Thermodynamic characterization of muscle tropomyosins from marine invertebrates

Comparative Biochemistry and Physiology, Part B 160 (2011) 64–71

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Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b

Thermodynamic characterization of muscle tropomyosins from marine invertebrates Hideo Ozawa, Shugo Watabe, Yoshihiro Ochiai ⁎, 1 Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo, 113-8657, Japan

a r t i c l e

i n f o

Article history: Received 11 March 2011 Received in revised form 12 June 2011 Accepted 15 June 2011 Available online 22 June 2011 Keywords: Circular dichroism Differential scanning calorimetry Thermostability Tropomyosin

a b s t r a c t Structural properties of invertebrate muscle tropomyosin (TM) have not been characterized in detail to date. TMs were thus purified from the mantle muscle of Japanese common squid Todarodes pacificus, the foot muscle of tokobushi abalone Haliotis diversicolor and the tail muscle of kuruma prawn Marsupenaeus japonicus, and investigated for their thermodynamic properties by circular dichroism (CD) spectrometry and differential scanning calorimetry (DSC). From the CD spectrometry data, the apparent melting temperature and the apparent free energy of unfolding at 20 °C were calculated to be 43.5 °C and 14.5 kJ/mol for the squid TM, 43.0 °C and 23.9 kJ/mol for the abalone TM, and 47.3 °C and 50.2 kJ/mol for the prawn TM, respectively. From the DSC data, the total free energy of unfolding at 20 °C was calculated to be 129 kJ/mol, 253 kJ/mol, and 271 kJ/mol for the squid, abalone, and prawn TMs, respectively. These results suggest that the thermal stability was in the order of prawn TM N abalone TM N squid TM. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Muscle tropomyosin (TM) consists of two parallel α-helical polypeptide chains of 284 residues with a molecular mass of about 33 kDa. TM polymerizes into a thread-like filament by head-to-tail interaction (Ooi et al., 1962; Sousa and Farah, 2002). Skeletal muscle TM is involved in the regulation of muscle contraction, by interacting with one troponin complex and seven actin molecules (McKillop and Geeves, 1993; Maytum et al., 1999; Gordon et al., 2000; Perry, 2001; Sakuma et al., 2006). TM is characterized by a coiled-coil structure formed by the knobsinto-holes packing at the interface between the two α-helices (Crick, 1953). In this structure, characteristic heptad repeats (designated abcdefg from the N-terminal side) are recognized. The a and d positions are frequently occupied by hydrophobic amino acids. At the e and g positions, ionic amino acids are mostly located, while the b, c and f positions tend to be occupied by hydrophilic amino acids. It has been reported that there is high correlation between the thermostability and the sum of the hydrophobic moments of the residues at the a and d positions (Greenfield

Abbreviations: CD, circular dichroism; Cp, denaturation heat capacity; DSC, differential scanning calorimetry; DTT, dithiothreitol; ΔGapp, the apparent free energy of unfolding at 20 °C; ΔGtotal, the total free energy of unfolding at 20 °C; ΔHn, enthalpy of unfolding for the “n”th transition; ΔHtotal, total enthalpy of unfolding; ΔSn, entropy of unfolding for the “n”th transition; TM, tropomyosin; TMn, midpoint of unfolding for the nth transition; TMapp, the apparent melting temperature. ⁎ Corresponding author. Fax: +81 54 337 0239. E-mail address: [email protected] (Y. Ochiai). 1 Present address: Department of Marine Science and Technology, Tokai University, Shizuoka 424-8610, Japan. 1096-4959/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2011.06.001

