Báo cáo khoa học: Glutamic acid residues in the C-terminal extension of small heat shock protein 25 are critical for structural and functional integrity pptx

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

shock protein
Correspondence
M. J. Walker, School of Biological Sciences,
University of Wollongong, Wollongong,
NSW 2522, Australia
Fax: +61 2 4221 4135
Tel: +61 2 4221 3439
E-mail:
J. A. Carver, School of Chemistry &
Physics, The University of Adelaide,
Adelaide, SA 5005, Australia
Fax: +61 8 8303 4380
Tel: +61 8 8303 3110
E-mail:
(Received 19 February 2008, revised 14
September 2008, accepted 29 September
2008)
doi:10.1111/j.1742-4658.2008.06719.x
Small heat shock proteins (sHsps) are intracellular molecular chaperones
that prevent the aggregation and precipitation of partially folded and
destabilized proteins. sHsps comprise an evolutionarily conserved region of
80–100 amino acids, denoted the a-crystallin domain, which is flanked by
regions of variable sequence and length: the N-terminal domain and the
C-terminal extension. Although the two domains are known to be involved
in the organization of the quaternary structure of sHsps and interaction
with their target proteins, the role of the C-terminal extension is enigmatic.
Despite the lack of sequence similarity, the C-terminal extension of mam-
malian sHsps is typically a short, polar segment which is unstructured and
highly flexible and protrudes from the oligomeric structure. Both the polar-
ity and flexibility of the C-terminal extension are important for the mainte-

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-

teins by sHsps occurs through exposed hydrophobic
regions, and the resultant complex is stabilized through
electrostatic interactions [13]. Target proteins are held
in a folding-competent conformation until conditions
are permissive for their refolding or degradation, with
the former requiring the input of another chaperone
protein, e.g. Hsp70 [14].
sHsps comprise three structural regions: the con-
served a-crystallin domain is flanked by an N-terminal
domain and a C-terminal extension, both of which are
of variable length and sequence. Overall homology
amongst sHsps is therefore low [15]. Although exten-
sive work has been undertaken to elucidate the func-
tions of the N-terminal domain and a-crystallin
domain, the role of the C-terminal extension is less
clear. Despite its variability, the C-terminal extension
is a short region which is polar, highly flexible and
unstructured, and extends freely from the sHsp oligo-
mer [16]. These general properties are essential for the
correct functioning of sHsps as molecular chaperones.
Removal of the C-terminal extension inhibits the chap-
erone activity of aA-crystallin and Xenopus Hsp30C
[17,18], and also leads to a decrease in the solubility of
Hsp25, aA-crystallin and Caenorhabditis Hsp16-2
[17,19,20].
The C-terminal extension acts as a solubilizer to coun-
teract the hydrophobicity associated with target protein
sequestration [21]. The flexibility of the C-terminal
extensions of Hsp25 and aA-crystallin is maintained in
the final sHsp–target protein complex, with the exten-

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

aB-crystallin subunits can be inhibited by the replace-
ment of glutamic acid residues in the a-crystallin domain
with other residues, possibly through decreased electro-
static interactions and increased electrostatic repulsion
[30]. Similarly, it is likely that the glutamic acid residues
in the C-terminal extension of Hsp25 are important for
electrostatic interactions with the solvent and potentially
other regions of sHsp.
Although some studies have examined the role of
residues in the C-terminal extensions of other sHsps,
notably aA- and aB-crystallin, the flexible regions of
these proteins are unique and distinct from that of
Hsp25. The a-crystallins contain negatively charged
residues only near the anchor point of the flexible
region to the domain core, whereas Hsp25 has glu-
tamic acid residues spaced along its flexible extension.
Investigation into the function of these uniquely
positioned residues in Hsp25 has not been performed
previously.
Site-directed mutagenesis has been used in this study
to produce Hsp25 alanine substitution mutants of the
glutamic acid residues in the C-terminal extension, i.e.
E190A, E199A and E204A. An additional mutant,
Q194A, was also prepared and included as a control.
The role of the C-terminal extension and each of the
mutated residues was investigated by comparison of
the structure and function of these mutants with those
of wild-type Hsp25. The glutamic acid residue mutants
displayed altered structure and impaired thermostabil-
ity and chaperone activity compared with the wild-type

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.

