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 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: tmacrae@dal.ca (Received 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 activity 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 franciscana 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 thermotolerance induction in Escherichia coli, protection of insulin and citrate synthase 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 fluorescence, 1-anilino-8-naphthalene-sulphonate binding and secondary structure, paralleled reductions in oligomerization and chaperoning Three-dimensional modeling of p26 based on wheat Hsp16.9 crystal structure indicated many similarities between the two proteins, including peptide loops associated with secondary structure elements Loop of p26 was deleted in the G variant with minimal effect on oligomerization and chaperoning, whereas loop 3, containing b-strand was smaller than the corresponding loop in Hsp16.9, which may influence p26 function 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 bordered 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 extension active in oligomer formation, promotion of solubility and chaperoning [11,14–16] Functions assigned to N- and C-terminal extensions vary, reflecting environmental demands on organisms in addition to the types of molecular tasks that different sHSPs must perform Generally speaking, sHSPs constitute the first line of defense in stressed cells, binding denatured proteins in a process requiring oligomer disassembly and 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 Y Sun and T H MacRae 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 interact with cell components such as membranes [29,30], the cytoskeleton [31–34], and intranuclear elements [35,36] When perturbed by mutation or post-translational 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 Ovoviviparous 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 condition 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 protects 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 denaturation [32] and confers thermotolerance on transformed bacteria [11,25] That p26 functions in more than one major cell compartment is indicated by reversible 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 residues in length and the C-terminal is 40, both with limited 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 FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS Small heat shock protein sequence motifs FGFGGFGGG-29 containing 10 glycines, as compared 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 arginine), 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 ⁄ polyacrylamide gels containing protein extracts from anhydrotetracycline (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 localization, 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 transfected 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 5231 Small heat shock protein sequence motifs Y Sun and T H MacRae Fig Multiple sequence alignment of representative sHSPs The amino acid sequences of selected sHSPs were analyzed by CLUSTAL W (1.82) Ap26, A franciscana 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 cerevisiae Hsp26, NP_009628 sHSP domains are indicated above the alignment and regions corresponding to the deleted residues are boxed Residue number is indicated on the right No residue (–), identical residues (*), conserved substitutions (:) and semiconserved substitutions (.) are indicated A M G R TS WT V C M G R TS WT nuclei whereas p26 lacking the R motif was in the nuclei of all transfected cells (Fig 3C) Oligomerization of p26 B D Fig Purification of bacterially produced p26 Cell-free extracts from transformed E coli BL21PRO induced with aTc were electrophoresed in SDS polyacrylamide gels and either stained with Coomassie 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 sample 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 5232 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 FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS Y Sun and T H MacRae A M G Small heat shock protein sequence motifs R TS WT V A B B C C Fig 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 ⁄ ⁄ TO ⁄ myc-His.A containing p26 cDNA inserts, electrophoresed in SDS ⁄ polyacrylamide 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 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 heat shock (Fig 5A) Thermotolerance levels FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS Fig 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 °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 HRPconjugated 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 indicated by numbered arrows 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 transformed bacteria was low (Fig 2A,B), this protein is superior to the other modified p26 versions in conferring thermotolerance p26 exhibits chaperone activity in vitro Purified WT p26 effectively prevented dithiothreitolinduced denaturation of insulin (Fig 5B) For example, 5233 Small heat shock protein sequence motifs Y Sun and T H MacRae Table Oligomerization of p26 The molecular mass of p26 oligomers 