Trimethylamine N-oxide suppresses the activity of the actomyosin motor

Biochimica et Biophysica Acta 1820 (2012) 1597–1604

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Trimethylamine N-oxide suppresses the activity of the actomyosin motor Ryusei Kumemoto, Kento Yusa, Tomohiro Shibayama, Kuniyuki Hatori ⁎ Department of Bio-Systems Engineering, Graduate School of Science and Engineering, Yamagata University, Yonezawa 992–8510, Japan

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Article history: Received 23 February 2012 Received in revised form 23 May 2012 Accepted 7 June 2012 Available online 15 June 2012 Keywords: Muscular protein ATP hydrolysis Motility Hydration Urea Osmolyte

a b s t r a c t Background: During actomyosin interactions, the transduction of energy from ATP hydrolysis to motility seems to occur with the modulation of hydration. Trimethylamine N-oxide (TMAO) perturbs the surface of proteins by altering hydrogen bonding in a manner opposite to that of urea. Hence, we focus on the effects of TMAO on the motility and ATPase activation of actomyosin complexes. Methods: Actin and heavy meromyosin (HMM) were prepared from rabbit skeletal muscle. Structural changes in HMM were detected using fluorescence and circular dichroism spectroscopy. The sliding velocity of rhodamine-phalloidin-bound actin filaments on HMM was measured using an in vitro motility assay. ATPase activity was measured using a malachite green method. Results: Although TMAO, unlike urea, stabilized the HMM structure, both the sliding velocity and ATPase activity of acto-HMM were considerably decreased with increasing TMAO concentrations from 0–1.0 M. Whereas urea-induced decreases in the structural stability of HMM were recovered by TMAO, TMAO further decreased the urea-induced decrease in ATPase activation. Urea and TMAO were found to have counteractive effects on motility at concentrations of 0.6 M and 0.2 M, respectively. Conclusions: The excessive stabilization of the HMM structure by TMAO may suppress its activities; however, the counteractive effects of urea and TMAO on actomyosin motor activity is distinct from their effects on HMM stability. General significance: The present results provide insight into not only the water-related properties of proteins, but also the physiological significance of TMAO and urea osmolytes in the muscular proteins of waterstressed animals. © 2012 Elsevier B.V. All rights reserved.

1. Introduction TMAO, a natural osmolyte found in certain water-stressed organisms such as sharks, has been known to counteract the deleterious effects of urea on protein stability [1]. TMAO and urea seem to affect the hydration layer on the surfaces of proteins [2]. Urea probably weakens the hydrogen bonds of the water surrounding proteins, whereas TMAO tends to interact with the surrounding water molecules rather than the protein surfaces themselves [3]. Consequently, the selective solvation of dehydrated proteins can occur in the presence of urea, whereas TMAO can induce the preferential hydration of proteins [4]. TMAO stabilizes the ternary structure of proteins and protects against urea-induced unfolding through direct interactions with urea [5,6]. Although a number of studies have focused on the effects of TMAO and urea on the structural stability and enzymatic activity of various

Abbreviations: ATP, adenosine-5′-triphosphate; bis-ANS, 4,4′-dianilino-1,1′binaphthyl-5,5′-disulfonic acid; BSA, bovine serum albumin; HMM, heavy meromyosin; PCA, perchloric acid; Pi, inorganic phosphate; TMAO, trimethylamine N-oxide ⁎ Corresponding author. Tel./fax: + 81 238 26 3727. E-mail address: [email protected] (K. Hatori). 0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2012.06.006

proteins [1,7–11], the mechanism underlying the influence of osmolytes on the interactions between proteins is still unclear. The behavior of water molecules and effects of hydration at the interface between actin filaments and myosin heads may contribute to the activity of proteins involved in muscle contraction [12–14]. This raises the question of how the activity of the actomyosin complex is altered when TMAO or urea perturbs the properties of water associated with these proteins. Ortiz-Costa et al. have previously reported that the ATPase activity of myosin subfragment-1 is inhibited in the presence of urea at concentrations above 1.0 M, but is restored in the presence of TMAO [15]. We have also shown that urea can weaken the affinity between actin filaments and myosin heads and that their motility and ATPase activation are decreased with increasing urea concentrations from 0–1.0 M [16]. However, whether TMAO counteracts the effects of urea on the functions of actomyosin complexes is still unknown. In the present study, we showed that both the sliding velocity of actin filaments on HMM molecules and the ATPase activation of HMM by actin filaments decreased with increasing TMAO concentrations and that TMAO affected the structural stability of HMM in a manner opposite to that of urea. In addition, the presence of TMAO was found to be sufficient to suppress the activities of actomyosin complexes, independently of the presence of urea. We have also