Expression and purification of wild-type and
mutant Hsp25 proteins
Hsp25 mutants were designed to investigate the impor-
tance of the negatively charged residues in the C-termi-
nal extension. Wild-type Hsp25 and the Q194A and
glutamic acid residue mutants were purified success-
fully, as confirmed by the observation of the correct
masses by ESI-MS (not shown).
CD spectroscopy of wild-type and mutant Hsp25
Far-UV CD spectroscopy was performed to determine
whether substitution of the glutamine or glutamic acid
residues resulted in any alteration to the overall sec-
ondary structure of Hsp25. A broad minimum at
217 nm was observed for all spectra (Fig. 2, Table 2),
indicative of the predominance of b-sheet structure
[33]. The estimation of secondary structure content
obtained by deconvolution of the spectra was consis-
tent with previous measurements [20], with wild-type
Hsp25 having secondary structure contents of 38%
b-sheet and 6% a-helix at 25 °C. A slight increase in
Fig. 1. C-terminal extension sequences of murine Hsp25 and the 10
human sHsps aligned at their IXI motifs [67,68]. The IXI motifs are
shown in italics and the known flexible regions of various sHsps, as
determined by NMR spectroscopy [26,31,76], are in bold. Residues
used in the tally for Table 1 are underlined. Accession numbers
were: Hsp27 (P04792), MKBP (Q16082), aA-crystallin (P02489),
aB-crystallin (P02511), Hsp20 (O14558), HspB7 (Q9UBY9), Hsp22
(Q9UJY1), HspB9 (Q9BQS6) and ODFP (Q14990).
Table 1. Frequency of amino acid residues in the flexible region of the C-terminal extensions of human and murine sHsps. Residues present
in the flexible region of the C-terminal extension of each of the eight human sHsps that contain a flexible region (underlined residues in

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

unlikely that the secondary structure is altered signifi-
cantly as a result of the mutations, i.e. there was little
difference in overall secondary structure between the
wild-type and mutant proteins.
Tryptophan fluorescence spectroscopy of
wild-type and mutant Hsp25
Tryptophan fluorescence depends strongly on the local
environment of the amino acid and is a sensitive probe
of conformation in the vicinity of tryptophan residues
[36]. Fluorescence spectroscopy was performed on
wild-type and mutant Hsp25 to detect any changes in
the environment of the tryptophan residues resulting
from the mutations. The tryptophan residues of Hsp25
are located in the N-terminal domain at positions 16,
22, 43, 46 and 52, and in the a-crystallin domain at
position 99. A fluorescence maximum (F
max
)of
approximately 4700 arbitrary units with a wavelength
at maximum fluorescence (k
max
) of 340.2 nm was
observed for wild-type Hsp25 (not shown). No shift in
k
max
was observed for the mutants. A shift in k
max
is
indicative of a change in the polarity of the tryptophan
environment [37]. Therefore, the overall tryptophan

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.

Glutamic acid mutants of Hsp25 A. M. Morris et al.
5888 FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS
exposed clustered hydrophobicity of the protein and, if
altered, indicates a perturbation in tertiary structure
[38]. Such probes bind noncovalently to regions on pro-
teins that contain exposed clusters of hydrophobic ami-
noacyl residues, resulting in an increase in fluorescence
[39]. ANS binding fluorescence of wild-type and mutant
Hsp25 reached a maximum at a final concentration of
85 lm ANS, and the fluorescence values presented
(Fig. 3, Table 2) are the means of the plateau region of
the ANS binding curves (75–95 lm) (not shown). Wild-
type Hsp25 resulted in an ANS binding fluorescence of
approximately 570 arbitrary units. The Q194A, E190A,
E199A and E204A mutants exhibited increases in ANS
binding fluorescence of approximately 27%, 45%, 53%
and 63%, respectively, compared with the wild-type
protein, indicating that all of the mutants have greater
clustered hydrophobicity exposed to the solvent, and
thus an altered tertiary structure.
Oligomer formation by wild-type and
mutant Hsp25
Size-exclusion chromatography was performed in order
to determine whether the glutamic acid substitutions
affected the oligomeric size of Hsp25. Wild-type Hsp25
eluted between the molecular weight markers thyro-
globulin (mass of 669 kDa) and apoferritin (mass of
443 kDa) (Fig. 4), with an average molecular mass of
613 ± 185 kDa, as calculated from the standard curve
(not shown), corresponding to an average oligomer of

