Báo cáo khoa học: Stability and fibril formation properties of human and fish transthyretin, and of the Escherichia coli transthyretin-related protein potx

Stability and fibril formation properties of human and fish transthyretin, and of the Escherichia coli transthyretin-related protein Erik Lundberg1, Anders Olofsson2, Gunilla T Westermark3 and A Elisabeth Sauer-Eriksson1 ˚ Department of Chemistry, Umea University, Sweden ˚ Department of Medical Biochemistry and Biophysics, Umea University, Sweden Division of Cell Biology, Diabetes Research Centre, Linkoping University, Sweden ă Keywords amyloid; fibril formation; HIU hydrolase; transthyretin; transthyretin-related protein Correspondence A E Sauer-Eriksson, Department of ˚ Chemistry, Umea University, SE-90187 ˚ Umea, Sweden Fax: +46 90 7865944 Tel: +46 90 7865923 E-mail: elisabeth.sauer-eriksson@chem umu.se (Received November 2008, revised 20 January 2009, accepted 26 January 2009) doi:10.1111/j.1742-4658.2009.06936.x Human transthyretin (hTTR) is one of several proteins known to cause amyloid disease Conformational changes in its native structure result in aggregation of the protein, leading to insoluble amyloid fibrils The transthyretin (TTR)-related proteins comprise a protein family of 5-hydroxyisourate hydrolases with structural similarity to TTR In this study, we tested the amyloidogenic properties, if any, of sea bream TTR (sbTTR) and Escherichia coli transthyretin-related protein (ecTRP), which share 52% and 30% sequence identity, respectively, with hTTR We obtained filamentous structures from all three proteins under various conditions, but, interestingly, different structures displayed different tinctorial properties hTTR and sbTTR formed thin, curved fibrils at low pH (pH 2–3) that bound thioflavin-T (thioflavin-T-positive) but did not stain with Congo Red (CR) (CR-negative) Aggregates formed at the slightly higher pH of 4.0–5.5 had different morphology, displaying predominantly amorphous structures CR-positive material of hTTR was found in this material, in agreement with previous results ecTRP remained soluble at pH 2–12 at ambient temperatures By raising of the temperature, fibril formation could be induced at neutral pH in all three proteins Most of these temperature-induced fibrils were thicker and straighter than the in vitro fibrils seen at low pH In other words, the temperature-induced fibrils were more similar to fibrils seen in vivo The melting temperature of ecTRP was 66.7 °C This is approximately 30 °C lower than the melting temperatures of sbTTR and hTTR Information from the crystal structures was used to identify possible explanations for the reduced thermostability of ecTRP Transthyretin (TTR) is a homotetrameric plasma protein that binds and transports the thyroid hormones 3,5,3¢-triiodo-l-thyronine and 3,5,3¢,5¢-tetraiodo-l-thyronine (thyroxine) and retinol by binding to the retinolbinding protein when it is loaded with retinol [1] TTR is mainly expressed in the adult liver, the choroid plexus of the brain, and the retina [2,3] TTR is involved in three amyloid diseases: familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy (FAC), and senile systemic amyloidosis (SSA) [4,5] Whereas SSA is associated with native TTR, point mutations, of which more than 80 have been identified, cause FAP and FAC [6] TTR mutations associated with familial amyloid diseases display a wide range of Abbreviations AFM, atomic force microscopy; BME, b-mercaptoethanol; CR, Congo Red; DSC, differential scanning calorimetry; ecTRP, Escherichia coli transthyretin-related protein; EM, electron microscopy; FAC, familial amyloidotic cardiomyopathy; hTTR, human transthyretin; rTTR, rat transthyretin; sbTTR, sea bream transthyretin; SSA, senile systemic amyloidosis; ThT, thioflavin-T; TLP, transthyretin-like protein; TRP, transthyretin-related protein; TTR, transthyretin FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS 1999 Stability and fibril formation of TTR and TRP E Lundberg et al diversity in age of onset, penetrance, and tissues affected [7,8] SSA is a geriatric disease affecting approximately 25% of the European Caucasian population over 80 years of age [4] Like FAC, SSA is characterized by heavy deposits of amyloid fibrils in the heart Structures of TTRs from different species have been studied [9], including human transthyretin (hTTR) [10–12], rat TTR (rTTR) [13], chicken TTR [14], and sea bream TTR (sbTTR) [15,16] Within the TTR family, fish TTR has the lowest sequence identity with hTTR (e.g sbTTR 52% [16,17], and lamprey TTR 47% [18]) The transthyretin-related proteins (TRPs) comprise a family of proteins recently shown to function as 5-hydroxyisourate hydrolases in the purine degradation pathway [19–23] These proteins are also referred to in the literature as transthyretin-like proteins (TLPs) However, to separate this family, whose members have the characteristic sequence motif YRGS at their C-terminal end, from