Thermal unfolding of smooth muscle and nonmuscle tropomyosin a-homodimers with alternatively spliced exons Elena Kremneva 1 , Olga Nikolaeva 2 , Robin Maytum 3 *, Alexander M. Arutyunyan 2 , Sergei Yu. Kleimenov 1 , Michael A. Geeves 3 and Dmitrii I. Levitsky 1,2 1 A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia 2 A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia 3 Department of Biosciences, University of Kent at Canterbury, UK Tropomyosins (Tm) are a family of actin-binding, a-helical coiled-coil proteins found in most eukaryotic cells [1]. They bind to actin cooperatively along the length of actin filaments and confer cooperativity upon the interaction of actin with myosin heads [2]. The Tm molecules are parallel homo- or hetero- dimers (encoded from the same or different genes) of two a-helical chains of identical length, although the length can vary according to isoform type. In mam- malian cells, alternative splicing produces a variety of muscle and nonmuscle isoforms from four differ- ent genes [1]. Muscle cells express two major iso- forms of Tm (a and b), each containing 284 residues. Smooth and skeletal muscles express differ- ent isoforms resulting from alternative splicing of the a and b genes. There are two major classes of a-Tm: long (or high relative molecular mass) Tm (284 residues) are expressed in muscle and nonmuscle cells whereas short (or low relative molecular mass) Tm (247 residues) are Keywords tropomyosin; actin; thermal unfolding; differential scanning calorimetry; circular dichroism Correspondence D.I. Levitsky, A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prosp. 33, 119071 Moscow, Russia Fax: +7095 9542732 E-mail: levitsky@inbi.ras.ru *Present address School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK (Received 14 September 2005, revised 2 December 2005, accepted 6 December 2005) doi:10.1111/j.1742-4658.2005.05092.x We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of a-tropomyosin (Tm) in comparison with that of smooth muscle Tm. These two nonmuscle Tm isoforms 5a and 5b differ internally only by exons 6b ⁄ 6a, and they both differ from smooth muscle Tm by the N-terminal exon 1b which replaces the muscle-specific exons 1a and 2a. We show that the presence of exon 1b dramatically decreases the measurable calorimetric enthalpy of the thermal unfolding of Tm observed with DSC, although it has no influence on the a-helix content of Tm or on the end-to- end interaction between Tm dimers. The results suggest that a significant part of the molecule of fibroblast Tm (but not smooth muscle Tm) unfolds noncooperatively, with the enthalpy no longer visible in the cooperative thermal transitions measured. On the other hand, both DSC and CD stud- ies show that replacement of muscle exons 1a and 2a by nonmuscle exon 1b not only increases the thermal stability of the N-terminal part of Tm, but also significantly stabilizes Tm by shifting the major thermal transition of Tm to higher temperature. Replacement of exon 6b by exon 6a leads to additional increase in the a-Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle a-Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule. Abbreviations CD, circular dichroism; DSC, differential scanning calorimetry; Tm, tropomyosin; smTm, recombinant smooth muscle Tm; Tm5a and Tm5b, recombinant fibroblast Tm isoforms with alternatively spliced exons 6b and 6a, respectively. 588 FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS found in nonmuscle cells [1,2]. In the short a-Tm iso- forms, a single exon (exon 1b, encoding residues 1–43) replaces the first two exons (exons 1a and 2 encoding residues 1–80) in long a-Tm (see Fig. 1). The two other alternatively spliced exons in a-Tm are exons 6 and 9. Possible relationships between the alternatively spliced exons and the functional properties of the a-Tm iso- forms have been addressed in previous studies. The actin binding properties are mainly determined by the two terminal regions, encoded by exons 1 and 9 [3]. Amino acid replacements in the region encoded by exon 2 of lobster muscle Tm altered end-to-end inter- action between Tm molecules and actin affinity of Tm [4]. The replacement of muscle-specific exon 6b by nonmuscle exon 6a in recombinant rat smooth muscle a-Tm was shown to increase the actin affinity of a-Tm [5], and the same exon exchange has a similar effect between fibroblast Tm5a and 5b isoforms [6]. This replacement in rat fibroblast a-Tm has been shown to increase the calcium sensitivity of the regulation of acto-myosin interaction in the presence of troponin [7]. Our aim in the present study was to determine how the alternatively spliced exons 1, 2, and 6 affect the structural properties of the a-Tm molecule. For this purpose, we have used the smooth muscle a-Tm (smTm) and the two a-Tm fibroblast isoforms Tm5a and Tm5b, as shown in Fig. 1. All of these a-Tm iso- forms have a smooth muscle-like exon 9d. Both of the fibroblast isoforms are shorter than smTm because they lack exon 2 due to replacement of muscle exons 1a and 2a by nonmuscle exon 1b. The short fibroblast isoforms Tm5a and Tm5b differ internally only by exon 6. Confusingly, Tm5a has exon 6b, whereas Tm5b possesses exon 6a (Fig. 1). Thus, comparison of Tm5a with smTm allows study of the effects of replacement of muscle exons 1a and 2a by nonmuscle exon 1b, and comparison of Tm5a with Tm5b allows the effects of exchange of exon 6 to be studied. Studies of