Glutamic acid residues in the C-terminal extension
of small heat shock protein 25 are critical for structural
and functional integrity
Amie M. Morris
1
, Teresa M. Treweek
2
, J. A. Aquilina
1
, John A. Carver
3
and Mark J. Walker
1
1 School of Biological Sciences, University of Wollongong, Australia
2 Graduate School of Medicine, University of Wollongong, Australia
3 School of Chemistry & Physics, The University of Adelaide, Australia
Small heat shock proteins (sHsps) are a family of
intracellular molecular chaperones defined by the pres-
ence of an evolutionarily conserved region of 80–100
amino acid residues, denoted the a-crystallin domain
[1]. Despite having a relatively small monomeric size
(12–43 kDa) [2], sHsps exist under physiological condi-
tions as large oligomers of up to 50 subunits and
1.2 MDa in mass [3,4]. sHsps are found in most cell
types in most organisms, and their expression is upreg-
ulated under a range of stress conditions, such as heat,
oxidative conditions, pH changes, infection and in
many disease states characterized by the formation of
Keywords
C-terminal extension; Hsp25; molecular
chaperone; protein aggregation; small heat

nance of sHsp solubility and for complexation with its target protein. In
this study, mutants of murine Hsp25 were prepared in which the glutamic
acid residues in the C-terminal extension at positions 190, 199 and 204
were each replaced with alanine. The mutants were found to be structurally
altered and functionally impaired. Although there were no significant dif-
ferences in the environment of tryptophan residues in the N-terminal
domain or in the overall secondary structure, an increase in exposed hydro-
phobicity was observed for the mutants compared with wild-type Hsp25.
The average molecular masses of the E199A and E204A mutants were
comparable with that of the wild-type protein, whereas the E190A mutant
was marginally smaller. All mutants displayed markedly reduced thermo-
stability and chaperone activity compared with the wild-type. It is con-
cluded that each of the glutamic acid residues in the C-terminal extension
is important for Hsp25 to act as an effective molecular chaperone.
Abbreviations
ADH, alcohol dehydrogenase; ANS, 8-anilinonaphthalene-1-sulfonate; sHsp, small heat shock protein.
FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS 5885
insoluble amyloid plaques, e.g. Alzheimer’s, Creutz-
feldt–Jakob and Parkinson’s diseases [5–8]. Increased
levels of sHsps, in particular aB-crystallin and Hsp27,
are observed in the brains of sufferers of these diseases
[9,10]. Hsp25 is the murine homologue of Hsp27.
Stress conditions can promote the partial unfolding
of proteins, which subsequently leads to the exposure
of hydrophobic residues [11]. This increase in surface-
exposed hydrophobicity encourages partially folded
proteins to mutually associate and potentially precipi-
tate [12]. sHsps prevent the aggregation of such pro-
teins by interacting with them and sequestering them
into a large complex. The recognition of target pro-

sions remaining solvent exposed. Under heat stress, the
extension of aB-crystallin has been shown to exhibit
reduced flexibility on sHsp–target complex formation,
implying that the extension may be involved in target
protein capture and have functions in addition to acting
as a solubilizer [22]. The oligomeric sizes of aA-crystal-
lin, bacterial Hsp16.3 and bacterial HspH are affected
by C-terminal extension removal [23,24], indicating that
the C-terminal extension is involved in the quaternary
structural arrangement of sHsps.
The alteration of the properties of the C-terminal
extension also leads to significant changes to the struc-
ture and function of sHsps. The chaperone activity of
Hsp30C is impaired when the polarity of the C-termi-
nal extension is reduced [25], and introduction of
hydrophobicity into the C-terminal extension of
aA-crystallin results in immobilization of the C-termi-
nal extension and reduced chaperone activity [21].
Conversely, an increase in the charge of the extension
of aA-crystallin results in no significant changes in
chaperone activity relative to wild-type aA-crystallin
[26,27], highlighting the importance of the polar resi-
dues in the C-terminal extension of sHsps.
The thermostability of proteins from thermophilic
organisms is related to electrostatic interactions through
the presence of polar and charged groups, as well as
hydrophobic and packing effects [28]. These proteins
typically have a higher proportion of polar and charged
residues, primarily glutamic acid and lysine, than their
mesophilic equivalents [29]. Interactions between

