
sequence. Recently, a number of accessory proteins have been identified
that affect the folding and subsequent assembly of proteins. These
include the protein isomerases catalyzing
cis-trans-isomerization of peptide bonds or disulfide
exchange (3, 4) and the polypeptide binding proteins termed as
"molecular chaperones" (5). The molecular mechanisms by which the
protein isomerases accelerate the rate-determining step in the folding
of proteins are understood but perhaps not in detail (3, 4).
Elucidating the mechanistic details underlying the efficient refolding
of proteins by chaperones now appears to be an important consideration
for defining how proteins fold in vivo. The GroEL and GroES
proteins from Escherichia coli are among the most detailed
characterized chaperones (5). Horwitz (6) and other workers (7-9) have
shown that
-crystallin acts as a molecular chaperone under various
denaturing conditions including thermal inactivation, UV irradiation,
or reduction of disulfide bonds. Until recently,
-crystallin was
believed to be a lens-specific protein; however, now it has been
reported to be present in many non-lenticular tissues (10, 11). The
expression of
-crystallin has been shown to be induced by thermal
(11) or hypertonic stress (12). Numerous studies provide evidence that
the ability of
-crystallin to suppress aggregation of damaged
proteins plays a crucial role in maintaining the transparency of the
ocular lens, and the failure of this function could contribute to the
development of cataracts (13, 14). Important recent development is the finding that
-crystallin shows extensive structural similarity with
small heat shock proteins that are known to act in vitro as
molecular chaperones (15, 16).
-Crystallin has been shown to be
functionally equivalent to the small heat shock proteins namely murine
Hsp25 and human Hsp27 in refolding of
-glucosidase and citrate
synthase in vitro (16). It was, however, unable to refold
rhodanase denatured in 6 M
GdmCl1 (17). Recently
-crystallin has been reported to bind the temperature-induced molten
globule state of proteins (18, 19) and prevent photoaggregation of
-crystallin by providing hydrophobic surfaces (8). Despite the
growing interest in the chaperone action of
-crystallin, little is
known about its mechanism of chaperoning. For this functional in
vitro analysis of
-crystallin we used xylose reductase (XR), from Neurospora crassa as the model system. This
oxidoreductase plays a crucial role in the fermentation of xylose to
ethanol (20) and has recently gained more importance due to its
application in the synthesis of xylitol, an acariogenic non-caloric
sweetener used in food products (21). Earlier we have undertaken
structure-function studies to understand the contribution of essential
amino acids in the catalytic mechanism of XR, an enzyme that belongs to
the important class of oxidoreductases; the conformation and
microenvironment of the active site has also been assessed using
fluorescent chemoaffinity labeling (22-24). Various studies on XR will
assist in its biotechnological exploitation. Experimental evidence
presented in this paper serves to implicate that the chaperone
-crystallin stabilizes the molten globule state of XR and thus
restrains the non-native conformer from exploring unproductive
pathways. Lowering the temperature to 4 °C or presence of ATP at
37 °C induces a conformational change in
-crystallin·XR-m
complex that is accompanied by a concomitant internalization of
hydrophobic surfaces previously exposed. This acts to reduce the
hydrophobic interactions that facilitates the dissociation of the
complex further allowing reconstitution of the active XR. This paper
reports for the first time the mechanism of
-crystallin-mediated
reconstitution of an active enzyme and the role of ATP in the function
of
-crystallin as a molecular chaperone.
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EXPERIMENTAL PROCEDURES |
Materials--
NADPH, ATP, AMP-PNP, ANS
(8-anilinonaphthalene-1-sulfonic acid),
-crystallin (c-4163), GdmCl
(guanidinium chloride) were purchased from Sigma and Centricon-100
microconcentrators were from Amicon. All chemicals used were of
analytical grade.
Xylose Reductase Purification and Assay--
Purification of the
Neurospora crassa xylose reductase was according to the
procedure described earlier (22). The enzyme activity was measured
spectrophotometrically at 28 °C by monitoring the decrease in NADPH
absorbance at 340 nm. The reaction mixture (1 ml) contained 50 mM sodium phosphate buffer, pH 7.2, 0.15 mM NADPH, and an appropriate amount of XR. The reaction was started by
addition of D-xylose (final concentration 250 mM). Enzyme units are defined as µmol of NADPH oxidized
per min. Protein concentration has been determined by the method of
Bradford (25) with bovine serum albumin as a standard. The molar
concentration of XR was calculated assuming a Mr
of 60,000 (22).
Denaturation/Renaturation Studies of XR--
All denaturation
and renaturation experiments were carried out in 50 mM
sodium phosphate buffer, pH 7.2. XR was incubated with varying
concentrations of GdmCl for 1.5 h at 28 °C, and the accompanying structural changes were investigated using fluorescence and circular dichroism measurements. Renaturation was initiated by
rapidly diluting the sample (XR at varying states of denaturation) in
the same buffer with or without
-crystallin, and the XR activity at
various times of refolding was measured. When the effect of adenine
nucleotides ATP, AMP-PNP, ADP, and AMP on the
-crystallin-assisted renaturation of XR was to be investigated these were added 1 h after initiation of renaturation process. Renaturation of XR was also
carried out by substituting
-crystallin with bovine serum albumin
(0.6 mg/ml). Other experimental details have been described in the
legends to figures.