thermal unfolding of Tm may provide valuable information on the structure of Tm both free in solution and bound to actin. Thermal unfolding of the Tm coiled-coil can be successfully studied by differ- ent methods such as CD, fluorescence, and DSC. Many authors have used DSC for detailed investiga- tion of the thermal unfolding of Tm from skeletal and smooth muscles [8–14]. Other authors successfully used CD to study the thermal unfolding of homo- and het- erodimers of skeletal [15,16] and smooth [17–19] muscle Tm, their mutants [20–23], and numerous coiled-coil model peptides corresponding to the N- and C-terminal parts of the Tm molecule [24–29]. CD measures whole the process of the unfolding of a-helical coiled-coil of Tm, whereas DSC generally gives reliable information only on the thermal unfolding of those parts of Tm which melt cooperatively with significant changes in enthalpy. On the other hand, DSC is capable of monitoring the thermal unfolding of Tm when bound to actin [13,14,30], whereas CD is of limited use in the presence of actin as the signal from the six- or sevenfold molar excess of actin dominates the signal. Fluorescent labels can also be used in the presence of actin but may report only the local unfolding of regions close to the label. Thus, each method has strong and weak sides, but combination of both the DSC and CD provides a powerful approach for structural characteri- zation of Tm and its interaction with actin. In this work we have used DSC and CD to character- ize the thermal unfolding of smTm, Tm5a, and Tm 5b. We have shown that exon replacements alter the stabil- ity of the entire Tm molecule. The replacement of mus- cle exons 1a and 2a in smTm by nonmuscle exon 1b in Tm5a (and Tm5b) dramatically decreases the total measurable calorimetric enthalpy of the thermal unfold- ing of Tm, although it has no influence on the a-helix content of Tm and on end-to-end interaction between Tm dimers. On the other hand, this replacement signifi- cantly stabilizes Tm, increasing the temperature of the cooperative thermal transitions of Tm. Replacement of exon 6b in Tm5a by exon 6a in Tm5b leads to addi- tional increase in the thermal stability of Tm. Results Thermal unfolding of recombinant Tm: DSC studies The excess heat capacity curves obtained for recombin- ant smTm, Tm5a, and Tm5b are presented in Fig. 2. Judging by the complete reproducibility of the calori- metric traces after cooling the sample within the DSC cell, the heat-induced unfolding of Tm was fully reversible. For both fibroblast Tms the major trans- ition takes place at a higher temperature than for Fig. 1. Exon structure of smooth muscle a-Tm and the two fibro- blast isoforms Tm5a and Tm5b with constitutive exons are shown in white, smooth muscle exons in light grey, and nonmuscle exons in dark grey. E. Kremneva et al. Thermal unfolding of nonmuscle tropomyosin FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS 589 smTm (T m ¼ 34.6 °C), by  5 °C for Tm5a (T m ¼ 39.3 °C) and by  8 °C for Tm5b (T m ¼ 42.7 °C). Surprisingly, the calorimetric enthalpy, DH cal , of the thermal unfolding of both Tm5a and Tm5b was much less than that for smTm, and it represented only  60% of the DH cal value for smTm (Table 1). Another difference between Tm5a, Tm5b and smTm is that both fibroblast Tm isoforms possess, in addition to the major sharp thermal transition and the shoulder at 25–35 °C, a broad low-cooperative transition at 55– 65 °C (Fig. 2). It is important to note that it was diffi- cult to reveal this broad high-temperature transition in our apparatus as it was small and difficult to distin- guish from the instrumental baseline. Therefore we used a specially developed method to avoid artefacts caused by subtraction of the instrumental baseline (see Experimental procedures). The heat capacity curves obtained in this way were subjected to deconvolution analysis (Fig. 3), which shows that the profiles can be decomposed into three separate thermal transitions (calorimetric domains). The first, the low-temperature transition at  33 °C (Fig. 3) represents 15–20% of the total calorimetric enthalpy (Table 1). The second transition, which repre- sents more than 60% of the total enthalpy, is similar in enthalpy but more stable in Tm5b (T m ¼ 42.7 °C) than in Tm5a (T m ¼ 39.3 °C). The third transition again represents  20% of the total enthalpy, and it is very similar for Tm5a and Tm5b (T m  57.5 °C) (Fig. 3, Table 1). Since the only major difference between the two Tms is in transition 2 this is likely to represent the unfolding of the region of Tm containing the alternately spliced exon 6. The heat capacity curve for smTm also reveals three transitions. However, the first and third transitions are broader and are not as clearly resolved from the second, main transition. SmTm and Tm5a are identical in struc- ture except for the alternative exons 1 and 2 therefore differences in the unfolding thermogram should reflect the role of these exons in thermal stability. As seen from Table 1 all three transitions for smTm are at a lower temperature than for Tm5a (by 2, 4.7, and 18.4 °C, respectively) while the total enthalpy of smTm unfolding is 1.6 times that of Tm5a. Note however, that the parti- tion of the total enthalpy between the three transitions remains at approximately 20, 60, and 20%, respectively. The major difference between the thermal unfolding of smTm and Tm5a is on the T m of the third trans- ition