Tài liệu Báo cáo khoa học: The effect of small molecules in modulating the chaperone activity of aB-crystallin against ordered and disordered protein aggregation pdf

The effect of small molecules in modulating the chaperone
activity of aB-crystallin against ordered and disordered
protein aggregation
Heath Ecroyd and John A. Carver
School of Chemistry and Physics, The University of Adelaide, Australia
Protein aggregation is the result of the mutual associa-
tion of partially folded intermediate states of a protein,
most likely via predominately hydrophobic interac-
tions. Protein aggregation can proceed via disordered
or ordered mechanisms: which mechanism predomi-
nates is thought to be determined by a number of fac-
tors, including the rate of unfolding, the amino acid
sequence of the protein, the experimental conditions
and the nature of the intermediate state(s) that form
[1,2]. Disordered aggregation results in amorphous
aggregates of protein, whilst ordered aggregation pro-
duces amyloid fibrils, long threadlike protein structures
that are rich in b-sheet and resistant to proteolytic deg-
radation. Protein misfolding, and in particular amyloid
fibril formation, is associated with a range of diseases,
including Alzheimer’s, Parkinson’s and Creutzfeldt-
Jakob diseases, type II diabetes and possibly cataracts
[3–5]. Protein aggregation is also responsible for inclu-
sion body formation, and therefore the ability to pre-
vent it would be of enormous benefit in recombinant
protein production, avoiding the need for resolubiliza-
tion of the aggregated and precipitated protein. Thus,
studies aimed at preventing protein aggregation are of
interest due to both their biomedical and biotechnolog-
ical applications.
In terms of biotechnological applications, small mol-

ecules such as guanidine and urea are well-established
suppressors of aggregation, and are often used to
Keywords
amyloid fibril; arginine; protein aggregation;
small heat-shock protein; aB-crystallin
Correspondence
H. Ecroyd, School of Chemistry and
Physics, The University of Adelaide,
Adelaide, SA 5005, Australia
Fax: +61 8 830 34358
Tel: +61 8 830 35505
E-mail:
(Received 12 November 2007, revised 16
December 2007, accepted 20 December
2007)
doi:10.1111/j.1742-4658.2008.06257.x
Protein aggregation can proceed via disordered or ordered mechanisms,
with the latter being associated with amyloid fibril formation, which has
been linked to a number of debilitating conditions including Alzheimer’s,
Parkinson’s and Creutzfeldt-Jakob diseases. Small heat-shock proteins
(sHsps), such as aB-crystallin, act as chaperones to prevent protein aggre-
gation and are thought to play a key role in the prevention of protein-mis-
folding diseases. In this study, we have explored the potential for small
molecules such as arginine and guanidine to affect the chaperone activity
of aB-crystallin against disordered (amorphous) and ordered (amyloid
fibril) forms of protein aggregation. The effect of these additives is highly
dependent upon the target protein undergoing aggregation. Importantly,
our results show that the chaperone action of aB-crystallin against aggrega-
tion of the disease-related amyloid fibril forming protein a-synucleinA53T
is enhanced in the presence of arginine and similar positively charged com-

pounds (such as lysine and guanidine). Thus, our results suggest that target
protein identity plays a critical role in governing the effect of small mole-
cules on the chaperone action of sHsps. Significantly, small molecules that
regulate the activity of sHsps may provide a mechanism to protect cells
from the toxic protein aggregation that is associated with some protein-
misfolding diseases.
Abbreviations
ANS, 8-anilino-1-naphthalene sulphonate; DTT, 1,4-dithiothreitol; Gdn, guanidine; RCMj-CN, reduced and carboxymethylated j-casein;
sHsp, small heat-shock protein; ThT, thioflavin T.
FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS 935
inhibit aggregation of expressed proteins or to
resolubilize proteins that have already aggregated into
inclusion bodies [6,7]. In suppressing aggregation, these
small molecules act by weakening the hydrophobic in-
termolecular interactions between unfolded or partially
folded protein intermediates that are responsible for
the aggregation process. The amino acid arginine is
also often employed as a suppressor of aggregation,
and is thought to facilitate correct folding of proteins
by destabilizing incorrectly folded structures [8,9].
However, high concentrations of guanidine, urea
and ⁄ or arginine are usually required for this purpose
and must be removed during purification of the recom-
binant protein.
In vivo, protein aggregation is prevented through the
action of a broad range of highly specialized proteins
known as molecular chaperones. One such chaperone is
a-crystallin, a small heat-shock protein (sHsp) that acts
to prevent protein aggregation intracellularly [10].
a-Crystallin is present in large concentrations in the eye

lens, where it is thought to provide stability and struc-
tural support to the other proteins present. It is made
up of two closely related subunits, aA- and aB-crystal-
lin, which exist at an approximate molar ratio of 3 : 1
in the mammalian lens. Moreover, aB-crystallin is
found at significant levels in other tissues, such as the
heart, kidney, muscle and brain, and its expression is
up-regulated in response to stress and pathological con-
ditions [11,12]. Recent studies have shown that signifi-
cant levels of aB-crystallin are found in protein
deposits such as those associated with disease [13,14].
The molecular chaperone action of aA- and aB-crystal-
lin is manifested by binding to partially unfolded or
misfolded target proteins, thus inhibiting their aggrega-
tion and precipitation. Whilst the chaperone action of
aB-crystallin against amorphously aggregating target
proteins has been well established, it is only recently
that studies have shown that aB-crystallin also acts to
prevent ordered amyloid fibril assembly [15–18].
Some studies have shown that structural perturba-
tion of a -crystallin and ⁄ or its two subunits (e.g.
through heating) enhances its chaperone activity
against amorphously aggregating target proteins [19–
21], presumably due to increased exposure of its
hydrophobic surfaces that facilitate target protein
binding [22]. In addition to temperature, other treat-
ments (e.g. reduction) [23,24] and post-translational
modifications (e.g. phosphorylation) [18,25,26] that
slightly perturb the structure of a-crystallin have been
shown to enhance the chaperone activity of the protein