protein, highlighting the importance of the negatively
charged glutamic acid residues in the C-terminal exten-
sion of Hsp25.
Glutamic acid mutants of Hsp25 A. M. Morris et al.
5886 FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS
Results
Sequence analysis of the C-terminal extensions
of mammalian sHsps
The C-terminal extensions of mammalian sHsps are
highly variable in length and sequence, yet they share
the characteristics of being polar, flexible and unstruc-
tured, suggesting that the types of residue present in
the C-terminal extension, rather than their sequence,
are important. This was investigated by analysing the
amino acid residues corresponding to the known flexi-
ble regions of aA- and aB-crystallin and Hsp25
[16,26,31] (Fig. 1). Proline is present in six of the eight
human sHsps that contain a flexible region (Fig. 1,
Table 1) and in seven of the corresponding murine
sHsps (not shown), and is a predominant residue in
the flexible regions of both human and murine sHsps.
The majority of residues present in the flexible regions
of the extensions (71% and 74% for human and mur-
ine, respectively) are those that have been shown to
promote disorder (Table 1) [32]. Although the C-termi-
nal extension of human Hsp27 contains an aspartic
acid residue, that of murine Hsp25 does not (Fig. 1).
Thus, apart from the C-terminal carboxyl group, the
three glutamic acid residues are the only source of
negative charge in the flexible extension of Hsp25.

Fig. 1) are tallied. Only the totals are given for murine sHsps. Residues that promote disorder are in bold and those that promote order are
in italic [32]. Residues are denoted as charged (+ or )), polar (p) or nonpolar (n).
Residue PAEKST C R L D QGVNYI M FWH
Charge nn) +p p p + n ) pnnpnnn nn +
Hsp27 153221 1 1 1 12
HspB2 536 1 1 11
aA-crystallin 212131
aB-crystallin 2323 1 1
Hsp20 31 1
Hsp22 1 2 1 1 1
HspB9 1 1 11
ODFP 4124 2 1 1 1 1211 1
Human total 17 16 14 8 7 6 5 4 4 3 3332221 10 0
Murine total 18 13 14 9 10 1 6 4 3 2 4543131 10 0
A. M. Morris et al.
Glutamic acid mutants of Hsp25
FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS 5887
negative ellipticity and flattening of the spectra were
observed for wild-type and mutant Hsp25 samples with
increasing temperature from 25 to 55 °C. The increase
in negative ellipticity at around 210 nm implies an
increase in a-helical content [33]. However, following
deconvolution of the spectra, changes to each of the
structural element proportions were less than 5%
between all spectra, and were deemed to be insignifi-
cant [34]. The overall increase in negative ellipticity at
higher temperatures is consistent with a slight increase
in or stabilization of secondary structure [35]. In com-
paring the mutants with wild-type Hsp25, the consis-
tency of the deconvolution data suggests that it is

environment was not significantly affected by substitu-
tion of the glutamic acid residues in the C-terminal
extension, which are all distant in primary structure
from the tryptophan residues.
8-Anilinonaphthalene-1-sulfonate (ANS) binding
fluorescence spectroscopy of wild-type and
mutant Hsp25
The binding of ANS and other hydrophobic probes to a
protein enables the comparative determination of the
Table 2. Summary of changes in structure and function of C-terminal Hsp25 mutants. Comparisons are made with wild-type Hsp25. Qualita-
tive comparisons are given for exposed clustered hydrophobicity, thermostability and chaperone activity (DC18, Hsp25 truncation mutant
lacking the C-terminal 18 residues; ND, not determined).
Hsp25 mutant Charge
Secondary
structure
Exposed clustered
hydrophobicity
Average molecular
mass Thermostability
Chaperone
activity Reference
Q194A No change No change 27% increase No change No change No change This study
E190A +1 No change 46% increase 53 kDa smaller Poor Decreased This study
E199A +1 No change 54% increase No change Poor Decreased This study
E204A +1 No change 65% increase No change Poor Decreased This study
DC18 No change Increased a-helix 31% decrease No change ND Decreased [20]
Fig. 2. Far-UV CD spectra of wild-type ( ), Q194A ( ), E190A ( ), E199A ( ) and E204A ( ) Hsp25 at 25, 37 and 55 °Cin
10 m
M sodium phosphate buffer (pH 7.5). No significant differences in secondary structure between wild-type and mutant proteins were
observed at any of the three temperatures.