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shown that TMAO counteracts the urea-induced inhibition of motility, but not ATPase activation.

2. Materials and methods 2.1. Reagents and proteins TMAO was purchased from MP Biomedicals, and tetramethylrhodamine-phalloidin and 4,4′-dianilino-1,1′-binaphthyl5,5′-disulfonic acid (bis-ANS) were purchased from Sigma-Aldrich. Other chemicals were obtained from Nacalai Tesque (Kyoto) and were of special reagent grade. Actin and myosin were prepared from rabbit skeletal muscle, and actin monomers were purified according to the method described by Spudich and Watt [17]. Myosin molecules were purified using the method described by Perry, and then digested with alpha-chymotrypsin to obtain HMM molecules [18].

2.2. Fluorescence spectroscopy for detecting structural change Fluorescence measurements of actin filaments stabilized by phalloidin and of HMM molecules were performed using bis-ANS, which responds to hydrophobic environments, as a fluorescent probe [19]. The bis-ANS dye was added to a solution at 25 °C containing 25 mM KCl, 25 mM HEPES (pH 7.4), 4 mM MgCl2, and 0.05 mg/mL protein (HMM or actin filament) to obtain a final bis-ANS concentration of 0.002 mg/mL (6 μM). Measurements were obtained with a spectrofluorometer (Hitachi, F-2500). Excitation was set at 360 nm (band pass 10 nm), and the emission was monitored between 420 and 600 nm. The spectral area was calculated as the sum of fluorescence intensities in the wavelength range mentioned above.

2.3. Circular dichroism (CD) spectroscopy CD spectra for HMM molecules were recorded on a CD spectrometer equipped with a Peltier thermostating cuvette holder (Jasco, J-820). The solutions were examined under similar conditions to those mentioned in Section 2.2. for fluorescence spectroscopy. Mean residue molar ellipticity was determined as the value at which the observed ellipticity was divided by both the optical pass length (0.5 cm) and the residue molar concentration (47.5 mM for 0.05 mg/mL HMM). Thermal unfolding transition was monitored by measuring the ellipticity at 222 nm as a function of temperature in the range of 25–85 °C (heating rate: 2 °C/min).

2.4. ATPase activity of acto-HMM complexes ATP hydrolysis was monitored by measuring the concentration of Pi using the malachite green method [20]. Just before use, the malachite green reagent (0.06 g malachite green, 2.46 g sodium molybdate, 0.1 g Triton-X in 200 mL of 1 M HCl) was mixed with an equal volume of 0.3 M PCA. The ATPase reaction was initiated by adding 50 μL of 2 mg/mL HMM to 950 μL of F-actin solution (0.3 mg/mL F-actin, 25 mM KCl, 25 mM imidazole–HCl [pH 7.4], 4 mM MgCl2, 1 mM ATP, and variable concentrations of urea or TMAO). The reaction was performed at 25 °C. At 5-min intervals, 20 μL aliquots were taken from each reaction mixture, and 2 mL of malachite green reagent was added for termination and coloration. Immediately after sampling, 200 μL of 34% sodium citrate solution (pH 8) was added, and the reaction mixture was agitated for few seconds. After incubation for 13 min at 25 °C, the absorbance of the sample was measured at 650 nm by spectrometry. ATPase activity was assessed as the molar ratio of Pi release to HMM molecules per second. We confirmed that TMAO and urea did not affect the determination of Pi concentration during the experimental procedure.