and Hitchcock-DeGregori, 1995). In addition, more than three consecutive residues in the a and d positions, consisting of Leu, Met, Ile, Val, Phe and Tyr, stabilize the coiled-coil (Kwok and Hodges, 2004). Marine invertebrate TMs have so far been purified from several species, namely, squid (Tsuchiya et al., 1980), scallop (Watabe and Hashimoto, 1980), sea urchin (Maekawa et al., 1989) and lobster (Miegel et al., 1992), whereas their deduced amino acid sequences are now available for several species such as mussel (Iwasaki et al., 1997), scallop (Hasegawa, 2001), and squid (Motoyama et al., 2006). Molluskan and arthropod muscle TMs, which comprise 284 amino acids like vertebrate muscle TMs, showed about 50% sequence identity with vertebrate TMs. Any crystal structure of invertebrate TM has not been determined yet, although only the low resolution of crystal structures of the full-length molecule (Whitby and Phillips, 2000) and the several regional structures of high resolution (Greenfield et al., 1998; Brown et al., 2001; Greenfield et al., 2003; Brown et al., 2005; Greenfield et al., 2006; Minakata et al., 2008) are available for vertebrate TMs. Compared to vertebrate TMs, marine invertebrate TMs showed high viscosity (Nishimura et al., 1997). Since the recombinant scallop TM, without N-terminal acetylation or a short peptide fused to the N-terminus, did not show high viscosity like vertebrate TMs, the high viscosity shown by marine invertebrate TMs would have originated from the stronger head-to-tail interaction (Inoue et al., 2004). TMs from invertebrate species are known as the major allergens of these animals, while TMs from vertebrate species are considered to be non-allergenic (Reese et al., 1999). Incidentally, the epitopes of brown shrimp Penaeus aztecus TM has been intensively studied (Ayuso et al., 2002a, 2002b), although the epitopes of invertebrate TMs are not necessarily common to each other. It has been reported that the stability against proteolytic digestion is a significant and valid indicator that distinguishes food

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allergens from non-allergens (Astwood et al., 1996), but such relationship is under debate for TMs (Fu et al., 2002; Taylor, 2003). Thermal denaturation profiles of proteins give crucial information for their structural stability closely related to their tertiary conformation. Especially in case of TMs, having quite a simple rod-like structure as described above, thermodynamic analysis is an excellent approach to understand the structural characteristics. The thermostability of vertebrate TMs has so far been measured for TMs from some species, such as walleye pollack, (Ochiai et al., 2003), bluefin tuna (Huang et al., 2004), white croaker, Atlantic salmon, pufferfish and zebrafish (Huang and Ochiai, 2005). In addition, by using five 30mer peptides, the regional stability differences of walleye pollack TM have been determined (Ozawa et al., 2009). Furthermore, the studies on fish and scallop TMs have suggested that the habitat temperature (or body temperature of poikilotherms) is not always correlated with the stability of TM (Huang and Ochiai, 2005; Ozawa et al., 2010). As described above, marine invertebrate TMs have unique properties unlike vertebrate counterparts. However, their thermodynamic properties have not been elucidated yet. Thus it would be of great interest to characterize the marine invertebrate TMs. In the present study, attempts were made to compare TMs from three marine invertebrate species based on the availability of amino acid sequences and easiness of live sample collection and protein purification. The purified TMs were investigated for their thermodynamic properties by circular dichroism (CD) spectrometry and differential scanning calorimetry (DSC). The results were discussed in relation with the differences in their primary structures and the thermodynamic properties of TMs studies to date by our group.

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gradient from 10 mM to 500 mM potassium phosphate buffer (pH 7.0) in the presence of 1 mM DTT. Fast skeletal muscle TM from fish (white croaker Pennahia argentata) was used as a control (Ochiai et al., 2001). The data of Yesso scallop Mizuhopecten yessoensis (scallop) and white croaker TMs were cited from the previous study (Ozawa et al., 2010). 2.2. CD spectrometry

2. Materials and methods

TMs were dissolved in and extensively dialyzed against 10 mM sodium phosphate buffer (pH 7.0) containing 0.1 M KCl, 0.1 mM DTT, and 0.001% NaN3. CD spectra were measured at 0.1 °C intervals ranging from 5 to 80 °C in the above medium with a J-720 spectropolarimeter (JASCO, Tokyo, Japan). A cuvette cell of 10 mm optical path length was used with constant N2 flux. Wavelength and protein concentration for measurement were 222 nm and 0.025 mg/ mL, respectively. α-Helical content was estimated assuming that the mean residue ellipticity at 222 nm, [θ]222, of poly L-glutamic acid is −36000 deg·cm2/ dmol when the substance is of completely helical structure (Yang et al., 1986; Wallimann et al., 2003). To compare the overall stability of TMs, two criteria were used; the apparent melting temperature (TMapp) and the apparent free energy of unfolding at 20 °C (ΔGapp). At TMapp, the value of [θ]222 corresponds to the midpoint between those at 5 and 80 °C. In the previous study, the apparent free energy of unfolding at 20 °C was calculated, and this free energy gave the negative value, although the absolute value is the same with ΔGapp. To evaluate ΔGapp, it was assumed that unfolding could be fit by a helix-coil transition. The CD spectrometry data, normalized to a scale of 0–1, were fit by the following equation (Greenfield and Hitchcock-DeGregori, 1995):

