# Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains

^{1}

^{2}

^{*}

## Abstract

**:**

## 1. Introduction

## 2. New Approach to Analysis of Irreversible Denaturation of Multidomain Proteins

#### 2.1. Theory

_{a}is the experimental energy of activation, R is the universal gas constant, T is the absolute temperature, and T

^{*}is the temperature at which k = 1 min

^{−1}(the dimension of k in this formula is min

^{−1}). The kinetics of denaturation is described by the equation:

^{ex}is the excess heat absorbed at denaturation. This dependence may be presented in an expended form:

_{a}and T* from the experimental thermogram. From Equations 4 and 6 we can obtain the expression for the rate constant k as a function of temperature:

_{i}is the enthalpy of denaturation of i-th domain. Preliminary incubation of such a protein at a certain constant temperature over some period of time just before the calorimetric experiment (denoted below in the text as annealing) results generally in partial denaturation of each domain. The shape of calorimetric peak for each domain remains unchanged, however the decrease in the registered enthalpy of the transition occurs: ΔH

_{i},

_{ann}= x

_{i}· ΔH

_{i}, where x

_{i}is a portion of i-th domain remaining in the native form after the annealing (0 < x

_{i}< 1). It follows herefrom that ${C}_{\text{p},\hspace{0.17em}i,\hspace{0.17em}\text{ann}}^{\text{ex}}(T)={x}_{i}\cdot {C}_{\text{p},\hspace{0.17em}\hspace{0.17em}i}^{\text{ex}}(T)$. Thus, thermogram of the whole protein is expressed as follows:

_{ann}gives us the x

_{i}value: x

_{i}= exp[–k

_{i}(T

_{ann}) · t

_{ann}], where k

_{i}(T

_{ann}) is the rate constant for denaturation of i-th domain at the temperature T

_{ann}calculated from the Arrhenius equation (2). Thus, x

_{i}is the function depending on t

_{ann}and T

_{ann}and parametrically depending on E

_{a,i}and ${T}_{i}^{*}$ (i.e., parameters of the Arrhenius equation for i-th domain).

_{i, N}≡ 1 is valid).

_{1,}

_{j}< x

_{2,}

_{j}< . . . < x

_{N, j}. From the theoretical point of view, if only one of parameters E

_{a}and T

^{*}for (i – 1)-th and i-th calorimetric domains has different values (otherwise these domains should be considered as a single domain), it is possible to select the temperature and time of annealing, at which x

_{i}

_{–1,}

_{j}≈ 0 (in practice, the inequality x

_{i}

_{–1,}

_{j}< 0.01 should be met) and x

_{i}

_{,}

_{j}≫ 0. From the predicate of the numeration of domains follows that under these conditions of incubation 1

^{st}, 2

^{nd}, . . . , (i – 2)-th domains are also annealed, i.e., x

_{1,}

_{j}, x

_{2,}

_{j}, . . . , x

_{i}

_{–2,}

_{j}≈ 0. Thus, it is possible to select the set of annealing conditions that makes the system of equations (13) transformed into the triangle form:

_{a}and T

^{*}for the most thermostable domain to be estimated. With a knowledge of parameters E

_{a}and T

^{*}, we can calculate x

_{N}(T) and k

_{N}(T). The optimization of the whole thermogram by thermogram corresponding to the most thermostable domain in the coordinates ${C}_{\text{p}}^{\text{ex}}(T)$ vs (k

_{N}(T) · x

_{N}(T)/ν) (see Equation 6) in this region of temperatures using the least squares method allows parameter ΔH

_{N}to be determined.

_{a, N}, ${T}_{N}^{*}$ and ΔH

_{N}. With a knowledge of these parameters, we simulate ${C}_{\text{p},\hspace{0.17em}\hspace{0.17em}N}^{\text{ex}}(T)$ and subtract it from the whole thermogram of protein. Then this procedure is repeated for the function $({C}_{\text{p}}^{\text{ex}}(T)-{C}_{\text{p},\hspace{0.17em}\hspace{0.17em}N}^{\text{ex}}(T))$, which is the overall thermogram of all domains except for the most thermostable domain. As a result we obtain parameters for (N – 1)-th domain, simulate function ${C}_{\text{p},\hspace{0.17em}\hspace{0.17em}\hspace{0.17em}N-1}^{\text{ex}}(T)$ and subtract it from the function ( ${C}_{\text{p}}^{\text{ex}}(T)-{C}_{\text{p},\hspace{0.17em}\hspace{0.17em}N}^{\text{ex}}(T)$). By repeating this procedure we estimate parameters for (N – 2)-th domain, (N – 3)-th domain and so on down to first calorimetric domain.