Circular Dichroism and Fluorescence Studies--
Circular
dichroism (CD) spectra were recorded on a JASCO J600 model
spectropolarimeter. Changes in the secondary structure of XR induced by
the denaturant GdmCl were monitored in the far UV region (200-250 nm)
using a 1-mm path length cell. The tertiary structure was monitored in
the near UV region (250-320 nm) using a 10-mm path length cell. The
enzyme concentrations in these experiments was 0.5 mg/ml. Mean residue
ellipticities [
] (expressed as degree cm2
dmol
1) were determined according to Ref. 26.
Fluorescence spectra were recorded with a Perkin-Elmer LS 50B
spectrofluorimeter equipped with a Julabo F20 water bath or Aminco
SPF-500 spectrofluorimeter. The excitation and emission wavelengths
have been mentioned in the legends to figures. Corrections due to inner
filter effect were made according to the formula Fc = F antilog
((Aex + Aem)/2), where
Fc and F are the corrected and
uncorrected fluorescence intensities, and Aex
and Aem are the sample absorbance at the
excitation and emission wavelengths, respectively (27).
Additional experimental details have been described in the text and
figure legends.
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RESULTS AND DISCUSSION |
Folding Intermediates of XR--
The CD spectrum of XR in the far
UV region (200-260 nm) exhibited a strong negative ellipticity in the
region 215-222 nm and a weaker one at 208 nm, characteristic of a
protein having an
-helix (data not shown). XR was incubated with
increasing concentrations of the denaturant GdmCl, and the changes in
the negative CD band in the far UV region were monitored. The mean
residue ellipticities obtained at 220 nm [
]220 were
normalized with respect to that in the absence of GdmCl and plotted
against the respective GdmCl concentration (Fig.
1). A decrease in the negative
ellipticity was observed with the addition of GdmCl, and at 1.4 M GdmCl the [
]220 decreased by almost 43%
of that in the absence of GdmCl. Further increase in the denaturant
resulted in a loss of negative ellipticity until there was a total loss
of structure of the CD band in 6 M GdmCl indicating a
considerable loss of secondary structure (Fig. 1). 6 M
GdmCl converted XR into unfolded polypeptides, and this state has been
referred to as XR-u. The CD spectrum of XR was also monitored in the
near UV region (250-320 nm) to study GdmCl-induced changes in the
environment of tryptophan and tyrosine side chains. In the absence of
the denaturant, the spectrum exhibited a broad negative band with a
double minimum at 278 and 285 nm. However, the presence of 1.4 M GdmCl resulted in a decrease in the negative ellipticity
in this region similar to that of the unfolded XR in 6 M
GdmCl indicating that the aromatic residues in this state were no
longer in an asymmetric environment (data not shown).

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Fig. 1.
Dependence of mean residue ellipticity of XR
at 220 nm on GdmCl. XR was incubated with varying concentrations
of GdmCl for 1.5 h at 28 °C in 50 mM sodium
phosphate buffer, pH 7.2, and the CD spectras in the far UV region
(200-250 nm) were recorded. Protein concentration was 0.5 mg/ml, and a
1-mm path length cell was used. The ellipticity values obtained were
normalized with respect to that in the absence of GdmCl.
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We used the fluorophore ANS to determine the relative amount of exposed
hydrophobic surfaces in the folding intermediates of XR. ANS is not
fluorescent in aqueous solutions (
em 525 nm); however,
on addition of proteins containing hydrophobic pockets its emission
maximum shifts to shorter wavelengths, and the emission intensity is
enhanced. As shown in Fig. 2 the binding
of ANS to XR was measured as a function of GdmCl. A maximum increase in the ANS fluorescence (
em 475 nm) was observed at 1.4 M GdmCl indicating maximum exposure of hydrophobic surfaces
in this state of XR. At higher concentrations of the denaturant, a
decrease in the intensity of the dye fluorescence was observed which
was accompanied by a shift in the
em toward red
indicating unfolding of XR (Fig. 2). ANS has been widely used to detect
the formation of molten globule-like intermediates in the folding
pathways of several proteins (28). This state is characterized to be as compact as the native protein with solvent-accessible hydrophobic regions and appreciable amount of secondary structure but no rigid tertiary structure (29, 30). It was thus evident from the CD studies
that at 1.4 M GdmCl XR retains substantial amount of secondary structure (Fig. 1) but very little tertiary structure (data
not shown). Altogether our CD and ANS binding studies revealed that at
1.4 M concentration of GdmCl XR partially unfolded to its
molten globule state which has been referred to as XR-m.

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Fig. 2.
GdmCl-dependent exposure of
hydrophobic surfaces of XR measured by ANS fluorescence. ANS
fluorescence intensity at 475 nm ( ) and max ( ) on
incubation of 0.25 µM GdmCl-treated XR (described in
legend to Fig. 1) with 10 µM ANS for 15 min. The
concentrations indicated are the final concentrations. The samples were
excited at 375 nm.