other protein sequences listed as TTR-like, we prefer to refer to them as TRPs [19,24] Sequence analysis of representative TTRs, TRPs and TLPs suggests that the three protein groups are not functionally related (Fig 1) The sequence identity between TRPs and TTRs is relatively low; Escherichia coli TRP (ecTRP) shares 30% and 35% sequence identity with hTTR and sbTTR, respectively (Fig 1) [19] Structures of TRP from several species have been determined, and despite their low sequence identity, the TTR and TRP structures were found to be very similar [24–27] In TTR amyloidoses, the normally folded, secreted protein cannot assemble into amyloid fibrils unless a preceding partial unfolding event occurs [28] Muta- tions that destabilize the native structure of TTR are known to lead to disease [29], but thermodynamic stability alone does not reliably predict the severity of the disease [30] Instead, thermodynamic data, combined with kinetic data, more reliably explain why only some mutations lead to severe pathologies [31] To understand the mechanism behind hTTR dissociation, misfolding, and amyloid formation, studies from other species have provided valuable information Like hTTR, rTTR forms amyloid-like fibrils in vitro after partial acid denaturation [32] rTTR shares 85% sequence identity with hTTR, which raises the question of how important tertiary similarities are, as opposed to sequence identity, for the ability of the protein to form fibrils In an attempt to answer this question, we have investigated the fibril-forming properties of sbTTR and ecTRP in vitro, and compared the results with those of hTTR Our results showed that hTTR, sbTTR and ecTRP can form fibrillar structures, but under different solution and temperature conditions Furthermore, depending on the conditions used, fibrils of different morphology were obtained Recent studies have shown that sbTTR binds thioflavin-T (ThT) at low pH, suggestive of amyloid [33] In our study, we verified that sbTTR forms fibrillar structures at low pH that are similar in shape to those of hTTR We also found that, even though  70% of the amino acids of ecTRP are different from the respective amino acids in hTTR and sbTTR, ecTRP has the ability to form Congo Red (CR)-positive fibrils in vitro if the temperature is increased sufficiently Similar findings for ecTRP were published while this work was in progress [34] The Fig Multiple sequence alignment of representatives of TRPs, TTRs, and TLPs There are 57 gene clusters in Caenorhabditis elegans, referred to as TTR-1 to TTR-57 in WORMBASE Some of these sequences were originally identified as being structurally TTR-like by Sonnhammar & Durbin [68] They seem to be functional and to influence aging in C elegans, and are referred to as TRPs [69] TTR-1 to TTR-57 are, however, not related to the YRGS TRP family [19], which is why we prefer to refer to them as TLPs The 57 TLP sequences present in C elegans are nematode-specific and share low sequence identity with each other; however, sequences TTR-18 to TTR-31 seem to comprise a subgroup that is also found in other nematodes (A) In this sequence alignment, TLP representatives from six nematodes have been aligned with representatives from TTR and TRP Identical and homologous residues within the three subgroups are marked in red and pink, respectively The first residue after signal sequence cleavage is highlighted in black Sequence numbering refers to mature proteins Similarity is defined as amino acid substitutions within one of the following groups: FYW, IVLMF, RK, QDEN, GA, TS, or HNQ Identical and similar residues within the TRP family are shown in dark and light green, those within the TTR family in dark and light blue, and those within the TLP subgroup in dark and light gray The secondary structure elements are based on hTTR [12] Residues lining the substrate-binding channel in TTRs [13] and TRPs [26] are marked with blue stars The assignment (*.:) shown below the sequences is directly from CLUSTALW2 [75] and refers to alignment of the six TLP sequences only Only two amino acids (proline and glycine) are conserved throughout the three groups Whereas residues at the ligand-binding sites are almost completely conserved in the TTR and TRP families, they are not conserved within the TLP group In contrast, the most conserved regions are found at sites corresponding to b-strands B and E in TLPs, and to a-helix E in TTRs and TRPs (B) Phylogenetic tree of TRPs, TTRs, and TLPs The tree was based on the multiple sequence alignment from (A) Caenorhabditis elegans TRP-R09H10.3 (GI:115532920); C elegans TRP-ZK697.8 (GI:115534555); Mus Musculus TRP (GI:81916776); Bacillus subtilis TRP (GI:3915561); Escherichia coli TRP (GI:3915454); Petaurus breviceps TTR (GI:1279636); Sminthopsis macroura TTR (GI:1279727); Gallus gallus TTR (GI:45384444); Homo sapiens TTR (GI:114318993); Sparus aurata TTR (GI:6648602); Heterodera glycines TLP (GI:8571913); Radopholus similis TLP (GI:145279861); Brugia malayi TLP (GI:170583879); Xiphinema index TLP (GI:55724912); C elegans TLP (TTR-30, GI:25153261); Caenorhabditis briggsae TLP (TTR-18 GI:187037056) 2000 FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS E Lundberg et al Stability and fibril formation of TTR and TRP A B TLP H glycines TLP R similis TLP B malayi TLP X index TLP C elegans TTR-30 TLP C briggsae TTR-18 TTR P breviceps TTR S macroura TTR G gallus TTR H sapiens TTR S aurata TRP E coli TRP C elegans R09H10.3 TRP C elegans ZK697.8 TRP M musculus TRP B subtilis FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS 2001 Stability and fibril formation of TTR and TRP E Lundberg et al results emphasize the potential for amyloid formation as a common property of all proteins, a feature that can sometimes even bring new functionality [35–37] The thermal stability of ecTRP was found by differential scanning calorimetry (DSC) to be approximately 30 °C lower than that of sbTTR and hTTR Comparative studies of structures from homologous thermophiles and mesophiles have revealed several factors that generally contribute to the intrinsic thermal stability of proteins [38–40] These include tighter hydrophobic packing of the protein core [41–43], increased electrostatic interactions on the surface of the protein [44,45], more prolines and alanines [46,47], and increased hydrogen bonding of the polypeptide chain [48–50] In addition, improved intersubunit contacts within oligomeric proteins contribute to protein stability [51,52] Here, we analyze the structural basis for the differences in thermostability of hTTR, sbTTR, and ecTRP Results Partial acid denaturation generates fibrils of hTTR and sbTTR Partial acid denaturation combined with turbidity assays is a method frequently used to induce and monitor the degree of protein aggregation and fibril formation of hTTR [28,53] Application of this method to the three proteins in our study showed that hTTR displayed an increase in turbidity at low pH, with a turbidity maximum at pH 4.5 (Fig 2) This is in agreement with previous results [28,53] For sbTTR, we measured only minor turbidity increases at the low pH range of 4.0–5.5, and for ecTRP, no effect was apparent over the entire pH range (2–12) (Fig 2) It should be noted that the pI of ecTRP is estimated to be 8.2, in contrast to those of hTTR and sbTTR, which are estimated to be 5.5–6.0 The samples treated at low pH were visualized with atomic force microscopy (AFM) to determine the morphologies of the protein aggregates Both hTTR and sbTTR displayed fibrils at pH 2.0–3.5 that were very similar in structure (Fig 3) For hTTR, small amounts of fibrillar structures were also formed at pH 4.0 The thickness of these structures was found to be 1.3 nm, which means that they were thinner than amyloid fibrils Their curved morphology agreed with previous observations of both hTTR and rTTR in vitro fibrils, suggesting that they most likely represented protofibrils [32,54–56] hTTR in vitro fibrils are reported to have widths varying between 2.8 [55] and 10 nm [54], and the thicker in vitro fibrils are believed to consist of up to five intertwined protofilaments [54] At the pH interval 4.0–5.5, predominantly amorphous aggregates, rather than fibrillar structures, were observed in the hTTR samples (Fig 3A) It is, however, not possible to quantitatively estimate the ratio of aggregates to fibrillar structures from the AFM images The hTTR and sbTTR samples were also visualized with electron microscopy (EM) The EM images were consistent with the morphologies that we elucidated from the AFM images, and verified that fibrillar structures were present in the protein samples at pH 4.5 (Fig 3B) To determine whether the lack of fibrillar structures in the hTTR and sbTTR samples at pH 4.5 could be an effect of the technique used for analysis (that is, the fibrils are unable to bind to mica gels at this pH), fibril-containing samples of hTTR formed at pH 2.0 were adjusted to pH 4.5 and incubated for various lengths of time AFM images of this material showed that the fibrils formed at pH 2.0 persisted at pH 4.5, thereby verifying that these fibrillar forms can bind to mica gels even at higher pH (data not shown) Tinctorial properties of fibrillar structures formed at low pH The protein fibrils and aggregates obtained by the partial acid denaturation experiments were tested for ThT, which is a fluorescent dye commonly used to Fig Turbidity assays for hTTR, sbTTR, and ecTRP The turbidity was measured at 330 nm after incubation of protein samples at 37 °C for 72 h 2002 FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS E Lundberg et al Stability and fibril formation of TTR and TRP A B a b a b C Fig (A) AFM images of hTTR (left) and sbTTR (right) The samples were incubated at 37 °C for 72 h Fibrils were present in samples incubated at pH 2.0–3.5, whereas aggregates were predominantly present in samples incubated at pH 4.5–5.5 No fibrils or