against amorphously aggregating target proteins. Of
particular note, low concentrations of denaturant, such
as guanidine hydrochloride (Gdn-HCl) enhance the
chaperone activity of a-crystallin against reduction-
induced amorphous aggregation of the insulin B-chain
[27]. Moreover, it was also shown that millimolar con-
centrations of arginine hydrochloride (Arg-HCl) had a
similar effect on the chaperone activity of aB-crystallin
[27], which was reported to occur via enhancement of
the dynamics of subunit assembly [28]. However, to
date there have been no reports of the effects of such
compounds on the chaperone activity of aB-crystallin
against ordered protein aggregation leading to fibril
formation.
In this study, we have explored the potential for
small molecules such as Arg-HCl and Gdn-HCl to
affect the chaperone activity of aB-crystallin against
disordered (amorphous) and ordered (amyloid fibril)
forms of protein aggregation. We report that the effect
of these additives on the chaperone action of aB-crys-
tallin is dependent on the target protein used, and
therefore the results highlight the need to assess the
activity of chaperone proteins against a variety of tar-
get proteins before drawing conclusions about their
generic effects. Of particular note, the results from this
study show that the chaperone action of aB-crystallin
against aggregation of the disease-related amyloid fibril
forming protein, a-synucleinA53T, is enhanced in the
presence of Arg-HCl and similar positively charged
compounds (such as Lys-HCl and Gdn-HCl). Fibril

formation by a-synuclein is causally linked to Lewy
body formation that occurs in diseases such as Parkin-
son’s, and the A53T mutant is associated with early-
onset Parkinson’s disease. Thus, our results suggest
that small molecules that act on sHsps in a similar
manner to Arg-HCl may provide a mechanism to pro-
tect cells from the toxic protein aggregation that is
associated with some protein-misfolding diseases.
Results
The effect of Arg-HCl on the chaperone activity of
aB-crystallin is target protein-specific
In order to investigate the effect of Arg-HCl on the
chaperone activity of aB-crystallin, we examined a
variety of model target proteins to determine the
generic effects of Arg-HCl. In particular, we used
both ordered (amyloid fibril-forming) and disordered
(amorphous) target protein aggregation systems. In
investigating the effect of Arg-HCl on the chaperone
action of aB-crystallin, we also looked at related mole-
cules, to investigate whether any observed effects were
specific to Arg-HCl. Thus, we also investigated the
effects of (a) glycine (Gly), to test whether any effects
were attributable to addition of an amino acid to the
Chaperone activity of aB-crystallin H. Ecroyd and J. A. Carver
936 FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS
solution; (b) lysine hydrochloride (Lys-HCl), to test
whether any effects were attributable to adding a basic
amino acid; and (c) Gdn-HCl, to test whether any
effects of Arg-HCl were attributable to the guanidini-
um group of the molecule. We tested each of these

compounds at low (10 mm), intermediate (100 mm)
and high (250 mm) concentrations unless otherwise
indicated. At these concentrations, the additives were
found to change the pH of the buffers used in these
aggregation assays by < 0.1 units. However, at very
high concentrations (e.g. > 500 mm), some of the
compounds had significant effects on the pH of these
buffers (i.e. increasing the pH by > 0.2 units). In
addition, for each assay we used concentrations of
aB-crystallin that only partially inhibited aggregation
of the target protein in order to enable the effects of
the compounds on the chaperone activity to be readily
interpreted.
Disordered (amorphous) aggregation systems
Reduction-induced aggregation of a-lactalbumin
Upon addition of 1,4-dithiothreitol (DTT), aggregation
and precipitation of a-lactalbumin commenced after
25 min and reached a plateau after 90 min. The
amount of DTT-induced aggregation of a-lactalbumin
was increased in a concentration-dependent manner by
the addition of Gly, such that, at 250 mm, light scat-
tering due to its precipitation had increased by
50 ± 7% [mean ± standard error of the mean
(SEM)], i.e. the calculated percentage protection value
was negative because this treatment increased the
amount of precipitation compared to that observed
when a-lactalbumin was incubated alone (Fig. 1A,C).
Lys-HCl had a similar concentration-dependent effect.
However, Arg-HCl had the opposite effect whereby
increasing concentrations of Arg-HCl decreased the

amount of precipitation, such that, at high concentra-
tions, it had decreased by 60 ± 3% compared to that
observed when a-lactalbumin was incubated alone.
Gdn-HCl had a more complex effect, whereby concen-
trations up to 100 mm increased the amount of light
scattering, but the high concentration (i.e. 250 mm)
decreased it (Fig. 1A,C). Whilst Gly, Lys-HCl and
Arg-HCl had no significant effect on the lag phase of
precipitation of a-lactalbumin (approximately 25 min),
Gdn-HCl decreased it to 15 min (Fig. 1A).
Addition of aB-crystallin at a 1.0 : 1.0 w ⁄ w ratio
of a-lactalbumin : aB-crystallin decreased the precipi-
tation of a-lactalbumin by 81 ± 8%. The ability of
A
C
B
Fig. 1. The effect of additives on the ability
of aB-crystallin to prevent the DTT-induced
aggregation of a-lactalbumin. a-Lactalbumin
(
, 0.5 mgÆmL
)1
) was incubated at 37 °Cin
50 m
M phosphate buffer, pH 7.2, containing
100 m
M NaCl with 20 mM DTT in (A) the
absence or (B) the presence of aB-crystallin
(0.5 mgÆmL
)1

