(Received for publication, February 21, 1996)
From the
-Crystallin, the major protein of the ocular lens, acts as
a molecular chaperone by suppressing the nonspecific aggregation of
damaged proteins. To investigate the mechanism of the interaction
between
-crystallin and substrate proteins, we prepared a
tryptophan-free mutant of human
A-crystallin and assessed the
conformation of thermally destabilized proteins captured by this
chaperone using fluorescence spectroscopy. The fluorescence emission
characteristics of bound substrates (rhodanese and
-crystallin)
and the results of fluorescence quenching experiments indicate that the
proteins captured by
-crystallin are characterized by a very low
degree of unfolding. In particular, the structure of rhodanese bound to
A-crystallin appears to be considerably more native-like compared
to that of the enzyme bound to the chaperonin GroEL. We postulate that
-crystallin (and likely other small heat shock proteins) recognize
preferentially the aggregation-prone conformers that occur very early
on the denaturation pathway. With its ability to capture and stabilize
these early non-native structures,
-crystallin appears to be
uniquely well suited to chaperone the transparency properties of the
ocular lens.
-Crystallin, the major protein of the vertebrate eye lens,
consists of two types of highly homologous 20-kDa subunits,
A and
B. The A and B chains noncovalently self-associate to form a large
macromolecular complex of approximately 40
subunits(1, 2) . Spectroscopic data provided strong
evidence that the secondary structure of
-crystallin is dominated
by
-sheets(3, 4) . Much less specific information
is available regarding the tertiary and quaternary structure of the
protein. While a number of structural models have been proposed for
-crystallin oligomers(2) , none of them is generally
accepted.
Believed for many years to be strictly lens-specific
proteins, B- and
A-crystallin have recently been found in
many nonlenticular
tissues(5, 6, 7, 8) . Furthermore,
B-crystallin has been associated with a number of
neurodegenerative disorders(5, 6, 8) .
Another important recent development is the rapidly growing evidence
that
-crystallin belongs to a family of small heat shock proteins
(sHSPs). (
)This is indicated by extensive structural
similarities between
-crystallin and other sHSPs(8, 9) as well as by the recent findings that
B-crystallin is
inducible by various stress conditions (10, 11) and
that
B- and
A-crystallin are able to confer cellular
thermoresistance(12, 13) . Despite the abundance of
sHSPs in both eukaryotic and prokaryotic organisms(14) , no
obvious function has been associated with these ubiquitous proteins. A
new light on the potential physiological role of
-crystallin and
related sHSPs has been shed by recent findings that these proteins act in vitro as molecular chaperones by preventing the aggregation
of other proteins under conditions of thermal stress or other
insults(1, 15, 16, 17, 18, 19, 20) .
The chaperone function of
-crystallin and other sHSPs is likely to
be of considerable importance in vivo. In the context of eye
research, it has been postulated that the ability of
-crystallin
to suppress the aggregation of damaged proteins may be critical for
maintaining the transparency of the ocular lens and that aging-related
deterioration of the chaperone function could contribute to the
development of cataracts(1, 2, 15) .
Despite the rapidly growing interest in the chaperone function of
-crystallin and related sHSPs, very little is known about the
mechanism by which these proteins interact with their substrates.
Recent results indicate that, in contrast to other known chaperones,
-crystallin has very low affinity for folding intermediates formed
during protein refolding reactions in vitro and that its
substrate specificity is limited to non-native structures that occur on
the denaturation pathway only(21) . However, the critically
important information about the specific conformational state(s) of the
non-native structures that are recognized by
-crystallin is still
missing. In this work, we have cloned and overexpressed
A-crystallin mutant in which the sole Trp residue has been
replaced with Phe. Fluorescence spectroscopy experiments with the
complexes formed between the Trp-free
A-crystallin and various
substrate proteins allowed us, for the first time, to gain a direct
insight into the conformation of proteins bound to this chaperone.
The fluorescence characteristics of tryptophan residues
depend strongly on the microenvironment and thus provide a sensitive
probe of the conformational state of proteins. Measurements of
intrinsic fluorescence properties of substrate proteins have been used
previously to study protein interaction with the chaperonin GroEL (26, 27, 28) and the heat shock protein
DnaJ(29) , providing critical information about the
conformation of folding intermediates that are captured by these
chaperones. Fluorescence studies with proteins bound to
-crystallin are hampered by the fact that, in contrast to
tryptophan-free GroEL and DnaJ,
-crystallin itself contains Trp
residues whose emission spectrum overlaps with those of substrate
proteins. Therefore, in order to facilitate characterization of the
conformational states of proteins bound to
-crystallin, we have
cloned and overexpressed in Escherichia coli the mutant of
A in which the sole Trp at position 9 was replaced with Phe. As
anticipated, the conservative Trp
Phe substitution had a
negligible effect on the structural and functional properties of the
protein. Thus, the Sephacryl S-300 size exclusion chromatography
profiles for the wild type
A-crystallin and the W9F mutant were
essentially identical. Furthermore, the mutation fully preserved the
chaperone function of the protein as indicated by a very similar
ability of both the wild type and Trp-free
A-crystallin to
suppress the thermal aggregation of rhodanese and
-crystallin
(data not shown).