aggregates were detected in the ecTRP samples, at any of the pH intervals tested (pH 2.0–12.0; data not shown) The white scale bar is 500 nm (B) EM images of fibrils of hTTR (a) and sbTTR (b) incubated at pH 4.5 (C) CR staining of hTTR incubated for days at pH 4.5 (a) shows fluorescence (at 594 nm) and (b) shows the characteristic apple-green birefringence with polarized light assess amyloid fibril formation [57,58], including hTTR amyloid [59,60] Pronounced emission at 482 nm, as a result of ThT binding, was seen for the human and sea bream samples incubated at pH 2.0 and 2.5, respectively (Fig 4) This is in agreement with the presence of the thin fibrils seen with AFM (Fig 3A) At a pH value around 3.0, both hTTR and sbTTR showed significantly reduced ThT binding as compared to that at lower pH However, at the slightly higher pH range of 3.5–4.5, hTTR formed structures that bound to ThT (Fig 4) This ThT-binding pattern correlates well with the increase in turbidity of hTTR observed at pH 4.5 (Fig 2) The sbTTR samples did not display increased ThT binding at pH 3.5–4.5, and demon- strated only a very small increase in turbidity at this pH range ecTRP did not react with ThT at any pH range tested The partially denaturated protein samples were also tested for CR staining, visualized with EM Generally, the ability of fibrils to bind CR and to display a characteristic apple-green birefringence under polarized light are the two most important criteria for detection of amyloid fibrils in vivo Such fibrils are called CR-positive, but the specificity of this test has recently been questioned [61,62] The material from hTTR was CR-positive at pH 4.0–4.5 (Fig 3C), in agreement with previous studies [28,53] Aggregates from sbTTR, on the other hand, were not found to be CR-positive at FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS 2003 Stability and fibril formation of TTR and TRP E Lundberg et al To determine whether the thin and curved protofibrils formed at pH 2.0 by hTTR and sbTTR could be induced to form amorphous aggregates or thicker filaments, the samples were readjusted to a pH of 4.5 Interestingly, even after weeks at °C, these samples contained the same type of protofibrils This implies that the low-pH-induced and ThT-binding fibrils are not readily converted to CR-positive fibrillar aggregates or thicker filaments, or at least not under these conditions Fiber formation induced by heating Fig ThT-binding assays Relative intensity of emission at 482 nm for samples incubated for 72 h at different pH The excitation wavelength was 440 nm The standard deviations between triplet samples for pH intervals from 7.0 to 2.0 and between double samples for pH intervals from 12.0 to 7.5 are shown Strong ThT binding is seen for hTTR and sbTTR at the lowest pH values, 2.0– 3.0 For hTTR, weaker ThT binding is also detected between pH 3.5 and pH 4.5 As expected, neither hTTR nor sbTTR samples incubated at pH 2.0 or 4.5 produce increased emission at 482 nm if ThT is not added, which means that the increase in emission is due to actual ThT binding A local ThT-binding minimum is seen at pH 3.0 for hTTR, even though fibrils are detected with AFM The reason for this is unknown The fiber thickness at pH 3.0 generally seems to be the same as for the fibers seen at lower pH ecTRP does not bind ThT at any pH level any pH range Also, the large amounts of small fibrillar hTTR and sbTTR structures, observed with AFM (Fig 3A) at pH 2.0–3.0, did not stain with CR Apparently, ThT is more promiscuous than CR in binding to thinner immature fibrillar structures of TTR A hTTR B 0.2 µm 2004 sbTTR Fibrillar structures of hTTR, sbTTR and ecTRP were obtained by heating the protein samples for 72 h without stirring at different temperatures Fibrils of hTTR have previously been reported at 75 °C [63] In this study, we obtained thick fibrils at 55 °C for hTTR and 65 °C for sbTTR and ecTRP (Fig 5A) Of these, only fibrils from ecTRP showed a strong ThT response (data not shown) These fibrils were verified to be CR-positive (Fig 5B) Protein stability measured by SDS/PAGE and DSC The propensity for TTR amyloid formation is coupled to tetramer dissociation The stability of the tetrameric structures of hTTR, sbTTR and ecTRP was analyzed by gel electrophoresis according to the method of Lai et al [53] (Fig 6) Unboiled samples from the acid denaturation experiment were run on gel electrophoresis in the presence of SDS and b-mercaptoethanol (b-ME) Whereas tetrameric hTTR dissociated at pH ecTRP Fig (A) AFM images of hTTR, sbTTR and ecTRP heated at 55 °C (hTTR) or 65 °C (sbTTR and ecTRP), respectively, for 72 h The fibril heights were estimated to be  2.8 nm for hTTR,  3.5 nm for sbTTR, and  4.0 nm for ecTRP The white scale bar is 500 nm (B) Left: EM image of ecTRP from the same sample as in (A) The material shows fluorescence (at 594 nm) (middle) and apple-green birefringence (right) after visualization with polarized light FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS E Lundberg et al Stability and fibril formation of TTR and TRP Fig DSC profiles of hTTR, sbTTR, and ecTRP The melting temperatures (Tm) were 97.8 °C for hTTR, 93.0 °C for sbTTR, and 66.7 °C for ecTRP Fig Analysis of tetramer stability by SDS ⁄ PAGE The samples were not boiled The tetrameric structures of sbTTR and ecTRP show increased stability at lower pH as compared to hTTR Notably, both hTTR and sbTTR are unaffected by the SDS ⁄ BME treatment, and remain either in a monomeric or a tetrameric state, depending on the pH of the protein buffer In contrast, SDS ⁄ b-ME treatment alone dissociates a fraction of the tetrameric ecTRP into the monomeric state at all pH values values lower than 5.0, as shown previously [53], sbTTR and ecTRP did not completely separate into their monomeric units until the pH values were below 4.6 and 3.0, respectively (Fig 6) In agreement with the partial acid denaturation experiments, the ecTRP tetrameric structure seems to be more resistant to variations of pH However, the SDS ⁄ b-ME treatment generally dissociated a larger fraction of the ecTRP protein material than of hTTR and sbTTR into monomeric structures This behavior is likely to reflect differences in the chemical composition of the proteins SDS is a detergent that readily dissolves hydrophobic molecules, whereas acid denaturation affects electrostatic interactions rather than the hydrophobicity of molecules Gel filtration chromatography of ecTRP in the presence and absence of 0.5% SDS in the running buffer produced monomers only when the SDS was included (data not shown) The thermal stability of hTTR, sbTTR and ecTRP was further studied by DSC (Fig 7) The measured thermal melting point (Tm) for hTTR was close to values previously reported [64,65] Even though tetrameric sbTTR seems to be more stable than hTTR at lower pH, we found that it was less stable than hTTR at physiological pH, with the Tm value for sbTTR being approximately 4.5 °C lower than that for hTTR Gilthead sea bream is an ectotherm whose normal habitat is the Mediterranean Sea One feature that generally defines cold-adapted proteins and distinguishes them from their mesophilic and thermophilic counterparts is their lower thermal stability [66] This could therefore be one explanation for the reduced stability that we observed for sbTTR as compared to hTTR More unexpected, however, was the low thermostability of the ecTRP protein, with a Tm value approximately 26 °C below the values for both sbTTR and hTTR (Fig 7) The inability of ecTRP to form fibrils at low pH can therefore not be directly correlated with the thermostability of its tetrameric and monomeric structures Thermostability and protein structures We have analyzed the structures of hTTR (Protein Data Bank code: 1F41 [12]), sbTTR (Protein Data Bank code: 1SN2, [16]) and ecTRP (Protein Data Bank code: 2G2N [24]) in an attempt to identify factors that contribute to their profound differences in stability The results are summarized in Table Introduction of alanines, prolines and aromatic residues can contribute to protein stability [46,47,52] hTTR and sbTTR have more alanine residues than ecTRP, 12 versus eight and 13 versus eight, respectively, which might contribute to entropic stabilization On the other hand, ecTRP has more aromatic residues than hTTR and sbTTR, 13 versus 12 and 13 versus FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS 2005 Stability and fibril formation of TTR and TRP E Lundberg et al Table Structural factors implicated in the thermostabilities of hTTR, sbTTR, and ecTRP Protein volume calculations were obtained using VOIDOO [72], and hydrogen bonds were calculated using WHATIF [73] hTTR Melting temperature (°C) Amino acid composition Ala Phe, Tyr, Trp Pro Glu, Asp Lys, Arg Salt bridgesa ˚ Protein volume (A3)b Monomer, Vm Tetramer, Vt DV = Vt ) 4Vm Hydrogen bondsc Monomer Dimer sbTTR ecTRP 97.8 93.0 66.7 12 12 17 12 13 11 14 10 13 12 13 11 490 45 760 )200 11 340 45 190 )170 11 810 47 100 )140 75 (12) 165 (20) 70 (11) 163 (20) 64 (4) 141 (10) a ˚ A distance less than or equal to A between charged groups defines an ion pair [74] b The molecular volumes were calculated ˚ using a probe with radius A, in order to obtain the protein volume per se [52] Before calculation, alternate conformations were removed, and the structures were truncated at their N-termini and C-termini, so that they structurally start and end at the same position Residues included from the four chains, A, B, C, and D, of the tetrameric structures were: hTTR, A10–A122, B10–B122, A¢11– A¢122, and B¢10–B¢122; sbTTR, A10–A122, B10–B122, C11–C122, and D10–D122; and ecTRP, A4–A114, B4–B114, C5–C114, and D4–D114 c The numbers in parentheses are the numbers of buried water molecules included in the calculation 11, respectively The backbone flexibility of the TTR structures is probably also reduced because of a higher proline content, which decreases the