), and the change in light scat-
tering at 340 nm was monitored over time.
For both (A) and (B), the additives were
250 m
M of Gly (d), Lys-HCl ()), Arg-HCl
(
) or Gdn-HCl (h). The buffer-only control
(r) is also shown in (A) and (B). (C) Percent-
age protection (mean ± SEM of four inde-
pendent experiments), calculated 90 min
after the start of the assay, when a-lactalbu-
min was incubated with increasing concen-
trations of the additives, in the absence (
)
or presence (
)ofaB-crystallin. The per-
centage protection that would be expected
assuming no influence of the additives on
the chaperone activity of aB-crystallin, calcu-
lated as described in Experimental proce-
dures, is also shown (j). The asterisks
indicate a significant (P < 0.05) decrease in
the chaperone ability of aB-crystallin in the
presence of that concentration of the addi-
tive.
H. Ecroyd and J. A. Carver Chaperone activity of aB-crystallin
FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS 937
aB-crystallin to protect against this precipitation was
significantly decreased in the presence of Gly, Lys-
HCl and Gdn-HCl, such that, when they were present

at high concentrations, aB-crystallin had no effect on
the amount of light scattering compared to that
observed when the additives were present alone
(Fig. 1C). In contrast, the chaperone action of aB-
crystallin against a-lactalbumin was maintained in the
presence of intermediate concentrations of Arg-HCl,
but was not further enhanced by it (Fig. 1C). The sig-
nificant decrease in the amount of precipitation in
the presence of high concentrations of Arg-HCl
in the absence of the chaperone precluded analysis of
the effect of this concentration on the protective
ability of aB-crystallin.
Reduction-induced aggregation of the insulin B-chain
Light scattering due to DTT-induced amorphous
aggregation and precipitation of the insulin B-chain
commenced after 10 min and reached a plateau after
45 min (Fig. 2A). The amount of precipitation was
increased in a concentration-dependent manner by Gly
and Lys-HCl compared to that observed when insulin
was incubated alone (Fig. 2A,C). Addition of Arg-HCl
(up to 250 mm) had a negligible effect on the amount
of precipitation. Similarly, low and intermediate con-
centrations of Gdn-HCl had no effect on the precipita-
tion of insulin; however, high concentrations (i.e.
250 mm) had a protective effect, decreasing the
amount of light scattering by 48 ± 2% (Fig. 2A,C).
None of the additives used affected the lag phase of
the aggregation.
When incubated in the presence of aB-crystallin
alone (at a 1.0 : 1.0 w ⁄ w ratio of insulin : aB-crystal-

lin), the precipitation of insulin was inhibited by
40 ± 4% (Fig. 2B,C). Only Arg-HCl significantly
(P < 0.05) enhanced this protective activity of aB-
crystallin, such that, at 250 mm Arg-HCl, the light
scattering due to precipitation of insulin was
decreased by 65 ± 8%. Low and intermediate con-
centrations of Gly had no effect on the chaperone
activity of aB-crystallin against this target protein,
but it was significantly reduced at 250 mm. A similar
trend was observed for Lys-HCl, with high concen-
trations significantly inhibiting the ability of aB-crys-
tallin to prevent precipitation (Fig. 2C). Gdn-HCl
had no effect on the chaperone activity of aB-crystal-
lin against the DTT-induced aggregation and precipi-
tation of insulin.
A
C
B
Fig. 2. aB-crystallin protects against the
DTT-induced aggregation of insulin, and this
activity is enhanced by Arg-HCl. Insulin
(
, 0.25 mgÆmL
)1
) was incubated at 37 °C
in 50 m
M phosphate buffer, pH 7.2, with
10 m
M DTT in (A) the absence or (B) the
presence of aB-crystallin (0.25 mgÆmL

)1
).
For other details, refer to the legend to
Fig. 1. In addition, the hash symbol (#) indi-
cates a significant (P < 0.05) increase in the
chaperone ability of aB-crystallin in the pres-
ence of that concentration of the additive.
Chaperone activity of aB-crystallin H. Ecroyd and J. A. Carver
938 FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS
Heat-induced aggregation of catalase
We used bovine catalase as the model substrate to test
the effect of the small molecules on the chaperone abil-
ity of aB-crystallin against a target protein undergoing
heat-stressed induced aggregation and precipitation.
Aggregation of catalase occurs at high temperatures,
i.e. 55 °C, and these studies aimed to investigate
whether these small molecules could further enhance
the well-characterized increase in the chaperone activ-
ity of aB-crystallin at high temperatures due to
changes in its tertiary structure [20,21]. The precipita-
tion of catalase commenced after 20 min, and the
increase in light scattering due to precipitation of the
protein reached a maximum after 90 min (Fig. 3A).
All of the additives tested increased the amount of
light scattering due to precipitation of catalase com-
pared to that observed when it was incubated alone.
Of these, Gdn-HCl had the most dramatic effect, with
250 mm Gdn-HCl increasing the amount of precipita-
tion of catalase by 190 ± 5% (Fig. 3A). The presence
of aB-crystallin at a 1.0 : 0.5 w ⁄ w ratio of cata-