which is destabilized by almost 20 °C. It is there- fore most likely to reflect the N-terminal part of the molecule dominated by the exchange of exon 1b for exons 1a and 2a. However all three calorimetric domains are destabilized by the exon change and the total enthalpy is increased. This suggests that the N-terminal part of the molecule is affecting the stabil- ity of the entire molecule. End-to-end interaction One possible reason for the differences in the thermal stability between smTm and Tm5a is that it is related Fig. 2. Temperature dependence of the excess heat capacity (C p ) of smTm, Tm5a, and Tm5b. The protein concentration was 1.2 mgÆmL )1 . Other conditions: 30 mM Hepes pH 7.3, 100 mM KCl, and 1 m M MgCl 2 . The heating rate was 1 KÆmin )1 . Table 1. Calorimetric parameters obtained from the DSC data for individual thermal transitions (calorimetric domains) of smTm, Tm5a, and Tm5b. The parameters were extracted from Fig. 3. The error of the given values of transition temperature (T m ) did not exceed ± 0.2 °C. The relative error of the given values of calorimetric enthalpy, DH cal , did not exceed ± 8%. Sample Transition 1 Transition 2 Transition 3 Total DH (kJÆmol )1 )DH (kJÆmol )1 ) T m (°C) DH (kJÆmol )1 ) T m (°C) DH (kJÆmol )1 ) T m (°C) smTm 150 30.9 515 34.6 125 39.0 790 Tm5a 100 32.9 310 39.3 85 57.4 495 Tm5b 75 33.5 300 42.7 110 57.9 485 Thermal unfolding of nonmuscle tropomyosin E. Kremneva et al. 590 FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS to different end-to-end interactions of the Tm species. Indeed, Tm5a not only differs from smTm by the sequence encoded by the N-terminal exon 1 (Fig. 1), but also because the recombinant smTm was expressed with an N-terminal Ala-Ser extension to substitute for the N-terminal acetylation of the native Tm [31]. The fibroblast Tm5a (and Tm5b) has a natural five-amino acid extension in exon 1b (Ala-Gly-Ser-Ser-Ser) in comparison to exon 1a [6,7,32]. These differences in the sequence might affect end-to-end interaction between Tm dimers and could influence the observed enthalpy of the thermal unfolding. The strength of the end-to-end interaction between Tm dimers can be estimated by viscometry, and the interaction is known to be highly sensitive to ionic strength [17,33]. We measured the viscosity and calori- metric enthalpy (total D H cal ) of smTm and Tm5a at dif- ferent ionic strengths as shown in Fig. 4. The relative viscosity of Tm5a was very similar to that of smTm at all ionic strengths (Fig. 4A). At 500 mm KCl the visco- sity was similar to that of water consistent with the absence of any significant polymerization of Tm. Over the range of ionic strengths studied, the DH cal value for Tm5a was consistently smaller by 40–50% than that of smTm (Fig. 4B). This means that end-to-end inter- actions of Tm play little role in the difference in the calorimetric enthalpy between smTm and fibroblast Tm5a. Thermal unfolding of recombinant Tm: CD studies The CD spectra of smTm, Tm5a, and Tm5b, which are shown in Fig. 5, possessed the double minima at 208 and 222 nm characteristic of the correctly folded, fully a-helical structure of Tm. The CD spectra of Tm5a and Tm5b were virtually identical to each other and to the spectrum of smTm. Fig. 3. Deconvolution analysis of the heat sorption curves of smTm (A), Tm5a (B), and Tm5b (C). Conditions were the same as in Fig. 2. Solid lines represent the experimental curves after subtrac- tion of instrumental and chemical baselines, and dotted lines repre- sent the individual thermal transitions (calorimetric domains) obtained from fitting the data to the nontwo-state model. Fig. 4. Effects of ionic strength on the relative viscosity (A) and calorimetric enthalpy DH cal (B) of smTm and Tm5a. The concen- tration of Tm was 0.5 mgÆmL )1 in viscosity experiments and 1.2 mgÆmL )1 in DSC experiments. E. Kremneva et al. Thermal unfolding of nonmuscle tropomyosin FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS 591 The thermal unfolding of the a-helix in smTm, Tm5a, and Tm5b was measured by the temperature dependence of the CD elipticity at 222 nm (Fig. 6). These CD studies were performed under similar condi- tions and at the same heating rate (1 °CÆmin )1 ) as the DSC measurements except that lower protein concen- tration (0.1 mgÆmL )1 ) and sodium phosphate buffer was used instead of Hepes. However, the buffer replacement had no significant influence on the ther- mal unfolding of any Tm measured by DSC (data not shown). Melting was fully reversible with a repeated melting curve being identical to the initial ones. The helix unfolding profile of recombinant smTm (Fig. 6A) agrees with previous CD studies of the native aa-homo- dimers of smooth muscle Tm [18,19]. The transition midpoint for smTm ( 34 °C) is similar to the max- imum temperature ( T m ¼ 34.6 °C) of the heat capacity curve measured by DSC under similar conditions (Fig. 2). The CD melting curve of Tm5a and 5b are similar and differ significantly from that for smTm, having broader (lower cooperativity) changes in ellipticity (Fig. 6A). The first derivative of the data, dh ⁄ dt, shows three well distinguished peaks on the profile of Tm5a (Fig. 6B), with the major peak at 39.6 °C and two small peaks at  30 °C and  50 °C similar to those observed in DSC. The CD melting profile of Tm5b also shows, like Tm5a, significant changes in ellipticity long before and well after the major peak at 41.2 °C (Fig. 6B). In