2.5. Sliding velocity of actin filaments on HMM molecules The prepared actin filaments were labeled with tetramethylrhodamine-phalloidin. HMM molecules were fixed on the collodion-coated surface of glass slides (Matsunami, No. 1, 24 ×50 mm) by a perfusion of HMM solution (0.05 mg/mL HMM, 25 mM KCl, 25 mM imidazole–HCl [pH 7.4], 4 mM MgCl2, 0.5% 2-mercaptoethanol) in the 0.1 mm separation between the slide and the cover glass (Matsunami, No. 1, 18 ×18 mm). Sixty seconds after perfusion, the solution was replaced with bovine serum albumin (BSA) solution (3 mg/mL bovine serum albumin, 25 mM KCl, 25 mM imidazole–HCl [pH 7.4], 4 mM MgCl2, 0.5% 2-mercaptoethanol) to remove unbound HMM molecules. The slide was subsequently perfused with 1 μg/mL of labeled actin solution. The sliding movement of actin filaments was examined immediately after replacement with ATP solution (25 mM KCl, 25 mM imidazole–HCl [pH 7.4], 4 mM MgCl2, 1 mM ATP, 0.5% 2-mercaptoethanol, 3 mg/mL glucose, 0.1 mg/mL glucose oxidase, 0.02 mg/mL catalase) containing TMAO with or without urea at various concentrations using a fluorescence microscope (Nikon, Diaphoto-TMD, objective DIC 100×oil) with the help of fluorescent equipment (Nikon, TMD-EF2) and optical interference filters optimized for rhodamine dyes (Omega, XF101-2). Each experiment was performed at 25 °C within approximately 10 min of the addition of the ATP solution. Fluorescent images were obtained using a highly sensitive camera (Hamamatsu, C2400-08) and recorded on a computer (Apple Co., Power Mac G3) through a video grabber board (Scion Co., LG-3). The sliding velocity of the actin filaments was determined using image analysis software (NIH, ImageJ) to measure the distance of movement at 0.5-s intervals. The sliding velocity of actin filaments was calculated by averaging 100 independent samples for each case. Spacing between the nearest-neighbor pixels in the images was 0.083 μm. To evaluate the distortions of actin filaments, both the contour length of a filament and the end-to-end distance of the same filament were measured in the presence and absence of TMAO. 2.6. Dissociation rate of actin filaments from HMM molecules In the absence of ATP, the rate of dissociation of an actin filament from an HMM molecule fixed on a glass surface was recorded using a fluorescent microscope. HMM solution (1 μg/mL) was dispersed on the collodion-coated slide glass. After 60 s, the solution was replaced with 10 mg/mL of BSA solution. Short actin filaments prepared by ultrasonication were then added. The final solution contained 25 mM KCl, 25 mM imidazole–HCl (pH 7.4), 4 mM MgCl2, 0.5% 2mercaptoethanol, 0.02 mg/mL catalase, 0.1 mg/mL glucose oxidase, 6 mg/mL glucose, 0.05 mg/mL hexokinase, and 1.0 M TMAO with or without 1.0 M urea. The dwell time between the binding of an actin filament to an HMM molecule and its subsequent detachment was measured. Two hundred filaments were recorded under each condition. Dissociation rate constants (koff) were determined by creating an exponential plot of the frequency distribution (N) versus dwell time (t), where N(t) = N0 exp (− koff t). 3. Results 3.1. Effect of TMAO on the structure of actin filaments and HMM molecules TMAO is a known stabilizer of protein structure. We therefore examined whether this osmolyte alters the ternary structure of actin and HMM molecules using fluorescence spectroscopy with a bis-ANS fluorescence probe. The fluorescence intensity of bis-ANS is greatly enhanced in nonpolar environments; therefore, when hydrophobic regions such as the ATP-binding regions of myosin heads or actin molecules are accessible to the bis-ANS probes, their fluorescence intensity is enhanced. Fig. 1A shows that the presence of 0–1.5 M TMAO resulted in a decrease in the

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3.2. Effects of TMAO and urea on the ATPase activity of acto-HMM complexes The ATPase activity of HMM molecules was also examined in the presence and absence of actin filaments. TMAO at concentrations up to 1.0 M was found to induce an approximately 2-fold increase in the