entropy of unfolding [46] hTTR and sbTTR have eight proline residues each, whereas ecTRP has only five The formation of salt bridges is another important contributor to the temperature stability of proteins [67] In agreement with this, there is a higher number of charged residues in the more thermostable TTR protein structures (Table 1) There are five salt bridges in hTTR and sbTTR, but only three in ecTRP Thermostable proteins are generally more tightly packed than their less thermostable homologs [42] The structural homologs hTTR, sbTRP and ecTRP have no internal cavities However, hTTR and sbTRP have two polar residues, Thr75 and His88 (hTTR numbering), buried within the hydrophobic core of the monomers The side chains of these residues form, together with the Ne1 atom of Trp79, hydrogen bonds with three or four buried water molecules [12,16] This probably increases packing density and contributes to stability [49] In the ecTRP structure, two phenylala2006 nines occupy the same position as His88 and Trp79 in hTTR, and consequently only one water molecule can bind at this site in ecTRP [24] The protein volumes of single subunits and tetrameric structures for the three proteins were investigated Our calculations show that there is a decrease in the protein volume of each monomer that is related to an increase in thermal stability Furthermore, there is a clear correlation between thermostability and molecular volume occupied by the tetramers (Table 1) The difference between the tetrameric volume and the volume of the corresponding number of monomeric units, DV, is negative in all cases, demonstrating that the protein density increases slightly upon tetramerization Analysis of the number of hydrogen bonds formed within monomers and dimers revealed pronounced differences between the three proteins As previously mentioned, the hTTR and sbTTR structures contain buried polar residues and water molecules These waters allow the formation of 10 more hydrogen bonds within their monomeric units, and about 20 more hydrogen bonds within their dimeric units, than in the ecTRP protein structure (Table 1) In addition, 14 and 15 hydrogen bonds are formed at the monomer–monomer interface of sbTTR and hTTR, whereas only eight are formed in ecTRP Five hydrogen bonds are formed across the dimer–dimer interface of both hTTR and sbTRP, whereas three are formed in ecTRP (Table 1) Discussion TTRs and TRPs are two protein families with similar structures but different functions, due to divergent evolution The TTRs, found only in vertebrates, function as retinoic acid and thyroid hormone carriers, whereas the TRPs, found predominantly in lower eukaryotes and prokaryotes, are enzymes that hydrolyze 5-hydroxyisourate [19,21–23] in the purine catabolic pathway The structural similarity between several TTR and TRP representatives has been verified by crystallographic studies [11,12,24–27] Members from both protein families have their active site positioned in the hydrophobic channel formed at the dimer–dimer interface of their homotetrameric structure The four amino acid sequence motif YRGS at the C-terminal end of the TRP sequences distinguishes them from other proteins annotated as TTR-like or TTR-related [19] These residues are involved in binding to substrate analogs [26] Other proteins with sequence homology to TRPs and TTRs exist, and we refer to these as TLPs TLPs have so far been found only in nematodes Some of these were identified as being structurally TTR-like in 1997 by FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS E Lundberg et al Sonnhammar & Durbin [68] The function of the TLPs is not known, but studies suggest that they are involved in the regulation of the nematode lifespan [69] From their sequences, however, it is clear that their function must be completely different from that of TTRs and TRPs (Fig 1) In the current study, the fibril-forming and amyloidforming properties of three representatives from the TTR and TRP families were investigated Amyloidogenic properties of proteins are linked to their stability and dissociation kinetics [31] Therefore, the stabilities of hTTR, sbTTR and ecTRP were analyzed by partial acid and thermal denaturation Some differences were apparent Analysis with SDS ⁄ PAGE showed that hTTR is less stable than the other proteins under acidic conditions, and dissociates into monomers when the pH falls below 5.0 sbTTR shows similar behavior, and is only marginally less sensitive to acidic pH, dissociating at a pH below 4.5 Interestingly, we found that ecTRP maintains its tetrameric structure even at very low pH values Different results were obtained when the protein samples were analyzed with DSC The melting point for ecTRP was determined to be 66.7 °C, which is more than 26 °C lower than those of both hTTR and sbTTR (Fig 7) Therefore, whereas previous SDS ⁄ PAGE analysis suggested that the tetrameric structure of ecTRP is more stable than that of hTTR [34], the DSC