2.1. Animals and isolation of TM

θ = ε1 α1 + ε2 α2

Live specimens of Japanese common squid Todarodes pacificus (referred to as squid later), tokobushi abalone Haliotis diversicolor (abalone) and kuruma prawn Marsupenaeus japonicus (prawn) were purchased at the Tokyo Central Wholesale Market, and transported to our laboratory on ice. All the following procedures were carried out at 0–4 °C, unless otherwise stated. Unlike vertebrate TMs, it was essential to modify purification procedures depending on the species as described below to improve the purity and recovery of each TM. Acetone dried powder was prepared from mantle muscle of the squid, foot muscle of the abalone, and tail muscle of the prawn. Extraction of TM was performed as described previously (Huang et al., 2004; Ozawa et al., 2010). Further purification was performed as follows under the conditions optimized depending on the species. For the squid TM, anion exchange chromatography using a Mono Q 5/50 GL column (0.5×5 cm; GE Healthcare, Amersham, UK) was carried out. Elution was performed by a linear gradient from 150 mM to 490 mM KCl containing 50 mM potassium phosphate buffer (pH 7.0) and 1 mM dithiothreitol (DTT). Saturated ammonium sulfate solution (pH 7.0) was added to make 45% saturation for the subsequent chromatography. Hydrophobic interaction chromatography was carried out using a TSKgel BioAssist Phenyl column (0.78×5 cm; Tosoh, Tokyo, Japan), and proteins absorbed to the column were eluted by linearly decreasing the saturation of ammonium sulfate from 45% to 0% for 20 column volumes in the presence of 20 mM potassium phosphate buffer (pH 7.0). For the abalone TM, isoelectric precipitation and ammonium sulfate fractionation were repeated twice. Anion exchange chromatography was carried out under the same condition for the squid TM. For the prawn TM, hydrophobic interaction chromatography was carried out by linearly decreasing the saturation of ammonium sulfate from 40% to 0% for 20 column volumes in the presence of 20 mM potassium phosphate buffer (pH 7.0). Then, anion exchange chromatography was carried out again and proteins adsorbed to the column were eluted by a linear

ð1Þ

where α1 = 1 = ð1 + K1 Þ and α2 = 1 = ð1 + K2 Þ

ð2Þ

ε1 and ε2 are the extinction coefficients for the CD spectrometry associated with each transition. K1 = exp½ðΔH1 = RT ÞfðT = TM1 Þ–lg; K2 = exp½ðΔH2 = RT ÞfðT = TM2 Þ−lg ð3Þ To calculate the values of enthalpy and midpoint of unfolding for the 1st and 2nd transitions (ΔH1, ΔH2, TM1, and TM2), the initial values of these parameters were estimated, and the unfolding equations were fit using Microsoft Excel 2007. The values of entropy of unfolding for the 1st and 2nd transition (ΔS1 and ΔS2) were determined at TM1 and TM2 of each transition using the following relationship: ΔS1;2 = ΔH = TM1;M2

ð4Þ

The obtained values were then used to estimate ΔGapp using the following relationship: ΔGapp = ε1 ðΔH1 –293:15ΔS1 Þ + ε2 ðΔH2 –293:15ΔS2 Þ

ð5Þ

2.3. DSC Thermal denaturation profiles of TMs were also analyzed with a differential scanning microcalorimeter (MicroCal model VP-DSC, Northampton, MA, USA) applying the raising speed of temperature at 1 °C/min from 5 to 90 °C. Progress baseline mode was adopted, assuming that each point reflects the extent of progress of the reaction, and independent denaturation of each domain was adopted. DSC data were analyzed for determination of total transition number, N, thermodynamic parameter, TMn, and ΔHn (n = 1, 2, …, N), using a

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software package Origin developed by MicroCal. The measurement was performed three times successively for each sample, and the results are shown as the 1st, 2nd, and 3rd scans, respectively. The total free energy of unfolding at 20 °C (ΔGtotal) was calculated as follows; ΔGtotal = ∑ðΔHn –293:15ΔSn Þ

