Báo cáo khoa học: Characterization of novel sequence motifs within N- and C-terminal extensions of p26, a small heat shock protein from Artemia franciscana potx

Characterization of novel sequence motifs within N- and
C-terminal extensions of p26, a small heat shock protein
from Artemia franciscana
Yu Sun and Thomas H. MacRae
Department of Biology, Dalhousie University, Halifax, Canada
The small heat shock proteins (sHSPs), characterized
by a conserved a-crystallin domain of approximately
90 residues and the ability to reversibly oligomerize,
constitute a distinctive molecular chaperone family
composed of monomers ranging in mass from 12 to
43 kDa [1–4]. The a-crystallin domain [5–7] is bor-
dered on one end by a variable N-terminal extension
involved in substrate interaction, oligomerization and
subunit dynamics [8–14], and on the other by a poorly
conserved, charged, highly flexible, C-terminal exten-
sion active in oligomer formation, promotion of solu-
bility and chaperoning [11,14–16]. Functions assigned
to N- and C-terminal extensions vary, reflecting envi-
ronmental demands on organisms in addition to the
types of molecular tasks that different sHSPs must per-
form. Generally speaking, sHSPs constitute the first
line of defense in stressed cells, binding denatured pro-
teins in a process requiring oligomer disassembly and
Keywords
molecular chaperone; p26 structure ⁄
function; small heat shock protein; stress
resistance; Artemia franciscana
Correspondence
T. H. MacRae, Department of Biology,
Dalhousie University, Halifax, N.S. B3H 4J1,
Canada

Fax: +1 902 494 3736
Tel: +1 902 494 6525
E-mail:
(Received 9 June 2005, revised 11 August
2005, accepted 16 August 2005)
doi:10.1111/j.1742-4658.2005.04920.x
The small heat shock proteins function as molecular chaperones, an activ-
ity often requiring reversible oligomerization and which protects against
irreversible protein denaturation. An abundantly produced small heat
shock protein termed p26 is thought to contribute to the remarkable stress
resistance exhibited by encysted embryos of the crustacean, Artemia francis-
cana. Three novel sequence motifs termed G, R and TS were individually
deleted from p26 by site-directed mutagenesis. G encompasses residues
G8–G29, a glycine-enriched region, and R includes residues R36–R45, an
arginine-enhanced sequence, both in the amino terminus. TS, composed of
residues T169–T186, resides in the carboxy-extension and is augmented in
threonine and serine. Deletion of R had more influence than removal of G
on p26 oligomerization and chaperoning, the latter determined by thermo-
tolerance induction in Escherichia coli, protection of insulin and citrate syn-
thase from dithiothreitol- and heat-induced aggregation, respectively, and
preservation of citrate synthase activity upon heating. Oligomerization of
the TS and R variants was similar, but the TS deletion was slightly more
effective than R as a chaperone. The extent of p26 structural perturbation
introduced by internal deletions, including modification of intrinsic fluores-
cence, 1-anilino-8-naphthalene-sulphonate binding and secondary structure,
paralleled reductions in oligomerization and chaperoning. Three-dimen-
sional modeling of p26 based on wheat Hsp16.9 crystal structure indicated
many similarities between the two proteins, including peptide loops associ-
ated with secondary structure elements. Loop 1 of p26 was deleted in the
G variant with minimal effect on oligomerization and chaperoning,

whereas loop 3, containing b-strand 6 was smaller than the corresponding
loop in Hsp16.9, which may influence p26 function.
Abbreviations
ANS, 1-anilino-8-naphthalene-sulphonate; aTc, anhydrotetracycline; CD, circular dichroism; sHSP, small heat shock protein; WT, wild type.
5230 FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS
where substrates are held in a folding-competent state
[7,10,14,17–21]. Subunit dynamics and chaperone
activity are closely related in sHSPs from yeast, plants
and bacteria, but less so in human aA-crystallin [22].
Substrate release from sHSPs and subsequent refolding
depend on ATP-requiring chaperones such as HSP70
[18,23,24]. The sHSPs confer stress tolerance on living
organisms [25], modulate apoptosis [26–28] and inter-
act with cell components such as membranes [29,30],
the cytoskeleton [31–34], and intranuclear elements
[35,36]. When perturbed by mutation or post-transla-
tional modification the sHSPs contribute to cataract
and desmin-related myopathy, among other diseases
[37–40].
The extremophile crustacean, Artemia franciscana,
populates aquatic environments of high salinity, where
they are subject to several stressors [41,42]. One adaptive
strategy exhibited by Artemia in response to its habitat
is to undertake different developmental pathways. Ovo-
viviparous development yields swimming embryos ready
to take advantage of favorable growth conditions. In
contrast, during oviparous development, embryos arrest
as gastrulae, encyst and enter diapause [42,43], a condi-
tion characterized by profound reduction in metabolic
activity and extreme stress resistance including anoxia

tolerance for several years [44–46]. Diapause-destined
embryos synthesize large amounts of a developmentally
regulated but stress-indifferent sHSP termed p26, which
peaks in encysted embryos and remains at high levels
until larvae emerge from cysts [42,47,48]. Composed of
20.8 kDa monomers, p26 forms oligomers as large as 34
subunits with a molecular mass approximating 700 kDa
[11,49]. p26 is thought to contribute to stress resistance
in encysted Artemia embryos by acting as a molecular
chaperone. In support of this proposal, the protein pro-
tects citrate synthase against heat-induced aggregation
and inactivation of its enzymatic activity and shields
insulin from dithiothreitol-induced denaturation in vitro
[11]. p26 also guards tubulin against heat-induced dena-
turation [32] and confers thermotolerance on trans-
formed bacteria [11,25]. That p26 functions in more
than one major cell compartment is indicated by reversi-
ble cytoplasmic to nuclear translocation in Artemia
embryos during development, upon exposure to stress
and by pH modulation in vitro [50–53].
As shown in this paper, the p26 a-crystallin domain
consists predominantly of b-strands arranged as a
b-sheet sandwich. The N-terminal extension is 60 resi-
dues in length and the C-terminal is 40, both with lim-
ited similarity to corresponding regions in other sHSPs
(Fig. 1). The extensions may determine distinct sHSP
properties and in this context the p26 N-terminus
possesses a novel peptide, 8-GGFGGMTDPWSDP
FGFGGFGGG-29 containing 10 glycines, as com-
pared to three or four glycines in similar locations of