lase : aB-crystallin inhibited the precipitation of cata-
lase by 71 ± 7% (Fig. 3B). This chaperone activity
was not affected by increasing concentrations of Gly,
but was completely abolished by intermediate and high
concentrations of Lys-HCl, and was inhibited by Gdn-
HCl in a concentration-dependent manner (Fig. 3B,C).
Intermediate concentrations (i.e. 100 mm) of Arg-HCl
significantly inhibited the ability of aB-crystallin to
prevent the precipitation of catalase; however, this
effect was not seen at high concentrations of Arg-HCl,
i.e. the chaperone activity of aB-crystallin was main-
tained in the presence of 250 mm Arg-HCl.
Ordered aggregation leading to amyloid fibril
formation
We employed two models to examine the effect of the
small molecules on the ability of aB-crystallin to pre-
vent amyloid fibril formation – a familial mutant of
the disease-related protein a-synuclein (i.e. a-synuclein-
A53T) and reduced and carboxymethylated j-casein
(RCMj-CN), both of which are natively disordered
proteins [29]. We employed these systems as they both
form fibrils at physiological pH and temperature
[30,31], and so can be used to examine the activity of
aB-crystallin without confounding factors such as low
pH or the presence of other denaturants, which are
often required in other amyloid fibril-forming systems.
A
C
B
Fig. 3. Heat-induced amorphous aggrega-

tion of catalase is increased by increasing
concentrations of the additives. Catalase
(
, 0.5 mgÆmL
)1
) was incubated at 55 °Cin
50 m
M phosphate buffer, pH 7.2, in (A) the
absence or (B) the presence of aB-crystallin
(0.25 mgÆmL
)1
). For other details, refer to
the legend to Fig. 1.
H. Ecroyd and J. A. Carver Chaperone activity of aB-crystallin
FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS 939
In both systems, fibril formation was assessed by an
in situ thioflavin T (ThT) fluorescence assay.
Amyloid fibril formation by RCMj-CN
Fibril formation by RCMj-CN, as monitored by an
increase in ThT binding, showed a gradual increase
over the time course of the assay (Fig. 4A). At the end
of the assay, electron micrographs of negatively stained
RCMj-CN fibrils showed them to be thread-like
structures, approximately 100–700 nm in length
(Fig. 6A,B), similar to those reported previously [30].
Addition of Gly slightly increased the degree of ThT
binding in a concentration-dependent manner, such
that, at 250 mm, there was an increase of 10 ± 1%
compared to that observed when RCMj-CN was
incubated alone (Fig. 4A,C). Lys-HCl, Arg-HCl and

Gdn-HCl all decreased the change in ThT fluorescence
associated with amyloid fibril formation by RCMj-CN
in a concentration-dependent manner, such that, at
250 mm of Arg-HCl and Gdn-HCl, the increase in
ThT was almost completely abolished (Fig. 4A), pre-
cluding analysis of the effect of these concentrations
on the chaperone activity of aB-crystallin (Fig. 4B,C).
None of the compounds had an effect on the morphol-
ogy of the amyloid fibrils formed (data not shown).
When incubated in the presence of aB-crystallin, the
change in ThT fluorescence associated with amyloid
fibril formation by RCMj-CN decreased by 30 ± 3%
(1.0 : 0.5 w ⁄ w ratio of RCMj-CN : aB-crystallin)
(Fig. 4B). The amino acids had no significant effect on
the chaperone activity of aB-crystallin against this
fibril-forming target protein (Fig. 4C). At 100 mm,
Gdn-HCl had a negative effect on the chaperone acti-
vity of aB-crystallin in preventing amyloid fibril forma-
tion by RCMj-CN.
Amyloid fibril formation by a-synucleinA53T
At 37 °C, the increase in ThT fluorescence associated
with fibril formation by a-synucleinA53T reached a
plateau after 140 h (Fig. 5A). Electron micrographs of
a-synucleinA53T at the end of the assay confirmed the
formation of fibrils, which were long (between 1 and
5 nm), straight and unbranched (Fig. 6C,D). Addition
of Gly and Lys-HCl at 250 mm increased both the rate
and magnitude of the change in ThT fluorescence asso-
ciated with fibril formation by a-synucleinA53T
(Fig. 5A,C). Overall, Arg-HCl had little effect on fibril

formation by a-synucleinA53T, whereas Gdn-HCl at
A
C
B
Fig. 4. Ordered aggregation of RCMj-CN
into amyloid fibrils is inhibited by aB-crystal-
lin but this activity is not affected by Arg-
HCl. The change in ThT fluorescence at
490 nm was used to monitor amyloid fibril
formation by RCMj-CN (
, 0.5 mgÆmL
)1
)in
(A) the absence or (B) the presence of aB-
crystallin (0.25 mgÆmL
)1
). For both (A) and
(B), RCMj-CN was incubated at 37 °Cin
50 m
M phosphate buffer, pH 7.2, without
shaking for 15 h in the presence of 250 m
M
of Gly (d), Lys-HCl ()), Arg-HCl ( ) or Gdn-
HCl (h). The buffer-only control (r) is also
shown. (C) Percentage protection data
(mean ± SEM of three independent experi-
ments), calculated 15 h after the start of the
assay, for RCMj-CN incubated with increas-
ing concentrations of the additives in the
absence (