26–27 subunits. The peak maxima of the Q194A,
E199A and E204A mutants eluted at volumes almost
identical to that of the wild-type, indicating very simi-
lar average oligomeric sizes to the wild-type (Table 2).
The small extra peak in the elution profile of E199A
represents a protein of less than 250 kDa in mass. The
oligomeric species of wild-type Hsp25 is in equilibrium
with a tetrameric form [40], and so the smaller species
may be a tetramer. Elution of the E190A mutant was
delayed slightly compared with elution of the wild-type
protein, with the elution peak corresponding to a
calculated average molecular mass of approximately
53 kDa smaller than wild-type Hsp25, and to an aver-
age oligomer of 24–25 subunits. Thus, with the excep-
tion of the E190A mutant, the oligomeric size of
Hsp25 was not affected by glutamine or glutamic acid
residue mutations.
Thermostability studies of wild-type and
mutant Hsp25
The thermostability of wild-type and mutant Hsp25
was investigated by monitoring the increase in light
scattering at 360 nm as a result of the formation of
large aggregates, followed by precipitation with
increasing temperature. Wild-type Hsp25 was very heat
stable and remained in solution up to temperatures of
100 °C (Fig. 5, Table 2). No precipitate was observed
Fig. 3. ANS binding fluorescence emission spectra of wild-type
(
), Q194A ( ), E190A ( ), E199A ( ) and E204A
(

tures above approximately 70 °C is consistent with an
increase in aggregate size [41]. The Q194A mutant
showed a light scattering profile comparable with that
of the wild-type protein. In marked contrast, the glu-
tamic acid residue mutants all precipitated out of solu-
tion within 2 °C of the onset of aggregation, i.e. at
approximately 68 °C for E190A and 70 °C for E199A
and E204A. Decreased light scattering after maximum
precipitation had been reached resulted from the pre-
cipitate sinking to the bottom of the cuvette and there-
fore not obscuring the light path [42]. Thus, the Q194A
mutant showed thermostability similar to that of wild-
type Hsp25, whereas the glutamic acid residue mutants
exhibited significantly decreased thermostability.
Functional chaperone activity assays of wild-type
and mutant Hsp25
The chaperone activity of wild-type and mutant Hsp25
was assessed by determining the ability of these proteins
to prevent the amorphous aggregation and precipitation
of target proteins under stress conditions. Assays were
performed with alcohol dehydrogenase (ADH) under
heat stress and insulin under reduction stress in the pres-
ence of varying concentrations of Hsp25.
Yeast ADH is a tetramer of four equal subunits
with a total molecular mass of 141 kDa [43]. Thermal
stress assays using this enzyme are commonly per-
formed at temperatures of 48–60 °C. The optimal rate
of precipitation of yeast ADH for monitoring precipi-
tation was found to be 55 °C (not shown), and the
inactivation and precipitation of yeast ADH at this

residue mutants, in particular the E204A mutant,
displayed reduced suppression at the lower ratio
(0.05 : 1.0), and the E190A mutant showed reduced
suppression at 0.25 : 1.0. At all ratios, the Q194A
mutant exhibited very similar levels of suppression of
insulin B chain precipitation to the wild-type protein.
Taken together, the thermal and reduction stress
assays demonstrate that each of the glutamic acid
mutants, in particular E190A and E204A, are signifi-
cantly less effective chaperones than is wild-type
Hsp25 (Table 2).
Discussion
Many proteins contain intrinsically disordered regions
that are necessary for their function [46]. Accordingly,
these regions have a higher frequency of disorder-pro-
moting residues [32,47]. Such is the case for the flexible
regions located at the extremity of the C-terminal
extensions of human and murine sHsps. Despite the
Fig. 5. Thermostability profiles of wild-type ( ), Q194A ( ),
E190A (
), E199A ( ) and E204A ( ) Hsp25. Samples
were prepared to a final concentration of 0.2 mgÆmL
)1
in 50 mM
sodium phosphate buffer (pH 7.3). The temperature was increased
at a rate of 1 °CÆmin
)1
. Wild-type Hsp25 and Q194A remained in
solution up to temperatures of 100 °C. By contrast, the E190A,
E199A and E204A mutants precipitated out of solution within 2 °C