_{a}, T

^{*}and ΔH for all calorimetric domains.

#### 2.2. Resolving Power of the Method. Selection of the Annealing Conditions

_{a}or T

^{*}for two domains are non-identical, the conditions of annealing may be selected at which x

_{i}

_{–1}≈ 0 and x

_{i}> 0. However, the acceptable values of x

_{i}

_{−1}and x

_{i}are the following: x

_{i}

_{−1}< 0.01 and x

_{i}> 0.1. Otherwise the determination of parameters of i-th domain becomes impossible. The acceptable time of annealing is at least 10 min. If time of annealing is less than 10 min, delayed heating of the sample to the temperature of incubation and following cooling inevitably lead to an unacceptably large error. In other words, the following conditions should be satisfied:

^{*}are about 320 K, after the substituting of the Arrhenius equation in the expressions for the corresponding rate constants and solving the system of inequalities (15) we obtain the following limitations. For the case of identical T

^{*}values the minimum reasonable difference between parameters E

_{a}is ∼150 kJ/mole. For the case of identical values of the activation energy the difference ${T}_{i}^{*}-{T}_{i-1}^{*}$ should not be less than 2 K.

_{ann}and t

_{ann}. For definiteness we fix the ratio x

_{i}

_{−1}/x

_{i}≡ A (with the requirements for x

_{i}

_{−1}and x

_{i}we obtain that A < 0.1). Finally we come to the system of inequalities:

_{ann}is a curve t

_{ann}= f(T

_{ann}) established by the conditions:

## 3. Results and Discussion

#### 3.1. Thermal Denaturation of Myosin S1 Isoforms Studied by DSC

_{a}, T

^{*}and ΔH

_{trans}(the area under the experimental ${C}_{\text{p}}^{\text{ex}}$ curve) parameters can be easily found. Taking into account the conditions of annealing, we can calculate the ΔH for this domain using equation:

_{ann}and T

_{ann}are the annealing time (in min) and temperature (in K), respectively. Knowing all three parameters of the calorimetric domain, we can simulate its thermogram. Figure 1A shows comparison of such a simulated thermogram of the most thermostable domain (red curve) with the thermogram of initial S1(A1) preparation obtained before the annealing procedure (black curve). Obviously, the thermogram of one of the calorimetric domains should be the part of the thermogram of the entire protein. It is seen from Figure 1A that this is not the case. Similar results were obtained for S1(A1) under low ionic strength conditions, as well as for S1(A2) independently of ionic strength (data not shown).

_{1}and k

_{2}obey the Arrhenius law (Equation 2), and both stages are accompanied by positive thermal effect (ΔH

_{1}, ΔH

_{2}> 0). Correspondingly, the thermogram of such a domain can be described as follows

_{N}is the portion of the native state and x

_{I}is the portion of the kinetic intermediate state:

_{0}. Obviously, before annealing, ${x}_{N}^{0}=1$ and ${x}_{I}^{0}=0$ (i.e., this domain is in its native state in all protein molecules). However, this is not the case for protein preparations which have undergone the annealing procedure. In this case, both ${x}_{N}^{0}$ and ${x}_{I}^{0}$ can be easily calculated by solving a system of differential equations corresponding to the model of thermal denaturation (19) and by finding parameters k

_{1}and k

_{2}from the Arrhenius equation (Equation 2). It is evident that, under some rather hard conditions of the annealing, we can obtain the protein preparation, in which ${x}_{N}^{0}\approx 0$ and ${x}_{I}^{0}\ne 0$. The thermogram for such type preparation can be described by the following equation:

_{0}on the DSC profile, for protein preparation that has not been subjected to annealing procedure. Therefore, when the above-proposed algorithm is applied for analysis of the high-temperature region of the S1 thermogram, we can erroneously consider the second stage of the thermal denaturation of the most thermostable S1 domain as independent calorimetric domain. This indirectly evidences the model of S1 thermal denaturation involving two calorimetric domains, one of which denatures in two stages thus yielding two thermal transitions. Since independent calorimetric domains often correspond to independent structural domains, two S1 calorimetric domains may reflect the thermal denaturation of the main S1 structural domains, the motor domain and the regulatory domain, and this may again indicate that the two-domain model of the S1 thermal denaturation is correct.