) or presence ( )ofaB-crystallin.
The percentage protection that would result
if there was no influence of the additives on
the chaperone activity of aB-crystallin, as
described in the Experimental procedures, is
also shown (j). The asterisk indicates
denotes a significant (P < 0.05) decrease in
the chaperone ability of aB-crystallin in the
presence of 100 m
M Gdn-HCl.
Chaperone activity of aB-crystallin H. Ecroyd and J. A. Carver
940 FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS
250 mm inhibited it by 53 ± 5%. This significant
decrease in the amount of aggregation in the presence
of high concentrations of Gdn-HCl precluded analysis
of the effect of this concentration when aB-crystallin
was also present. Therefore, we also tested Gdn-HCl at
100 mm in these studies (Fig. 5), and this concentration
was found to inhibit fibril formation by a-synuclein-
A53T by 21 ± 2%. None of the compounds were
found to have an effect on the morphology of the fibrils
formed by a-synucleinA53T (data not shown), and thus
A
C
B
Fig. 5. Amyloid fibril formation by a-synucle-
inA53T is inhibited by aB-crystallin, and this
chaperone activity is enhanced by Lys-HCl,
Arg-HCl and Gdn-HCl. Fibril formation was
induced by incubating a-synucleinA53T (

;
2.0 mgÆmL
)1
) with constant shaking at
37 °Cin50m
M phosphate buffer, contain-
ing 100 m
M NaCl, pH 7.4, for 5 days either
in (A) the absence or (B) the presence of
aB-crystallin (0.4 mgÆmL
)1
) and either
250 m
M of Gly (d), 250 mM of Lys-HCl ()),
250 m
M of Arg-HCl ( ) or 100 mM of Gdn-
HCl (h). The buffer-only control (r) is also
shown. (C) Percentage protection (mean ±
SEM of three independent experiments) for
a-synucleinA53T incubated with the addi-
tives in the absence (
) or presence of aB-
crystallin (
) was calculated using data from
the 160 h time point. The percentage pro-
tection that would result if there was no
influence of the additives on the chaperone
activity of aB-crystallin is also shown (j),
the hash symbol (#) denotes a significant
(P < 0.05) increase in the chaperone ability

of aB-crystallin in the presence of the addi-
tive. Note that the concentration of Gdn-HCl
used in this experiment is 100 m
M.
AB
CD
Fig. 6. Amyloid fibrils formed by the
ordered aggregation of RCMj-CN and
a-synucleinA53T. Electron micrographs of
RCMj-CN (0.5 mgÆmL
)1
, A and B) and
a-synculeinA53T (2.0 mgÆmL
)1
, C and D)
500 lgÆmL
)1
) following incubation at 37 °C
in 50 m
M phosphate buffer, pH 7.2, for 15 h
and 50 m
M phosphate buffer containing
100 m
M NaCl, pH 7.4, for 5 days, respec-
tively. The scale bars represent 1 lm (A, C)
and 0.2 lm (B, D).
H. Ecroyd and J. A. Carver Chaperone activity of aB-crystallin
FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS 941
the change in ThT fluorescence is interpreted to be
directly attributable to a change in the number of fibrils

formed in the presence of these additives. In the
presence of aB-crystallin (1.0 : 0.2 w ⁄ w ratio of
a-synucleinA53T : aB-crystallin), the increase in ThT
fluorescence associated with fibril formation by a-synu-
cleinA53T was decreased by 46 ± 3% (Fig. 5B,C). Gly
had no significant effect on the chaperone activity of
aB-crystallin in preventing the increase in ThT fluores-
cence associated with fibril formation by a-synuclein-
A53T, but both Lys-HCl and Arg-HCl were found to
significantly increase its chaperone activity, such that,
at 250 mm, the percentage protection was increased
to 27 ± 3% (Lys-HCl) and 99 ± 4% (Arg-HCl)
(Fig. 5C). Similarly, at 100 mm, Gdn-HCl also signifi-
cantly increased the chaperone activity of aB-crystallin
(84 ± 4%) against this target protein.
The effect of Arg-HCl on the structure and
assembly of aB-crystallin
We investigated whether the effects of these additives
on the chaperone action of aB-crystallin were attribut-
able to changes in the quaternary structure and oligo-
merization of the protein. We found that, at 250 mm,
none of the compounds had a significant effect on the
oligomeric size of aB-crystallin as assessed by size-
exclusion chromatography (Fig. 7A) (i.e. in either the
absence or presence of the compounds, aB-crystallin
was found to elute with an apparent mass of 580 kDa,
which corresponds to the mass of the oligomer
reported previously [32]). We also found no significant
differences in the accessibility of exposed hydrophobic
clusters, as assessed by ANS fluorescence (Fig. 7B), or

solvent accessibility of the N-terminal tryptophan resi-
dues (Trp9 and Trp60), as assessed by intrinsic fluores-
cence (data not shown), in the presence of these
compounds. Thus, it appears that the additives may
cause subtle changes in the structure of both the target
protein and aB-crystallin that lead to changes in the
chaperone activity of aB-crystallin for some target pro-
teins but not others.
Discussion
We have investigated the effect of Arg-HCl on the
chaperone activity of aB-crystallin against various tar-
get proteins undergoing either disordered (amorphous)
or ordered (i.e. amyloid fibril formation) aggregation.
We show that the effect of these compounds on the
chaperone activity of aB-crystallin is dependent on
the target protein undergoing aggregation. Thus, our
results highlight the need to consider a number of
aggregation systems in order to assess the effect of var-
ious additives and ⁄ or modifications on the overall
activity of chaperone proteins. Of the target proteins
tested, Arg-HCl was found to specifically increase the
activity of aB-crystallin against DTT-induced precipi-
tation of insulin at intermediate and high concentra-
tions, and it also increased the activity of aB-crystallin
in preventing the aggregation leading to amyloid fibril
formation by a-synucleinA53T when used at high
concentrations. With regard to the latter result, the
increase in chaperone activity resulting in the inhibi-
tion of fibril formation by a-synucleinA53T was not
specific for Arg-HCl as Lys-HCl and Gdn-HCl showed