In general, when the protein unfolds, it exposes
buried tryptophans to the aqueous solvent; this results in the shift of
the fluorescence emission maximum toward longer wavelength. The
fluorescence spectrum of native rhodanese has a maximum at 332 nm (Fig. 1A, trace 1) and is consistent with a
largely apolar environment of eight Trp residues. Upon unfolding in 6 M guanidine HCl, the fluorescence maximum of rhodanese shifts
to 352 nm, a wavelength characteristic of tryptophans fully exposed to
water. The spectrum of rhodanese bound to W9F A-crystallin has a
maximum at 337 nm (Fig. 1A, trace 2). A small
(5 nm) red-shift in the fluorescence spectrum indicates that the
tertiary structure of the chaperone-bound enzyme is somewhat looser
that that of the native form; however, the average environment of Trp
residues in the bound protein appears to be still fairly hydrophobic.
Notably, the position of the emission maximum for rhodanese associated
with
A-crystallin is much closer to that observed for the native
enzyme than for its chemically denatured form. The fluorescence
properties of
-crystallin bound to
A-crystallin were very
similar to those of rhodanese.
-Crystallin contains four Trp
residues which are uniformly distributed throughout the molecule. As
shown in Fig. 1B, unfolding of bovine
-crystallin
in guanidine HCl results in a 23 nm red-shift of the fluorescence
maximum (from 330 nm in the native form to 353 nm).
-Crystallin
bound to
A-crystallin shows an emission maximum at 337 nm, i.e. red-shifted only by 7 nm from that of its native state.
These observations suggest that the conformation in which both proteins
are captured by
-crystallin is characterized by a relatively low
degree of unfolding. Furthermore, the conformation of
-crystallin-associated proteins appears to be remarkably stable;
there is very little change (2-3 nm) in fluorescence maxima of
bound rhodanese or
-crystallin upon heating of the complexes to
temperatures well above those at which free proteins undergo thermal
denaturation (Table 1).
Figure 1:
Fluorescence
emission spectra of rhodanese (A) and -crystallin (B). 1, native protein; 2, protein bound to
W9F
A-crystallin; 3, protein unfolded in 6 M guanidine HCl. Spectra were recorded at 25
°C.
Further insight into the tertiary
structure of -crystallin-bound substrate proteins may be obtained
from fluorescence quenching experiments. Acrylamide and iodide were
used as a complementary set of water-soluble quenchers of tryptophan
fluorescence. Neutral acrylamide, although relatively polar, has the
ability to penetrate into protein interior(25) . Iodide is a
highly hydrated, negatively charged molecule; its quenching ability is
limited almost exclusively to surface-exposed tryptophans and also
depends on the location of the neighboring charged groups. In general,
the interpretation of fluorescence quenching data for multitryptophan
proteins is complicated by factors such as heterogeneous emission and
varied accessibility of individual tryptophan residues to the
quenchers. Therefore, we have used in our analysis the effective
Stern-Volmer constant (K
)
; this
effective parameter represents weighted average of the quenching
constants of individual Trp residues and may contain contributions from
both dynamic and static quenching processes(25) . Fig. 2shows representative Stern-Volmer plots for the quenching
of Trp fluorescence of rhodanese and
-crystallin under various
experimental conditions. The quenching parameters (K
)
and f
are
summarized in Table 1. Unfolding of both rhodanese and
-crystallin in 6 M guanidine HCl at 25 °C results in
a greatly increased solvent exposure of Trp residues, as indicated by
about a 6-fold increase in (K
)
for
acrylamide. This may be contrasted with only a 2-fold increase of the
above parameter observed for the proteins bound to
-crystallin. A
very similar trend may be found when the characteristics of iodide
quenching are compared, although in this case the analysis is somewhat
more complicated since only a fraction of the emission arises from
iodide-quenchable fluorophores (as indicated by a downward curvature of
the respective Stern-Volmer plots). For both proteins, the fraction of
iodide-accessible tryptophans in
-crystallin-bound conformers is
larger compared to the native states. However, the increase in (K
)
values for bound proteins is
very modest, especially when compared with that observed upon unfolding
of the proteins in guanidine HCl. Altogether, the results of the
quenching experiments support the notion that the structure of
-crystallin-bound proteins is characterized by a relatively low
degree of unfolding. Although somewhat loosened, this structure is
clearly closer to the native conformation than that of chemically
denatured proteins. It is also worthwhile noting that heating of the
complexes to temperatures as high as 75 °C has only a small effect
on the quenching parameters of bound proteins. This is consistent with
the lack of major changes in
at high temperatures
and further indicates that the conformation of
-crystallin-associated proteins is highly stable.
Figure 2:
Stern-Volmer plots for the quenching of
the fluorescence of rhodanese (A) and -crystallin (B). Top panel for each protein represents iodide
quenching, and the bottom panel represents acrylamide
quenching.
, native protein; +, protein unfolded in 6 M guanidine HCl;
, protein bound to W9F
A-crystallin.
Quenching data were obtained at 25 °C.