ð6Þ

2.4. Protein concentration determination and SDS-PAGE Protein concentration was determined by bicinchoninic acid (BCA) method (Smith et al., 1985) using bovine serum albumin as a standard. SDS-PAGE was performed according to Laemmli (1970) using 15% polyacrylamide slab gels. Protein molecular weight markers were purchased from Sigma Aldrich (SDS-7, St. Louis, MO, USA). 2.5. Bioinformatic analysis The amino acid sequence of TMs in the NCBI database were used: BAE54431 for the squid, AAG08987 for the abalone, BAF47263 for the prawn, BAB20881 for white croaker, BAA20455 for the scallop striated muscle, and BAB17857 for the scallop smooth muscle. Propensity for α-helix formation was used to estimate α-helix formation ability based on the amino acid sequence (Chou and Fasman, 1978). Hydrophobicity at the a and d positions on heptad repeat was estimated according to Kyte and Doolittle (1982). The secondary structure prediction was performed using the GOR IV program (Garnier et al., 1996). In this program, the predicted secondary structure is basically one of the highest probabilities, compatible with a predicted αhelix segment of at least four residues and a predicted extended segment of at least two residues. The average probability score for αhelix at each amino acid position was also considered. The prediction of coiled-coil score was performed using the COILS program (Lupas et al., 1991), with a window width of 14, MTIDK as a matrix, and no weighing of position. To calculate the correlation between the values obtained by DSC, CD, or bioinformatic analysis, the values of squid, abalone, prawn scallop (striated and smooth muscle), fish (white croaker) TMs were used. The correlation values from 0 to 0.2, from 0.2 to 0.4, from 0.4 to 0.7, from 0.7 to 0.9, and from 0.9 to 1.0 were regarded as very weak, weak, moderate, strong, and very strong, respectively. 3. Results and discussion 3.1. TM purification The squid and abalone TMs were obtained in the 50–70% and 45– 55% ammonium sulfate saturation fractions, respectively, while the prawn TM was obtained in the supernatant of 40% saturation. In case of the squid TM, KCl concentration of the crude extract was decreased to 0.1 M and centrifuged before isoelectric precipitation, in order to precipitate contaminating paramyosin. TMs from the squid, abalone, and prawn were successfully purified to homogeneity (Supplementary Fig. 1). Purification conditions were modified depending on the species because the same condition could not be applicable to all the TMs. The contaminating proteins were also found to be different among species. Since the purification was carried out only under mild conditions, the effects of different procedures on the stability of TMs were considered to be negligible. 3.2. CD spectrometry The values of TMapp and ΔGapp were calculated for the squid, abalone, and prawn TMs (Fig. 1, Table 1). The data of the scallop and white croaker TMs in Table 1 were cited from our previous study (Ozawa et al., 2010).