similar effects (Fig. 5C).
A number of studies have indicated that small mole-
cules, including common metabolites such as pante-
thine and glutathione [33], can increase the chaperone
activity of a-crystallin. We confirm here previous
results showing that high concentrations of Arg-HCl
Fig. 7. The additives have no effect on the oligomeric size of aB-
crystallin (A) or its ability to bind ANS (B). (A) aB-crystallin
(1.0 mgÆmL
)1
), in the absence or presence of 250 mM of the addi-
tives, was loaded on to a Superdex 200HR 10 ⁄ 30 column and
eluted in 50 m
M phosphate buffer, pH 7.2, at a flow rate of
0.4 mLÆmin
)1
. Calibration of the column was performed using (1)
blue dextran, void; (2) thyroglobulin, 670 kDa; (3) c-globulin,
158 kDa; (4) ovalbumin, 44 kDa; (5) myoglobulin, 17 kDa. (B) ANS
fluorescence of aB-crystallin (0.1 mgÆmL
)1
)in50mM phosphate
buffer, pH 7.2, alone (
) or in the presence of 250 mM of Gly (d),
Lys-HCl ()), Arg-HCl (
) or Gdn-HCl (h), monitored following exci-
tation at 350 nm. The samples were maintained at 37 °C for
30 min before the fluorescence spectra were obtained.
Chaperone activity of aB-crystallin H. Ecroyd and J. A. Carver
942 FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS

(> 100 mm) increase the chaperone activity of
aB-crystallin against the DTT-induced precipitation of
insulin [27,28]. These studies also showed that 100 mm
Arg-HCl increases the chaperone activity of a-crystal-
lin against the thermally induced aggregation of f-crys-
tallin at 43 °C [27]. Our results indicate that this effect
of Arg-HCl is not limited to proteins undergoing dis-
ordered (amorphous) aggregation, as Arg-HCl also
increases the ability of aB-crystallin to reduce amyloid
fibril formation by a-synucleinA53T. This result is sig-
nificant due to the association of this type of protein
aggregation with disease. Lys-HCl and Gdn-HCl also
enhanced the chaperone activity of aB-crystallin
against this fibril-forming protein, implying that it is
the common positively charged group that plays a role
in increasing the activity of aB-crystallin against this
target protein. To our knowledge, this is the first study
that has investigated the effects of small molecules,
such as amino acids and Gdn-HCl, on the chaperone
function of sHsps against amyloid fibril-forming target
proteins. Whilst the concentrations used in these stud-
ies are high, the results suggest that small molecules
such as these may represent important therapeutic
leads for increasing the protective ability of chaperone
proteins against disease-related amyloid fibril forma-
tion.
Interestingly, none of the compounds tested
increased the chaperone activity of aB-crystallin
against amyloid fibril formation by RCMj-CN, a
milk-derived protein that readily forms fibrils under

conditions of physiological pH and temperature. The
differences in the effect of the small molecules on the
chaperone activity of aB-crystallin against the two
amyloid fibril-forming target proteins may be attribut-
able to differences in the rate of fibril formation
(RCMj-CN forms fibrils much more rapidly than
a-synucleinA53T) or the nature of the amyloidogenic
intermediate(s) with which aB-crystallin interacts.
Moreover, we found no generic effect of each com-
pound on the chaperone activity of aB-crystallin.
We have previously shown that phosphorylation of
aB-crystallin, which occurs under conditions of cellular
stress [34,35], also has a differential effect on its chap-
erone activity, increasing the activity against some
target proteins, but decreasing it against others [18].
Thus, we conclude that aB-crystallin most likely
employs various methods of binding (or binding
modes) in order to prevent the aggregation of stressed
proteins. Some of these binding modes (or binding
sites) are favoured by phosphorylation or interaction
with compounds such as Arg-HCl, whilst others are
either not affected or are perturbed. Studies using
destabilized T4 lysozyme mutants have shown that
both aA- and aB-crystallin possess at least two binding
modes, and that these are influenced by both external
factors (e.g. changes in temperature and pH) and
intrinsic factors (e.g. mutation and phosphorylation)
[23,26,36]. Various binding modes may facilitate the
interaction of aB-crystallin with the various intermedi-
ates formed during the aggregation process of diverse

targets. It may also enable the chaperone protein to
better cope with the various types of stresses experi-
enced by cells that cause proteins to unfold.
Of course, the effect of compounds such Arg-HCl
and Gdn-HCl may be also due to changes that they
induce in the stability and ⁄ or intermediate states of the
target protein itself. The denaturant effect of guanidine
on proteins is well established; it decreases the stability
of the native protein but also suppresses aggregation
by weakening the hydrophobic intermolecular interac-
tions between the unfolded states of a protein (i.e.
increasing the solubility of the unfolded state). In con-
trast, arginine has been shown to suppress aggregation
of some proteins by acting on the unfolded state of the
protein and increasing the reversibility of unfolding
[37]. Arginine had no effect on the stability of the pro-
tein’s native state, although it may also interact with it
[37]. This effect of arginine on protein aggregation has
been attributed to the guanidinium group of the
compound, which, through electrostatic interactions,
prevents the intermolecular interactions leading to
aggregation [37–39]. However, its effects vary from
protein to protein [9]. This is clearly evident from our
studies in which, even at low concentrations, the aggre-
gation of target proteins examined was affected by the
compounds used, and this varied for different target
proteins (e.g. whilst Arg-HCl at 250 mm had little
effect on the aggregation of insulin or a-synucleinA53T
alone, it dramatically increased the aggregation of cat-
alase and a-lactalbumin but significantly decreased the