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Chaperone-assisted Renaturation of XR--
Attempts to refold XR
from the XR-u state in the absence and presence of
-crystallin were
unsuccessful (Fig. 3). Similar results
were obtained for progressively less denatured states (XR denatured
with 2-4 M GdmCl). Further investigations were carried out
to study the influence of
-crystallin on the renaturation of XR-m.
The refolding of XR-m was initiated at 28 °C in the absence/presence of
-crystallin; after 30 min the samples were shifted to varying temperatures (Fig. 3), and the XR activity recovered at different time
intervals was measured. As shown in Fig. 3, XR-m lacked the ability to
spontaneously reconstitute active XR. However, in the presence of
-crystallin the renaturation process at 4 °C followed a sigmoidal
time course. As can be observed from inset of Fig. 3, there
was no measurable XR activity for the first 15 min (lag phase). Thus
similar to renaturation of oligomeric proteins (2), inactive XR
monomers may be produced in an early folding step which then undergo
additional folding and/or association prior to the assembly of XR into
active oligomers. The rate of reactivation beyond the lag phase was
slow, and a maximum 55% of the XR activity was recovered in 6 h.
A value of 112 min was observed for t1/2, where the
activity recovered was half of the maximal extent. At 28 °C, the
renaturation process yielded a maximum 14% of the XR activity, whereas
the values for the lag phase and t1/2 were similar to that observed at 4 °C.
-Crystallin, however, failed to
reconstitute active XR at 37 °C (Fig. 3). These temperature-shift
experiments thus revealed that the complex of
-crystallin and the
bound XR is stable at 37 °C but is cold-labile since lowering the
temperature of the renaturation process from 28 to 4 °C resulted in
the reconstitution of active XR. Unlike the observations at 28 °C,
-crystallin failed to reconstitute active XR when the refolding
process was initiated at 4 °C.

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Fig. 3.
Time course of renaturation of XR in the
absence or presence of -crystallin. XR at a concentration of 25 µM was incubated with 6 or 1.4 M GdmCl for
1.5 h at 28 °C to yield XR-u and XR-m, respectively. The
renaturation process was initiated at the same temperature by diluting
10 µl of the sample into a final volume of 1 ml of 50 mM
sodium phosphate buffer, pH 7.2, with or without -crystallin (final
concentration 0.6 mg/ml). After 30 min the samples were kept at varying
temperatures, and 100-µl aliquots of the refolding solution were
withdrawn at various times of refolding and assayed for XR activity as
described under "Experimental Procedures." represents refolding
of XR-m in the absence of -crystallin and in its presence when the
refolding solution was shifted from 28 °C to the following
temperatures: 4 °C ( ), 28 °C ( ), and 37 °C ( ). and represents the refolding of XR-u and progressively less
denatured states of XR (XR denatured with 2-4 M GdmCl) in
the absence and presence of -crystallin under the experimental
conditions described above. The percentage activity recovered was
determined with reference to native XR. The inset shows the
early time course of -crystallin-assisted renaturation demonstrating
the lag phase (first 15 min).
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The
-crystallin-mediated renaturation of XR was examined as a
function of the chaperone concentration. As shown in Fig.
4, 5% of the original XR activity was
recovered at the lowest concentration of
-crystallin (0.05 mg/ml).
The extent of renaturation increased in a
concentration-dependent manner, and a maximum 55-57% of
the original activity was recovered at
-crystallin concentration of
0.6-0.8 mg/ml. The concomitant increase in the extent of renaturation with an increase in
-crystallin can be attributed to simple mass action effects, wherein an increase in the
-crystallin concentration would increase the collisional frequency so as to favor the formation of
-crystallin·XR-m complex as opposed to forming non-native XR.
To test the specificity of
-crystallin, the renaturation of XR-m was
also investigated in the presence of bovine serum albumin alone (0.6 mg/ml), under the conditions described for renaturation with
-crystallin. It was observed that unlike
-crystallin bovine serum
albumin failed to mediate the reconstitution of active XR.

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Fig. 4.
Reactivation of XR-m at varying
concentrations of -crystallin. XR-m was renatured (as described
in the legend to Fig. 3) in the presence of varying concentrations of
-crystallin. After 30 min the samples were kept at 4 °C, and
aliquots withdrawn after 6 h were assayed for XR activity. The
percentage activity recovered is with respect to native XR.
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Dependence of Renaturation Process on the Time Interval between
Initiation of Refolding and Addition of
-Crystallin--
-Crystallin mediated reconstitution of active
XR only from its XR-m state (Fig. 3), indicating that the chaperone
probably traps the oxidoreductase in a conformation resembling the
molten globule. Evidence for this observation was provided by delay
experiments wherein refolding of XR-m was initiated in a solution
lacking
-crystallin which was then added at the indicated times. As
shown in Fig. 5, a concomitant decrease
in the ability of
-crystallin to reconstitute active XR from the
XR-m state was observed with an increase in the time between the
dilution of XR-m and the addition of
-crystallin. The increase in
XR-m concentration also resulted in a progressive decrease in the yield
of reconstituted XR (Fig. 5) indicating that the loss of recoverable XR
was due to aggregation and not due to some irreversible isomerization.