The α-helical contents at 5 °C and 80 °C were estimated to be 100% and 12% for the squid TM, 82% and 9% for the abalone TM, 85% and 10% for the prawn TM, respectively. The α-helical content of 100% at 5 °C for the squid TM might have been overestimated because TMs generally do not show perfect heptad repeats and some breakage of α-helix is contained (Whitby and Phillips, 2000). The value of −d (θcalculated)/dT gave the maximum, where α-helix contents decreased at the highest rate, namely, at 52.4 °C, 58.5 °C, and 48.2 °C for the squid, abalone, and prawn TMs, respectively. The value gave the maximum at 41.7 °C, 36.7 °C, 30.7 °C for the white croaker, scallop striated muscle TM, and scallop smooth muscle TMs, respectively (Ozawa et al., 2010). The temperatures, where the values gave the maximum, were around TM2 in good agreement with the fact that the second transitions of these TMs showed larger ΔH value than the first transition (Table 1). TMs showed multiple transitions in CD measurement, and it was difficult to evaluate the stability just by comparing the denaturation curves of the measurements. Thus, TMapp and ΔGapp were used to characterize in detail the thermostability differences of TMs. The thermostability of TM was determined by TMapp, and ΔGapp, when TMapp is comparable. The TMapp values suggested that the squid and abalone TMs have comparable stability, but slightly lower than that of the prawn TM. The difference in ΔGapp has revealed that the abalone TM is more stable than the squid TM mainly due to the higher ΔH2 value of the abalone TM. The stability is the prawn TM N abalone TM, white croaker TM N squid TM N scallop smooth muscle TM N scallop striated muscle TM. In the analysis for CD measurement, two transitions have been presumed with each ε value indicative of the α-helical fraction for unfolding. For the squid and abalone TMs, ε1 was larger than ε2, suggesting that the decrement of α-helical content in the lower temperature transition was much larger than that in the higher temperature transition (Table 1). The values of ΔH1 and ΔH2 for the abalone TM were comparable to those of the prawn TM, and TM1 and TM2 of the abalone TM showed higher values than those of the prawn TM. However, ΔGapp value of the prawn TM was much larger than that of the abalone TM. It was due to the large ε2 value and a drastic decrement of the normalized [θ]222 value at around 48 °C of the prawn TM (Fig. 1). 3.3. DSC In the case of squid TM, the values of total enthalpy of unfolding (ΔHtotal) were moderately reduced to 85.0% and 89.5% at the 2nd and 3rd scans, compared with that of the 1st scan (Table 2). In the case of abalone TM, the values of ΔHtotal were markedly reduced to 50.1% and 38.8% at the 2nd and 3rd scans, respectively (Table 2). In the case of prawn TM, the values of ΔHtotal were markedly reduced to 64.4% and 50.0% at the 2nd and 3rd scans, respectively (Table 2). The values of ΔHtotal for the scallop striated muscle TM were reduced to 93.0% and 87.5%, and those for the smooth muscle were reduced to 82.2% and 74.8% at the 2nd and 3rd scans, respectively (Ozawa et al., 2010). On the other hand, the values of ΔHtotal for white croaker TM were reduced to 87.1% and 77.4% at the 2nd and 3rd scans, respectively (Ozawa et al., 2010). For the squid, scallop, and white croaker TMs, the reduction of ΔHtotal in the 2nd and 3rd scans was low compared to the 1st scan, in contrast to the abalone and the prawn TMs. The abalone and prawn TMs showed higher value of ΔHtotal in the 1st scan compared to the other TMs in the present study as well as in our previous study (Ozawa et al., 2010). It is likely that the higher values of ΔHtotal in the 1st scan resulted in the marked reduction in the 2nd and 3rd scans. The value of ΔHtotal would reflect the degree of hydrophobic core packing, which reflects the inaccessibility of water molecules. Thus, the hydrophobic cores of prawn and abalone TMs seemed to be tightly packed, and this packing was not restored properly even after cooling. Compared to the 3rd scans of these TMs,

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Fig. 1. Circular dichroism (CD) spectrometry analysis on the temperature dependent unfolding of tropomyosins from squid, abalone, and prawn. In left panels, [θ]measured (dot) was obtained by normalizing CD spectrometry data; in right panels, [θ]calculated (line) was obtained based on the analysis. Two independent helix-coil transitions were assumed.

the ΔHtotal in the 1st scans was more variable, although the physiological importance of this phenomenon is to be explained. In the case of squid TM, 4 peaks were observed in all the scans (Fig. 2, Table 2). In the case of abalone TM, 7, 5, and 5 peaks were obtained in the 1st, 2nd, and 3rd scans, respectively (Table 2). In the case of prawn TM, 7, 5, and 4 peaks were obtained in the 1st, 2nd, and 3rd scans, respectively (Fig. 2, Table 2). The scallop striated and smooth muscle TMs exhibited 5 and 4 peaks, respectively, in all the scans (Ozawa et al., 2010). On the Table 1 Estimation of the thermodynamic parameters for the unfolding of tropomyosins based on the ellipticity at 222 nm as a function of temperature. Species

ΔH1 ΔH2 TM1

TM2

ε1

ε2

ΔGapp TMapp

Squid Abalone Prawn Scallop (striated muscle)a Scallop (smooth muscle)a Fish (white croaker)a

103 126 110 146 136 84

53.1 58.4 48.2 30.7 36.4 41.7

0.610 0.799 0.347 0.395 0.281 0.506

0.390 0.201 0.653 0.605 0.719 0.494

14.5 23.9 50.2 13.4 31.9 24.1

272 751 850 545 808 640

38.5 40.1 32.5 29.3 28.3 37.3

43.5 43.0 47.3 30.5 36.0 41.2

ΔH1 and ΔH2 are the enthalpies in kJ/mol for unfolding of the helix-coil transition. TM1 and TM2 are the observed midpoints (°C) of corresponding transition. The symbols ε1 and ε2 are the fractions of the tropomyosin molecule folded in corresponding transition, ΔGapp is the apparent free energy for unfolding at 20 °C and TMapp is the temperature (°C) at which the ellipticity, normalized to a scale of 0–1, is equal to 0.5. a The values of scallop and white croaker tropomyosins were cited from Ozawa et al. (2010).