results showed that ecTRP is significantly less thermostable than either hTTR or sbTTR Comparison of the crystal structures of hTTR, sbTTR and ecTRP highlights a number of structural differences that are consistent with the current explanations of thermal stability in proteins Noteworthy is the reduced number of negatively charged residues in the ecTRP structure This could possibly also explain its structural stability at low pH Furthermore, the thermostable TTR proteins have more hydrogen bonds and ion pairs, and their structures are more densely packed than that of ecTRP Thus, it seems that the differences in thermostability are mainly due to the presence of specific polar and charged residues in hTTR and sbTTR, which form additional hydrogen bonds that stabilize their monomeric subunits as well as their monomer–monomer and dimer–dimer interfaces In agreement with previous reports, TRP is amyloidogenic; fibrils form upon heating of the protein sample ([34] and this study) Whereas the previous study reported an amyloid-inducing protocol for TRP that involves heating at 24 °C at pH 5.8 with stirring, our protocol involves heating at 65 °C at pH 7.4 for days without stirring This temperature was chosen because it was shown by DSC to be the Tm of the protein These fibrils are amyloidogenic, as determined from their Stability and fibril formation of TTR and TRP tinctorial properties in the ThT-binding and CR-binding assays hTTR and sbTTR could also be induced to form fibrils by heating (Fig 5) Generally, fibrils obtained from the heating procedure were thicker for all proteins, and exhibited a straighter morphology than that of the short and curved fibrils formed at low pH by sbTTR and hTTR We did not detect any fibrous material in samples incubated at 24 and 37 °C at pH 5.8, with or without stirring However, the discrepancy with the previously described protocol [34] could be due to subtle changes in sample preparation or experimental procedure, which can drastically impact on fibril formation Interestingly, misfolded and aggregated ecTRP material has been shown to be toxic for neuroblastoma cells, although the soluble protein is not [34] In conclusion, sbTTR has properties similar to those of hTTR in terms of tetramer dissociation and fibril formation Generally, where fibrils were observed for hTTR, fibrils of similar morphology were observed also for sbTTR, after some minor adjustments of the fibrillization protocol We did not detect any CR-positive fibrils of sbTTR at pH 4.5–5.5 This suggests that hTTR forms amyloid fibrils by partial acid dissociation more readily than sbTTR The result does not exclude the possibility that sbTTR can form CR-positive fibrils at low pH, but more samples need to be examined, or the concentration of protein needs to be increased ecTRP shares 30% sequence identity with hTTR In agreement with previous reports [34], we detected CR-positive fibrils of ecTRP induced by heating These fibrils are similar both in shape and in dimension to the fibrils of hTTR and sbTTR formed by heating The thick and straight morphology of heat-induced fibrils of hTTR, sbTTR and ecTRP is similar to that of amyloid fibrils in vivo We have so far not been able to convert thin and ThT-positive protofibrils of hTTR and sbTTR, formed at low pH, to thicker and CR-positive structures, suggesting that the kinetics are very slow This suggests that the TTR amyloid architecture is not the result of only one highly stringent assembly of structures In the past, the propensity of proteins to form fibrillar structures has most often been associated with disease Recently, however, examples have been presented where conformational changes and fibril formation are associated with an advantageous gain of function [37] It is not clear whether the fibril formation properties of the TRP family are associated with any gain of function or whether they even have any biological implications whatsoever In vivo fibrils have only been reported with hTTR and rTTR, and it would be interesting to know whether TTR from other species, as well as members of the TRP family, can form fibrils in vivo FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS 2007 Stability and fibril formation of TTR and TRP E Lundberg et al all samples were thoroughly vortexed to disperse aggregated material before analysis by absorbance measurements at 330 nm in a standard UV cell Experimental procedures Protein expression and purification hTTR was expressed as previously described [70] In brief, competent E coli BL21 cells were transformed with the pET3a vector containing the hTTR construct, and plated onto LB agar plates containing 50 lgỈmL)1 carbenicillin Bacteria were grown in LB medium supplemented with 50 lgỈmL)1 carbenicillin at 37 °C At A600 nm = 0.4, cells were induced with 0.2 mm isopropyl thio-b-d-galactoside for h, harvested by centrifugation for 20 minutes at 2800 g, and stored at )20 °C The sbTTR gene placed in a pET24d vector [16] was expressed using a similar protocol as for hTTR, but with 50 lgỈmL)1 kanamycin as the antibiotic After induction with isopropyl thio-b-d-galactoside, the cells were grown overnight at 30 °C Similar purification protocols were used for hTTR and sbTTR Frozen cells were thawed in 20 mm Tris ⁄ HCl (pH 8.0) and 50 mm NaCl, and lysed by sonication in the presence of DNase I Cell debris was removed by ultracentrifugation (120 000 g for 40 min) at °C The lysate was filtered through a 0.2 lm syringe filter (Millipore Corporation, Bedford, MA, USA), and purified on a Q-Sepharose Fast Flow anion exchange column (GE Healthcare, Uppsala, Sweden) equilibrated with 20 mm Tris ⁄ HCl (pH 8.0) and 50 mm NaCl, and eluted with a linear gradient (0.1–1 m NaCl in 20 mm Tris ⁄ HCl, pH 8.0) TTR fractions were pooled and concentrated (Centriprep-10; Amicon Inc., Beverly, MA, USA), and then further purified by gel filtration on a HiLoad 16 ⁄ 60 Superdex-75 (GE Healthcare) column with buffer containing 20 mm Tris ⁄ HCl (pH 6.8) and 50 mm NaCl Pure TTR fractions were pooled, concentrated to mgỈmL)1 (Centriprep-3; Amicon), and stored at )20 °C ecTRP was cloned, expressed and purified as previously described [19], using 50 mm Tris ⁄ HCl (pH 7.0) and 200 mm NaCl as buffer in the final gel filtration step The pure ecTRP fractions were pooled and stored at )20 °C Partial acid denaturation Denaturation studies were performed according to a previously described protocol for hTTR [53] hTTR, sbTTR and ecTRP were dialyzed against mm NaH2PO4 ⁄ Na2HPO4 (pH 7.4) and 20 mm NaCl, and mixed to a final concentration of 0.2 mgỈmL)1 ( 3.5 lm tetramer), corresponding to the TTR concentration in human plasma, at pH 2.0–12.0, at intervals of 0.5 pH units The buffers used gave a final concentration of 50 mm glycine–HCl (pH 2.0–2.5), or 50 mm sodium acetate (pH 3.0–5.5), or 50 mm sodium phosphate (pH 6.0–8.0), or 50 mm Hepes (pH 8.5–9.0), or 50 mm CAPSO (pH 9.5–10.0), or 50 mm CAPS (pH 10.5– 12.0) All buffers included 100 mm potassium chloride, mm EDTA, and mm dithiothreitol After 72 h at 37 °C, 2008 Visualization AFM Following the turbidity measurements, the protein samples were examined by AFM Samples were diluted 10-fold with H2O, and then lL of the diluted sample solutions was applied to freshly cleaved ruby red mica (Goodfellow, Cambridge, UK) The material was allowed to adsorb for 10 s, washed three times with 100 lL of distilled water, and air dried The surface was analyzed with a Nanoscope IIIa multimode atomic force microscope (Digital Instruments, Santa Barbara, CA, USA), using Tapping Mode in air A silicone probe was oscillated at around 270 kHz, and images were collected at an optimized scan rate corresponding to 1–4 Hz The images were flattened and presented in height mode using nanoscope software (Digital Instruments) EM Negative staining for EM was performed on the same samples used in the AFM studies For this purpose, the material was centrifuged at 16 000 g for 30 min, after which most of the supernatant was removed and 200 lL of distilled water was added The material was vortexed, and aliquots of 3–5 lL were applied to Formvar-coated copper grids Contrast was achieved with 2% uranyl acetate in 50% ethanol, and the material was studied at 100 kV in a Jeol 1230 electron microscope (Jeol, Akishima, Tokyo, Japan) CR-binding studies For analysis with CR, 1–2 lL of diluted, vortexed samples were applied to microscope slides and air dried CR staining was performed according to Puchtler et al [71], and examined by light microscopy The presence of amyloid was verified by the green birefringence in polarized light and with red fluorescence in a microscope equipped with filters for wavelengths at 560 nm (excitation) and 590 nm (emission) ThT-binding studies Protein samples incubated at 37 °C for 72 h were vortexed, and 25 lL aliquots were mixed with 173 lL of a buffer containing 100 mm sodium phosphate, 100 mm potassium chloride (pH 7.6), and lL of ThT stock solution (1 mm ThT in 10 mm sodium phosphate, pH 7.4) The samples were excited at 440 nm, and the emission at 482 nm was recorded FEBS Journal 276 (2009) 1999–2011 ª 2009 The Authors Journal compilation ª 2009 FEBS E Lundberg et al Tetramer stability SDS/PAGE hTTR protein, sbTTR protein and ecTRP that had been subjected to partial acid denaturation at various pH values were analyzed by SDS ⁄ PAGE on 8–25% gradient polyacrylamide gels, using the Phast system (GE Healthcare), and stained with Coomassie Brilliant Blue The loading buffer included 2.5% SDS, 5% b-ME, and 0.01% bromophenol blue DSC Protein samples at a concentration of 1.5 mgỈmL)1 ( 25 lm tetramer) of hTTR, sbTTR and ecTRP were dialyzed against NaCl ⁄ Pi Prior to DSC experiments, the samples were vacuum degassed for 15 at room temperature DSC measurements were performed on a VP-DSC calorimeter (MicroCal, Inc., Northampton, MA, USA) at a heating rate of 10 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