These results reveal that when XR-m is diluted into a solution
containing
-crystallin two competitive processes occur, namely
aggregation or formation of
-crystallin·XR-m binary complex. Since
aggregation is a second-order process it can be much faster than
first-order folding (31) and hence predominant with increasing
concentrations of XR-m. The delay experiments also revealed the
inability of
-crystallin to redissolve XR aggregates formed in its
absence.

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Fig. 5.
The lability of XR-m in the absence of
-crystallin and the concentration dependence of its decay
aggregation. Renaturation of varying concentrations of XR-m was
initiated at 28 °C in 50 mM sodium phosphate buffer, pH
7.2, lacking -crystallin. The chaperone (final concentration 0.6 mg/ml) was then added at the indicated times. The refolding solution
was stirred vigorously to ensure rapid mixing. After 30 min, the
samples were kept at 4 °C; aliquots were withdrawn after 6 h
and assayed for XR activity. The final concentrations of XR-m in the
refolding solution were 200 nM ( ), 300 nM
( ), and 400 nM ( ). The percentage activity recovered
was determined with reference to equal concentration of native XR. The
data are normalized with respect to the time 0 points for each XR-m
concentration.
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-Crystallin Forms a Complex with Folding
Intermediate--
Fluorescence studies were performed to confirm that
the XR bound to
-crystallin exists in the molten globule state. The
tryptophanyl fluorescence of native, denatured, and
-crystallin-bound XR is shown in Fig.
6. Native XR exhibited an emission
maximum at 326 nm, whereas in 6 M GdmCl the emission
maximum was shifted to 350 nm which corresponds to the fluorescence
maximum of tryptophan in aqueous solution. The XR bound to
-crystallin exhibited an emission maximum at 334 nm indicating that
the tryptophans in the bound form of XR are more exposed to the solvent
than the native enzyme. The increase in fluorescence intensity of the
-crystallin-bound XR may be attributed to the denaturant-induced
changes in the microenvironment of the tryptophans of XR or may be due
to interactions of the partially unfolded protein with
-crystallin.
Altogether these results revealed that the conformation of XR bound to
-crystallin is neither native-like nor completely unfolded but a
partially folded intermediate resembling the molten globule. Thus by
sequestering the molten globule state of XR in the form of a stable
binary complex,
-crystallin is able to suppress their interaction
that would otherwise lead to aggregation.

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Fig. 6.
Tryptophanyl fluorescence of free and
-crystallin-bound XR. XR at a concentration of 25 µM was incubated with 1.4 M GdmCl for
1.5 h, and a further 10 µl of the sample was diluted into a
final volume of 1 ml in 50 mM phosphate buffer, pH 7.2, containing 0.6 mg/ml -crystallin at 37 °C. After 30 min
incubation the refolding solution was diluted to 2 ml, and the
tryptophanyl fluorescence was recorded. The fluorescence spectrum of
bound XR (---) without contribution from -crystallin was obtained on
subtracting the spectrum of -crystallin·XR-m complex from that of
-crystallin. - - - and -·- represent the fluorescence spectra of
native and denatured XR in the absence or presence of 6 M
GdmCl, respectively. All samples were excited at 295 nm.
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Temperature Dependence of the Exposure of Hydrophobic Surfaces of
-Crystallin·XR-m Complex--
The temperature dependence of the
hydrophobic interactions in protein folding has been studied earlier by
Baldwin (32). Maximum stabilization of these interactions is observed
at high temperature where the enthalpy is the dominating factor in
determining the stability, and as the temperature is decreased the
interactions are weakened. Our temperature-shift experiments (Fig. 3)
revealed an increase in the
-crystallin-mediated reconstitution of
XR with the decrease in the temperature of the refolding solution implying that the hydrophobic interactions play a crucial role in the
formation of
-cystallin·XR-m complex.
Attempts were made to correlate temperature-mediated alterations
in the hydrophobic surfaces of the
-crystallin·XR-m complex to
reconstitution of active XR, using ANS as a probe for apolar binding
sites whose fluorescence is dependent on the hydrophobicity of the
environment. As shown in Fig. 7, presence
of
-crystallin·XR-m complex incubated at 37 °C resulted in a
blue shift in the ANS fluorescence from 525 to 475 nm accompanied by an
increase in fluorescence intensity; however, in the presence of the
complex incubated at 4 °C a 35% decrease in the dye fluorescence
was observed compared with that at 37 °C (Fig. 7). These results
indicate that at 37 °C the complex exists in a state with
hydrophobic binding sites that are accessible to ANS; however, a
decrease in the incubation temperature to 4 °C probably mediates a
conformational change in the complex that is accompanied by
internalization of the hydrophobic surfaces previously exposed. This
further acts to weaken the hydrophobic interactions holding the
-crystallin·XR-m complex and thus reduces the affinity of
-crystallin for the substrate protein further, allowing
reconstitution of active XR (Fig. 3). This observation also explains
the cold lability of
-crystallin·XR-m complex and the inability of
-crystallin to reconstitute active XR when refolding was initiated
at 4 °C.

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Fig. 7.