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Concentration of TMAO [M] Fig. 1. Spectral area of the fluorescence of bis-ANS bound to HMM molecules (A) and actin filaments (B) in the presence of TMAO and urea. Filled circle, open circle, and open triangle denote the absence of urea, the presence of 0.6 M urea, and the presence of 1.0 M urea, respectively. Excitation was set at 360 nm, and the emission was monitored between 420 and 600 nm. Spectral areas for each condition were normalized to the original area in the absence of both osmolytes. Error bars indicate the standard deviation estimated from 3 independent experiments performed under similar conditions. The horizontal dotted line indicates the original level in the absence of both osmolytes.

bis-ANS fluorescence intensity of HMM molecules. Furthermore, whereas 0.6 M urea enhanced the fluorescence intensity, this effect was absent in the presence of 0.3 M TMAO. In the case of phalloidin-stabilized actin filaments, the presence of TMAO did not change the bis-ANS intensity, whereas the presence of 0.6 M urea decreased the intensity to approximately 90% of the original level (Fig. 1B). In the case of actin filaments, this decrease in intensity was independent of TMAO concentration. Because bis-ANS may act as a tightening agent or exhibit chaperonlike activity for certain proteins [21,22], we next examined whether bisANS itself induced structural changes in HMM using CD spectroscopy. Fig. 2A shows the CD spectra of HMM in the presence of either bis-ANS or TMAO. In the wavelength range from 250 nm to 220 nm, the spectrum of HMM in the presence of bis-ANS was very similar to that without the dye (control). Furthermore, the presence of TMAO did not significantly alter the spectrum of HMM in this wavelength range. Fig. 2B shows the changes in ellipticity of HMM that occurred in the presence of bis-ANS or TMAO in response to elevated temperature. In the absence of both bis-ANS and TMAO, the value of ellipticity at 222 nm increased from approximately −15.7 to −4.9 kdeg cm2 dmol− 1 as the temperature increased from 25 °C to 85 °C. The transition temperature (Tm) at the midpoint between the upper and lower limits of the observed ellipticity

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was estimated to be 50 °C in the control. The Tm was also 50 °C in the presence of bis-ANS. In contrast, the response of ellipticity of HMM to temperature varied in the presence of 1.5 M TMAO, resulting in a higher Tm (61 °C) than was observed with the control. The Tm of HMM was also found to be dependent on the concentrations of TMAO and urea in the solution (Fig. 3), whereby the transition temperature increased from 50 °C to 58 °C with an increase in TMAO concentration from 0 M to 1.0 M. On the other hand, the presence of 0.6 M and 1.0 M urea in the absence of TMAO induced a decrease in Tm to 46 °C and 44 °C, respectively. The decreased Tm values observed in the presence of 0.6 M and 1.0 M urea were, however, recovered by the addition of approximately 0.3 M and 0.5 M TMAO, respectively. These results clearly indicate that TMAO induces the stabilization of the HMM structure. In the case of phalloidin-bound actin filaments, an increase in temperature to 70 °C induced an increase in ellipticity in the presence of TMAO and a decrease in ellipticity in the presence of urea (data not shown). The change in ellipticity induced by 0.6 M urea could again be recovered to normal levels with the addition of 0.3 M TMAO.

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Temperature [oC] Fig. 2. CD spectra (A) and temperature-dependence of ellipticity (B) of HMM in the presence of either bis-ANS or TMAO. Solid line, dotted line, and dashed line indicate the control (neither bis-ANS nor TMAO), the presence of 6 μM bis-ANS, and the presence of 1.5 M TMAO, respectively. In panel B, filled circles, open triangles, open circles, and filled triangles correspond to the control, the presence of bis-ANS, the presence of TMAO, and the presence of both bis-ANS and TMAO, respectively. Unfolding transition temperatures for control, for bis-ANS, and for TMAO were estimated to be 50.0 °C, 49.8 °C, and 61.4 °C, respectively.