ordered aggregation leading to fibril formation by
RCMj-CN). As such, consideration not only for the
effect of compounds on the activity of the chaperone
protein, but also its destabilized target, must be taken
into account when examining the effect of an additive
on the activity of chaperone proteins.
We have shown that the mechanism by which the
tested molecules influence the activity of aB-crystallin
is not through gross quaternary structural changes
(as assessed by size-exclusion chromatography; see
Fig. 6A) or changes in exposure of the tryptophan resi-
dues or clustered regions of exposed hydrophobicity
(as assessed by intrinsic and ANS fluorescence) of the
protein. With regard to the effect of Arg-HCl on the
mass of a
B-crystallin, a previous study [27], using glyc-
erol sedimentation, reported that 300 mm Arg-HCl
H. Ecroyd and J. A. Carver Chaperone activity of aB-crystallin
FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS 943
resulted in a decrease in the size of aB-crystallin, which
implies that, at higher concentrations than used in this
study, Arg-HCl may have a significant effect on the
quaternary structure of aB-crystallin. However, at
250 mm, we found that the effect of these additives on
the mass of aB-crystallin is negligible, and these data
are in agreement with previous work using Gdn-HCl
at the same concentration [40,41]. Previous studies
employing both near and far-UV circular dichroism
have also reported that there is little effect of Arg-HCl
on the overall secondary or tertiary structure of a-crys-

tallin, but that Arg-HCl mediates an increase in sub-
unit exchange and destabilization of the overall
structure of a-crystallin (as assessed by denaturation
with urea) [28]. Arginine’s side chain, the guanidinium
group, is able to interact with a number of functional
groups, including the aromatic side chains of some
amino acids, through a stacking mechanism [42]. The
interaction of arginine with aromatic amino acids of
aB-crystallin may facilitate its effects. Our results sug-
gest that an increase in subunit exchange in the pres-
ence of Arg-HCl may only be important in enhancing
the chaperone activity of sHsps against certain target
proteins. Moreover, these are likely to be limited to
those situations in which the chaperone forms only a
transient complex with the target protein, such as
has been described for the amorphous aggregation of
a-lactalbumin [43] and amyloid fibril formation by
apoC-II [16], as we found no evidence that the overall
ability of aB-crystallin to suppress the aggregation of
these target proteins was the same after extended time
periods.
In summary, our results show that the effect of
small compounds (such as Arg-HCl) on the chaperone
activity of aB-crystallin is highly dependent on the
aggregating target protein. Significantly, we found that
Arg-HCl, Lys-HCl and Gdn-HCl increased the ability
of aB-crystallin to prevent the ordered aggregation
leading to amyloid fibril formation of a mutant form
of the Parkinson’s disease-related protein a-synuclein
(i.e. a-synucleinA53T). These results suggest that, due

to their action on molecular chaperone proteins, bio-
logically compatible small molecules, such as Arg-HCl,
may be potential candidates as therapeutic agents in
the treatment of protein-misfolding diseases.
Experimental procedures
Materials
Bovine j-casein was obtained from Sigma Chemical Co.
(St Louis, MO, USA), and was reduced and carboxymethy-
lated (RCMj-CN) prior to use as described previously [44].
Thioflavin T (ThT), 8-anilino-1-napthalene sulfonate (ANS)
and b-mercaptoethanol, Arg-HCl, Gdn-HCl, Lys-HCl and
Gly were also obtained from Sigma. The vector pET24d(+)
(Novagen, Madison, WI, USA) containing the gene for
expression of human aB-crystallin was a kind gift from
W. de Jong and W. Boelens (University of Nijmegen, Neth-
erlands), and the vector pRSETB (Invitrogen, Carlsbad,
CA, USA) containing the human a-synucleinA53T gene
was a kind gift from R. Cappai (University of Melbourne,
Australia). The aB-crystallin and a-synucleinA53T proteins
were expressed and purified as described previously [45,46].
SDS–PAGE analysis of the purified proteins indicated that
they contained < 5% contaminating proteins. The concen-
trations of proteins used in these studies were determined
by spectrophotometric methods using a Cary 5000 UV-Vis-
NIR spectrophotometer (Varian, Melbourne, Australia),
and calculated extinction coefficients based on amino acid
sequences. All the buffers in these experiments were passed
through a 0.2 lm filter prior to use.
Intrinsic and extrinsic fluorescence
Intrinsic tryptophan fluorescence spectra of aB-crystallin

(0.1 mgÆmL
)1
in 50 mm phosphate buffer, pH 7.2), in the
presence or absence of the amino acids or Gdn-HCl, were
recorded using a Cary Eclipse fluorescence spectrophotome-
ter (Varian) equipped with temperature control and using a
cuvette with a 1 cm path length. The excitation wavelength
was set at 295 nm, and fluorescence emission was moni-
tored between 300 nm and 400 nm. The excitation and
emission slit widths were set at 5 nm. Samples were main-
tained at 37 °C for 30 min before being assayed.
For the ANS binding studies, a stock solution of meth-
anolic ANS (100 mm) was diluted 1000-fold into a
0.1 mgÆmL
)1
protein solution in 50 mm phosphate buffer,
pH 7.2. Emission fluorescence spectra were monitored
(400–600 nm) following excitation at 350 nm. The excita-
tion and emission slit widths were set at 5 nm. Samples
were maintained at 37 °C for 30 min before being assayed.
Chaperone activity assays
To test the relative chaperone activity of aB-crystallin in
the presence or absence of the additives, we monitored the
aggregation and ⁄ or precipitation of various target proteins
using either ThT fluorescence or turbidity assays (see
below). The effect of the additives on aggregation of the
target protein (in the absence and presence of aB-crystallin)
was assessed at the end of each assay by calculating the
percentage protection using the formula:
% protection ¼ 100 Â

