(Received for publication, May 2, 1995; and in revised form, June 9, 1995)
From the
-Crystallin, a multimeric protein present in the eye lens,
is shown to have chaperone-like activity in preventing thermally
induced aggregation of enzymes and other crystallins. We have studied
the rapid refolding of
-crystallin, and compared it with other
calf eye lens proteins, namely
- and
-crystallins.
-Crystallin forms a clear solution upon rapid refolding from 8 M urea. The refolded
-crystallin has native-like
secondary, tertiary, and quaternary structures as revealed by circular
dichroism and fluorescence characteristics as well as gel filtration
and sedimentation velocity measurements. On rapid refolding,
- and
-crystallins aggregate and form turbid solutions. The presence of
-crystallin in the refolding buffer marginally increases the
recovery of
- and
-crystallins in the soluble form. However,
unfolding of these crystallins together with
-crystallin using 8 M urea and subsequent refolding significantly increases the
recovery of these proteins in the soluble form. These results indicate
that an intermediate of
-crystallin formed during refolding is
more effective in preventing the aggregation of
- and
-crystallins. This supports our earlier hypothesis (Raman, B., and
Rao, C. M.(1994) J. Biol. Chem. 269, 27264-27268) that
the chaperone-like activity of
-crystallin is more pronounced in
its structurally perturbed state.
-Crystallin is a multimeric protein, made up of the two
homologous gene products of
and
,
present in high concentration in the eye lens.
is
also known to occur in non-lenticular tissues such as heart, muscle,
and kidney(1, 2, 3, 4) . Its
expression can be induced by thermal (5) or hypertonic
stress(6) . It is structurally related to small heat shock
proteins and behaves in several ways like the small
Hsps(
)(5, 7, 8, 9) .
-Crystallin is heat-stable up to 100 °C(10) .
Studies on -crystallin over several years have been reviewed
recently by Groenen et al.(11) .
-Crystallin has
been considered as a structural protein; the quaternary structure of
this crystallin has received much attention in order to gain an insight
into its packing in the transparent eye lens. However, the
understanding of the tertiary and quaternary structure of
-crystallin is still not complete. Identification of
-crystallin in several other tissues suggests that it might have a
role to play in other functions. Like some
Hsps(12, 13, 14) , it is known to interact
with membrane (15, 16) and cytoskeletal elements (17, 18) and to modulate the intermediate filament
assembly(19) . Mixed aggregates of
-crystallin
and small Hsps have been observed in
vivo(20, 21) .
-Crystallin and Hsp25 form
mixed aggregates in vitro and resemble each other in secondary
structure and in stability toward urea denaturation(9) .
-crystallin has been shown to prevent thermal aggregation of
- and
-crystallins and other proteins(22) .
-Crystallin, like the small heat shock proteins Hsp25
and Hsp27, has been shown to facilitate the refolding of citrate
synthase and
-glucosidase in vitro(23) . It forms
a complex with carbonic anhydrase upon heat denaturation of the enzyme (24) . Recently, it has been shown to bind to ATP, but not to
ADP or GTP (25) , and possess autokinase activity(26) .
It is interesting to note that several years ago Bloemendal et al. showed that
- and
-crystallins form a complex at high
concentrations, whereas
-crystallin remains
uncomplexed(27) .
We had earlier investigated the
chaperone-like activity of -crystallin toward the photoaggregation
of
-crystallin (28) and the aggregation of insulin B chain
upon reduction of interchain disulfide bonds of insulin(29) .
It does not prevent aggregation of proteins at low temperatures but
does so at temperatures above 30 °C. We have hypothesized that
-crystallin prevents aggregation of non-native structures by
providing appropriately placed hydrophobic surfaces. A structural
transition above 30 °C enhances the protective ability of
-crystallin, perhaps by increasing or reorganizing the hydrophobic
surfaces. A structurally perturbed state, thus, is important in the
chaperone-like activity of
-crystallin. We now report on the rapid
refolding process of
-,
-, and
-crystallins in the
context of the chaperone-like activity of
-crystallin. Our results
suggest that an intermediate of
-crystallin, formed during its
refolding, is substantially more effective in preventing the
aggregation of
- and
-crystallins.
s values were obtained
by applying appropriate corrections for temperature and solvent
viscosity.