other sHSPs. Additionally, six arginines occur in the
sequence 36-RPFRRRMMRR-45. The p26 C-terminal
extension encompasses 12 serine ⁄ threonine residues
in the peptide 169-TTGTTTGSTASSTPARTT-186.
These unusual regions were deleted by site-directed
mutagenesis in order to examine their contribution to
p26 structure and function and ascertain their role in
Artemia stress resistance.
Results
Mutagenesis and purification of p26 produced
in E. coli
Alignment of sHSPs from several species, a selection
of which is shown (Fig. 1), demonstrated two novel
sequence motifs in the p26 N-terminal extension and
another in the C-terminus. The deletion of these
motifs, termed G (multiple glycine), R (multiple argin-
ine), and TS (multiple threonine ⁄ serine), was confirmed
by sequencing and the modified cDNAs were cloned in
expression vectors. In addition to the p26 sequence,
each bacterial expression vector contained DNA from
the original p26-3-6-3 template clone that encoded a
short N-terminal peptide (PRAAGIRHELVLK) and
the His-tag. Bands corresponding in size to p26 were
just visible in Coomassie blue stained SDS ⁄ polyacryl-
amide gels containing protein extracts from anhydro-
tetracycline (aTc)-induced bacteria transformed with the
G and R constructs, but not the TS construct, however,
all extracts contained polypeptides that reacted with
anti-p26 antibody (Fig. 2A,B). Upon purification, single
bands of the expected size were observed in stained gels

and these polypeptides were recognized on western blots
by antibody to p26 (Fig. 2C,D).
p26 synthesis and localization in mammalian
cells
In order to examine oligomerization and cell localiza-
tion, both interesting in the context of Artemia embryo
development and sHSP function, mammalian cells were
transfected with p26 cDNA. Immunoprobing of western
blots revealed p26 in protein extracts from transiently
transfected COS-1 cells, with the yield of TS somewhat
lower than for the other variants (Fig. 3A,B). The trans-
fected cells stained strongly with anti-21 antibody
(Fig. 3C). Wild-type (WT) p26 localized exclusively to
the cytoplasm of transfected cells, whereas all modified
versions of p26 occurred in both the cytosol and nuclei.
The p26 variants G and TS were found in only some
Y. Sun and T. H MacRae Small heat shock protein sequence motifs
FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS 5231
nuclei whereas p26 lacking the R motif was in the nuclei
of all transfected cells (Fig. 3C).
Oligomerization of p26
As revealed by sucrose density gradient centrifugation,
oligomers with the largest mass and greatest number of
monomers were produced in bacteria expressing WT
p26 (Fig. 4A,B; Table 1). Oligomers formed in bacteria
with G variants of p26 were somewhat smaller than WT
p26, followed by R and TS which were very similar.
Purification of p26 from bacterial extracts had no effect
on oligomer mass. Except for WT p26, the maximum
monomer number was greater for oligomers assembled

in mammalian cells than bacteria (Fig. 4; Table 1).
Additionally, in contrast to the situation with bacteria,
the maximum monomer number for oligomers produced
by G and WT p26 in mammalian cells was the same.
Maximum monomer numbers for oligomers of R and
TS p26 produced in mammalian cells were identical and
somewhat smaller than for wild type.
Fig. 1. Multiple sequence alignment of
representative sHSPs. The amino acid
sequences of selected sHSPs were ana-
lyzed by C
LUSTAL W (1.82). Ap26, A. francis-
cana p26, AAB87967; HCRYAA, Homo
sapiens aA-crystallin, P04289; HCRYAB,
H. sapiens aB-crystallin, P02511; HHSP27,
H. sapiens Hsp27, NP_001532; MHSP25,
Mus musculus Hsp25, JN0679; DHSP26,
Drosophila melanogaster Hsp26, P02517;
CHSP16-1, Caenorhabditis elegans Hsp16–
1, P34696; YHSP26, Saccharomyces cere-
visiae Hsp26, NP_009628. sHSP domains
are indicated above the alignment and
regions corresponding to the deleted resi-
dues are boxed. Residue number is indica-
ted on the right. No residue (–), identical
residues (*), conserved substitutions (:)
and semiconserved substitutions (.) are
indicated.
A
MGRTSWTV

MG R
TS
WT
C
BD
Fig. 2. Purification of bacterially produced p26. Cell-free extracts
from transformed E. coli BL21PRO induced with aTc were electro-
phoresed in SDS polyacrylamide gels and either stained with Coo-
massie blue (A) or blotted to nitrocellulose and reacted with
antibody to p26 (B). Proteins purified by affinity chromatography
were electrophoresed in SDS polyacrylamide gels and either
stained with Coomassie blue (C), or blotted to nitrocellulose and
reacted with antibody to p26 (D). All lanes received 10 lL of sam-
ple. Lane V, vector lacking p26 cDNA; lane M, molecular mass
markers of 97, 66, 45, 31, 21 and 14 kDa; other lanes received
wild-type or modified p26 as indicated. Arrow, p26.
Small heat shock protein sequence motifs Y. Sun and T. H MacRae
5232 FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS
p26 confers thermotolerance on transformed
bacteria
E. coli expressing WT p26 were more resistant to heat
stress than bacteria expressing modified versions of the
protein, and all transformed bacteria were significantly
more thermotolerant than those containing only the
pPROTet.E233 vector which failed to survive the
60 min heat shock (Fig. 5A). Thermotolerance levels
induced by expression of G and TS were similar to
each other (P > 0.05) and significantly higher than the
thermotolerance conferred by variant R (P<0.05).
However, because the amount of TS p26 in trans-