The
fluorescence of bis-ANS is strongly dependent on the polarity of the
environment: it is very weak in water and increases dramatically upon
binding to hydrophobic sites of proteins. This compound has been widely
used as a probe to assess the exposure of hydrophobic surfaces in
proteins(19, 30) . As shown previously, bis-ANS has
considerable affinity for -crystallin; this indicates the presence
of surface-exposed hydrophobic patches(19) . In contrast,
essentially no binding of the probe could be detected to native
rhodanese,
- and
-crystallin. However, when the
same proteins were bound to the chaperone, the fluorescence of bis-ANS
in the presence of the complexes was substantially higher than that for
-crystallin alone. Furthermore, the increase in fluorescence
intensity appeared to correlate with the amount of bound substrates (Fig. 3). This strongly indicates that, in contrast to the
native states, the conformation of chaperone-associated proteins is
characterized by high affinity for the hydrophobic probe.
Figure 3:
Fluorescence intensity at 25 °C of
bis-ANS (20 µM) in the presence of free substrate proteins
at a concentration of 0.1 mg/ml (first bar in each panel), A-crystallin (second bar), and
A-crystallin complexes with substrate proteins (last two
bars). The numbers indicate the molar ratio of
A to
the substrate protein in a given complex. The concentration of
A-crystallin in each case was 0.1 mg/ml. Prior to incubation with
the probe, control
A-crystallin was subjected to the same thermal
treatment as used to prepare a given complex (see ``Materials and
Methods'').
Employing site-directed mutagenesis and fluorescence
spectroscopy, we have explored the structural properties of non-native
proteins that are bound to A-crystallin. The present data clearly
show that the fluorescence characteristics of Trp residues in
-crystallin-bound conformers are much closer to those of native
proteins than the fully unfolded ones. This strongly suggests that
-crystallin stabilizes aggregation-prone proteins in a
conformation which, although compromised, remains relatively compact
and is characterized by a low degree of unfolding.
The specific
conformational features of substrate proteins bound to various
functionally different chaperones have been the subject of many recent
studies (for review see (31) and (32) ). For example,
DnaK and PapD are believed to bind polypeptides in an extended
conformation. In contrast, substrate proteins bound to the chaperones
GroEL and DnaJ appear to be in a partially folded conformation, often
characterized as a molten globule state with a largely preserved
secondary structure and a collapsed, highly flexible tertiary
structure(26, 27, 28, 29, 32) .
It is informative to compare the fluorescence characteristics of the
same substrate, rhodanese, bound to -crystallin and the two latter
chaperones.The fluorescence emission maximum for rhodanese bound to
GroEL or DnaJ occurs at 342-343 nm(26, 29) , i.e. it is red-shifted by 10-11 nm compared to that of
the native enzyme. In contrast,
maximum for
rhodanese associated with
-crystallin occurs at a wavelength as
low as 337 nm, indicating a substantially more hydrophobic
(native-like) environment of Trp residues as compared with that of
rhodanese bound to GroEL or DnaJ. A more compact (less accessible)
tertiary structure of the substrate protein associated with
-crystallin than that bound to GroEL is further supported by the
quenching data for rhodanese stabilized by these different chaperones (cf. data for GroEL in Refs. 26 and 27 and present results for
-crystallin). A potential complication in the interpretation of
fluorescence data for substrate proteins is caused by the possibility
that the chaperone itself could affect the environment of tryptophan
residues by shielding them from the aqueous environment. This indeed
may be a factor for GroEL-bound proteins, in which case a single
molecule of a substrate is locked in the central cavity of the
oligomeric chaperone(33) . However, since
-crystallin can
accommodate as many as 40 substrate molecules per 800-kDa oligomer (one
substrate protein per subunit), geometric considerations point to the
location of substrate proteins on the surface of the chaperone. In this
case, the role of shielding effects mentioned above would be much less
significant.
We and others have recently shown that -crystallin
has substrate specificity strikingly different from other chaperones:
it binds non-native protein structures formed on the denaturation
pathway, but apparently has no affinity for intermediates formed on the
refolding pathway(21, 34) . Our present results
complement the previous findings by providing crucial information about
the conformational state of proteins captured by this chaperone. In
view of these combined data, one can postulate that
-crystallin
preferentially recognizes non-native structures characterized by an
increased surface hydrophobicity but a remarkably low degree of
unfolding, i.e. the aggregation-prone conformers that occur very early on the denaturation pathway. Such a unique
substrate specificity would be fully consistent with the putative
physiological role of
-crystallin as a ``junior
chaperone'' specifically designed to suppress irreversible
aggregation of proteins under stress conditions. The above function of
-crystallin may be of particular importance in the ocular
lens(15) . Major physiologically relevant factors known to
induce aggregation of lens proteins include ultraviolet radiation and
oxidative stress. While the above insults are highly unlikely to cause
extensive unfolding of protein molecules, they may induce perturbations
in the tertiary structure, leading to an increased surface
hydrophobicity and, eventually, protein aggregation (17) . With
its ability to capture and stabilize these early non-native structures,
-crystallin appears to be uniquely well suited to chaperone the
transparency properties of the ocular lens.