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ATPase activity of HMM in the absence of actin filaments (Fig. 4A), although in the presence of 0.6 M urea, ATPase activity was nearly independent of TMAO concentration. In contrast, the ATPase activity of acto-HMM was drastically decreased from 1.3 to 0.6 s − 1 as TMAO concentration increased to 0.2 M (Fig. 4B). In this case, the decreased level of ATPase activity observed in the presence of 0.6 M urea was further reduced following the addition of increasing concentrations of TMAO. Similarly, in the presence of 1.0 M urea, the ATPase activity of the HMM molecules decreased considerably as TMAO concentration increased, ultimately reaching 0.3 s− 1. To determine the kinetic parameters Km and Vmax, the dependence of ATPase activation on actin concentration was also examined. Fig. 5 shows that the Km of solutions in the presence of 0.2 M TMAO was somewhat higher than that in the absence of osmolytes, whereas the Vmax was lowered to half the original value. This result indicates that TMAO exhibits noncompetitive inhibition of ATPase activation. The presence of both TMAO and urea induced a further reduction in Vmax and an increase in Km.

3.3. Effects of TMAO and urea on the motility of actin filaments on HMM molecules The sliding movement of rhodamine-phalloidin-bound actin filaments propelled by HMM with ATP hydrolysis was observed under a fluorescence microscope. Fig. 6 shows the velocities of the filaments at various concentrations of TMAO and urea. The presence of 0–1.5 M TMAO in the absence of urea was found to gradually decrease sliding velocity from 5 to 0 μm/s, with the velocity being completely suppressed by 1.5 M TMAO. Although velocity was also decreased with an increase in urea concentration, the decrease in velocity observed in the presence of 0.6 M urea was somewhat restored by the addition of 0–0.3 M TMAO; however, the complete suppression of velocity by 1.0 M urea could no longer be offset by TMAO in the concentration range of 0–1.5 M. Other aspects of actin filaments were also examined in the presence of TMAO. For instance, the actin filaments appeared to shrink considerably in the presence of TMAO under our in vitro motility assay conditions when compared to those in samples without TMAO (Fig. 7A, B). In other words, it is likely that some bending or winding partially occurs within single filaments in this situation. Fig. 7C shows that the presence of 1.5 M TMAO also increased the fraction of filaments with a much shorter end-to-end distance than contour length, which is suggestive of distortion of the filaments.

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Concentration of TMAO [M] Fig. 4. ATPase activities of HMM alone (A) and acto-HMM (B) in the presence of TMAO and urea. Filled circle symbols correspond to findings in the absence of urea. Open circle and open triangle symbols correspond to findings in the presence of 0.6 M urea and 1.0 M urea, respectively. Error bars indicate the standard deviation estimated from 3 independent experiments performed under similar conditions.

Finally, the dwell time for the dissociation of single actin filaments from individual HMM molecules in the absence of ATP was directly measured using a fluorescence microscope. Dissociation rate constants were estimated from the relationship between dwell time and event number. Fig. 8A shows that both TMAO and urea induced an increase in the dissociation rate constant, with TMAO unexpectedly eliciting a greater increase than urea. However, the coexistence of TMAO and urea mitigated the individual effects of the osmolytes (Fig. 8B).

4. Discussion 4.1. Stabilization of the HMM structure and suppression of acto-HMM activity in the presence of TMAO In general, a high concentration of urea denatures proteins, whereas TMAO prevents this urea-induced denaturation. At low concentrations (below 1.0 M), urea induces a modest, reversible loosening of the HMM structure [16,23]. In the present study, using a bis-ANS fluorescence method, we showed that the presence of urea in a solution containing HMM increased the fluorescence intensity of the sample, whereas TMAO decreased the intensity. This result indicates that the effect of TMAO on the HMM structure is opposite to that of urea. However, the bis-ANS fluorescence change of actin filaments in the presence of

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Concentration of actin [mg/ml] Fig. 5. Dependence of ATPase activity on actin concentration in the presence of 0.2 M TMAO (open circle) or both 0.6 M urea and 0.2 M TMAO (open triangle). The filled circle symbols denote the absence of both osmolytes (control). Data are the average of 3 independent experiments. Each line is a fit to the Michaelis–Menten equation. Inset: Double reciprocal plot of the same data. Maximum activities detected in the 0 M TMAO (control), 0.2 M TMAO, and 0.2 M TMAO with 0.6 M urea samples were estimated to be 8.6 s− 1, 4.2 s− 1, and 1.3 s− 1, respectively, whereas Km values were determined to be 34 μM, 47 μM, and 16 μM, respectively.

