Báo cáo khoa học: Betaine protects urea-induced denaturation of myosin subfragment-1 pptx

Betaine protects urea-induced denaturation of myosin subfragment-1 Susana Ortiz-Costa 1,2 , Martha M. Sorenson 2 and Mauro Sola-Penna 1 1 Laborato ´ rio de Enzimologia e Controle do Metabolismo, Departamento de Fa ´ rmacos, Universidade Federal do Rio de Janeiro, Brazil 2 Instituto de Bioquı ´ mica Me ´ dica, Universidade Federal do Rio de Janeiro, Brazil Osmolytes are osmotically active solutes that can be grouped into four major classes: polyols (sugar and sugar derivatives), amino acids (and their derivatives), methyl ammonium compounds and urea [1,2]. Often, they are further classified as ‘compatible’ or ‘counter- acting’ on the basis of their ability to affect the func- tional activity of proteins. Compatible osmolytes increase protein stability against denaturation with little or no effect on their function [1–20], whereas counteracting osmolytes have, in addition, the ability to offset the deleterious effects of denaturants on the catalytic activity [1,3,6–8,11–13,17–25]. The counteract- ing solute effect can be viewed as a situation in which the tendency of a denaturant to unfold the protein structure is counterbalanced by a tendency of the sol- ute to minimize the protein surface area in contact with water [18–20,25]. Myosin (EC 3.6.4.1) belongs to the restricted family of motor proteins that transform the chemical energy of ATP into mechanical work. The motor function of myosin is located in its two globular heads, which can be separated by proteolysis as subfragment-1 (S1), equipped with separate sites for actin binding and hydrolysis of ATP [26]. Crystallographic determination of the S1 structure and electron microscopic studies have contributed a great deal to our understanding of the structure and mapping of the functional subdo- mains [27,28]. Despite the extensive knowledge about the structure, our understanding of the folding path- way is limited, and experiments on protein denatur- ation–renaturation may help us to understand how a multidomain oligomeric protein, such as the myosin head, folds, and how its subunits and domains acquire stability. The portion of myosin studied in this work is the myosin head or myosin S1 obtained after chymotryptic proteolysis. It consists of two nonidentical essential light chains with a molecular mass of 17–23 kDa and Keywords methylamine; muscle; myosin; protection; urea Correspondence M. Sola-Penna, Laborato ´ rio de Enzimologia e Controle do Metabolismo (LabECoM), Departamento de Fa ´ rmacos, Faculdade de Farma ´ cia, Universidade Federal do Rio de Janeiro, Ilha do Funda˜o, Rio de Janeiro – RJ, 21941-590, Brazil Fax ⁄ Tel: +55 21 2260 9192 ext 251 E-mail: maurosp@ufrj.br (Received 30 January 2008, revised 19 March 2008, accepted 30 April 2008) doi:10.1111/j.1742-4658.2008.06487.x We have demonstrated previously that urea inhibits the activity and alters the tertiary structure of skeletal muscle myosin in a biphasic manner. This was attributed to differential effects on its globular and filamentous por- tion. The inhibition of catalytic activity was counteracted by methylamines. With the aim of comprehending the effects of urea on the catalytic (globu- lar) portion of myosin, this study examines the effects of urea and the countereffects of betaine on the catalytic activity and structure of myosin subfragment-1. It is shown that urea inactivates subfragment-1 in parallel with its ability to induce exposure of the enzyme hydrophobic domains, as assessed using intrinsic and extrinsic fluorescence. Both effects are counter- acted by betaine, which alone does not significantly affect subfragment-1. Urea also enhances the accessibility of thiol groups, promotes aggregation and decreases the a-helix content of S1, effects that are also counteracted by betaine. We conclude that urea-induced inactivation of the enzyme is caused by partial unfolding of the myosin catalytic domain. Abbreviations bis-ANS, 4,4¢-bis(1-anilinonaphthalene 8-sulfonate); Nbs 2, 5,5¢-dithiobis(2-nitrobenzoic acid); nd-PAGE, nondenaturing PAGE; S1, subfragment-1 of skeletal muscle myosin II; TMAO, trimethylamine-N-oxide. 