formed bacteria was low (Fig. 2A,B), this protein is
superior to the other modified p26 versions in confer-
ring thermotolerance.
p26 exhibits chaperone activity in vitro
Purified WT p26 effectively prevented dithiothreitol-
induced denaturation of insulin (Fig. 5B). For example,
A
B
C
MGRTSWTV
Fig. 3. p26 synthesis and localization in transfected COS-1 cells.
Equal volumes of cell-free extracts were obtained from COS-1 cells
transiently transfected with the vector pcDNA ⁄ 4 ⁄ TO ⁄ myc-His.A
containing p26 cDNA inserts, electrophoresed in SDS ⁄ polyacryl-
amide gels and either stained with Coomassie blue (A) or blotted to
nitrocellulose and stained with antibody to p26 (B). Lane V, vector
lacking p26 cDNA; lane M, molecular mass markers of 97, 66, 45,
31, 21 and 14 kDa; other lanes received wild-type or modified p26
as indicated. (C) Transiently transfected COS-1 cells were incubated
with antibody to p26 followed by FITC-conjugated goat antirabbit
IgG antibody (green). Nuclei were stained with propidium iodide
(red). p26 variants are indicated in the figure. The bar represents
100 lm and all figures are the same magnification.
A
B
C
Fig. 4. p26 oligomer formation. Bacterially produced p26 either
before (A) or after (B) purification and p26 in COS-1 cell extracts
(C) were centrifuged at 200 000 g for 12 h at 4 °C in 10–50%
continuous sucrose gradients. Samples from gradient fractions

were electrophoresed in SDS ⁄ polyacrylamide gels, blotted to
nitrocellulose and reacted with antibody to p26 followed by HRP-
conjugated goat antirabbit IgG. The top of each gradient is to the
right and fractions are numbered across the top. The molecular
mass markers, a-lactalbumin, 14.2 kDa; carbonic anhydrase,
29 kDa; bovine serum albumin, 66 kDa; alcohol dehydrogenase,
150 kDa; apoferritin, 443 kDa; and thyroglobulin, 669 kDa are indi-
cated by numbered arrows.
Y. Sun and T. H MacRae Small heat shock protein sequence motifs
FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS 5233
WT p26 inhibited insulin aggregation by 39% after
30 min at 0.1 lm, and almost completely at 1.6 lm,a
0.4 : 1 monomer to monomer molar ratio of chaperone
to substrate. Mutant R was the least effective, whereas
G and TS provided an intermediate level of protection,
with G moderately more effective at higher concentra-
tions. Bovine serum albumin (BSA) and IgG at 1.6 lm
failed to inhibit insulin aggregation (not shown). Puri-
fied, bacterially produced WT p26 also exhibited the
greatest ability to shield citrate synthase from heat-
induced denaturation while mutant R had the least,
although all mutants provided protection (Fig. 5C). At
600 nm WT p26, representing a chaperone to target
molar ratio of 4 : 1 (p26 monomer to citrate synthase
dimer), citrate synthase aggregation was inhibited
almost completely for 1 h at 43 °C (Fig. 5C), a result
similar to that obtained with p26 purified from
Artemia (not shown). At 37.5 nm, where the molar
ratio of WT p26 to citrate synthase was 1 : 4, heat-
induced turbidity was reduced by 46% after 1 h at

Table 1. Oligomerization of p26. The molecular mass of p26 oligo-
mers was determined by sucrose density gradient centrifugation.
Monomer mass refers to the molecular mass of p26 polypeptides.
Oligomer mass range represents the smallest to largest oligomers
observed while oligomer mass maximum refers to the mass of the
largest oligomer. Maximum monomer number refers to monomer
number in the largest oligomer.
p26 mutant
Monomer
mass (kDa)
Oligomer mass
Maximum
monomer
number
Range
(kDa)
Maximum
(kDa)
E. coli
G 23.7 14.2–443 443 19
R 24.1 14.2–300 300 12
TS 23.8 14.2–300 300 13
WT 25.5 29.0–669 669 26
COS-1 cells
G 18.7 14.2–443 443 24
R 19.4 14.2–300 300 16
TS 19.1 14.2–300 300 16
WT 20.8 14.2–500 500 24
015
8

0
0.01
0.02
0.03
0.04
0.05
0.06
AB
CD
7
6
5
4
3
2
1
0
70
60
50
40
30
20
10
0
30 45
Time (min)
E. coli thermotolerance
Insulin aggregation
Citrate synthase inactivation

CS activity (umole citrate/mg protein/min
Citrate synthase aggregation
Log
10
of CFU/ml
A
400

A
360

Time (min)
p26 (nm)
p26 (µM)
60
No p26
No p26
WT
G
TS
R
WT
G
TS
R
G
TS
WT
No p26
R

G
TS
WT
No p26
R
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60
1200
0.1
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
600 300 150 75 37.5
1.6 0.8 0.4 0.2 0.1
Fig. 5. Chaperone activity of p26. (A) Transformed E. coli was incubated at 54 °C, diluted, plated in duplicate on LB agar and colonies were
counted after incubation overnight at 37 °C. (B) Purified, bacterially produced p26 was incubated with insulin for 30 min in the presence of
dithiothreitol and solution turbidity was measured at 400 nm. The p26 variants are in the same order in each histogram group. (C) Purified,
bacterially produced p26 at 600 n
M was heated at 43 °C for 1 h with 150 nM citrate synthase, and solution turbidity was measured at
360 nm. The A
360
values were multiplied by 1000. (D) Citrate synthase at 150 nM was heated at 43 °C for 1 h in either the absence or the
presence of p26 and enzyme activity was determined. The p26 variants are in the same order in each histogram group. Results in all experi-
ments are averaged from three independent experiments.