Temperature-dependent exposure of
hydrophobic surfaces of -crystallin·XR-m complex measured by ANS
binding. Renaturation of 250 nM XR-m was initiated at
37 °C in 50 mM phosphate buffer containing 0.6 mg/ml
-crystallin preincubated at the same temperature for 2 h. After
30 min the samples were incubated at 37 °C or shifted to 4 °C;
furthermore, ANS (final concentration 100 µM) was added
after 12 h, and the fluorescence was recorded at the respective
temperatures (maintained ±0.2 °C by Julabo F20 temperature water
bath) at 1 h incubation, with the excitation wavelength fixed at
375 nm. The bottom spectrum represents that of 100 µM ANS alone.
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Fluorescent Chemoaffinity Labeling of XR Renatured in the Presence
of
-Crystallin--
Fluorescent chemoaffinity labeling studies were
performed using o-phthalaldehyde as the chemical initiator
to shed some light on the conformation of XR renatured in the presence
of
-crystallin. Chemoaffinity labeling is a powerful technique and
combines some of the advantages associated with the photoactivated and
electrophilic affinity labeling. o-Phthalaldehyde is a
bifunctional agent that cross-links SH and NH2 groups
situated in close proximity to form an isoindole derivative that
exhibits strong fluorescence (33). XR reacts with
o-phthalaldehyde resulting in the formation of fluorescent
XR-isoindole derivative at the active site (
ex 338 nm;
em 410 nm) (23, 24). As shown in Fig.
8, incubation of the
-crystallin
renatured XR with o-phthalaldehyde resulted in the formation
of XR-isoindole derivative as observed with the native enzyme. However,
XR when renatured in the absence of
-crystallin failed to form the
derivative. These results suggest that
-crystallin mediates
refolding of XR to a conformation similar to that of the native
enzyme.

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Fig. 8.
Isoindole fluorescence of
-crystallin-renatured XR on reaction with
o-phthalaldehyde. XR-m was renatured as described in
legend to Fig. 3. After 30 min the refolding solution was further
incubated at 4 °C for 6 h, when maximum XR activity was
recovered. The sample was repetitively filtered through Centricon-100
microconcentrators (Amicon) with 100-kDa cut-off to separate the
released XR (filtrate) from -crystallin (retentate). Furthermore, 10 µl of 3 mM o-phthalaldehyde was added to 2 ml
of the filtrate, and the spectra were recorded after 30 min, with the
excitation wavelength fixed at 338 nm. Similar experiments were
repeated for renaturation of XR-m in the absence of -crystallin. --- and - - - represent the isoindole spectra of XR renatured from XR-m
state in the presence and absence of -crystallin,
respectively.
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Effect of Adenine Nucleotides and XR Substrates on
-Crystallin
mediated Renaturation--
The inability of
-crystallin to
reconstitute active XR at 37 °C (Fig. 3) cannot be attributed to
lack of binding of the chaperone to XR as shown in Fig. 6 but may be
due to either an inability to release the bound XR at high temperature
or due to the polypeptide being released in a temperature-sensitive
conformation. Earlier the role of ATP in the renaturation of the
proteins rhodanese, ribulose-bisphosphate carboxylase/oxygenase, and
-protein (34-36) by the chaperone GroEL has been reported. Recently
evidence for the binding of ATP to
-crystallin was provided by
31P NMR spectroscopy (37) and fluorescence studies (38).
Hence, studies were undertaken to find out if ATP played any role in the chaperone function of
-crystallin. For this functional in vitro analysis refolding of XR-m was initiated in a buffer
containing
-crystallin at 37 °C and further incubated in the
absence/presence of ATP. As shown in Fig.
9,
-crystallin mediated reconstitution of active XR was not observed in the absence of ATP. However, with ATP
the renaturation process followed a sigmoidal time course, and values
of 10 and 53 min were observed for the lag time (Fig. 9
inset) and t1/2 (where the activity
regain was half of the maximal extent), respectively. A maximum 22% of
the XR activity was recovered in 1.5 h, and a further decrease in
the yield may be attributed to temperature-mediated inactivation of the
released XR.

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Fig. 9.
Influence of ATP and XR substrates on the
-crystallin-mediated reactivation of XR. Renaturation of XR-m
(25 µM) was initiated at 37 °C by diluting 10 µl of
the sample into a final volume of 1 ml of phosphate buffer, pH 7.2, with -crystallin 0.6 mg/ml preincubated for 2 h at 37 °C.
After 1 h the following additions were made, no nucleotide ( ),
ATP (1 mM) ( ), NADPH (0.5 mM) ( ), ATP (1 mM) + NADPH (0.5 mM) ( ), and xylose (250 mM) ( ); furthermore, at the times indicated, 100-µl
aliquots of the refolding solution were withdrawn and added to the
assay mixture to determine the XR activity as described under
"Experimental Procedures." represents the renaturation of XR-u
(XR at a concentration of 25 µM denatured with 6 M GdmCl) in the presence of the nucleotides/XR substrates
as described above. The percentage activity recovered is with respect
to the control containing native XR. The inset shows the
early time course of -crystallin-assisted renaturation demonstrating
the lag phase of 10 min for the renaturation process in the presence of
ATP ( ) or ATP + NADPH ( ).