3388 FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS one heavy chain (approximately 95 kDa). In a previ- ous study [29], we investigated the counteracting effects of methylamines against urea-induced changes in skele- tal muscle myosin. We showed that myosin loses its ATPase activity when the assay is performed in the presence of 2 m urea. However, when the assay is per- formed in the presence of 2 m urea combined with 1 m methylamine, the myosin ATPase activity is preserved. Nevertheless, these modifications in ATPase activity by urea were not directly correlated with the modifica- tions in myosin structure detected by intrinsic fluores- cence spectroscopy [29]. In the present work, we studied the ability of betaine (an amino acid derivative of methylamine) to counteract the effects of urea on the function and structure of S1. Our purpose was to study, in more detail, the protective effect of betaine on the function of skeletal muscle myosin, and, for this, we used the motor domain (S1). We investigated the effects of urea and betaine on K + EDTA ATPase activity, intrinsic fluorescence emission, binding of the hydrophobic fluorescent probe 4,4¢-bis(1-anilinonaph- thalene 8-sulfonate) (bis-ANS), aggregation, exposure of thiol groups and far-UV CD of S1. The addition of urea revealed that slight changes in the structure of S1 could account for the loss of its functional activity. In addition, betaine was able to protect against these small changes in structure, thereby preserving the enzyme’s catalytic activity. Results and Discussion The effects of urea and betaine on S1 ATPase activity were evaluated (Fig. 1). Urea inhibits S1 ATPase activ- ity in a dose-dependent manner (Fig. 1A, open circles), presenting an I 0.5 value of 1.32 ± 0.04 m and a coop- erativity index (n) of 5.3 ± 0.9, calculated as described in Materials and methods. When the same experiment is performed in the presence of 1 m betaine, the inhibi- tory effects of urea are attenuated (Fig. 1A, filled cir- cles): I 0.5 increases to 2.11 ± 0.04 m (P < 0.05, Student’s t-test) without a significant change in the cooperativity index (n) (6.7 ± 1.0; P > 0.05, Student’s t-test). These results are similar to those obtained for the effects of urea and betaine on native myosin [29]. Thus, although urea promotes significant changes in the myosin rod [29], the observed effects on ATPase activity are exclusively the result of alterations in the globular head domain. The effects of 2 m urea on S1 ATPase activity are counteracted by increasing concentrations of betaine (Fig. 1B, filled squares). Betaine alone promotes a slight decrease in activity, so that the remaining activ- ity is 93.6 ± 0.9% and 68.7 ± 0.5% of the original value in the presence of 1 and 2 m betaine, respectively (Fig. 1B, open squares). There is no significant differ- ence in the S1 ATPase activity when the enzyme is incubated with 1–2 m betaine in the absence or pres- ence of 2 m urea, indicating that the denaturant has no effect on the enzyme catalytic activity in the presence of these concentrations of betaine, consistent with the known counteracting effects of methylamines on urea-induced inactivation and denaturation of other proteins using a 2 : 1 concentration ratio of urea to methylamines [1]. In order to understand the effects of urea on S1 ATPase activity at the structural level, we measured the intrinsic fluorescence emission of S1 in the presence of increasing concentrations of urea (Fig. 2). The intrinsic fluorescence of S1 is altered in the presence of increasing concentrations of urea (Fig. 2A). Urea pro- motes a dose-dependent decrease in fluorescence inten- sity which can be evaluated by means of the spectral Fig. 1. Effects of urea and betaine on S1 ATPase activity. S1 ATPase activity was assessed as described in Materials and methods in the presence of the concentrations of urea and betaine indicated for each panel. Plotted values are the mean ± SE of at least four independent experiments performed in duplicate. Full lines are the results of nonlinear regression as described in Materials and methods. S. Ortiz-Costa et al. Betaine protects subfragment-1 against urea FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS 3389 area (Fig. 2B, open circles). The I 0.5 value calculated for this phenomenon is 1.34 ± 0.09 m, which is not statistically different from the I 0.5 value calculated for the urea-induced inhibition of S1 ATPase (P > 0.05, Student’s t-test), suggesting that the effects of urea on the enzyme activity are a result of alterations in its structure. However, the presence of 1 m betaine does not counteract the effects of urea on S1 fluorescence (Fig. 2B, filled circles): the calculated value of I 0.5 is 1.45 ± 0.11 m, not statistically different from the value obtained in the absence of betaine (P > 0.05, Student’s t-test). The center of mass of the intrinsic fluorescence spectrum is not affected by urea up to 4 m, being shifted to the red region of the spectrum in the presence of 8 m urea (Fig. 2B, inset). This red shift is also not counteracted by 1 m betaine (data not shown). The center of mass of intrinsic fluorescence spectra has been used to evaluate protein unfolding or dissoci- ation [9,16,17,30–33]. However, in many cases, changes in this parameter do not correlate with changes in enzyme catalytic activity, which decreases under condi- tions in which no changes are observed in the center of mass [7–12,16]. Nevertheless, the attenuation of the flu- orescence intensity is generally correlated with the exposure of protein tryptophan residues to a more polar environment, thus affording a means of evaluat- ing perturbations in tertiary structure [34]. Our data clearly show that the effects of urea on S1 ATPase activity occur in parallel with its effects on the intrinsic fluorescence emission. Despite the coincident I 0.5 values for the effects of urea on both the ATPase activity and intrinsic fluorescence emission, the fact that betaine does not counteract the decrease in fluorescence emis- sion does not support the hypothesis that urea inhibi- tory effects are only a result of the exposure of tryptophan residues. As a means of evaluating other tertiary structural changes related to the effects of urea on S1 ATPase activity and its counteraction by betaine, we assessed the binding of bis-ANS. This fluorescent dye binds to distinct hydrophobic regions of S1, including its cata- lytic ATP binding site [35], increasing the fluorescence AB Fig. 2. Area and center of mass of intrinsic fluorescence spectra of S1 in the presence of urea and betaine. Experiments were performed as described in Materials and methods. (A) Intrinsic fluorescence spectra of S1 in the absence (control) and presence of the concentrations of urea indicated in the figure. This is a representative experiment of a series of four independent experiments. (B) Calculated spectral area of S1 in the presence of the concentrations of urea indicated on the abscissa, in the absence (filled circles) and presence (open circles) of 1 M betaine. Experiments shown in (A) and (B) are representative of a series of four experiments presenting similar results. Inset: center of mass of S1 intrinsic fluorescence spectra obtained in the presence of the concentrations of urea indicated on the abscissa. Plotted values are the mean ± SE of four independent experiments. Fig. 3. bis-ANS binding to S1 in the presence of urea and betaine. Experiments were performed as indicated in Materials and meth- ods in the presence of the concentrations of urea indicated on the abscissa with and without 1 M betaine. Plotted values are the mean ± SE of four independent experiments. Inset: representative spectrum of bis-ANS alone (dotted line) or in the presence of S1 (full line). Betaine protects subfragment-1 against urea S. Ortiz-Costa et al. 3390 FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS emission of the dye (Fig. 3, inset: dotted line, without S1; full line, with S1). Urea up to 2 m induces a dose- dependent increase in bis-ANS fluorescence, which is not observed in the presence of 1 m betaine (Fig. 3). This result indicates that urea is promoting the expo- sure of S1 hydrophobic domains, allowing bis-ANS to bind to the protein. This phenomenon is prevented by the presence of 1 m betaine in the medium, which pro- tects the S1 tertiary structure against this unfolding promoted by urea. Higher concentrations of urea (3–8 m) induce a progressive decrease in the bis-ANS fluorescence (Fig. 3), indicating that these urea concen- trations are disrupting S1 hydrophobic domains. The presence of 1 m betaine does not counteract this effect, and the fluorescence signal decreases in the presence and absence of the methylamine (Fig. 3). The exposure of protein hydrophobic groups to a polar environment often favors the formation of pro- tein aggregates. In order to assess this possibility, S1 was analyzed by electrophoresis by nondenaturing PAGE (nd-PAGE) in the absence and presence of urea and betaine. In the absence of additives, nd-PAGE reveals a single band at the top of