Small heat shock protein sequence motifs Y. Sun and T. H MacRae
5234 FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS
43 °C (Fig. 1, supplemental data). Mutants G, TS and
R followed in decreasing order of activity. Although
none of the mutants with internal deletions completely
inhibited the aggregation of citrate synthase at 600 nm,
even the least effective chaperone offered significant
protection (Fig. 5C). BSA and IgG at 600 nm provided
almost no protection when heated with citrate synthase
(not shown). WT p26 was the most effective in guard-
ing citrate synthase against heat induced inactivation,
followed by G and TS which were very similar. p26 R
exhibited the least protection at each concentration
tested (Fig. 5D). Even when mutated, p26 provided a
significant level of protection to citrate synthase at
600 nm, ranging from 34% for R to 52% for G and
TS, as compared to 80% for WT. The activity of cit-
rate synthase in the absence of heating was 0.09 lmol
citrateÆmg protein min
)1
.
p26 intrinsic fluorescence and surface
hydrophobicity
The maximum emission peak of bacterially produced
WT p26 was 344 nm, shifted when compared to the
value of 352 nm for p26 from encysted Artemia
embryos (Fig. 6A). In comparison, all mutants exhi-
bited reduced emission intensities, with the peak of G
and TS at 348 nm and R at 360 nm. The fluorescence
intensity of R was the lowest and it was red-shifted in

comparison to other samples. The extent of tertiary
structure modification as indicated by intrinsic fluores-
cence paralleled reductions in chaperone activity for
each p26 variant. WT p26 from bacteria and Artemia
had similar 1-anilino-8-naphthalene-sulphonate (ANS)
binding capacities at 25 °C and 43 °C and these were
greater than for any modified p26 variant (Fig. 6B). R
exhibited the lowest ANS binding, with fluorescence
intensity 39% of bacterial WT p26 at 25 °C and 29%
at 43 °C. The fluorescence of each sample increased
when the temperature was elevated, with emission
from Artemia p26 at 43 °C 74% higher than at 25 °C.
However, temperature-dependent increases in fluores-
cence intensity of modified p26 variants were less than
for WT, and as an example, the increase for R was
only 28%. Decreases in ANS binding paralleled the
loss of chaperone activity.
p26 secondary structure
Far-UV circular dichroism (CD) spectra of WT p26
purified from transformed bacteria and Artemia cysts
had peak positive and negative values at 194 nm and
214 nm, respectively, these characteristic of b-sheets
(Fig. 7). The p26 mutants each exhibited a positive
peak early in the CD scan which was not shown by
WT p26 and a second peak shared with WT p26 at
194 nm. The mutated versions of p26 all possessed a
wide maximal negative reading, encompassing approxi-
mately 208 nm to 220 nm. In comparison to WT p26,
R exhibited the greatest change in CD spectra (Fig. 7;
Table 2). The calculated secondary structure elements

show decreased b-structure and increased a-helical
constituents for the variants, with these most pro-
nounced for R.
Modeling of p26 structure
Sequence identity between a-crystallin domains
allowed generation of a p26 model based on wheat
G
TS
p26 Art
p26 Bac
R
GTS
p26 Art
p26
25°C
43°C
p26 Bac
R
310
1600
1400
1200
1000
800
600
400
200
0
25
A

B
20
15
10
5
0
316 322 328 334 340
Emission wavelength (nm)
Relative fluorescence
Relative fluorescence
346 352 358 364 370 376 382 388 394 400
Fig. 6. p26 tertiary structure. (A) Purified p26 was diluted in 10 mM
NaH
2
PO
4
, pH 7.1–0.06 mgÆmL
)1
and intrinsic fluorescence was
measured at an excitation wavelength of 280 nm with a 2 nm band
pass and fluorescence emission was detected over 310–400 nm.
(B) Purified p26 at 0.06 mgÆmL
)1
in 10 mM NaH
2
PO
4
, pH 7.1 was
oversaturated with ANS and fluorescence was measured with an
excitation wavelength of 388 nm and a band pass of 8 nm, and an

emission wavelength of 473 nm with a band pass of 8 nm. Meas-
urements were carried out at either 25 °C (grey) or 43 °C (black).
Fluorescence generated by buffer containing ANS but no p26 was
subtracted from each sample. p26 Bac and p26 Art were WT sam-
ples obtained from transformed E. coli and Artemia, respectively.
Each spectrum was recorded in duplicate using two independent
sample preparations.
Y. Sun and T. H MacRae Small heat shock protein sequence motifs
FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS 5235
Hsp16.9 crystal structure (Fig. 8), although the lack of
correspondence between C-terminal extensions preclu-
ded inclusion of p26 residues A159 to A192. Extrapo-
lation from the structural characteristics of Hsp16.9
suggests each p26 monomer contains an N-terminal
region potentially buried within oligomers, an a-crys-
tallin domain and a solvent-accessible C-terminal
extension [6]. Eight b-strands of p26 are organized into
two antiparallel b-sheets and four loop regions were
observed in the protein. Loop 1 contains residues
22-GFGGFGGGMDL-32 and is located towards the
center of the amino-terminal region, whereas loop 2,
encompassing residues 58-TPGLSR-63 is adjacent
to the a-crystallin domain. Loop 3 (102-SDEYGH-
VQRE-111) protrudes from the a-crystallin domain
and corresponds to the larger Hsp16.9 loop containing
b-strand 6. Loop 4 (132-SSDGV-136) equates to the
sequence connecting b8 and b9 of Hsp16.9. Residues
153-IVPITP-158 of the p26 C-terminal extension
are included in the model and within this sequence
residues V154 and I156 correspond to I147 and I149

of Hsp16.9.
Discussion
p26 contains two novel N-terminal sequences, one
enriched in glycine and the other in arginine. Addition-
ally, the C-terminus contains an unusual stretch of
residues endowed with threonine and serine. These
regions are interesting because the characteristics of
different sHSPs depend on the properties of their vari-
able N- and C-terminal extensions. For example, the
unusual sequence motifs may either enhance p26 oligo-
merization, possibly through stabilization of dimer for-
mation, or promote chaperoning, thus boosting stress
resistance in encysted embryos. To investigate these
and related questions, the three sequences were deleted
from p26 by site-directed mutagenesis and the resulting
proteins characterized.
p26 forms poly-disperse oligomers with a maximum
mass of 669 kDa and composed of 32 subunits [49],
properties common to many other sHSPs [54,55], but
contrasting mono-disperse sHSPs from wheat [6] and
the hyperthermophilic Archaea, Methanococcus janna-
schii [5]. The maximum monomer number for WT p26
oligomers synthesized in E. coli and COS-1 cells was
slightly less than for p26 from Artemia, but more than
for any mutated p26. Of the modified p26 versions, G
formed oligomers with the greatest number of mono-
mers and in transfected COS-1 cells monomer number
was the same as for WT p26, showing this internal
deletion had no effect on oligomer mass. A portion of
the p26 G peptide shares sequence similarity with an