respective wild-type proteins [17,53,54] The secondary
structure of wild-type and mutant Hsp25 did not
change significantly from 25 to 55 °C, consistent with
previous findings that Hsp25, a-crystallin and IpbB
resist changes to secondary structure with increasing
temperatures up to approximately 60 °C [50,55,56].
The secondary structure of Hsp25 has also been shown
to be stable under mildly denaturing conditions [40],
and temperatures of at least 60 °C are required for a
loss of b-sheet structure [40].
Fig. 6. Chaperone activity of wild-type and mutant Hsp25, as measured by the suppression of precipitation of ADH under thermal stress.
Ratios represent the molar concentration of Hsp25 monomers to ADH subunits. Assays were performed at 55 °Cin50m
M sodium phos-
phate buffer (pH 7.3) containing 0.02% NaN
3
. Traces are the average of duplicates. The precipitation of ADH was completely suppressed at
an Hsp25 : ADH ratio of 1.4 : 1.0. The Q194A mutant showed comparable chaperone activity with the wild-type protein. Each of the
glutamic acid residue mutants displayed a decrease in chaperone activity compared with wild-type Hsp25.
A. M. Morris et al. Glutamic acid mutants of Hsp25
FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS 5891
Although the secondary structure of Hsp25 was not
altered as a result of the mutations, significant differ-
ences in exposed hydrophobicity, as indicated by
ANS binding, were observed, suggesting that the
same elements of secondary structure were adopted,
but that the subunits were arranged differently from
that of the wild-type protein. Because five of the six
tryptophan residues in Hsp25 are located in the
N-terminal domain, the comparable overall trypto-
phan exposure in the mutants compared with the

at an Hsp25 : insulin ratio of 0.5 : 1.0. The Q194A mutant showed comparable chaperone activity with the wild-type protein. Each of the
glutamic acid residue mutants displayed a decrease in chaperone activity compared with wild-type Hsp25.
Glutamic acid mutants of Hsp25 A. M. Morris et al.
5892 FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS
IXI motif, it is possible that substitution of this residue
disrupts the interaction between the IXI motif and the
hydrophobic groove, resulting in an altered oligomeric
structure. The formation of large sHsp oligomers is
also dependent on interactions between N-terminal
domains [23], which are not affected by mutations in
the C-terminal extension. The comparability of oligo-
meric sizes between the mutants and wild-type Hsp25
clearly demonstrates this.
In mammalian sHsps, the presence of a flexible, sol-
vent-exposed C-terminal extension helps to counteract
the large amount of hydrophobicity exposed by the
remainder of the protein [15,26]. Removal of the
C-terminal extension results in a decrease in thermo-
stability of Hsp25, aA-crystallin, Xenopus Hsp30C and
Caenorhabditis Hsp16-2 [17–20]. The drastically
reduced thermostability of the glutamic acid residue
mutants demonstrates the importance of each of the
negatively charged residues in the C-terminal extension
in maintaining the solubility of the Hsp25 oligomer.
Similarly, the introduction of hydrophobicity into the
C-terminal extension of aA-crystallin results in a
decrease in the thermostability of this sHsp [21]. These
data suggest that relatively modest alterations to the
C-terminal extensions of sHsps, resulting in a decrease
in polarity, are sufficient to disrupt the ability of the

type Hsp25 and the glutamic acid residue mutants of
Hsp25 were stable in solution at the temperatures at
which these assays were performed. Recognition of and
interaction with target proteins by sHsps is largely
hydrophobic in nature [13]. On this basis, it would be
expected that the Hsp25 mutants, with increased surface
hydrophobicity, would display enhanced chaperone
ability compared with the wild-type protein [51].
However, the structural changes associated with the
mutations appear to have a greater influence on the
chaperone activity than simply the degree of exposed
hydrophobicity [62].
Although conclusive identification of the chaperone
binding sites of sHsps remains elusive, there is evidence
that the binding of target proteins occurs in the groove
between monomers, and involves a b-sheet region
located at the beginning of the a-crystallin domain cor-
responding to residues 70–88 in aA-crystallin [63,64].
The changes in tertiary structure observed for the
mutants, as evidenced by the alteration in exposed
hydrophobicity, could result in the disruption of bind-
ing sites, and thus hindered recognition and sequestra-
tion of target proteins. These changes may also inhibit
stabilization by electrostatic interactions, resulting in
less effective target protein sequestration. The decrease
in polarity of the C-terminal extension may also facili-
tate interaction between the extension and hydro-
phobic chaperone binding sites, resulting in the
binding sites being less accessible to the target protein,
leading to a decrease in target protein binding [26].