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
(

) Hsp25 and buffer ( ). Experiments were performed at
25 °C with 85 l
M ANS and an excitation wavelength of 387 nm.
Samples were prepared to a final concentration of 5 l
M in 50 mM
sodium phosphate buffer (pH 7.3) containing 0.02% NaN
3
.
Increases in maximum fluorescence of 27, 45, 53 and 63% were
observed for the Q194A, E190A, E199A and E204A mutants,
respectively, in comparison with wild-type Hsp25.
Fig. 4. Size-exclusion chromatography FPLC of wild-type ( ),
Q194A (
), E190A ( ), E199A ( ) and E204A ( )
Hsp25. Samples were prepared to a final concentration of 30 l
M in
50 m
M sodium phosphate buffer (pH 7.3) containing 0.02% NaN
3
,
with 100 lL being loaded onto the column. The peak positions of
the elution of molecular standards are indicated at the top of the
graph. The elution of wild-type Hsp25 corresponds to an average
molecular mass of 613 kDa. No significant differences in mass
were observed for the Q194A, E199A and E204A mutants. The
E190A mutant eluted at a volume corresponding to an average
molecular mass of 560 kDa.
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 5889
and the small increase in light scattering at tempera-

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

temperature have been well characterized [44]. This
temperature was also well below the onset of aggrega-
tion and precipitation for wild-type and mutant Hsp25
proteins, as shown by thermostability studies. Any
precipitation observed was therefore not attributable
to Hsp25 instability.
Complete suppression [45] of yeast ADH precipita-
tion was observed for wild-type Hsp25 at a molar ratio
of 1.4 : 1.0 Hsp25 : ADH (Fig. 6). A decrease in sup-
pression of ADH precipitation was observed at this
ratio for all of the glutamic acid residue mutants. At
all other ratios, the E199A mutant showed minor
reductions in suppression of ADH precipitation com-
pared with wild-type Hsp25, whereas the E190A and
E204A mutants showed markedly reduced suppression.
The Q194A mutant showed similar levels of suppres-
sion of aggregation to the wild-type protein at all
ratios.
Precipitation of insulin can be initiated by the addi-
tion of a reducing agent, such as dithiothreitol, which
cleaves the disulfide bonds between the A and B chains
of insulin, resulting in the aggregation and precipita-
tion of the B chain. Reduction stress assays are advan-
tageous over thermal stress assays as they can be
performed at physiological temperatures, i.e. 37 °C.
The precipitation of insulin under reduction stress was
completely suppressed by wild-type Hsp25 at a molar
ratio of 0.5 : 1.0 Hsp25 : insulin (Fig. 7), with all
mutants showing comparable suppression of insulin
precipitation at this ratio. All of the glutamic acid

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

of the onset of precipitation at 68 °C for E190A and 70 °C for
E199A and E204A.
Glutamic acid mutants of Hsp25 A. M. Morris et al.
5890 FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS
low sequence similarity, these regions are abundant in
disorder-promoting residues, such as proline [48]. The
hydrophilicity, lack of structure and associated flexibil-
ity are essential for the solubilizing role of the exten-
sion in the sHsp and complexation of the sHsp with
target proteins. The unstructured and highly dynamic
nature of this flexible region also ensures that it does
not interfere with or block the preceding conserved
IXI motif, which is important in subunit–subunit inter-
actions [49].
Despite the low sequence similarity throughout the
sHsp family, Hsp25 has a very similar, predominantly
b-sheet, secondary structure to that of other sHsps,
including mammalian aA- and aB-crystallin and bacte-
rial IpbB [50,51]. The glutamic acid residue substitu-
tions did not affect the secondary structure of Hsp25,
indicating that these residues, which are part of an
unstructured region, are not important for the determi-
nation of this level of structure. In support of this con-
clusion, mutants of aA- and aB-crystallin, in which the
C-terminal extensions were swapped, have been shown
to have secondary structures similar to each other and
to their wild-type counterparts [52]. Similarly, the
removal of the C-terminal extension of aA-crystallin,
aB-crystallin and bacterial Hsp16.3 produces proteins
with secondary structure comparable with that of the