the 6% gel (Fig. 4, lane 1). In the presence of increasing concentrations of urea (0.5–8 m), this band is progressively replaced by a band at the top of the 3.6% gel, indicating the forma- tion of aggregates that do not enter the 3.6% gel (Fig. 4, lanes 2–7). Betaine, which alone does not sig- nificantly alter the nd-PAGE pattern of S1 (Fig. 4, lanes 13–15), at a 2 : 1 concentration ratio of urea to betaine, partially protects S1 from the urea-induced formation of aggregates (Fig. 4, lanes 9, 11 and 12). Comparing lane 5 (in the presence of 2 m urea) and lane 9 (in the presence of 2 m urea and 1 m betaine), it can be seen that the S1 aggregate formed at the top of the 3.6% gel in lane 5 is not present in lane 9. Con- versely, the loss of native S1 at the top of the 6% gel in lane 5 does not occur in lane 9. Similar results are obtained with 4 m urea in the absence (lane 6) and presence (lane 12) of 2 m betaine, and with 8 m urea in the absence (lane 7) and presence (lane 11) of 4 m betaine. It is important to note that, in the presence of 1or4m urea, both with 1 m betaine, the electropho- retic patterns are no different from the results obtained in the presence of 1 or 4 m urea alone, indicating the need for a 2 : 1 concentration ratio of urea to betaine. Taken together, these results indicate that betaine pre- vents S1 from the unfolding and aggregation induced by urea. In order to better understand the kinetics of urea- induced S1 aggregation, we performed light scattering measurements of S1 as a function of incubation time in the presence of 1 m betaine, 2 m urea or both together in the medium. Corroborating the results presented in Fig. 4, light scattering experiments revealed that S1 aggregates in the presence of 2 m urea (Fig. 5, open triangles), an effect that is counteracted by the simul- taneous presence of 1 m betaine (Fig. 5, filled triangles). A small but significant increase in light scattering can be observed when S1 is incubated in the presence of 1 m betaine alone (Fig. 5, open circles). From a comparison of lanes 1 (control) and 13 (1 m betaine) in Fig. 4, it can be seen that a weak band is visualized in the 3.6% gel, which was neglected when analyzing this result alone. Nevertheless, the results presented in Fig. 5 suggest that betaine induces, to a much lesser extent when compared Fig. 4. nd-PAGE of S1 in the presence of urea and betaine. Experi- ments were performed as described in Materials and methods in the presence of the urea and betaine concentrations indicated in the figure. This is a representative experiment of a series of three. Fig. 5. Light scattering of S1 in the presence of urea and betaine. Experiments were performed as indicated in Materials and meth- ods in the presence of the concentrations of urea indicated in the figure and in the absence (filled circles, open triangles) or presence (open circles, filled triangles) of 1 M betaine. Plotted values are the mean ± SE of four independent experiments. S. Ortiz-Costa et al. Betaine protects subfragment-1 against urea FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS 3391 with urea, the aggregation of S1. This aggregation is probably not caused by the same mechanism as that of urea as, under the same conditions, S1 is still active in the presence of betaine, but not in the presence of urea alone (see Fig. 1). Moreover, it is possible that the beta- ine-induced aggregation of S1 helps protect the enzyme against the deleterious effects of urea, preventing the lat- ter osmolyte from interacting with some domains of S1. It has been shown that guanidinium hydrochloride- induced unfolding of S1 increases the accessibility of the reactive thiol groups of the enzyme prior to its complete unfolding [36]. In order to determine whether betaine counteracts the initial steps of urea-induced S1 unfolding, we assessed the effects of urea on the acces- sibility of S1 thiol groups to 5,5¢-dithiobis(2-nitroben- zoic acid) (Nbs 2 ) (Fig. 6). Urea increases the accessibility of S1 thiol groups, in a dose-dependent manner, from 2 molÆmol )1 in the absence of urea to 4 molÆmol )1 in the presence of 2 m urea (Fig. 6, open circles). These results are similar to those obtained with guanidinium chloride [36], and are consistent with the concept that the effects of urea on S1 catalytic activity are a result of the initial stages of unfolding of the enzyme. Moreover, 1 m betaine totally prevents this unfolding