Our understanding of protein folding, particularly in vivo, is far from complete despite the considerable progress made over the last two decades. According to the generally accepted paradigm, all the information required for protein folding resides in its primary sequence, and protein folding is an unassisted, spontaneous process. However, many proteins appear to require external assistance, mostly through other proteins, to fold and assemble to their functional forms. Chaperones that provide the required assistance (33, 34, 35) prevent the unfavorable interactions in the folding pathway of a protein. Hydrophobic interactions are known to play a crucial role in such molecular processes.
Recently we have shown that the chaperone-like activity
of -crystallin is enhanced when its quaternary structure is
perturbed. In order to get a better insight into the chaperone-like
activity of
-crystallin, we have studied the refolding properties
of
-,
-, and
-crystallins using the rapid refolding
method. This is one of the widely used methods (36, 37, 38) to study the refolding
properties of a protein where the protein in its unfolded state is
rapidly transferred to the condition(s) that favor its folding. In the
present study, the crystallins were subjected to refolding at different
concentrations. Usually proteins at high concentration tend to
aggregate upon rapid refolding. Fig. 1A represents the
turbidity profiles of crystallins as a function of the amount of
protein taken for the refolding. Interestingly,
-crystallin
remains clear upon rapid refolding in the concentration range studied,
while
- and
-crystallins aggregate and yield turbid solution.
Turbidity increases with concentration. We have observed that refolding
of
-crystallin even at a concentration of 13 mg/ml yields a clear
solution. The amount of protein recovered in the soluble form, after
removing the precipitate by centrifugation, is shown in Fig. 1B. As evident from the figure, more than 90% of
-crystallin is recovered in the soluble form, while the other two
crystallins kinetically partition to aggregate in a
concentration-dependent manner. The recovery of
-crystallin
appears to be comparatively lower. This may be because
-crystallin
is more prone to aggregation or because of other factors such as
preferential adsorption to the surface of the reaction tube.
Figure 1:
Rapid refolding of - (
),
- (
),
- (
), and
-
(
) crystallins. A, turbidity profile upon refolding at
different concentrations of crystallins. Turbidity was measured as the
optical density at 500 nm. B, variation of recovery of the
crystallins in soluble form as a function of concentration. Percentage
recovery of proteins is with respect to the amount taken for refolding.
Concentrations of the crystallins are those obtained after dilution
into refolding buffer.
Since
the refolding of -crystallin yields a clear solution, we performed
CD and fluorescence spectroscopy to determine whether the refolded
-crystallin attains its native conformation. Fig. 2A shows the far UV CD spectra of native and refolded
-crystallin. These spectra overlap within the experimental error,
suggesting that it regains almost all of its secondary structure.
Comparison of near UV CD spectra of native and refolded
-crystallin (Fig. 2B) suggests that tertiary
structure is also regained. It is also known that
-crystallin
regains secondary and tertiary structures upon slow refolding (where
the denaturant is removed by dialysis)(39, 40) .
Figure 2:
Circular dichroism of native (-) and
refolded (at 1 mg/ml)(- - -) -crystallin. A, far UV CD
spectra. B, near UV CD spectra.
The fluorescence emission maxima of native and refolded
-crystallins, upon excitation at 295 nm, do not differ; they are
336 and 336.6 nm, respectively. The synchronous spectrum of a mixture
of fluorophores or fluorophore in different local microenvironment is
likely to be more informative than the normal emission or excitation
spectrum(41) . Synchronous scan might be expected to provide a
signature and reflect minor structural perturbations. An earlier study
on
-,
-, and
-crystallins using synchronous fluorescence
scanning by one of us (32) suggests that the synchronous scan
spectra of these crystallins are distinguishable and appear to provide
a specific signature. The synchronous scan spectra of the native and
refolded
-crystallin superimpose on each other as shown in Fig. 3, indicating that the microenvironments of the aromatic
amino acids are indistinguishable in both cases.