_{a}) and special temperature (T

^{*}, i.e., the temperature at which the rate constant is equal to 1 min

^{−1}).

^{*}of the least thermostable calorimetric domain (transition 1 in Figures 2, 3 and domain I (or transition 1) in Table 1). This parameter is lower by 1.5–2.0 K in the case of S1(A1) in comparison with S1(A2) (Table 1).

^{*}parameter of domain I, we have performed additional optimization procedure, as follows. All calorimetric parameters (except only T

^{*}of domain I) were allowed to change and domains’ I T

^{*}values were set equal to the average value among S1(A1) and S1(A2) (318.3 K under high ionic strength conditions) and frozen. The parameters obtained after such an optimization were used to simulate the DSC profiles of S1 isoforms after control annealing (17 min at 44 °C) (curves 3 on Figure 4), and these simulated DSC curves were compared with the experimentally obtained DSC profiles (curves 1 on Figure 4). Figure 4 also shows the DSC curves simulated using all the parameters given in Table 1 (curves 2). It is seen from Figure 4 that curves 2 almost completely coincide with experimentally obtained curves 1, whereas curves 3 simulated according to assumption of domains’ I T

^{*}values equality are quite different from curves 1. Thus, we can conclude that this assumption is erroneous, and therefore the difference in domains’ I T

^{*}values between S1 isoforms is reliable. This means that domain I is more thermostable in S1(A2) than in S1(A1). It should be noted that this effect is independent of ionic strength conditions as it is observed both at low ionic strength and in the presence of 100 mM KCl.

#### 3.2. Thermally-Induced Changes in the Intrinsic Fluorescence of S1 Isoforms

_{320}and I

_{365}are fluorescence intensities at λ

_{em}= 320 and 365 nm, respectively) from 1.05–1.07 to 1.30–1.35. This means that the environment of tryptophan residues (at least some of them) becomes more hydrophobic after irreversible thermal denaturation. The only explanation for this strange behavior of S1 is that its irreversible thermal denaturation cannot be simply considered as trivial unfolding. Apparently, we are still far from detailed understanding of the mechanism of irreversible thermal denaturation of proteins.

_{min}and A

_{max}represent minimal and maximal values of the parameter A, respectively) and compared the fraction of conversion from native to denatured state calculated using this fluorescent parameter with that obtained from the DSC data for calorimetric domains of S1 (Figure 8). It is clearly seen that, for both S1 isoforms, the temperature-induced changes of parameter A occur within the temperature region corresponding to calorimetric transitions 2 and 3, i.e., in the region of thermal denaturation of the second calorimetric domain, while no changes of parameter A are observed in the region of thermal denaturation of calorimetric domain I (thermal transition 1) (Figure 8). Taking into account the fact that all tryptophan residues are localized in the motor domain of the S1 molecule (Figure 5), our results allow to identify the second calorimetric domain (involving thermal transitions 2 and 3) as the motor domain and calorimetric domain I—as the regulatory domain.

#### 3.3. Heat-Induced Inactivation of S1 ATPase

_{a}and T

^{*}obtained from the DSC studies (Table 1). Comparison of these rate constants with those calculated for ATPase inactivation of S1 isoforms is presented in Figure 10B, C. It is clearly seen from this comparison that the rate constants of ATPase inactivation are very close to those calculated for thermal denaturation of calorimetric domain II. Since the ATPase site is localized in the motor domain of the S1 molecule (Figure 5), these results, in good agreement with those obtained from experiments on S1 intrinsic tryptophan fluorescence (Figures 7 and 8), are in favor of correspondence of calorimetric domain II to the motor domain of S1.

#### 3.4. Heat-Induced Aggregation of S1 Isoforms

_{h}) of the formed aggregates, did not depend on the S1 concentration within a rather wide range, from 0.125 to 2.0 mg/mL (data not shown). This effect was also observed with separate S1 isoforms. Figure 11 shows, in semi-logarithmic coordinates, the typical dependences of R

_{h}value for both S1 isoforms on the time of their incubation at 44 °C. These dependences are well described by the following exponents:

_{0}is the delay time before the beginning of R

_{h}growth (i.e., lag period of the aggregation), ${R}_{\text{h}}^{0}$ is the initial hydrodynamic radius (at t = t