TMAO was at a barely detectable level. Further, CD spectroscopy showed that the spectrum for HMM in the presence of bis-ANS, as well as its temperature dependence, were indistinguishable from HMM in the absence bis-ANS, suggesting that bis-ANS had little effect on the secondary structure of HMM and its stability under our assay conditions. In addition, the finding that TMAO did not alter the CD spectrum of HMM indicates that the secondary structure of this protein was maintained in its native form. Therefore, the TMAO-induced decrease in the bis-ANS intensity of HMM may reflect decreased hydrophobicity around bis-ANS bound to HMM because of further peripheral hydration of HMM in the presence of TMAO. At the same time, the increase in the unfolding transition temperature observed by CD spectroscopy suggests that TMAO induced the stabilization of the structure of HMM, which is similar to the effects of TMAO on lysozyme at neutral pH as previously reported by Singh et al. [11]. The ATPase activity of HMM in the absence of actin filaments was also shown to be enhanced by TMAO in this study, which is consistent with the results of a previous report [15]. The stabilization of the HMM structure may contribute to the ATPase activity. In contrast, actin-activated ATPase activity was drastically decreased with increasing TMAO concentrations. Furthermore, the fact that Vmax declined with little change of Km in the presence of TMAO indicates that TMAO may suppress the rate-limiting step of the ATPase cycle. This behavior differs from that of urea, which is more effective at increasing Km than at varying Vmax [16]. The rate-limiting step for the formation of the actomyosin complex seems to occur in the transition from a weak to strong binding state following the force generating step, in which the interaction is mainly hydrophobic [24,25]. Because TMAO does not affect hydrophobic interactions [26], it is unlikely to affect the affinity of actomyosin complexes during this transition state. In view of the stabilization of the HMM structure by TMAO, the conformational change of the myosin heads during the rate-limiting step may not be able to readily take place in the presence of TMAO. In addition to the inhibition of the actin-activated ATPase activity of HMM, the sliding velocity was also decreased with an increase in TMAO concentration. However, at a TMAO concentration of approximately 0.2 M, the degree of the decrease in the ATPase activity (52%)

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was greater than the 30% decrease in velocity. This difference can be explained by the relationship between enzymatic and motile activities. In fact, Amitani et al. have previously proposed that the sliding velocity in various actomyosin systems is proportional to the square root of Km multiplied by the Vmax of actin-activated ATPase activity [27]. This relationship indicates that the decreasing rate of velocity is lower than the decrease in Vmax if Km is unchanged. Actin filaments in the presence of 1.5 M TMAO were also observed to undergo winding without movement under our in vitro motility conditions. This feature was unique to samples containing TMAO. In this situation, actin filaments remained bound to HMM, although they failed to move. If TMAO causes the force-generating step to become slower, the uniformity of the driving forces may collapse within single filaments, resulting in deformation of the filaments such as winding distortions. Unexpectedly, the rigor of actomyosin complex binding was also found to be weakened by the presence of TMAO, although the actin filaments were shown to be bound to HMM in the presence of ATP. Therefore, the effect of TMAO on the binding of actin filaments to HMM in the absence of ATP may be distinct from its effect on binding during ATP hydrolysis. Here, we attempted to compare our findings with the thermodynamic behavior of actomyosin molecules reported by other groups. For instance, it has been reported that binding between actin filaments and the heads of skeletal muscle myosin II occurs through an endothermic process (positive enthalpy change) in the absence of ATP [28]. Thus, this spontaneous binding process must be compensated for by positive changes in entropy, such as increases in dehydration and degrees of freedom. The increased dissociation rate of actomyosin that was observed to occur in response to TMAO in the absence of ATP in this study may be because of the reinforcement of HMM structural stability by TMAO, which could act against the endothermic process because of the accompanying changes in intramolecular interactions. In contrast, the TMAO-enhanced ATPase activity of HMM in the absence of actin filaments can be explained by the facilitation of the Pi release process, which is accompanied by decreases in enthalpy and entropy [29]. With respect to actomyosin complexes, the TMAO-induced decreases in ATPase activation and motility may result from the inhibition of the myosin isomerization step that occurs before Pi release, in which the increases in enthalpy and entropy take place. If this is the case, the endothermic processes that occur with the increase in entropy may be particularly