were tallied. The C-terminal extensions of the equivalent
murine sHsps were similarly analysed.
Site-directed mutagenesis of pAK3038-Hsp25
Site-directed mutagenesis was performed using the Quik-
Change
Ò
system (Stratagene, La Jolla, CA, USA), accord-
ing to the manufacturer’s instructions, except that 14 cycles
were used (Cooled-Palm 96, Corbett Research, Mortlake,
NSW, Australia). All primers were synthesized by Sigma
Genosys (Castle Hill, NSW, Australia). The primer pairs
for site-directed mutagenesis were as follows: 5¢-TTCGA
GGCCCGCGCCGCAATTGGGGGCCCAGAA-3¢ and 5¢-
TTCTGGGCCCCCAATTGCGGCGCGGGCCTCGAA-3¢
for E190A, 5¢-ATTCCGGTTACTTTCGCGGCCCGCGC
CCAAATT-3¢ and 5¢-AATTTGGGCGCGGGCCGCGA
AAGTAACCGGAAT-3¢ for E190A, 5¢-CAAATTGGGGG
CCCAGCAGCTGGGAAGTCTGAA-3¢ and 5¢-TTCAGA
CTTCCCAGCTGCTGGGCCCCCAATTTG-3¢ for E199A,
and 5¢-GAAGCTGGGAAGTCTGCACAGTCTGGAGCC
AAG-3¢ and 5¢-CTTGGCTCCAGACTGTGCAGACTTCC
CAGCTTC-3¢ for E204A. Mutated codons are shown in
italic type. Dimethylsulfoxide was added to a final concen-
tration of 5% (v ⁄ v) to reactions in which strong secondary
interactions were likely, as advised by the supplier. Success-
ful mutagenesis was confirmed by DNA sequence analysis
of the forward and reverse strands with BigDyeÔ Termina-
tor Ready Reaction Mix (Applied Biosystems, Foster City,
CA, USA) on a Prism 377 DNA sequencer (Applied
Biosystems) using the primers 5¢-TCTCGGAGATCC

and dithiothreitol was added to a final concentration of
50 mm. The sample was incubated at room temperature for
30 min before being loaded onto a Sephacryl S-300HR
(Pharmacia, Uppsala, Sweden) column with a volume of
approximately 470 mL. Recombinant Hsp25 eluted in the
first peak with 50 mm Tris ⁄ HCl buffer (pH 8.0) containing
1mm EDTA and 0.02% (w ⁄ v) NaN
3
. Fractions containing
Hsp25 were concentrated, dialysed exhaustively against,
or exchanged into, MilliQ water and lyophilized. Both
chromatographic steps were performed at 4 °C. The purity
of recombinant proteins was confirmed by nanoscale
ESI-MS.
Far-UV CD spectroscopy
CD spectra were acquired on a J-810 spectropolarimeter
(Jasco, Tokyo, Japan) with an attached Peltier temperature-
controlled water circulator. Samples were prepared in
10 mm phosphate buffer (pH 7.5) to a final concentration
of 10–15 lm and filtered through a 0.22 lm Minisart filter.
Spectra were recorded at 25, 37 and 55 °C, and are accu-
mulations of 16 scans recorded from 190 to 250 nm with a
path length of 1 mm. The sample concentration was deter-
mined using a bicinchoninic acid assay (Sigma-Aldrich). An
estimation of secondary structure composition was
performed using the cdsstr program [70–72] in the
DICHROWEB Online Circular Dichroism Analysis suite
[73,74].
Intrinsic tryptophan fluorescence and ANS
binding fluorescence spectroscopy

¨
KTAÔFPLCÔ system (Amersham Biosciences, Little
Chalfont, Buckinghamshire, UK). Samples were prepared
to a final concentration of 30 lm, as determined by A
280
values, with 100 lL being loaded onto the column. Protein
was eluted at 0.5 mLÆmin
)1
with 50 mm phosphate buffer
(pH 7.3) containing 0.02% (w ⁄ v) NaN
3
and detected at
280 nm. The column was calibrated with blue dextran
(2 MDa), thyroglobulin (669 kDa), apoferritin (443 kDa)
and catalase (250 kDa). Standard deviations are given as
the oligomeric range at half peak height [69].
Thermostability studies
Hsp25 solutions were prepared at 0.2 mgÆmL
)1
, as deter-
mined by A
280
values, in 50 mm phosphate buffer (pH 7.3)
containing 0.02% (w ⁄ v) NaN
3
. Samples were heated from
25 to 100 °Cat1°CÆmin
)1
in 1 mL quartz cuvettes with a
1 cm path length in a Cary-500 Scan UV-Vis-NIR spectro-

cal Research Council of Australia and the Australian
Research Council.
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