_{0}), and t

_{2R}is the time interval over which the hydrodynamic radius of the protein aggregates increases twofold. The parameter t

_{2R}characterizes the rate of aggregation [27]. The higher the t

_{2R}value, the lower is the rate of aggregation. One can see that straight lines obtained at different protein concentrations are in parallel and differ from each other only by shift along the abscissa axis (Figure 11). The observed shift of the lines is suggested to be due to some uncertainty caused by preliminary heating of the protein solution in the instrument from storage temperature (4 °C) to 44 °C, i.e., the temperature, at which the kinetics of R

_{h}growth was recorded. We performed these experiments several times and only the slope of the lines was reproducible but not their absolute location on the abscissa axis. The fact that lines in both Figure 11A and Figure 11B have the same slope indicates that the parameter t

_{2R}is the same for all the concentrations studied. Optimal values of this parameter have been found as described in Experimental Section (see subsection 4.7). Within the error limit (confidence interval of 95%), these parameters were identical at all protein concentrations and equal to 4.5 ± 0.4 min for both S1 isoforms. Hence, we can conclude that the S1 aggregation is limited by some first-order reaction. For better understanding the mechanism of S1 thermal aggregation, we investigated the dependence of the t

_{2R}value on temperature. Figure 12 shows, in semi-logarithmic coordinates, the dependences of R

_{h}value for the both S1 isoforms on the time of their incubation at different temperatures. It is clearly seen that the rate of aggregation increases upon the increase in the temperature. At all temperatures studied, the kinetic curves of R

_{h}growth are well described by Equation 26, thus allowing us to calculate the t

_{2R}values at different temperatures. The temperature dependences of the t

_{2R}values for both S1 isoforms are presented on Figure 13 (black lines). These dependences are compared with those of the half-life values

_{2R}values coincide with the t

_{0.5}values calculated for the first stage of thermal denaturation of calorimetric domain II, which was identified above as the motor domain of the S1 molecule.

_{a}and T

^{*}of the Arrhenius equation (Equation 2) for the R

_{h}growth process of S1 aggregates. The E

_{a}value was equal to 430 ± 50 kJ/mole for S1(A1) and 440 ± 70 kJ/mole for S1(A2), and T

^{*}was equal to 320.8 ± 0.6 K for both S1 isoforms. It is seen that both these parameters agree with those obtained from DSC experiments for the first stage of denaturation of calorimetric domain II (see Table 1). Thus, just this reaction, i.e., the first stage of denaturation of calorimetric domain II, limits the overall process of S1 thermal aggregation under high ionic strength conditions (in the presence of 100 mM KCl).

_{0}value), we also investigated the temperature dependences of R

_{h}growth upon heating the S1 isoforms with constant rate of 1 °C/min under high ionic strength conditions (in the presence of 100 mM KCl). These dependences were the same for both S1 isoforms and independent of the protein concentration within the range from 0.25 to 1.0 mg/mL up to R

_{h}values of 3000 nm (data not shown). Figure 14 shows initial parts of the temperature dependences of R

_{h}growth (up to 250 nm) in comparison with fractions of conversion for calorimetric transitions 1, 2, and 3 obtained from DSC experiments (these curves were calculated as it was described in the legend to Figure 8). It is clearly seen that for both S1(A1) and S1(A2) a few degrees delay is observed between the process of R

_{h}fast growth and the first stage of thermal denaturation of the calorimetric domain II (transition 2) that limits the overall aggregation process. This observation leads to the conclusion that nucleation (i.e., formation of start aggregates that are seeds for following aggregation [27]) is an important stage of the overall process of the S1 thermal aggregation. Obviously, formation of necessary amount of these seeds needs some time, which accounts for the observed delay in the aggregation process.

_{h}fast growth (black points and curve in Figure 14), and the replacement of the red shift by the blue shift at higher temperature region. Thus, the temperature dependence of parameter A would be non-monotone. However, it is clearly seen from Figure 8 that parameter A grows with temperature monotonely. Moreover, the changes in parameter A occur in the temperature interval between calorimetric transitions 2 and 3, so slightly outstrip the growth of the aggregates. Despite the fact that described investigations of temperature dependences of S1 fluorescent properties and its thermal aggregation were carried out at different protein concentration (see Experimental Section), we suggest that the comparison of intrinsic fluorescence data and DLS data is reasonable. We observed that pronounced aggregation of both S1 isoforms occurred in the same temperature intervals for both high and low (0.05 mg/mL, as in the fluorescence studies; data not shown) S1 concentrations. Moreover, the earlier investigations revealed that the blue shift of S1 intrinsic fluorescence spectrum also takes place at protein concentration of 0.5 mg/mL and it also has a monotonic character [16]. Therefore, we have to conclude that observed blue shift of S1 intrinsic fluorescence spectrum is due to changes in the protein molecule occurring in the process of its irreversible thermal denaturation but not its aggregation.