effect of 2 m urea (Fig. 6, filled circles), consistent with its protection of S1 activity at this con- centration (Fig. 1A). So far, our findings reveal that urea inactivates S1 ATPase as a result of the initial changes in the enzyme tertiary structure promoted by this compound. Furthermore, both structural and catalytic changes are counteracted by betaine at a 2 : 1 concentration ratio of urea to betaine. The effects of urea and methylamines on the acces- sibility of thiol groups have been evaluated by Yancey and Somero [37] using another enzyme: bovine liver glutamate dehydrogenase. These authors found similar results, with urea increasing the accessi- bility of thiol groups and trimethylamine-N-oxide (TMAO) counteracting this effect. Nevertheless, they showed that TMAO alone diminished the accessibility of thiol groups in the enzyme, an effect that was not observed using betaine and S1 (see Fig. 6). However, in our previous work, we have found that TMAO is more effective than betaine in protecting myosin activity against the deleterious effects of urea [29]. Although we did not observe any difference between the effects of betaine and TMAO on the myosin fluo- rescence spectra, Go ¨ ller and Galinsky [38], studying the rabbit muscle lactate dehydrogenase, demon- strated that TMAO was more effective than betaine in protecting the enzyme against denaturation, as measured by intrinsic fluorescence. These results sug- gest that, as stated for the protection conferred by carbohydrates on many enzymes [8], the degree of protection provided by distinct methylamines depends on the nature of the enzyme, and must be evaluated on a case-by-case basis. In order to investigate whether urea and betaine affect the secondary structure of S1, we recorded the far-UV CD spectra from S1 in the presence of urea and betaine. Figure 7A shows that increasing concen- trations of urea progressively decrease the a-helix con- tent of S1, as observed at 222 nm. Betaine alone does not significantly alter the S1 CD spectrum, although it counteracts the effect of urea observed through this technique (Fig. 7B). About one-half of the total effect of urea on the ellipticity at 222 nm occurs when urea is increased to 2 m (Fig. 7A, inset) and, at this concen- tration of urea, 1 m betaine (purple triangle) blocks the effect. These results indicate that folding intermedi- ates may be formed during urea titration into the solu- tion containing S1, and that these intermediates may not be produced in the presence of betaine. Thus, beta- ine counteracts the effects of urea by preventing the formation of folding intermediates which favor the denaturation of the enzyme. Together, our results indicate that urea-induced S1 inactivation and its counteraction by betaine are caused by changes in the S1 structure at secondary and tertiary levels. Although many reports have not revealed a direct correlation of distinct enzyme inacti- vation and unfolding [7–12,36,39–41], the present work shows that, for myosin S1, this correlation is quite precise, at least for secondary structure and early unfolding steps. Moreover, it is clear from the current Fig. 6. Nbs 2 titration of S1 in the presence of urea and betaine. Experiments were performed as indicated in Materials and meth- ods in the presence of the concentrations of urea indicated on the abscissa and in the absence (open circles) or presence (filled circles) of 1 M betaine. Plotted values are the mean ± SE of four independent experiments. Betaine protects subfragment-1 against urea S. Ortiz-Costa et al. 3392 FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS results that intrinsic fluorescence measurements may not be an accurate technique to evaluate the initial loss of S1 tertiary structure, which is better assessed by other methods, such as the accessibility of thiol groups, extrinsic fluorescence and nondenaturing gel electrophoresis. Thus, we conclude that the protection granted by betaine against S1 inactivation by urea is a result of the prevention of secondary structure loss and the initial unfolding process. Materials and methods Materials Urea, betaine, ATP (Grade I), a-chymotrypsin (type I-S), Nbs 2 , phenylmethanesulphonyl fluoride, Hepes and the dialysis membranes were obtained from Sigma (St Louis, MO, USA). The DE-52 anion-exchange resin was from Whatman (Clifton, NJ, USA). All other reagents used were of analytical grade. [ 32 P]Pi was