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 Oligomer mass Monomer mass (kDa) Maximum monomer number 14.2–443 14.2–300 14.2–300 29.0–669 443 300 300 669 19 12 13 26 18.7 19.4 19.1 20.8 E coli G R TS WT COS-1 cells G R TS WT Maximum (kDa) 23.7 24.1 23.8 25.5 p26 mutant Range (kDa) 14.2–443 14.2–300 14.2–300 14.2–500 443 300 300 500 24 16 16 24 E coli thermotolerance A WT p26 inhibited insulin aggregation by 39% after 30 at 0.1 lm, and almost completely at 1.6 lm, a 0.4 : 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 concentrations Bovine serum albumin (BSA) and IgG at 1.6 lm failed to inhibit insulin aggregation (not shown) Purified, bacterially produced WT p26 also exhibited the greatest ability to shield citrate synthase from heatinduced 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 : (p26 monomer to citrate synthase dimer), citrate synthase aggregation was inhibited almost completely for 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 : 4, heatinduced turbidity was reduced by 46% after h at B Insulin aggregation 0.05 G R TS WT No p26 0.06 0.04 WT A400 Log10 of CFU/ml G TS R No p26 0.03 0.02 0.01 0 15 30 45 60 1.6 0.8 Time (min) No p26 60 A360 50 40 30 R 20 TS 10 G WT 0 12 16 20 24 28 32 36 40 44 48 52 56 60 Time (min) 0.2 0.1 Citrate synthase inactivation 0.1 0.09 0.08 0.07 0.06 0.05 G R TS WT No p26 D 70 CS activity (umole citrate/mg protein/min Citrate synthase aggregation C 0.4 p26 (µM) 0.04 0.03 0.02 0.01 1200 600 300 150 75 37.5 p26 (nm) Fig 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 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 nM was heated at 43 °C for h with 150 nM citrate synthase, and solution turbidity was measured at 360 nm The A360 values were multiplied by 1000 (D) Citrate synthase at 150 nM was heated at 43 °C for 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 experiments are averaged from three independent experiments 5234 FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS Y Sun and T H MacRae 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 exhibited 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 fluorescence 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 fluorescence 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 FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS A 1600 p26 Bac p26 Art Relative fluorescence 1400 1200 G 1000 800 TS 600 R 400 200 310 316 322 328 334 340 346 352 358 364 370 376 382 388 394 400 Emission wavelength (nm) 25 20 15 25°C 43°C B Relative fluorescence 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 guarding 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 citrate synthase in the absence of heating was 0.09 lmol citratmg protein min)1 Small heat shock protein sequence motifs 10 G R TS p26 Bac p26 Art p26 Fig p26 tertiary structure (A) Purified p26 was diluted in 10 mM NaH2PO4, pH 7.1–0.06 mgỈmL)1 and intrinsic fluorescence was measured at an excitation wavelength of 280 nm with a nm band pass and fluorescence emission was detected over 310–400 nm (B) Purified p26 at 0.06 mgỈmL)1 in 10 mM NaH2PO4, pH 7.1 was oversaturated with ANS and fluorescence was measured with an excitation wavelength of 388 nm and a band pass of nm, and an emission wavelength of 473 nm with a band pass of nm Measurements 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 samples obtained from transformed E coli and Artemia, respectively Each spectrum was recorded in duplicate using two independent sample preparations 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 approximately 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 pronounced for R Modeling of p26 structure Sequence identity between a-crystallin domains allowed generation of a p26 model based on wheat 5235 Small heat shock protein sequence motifs Y Sun and T H MacRae residues V154 and I156 correspond to I147 and I149 of Hsp16.9 25 20 15 CD [m deg] 10 Discussion 180 184 188 192 196 200 204 208 212 216 220 224 228 232 236 240 244 248 252 256 260 –5 –10 –15 –20 Wavelength (nm) Fig Far-UV circular dichroism Each spectrum represents the average of three scans obtained with purified bacterially produced p26 dissolved in 10 mM NaH2PO4, pH 7.1, at 0.2 mgỈmL)1 The absorption data were expressed as molar ellipticity in degrees cm2Ỉdmol (m deg) 1, WTp26 from bacteria; 2, WTp26 from Artemia; 3, G; 4, R; 5, TS Table Secondary structure elements of p26 Secondary structure elements of p26 were calculated with the CDNN v2.1 deconvolution 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 b-Antiparallel b-Parallel b-Turn Random coil 21.8 17.8 9.8 17.1 33.5 24.9 15.6 9.4 17.4 32.7 22.2 16.4 9.5 17.5 34.4 17.7 21.6 10.3 16.5 34.0 17.7 23.0 10.0 16.8 32.5 Hsp16.9 crystal structure (Fig 8), although the lack of correspondence between C-terminal extensions precluded inclusion of p26 residues A159 to A192 Extrapolation from the structural characteristics of Hsp16.9 suggests each p26 monomer contains an N-terminal region potentially buried within oligomers, an a-crystallin 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 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 (102-SDEYGHVQRE-111) protrudes from the a-crystallin domain and corresponds