general, these CD data are in very good agreement with DSC results presented above (Figs 2 and 3). Both DSC and CD show that the magnitude of the main thermal transition is much higher for smTm than for Tm5a and Tm5b. Thus, both methods show a marked difference in the thermal unfolding between smooth and nonmuscle Tms. DSC studies of the thermal unfolding of Tm in the presence of F-actin Previous studies have shown that DSC can be success- fully used for studies of the actin-induced changes in the thermal unfolding of Tm [13,14,30]. Interaction of smooth muscle Tm with F-actin caused a 2–6 °C shift in the Tm thermal transition to higher temperature, depending on the Tm : actin molar ratio [13]. In the present work, we apply this approach to characterize the thermal unfolding of smTm, Tm5a, and Tm5b complexed with F-actin. DSC experiments with Tm–F-actin complexes were performed in the presence of excess of Tm, i.e. under conditions where actin filaments should be fully satur- ated with Tm molecules. The character of the thermal Fig. 5. CD spectra in the far-UV region of smTm, Tm5a, and Tm5b. The spectra were measured at 20 °C. Protein concentration was 0.1 mgÆmL )1 in all cases. Other conditions: 50 mM sodium phos- phate buffer pH 7.3 containing 100 m M NaCl and 1 mM MgCl 2 . Fig. 6. The thermal unfolding profiles of smTm, Tm5a, and Tm5b as measured by CD. (A) The temperature dependence of a-helix content measured as the ellipticity at 222 nm. The heating rate was 1 KÆmin )1 . The protein concentration was 0.1 mgÆmL )1 in all cases. The heating rate was 1 KÆmin )1 . Other conditions: 50 mM sodium phosphate buffer pH 7.3 containing 100 mM NaCl, 1 mM MgCl 2 and 1 mM b-mercaptoethanol. (B) First derivative profiles for the data shown in (A). Thermal unfolding of nonmuscle tropomyosin E. Kremneva et al. 592 FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS denaturation of Tm was noticeably changed when bound to F-actin. This is reflected in the appearance of a new highly cooperative thermal transition at higher temperature (Fig. 7). The interaction of Tm with actin had no effect on the thermal denaturation of F-actin stabilized by phalloidin, which denatures irreversibly at much higher temperature (80 °C), as was previously shown for smooth and skeletal muscle Tms [13,14]. Thus, after heating of the Tm–F-actin complex to 90 °C (i.e. after complete irreversible dena- turation of actin) and subsequent cooling, only the peaks corresponding to the thermal denaturation of free Tm were observed during a second heating (dashed line curves on Fig. 7). Thus, we conclude that the appearance, in the presence of F-actin, of new peak at higher temperature reflects the actin-induced changes in the thermal unfolding of Tm. This peak on Fig. 7 is named as peak 2, whereas the peak named as peak 1 corresponds to the thermal unfolding of non- actin-bound Tm in the presence of F-actin, and peak 3 corresponds to the thermal unfolding of free Tm dur- ing re-heating after complete irreversible denaturation of actin. It should be noted that in this case we did not analyse the small thermal transitions of Tm5a and Tm5b at  57.5 °C (Figs 2 and 3), because irreversible thermal denaturation of F-actin began in this temper- ature range. In the case of smTm, the actin-induced shift in the thermal transition (i.e. the difference in T m between peak 2 and peak 3) was equal to 3.8 °C, while this effect was less pronounced for Tm5a (T 2 ) T 3 ¼ 3 °C) and Tm5b (T 2 ) T 3 ¼ 1.4 ° C) (Table 2). However, it is clearly seen on Fig. 7 that the actin-induced increase in the enthalpy of thermal unfolding of Tm5a and Tm5b is more pronounced than for smTm. Indeed the enthalpy of the actin-induced peak 2 (DH 2 ) for Tm5a is now almost exactly six-sevenths of the value for smTm consistent with similar enthalpy of unfolding per unit length. To calculate the actin-induced increase in enthalpy we measured the enthalpy of the actin- induced peak 2 (DH 2 ) and determined the difference between DH 2 and the enthalpy of free Tm (DH 3 ) with the enthalpy of peak 1 subtracted (DH 3 ) DH 1 ) (Table 2). (It is noteworthy that in the absence of actin the enthalpy of reversible unfolding of Tm did not sig- nificantly change even after heating to 90 °C). As a result, interaction with F-actin increased the enthalpy of the thermal unfolding by 220–240 kJÆmol )1 for Fig. 7. Thermal denaturation of smTm (A), Tm5a (B), and Tm5b (C) complexed with phalloidin-stabilized F-actin. A temperature region above 55 °C corresponding to irreversible thermal denaturation of F-actin stabilized by phalloidin is not shown. Accordingly, small ther- mal transitions of Tm5a and Tm5b at  57.5 °C were not analysed in this case. Curves shown by dashed lines (peak 3) were obtained by reheating the same samples after the first heating to 90 °Cand following cooling to 5 °C. The heating rate was 1 KÆmin )1 . The peaks 1, 2, and 3 are described in the text. In C peak 1 is highligh- ted as a dotted line showing the curve fit. Concentration of Tm was 10 l M for smTm and 15 lM for Tm5a and Tm5b. Other condi- tions: 46 l M F-actin, 70 lM phalloidin, 30 mM Hepes, pH 7.3, 100 m M KCl, 1 mM MgCl 2 ,and1mM b-mercaptoethanol. Table 2. Parameters of the thermal transitions observed by DSC for smTm, Tm5a, and Tm5b in the presence of F-actin and of heat- induced dissociation of the Tm–F-actin complexes. The calorimetric parameters, T m and DH cal , were extracted from Fig. 