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-

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

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

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

), 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

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

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-

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

) 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 (

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

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

(> 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

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

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

(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

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

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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