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

wild-type protein indicates that the structure of the
N-terminal domain is maintained, at least in the
vicinity of the tryptophan residues. The increase in
exposed clustered hydrophobicity observed for the
mutants is therefore likely to arise from rearrange-
ments of secondary structural elements in the a-crys-
tallin domain.
The slightly reduced oligomeric size of the E190A
mutant compared with the wild-type suggests that the
E190 residue is important for the correct formation of
the quaternary structure of Hsp25. Examination of the
crystal structures of Hsp16.9 and Hsp16.5, which do
not have flexible C-terminal extensions, shows that the
conserved IXI motif (residues 185–187 in Hsp25) forms
hydrophobic contacts with a groove between b-strands
in the a-crystallin domain of another monomer, and
that this interaction is essential for the oligomerization
of sHsps [57]. Truncation from the C-terminus of
aA-crystallin to remove the IXI motif renders the pro-
tein unable to form oligomers [58], indicating that
interactions involving the IXI motif are also essential
for the oligomerization of mammalian sHsps contain-
ing a flexible C-terminal extension. Because of the
proximity between the E190 residue of Hsp25 and the
Fig. 7. Chaperone activity of wild-type and mutant Hsp25, as measured by the suppression of precipitation of insulin under reduction stress.
Ratios represent the molar concentration of Hsp25 monomers to insulin molecules. Assays were performed at 37 °Cin50m
M sodium phos-
phate buffer (pH 7.3) containing 0.02% NaN
3
. Traces are the average of triplicates. The precipitation of insulin was completely suppressed

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

C-terminal extensions to efficiently act as solubilizers.
At temperatures above 60 °C, Hsp25 and the a-crys-
tallins undergo changes in their tertiary structure that
result in the exposure of hydrophobic regions [56,59].
The onset of precipitation of the glutamic acid residue
mutants of Hsp25 corresponds approximately to this
temperature. The temperature-induced increase in
hydrophobic exposure did not induce the precipitation
of wild-type Hsp25, although a small increase in light
scattering implies the formation of larger aggregates
[41]. Thermostable proteins display more effective bur-
ial of hydrophobic regions than do less thermostable
proteins [28]. The increase in exposed hydrophobicity
associated with the mutations, coupled with the
temperature-induced increase in hydrophobicity, is
consistent with the poor thermostability observed for
the glutamic acid residue mutants.
The glutamic acid residue mutants showed reduced
chaperone activity compared with wild-type Hsp25
towards target proteins under different assay conditions,
a property that has also been observed recently for wild-
type and mutant forms of aB-crystallin [60,61]. The
E190A and E204A mutants performed poorly compared
with the wild-type protein in both assays, most notably
at the lower Hsp25 : target protein ratios used. The
E199A mutant showed somewhat decreased chaperone
activity towards ADH under heat stress, but performed
poorly at lower ratios towards insulin under reduction
stress. These functional differences were not a result of
the lack of solubilization of the chaperone, as both wild-

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].

In summary, the three negatively charged glutamic
acid residues in the C-terminal extension of Hsp25
(E190, E199 and E204) are essential for the correct
structure and function of this sHsp. These residues
contribute to the polarity of the extension and pro-
mote its disorder, ensuring that the C-terminal exten-
sion remains unstructured and solvent exposed, and
therefore able to perform its solubilizing role in the
sHsp and in the complexes formed with target proteins
during chaperone action. Indeed, the presence of sig-
nificant regions of structural disorder is a common
characteristic of molecular chaperones, and is integral
to their effective chaperone action [65]. Despite an
alteration in exposed hydrophobicity, the Q194A
mutant showed comparable oligomeric size and func-
tional properties to those of wild-type Hsp25. Thus,
residues in the flexible region of the C-terminal exten-
sion are not equally important for Hsp25 to perform
its role as a molecular chaperone, emphasizing the
importance of the glutamic acid residues.
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 5893
Experimental procedures
Sequence analysis of the C-terminal extension of
mammalian sHsps
The C-terminal extensions of the 10 human sHsps [66] were
aligned according to their IXI motifs, when present. When
absent, the alignments were based on those of Fontaine
et al. [67] and Franck et al. [68]. Residues that aligned with
the known flexible regions of aA- and aB-crystallin [16]