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Concentration of TMAO [M] Fig. 6. Sliding velocities of rhodamine-phalloidin-bound actin filaments on an HMMcoated glass surface in the presence of TMAO and urea. Filled circle symbols denote findings in the absence of urea. Open triangle, filled triangle, open circle, and filled square symbols denote findings in the presence of 0.1 M, 0.3 M, 0.6 M, and 1.0 M urea, respectively. Error bars indicate the standard deviation estimated from 3 independent experiments performed under similar conditions. For each experiment, the results from 100 filaments were averaged.

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Fig. 7. Typical fluorescence images of rhodamine-phalloidin-bound actin filaments on HMM-coated glass surfaces in the absence (A) and the presence of 1.5 M TMAO (B). The scale bar indicates the distance of 20 μm. The contour length versus the end-to-end distance of actin filaments (C). Filled circle and open circle symbols denote findings without osmolytes and in the presence of 1.5 M TMAO, respectively.

suppressed by TMAO through its stabilization of the HMM structure and the configuration or internal strain of motor domains due to peripheral hydration. 4.2. Counteractive effects of TMAO and urea on acto-HMM motor activity The ability of TMAO to counteract the actions of urea has been widely investigated because the interaction between proteins and osmolytes that intervene in hydrogen bonding is thought to characterize the nature of protein folding and stability. Our fluorescence study revealed that the destabilization of the HMM structure induced by 0.6 M urea was recovered with 0.3 M TMAO. Likewise, CD spectroscopy showed that approximately 0.3 M TMAO was able to recover the decrease in the Tm of HMM elicited by 0.6 M urea. In addition, TMAO-enhanced HMM ATPase activity was returned to its original level when 0.6 M urea was added. Although the affinity of actin filaments for HMM under rigorous binding conditions was decreased by the presence of either urea or TMAO, this affinity was recovered in the presence of both TMAO and urea. Thus, it was confirmed that a 2:1 ratio of urea to TMAO is highly effective for their counteraction, as has been widely shown in various previous reports [1,6,8,30]. These data suggest that the counteraction of the effects of urea by TMAO primarily occurs when HMM is inactivate, in the absence of either actin filaments or ATP. Furthermore, counteractive effects were also found in the sliding velocity of actin filaments in the presence of 0.6 M urea and 0.2–0.3 M TMAO (nearly a 2:1 ratio), although TMAO could not induce the recovery of motility with other concentrations of urea. Moreover, the absolute velocity of actin filaments in the presence of 0.6 M urea and 0.2 M TMAO was lower than in the presence of 0.2 M TMAO alone, indicating that, under our experimental conditions, the counteractive effects of TMAO did not achieve complete recovery of the motile activity of the actomyosin motor after it was impaired by urea. Moreover, when the actin concentration-dependence of ATPase activity was examined, the value of Vmax was shown to decrease even in the presence of both TMAO and urea. The counteraction of the effects of urea by TMAO was not observed in the process of ATPase activation. The additive inhibitory effect of urea and TMAO on ATPase activation, rather than counteraction, could be because of the specific characteristics of the actomyosin motor, similar to what has been observed with regard to aldose reductase activity [31]. A comparison between the concentration dependence of the effects of urea and TMAO on the sliding velocity of actin filaments