7. The parame- ters T 1 , T 2 , T 3 , DH 1 , DH 2 , and DH 3 correspond to peaks 1, 2, and 3 described in the text. Concentration of Tm was 10 l M for smTm and 15 l M for Tm5a and Tm5b; concentration of phalloidin-stabil- ized F-actin was 46 l M. The error of the T m values did not exceed ± 0.2 °C. The relative error of the DH cal values did not exceed ± 10%. The values of T diss were calculated from light-scat- tering data presented in Fig. 8. The error of the T diss values did not exceed ± 0.2 °C. Sample T diss (°C) T m (°C) DH cal (kJÆmol )1 ) T 1 T 2 T 3 DH 1 DH 2 DH 3 DH 2 –(DH 3 –DH 1 ) smTm 38.5 33.8 38.3 34.5 120 685 680 125 Tm5a 43.2 38.7 43 40 65 575 400 240 Tm5b 43.9 40.3 44.4 43 60 530 370 220 E. Kremneva et al. Thermal unfolding of nonmuscle tropomyosin FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS 593 Tm5a and Tm5b, and by only 125 kJÆmol )1 for smTm (Table 2). Thermally induced dissociation of Tm–F-actin complexes Previous studies have shown that Tm dissociates from F-actin on heating, and this process can be studied by light scattering measurements [13,14,34]. To examine the thermal dissociation of the Tm– F-actin complexes, we measured the temperature dependence of light scattering for the complexes of phalloidin-stabilized F-actin with smTm, Tm5a, and Tm5b. These measurements were performed under conditions identical to those of the DSC experiments presented in Fig. 7. When heated below the denatur- ation temperature of actin under these conditions, dissociation of the Tm–F-actin complexes was revers- ible, as the light scattering intensity increased during cooling after the first heating and decreased again during the second heating. The fitted curves to the normalized light scattering changes of dissociation of the Tm–F-actin complexes are shown in Fig. 8. The temperature of midpoint of dissociation (T diss ), i.e. the temperature at which a 50% decrease in light scattering occurs, are presented in Table 2 and com- pared with the maximum temperature of the actin- induced transition 2 (T 2 ) for the same samples studied by DSC. This comparison shows a very good correlation between the T diss and T 2 (Table 2). We therefore conclude that actin-induced changes in the thermal denaturation of Tm (i.e. the appearance of the actin-induced peak 2) are associated with dissoci- ation of Tm from F-actin. This confirms the DSC data showing that both fibroblast Tms, Tm5a and Tm5b, dissociate from F-actin at higher temperature (T diss  43.5 °C) than smTm (T diss ¼ 38.5 °C). Discussion In the present work we applied DSC combined with CD to characterize the thermal unfolding of recombin- ant fibroblast Tms, Tm5a and Tm5b, in comparison with that for smooth muscle Tm. The thermal unfold- ing of smooth muscle Tm isoforms has been investi- gated in detail by DSC [11–13] and by CD [17–19] and the results presented here agree with these earlier works. Thus, smTm expressed with the addition of Ala-Ser to the N-terminus is a good mimic of native acetylated Tm. The thermal unfolding of nonmuscle fibroblast Tms has not been studied by DSC before. The two a-Tm fibroblast isoforms Tm5a and 5b are identical in seven of their eight exons and differ internally only by exon 6 (Fig. 1). The short 25-residue sequences encoded by exons 6b and 6a do not differ in stabilizing or destabil- izing clusters defined by Hodges and coworkers in the hydrophobic core [35–37], both containing only one stabilizing cluster of five residues. However, sequence analysis of exon 6 using coiled-coil prediction software [38] suggests that the coiled-coil propensity of exon 6b (Tm5a) is lower than that of exon 6a (Tm5b) [7]. The DSC data presented are consistent with this prediction, showing that the replacement of exon 6b in Tm5a by exon 6a in Tm5b increases the thermal stability of the major thermal transition by 3.4 °C. In contrast the exon swap has no appreciable influences on the a-helix content at 20 °C (Fig. 5) and on the total calorimetric enthalpy of the thermal unfolding (Table 1). Previous CD studies also showed an increased thermal stability of a recombinant smooth muscle a-Tm with exon 6b replaced by exon 6a [5,39]. Previous studies of thermal unfolding of skeletal Tm have allowed assignment of the thermal transitions to specific regions of Tm for skeletal a-Tm [8,9,14]. Ther- mal unfolding of smTm (Fig. 3A) is quite different from that of skeletal Tm measured by DSC under the same conditions, skeletal Tm has for example two Fig. 8. Normalized temperature dependence of dissociation of the F-actin complexes with smTm, Tm5a, and Tm5b. 100% corres- ponds to the difference between light scattering of the Tm–F-actin complexes measured at 25 °C and that of pure F-actin stabilized by phalloidin which was temperature independent within the temper- ature range used. A decrease in the light-scattering intensity reflects dissociation of the Tm–F-actin complexes. Conditions were the same as for DSC experiments presented in Fig. 7. The heating rate was 1 KÆmin )1 . Thermal unfolding of nonmuscle tropomyosin E. Kremneva et al. 594 FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS thermal unfolding transitions of similar enthalpy compared to one major and two minor transitions observed for smTm [14]. The less stable of the two thermal transitions for skeletal Tm has been assigned to the C-terminal part of the molecule but the C-ter- minal region of the two Tms differ at exon 9 (and at the