_{0}in the Equation 26. The following growth of the start aggregates is realized by the attachment of the individual S1 molecules, whose motor domains are on the stage of kinetic intermediate (see scheme 19), to these aggregates. It should be noted that the rate of this sticking process is much higher than the rate of the first stage of thermal denaturation of the S1 motor domain, which is the rate-limiting step for the whole process of the growth of aggregates. Further denaturation of the motor domain (i.e., the second stage of this process) probably occurs after inclusion of such partially denatured S1 molecules into the aggregates.

## 4. Experimental Section

#### 4.1. Protein Preparations

^{1%}at 280 nm of 7.5 cm

^{−1}. S1 preparation was separated into S1(A1) and S1(A2) isoforms by means of ion exchange chromatography on a column of SP-trisacryl [28]. All proteins were homogeneous according to SDS-PAGE [29]. Both preparations of S1 isoforms were homogeneous and no proteolysis of either heavy or light chains was observed.

#### 4.2. Differential Scanning Calorimetry (DSC)

_{2}in the presence or absence of 100 mM KCl. In order to check the reversibility of thermal denaturation after the first scan and subsequent cooling, the protein samples were reheated. Thermal denaturation of both S1 isoforms was fully irreversible. Calorimetric traces were corrected for instrumental background using special DSC approach described earlier [33]. Briefly, DSC measurements were performed not only in the 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 sample in the reference cell and the buffer in the sample cell. This inverted curve was then subtracted from the curve obtained by the usual way. The above DSC approach allowed us to subtract the instrumental baseline with very high precision [33]. Annealing procedures were performed by pre-heating the protein samples at appropriate temperature for definite period of time just before the DSC measurements (the temperature and time for these procedures were calculated as described in Section 2). All calorimetric traces were subjected to time response correction as described in [34] before the further analysis. Chemical base line correction was performed using the approach described below.

#### 4.3. Estimation of Chemical Base Line in DSC Experiments

_{p, i}(T)). Besides, to decompose Equation 30 into its components namely the equations of a type of Equation 28, the mechanism of thermal denaturation should be established. To work out this problem, we need the function ${C}_{\text{p}}^{\text{ex}}(T)$ to be known.

_{p, i}(T) to the corresponding coefficient of ∑

_{i}ΔC

_{p, i}(T) are identical and equal to the ratio ΔH

_{i}/∑

_{i}ΔH

_{i}. The above-mentioned assumption has a definite sense. Actually, the more bonds are broken during a given transition, the higher is the contribution of this transition in the overall enthalpy of a whole process. However, the more broken bonds, the more new degrees of freedom appear and, correspondingly, the higher is the heat capacity jump. It should be noted, however, that this relationship may be not linear.

_{p}value, the inaccuracy introduced in the ${C}_{\text{p}}^{\text{ex}}(T)$ value by this approximation should be not high.

_{p}(T). The first point (T

_{1}) corresponds to the beginning of the linear part of the thermogram before the peak of excess heat absorption, the second point (T

_{2}) corresponds to the beginning of the peak and simultaneously the end of the linear part before the peak, the third point (T

_{3}) corresponds to the end of the peak and the beginning of the linear part after the peak and the fourth point (T

_{4}) corresponds to the end of the linear part after the peak. The linear part before the peak is taken as ${C}_{\text{p}}^{N}(T)$. The linear part after the peak is taken as ${C}_{\text{p}}^{D}(T)$. Further we determine the corresponding coefficients of the linear approximation of ${C}_{\text{p}}^{N}(T)$ and ${C}_{\text{p}}^{D}(T)$. The obtained coefficients are used for the calculation of ${C}_{\text{p}}^{N}(T)$ and ${C}_{\text{p}}^{D}(T)$ in the temperature interval form first to fourth point (the calculated values of these functions are designated as ${C}_{\text{p},\hspace{0.17em}\text{calc}}^{N}(T)$ and ${C}_{\text{p},\hspace{0.17em}\text{calc}}^{D}(T)$, respectively). As a null approximation the chemical base line (designated as C