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The effect of XR substrates NADPH and xylose on the
-crystallin-mediated renaturation of XR-m was investigated. It was
observed that the percentage of XR activity recovered in the presence
of ATP and NADPH in 1.5 h was approximately 2.5-fold higher than that observed in the presence of ATP alone (Fig. 9). However, the lag
time (10 min) (Fig. 9 inset) and t1/2 (53 min) values observed in both cases were identical indicating that the
presence of NADPH did not alter the rate of the initial slow reaction
(refolding of monomer and/or correct formation of dimer) nor the
overall rate. The phosphorylated coenzyme, however, failed to mediate the reconstitution of active XR in the absence of ATP (Fig. 9). This
ruled out the possibility of interaction of NADPH with XR bound to
-crystallin and altered the equilibrium between the bound and free
enzyme. However, this may also be unlikely because the bound XR is in a
non-native state (Fig. 6) and lacks enzymatic activity. In light of
these data we propose that the release of
-crystallin-bound XR is
mediated by ATP, and the phosphorylated coenzyme NADPH traps the free
XR in a conformation stable at the physiological temperature. The XR
substrate xylose failed to exert its effect on
-crystallin-mediated
reconstitution of active XR in the presence of ATP (Fig. 9). This may
be due to the fact that the enzyme follows an iso-ordered bi bi
mechanism (22) wherein xylose binds to XR·NADPH binary complex.
The effects of adenine nucleotides upon the
-crystallin-mediated
reconstitution of active XR were investigated. As shown in Fig.
10, addition of AMP-PNP, an ATP analog
with a nonhydrolyzable
-
bond, resulted in a maximum 18% of XR
activity in 1.5 h from its partially folded state compared with
the maximum activity recovered in the presence of ATP. Further
increases in the incubation period resulted in a decline in the yield
of reconstituted XR which can be attributed to the thermal inactivation
of the free XR. Hence, the experiments were repeated wherein the
coenzyme NADPH was added 30 min after the addition of the adenine
nucleotides ATP/AMP-PNP so as to stabilize the released XR. Under these
conditions a maximum 53% of XR activity was recovered in the presence
of AMP-PNP in 12 h compared to the maximum observed in the
presence of ATP in 5 h and which did not increase with further
incubation. These results indicated differential ability of the adenine
nucleotides ATP and AMP-PNP to reconstitute active XR which may be
attributed to their different binding constants.
-Crystallin-mediated reconstitution of active XR was not observed in
the presence of the adenine nucleotides AMP or ADP (Fig. 10), implying
the involvement of the P
of ATP upon binding to
-crystallin·XR-m complex. Altogether, these results supported the
notion that ATP hydrolysis is not a prerequisite for release of XR
bound to
-crystallin since the nonhydrolyzable analogue AMP-PNP was
capable of reconstitution of the active XR. Instead, the release of XR
may be mediated in part through the binding of ATP or AMP-PNP producing
a similar conformational change in the chaperone which weakens its
interactions with XR, further allowing reconstitution of the active
enzyme. In contrast to 37 °C (Fig. 9) the
-crystallin-mediated
renaturation of XR-m was observed in the absence of ATP at 4 °C
(Fig. 3). Also, when the renaturation was initiated at 37 °C and
later the temperature shifted to 4 °C a maximum 55% of the original
XR activity was recovered in 24 h. These differences in the ATP
requirement support the notion that the role of adenine nucleotide
would seem to be linked with the need to provide a rapid dissociation
pathway for the
-crystallin·XR-m complex at 37 °C and is not
essential for correct folding of XR. In light of these data it is
tempting to speculate that a weak interaction exists between
-crystallin and the non-native XR. Tightly bound substrate proteins
would probably reduce the flexibility of
-crystallin; hence, the
binding of ATP alone or its hydrolysis would not be sufficient to
induce conformational change necessary for the dissociation of the
-crystallin-protein complex.

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Fig. 10.
Influence of adenine nucleotides on the
-crystallin-mediated reactivation of XR. Renaturation of XR-m
described in the legend to Fig. 9 was repeated in the presence of the
following nucleotides: a, no nucleotide; b, ATP;
c, AMP-PNP; d, ATP + NADPH; e, AMP-PNP + NADPH; f, ADP; and g, AMP. The recoveries were
determined after incubation for 3 h except for the samples
d and e, where the recoveries were determined
after 12 h. The final concentrations of the nucleotides in the
various refolding solutions were 1.0 mM each of adenine
nucleotides and 0.5 mM of NADPH. The percentage of activity
recovered is with respect to the control with native XR. In case of
samples d and e, the native XR was completely
inactivated in 12 h; therefore, the XR activity recovered is with
respect to the activity of native enzyme incubated with 0.5 mM NADPH under identical conditions.