N-terminal region of wheat Hsp16.9 (Fig. 8A), and
residues 7–13 of the Hsp16.9 sequence, containing the
WD ⁄ EPF motif as 10-FDPF-13, replace the dimer-
stabilizing b -strand 1 of M. jannaschii Hsp16.5 [5,6].
The WD ⁄ EPF motif also occurs in Chinese hamster
Hsp27 and when deleted, chaperone activity and oligo-
merization decline significantly [13]. In p26, D ⁄ EP is
replaced by GG, potentially reducing the importance
of the motif in dimer formation and explaining why
the G deletion has little effect on oligomerization and
chaperoning. The sequences, 20-SRLFDQFFG-28 and
21-SRLFDQFFG-29, found, respectively, in human
aA- and aB-crystallin, and possibly analogous to
residues 7-SNVFD-11 in wheat Hsp16.9, are important
in oligomer dynamics, assembly and stability [19], but
the peptide is replaced in p26 by 20-FGFGGFGGG-
28 (Fig. 1). The advantages of replacing functionally
important motifs in the a-crystallins with a glycine-
enriched sequence in p26, lacking in apparent struc-
tural and functional attributes, is unknown. Deleting
Table 2. Secondary structure elements of p26. Secondary structure
elements of p26 were calculated with the CDNN v2.1 deconvolu-
tion program. p26 Bac and p26 Art refer to WT p26 purified from
transformed E. coli and Artemia, respectively.
Structural
element
G
(%)
R
(%)

TS
(%)
p26 Bac
(%)
p26 Art
(%)
a-Helix 21.8 24.9 22.2 17.7 17.7
b-Antiparallel 17.8 15.6 16.4 21.6 23.0
b-Parallel 9.8 9.4 9.5 10.3 10.0
b-Turn 17.1 17.4 17.5 16.5 16.8
Random coil 33.5 32.7 34.4 34.0 32.5
–20
Wavelength (nm)
1
2
3
4
5
CD [m deg]
–15
–10
–5
180 184 188 192 196 200 204 208 216212 220 224 228 232 236 240 244 248 252 256 260
0
5
10
15
20
25
Fig. 7. Far-UV circular dichroism. Each spectrum represents the

average of three scans obtained with purified bacterially produced
p26 dissolved in 10 m
M NaH
2
PO
4
, pH 7.1, at 0.2 mgÆmL
)1
.The
absorption data were expressed as molar ellipticity in degrees
cm
2
Ædmol (m deg). 1, WTp26 from bacteria; 2, WTp26 from Arte-
mia;3,G;4,R;5,TS.
Small heat shock protein sequence motifs Y. Sun and T. H MacRae
5236 FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS
the positively charged, arginine-enriched sequence
reduced maximum monomer number approximately
33% in transfected COS-1 cells and more than 50% in
transformed bacteria, suggesting a role for the motif in
oligomer assembly, although nonspecific disruption of
p26 structure through removal of several negatively
charged residues cannot be discounted as the cause
of oligomer destabilization. Truncation experiments
revealed previously that the N-terminal extension
modulates p26 oligomer assembly [11], and the current
work shows that within this region the arginine-
enriched motif is more important than the glycine-
containing sequence.
Characterization of the TS mutant revealed a role

for the C-terminal threonine ⁄ serine enriched stretch
in oligomerization. Previously, removing 10 C-terminal
residues, A183-A192, which includes T185 and T186 of
the TS sequence, had little effect on p26 oligomeriza-
tion, but deleting the entire C-terminal extension,
including the TS sequence and the conserved I ⁄ V-X-
I ⁄ V motif (154-VPI-156 in p26) had more impact [11].
Eliminating the I ⁄ V-X-I ⁄ V motif from representative
members of the B. japonicum sHSP family, or substitu-
ting alanine for either or both isoleucines, reduces oligo-
mer size and chaperone activity [56], as occurs upon
mutating Val143 of Synechocystis Hsp16.6 [57].
A
B
C
D
Fig. 8. Comparative modeling of p26. (A) Amino acid sequences of p26 and Hsp16.9 from wheat were aligned by CLUSTAL W (1.82). Ap26,
p26, AAB87967; WHsp16.9, wheat Hsp16.9, 1GME_A. The secondary structure elements predicted with the PHD_ s program are depicted
above the wheat Hsp16.9 sequence and below the p26 sequence. Amino acid residues in red, small and hydrophobic; blue, acidic; magenta,
basic; green, contain a hydroxyl or amine group. –, no amino acid residue; *, identical residues; :, conserved substitution; ., semiconserved
substitution. Residue number is indicated on the right. (B) Wheat Hsp16.9 was used as template for sequence alignment with the program
of Swiss-Model. The p26 residues A2-P158, corresponding to S2-G151 of Hsp16.9, were used for modeling and the result is shown as a
monomer, with C-terminal residues A159-A192 deleted for alignment optimization. The returned three-dimensional model was enhanced
with the ‘Improve Fit’ function of Swiss-PdbViewer and sent for second round modeling. Internal deletions G and R are indicated by labeled
arrows. The arrows labeled A2 and P158 position the first and last amino acid residues, respectively. The N-terminal region, a-crystallin
domain and C-terminal extension are indicated by labeled arrows. Loops within the p26 monomer are boxed in A and indicated with arrows
in C, as is the large loop of the wheat Hsp16.9 monomer corresponding to p26 loop 3 (D).
Y. Sun and T. H MacRae Small heat shock protein sequence motifs
FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS 5237
However, substituting glycine for the isoleucines and