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

GACAGA-3¢ and 5¢-CTTTCGGGCTTTGTTAGCAG-3¢,
respectively.
Expression and purification of wild-type and
mutant Hsp25
pAK3038-Hsp25 was a gift from M. Gaestel (Institute of
Biochemistry, Hannover, Germany). DNA was transformed
into electrically competent BL21(DE3) Escherichia coli
before expression. Expression and purification of murine
Hsp25 and mutants were performed according to the
method described by Horwitz et al. [69] with minor
changes. Transformed cells were grown in Luria–Bertani
medium containing 0.4% (w ⁄ v) glucose and 100 lgÆmL
)1
ampicillin to select for pAK3038-Hsp25. Protein expression
was induced with 0.4 mm isopropyl thio-b-d-galactoside.
Cells were harvested by centrifugation and lysed as
described. After ultracentrifugation, dithiothreitol, polyeth-
yleneimine and EDTA were added to the supernatant to
final concentrations of 10 mm, 0.12% (v ⁄ v) and 1 mm,
respectively, and the lysate was incubated and centrifuged
as described. The final supernatant was filtered through a
0.22 lm Minisart filter (Sartorius, Epsom, UK) before
being loaded onto a DEAE-Sephacel (Sigma-Aldrich,
St Louis, MO, USA) column with a volume of approxi-
mately 90 mL. Recombinant Hsp25 was eluted with
100 mm NaCl in 20 mm Tris ⁄ HCl buffer (pH 8.5) contain-
ing 1 mm EDTA and 0.02% (w ⁄ v) NaN
3
. Fractions con-
taining Hsp25 were concentrated to approximately 5 mL

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

All fluorescence studies were performed at 25 °C using an
F-4500 fluorescence spectrophotometer (Hitachi High-Tech-
nologies, Tokyo, Japan) with a Thermomix temperature-
controlled water circulator (B. Braun, Melsungen,
Germany). Samples were prepared in 50 mm phosphate
buffer (pH 7.3) containing 0.02% (w ⁄ v) NaN
3
to a final
concentration of 5 lm, as calculated from A
280
values of
the samples, an extinction coefficient of 1.87 for a
1mgÆmL
)1
solution of Hsp25 [75] and molecular mass. An
Glutamic acid mutants of Hsp25 A. M. Morris et al.
5894 FEBS Journal 275 (2008) 5885–5898 ª 2008 The Authors Journal compilation ª 2008 FEBS
excitation wavelength of 295 nm was used for intrinsic
fluorescence, with emission spectra recorded from 300 to
450 nm. ANS binding fluorescence was performed with an
excitation wavelength of 387 nm. Sequential aliquots of
freshly prepared ANS were added to the samples and mixed
thoroughly until the emission fluorescence at 479 nm
reached a maximum. Emission spectra were then recorded
from 400 to 650 nm.
Size-exclusion chromatography
Size-exclusion chromatography was performed at room
temperature on a BioSep-SEC-S 4000 column (Pheno-
menexÒ, Torrance, CA, USA) connected to an
A

¨
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-

photometer (Varian, Palo Alto, CA, USA) with a Cary
temperature controller. Protein precipitation was monitored
by light scattering at 360 nm.
Chaperone activity assays
Thermally induced precipitation of 2 lm yeast ADH in the
presence of increasing concentrations of Hsp25 was per-
formed at 55 °C in a Hewlett Packard Diode Array UV-Vis
spectrophotometer (Agilent Technologies, Forest Hill, Vic.,
Australia) connected to a Thermomix temperature-con-
trolled water circulator (B. Braun). Samples were prepared
in 50 mm phosphate buffer (pH 7.3) containing 0.02%
(w ⁄ v) NaN
3
. Protein precipitation was monitored by light
scattering at 360 nm. Assays were performed in duplicate.
The precipitation of 45 lm insulin from bovine pancreas in
the presence of increasing concentrations of Hsp25 was per-
formed at 37 °C in a 96-well plate in a FLUOstar OPTIMA
plate reader (BMG LABTECH GmbH, Offenburg, Ger-
many). Samples were prepared in 50 mm phosphate buffer
(pH 7.3) containing 0.02% (w ⁄ v) NaN
3
. Precipitation was
initiated with the addition of dithiothreitol to a final concen-
tration of 20 mm and monitored by light scattering at
360 nm. Assays were performed in triplicate.
Acknowledgements
We are grateful to Professor Matthias Gaestel for pro-
viding the plasmid pAK3038. This work was supported
by grants to JAC from the National Health and Medi-

cal Research Council of Australia and the Australian
Research Council.
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