obtained from Instituto de Pesquisas Energe ´ t- icas e Nucleares (Sa ˜ o Paulo, Brazil). [c- 32 P]ATP was purchased from New England Nuclear (Boston, MA, USA) or synthesized as described in [42]. Protein purification Skeletal muscle myosin was prepared from chicken breast according to [43]. The purity (> 99%) was checked by SDS-PAGE [44]. Purified myosin was stored at )20 °Cina solution consisting of 300 mm KCl, 50 mm imidazole (pH 7.0), 1 mm dithiothreitol and 50% (v ⁄ v) glycerol. Before use, the protein was diluted 12-fold in ice-cold water, centrifuged and resuspended in 600 mm KCl and 50 mm imidazole (pH 7.0). S1 was prepared from purified myosin by digestion with chymotrypsin for 10 min at room temperature [45]. Phen- ylmethanesulphonyl fluoride was added to stop the reaction and the suspension was centrifuged to remove the rod region. The supernatant was loaded onto a Whatman DE-52 column (3.5 cm · 20 cm) equilibrated with 50 mm imidazole buffer (pH 7.0) and 1 mm dithiothreitol, and eluted with a linear NaCl gradient (0–200 mm NaCl ⁄ 50 mm imidazole, pH 7.0). Fractions containing the cleaved heavy chain ($ 95 kDa), together with light chain 1 or light chain 3, were pooled and concentrated nearly 15-fold by ultrafiltration under nitrogen across an Amicon YM 30 membrane (Millipore, Billerica, MA, USA). After dialysis against 50 mm KCl, 10 mm imidazole (pH 7.0), 20 mm potassium phosphate buffer (pH 7.0), 0.2 mm EDTA and 1mm dithiothreitol, the protein was diluted to 50% using glycerol and stored under liquid nitrogen in small aliquots. Before use, S1 was dialyzed against 50 mm KCl containing 50 mm imidazole or 50 mm Hepes (pH 7.0) for measurement of ATPase activity or fluorescence, respectively, and was centrifuged for 25 min at 350 000 g to eliminate aggregates. The myosin concentration was estimated by the biuret assay [46]. S1 was determined spectrophotometrically, assuming an absorption coefficient of A 1% 280 = 7.5 cm )1 . ATPase activity The assay medium used to measure the K + EDTA ATPase activity of S1 contained 0.05 mgÆmL )1 protein, 3 mm ATP, 600 mm KCl, 50 mm imidazole (pH 7.0) and 5 mm EDTA. The hydrolysis of ATP was measured at 30 °C after 10 min of incubation with osmolytes (urea or betaine). The effect of increasing urea concentration was measured in the absence or presence of 1 m betaine. The reactions were started by the addition of [c- 32 P]ATP and quenched with activated charcoal suspended in 0.1 m HCl. The reaction rate was measured by taking samples between 2 and 8 min on the linear portion of the progress curve. After centrifu- gation, an aliquot containing [ 32 P]Pi was applied to filter paper, dried and counted for radioactivity in a liquid A B Fig. 7. CD spectra of S1 in the presence of urea and betaine. Experiments were performed as indicated in Materials and meth- ods in the presence of the concentrations of urea or betaine indi- cated in the figure. Inset: ellipticity measured at 222 nm in the absence (black circle) or presence of 0.5 (red circle), 1 (green cir- cle), 2 (yellow circle), 4 (blue circle) or 8 M (magenta circle) urea, 1 M betaine (cyan triangle) or both 2 M urea and 1 M betaine (purple triangle). This is a representative experiment of a series. S. Ortiz-Costa et al. Betaine protects subfragment-1 against urea FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS 3393 scintillation counter. The velocity of hydrolysis was calcu- lated by linear regression. Appropriate blanks were carried out in duplicate as described previously [29]. Kinetic parameters for the inhibition of S1 ATPase activ- ity by urea were calculated by fitting the experimental data to obtain the parameters of the following equation: R a ¼ 100  I n 0:5 I n 0:5 þ½urea n ð1Þ where R a is the residual relative ATPase activity at a given concentration of urea ([urea]), I 0.5 is the [urea] value that promotes 50% of maximal inhibition and n is the cooper- ativity index for this phenomenon. Fluorescence experiments The intrinsic fluorescence measurements were carried out using an F-4500 spectrofluorimeter (Hitachi, Tokyo, Japan). The excitation wavelength was 280 nm, and emission spec- tra were recorded between 300 and 400 nm. Assays were performed at 30 °C in a medium containing 0.05 mgÆmL )1 S1, 600 mm KCl, 50 mm Hepes (pH 7.0) and 5 mm EDTA in the absence or presence of increasing concentrations of each osmolyte (urea or betaine) or increasing urea concen- trations