(Fig. 9A) showed that TMAO induced greater inhibition of motility than urea at concentrations below 0.6 M. In this case, the counteraction of the effects of urea by TMAO was not observed, because motility was more strongly inhibited by TMAO than by urea. At a urea concentration of 0.6 M, the inhibitory strength of urea became equal to that of TMAO, and counteractive effects on motility were observed again. At 1.0 M urea, motility could no longer be recovered by 0–1.0 M TMAO because urea was more effective than TMAO at this concentration. On the other hand, actin-activated ATPase activity remained decreased in the presence of both osmolytes because TMAO was more effective in decreasing this activity than urea at all concentrations tested (Fig. 9B). We therefore speculate that the dynamic motility inherent to the force generation process may be because of both the mobility of water molecules on the actomyosin surface and the actions that occur through the ATPase activation process, such as the structural changes of myosin heads. At this point, we cannot clearly account for the differences observed in the counteractive effects of TMAO and urea on the HMM structure and actomyosin interactions. Actomyosin interactions seem to be more sensitive to TMAO than their structural stability. Although our spectrum data showed the same optical signals with and without urea–TMAO at a 2:1 molar ratio, the molecular dynamic properties of HMM molecules can be modified by TMAO or urea by hydration or solvation in their local regions. More details on the conditions associated with actomyosin complexes, TMAO, and urea-containing waters are necessary to interpret the nonlinearity of the counteractive effects of TMAO and urea on motility and ATPase activation. From a biological standpoint, these osmolytes are physiologically important in water-stressed animals. In particular, concentrations of urea up to 0.6 M and TMAO up to 0.3 M (at an approximately 2:1 molar ratio) exist in the muscular tissues of cartilaginous marine fishes such as dogfishes and rays. These concentration ranges are in accord with the range in which the recovery of the motility of actomyosin motors is possible, although we examined actomyosin obtained from rabbit skeletal muscle. Therefore, the presence of these osmolytes at relatively low concentrations (below 1.0 M), at which the unfolding of the ternary structure of proteins cannot occur, may be able to fundamentally modulate the actomyosin interactions that accompany hydration changes. However, the effects of TMAO and urea on muscular activities may be somewhat controlled by other factors, given the presence of a variety of proteins and ions in the tissues.

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Dwell time [s] Fig. 8. Dwell time of single actin filaments bound to individual HMM molecules in the absence of ATP. Filled circle symbols denote findings in the absence of both urea and TMAO. Filled triangle, open circle, and open triangle denote findings in the presence of 1.0 M urea, 1.0 M TMAO, and both 1.0 M urea and 1.0 M TMAO, respectively. Each line is a fit to the equation N(t) = N0 exp (− kofft). The dissociation rate constants (koff) determined under control conditions, in the presence of 1.0 M urea, in the presence of 1.0 M TMAO, and in the presence of a mixture of these osmolytes were estimated at 0.026 s− 1, 0.036 s− 1, 0.067 s− 1, and 0.032 s− 1, respectively.

Fig. 9. Comparison of concentration dependence of the effects of urea and TMAO on the sliding velocity of actin filaments (A) and actin-activated ATPase activity (B). Filled circles and open circles denote findings from samples containing TMAO and urea, respectively. Sliding velocities and actin-activated ATPase activities for each condition were normalized to the original velocity and original activity in the absence of osmolytes, respectively (see data in Figs. 4 and 6). Additional lines indicate reciprocal fit curves without rational explanation.

Conflict of interest None declared.

5. Conclusion In this study, we showed that TMAO essentially decreased the sliding velocity and ATPase activation of acto-HMM complexes, although this osmolyte induced the stabilization of the HMM structure and enhanced the ATPase activity of HMM alone. The activity and interactions of acto-HMM complexes may be suppressed in response to TMAO because of a decrease in their molecular dynamics that results from structural stabilization. Furthermore, TMAO was shown to counteract the deleterious effects of urea on the structure of HMM. Nonetheless, the urea-induced decrease in actin-activated ATPase activity could not be recovered by the addition of TMAO. Although 0.2 M TMAO was observed to counteract the effects of 0.6 M urea on motility, the absolute value of velocity in this condition was lower than that observed in the presence of 0.2 M TMAO alone. Moreover, the counteraction of the effects of urea by TMAO did not occur in the concentration range in which TMAO was more effective at suppressing motility and ATPase activation than urea. This phenomenon was not coupled with the recovery of stability of the HMM structure.

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