_{CBL}(T)) is taken equal to ${C}_{\text{p},\hspace{0.17em}\text{calc}}^{N}(T)$ in the interval from T

_{1}to T

_{2}. In the interval from T

_{3}to T

_{4}the chemical base line is taken equal to ${C}_{\text{p},\hspace{0.17em}\text{calc}}^{D}(T)$. As for the interval from T

_{2}to T

_{3}, the chemical base line is assumed to be the straight line connecting the second and third points on the experimental curve of heat absorption. Then we estimate the null approximations for ΔH and ξ(T):

_{1}to T

_{4}is calculated by the formula:

_{p, i}was independent of temperature. In our opinion, this approximation is too rough. This was the reason for the elaboration of a new modification of the method for the estimation of the chemical base line.

#### 4.4. Intrinsic Fluorescence

_{2}in the presence or absence of 100 mM KCl. Fluorescence was excited at 297 nm (slit width 5 nm) and recorded in the range of 310–395 nm (slit width 2.5 nm). The proteins samples were heated with constant rate of 1 °C/min from 20 °C to 70 °C, and the fluorescence intensities at 320 nm and 365 nm were recorded. The position and form of the fluorescence spectra were characterized by parameter A (see Equation 24) [36–38]. In order to qualitatively analyze the temperature dependence of intrinsic fluorescence we used the fluorescence phase plots (the dependence of fluorescence intensity at 365 nm on the intensity of fluorescence at 320 nm obtained at different temperatures) [23].

#### 4.5. ATPase Inactivation

_{2}and 100 mM KCl. S1(A1) or S1(A2) aliquots were heated at appropriate temperature for appropriate periods of time, then cooled and subjected to ATPase measurements. Experiments were performed at different temperatures within the range from 39 °C to 47 °C. The ATPase activity of S1 isoforms (K

^{+}-EDTA-ATPase) was determined by P

_{i}release [39] at 25 °C in the medium containing S1 (0.04 mg/mL), 1 mM ATP, 0.5 M KCl, 5 mM EDTA and 50 mM Tris-HCl (pH 7.5). Reaction was initiated by addition of ATP and stopped after 10 min of incubation by addition of HClO

_{4}to final concentration of 2.5%.

#### 4.6. Dynamic Light Scattering (DLS)

_{h}) upon incubation of S1 in 20 mM Hepes (pH 7.3) containing 1 mM MgCl

_{2}and 100 mM KCl. The kinetics of S1 isoforms’ aggregation was studied either at constant temperature of 44 °C and different protein concentrations, from 0.25 to 1.0 mg/mL, or at constant protein concentration of 0.5 mg/mL and different temperatures, from 39 °C to 47 °C. Temperature dependences of R

_{h}growth were studied upon heating of S1 isoforms’ preparations with constant rate of 1 °C/min in the same buffer as in the kinetic experiments.

#### 4.7. Calculation Procedures

_{a}and T

^{*}values calculated for the same transition of the same S1 preparation never exceeded 40 kJ/mole and 0.4 K, respectively. The main source of the enthalpy error is inexact subtraction of chemical base line. Selection of different linear segments on thermograms before and after the peak of excess heat absorption results in slightly different values of calorimetric enthalpy. In our experiments this difference never exceeded 10%. Thus, we assumed the following error values for the calorimetric parameters presented: 0.4 K for T

^{*}, 40 kJ/mole for E

_{a}and 10% of the value for ΔH.

_{2R}parameters for the S1 aggregates growth were found with 95% confidence interval, using the Trust-Region algorithm embedded into the Curve Fitting Toolbox in the Matlab environment.

_{a}and T

^{*}values given in Table 1. The errors of these rate constants and half-life times were calculated as indirect errors caused by the errors in calculated calorimetric parameters (shown in Table 1) and by the error in incubation temperature that was assumed to be equal to 0.5 °C.

_{h}growth were also estimated using the Trust-Region algorithm embedded into the Curve Fitting Toolbox in the Matlab environment. The errors of these parameters were calculated as indirect errors caused by the errors in calculated values of the t

_{2R}parameters and by the error in incubation temperature that was assumed to be equal to 0.5 °C.