other hand, white croaker TM showed 5, 5, and 4 peaks in the 1st, 2nd, and 3rd scans, respectively (Ozawa et al., 2010). The decrease in number of the peak in the 2nd and 3rd scans, which were observed except for the squid and the scallop TMs, suggest that the hydrophobic core in some regions could not be refolded properly or some regions were non-specifically bound after heat denaturation. The temperature with the maximum denaturation heat capacity (Cp) value is shown in Table 2. The scallop striated muscle TM gave the maximum values at 28.5 °C, 28.7 °C and 28.2 °C in the 1st, 2nd and 3rd scans, respectively, whereas the smooth muscle TM did at 35.4 °C, 35.2 °C and 35.2 °C in the 1st, 2nd and 3rd scans, respectively (Ozawa et al., 2010). On the other hand, white croaker TM gave the maximum value at 42.8 °C, 42.7 °C and 42.2 °C for the 1st, 2nd and 3rd scans, respectively (Ozawa et al., 2010). The values of ΔGtotal in the 1st, 2nd and 3 rd scans for the squid, abalone and prawn are shown in Table 2. The ΔGtotal in the 1st, 2nd and 3 rd scans were calculated to be 72, 66 and 60 kJ/mol for the scallop striated muscle TM, 110, 90 and 79 kJ/mol for the scallop smooth muscle TM, and 139, 125 and 116 kJ/mol for white croaker TM in our previous study (Ozawa et al., 2010). Based on ΔGtotal in the 1st scan, the stability is thought to be in the order of the prawnN the abaloneN white croakerN the squidN the scallop smooth muscleN the scallop striated muscle TMs. In relation with these multiple transition steps in thermal denaturation of TMs, Potekhin and Privalov (1982) showed that TM contains seven cooperative blocks. Each block is considered to show a different thermal denaturation pattern.

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Table 2 Estimation of the thermodynamic parameters for the unfolding of squid, abalone, and prawn tropomyosins based on DSC as a function of temperature. Squid

ΔGtotal (kJ/mol) ΔHtotal (kJ/mol) Temperature (°C) at Cp maximum TM1 (°C) ΔH1 (kJ/mol) TM2 (°C) ΔH2 (kJ/mol) TM3 (°C) ΔH3 (kJ/mol) TM4 (°C) ΔH4 (kJ/mol) TM5 (°C) ΔH5 (kJ/mol) TM6 (°C) ΔH6 (kJ/mol) TM7 (°C) ΔH7 (kJ/mol)

Abalone

Prawn

1st scan

2nd scan

3rd scan

1st scan

2nd scan

3rd scan

1st scan

2nd scan

3rd scan

129 1.44 × 103 51.4 37.9 301 44.6 214 51.2 518 57.5 404 – – – – – –

109 1.22 × 103 51.1 38.0 195 41.9 249 50.8 445 57.8 332 – – – – – –

112 1.29 × 103 50.9 35.0 211 43.0 298 50.6 440 57.8 337 – – – – – –

253 3.15 × 103 56.3 32.0 343 36.2 431 40.5 435 44.8 469 49.8 453 54.1 395 56.9 620

134 1.58 × 103 56.0 35.1 317 43.0 352 50.2 405 56.0 502 – – – – – –

106 1.22 × 103 55.6 36.0 228 43.1 224 50.1 325 55.5 444 – – – – – –

271 3.62 × 103 47.5 30.4 341 35.2 418 39.7 428 44.7 491 47.5 633 47.8 920 53.4 387

186 2.33 × 103 47.4 34.2 266 40.1 272 52.8 362 45.8 540 47.7 885 – – – –

152 1.84 × 103 47.3 36.6 223 45.2 467 47.5 814 52.8 332 – – – – – –

–, Not determined.