N-terminal exons 1 and 2). Thus comparison between skeletal and smTm isoforms does not allow the assignment of the transitions of smTm. Comparison between the three Tm molecules studied here can allow assignment of the three thermal trans- itions observed to specific regions of the Tm. The observation that only the main thermal transition is affected by exon 6 in Tm5a and 5b suggests that trans- ition 2 represents the central part of the Tm including exon 6. Note however, that the enthalpy of transition 2 is almost identical for Tm 5a and 5b. Transition 3 is stabilized by almost 20 °C when the N terminal exons 1a and 2a of smTm are replaced by exon 1b in the fibroblast Tm suggesting that transition 3 represents unfolding of the N-terminal part of the Tm. In support of this are the CD data on model peptides showing that the peptide mimicking exon 1b is much more ther- mostable than the peptide corresponding to exon 1a [26,27,29]. Transition 1 is the least stable of the ther- mal transitions and is similar in all three Tms. There- fore this transition cannot be unambiguously assigned. It might correspond to the C-terminal part of Tm. In favour of this assumption are the CD data showing that model peptides corresponding to the C-terminal exon 9d are much less thermostable than the N-ter- minal peptides [29]. In contrast, however, the data of Paulucci et al. [40] show that the C terminus of Tm (in particular the last 24 residues) is crucial for the stabil- ity of Tm. The increased thermal stability of transition 3 in Tm5a and Tm5b in comparison with smTm can be explained in part by recently proposed theory of Hodges and coworkers [35–37] that the thermal stabil- ity of a-helical coiled-coil depends on the presence of stabilizing and destabilizing clusters in its hydrophobic core. They defined these clusters as three or more con- secutive core-forming a and d residues in a heptad repeat motif (abcdefg) n of either stabilizing (Leu, Ile, Val, Met, Phe, and Tyr) or destabilizing (the remaining 13 amino acids) residues, and postulated the presence of very long destabilizing cluster (seven residues) in the hydrophobic core of N-terminal part of muscle Tm encoded by exons 1a and 2a [37]. The sequence of fibroblast Tm5a [41], being analysed in the same way, contained only a short destabilizing cluster (just three residues) in the hydrophobic core of the N-terminal region encoded by exon 1b. The shortening of the destabilizing cluster from seven to three residues might stabilize the transition 3 assigned to the N-terminal part of the Tm. SmTm is  15% larger than the fibroblast Tm (due to inclusion of exon 2) and according to the CD spec- tra the helical content of the two proteins appears sim- ilar at 20 °C. Therefore the simple prediction is that the two proteins should have a similar enthalpy of unfolding (differing by 15% at most), yet the data in Table 1 shows that the total energy of unfolding of SmTm is 1.6-fold larger than that of Tm5a. The pro- portion of the total enthalpy in the three domains of each is very similar at 20 : 60 : 20, so this increase in enthalpy is not simply due to the change in the stabil- ity of the N-terminal part of the molecule, but a change in the observed enthalpy of the whole mole- cule. There are two possible explanations for the increased enthalpy in smTm compared to Tm5a, either the addition of exon 2 influences enthalpy of the whole molecule or there is some ‘unseen’ enthalpy in the Tm5a. Evidence that specific regions of Tm can have long- range effects on the stability of the whole molecule have been reported previously [40,42,43]. A recent study by Singh and Hitchcock-DeGregori [22] has shown that mutations in a region at the C-terminal end of exon 2b in a-Tm2 (identical to smTm except exon 2b vs. 2a) can cause a change in the melting pro- file of several unfolding transitions. The mutations which caused either an increase or decrease in mid- point of the melting transition could both result in an apparent major decrease in total enthalpy of un- folding. ‘Unseen’ enthalpy may be the consequence of broad noncooperative unfolding of parts of the molecule, giv- ing a very slight slope to the heat capacity curve. This proceeds over a broad temperature range and can be difficult to precisely measure by DSC. Noncooperative melting was earlier observed by DSC for some muscle proteins (e.g. troponin T, troponin I [30], and calponin [44]), which did not exhibit any detectable thermal transitions upon heating up to 100 °C. The assumption that noncooperative unfolding takes place for some parts of nonmuscle Tm is corroborated by our DSC studies on the complexes of Tm with F-actin. Interaction with F-actin significantly increases the thermal stability and unfolding enthalpy of Tm, and reduces the difference in total enthalpy seen between smTm and nonmuscle Tm isoforms 5a and 5b (Fig. 7, Table 2). In particular the enthalpy of peak 2 for Tm 5a, corresponding to the actin-bound Tm, is almost exactly six-sevenths of that of smTm as predic- ted on a simple size comparison. This suggests that the E. Kremneva et al. Thermal unfolding of nonmuscle tropomyosin FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS 595 stabilizing of Tm on actin results in almost all of the unfolding occurring as a single cooperative process simultaneous with dissociation from actin. It was con- cluded from our previous studies that F-actin prevents the actin-bound Tm from thermal