valines of the I ⁄ V-X-I ⁄ V motifs in human a-A and
a-B crystallins has no effect, although fluorescence
resonance energy transfer (FRET) indicated a role in
inter-subunit interactions [58]. The long p26 C-terminal
extensions may strap neighboring monomers together
and reducing the length of this region by 18 residues,
as occurs in the TS mutation, would weaken oligomer
structure, leading to reduced mass. Moreover, by pro-
moting oligomerization the extended p26 C-terminal
sequence, including TS, has the potential to enhance
Artemia embryo survival upon exposure to stress,
wherein structural stability of molecular chaperones
could be an asset.
Bacterially produced WT p26 prevented heat
induced citrate synthase aggregation and chemically
induced insulin denaturation at molar ratios of p26 to
substrate similar to those of other sHSPs [59–63]. The
p26 variants with internal deletions exhibited reduced
chaperoning, with R the least capable, but each
mutant retained considerable activity, suggesting chap-
eroning is relatively insensitive to structural change.
The fluorescence intensity of all p26 variants was
reduced in comparison to WT, implying modified ter-
tiary structure. The G deletion removed one of two
p26 tryptophans, accounting for some of the change
with this mutation. Surface hydrophobicity affects
chaperone–substrate interaction [64], and R exhibited
the lowest ANS fluorescence, followed by TS and then
G. The changes are consistent with the intrinsic fluor-
escence spectra and demonstrate that R, the poorest

chaperone, has the least hydrophobicity available for
substrate interaction. As determined by far-UV CD, R
underwent the greatest secondary structure changes,
although all variants were similar. If the N-terminal
extension is at least partially buried in oligomers,
removal of several arginines may leave unpaired negat-
ively charged residues that perturb packing and disrupt
b-strands within the nearby a-crystallin domain. The
similarities in intrinsic fluorescence, surface hydro-
phobicity and CD measurements for WT p26 from
bacteria and Artemia cysts indicate results obtained
with one are representative of the other.
Selected sHSPs enter nuclei, although their mission
remains elusive. Hsp20 migrates into the nuclei of cul-
tured rat neonatal cardiac myocytes during heat stress
[65]. aB-Crystallin and Hsp27 are nuclear speckle
components in unstressed, transcriptionally active cells,
and Hsp27 is also found in the nucleolus, implying
similar but not completely overlapping roles for these
proteins [66]. p26 occurs in Artemia nuclei during dia-
pause and stress and may stabilize nuclear matrix
proteins [52,53], but how p26 moves into nuclei is
unknown. The arginine-enriched region appears not to
function as a nuclear localization signal because all
COS-1 cells transfected with the R variant have intra-
nuclear p26, this contrasting WT p26 and the remain-
ing modified versions of the protein, one of which
experienced the same oligomer mass reduction as R. In
previous work, p26 reduced in oligomer size due to
C-terminal truncation remained in the cytoplasm [11],

whereas p26 mutant R114A, existing as oligomers sim-
ilar in size to those produced by WT p26, readily
entered nuclei (unpublished data). Remembering that
translocation may occur differently in transfected
mammalian cells vs. Artemia embryos p26 nuclear
migration apparently depends on a mechanism other
than oligomer mass reduction, although transient dis-
sociation into small oligomers as a prerequisite for
translocation is possible.
Sufficient sequence similarity existed to model p26
residues A2-P158 on the crystal structure of wheat
Hsp16.9 [6], permitting protein comparison and reveal-
ing if G and R reside in regions possessing structural
and functional characteristics defined by crystallization
studies. The TS deletion fell outside the compared
sequences and was not modeled, however, the region
may contribute to stability by increasing intersubunit
contacts in oligomers. p26 possesses four short loops
and of these loop 3 containing b-strand 6 is smaller
than the equivalent loop in Hsp16.9. b-strand 6 in loop
3 of Hsp16.9 stabilizes monomer–monomer interaction
at the dimer interface [6], but the p26 loop may be too
short to accomplish this, a result shown for a-crystal-
lins and other sHSPs from animals, as opposed to
plant and bacterial sHSPs [6,8,67]. Additionally,
removal of p26 loop 1 (residues 22-GFGGFGGG-
MDL-32), as occurred in G, had little effect on olig-
omerization and chaperone activity, suggesting limited
involvement of loop 1 in protein stability and function.
Loops 2 and 4 were not affected by internal deletions,

nor were equivalent loops apparent in Hsp16.9 for
comparison, leaving their roles undetermined.
Experimental procedures
Site-directed mutagenesis of p26 cDNA
Internal deletions of p26 were generated by site-directed
mutagenesis with the QuikChange
TM
Site-directed Muta-
genesis kit (Stratagene, La Jolla, CA, USA), using
pRSET.C-p26–3-6-3 as template [68], and designated prim-
ers (Table 3). PCR mixtures were incubated 30 s at 95 °C
prior to 12 cycles of 30 s at 95 °C, 1 min at 55 °C and
8 min at 68 °C. Resulting DNAs were digested for 1 h at
37 °C with DpnI and used to transform Escherichia coli
Small heat shock protein sequence motifs Y. Sun and T. H MacRae
5238 FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS
XL1-blue supercompetent cells (Stratagene). p26 cDNA
inserts were recovered by digestion with BamHI and XhoI,
electrophoresis in agarose and purification with the GFX
TM
PCR DNA and Gel Band purification kit (Amersham
Biosciences, Piscataway, NJ, USA) before cloning in the
eukaryotic expression vector pcDNA4 ⁄ TO ⁄ myc-His.A
(Invitrogen, San Diego, CA, USA) and transformation of
E. coli DH5a (Invitrogen, Carlsbad, CA, USA). p26
cDNAs were also cloned into pPROTet.E233 (Clontech
Laboratories, Inc, Palo Alto, CA, USA), a prokaryotic
expression vector containing a His-tag using the procedure
just described. Cloned inserts were sized by restriction
digestion followed by electrophoresis in 1% agarose and