3.4. The comparison in the thermodynamic parameter between CD spectrometry and DSC It is likely that, for proteins like TM, second structures are critical for their overall structures. The thermal stability parameters of TM (ΔGapp, TMapp, and ΔGtotal) and predictions (helix propensity, helical score, and coiled-coil score) seem to be correlated as shown in Tables 3 and 4. The temperature with the maximum value of −d(θcalculated)/dT by CD spectrometry was in good agreement with the maximum Cp value

obtained by DSC, as the cases of the scallop and white croaker TMs (Ozawa et al., 2010). Although the values of ΔHtotal obtained by the 1st scan of the CD spectrometry and DSC in the present and previous studies (Ozawa et al., 2010) showed strong correlation (correlation coefficient being 0.77), ΔHtotal values by DSC were much larger than those by CD spectrometry. This might have originated from the difference in detectable numbers of transition unit; namely, two or three by CD spectrometry, while more by DSC measurement. The values of ΔGtotal by DSC also showed strong

Fig. 2. DSC scans of tropomyosins from squid, abalone, and prawn. The subsequent deconvolution analysis and the sum of subsequent analysis are shown with thin and thick lines, respectively.

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Table 3 The thermostability and secondary structure predictions of tropomyosins. Species

ΔGapp (kJ/mol) by CD

TMapp (°C) by CD

ΔGtotal (kJ/mol) by DSC

Helix propensitya

Hydrophobicityb

Helical scorec

Coiled-coil score (%)d

Squid Abalone Prawn Scallop (striated muscle)e Scallop (smooth muscle)e Fish (white croaker)e

14.5 23.9 50.2 13.4 31.9 24.1

43.5 43.0 47.3 30.5 36.0 41.2

129 253 271 72 110 139

1.16 1.17 1.16 1.14 1.15 1.18

2.06 1.97 2.01 2.05 1.97 2.06

702 728 755 678 710 753

83.8 86.6 89.2 85.6 86.9 86.9

a b c d e

Chou and Fasman (1978). Per residue. Kyte and Doolittle (1982). At the a and d residues and per residue. Calculated by GOR IV. Estimated by COILS. The values of scallop and white croaker tropomyosins were cited from Ozawa et al. (2010).

correlation with the values of TMapp and moderate one with ΔGapp obtained by CD spectrometry (Tables 3, 4). The order of TM stability estimated by CD and DSC did not show any discrepancy, although the stability difference between the squid and white croaker TMs was not detectable by CD. 3.5. Primary structures associated with molecular phylogeny and αhelical structures Although the amino acid identity among vertebrate muscle TMs or among Yesso scallop TM isoforms was above 90% (Huang and Ochiai, 2005; Ozawa et al., 2010), the identity among TMs in the present experiment was low (60–80%) (Fig. 3). To obtain the relationship between amino acid replacement and stability, some bioinformatical analyses were performed as described below. Helix propensity showed marginal variance among the species, although it showed moderate correlation with ΔGtotal and TMapp, but very weak one with ΔGapp (Table 4). It has been reported that the thermal stability can be estimated by the hydrophobicity at the a and d positions (Greenfield and Hitchcock-DeGregori, 1995). But, hydrophobicity per residue at the a and d positions of heptad repeat showed a very weak negative correlation with TMapp and moderate negative correlation with ΔGapp and ΔGtotal (Table 4). They used many chimera TMs and evaluated the effect of the residues at the a and d positions of exons to the stability, but, in the present study, TMs has little relevancy in the sequence for each other, partly because of low amino acid identity. The ratio of α-helical residues predicted by GOR IV was 88.4%, 88.7% and 89.1% for the squid, abalone and prawn TMs, respectively. The values were 87.3%, 90.9% and 92.2% for the scallop striated muscle, the scallop smooth muscle and white croaker TMs, respectively (Ozawa et al., 2010). The value is not a good parameter for the thermostability, because this showed only a weak or very weak correlation with ΔGapp (0.28), TMapp (0.17) and ΔGtotal (−0.02). But, the helical scores (the full score being 1000) averaged for each amino acid by GOR IV (Table 3) showed strong correlation with ΔGapp, TMapp, and ΔGtotal (Table 4).