denaturation, which only occurs upon dissociation of Tm from F-actin [14,45]. As a result, Tm unfolds at higher temperature and with much higher cooperativity. This allows the ‘lost’ enthalpy of parts which unfold noncooperatively at temperatures below T diss in the absence of actin, to be ‘recovered’ in the highly cooperative dissociation transition. It is possible that smTm also has some ‘lost’ enthalpy, but it is likely to be much less than in non- muscle Tm isoforms, which demonstrate in the pres- ence of F-actin much more pronounced ‘recovery’ of enthalpy (by 70%) than smTm (by only 20%) (Table 2). The recent CD and DSC data of Dragan and Priv- alov on the thermal unfolding of a leucine zipper which, like Tm, is an a-helical, double-stranded coiled-coil [46] support noncooperative transitions in the unfolding pathway. These authors showed that the initial almost linear change of leucine zipper ellip- ticity prior to the sigmoidal change (that is very sim- ilar to those of Tms in Fig. 6A) cannot be regarded as a trivial optical effect but is associated with some temperature-induced conformational changes of the dimeric molecule from the very beginning of its heat- ing. They concluded, that the enthalpy of cooperative unfolding that is associated with dissociation of the two strands and is observed as a cooperative thermal transition by DSC, does not represent the full enthalpy of unfolding of the molecule. The full enthalpy also includes the enthalpy of all predissocia- tion changes, which comprises almost 40% of the total enthalpy. These temperature-induced changes in protein structure, that occur before the cooperative separation of strands, are believed to be associated with some rearrangements in the coiled-coil, and they are highly sensitive to modifications of the N termi- nus [46]. It seems possible that somewhat similar structural changes may also occur in nonmuscle Tm isoforms due to replacement of the N-terminal muscle exon 1a by nonmuscle exon 1b. The noncooperative unfolding of a significant part of the nonmuscle Tm molecule may suggest that this part (or these parts) of the molecule becomes more flexible due to replacement of the N-terminal muscle exons 1a and 2a by nonmuscle exon 1b. Higher flexibility may affect actin-binding (since Tm must match the actin periodicity to bind effectively and flexibility may be important in the ability of Tm to change position on the actin surface [22,47,48]). This could explain in part why the replacement of exon 1a by exon 1b strongly increases the affinity of Tm for actin [3] despite having little effect on Tm end-to-end interactions as shown by the Tm polymerization measured by viscosity (Fig. 4A). In conclusion, the data presented here comparing the three tropomyosins is compatible with previous reports that suggest there is no simple correlation between specific ‘domains’ of Tm and the overall stability of the molecule but there are long-range cooperative effects on structure. Furthermore, in some cases local confor- mation changes and unfolding occur as low enthalpy noncooperative transitions that are not easily detected by DSC. This missing enthalpy becomes apparent when the Tm dissociates from actin and unfolds as a single highly cooperative process. Understanding the nature and location of the noncooperative unfolding regions will be important to understand the way in which the exons changes affect the stability of remote unfolding domains. In the future, definitive identification of specific regions of Tm with particular unfolding transi- tions could be facilitated by the use of labels to report local unfolding events. Experimental procedures DNA constructs Clones of rat fibroblast tropomyosins 5a and 5b were amplified from PET8c (gift from M. Gimona and D. Helf- man) using PCR primers designed to introduce NdeI and BamHI restriction sites for cloning into pJC20. The sequences for the primers used were 5¢-GGAATTCCA TATGGCGGGTAGCAGCTCGCTGGCG-3¢ (5¢-forward primer) and 5¢-CGCGGATCCTCACATGTTGTTTAGCT CCAGTAAAG-3¢ (3¢-reverse primer). Identical primers were used for TPM5a and TPM5b as they differ only by an internal alternatively spliced exon 6 (see Fig. 1). The smooth muscle clone was amplified from a full-length clone which also contained the 5¢ UTR in pGem4 (gift from C. Smith, Cambridge), using PCR primers again designed to produce NdeI and BamHI restriction sites. The 5¢ forward primer also introduced bases coding for a three amino acid Met-Ala-Ser N-terminal extension to substitute for the lack of N-terminal acetylation. The sequence for the N-terminal 5¢ forward primer was 5¢-GGAATTCCATATGGCGAGC ATGGACGCCATCAAGAAGAAGATGC-3¢. As smTm uses the same C-terminal exon 9d as Tm5a and Tm5b, the same 3¢ reverse primer was used. The ligated plasmids were transformed into Escherichia coli XL-1 Blue for plasmid replication. The entire coding regions of the constructs were verified by automatic DNA sequencing on Applied Biosystems 373A sequencer (Applied Biosystems, Foster City, CA, USA) using a dye-based PCR sequencing reaction. Thermal unfolding of nonmuscle tropomyosin E. Kremneva et al. 596 FEBS Journal 273 (2006) 588–600 ª 2006 The Authors Journal compilation ª 2006 FEBS Expression and purification of recombinant tropomyosins For expression, all the clones were transformed in the BL-21 DE3 (pLys) cells and expressed and purified as pre- viously described [7,49] with some modifications. In brief, 1-L cultures were grown to