ðDI
c
À DI
s
Þ
DI
c
where DI
c
and DI
s
represent the change in absorbance or
ThT fluorescence for the target protein in the absence
Chaperone activity of aB-crystallin H. Ecroyd and J. A. Carver
944 FEBS Journal 275 (2008) 935–947 ª 2008 The Authors Journal compilation ª 2008 FEBS
(control) and presence of the additives (± aB-crystallin),
respectively. All experiments were independently repli-
cated at least three times, and the results are shown as
means ± SEM. In each experiment, conclusions regarding
the effects of the additives on the chaperone activity of
aB-crystallin were drawn based on the measured percentage
protection when both the additive and chaperone were
present compared to the theoretical percentage protection
that would result if there was no influence of the additive
on the chaperone activity of aB-crystallin (i.e. the effect of
the additive on the aggregation of the target protein is inde-
pendent and different from that of the chaperone). The
standard errors associated with this theoretical percentage
protection were calculated from the standard errors
associated with the means of the measured values (i.e. by

calculating the square root of the sum of the associated
individual errors squared). The maximum percentage pro-
tection in these experiments is therefore 100%, i.e. complete
inhibition of an increase in light scattering or ThT fluores-
cence.
ThT assays
The formation of amyloid fibrils by RCMj-CN
(0.5 mgÆmL
)1
) and a-synucleinA53T (2.0 mgÆmL
)1
) was
monitored using a ThT binding assay method [47] devel-
oped for a 96-well microtitre plate format and adapted as
described previously [18]. Briefly, fibril formation by
RCMj-CN was monitored in real time by incubation at
37 °Cin50mm phosphate buffer, pH 7.2, without shaking
for 15 h. Single-point ThT readings were taken for a-synu-
cleinA53T during incubation at 37 °Cin50mm phosphate
buffer containing 100 mm NaCl, pH 7.4, and 0.02%
sodium azide for 5 days. The microtitre plate containing
a-synucleinA53T was subjected to constant shaking between
readings. All samples were incubated with 10 lm ThT,
which did not affect fibril formation for either protein.
Fluorescence levels were measured with a Fluostar Optima
plate reader (BMG Labtechnologies, Melbourne, Australia)
with a 440 ⁄ 490 nm excitation ⁄ emission filter set, and the
change in ThT fluorescence is reported. The change in ThT
fluorescence in the absence of the target protein was negli-
gible for each assay. The percentage protection for the ThT

assays was calculated using the data from the 15-h (RCMj-
CN) or 160-h (a-synucleinA53T) time points.
Turbidity assays
Light scattering at 340 nm was measured and recorded
using a Fluostar Optima plate reader (BMG Labtechnolo-
gies). For each assay, the plate was shaken for 5 s after
each cycle. The change in light scattering at 340 nm for
each sample is reported. The change in light scattering in
the absence of the target protein was negligible for each
assay. For the heat-induced amorphous aggregation assay,
bovine liver catalase (0.5 mgÆmL
)1
) was incubated at 55 °C
in 50 mm phosphate buffer, pH 7.2. For the reduction-
induced amorphous aggregation assays, a-lactalbumin
(0.5 mgÆmL
)1
) or bovine insulin (0.25 mgÆmL
)1
) were incu-
bated at 37 °Cin50mm phosphate buffer, pH 7.2, contain-
ing 100 mm NaCl, and aggregation and precipitation was
initiated by addition of DTT to a final concentration of
20 mm (a-lactalbumin) or 10 mm (insulin). The percentage
protection for these amorphous aggregation assays was cal-
culated using the data from the 90 min time point.
Size-exclusion FPLC
Size-exclusion chromatography of aB-crystallin (100 lLof
a 1.0 mgÆmL
)1

solution), in the presence or absence of the
additives (250 mm), was performed on a Superdex 200HR
10 ⁄ 30 column (Amersham Biosciences, Little Chalfont,
UK). Samples were eluted at a flow rate of 0.4 mLÆmin
)1
with 50 mm phosphate buffer, pH 7.2, containing the corre-
sponding amino acid or Gdn-HCl at 250 mm. The column
was calibrated using gel filtration markers (Bio-Rad, Hemel
Hampstead, UK).
Transmission electron microscopy
Samples were prepared for transmission electron micros-
copy as described previously [4]. Briefly, Formvar and
carbon-coated nickel electron microscopy grids (SPI Sup-
plies, West Chester, PA, USA) were prepared by the addi-
tion of 2 lL of protein sample, washed with 3 · 10 lLof
Milli-Q water and negatively stained with 10 lL of uranyl
acetate (2% w ⁄ v). Samples were viewed using a Philips
CM100 transmission electron microscope (Philips, Eindho-
ven, the Netherlands) at a magnification range of 10 500–
96 000 using an 80 kV excitation voltage.
Acknowledgements
We thank Mr Ying Xiao for performing preliminary
experiments involved in this work. This work was sup-
ported by grants (to JAC) from the National Health
and Medical Research Council (NHMRC) of Australia
and the Australian Research Council (ARC). HE is
supported by an NHMRC Peter Doherty postdoctoral
training fellowship.
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