sequenced (DNA Sequencing Facility, Center for Applied
Genomics, Hospital for Sick Children, Toronto, Ontario,
Canada).
Purification of bacterially produced p26
Recombinant pPROTet.E233 plasmids were transformed
into E. coli BL21PRO (Clontech Laboratories, Inc.,
Mississauga, ON, Canada) which were induced with aTc
(Clontech Laboratories) at 100 ngÆmL
)1
. Bacterial cell-free
extracts were prepared and p26 was purified with BD
TALON resin (BD Biosciences Clontech) following manu-
facturer’s instructions. Purified p26 was dialyzed 4 h at
room temperature against 10 mm NaH
2
PO
4
, pH 7.1 with
one change of buffer, and then overnight at 4 °C before
concentration in CentriprepYM-10 centrifugal filter devices
(Amicon Bioseparations, Billerica, MA, USA).
SDS/polyacrylamide gel electrophoresis and
protein immunodetection
Protein samples electrophoresed in 12.5% SDS ⁄ polyacryla-
mide gels were either stained with Coomassie Brilliant Blue
R-250 (Sigma, St Louis, MO, USA) or blotted to nitrocel-
lulose (Bio-Rad, Hercules, CA, USA) and stained with 2%
Ponceau-S (Sigma) in 3% (v ⁄ v) trichloroacetic acid to
assess transfer. Blots were incubated 45 min in 5% low fat
milk powder in TBS ⁄ Tween [10 mm Tris, pH 7.4, 0.14 m

NaCl, 0.1% (v ⁄ v) Tween 20], followed by 30 min at room
temperature with either anti-p26 Ig [68] in high salt Tween
buffer (HST) (10 mm Tris, pH 7.4, 1 m NaCl, 0.5% (v ⁄ v)
Tween (20) or Omni-probe (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA, USA), a monoclonal antibody recog-
nizing the His
6
tag. Blots were washed then incubated for
30 min with either horseradish peroxidase (HRP)-conju-
gated goat anti-(rabbit IgG) Ig or HRP-conjugated goat
anti-(mouse IgG) Ig (Jackson ImmunoResearch, West
Grove, PA, USA) in HST. Antibody-reactive proteins were
detected with Western Lightning Enhanced Chemilumines-
cence Reagent Plus (PerkinElmer Life Sciences, Boston,
MA, USA).
p26 synthesis in mammalian cells
COS-1 cells were transiently transfected with p26-contain-
ing plasmids in SuperFect
TM
(Qiagen, Mississauga, ON,
Canada) and cell-free extracts were prepared (11). Equal
volumes of protein extracts from cells transfected with
the different cDNA constructs were electrophoresed in
SDS ⁄ PAGE and either stained with Coomassie Brilliant
Blue or blotted to nitrocellulose for p26 immunodetection.
p26 was localized in transfected COS-1 cells with anti-p26
antibody [68] followed by incubation with fluorescein iso-
thiocyanate-conjugated goat anti-(rabbit IgG) Ig (Jackson
ImmunoResearch) and nuclei were stained with propidium
iodide [11]. Rinsed cover-slips were inverted on Vecta-

shield
TM
mounting medium (Vector Laboratories, Burlin-
game, CA, USA), and examined with a Zeiss 410 inverted
confocal laser scanning microscope.
p26 oligomerization
Samples containing p26 were centrifuged at 200 000 g for
12 h at 4 °C in 10 mL continuous 10–50% (w ⁄ v) sucrose
gradients in 0.l m Tris ⁄ glycine buffer, pH 7.4. Gradients
were fractionated and 15 lL from each fraction was elec-
trophoresed in 12.5% SDS polyacrylamide gels before blot-
ting to nitrocellulose for p26 immunodetection. A p26
molecular mass of 20.8 kDa, determined by generunner
(version 3.05, Hastings Software, Inc.), was used to calcu-
late the number of monomers in oligomers, with correc-
Table 3. Primers for site-directed mutagenesis of p26. Internal deletions of p26 cDNA were generated by site-directed mutagenesis using
the listed primers. The p26 regions which encompass mutations are indicated in the left column. G, amino acid residues G8–G29 were dele-
ted; R, residues R36–R45 are lost; TS, residues T169–T186 are missing.
p26 region Mutation Primer sequence
N-terminal extension G 5¢-GGCACTTAACCCATGGTACATGGACCTTGATATTGAC-3¢
5¢-GTCAATATCAAGGTCCATGTACCATGGGTTAAGTGCC-3¢
R5¢-GGACCTTGATATTGACGGTCCAGATACC-3¢
5¢-GGTATCTGGACCGTCAATATCAAGGTCC-3¢
C-terminal extension TS 5¢-GGATTGAAGGGGGAAGATCAGGAGGTGC-3¢
5¢-GCACCTCCTGATCTTCCCCCTTCAATCC-3¢
Y. Sun and T. H MacRae Small heat shock protein sequence motifs
FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS 5239
tions for amino acid deletions. a-Lactalbumin (14.2 kDa),
carbonic anhydrase (29 kDa), BSA (66 kDa), alcohol dehy-
drogenase (150 kDa), apoferritin (443 kDa), and thyroglo-