Figure 3:
Synchronous fluorescence spectra of native
(-) and refolded(- - -) -crystallin.
was set at
40 nm. Both excitation and emission band passes were set at 3 nm. I
= normalized fluorescence intensity
(arbitrary units);
= excitation wavelength
(nm).
The shift in the
fluorescence emission wavelength upon excitation with the wavelength of
red edge of the absorption spectrum of a fluorophore is termed red edge
excitation shift. This effect is usually observed for polar
fluorophores in motionally restricted
media(42, 43, 44, 45, 46, 47, 48) .
Single-tryptophan proteins(46) , crystallins in intact eye lens
and in solutions(47, 48) , have been studied using
this technique. The magnitude of the shift in the observed emission
maximum can be correlated to the restriction that the microenvironment
imposes on the fluorophore. Both the native and the refolded
-crystallin show a 6-nm red edge excitation shift (336 to 342 nm)
upon shifting the excitation wavelength from 295 to 305 nm. This
indicates that the microenvironments around the tryptophans in the
native and the refolded
-crystallin (or at least its influence on
the mobility of the tryptophans) are identical.
Changes are known to
occur in the quaternary structure of -crystallin when it is
subjected to temperature perturbation and on urea
treatment(39, 49, 50, 51) . To find
out the state of the quaternary structure of the refolded protein, we
have performed gel filtration chromatography and sedimentation velocity
measurement. Analysis of the elution profiles (Fig. 4) of the
native and refolded
-crystallin suggests that the refolded
-crystallin might form a marginally smaller multimer compared with
the native form. The sedimentation coefficient (s
value) of the native
-crystallin, determined to be 17.1 S,
is in good agreement with the reported value for the
form(49) . The sedimentation pattern of the refolded
-crystallin shows a single peak (Fig. 5) with a S
value of 16.1, suggesting that the size of the refolded
-crystallin is marginally smaller than that of the native
-crystallin. Packing alterations, if any, without significant
shape and size changes might not be detected in the sedimentation
studies.
Figure 4:
Elution profile of native () and
refolded (
)
-crystallin on a Bio-Gel A-1.5m column (see
``Materials and Methods'' for details). The amount of protein
loaded was 1 mg in both cases. The elution positions of blue dextran
(2000 kDa) (A); thyroglobulin (670 kDa) (B); and
lysozyme (14.4 kDa) (C) are indicated by arrows.
Figure 5:
Sedimentation pattern of native (A) and refolded (B) -crystallin (see
``Materials and Methods'' for
details).
These results indicate that -crystallin regains most
of its three-dimensional structure upon rapid refolding and that it
does not need any external assistance. It is noteworthy that RNase A,
which is known to refold completely to its active form at low
concentrations (<0.1 mg/ml), misfolds and aggregates upon refolding
at concentrations higher than 0.1 mg/ml(38) .
-Crystallin,
on the other hand, refolds to its native state even at concentrations
as high as 13 mg/ml. This is significant and might be relevant to its
chaperone-like activity.
Several proteins need the assistance of
molecular chaperones to fold correctly to their native state. Whether
chaperones themselves need such assistance is not clear. Few studies
have attempted to answer this question. Chemically synthesized GroES
not only folds and assembles properly but also shows complete activity
in refolding of rubisco in the presence of GroEL(52) .
Urea-unfolded GroES regains its native assembly upon removal of the
denaturant(52) . GroEL, dissociated to its monomeric form by
urea, needs Mg-ATP to reassemble to its native polymeric form, and the
presence of native GroEL assembly enhances the process of such
reassembly(53) . Newly synthesized mitochondrial chaperonin
Hsp60 monomers do not assemble in the absence of preexisting native
protein assembly(54) . These studies have led to the concept of
``self-chaperoning.'' Thus, it appears that heat shock
proteins and chaperones have the tendency to fold spontaneously to
their native structure without any external assistance, and some
multimeric chaperones such as GroEL chaperone their own assembly. The
mechanism by which -crystallin refolds properly is not known. The
possibilities are: (i) the individual subunits refold and then assemble
to form large structures; (ii) the domains of each subunit that need to
interact for multimerization refold first, and subsequently the other
domains fold; and (iii) both folding and multimerization occur
simultaneously. Further studies are required to verify whether there is
a general preference for any of these possibilities.