combined with 1 m betaine. The osmolytes (solid powder) were added to the same sample in increasing quan- tities, followed by a 10 min incubation period in which no further changes were observed, as described previously [29]. The fluorescence intensity and osmolyte concentrations were corrected for changes in sample volume after addition of the chemical agent. The spectral area and spectral center of mass of the fluo- rescence spectra were calculated using sigmaplot 10.0 (Systat, San Jose, CA, USA), employing the following equations: Area ¼ X k  I k ð2Þ and Centre of mass ¼ X ðk  I k Þ= X I k ð3Þ where S is the sum over all wavelengths (k) 300–400 nm and I k is the fluorescence intensity at a given k [29–32,34]. In order to evaluate the binding of bis-ANS to S1, 2.5 lm of the dye was added to the same buffer as used for the intrinsic fluorescence measurements with 0.5 lm protein (0.05 mgÆmL )1 ). The excitation was set at 360 nm and the fluorescence emission was measured in the range 400– 600 nm; measurements were performed 10 min after all additions. Light scattering experiments Light scattering measurements were performed in a Jasco spectrofluorimeter (Jasco Corporation, Tokyo, Japan) in the same medium as described above for intrinsic fluorescence, in the absence and presence of 2 m urea, 1 m betaine or both. Appropriate reference spectra were subtracted from the data to correct for background interference, which was always less than 5% of the total signal. The incident light wavelength was set at 510 nm, and scattering was recorded at 90° between 500 and 520 nm. The spectral area was cal- culated using Eqn (2), and plotted values represent the area of the light scattering experiment at each incubation time divided by the area obtained before the additions. Reactivity of thiol groups The reactivity of thiol groups in S1 was measured by moni- toring, at 405 nm, the time course of reaction of the protein (2.5 lm) with Nbs 2 (90-fold excess over S1) in the presence of 600 mm KCl, 50 mm Hepes (pH 7.0) and 5 mm EDTA at 30 °C. The reaction was followed for 20 min, at 6 s intervals, using a Thermomax Microplate Reader (Molecu- lar Devices, Menlo Park, CA, USA). For urea denaturation studies and counteracting effects, stock solutions of 10 m urea and 5 m betaine, prepared in the same buffer as described above, were varied to give the appropriate final osmolyte concentrations. CD spectra CD spectra were recorded on a Jasco J-715 (Jasco Corpora- tion). The results were expressed as the mean residue molar ellipticity [h] (degÆcm 2 Ædmol )1 ) and calculated using the following equation: ½h¼ð½h obs  MRWÞ=ð10lcÞð4Þ where [h] obs is the observed ellipticity (mdeg), MRW is the mean residue molecular mass (molecular mass of the poly- peptide divided by the number of amino acid residues), c is the peptide concentration (mgÆmL )1 ) and l is the optical path length (0.1 cm). CD spectra were the average of three scans obtained by collecting data at 0.2 nm intervals from 260 to 200 nm. Stock protein solutions were prepared in 600 mm KCl, 50 mm Hepes (pH 7.0) and 5 mm EDTA. Stock solutions of 10 m urea and 5 m betaine were pre- pared in the same buffer. The ratios of buffer and osmolyte solutions added were varied to give the appropriate final osmolyte concentrations, and protein stock solution was added to give a final concentration of 0.05 mgÆmL )1 (0.5 lm). Nondenaturing electrophoresis nd-PAGE was performed in a Mini-Protean 3 system (Bio- Rad Laboratories, Hercules, CA, USA) according to [47]. Stacking (3.6% polyacrylamide) and running (6% poly- acrylamide) gels were prepared according to [47]. S1 samples Betaine protects subfragment-1 against urea S. Ortiz-Costa et al. 3394 FEBS Journal 275 (2008) 3388–3396 ª 2008 The Authors Journal compilation ª 2008 FEBS were pre-incubated for 10 min in the absence (control) or presence of distinct concentrations of urea and ⁄ or betaine, as indicated; 15 lg of protein was submitted to electro- phoresis for 1.5 h at constant voltage (200 V). After the runs, samples were stained using Coomassie blue dye, as described previously [48]. 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Betaine protects urea-induced denaturation of myosin subfragment-1 Susana Ortiz-Costa 1,2 , Martha M aim of comprehending the effects of urea on the catalytic (globu- lar) portion of myosin, this study examines the effects of urea and the countereffects of