to the larger Hsp16.9 loop containing b-strand Loop (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 5236 p26 contains two novel N-terminal sequences, one enriched in glycine and the other in arginine Additionally, 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 variable N- and C-terminal extensions For example, the unusual sequence motifs may either enhance p26 oligomerization, possibly through stabilization of dimer formation, 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 jannaschii [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 monomers 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 dimerstabilizing b-strand of M jannaschii Hsp16.5 [5,6] The WD ⁄ EPF motif also occurs in Chinese hamster Hsp27 and when deleted, chaperone activity and oligomerization 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-FGFGGFGGG28 (Fig 1) The advantages of replacing functionally important motifs in the a-crystallins with a glycineenriched sequence in p26, lacking in apparent structural and functional attributes, is unknown Deleting FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS Y Sun and T H MacRae Small heat shock protein sequence motifs A C B D Fig 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 (D) 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 arginineenriched motif is more important than the glycinecontaining sequence FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS 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 oligomerization, but deleting the entire C-terminal extension, including the TS sequence and the conserved I ⁄ V-XI ⁄ 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 substituting alanine for either or both isoleucines, reduces oligomer size and chaperone activity [56], as occurs upon mutating Val143 of Synechocystis Hsp16.6 [57] 5237 Small heat shock protein sequence motifs 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 promoting 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 chaperoning is relatively insensitive to structural change The fluorescence intensity of all p26 variants was reduced in comparison to WT, implying modified tertiary 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 fluorescence 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 negatively charged residues that perturb packing and disrupt b-strands within the nearby a-crystallin domain The similarities in intrinsic fluorescence, surface hydrophobicity 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 cultured 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 diapause and stress and may stabilize nuclear matrix proteins [52,53], but how p26 moves into nuclei is 5238 Y Sun and T H MacRae 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 intranuclear p26, this contrasting WT p26 and the remaining 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 similar 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 dissociation 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 revealing 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 containing b-strand is smaller than the equivalent loop in Hsp16.9 b-strand in loop 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-crystallins and other sHSPs from animals, as opposed to plant and bacterial sHSPs [6,8,67] Additionally, removal of p26 loop (residues 22-GFGGFGGGMDL-32), as occurred in G, had little effect on oligomerization and chaperone activity, suggesting limited involvement of loop in protein stability and function Loops and 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 QuikChangeTM Site-directed Mutagenesis kit (Stratagene, La Jolla, CA, USA), using pRSET.C-p26–3-6-3 as template [68], and designated primers (Table 3) PCR mixtures were incubated 30 s at 95 °C prior to 12 cycles of 30 s at 95 °C, at 55 °C and at 68 °C Resulting DNAs were digested for h at 37 °C with DpnI and used to transform Escherichia coli FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS Y Sun and T H MacRae Small heat shock protein sequence motifs Table 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 deleted; 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¢ 5¢-GGACCTTGATATTGACGGTCCAGATACC-3¢ 5¢-GGTATCTGGACCGTCAATATCAAGGTCC-3¢ 5¢-GGATTGAAGGGGGAAGATCAGGAGGTGC-3¢ 5¢-GCACCTCCTGATCTTCCCCCTTCAATCC-3¢ R C-terminal extension TS XL1-blue supercompetent cells (Stratagene) p26 cDNA inserts were recovered by digestion with BamHI and XhoI, electrophoresis in agarose and purification with the GFXTM 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 ngỈmL)1 Bacterial cell-free extracts were prepared and p26 was purified with BD TALON resin (BD Biosciences Clontech) following manufacturer’s instructions Purified p26 was dialyzed h at room temperature against 10 mm NaH2PO4, pH 7.1 with one change of buffer, and then overnight at °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 ⁄ polyacrylamide gels were either stained with Coomassie Brilliant Blue R-250 (Sigma, St Louis, MO, USA) or blotted to nitrocellulose (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 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 at room FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS temperature with either