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Dependence of
-Crystallin-assisted Renaturation on the Order of
Addition of Partially Folded XR and ATP to
-Crystallin--
The
highly selective nature of protein-ligand interaction provides a
sensitive mechanism for the modulation of protein activity. Experiments
were carried out to define the role of
-crystallin-ATP interaction
on the structure and mechanism of action of the chaperone
-crystallin. As shown in Fig. 11 the
order of addition of ATP and XR-m to
-crystallin resulted in a
difference in the
-crystallin-mediated renaturation profiles. The
renaturation process of XR-m carried out in the presence of preformed
-crystallin·ATP complex followed a sigmoidal time course, and
values of 15 and 40 min were obtained for the lag phase (Fig. 11
inset) and t1/2, respectively. However, when
-crystallin·XR-m complex was allowed to form prior to
addition of ATP the renaturation process had a t1/2
of 53 min and an initial lag time of 10 min (Fig. 11,
inset). Although the value of t1/2 was increased by 13 min in the latter case, the final extent of XR activity
recovered was 5.5-fold higher than that observed with the preformed
-crystallin·ATP complex. This indicated that the ATP-free form of
-crystallin mediates reconstitution of active XR more effectively
than the ATP-bound form.

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Fig. 11.
Influence of changes in the order of
addition of XR-m and ATP to -crystallin, on the
-crystallin-mediated renaturation of XR. Refolding of XR-m at
37 °C (described in the legend to Fig. 9) in the presence of the
following: (a) -crystallin followed by addition of ATP
after 1 h ( ); (b) -crystallin preincubated with ATP
for 1 h ( ); and (c) in the presence of
-crystallin alone ( ) or in its absence ( ). 0.5 mM
NADPH was added to all the samples. The recovery of XR activity was
determined as described earlier in the legend to Fig. 9. The
concentrations of XR-m, -crystallin, and ATP in the refolding
solution were 250 nM, 0.6 mg/ml, and 1 mM,
respectively. The inset shows the early time course of
renaturation demonstrating the lag phase values of 10 and 15 min for
the samples a and b, respectively.
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The structural differences in ATP-free and -bound forms of
-crystallin·XR-m were probed by fluorescence spectroscopy using the hydrophobic probe ANS. As shown in Fig.
12, ANS exhibited an emission maximum
at 525 nm; however, in the presence of
-crystallin·XR-m its
fluorescence intensity increased and the emission maximum shifted to
475 nm characteristic of the transfer of ANS into a hydrophobic
environment. Furthermore, a concomitant decrease in the intensity of
the dye fluorescence (
em 475 nm) was observed in the
presence of increasing concentrations of ATP (Fig. 12). These results
imply that binding of the adenine nucleotide to
-crystallin·XR-m
complex induces a conformational change that is accompanied by a
concomitant internalization of hydrophobic surfaces previously exposed.
This acts to reduce the hydrophobic interactions and thus the affinity
of the chaperone for the substrate protein further allowing
reconstitution of the active XR. Evidence for the ATP-
-crystallin
binding has earlier been provided by 31P NMR spectroscopy
(37) and fluorescence studies (38). ATP failed to influence the
tryptophanyl fluorescence of XR in 1.4 M GdmCl (XR-m)
indicating inability of the adenine nucleotide to bind XR-m (data not
shown). Altogether these results imply that in the presence of
-crystallin·XR-m complex, ATP binds
-crystallin and not the
bound XR which is quite likely because as shown in Fig. 6 the
-crystallin-bound XR exists in a non-native state. In light of these
data we propose that
-crystallin operates by providing hydrophobic
surfaces that interact with the molten globule state of XR, and the
hydrophobic interactions play an important role in the formation of
-crystallin·XR-m complex.

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Fig. 12.
Exposure of hydrophobic surfaces in the
presence of ATP-free or -bound -crystallin·XR-m complex measured
by ANS binding. Spectra for ANS (final concentration 100 µM) at 37 °C in 50 mM sodium phosphate
buffer, pH 7.2 (bottom spectrum), or presence of
-crystallin·XR-m complex (ATP-free) (---) or -crystallin·XR-m
complex in presence of ATP (ATP-bound) at the following concentrations:
0.05 mM (---), 0.2 mM (- - -), 0.6 mM (-·-), 1.0 mM (-··-). The ATP-free
-crystallin·XR-m complex was prepared as described in legend to
Fig. 6, whereas the ATP-bound form was obtained after further
incubation with the respective concentrations of the adenine nucleotide
(mentioned above) for 2 h at 37 °C. The excitation wavelength
was fixed at 375 nm.
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Conformational changes have been proposed to play a major role in the
binding of folding intermediates and in the discharge of polypeptides
from molecular chaperones. One of the signals for inducing such
structural changes is the hydrolysis of ATP as reported in case of the
chaperone DnaK (39) and GroEL (34, 35). However, reports are also
available wherein the chaperones GroEL (36) and BiP (40) do not require
ATP hydrolysis. Instead, the mere binding of the adenine nucleotide to
the chaperone induces a typological change in the chaperone that
weakens its interaction with the bound protein. This acts to release
the protein, further allowing it to assume its native state. Our
investigations reveal that the mechanism of chaperoning of
-crystallin also requires the binding of ATP to the chaperone and
not its hydrolysis.