bulin (669 kDa) (Sigma) were centrifuged separately and
their locations in sucrose gradients determined by measur-
ing the absorbance of fractions at 280 nm.
p26-induced thermotolerance
Transformed E. coli were incubated overnight with shaking
at 37 °C in 2 mL of Luria–Bertani medium containing
spectinomycin, chloramphenicol and aTc (50, 34 and
100 ng.ml
)1
respectively). The cultures, diluted 1 : 10 in
fresh LB, were then incubated at 54 °C with 100 lL sam-
ples removed periodically, plated in duplicate on LB agar
and incubated at 37 °C. Colonies were counted after 24 h
and experiments were done in triplicate. Between data
groups, two-sample t-tests were performed at a confidence
level of 95% with the statistical software MINITAB
14.12.0 (Minitab Inc., State College, PA, USA) to evaluate
the significance of difference which was accepted at
P < 0.05 level. To ensure heat shocked bacteria contained
p26, aTc-induced cells were homogenized before heating
and electrophoresed in SDS ⁄ polyacrylamide gels, followed
by p26 immunodetection on nitrocellulose membranes.
Chaperone activity of p26 in vitro
Citrate synthase (Sigma) at 150 nm in 40 mm Hepes ⁄ KOH
buffer, pH 7.5 was heated at 43 °C with purified, bacterially
produced p26. The molarities of citrate synthase and p26
were based on dimer and monomer molecular masses,
respectively, and solution turbidity was monitored at
360 nm with a SPECTRAmax PLUS spectrophotometer
(Molecular Devices). Citrate synthase enzyme activity was

measured in reaction mixtures containing 940 lLofTE
(50 mm Tris ⁄ HCl, pH 7.5, 2 mm EDTA), 10 lLof10mm
oxaloacetic acid (Sigma), 10 lLof10mm 5,5¢-dithiobis(2-
nitrobenzoic acid) (Sigma) and 30 lLof5mm acetyl-CoA
(Sigma). The reaction was initiated at 25 °C by adding
10 lL of 150 nm citrate synthase and monitored at 412 nm.
Insulin (Sigma) at 4.0 lm in 10 mm phosphate buffer,
100 mm NaCl, pH 7.4 was mixed with p26, dithiothreitol
(Sigma) was added to 20 mm and solution turbidity was
measured at 400 nm for 30 min at 25 °C.
p26 intrinsic fluorescence, ANS-binding capacity
and secondary structure
Purified p26 was diluted to 0.06 mgÆmL
)1
in 10 mm
NaH
2
PO
4
, pH 7.1 and fluorescence spectra were measured
at 25 °C with a SPECTRAmax GEMINIXS fluorescence
spectrophotometer (Molecular Devices). The emission
wavelength was initially 340 nm with a 2 nm band pass and
fluorescence excitation was detected from 250 to 310 nm.
Subsequently, excitation wavelength was 280 nm and fluor-
escence emission was detected from 310 to 400 nm. Spectra
were recorded in duplicate using independently prepared
samples.
To examine surface hydrophobicity 2 lL of ANS
(Molecular Probes, Eugene, OR, USA) at 8.0 mm in 10 mm

NaH
2
PO
4
, pH 7.1 was added to 198 lL of purified, bacteri-
ally produced p26 at 0.06 mgÆmL
)1
and mixtures were incu-
bated for 5 min at either 25 °Cor43°C. The excitation
wavelength was 388 nm with band pass of 8 nm and the
emission wavelength was 473 nm with band pass of 8 nm.
An AMINCO Bowman series z luminescence spectrometer
(AMINCO, Rochester, NY, USA) equipped with a thermo-
stated circular water-bath was employed, and conditions
were chosen to minimize inner filter effects.
Far-UV CD spectra were recorded at 25 °C for purified
p26 at 0.2 mgÆmL
)1
in 10 mm NaH
2
PO
4
, pH 7.1, in a
JASCO J-810 spectropolarimeter (Japan Spectroscopic,
Tokyo, Japan). A 0.1-cm path length quartz cuvette was
used and three scans over 180–260 nm were averaged per
spectrum. Bandwidth was 2 nm and all scans were correc-
ted for buffer and smoothed to eliminate background noise.
Secondary structure parameters were calculated with the
cdnn v2.1 deconvolution program (Martin-Luther-Univer-

sita
¨
t Halle-Wittenberg, Germany).
Modeling of p26 three-dimensional structure
The Swiss-Model Protein Modeling Server (version 36.0003,
Biozentrum University Basel, Basel; Swiss Institute of Gen-
eva, Switzerland; R & D S.A., Raleigh, NC, USA) [69–71]
was employed to model p26 three-dimensional structure
using wheat Hsp16.9 (ExPDB entry code: 1GME) as tem-
plate with the function of Swiss-Model. The returned three-
dimensional model was improved with the Improve Fit func-
tion of Swiss-PdbViewer (version 3.7, GlaxoSmithKline R &
D, Geneva, Switzerland) and submitted for a second round
of modeling. The validity of the model was confirmed by
application of Verify3D [72]. p26 three-dimensional struc-
ture, shown as a monomer, encompasses residues A2-P158,
corresponding to residues S2-G151 of Hsp16.9.
Acknowledgements
The assistance of Dr Neil Ross and Dr Steve Bearne
with biophysical measurements is gratefully acknow-
ledged. The research was funded by a Natural Sciences
and Engineering Research Council of Canada Discovery
Grant, a Nova Scotia Health Research Foundation ⁄
Canadian Institutes of Health Regional Partnership
Plan Grant, and a Heart and Stroke Foundation of
Nova Scotia Grant to THM. YS was the recipient of a
NSHRF Student Fellowship.
Small heat shock protein sequence motifs Y. Sun and T. H MacRae
5240 FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS
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Supplementary material
The following Supplementary material is available for
this article online:
Supplemental Fig. 1. p26 prevents heat-induced citrate
synthase aggregation. Purified, bacterially produced
p26 was heated at 43 °C for 1 h with 150 nm citrate
synthase, and solution turbidity was measured at 360
nm p26 concentrations were (A) 1200 nm; (B) 600 nm;
(C) 300 nm; (D) 150 nm; (E) 75 nm; (F) 37.5 nm. The
curves are labeled in (A) and they occupy the same
relative position in all graphs.
Y. Sun and T. H MacRae Small heat shock protein sequence motifs
FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS 5243