anti-p26 Ig [68] in high salt Tween buffer (HST) (10 mm Tris, pH 7.4, m NaCl, 0.5% (v ⁄ v) Tween (20) or Omni-probe (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), a monoclonal antibody recognizing the His6 tag Blots were washed then incubated for 30 with either horseradish peroxidase (HRP)-conjugated 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 Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Boston, MA, USA) p26 synthesis in mammalian cells COS-1 cells were transiently transfected with p26-containing plasmids in SuperFectTM (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 isothiocyanate-conjugated goat anti-(rabbit IgG) Ig (Jackson ImmunoResearch) and nuclei were stained with propidium iodide [11] Rinsed cover-slips were inverted on VectashieldTM mounting medium (Vector Laboratories, Burlingame, 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 °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 electrophoresed in 12.5% SDS polyacrylamide gels before blotting 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 calculate the number of monomers in oligomers, with correc- 5239 Small heat shock protein sequence motifs tions for amino acid deletions a-Lactalbumin (14.2 kDa), carbonic anhydrase (29 kDa), BSA (66 kDa), alcohol dehydrogenase (150 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa) (Sigma) were centrifuged separately and their locations in sucrose gradients determined by measuring the absorbance of fractions at 280 nm p26-induced thermotolerance Transformed E coli were incubated overnight with shaking at 37 °C in mL of Luria–Bertani medium containing spectinomycin, chloramphenicol and aTc (50, 34 and 100 ng.ml)1 respectively) The cultures, diluted : 10 in fresh LB, were then incubated at 54 °C with 100 lL samples 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 Y Sun and T H MacRae fluorescence excitation was detected from 250 to 310 nm Subsequently, excitation wavelength was 280 nm and fluorescence emission was detected from 310 to 400 nm Spectra were recorded in duplicate using independently prepared samples To examine surface hydrophobicity lL of ANS (Molecular Probes, Eugene, OR, USA) at 8.0 mm in 10 mm NaH2PO4, pH 7.1 was added to 198 lL of purified, bacterially produced p26 at 0.06 mgỈmL)1 and mixtures were incubated for at either 25 °C or 43 °C The excitation wavelength was 388 nm with band pass of nm and the emission wavelength was 473 nm with band pass of nm An AMINCO Bowman series z luminescence spectrometer (AMINCO, Rochester, NY, USA) equipped with a thermostated 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 mgỈmL)1 in 10 mm NaH2PO4, 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 nm and all scans were corrected for buffer and smoothed to eliminate background noise Secondary structure parameters were calculated with the cdnn v2.1 deconvolution program (Martin-Luther-Universitat Halle-Wittenberg, Germany) ă 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 lL of TE (50 mm Tris ⁄ HCl, pH 7.5, mm EDTA), 10 lL of 10 mm oxaloacetic acid (Sigma), 10 lL of 10 mm 5,5¢-dithiobis(2nitrobenzoic acid) (Sigma) and 30 lL of mm 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 at 25 °C p26 intrinsic fluorescence, ANS-binding capacity and secondary structure Purified p26 was diluted to 0.06 mgỈmL)1 in 10 mm NaH2PO4, 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 nm band pass and 5240 Modeling of p26 three-dimensional structure The Swiss-Model Protein Modeling Server (version 36.0003, Biozentrum University Basel, Basel; Swiss Institute of Geneva, 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 template with the function of Swiss-Model The returned threedimensional model was improved with the Improve Fit function 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 structure, 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 acknowledged 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 FEBS Journal 272 (2005) 5230–5243 ª 2005 FEBS Y Sun and T H MacRae References Narberhaus F (2002) 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Ouellette BFF (2005) Protein structure prediction and analysis In Bioinformatics: a Practical Guide to the Analysis of Genes and Proteins, 3rd edn, pp 223–252 John Wiley & Sons, Inc, Hoboken, NJ Supplementary material The following Supplementary material is available for this article online: Supplemental Fig p26 prevents heat-induced citrate synthase aggregation Purified, bacterially produced p26 was heated at 43 °C for 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 5243 ... 5¢-GGCACTTAACCCATGGTACATGGACCTTGATATTGAC-3¢ 5¢-GTCAATATCAAGGTCCATGTACCATGGGTTAAGTGCC-3¢ 5¢-GGACCTTGATATTGACGGTCCAGATACC-3¢ 5¢-GGTATCTGGACCGTCAATATCAAGGTCC-3¢ 5¢-GGATTGAAGGGGGAAGATCAGGAGGTGC-3¢ 5¢-GCACCTCCTGATCTTCCCCCTTCAATCC-3¢... 11222–11228 11 Sun Y, Mansour M, Crack JA, Gass GL & MacRae TH (2004) Oligomerization, chaperone activity, and nuclear localization of p26, a small heat shock protein from Artemia franciscana J Biol Chem... Journal 272 (2005) 5230–5243 ª 2005 FEBS Y Sun and T H MacRae Small heat shock protein sequence motifs A C B D Fig Comparative modeling of p26 (A) Amino acid sequences of p26 and Hsp16.9 from wheat