As stated
earlier, -crystallin undergoes photoaggregation, and
-crystallin prevents this aggregation by forming a
complex(28) ; it is also known to prevent the thermal
aggregation of
- and
-crystallins(22) . Our results (Fig. 1) show that rapid refolding of
- and
-crystallins also leads to aggregation and turbidity. We have,
therefore, studied the effect of
-crystallin on this aggregation
of
- and
-crystallins. The recovery of these proteins in the
soluble form increases to some extent when
-crystallin is present
in the refolding buffer. The recovery of these proteins with the
increasing concentration of
-crystallin is shown in Fig. 6.
The increase in the
-crystallin concentration marginally enhances
the recovery of
- and
-crystallin as shown in the figure.
Even a 10-fold excess of
-crystallin does not improve the recovery
of
-crystallin to more than 25-30%.
Figure 6:
Refolding of - (
),
- (
), and
- (
) crystallins in the
presence of native
-crystallin. The percentage recovery of protein
is with respect to the amount of protein taken for refolding, which is
0.3 mg. The amount of
-crystallin in the refolding buffer was
varied to get different [
]/[p] (w/w) ratios.
[p] = Concentration of
- or
-crystallin.
Interestingly, when
- or
-crystallins are denatured together with
-crystallin and then subjected to refolding, the recovery of
- or
-crystallin in the solution is increased significantly. Fig. 7A represents the turbidity profile of solutions
obtained by refolding of 0.3 mg of
- or
-crystallin together
with increasing amounts of
-crystallin; the percentage recovery of
these proteins is shown in Fig. 7B. Even at a 5 times
lesser concentration (w/w),
-crystallin is able to almost
completely prevent the aggregation of
-crystallin. Recovery of
-crystallin changes from 20 to 60% ( Fig. 6and Fig. 7B). These results suggest that the native
-crystallin is less effective in preventing the aggregation of
- or
-crystallin. On the other hand, when
-crystallin is
refolded together with
- or
-crystallin, it prevents the
aggregation of
- or
-crystallin very significantly. We
believe that an intermediate state (or assembly) of
-crystallin
formed in its refolding pathway is more effective in preventing the
aggregation of the other two crystallins when compared with the native
protein. In other words
-crystallin is more functional in its
structurally perturbed state in preventing the aggregation of other
crystallins. This supports our hypothesis that
-crystallin
prevents aggregation of non-native structures by providing
appropriately placed hydrophobic surfaces and that a structural
perturbation enhances the protective ability of
-crystallin. The
structurally perturbed state, thus, is important in the chaperone-like
activity of
-crystallin.
Figure 7:
Refolding of - (
),
- (
), and
- (
) crystallins together
with
-crystallin. A, turbidity profile upon refolding of
mixtures of
- and
-crystallins or
- and
-crystallins at different ratios. Turbidity was measured as
optical density at 500 nm. B, recovery of
- or
-crystallins in soluble form as a function of
[
]/[p] ratios. The percentage recovery of
protein is with respect to the amount of protein taken for refolding,
which is 0.3 mg. The amount of
-crystallin was varied to get
different [
]/[p] (w/w) ratios. [p]
= Concentration of
- or
-crystallin.
On the basis of our results, we
conclude that -crystallin rapidly refolds and regains most of the
secondary, tertiary, and quaternary structures. This might be a common
property of chaperones and heat shock proteins. The other eye lens
crystallins such as
- and
-crystallins aggregate and
precipitate out upon such rapid refolding. Native
-crystallin
prevents this aggregation to a lesser extent. However, if
- or
-crystallin is denatured together with
-crystallin and then
subjected to the refolding process,
-crystallin almost completely
prevents the aggregation of other crystallins, suggesting the
importance of a structurally perturbed state. These results should
prove useful in understanding intercrystallin interactions as well as
the mechanism of the chaperone-like activity of
-crystallin.