late-exponential phase and induced overnight with 0.4 mm isopropyl-1-thio-b-d-gal- actopyranoside. Cells were harvested, resuspended in 60 mL lysis buffer (20 mm Tris pH 7.0, 150 mm NaCl, 5mm EDTA, 5 mm MgCl 2 ), and lysed by sonication (two 2-min pulses separated by 1-min rest phase). The majority of E. coli proteins were precipitated by heating to 80 °C for 10 min, and the precipitated protein and cell debris were removed by centrifugation. Then solution was incubated with 5 mgÆL )1 DNase and 10 mgÆL )1 RNase for 1.5 h. The soluble Tm was then isoelectrically precipitated at pH 4.5 using 0.3 m HCl. The precipitate was pelleted and resus- pended in 10 mL running buffer (5 mm potassium phos- phate pH 7.0, 100 mm NaCl). This was then further purified using a 5 mL Hi-trap Q column (Amersham) and eluted with a 200–500 mm NaCl gradient, with the Tm elut- ing at 250–450 mm salt. Fractions were analysed by SDS ⁄ PAGE [50], pooled, and concentrated by isoelectric precipitation. Extinction coefficients for recombinant pro- teins were calculated from the sequences using the software antheprot (G. Deleage, IBCP-CNRS). Protein concen- trations were estimated using extinction coefficients E 1% at 280 nm of 1.41 cm )1 for smTm and 1.61 cm )1 for fibroblast Tm 5a ⁄ 5b, and molecular masses of 32834.8, 28557.9, and 28697.2 Da for smTm, Tm5a, and Tm5b, respectively. Protein molecular masses were determined by electro- spray mass spectrometry to confirm that the expressed Tms had the correct size. Small (50 lL) stock samples were dia- lysed overnight against 30 mm Hepes pH 7.3 containing 100 mm KCl and 1 mm MgCl 2 , and applied to a Finnegan Mat LCQ ion-trap mass spectrometer fitted with a nano- spray device. Predicted molecular masses for proteins were calculated using the AnTheProt with Delta Mass (ABRF) used to determine mass differences among the Tm species. Relative molecular masses determined by MS for smTm, Tm5a, and Tm5b were in good correspondence with the predicted masses. Before experiments, all Tm samples were incubated with 20 mm b-mercaptoethanol at 60 °C for 60 min. Such treat- ment results in Tm species in completely reduced state [14]. To maintain the reduced Tm species, 1 mm b-mercapto- ethanol was added to the samples. Preparation of actin Rabbit actin was prepared by the method of Spudich and Watt [51]. Its molar concentration was determined by its absorbance at 290 nm using an E 1% of 6.3 cm )1 and a molecular mass of 42 kDa. F-actin polymerized by the addition of 4 mm MgCl 2 and 100 mm KCl was further sta- bilized by the addition of a 1.5-fold molar excess of phalloi- din (Sigma). Viscosity measurements Measurements were carried out at 18.5 °C using an Ostow- ald type capillary viscosimeter (Institute for Biological Instrumentation, Puschino, Russia), with a buffer outflow time of 27.6 s. Before measurements, proteins (0.5 mgÆmL )1 ) were dialysed against 30 mm Hepes pH 7.3 containing 100 mm KCl and 1 mm MgCl 2 . Differential scanning calorimetry DSC experiments were performed on a DASM-4 m differ- ential scanning microcalorimeter (Institute for Biological Instrumentation, Pushchino, Russia) as described earlier [12–14,30]. All measurements were carried out at a scanning rate of 1 KÆmin )1 in either 30 mm Hepes, pH 7.3, or 50 mm sodium phosphate, pH 7.3, both containing 100 mm KCl and 1 mm MgCl 2 . The solution also contained 1 mm b-mercaptoethanol to prevent disulfide cross-linking between the chains in the Tm homodimers. In the case of Tm– F-actin complexes, the final concentration of F-actin was 46 lm. F-actin was stabilized by the addition of a 1.5-fold molar excess of phalloidin (Sigma) to obtain a better separ- ation of the thermal transitions of actin-bound Tm and F-actin [13,14]. The reversibility of the thermal transitions was assessed by reheating of the sample immediately after cooling from the previous scan. The calorimetric traces were corrected for the instrumental background by sub- tracting a scan with buffer in both cells. In some cases, to reveal small and low-cooperative thermal transitions in Tm5a and Tm5b, a special DSC approach was applied as follows. DSC measurements were performed not only by usual way, when the protein was placed into the sample cell and the buffer was placed into the reference cell, but also vice versa, with the same protein in the reference cell and the buffer in the sample cell. As a result, in last case the protein peak on the DSC curve turned over. This curve with inverted protein peak was then subtracted from the curve obtained by usual way. This procedure completely eliminated the instrumental baseline and doubled the ampli- tude of the protein signal. The resulting curve was then divided by two. The point is that the instrumental baseline is the own property of each calorimeter, which is independ- ent of the procedures described above. The above DSC approach allows us to subtract the instrumental baseline without its direct measurement, and to avoid all possible artefacts caused by the measurement of instrumental base- line and by its following subtraction from the DSC profile of the protein. 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Thermal unfolding of smooth muscle and nonmuscle tropomyosin a-homodimers with alternatively spliced exons Elena Kremneva 1 ,. comparison of Tm5a with smTm allows study of the effects of replacement of muscle exons 1a and 2a by nonmuscle exon 1b, and comparison of Tm5a with Tm5b