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

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

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

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

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

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

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

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

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

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

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
(%)

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

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

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,

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

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-

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

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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