Earlier studies have indicated that chaperones functioned
post-translationally before the formation of the folded functional enzyme. The present investigation was carried out to gain some insight
into the conformation of XR interacting with the chaperone
-crystallin and the mechanistic details underlying the
reconstitution of active enzyme. The conditions for the unfolding of
native XR were sought in the belief that the unfolded enzyme or its
folding intermediates would serve as a substrate for the
-crystallin-mediated reconstitution of active XR. Our denaturation
studies using the structure-perturbing agent GdmCl revealed that the
folding of XR involves an intermediate that resembles the molten
globule. The existence of molten globule like intermediates has been
demonstrated with several proteins, and these intermediates are known
to be involved in various cellular functions such as membrane
translocation of proteins (41, 42), chaperone-assisted protein folding
(5), and also in various genetic diseases (43, 44). Interest in such
intermediates is strong since they have been proposed to be an
obligatory intermediate formed early in the folding pathway (45). A
common feature of the molten globule state is the exposure of
hydrophobic surfaces that lead to aggregation of proteins during folding. Our in vitro studies using XR revealed that the
chaperone
-crystallin operates by interacting with the hydrophobic
regions that appear on the surface of molten globule state of XR. This reduces the concentration of the free partially folded XR (XR-m) during
renaturation and thus prevents loss of enzyme activity due to their
hydrophobic aggregation. Lowering the temperature to 4 °C or the
presence of ATP at 37 °C induces a conformational change in the
-crystallin·XR-m complex that is accompanied by a concomitant
internalization of previously exposed hydrophobic surfaces. This acts
to reduce the hydrophobic interactions involved in the formation of the
complex and thus the affinity of the chaperone for the substrate
protein further allowing reconstitution of the active XR. The results
presented here are consistent with the notion that the complete folding
of XR resulting in the formation of catalytically active dimer does not
occur while it is bound to the surface of
-crystallin. Our
investigation reveals for the first time the mechanism of
-crystallin-mediated reconstitution of an active enzyme, and the
role of temperature and ATP in its mechanism of chaperoning. Earlier it
has been reported that
-crystallin does not prevent the
photoaggregation of
-crystallin at low temperatures. However, it can
do so at temperatures above 30 °C (8). Our present investigation
also supports this view, since
-crystallin-mediated reconstitution
of XR was observed when the refolding process was initiated at 28 and
37 °C and not when initiated at 4 °C which is attributed to the
inability of the chaperone to prevent aggregation of XR-m at low
temperature.
Delay experiments revealed the inability of
-crystallin to dissolve
XR aggregates formed in its absence implying that the chaperone
-crystallin should be present during stress conditions. The
dependence of protein aggregation reactions on temperature and
concentration is known. Our results support the notion that one of the
functions of
-crystallin in vivo may be to protect non-native protein from intracellular aggregation during high rate of
protein synthesis and/or thermal stress. The inability of XR-u and XR-m
to spontaneously reconstitute active XR under the conditions used in
the present investigation is due to the fact that aggregation competes
with the correct folding pathway. The kinetic competition between
refolding and aggregation has been reported to be a major determinant
for lower yields or irreversibility in refolding of proteins in
vitro (46). However, refolding of XR may be possible under
different experimental conditions, but regardless of this observation
we are left with the fact that presence of
-crystallin resulted in a
substantial amount of reconstitution of active XR from the XR-m
state.
It has been reported that in E. coli a cascade of molecular
chaperones mediate folding of proteins. The chaperone DnaK interacts with polypeptides in their extended conformation and prevents premature
misfolding and aggregation after which GroEL stabilizes folding
intermediates resembling the molten globule and mediates proper
folding. The transfer of DnaK/DnaJ-bound protein to GroEL requires GrpE
as the coupling factor (47). Such a mechanism is likely to exist in
eukaryotes also (47-49). Our investigation reveals that
-crystallin
is able to reconstitute XR via interaction with its non-native
conformer characterized by an increased surface hydrophobicity but a
remarkably low degree of unfolding. The inability of
-crystallin to
reconstitute XR from its extended conformation implies that in
vivo other chaperones may be involved in binding to the unfolded
polypeptides and prevent premature misfolding and aggregation, whereas
the proper folding and assembly may depend on the subsequent transfer
of the partially folded polypeptide to
-crystallin. Further evidence
is provided by the observation that
-crystallin prevents aggregation
of lens proteins induced by oxidative stress and UV radiation. These
conditions are not likely to unfold protein molecules completely but
induce formation of partially folded state with hydrophobic surfaces
that result in its aggregation (19). For many years
-crystallin was
thought to be a lens-specific structural protein where it played a role to facilitate proper transmission of light. However, recently
-crystallin has been demonstrated to be present in various
non-lenticular tissues such as brain, spleen, and heart and also found
in NIH 3T3 cells expressing Ha-ras and v-mos (10,
11).
-Crystallin has been reported to be induced by thermal or
hypertonic stress (11, 12), and its expression is markedly increased in
a number of neurological diseases such as Creutzfeld-Jacob disease,
Alexander disease, and Lewy body disease (50-52). Our present
investigation on
-crystallin adds to the information available on
its chaperone function which may assist to shed some light on its
diverse roles in vivo.
We thank Prof. S. Mitra, Tata Institute of
Fundamental Research, Bombay, for permitting the use of CD facility and
Dr. K. N. Ganesh, National Chemical Laboratory, for the use of
spectrofluorometer.