The coiled-coil formation (%) estimated by COILS showed very strong correlation with ΔGapp and moderate one with ΔGtotal, but weak one with TMapp (Table 4). In addition, this value showed a strong correlation with ΔHtotal by CD (0.88) and DSC (0.82). From the correlations, helical scores by GOR IV and coiled-coil scores by COILS seemed to be good prediction parameters of thermal stability (Table 4). On the other hand, helix propensity and hydrophobicity were much less reliable, because helix propensity showed marginal variation (Table 3) and hydrophobicity showed a negative correlation with the set of thermostability index as obtained by CD and DSC (Table 4). 3.6. Amino acid residues involved in structural stability The relationships between the stability parameters of TM (ΔGapp, TMapp, and ΔGtotal) and their structural predictions (helix propensity, helical score, and coiled-coil score) are shown in Table 4. These predictions were then used for determination of the amino acid residue replacements responsible for the thermostability difference. The amino acid residues involved in the thermostability difference of TMs among species were presumed (Fig. 3). By the program GOR IV or COILS, not only the N- and C-termini regions but also the regions around the 130th residue of vertebrate and marine invertebrate TMs seemed to be unstable. At around the 50th residue, the helical score by GOR IV was low except for the prawn TM. This suggests that the high stability of the prawn TM might be partly due to the sequence at around this residue. Furthermore, there are many amino acid replacements around the 50th–53rd residues. The helical propensity in this region was not so small for the prawn TM (Supplementary Table 1). Thus, the substitutions in this region would stabilize the prawn TM. GOR IV analysis further revealed that the substitution at the 50th residue affects the stability difference (Supplementary Table 2). The substitution at the 51st residue would affect the difference in the stability between the prawn and other invertebrate TMs, except for the abalone counterpart. The substitution at the 52nd residue would increase the stability of the prawn, compared to the other vertebrate or invertebrate TMs. The substitution at the 53rd residue to Leu would

Table 4 Correlation between the thermostability and secondary structure prediction of tropomyosins.

TMapp (°C) by CD ΔGtotal (kJ/mol) by DSC Helix propensitya Hydrophobicityb Helical scorec Coiled-coil score (%)d

ΔGapp (kJ/mol)

TMapp (°C)

ΔGtotal (kJ/mol)

Helix propensitya

Hydrophobicityb

Helical scorec

0.55 0.68 0.13 − 0.43 0.71 0.91

0.82 0.68 − 0.06 0.76 0.33

0.50 − 0.43 0.71 0.62

0.13 0.78 0.17

− 0.10 − 0.42

0.71

To calculate correlations, the values of scallop and white croaker tropomyosins (Ozawa et al., 2010) were used. a Chou and Fasman (1978). Per residue. b Kyte and Doolittle (1982). At the a and d residues and per residue. c Calculated by GOR IV. d Estimated by COILS.

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Fig. 3. Amino acid sequence alignment of tropomyosin. Accession numbers are: rabbit (AAB34957), white croaker (BAB20881), Japanese common squid (BAE54431), tokobushi abalone (AAG08987), kuruma prawn (BAF47263), Yesso scallop Mizuhopecten yessoensis striated muscle (BAA20455) and smooth muscle (BAB17857) tropomyosins. The positions where the helical scores by GOR IV are lower than 0.6 are underlined. The positions where the coiled-coil scores by COILS are lower than 0.8 are shaded. The heptad repeats (abcdefg) are indicated in the first lines. Abbreviations: st, striated muscle; sm, smooth muscle. Identical residues with that of rabbit tropomyosin are indicated by dots.

also increase the stability of TM. In addition, it is possible that the high stability of the prawn TM is related with a higher coiled-coil score around the 165th and 215th residues compared to the squid or scallop striated muscle TMs and the other invertebrate TMs, respectively. At around the 165th residue, only the squid and scallop striated muscle TMs, which had lower values of ΔGapp, gave very low coiledcoil scores, and both TMs have Phe instead of Tyr at the 162nd residue. The substitution of Phe by Tyr at the 162nd residue in the squid and scallop striated muscle TMs increased coiled-coil scores by 0.9% and 0.5%, respectively, although the difference in the side chains between these two amino acids is only the phenolic hydroxyl group in Tyr. The helical scores by GOR IV suggest that the region around the 200th residue of invertebrate TMs is in a random coil state, which may be partly due to the substitution of Thr by Gly at the 201st residue. The prawn TM showed a high coiled-coil score at around the 215th residue, compared to the other invertebrate TMs. Since there are other substitutions in this region among marine invertebrate TMs, it was difficult to pinpoint all the residues responsible for the thermal stability differences. In conclusion, TMs from three marine invertebrate species were thermodynamically characterized in the present study. This is the first report on the detailed thermal denaturation profiles of invertebrate TMs. It is very unlikely that the animals in nature experience such high temperatures as applied in the present study. The comparative study of the thermal stability differences of TMs would be important to know the effect of sequence differences and amino acid replacement(s) on the structural stability differences of TMs. Thermal stability differences of TMs could reflect their physiological ones, but at the moment, it is difficult to relate these differences. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (KAKENHI #19380119 to Y.O.).

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