## 5. Conclusions

## Acknowledgments

## References

- Rayment, I; Rypniewski, WP; Schmidt-Base, K; Smith, R; Tomchick, DR; Benning, MM; Winkelmann, DA; Wesenberg, G; Holden, HM. Three-dimensional structure of myosin subfragment 1: a molecular motor. Science
**1993**, 261, 50–58. [Google Scholar] - Rayment, I. The structural basis of the myosin ATPase activity. J. Biol. Chem
**1996**, 271, 15850–15853. [Google Scholar] - Uyeda, TQ; Abramson, PD; Spudich, JA. The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc. Natl. Acad. Sci. USA
**1996**, 93, 4459–4464. [Google Scholar] - Weeds, AG; Taylor, RS. Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin. Nature
**1975**, 257, 54–56. [Google Scholar] - Frank, G; Weeds, AG. The amino-acid sequence of the alkali light chains of rabbit skeletal muscle myosin. Eur. J. Biochem
**1974**, 44, 317–334. [Google Scholar] - Wagner, PD; Slayter, CS; Pope, B; Weeds, AG. Studies on the actin activation of myosin subfragment-1 isoenzymes and the role of the myosin light chains. Eur. J. Biochem
**1979**, 99, 385–394. [Google Scholar] - Chalovich, JM; Stein, LA; Greene, LE; Eisenberg, E. Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide. Biochemistry
**1984**, 23, 4885–4889. [Google Scholar] - Sutoh, K. Identification of myosin-binding sites on the actin sequence. Biochemistry
**1982**, 21, 3654–3661. [Google Scholar] - Trayer, IP; Trayer, HR; Levine, BA. Evidence that the N-terminal region of A1-light chain of myosin interacts directly with the C-terminal region of actin. A proton magnetic resonance study. Eur. J. Biochem
**1987**, 164, 259–266. [Google Scholar] - Hayashibara, T; Miyanishi, T. Binding of the amino-terminal region of myosin alkali 1 light chain to actin and its effect on actin-myosin interaction. Biochemistry
**1994**, 33, 12821–12827. [Google Scholar] - Andreev, OA; Saraswat, LD; Lowey, S; Slaughter, C; Borejdo, J. Interaction of the N-terminus of chicken skeletal essential light chain 1 with F-actin. Biochemistry
**1999**, 38, 2480–2485. [Google Scholar] - Pliszka, B; Redowicz, MJ; Stepkowski, D. Interaction of the N-terminal part of the A1 essential light chain with the myosin heavy chain. Biochem. Biophys. Res. Commun
**2001**, 281, 924–928. [Google Scholar] - Borejdo, J; Ushakov, DS; Moreland, R; Akopova, I; Reshetnyak, Y; Saraswat, LD; Kamm, K; Lowey, S. The power stroke causes changes in the orientation and mobility of the termini of essential light chain 1 of myosin. Biochemistry
**2001**, 40, 3796–3803. [Google Scholar] - Lowey, S; Saraswat, LD; Liu, H; Volkmann, N; Hanein, D. Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins. J. Mol. Biol
**2007**, 371, 902–913. [Google Scholar] - Mrakovcic-Zenic, A; Oriol-Audit, C; Reisler, E. On the alkali light chains of vertebrate skeletal myosin. Nucleotide binding and salt-induced conformational changes. Eur. J. Biochem
**1981**, 115, 565–570. [Google Scholar] - Levitsky, DI; Nikolaeva, OP; Vedenkina, NS; Shnyrov, VL; Golitsina, NL; Khvorov, NV; Permyakov, EA; Poglazov, BF. The effect of alkali light chains on the thermal stability of myosin subfragment 1. Biomed. Sci
**1991**, 2, 140–146. [Google Scholar] - Abillon, E; Bremier, L; Cardinaud, R. Conformational calculations on the Ala14-Pro27 LC1 segment of rabbit skeletal myosin. Biochim. Biophys. Acta
**1990**, 1037, 394–400. [Google Scholar] - Kurganov, BI; Lyubarev, AE; Sanchez-Ruiz, JM; Shnyrov, VL. Analysis of differential scanning calorimetry data for proteins. Criteria of validity of one-step mechanism of irreversible protein denaturation. Biophys. Chem
**1997**, 69, 125–135. [Google Scholar] - Levitsky, DI; Khvorov, NV; Shnyrov, VL; Vedenkina, NS; Permyakov, EA; Poglazov, BF. Domain structure of myosin subfragment-1. Selective denaturation of the 50 kDa segment. FEBS Lett
**1990**, 264, 176–178. [Google Scholar] - Levitsky, DI; Shnyrov, VL; Khvorov, NV; Bukatina, AE; Vedenkina, NS; Permyakov, EA; Nikolaeva, OP; Poglazov, BF. Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1. Eur. J. Biochem
**1992**, 209, 829–835. [Google Scholar] - Levitsky, DI. Domain Structure of the Myosin Head. In Soviet Scientific Reviews Section D: Physico-Chemical Biology; Harwood Academic Publishers GmbH: Newark, NJ, USA, 1994; Volume 12, pp. 1–53. [Google Scholar]
- Tong, SW; Elzinga, M. Amino acid sequence of rabbit skeletal muscle myosin. 50-kDa fragment of the heavy chain. J. Biol. Chem
**1990**, 265, 4893–4901. [Google Scholar] - Permyakov, EA; Burstein, EA. Some aspects of studies of thermal transitions in proteins by means of their intrinsic fluorescence. Biophys. Chem
**1984**, 19, 265–271. [Google Scholar] - Kuznetsova, IM; Stepanenko, OV; Stepanenko, OV; Povarova, OI; Biktashev, AG; Verkhusha, VV; Shavlovsky, MM; Turoverov, KK. The place of inactivated actin and its kinetic predecessor in actin folding-unfolding. Biochemistry
**2002**, 41, 13127–13132. [Google Scholar] - Khanova, HA; Markossian, KA; Kurganov, BI; Samoilov, AM; Kleimenov, SYu; Levitsky, DI; Yudin, IK; Timofeeva, AC; Muranov, KO; Ostrovsky, MA. Mechanism of chaperone-like activity. Suppression of thermal aggregation of beta-L-crystallin by alpha-crystallin. Biochemistry
**2005**, 44, 15480–15487. [Google Scholar] - Markossian, KA; Khanova, HA; Kleimenov, SYu; Levitsky, DI; Chebotareva, NA; Asryants, RA; Muronetz, VI; Saso, L; Yudin, IK; Kurganov, BI. Mechanism of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. Biochemistry
**2006**, 45, 13375–13384. [Google Scholar] - Markossian, KA; Yudin, IK; Kurganov, BI. Mechanism of suppression of protein aggregation by alpha-crystallin. Int. J. Mol. Sci
**2009**, 10, 1314–1345. [Google Scholar] - Trayer, HR; Trayer, IP. Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states. Biochemistry
**1988**, 27, 5718–5727. [Google Scholar] - Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature
**1970**, 227, 680–685. [Google Scholar] - Nikolaeva, OP; Orlov, VN; Bobkov, AA; Levitsky, DI. Differential scanning calorimetric study of myosin subfragment 1 with tryptic cleavage at the N-terminal region of the heavy chain. Eur. J. Biochem
**2002**, 269, 5678–5688. [Google Scholar] - Shakirova, LI; Mikhailova, VV; Siletskaya, EI; Timofeev, VP; Levitsky, DI. Nucleotide-induced and actin-induced structural changes in SH1-SH2-modified myosin subfragment 1. J. Muscle Res. Cell Motil
**2007**, 28, 67–78. [Google Scholar] - Markov, DI; Pivovarova, AV; Chernik, IS; Gusev, NB; Levitsky, DI. Small heat shock protein Hsp27 protects myosin S1 from heat-induced aggregation, but not from thermal denaturation and ATPase inactivation. FEBS Lett
**2008**, 582, 1407–1412. [Google Scholar] - Kremneva, E; Nikolaeva, O; Maytum, R; Arutyunyan, AM; Kleimenov, SYu; Geeves, MA; Levitsky, DI. Thermal unfolding of smooth muscle and non-muscle tropomyosin alpha-homodimers with alternatively spliced exons. FEBS J
**2006**, 273, 588–600. [Google Scholar] - Lopez Mayorga, O; Freire, E. Dynamic analysis of differential scanning calorimetry data. Biophys. Chem
**1987**, 27, 87–96. [Google Scholar] - Filimonov, VV; Potekhin, SA; Matveev, SV; Privalov, PL. Thermodynamic analysis of scanning microcalorimetry data. 1. Algorithms for deconvolution of heat absorption curves. Mol Biol (Mosk)
**1982**, 16, 551–562. (in Russian). [Google Scholar] - Turoverov, KK; Haitlina, SYu; Pinaev, GP. Ultra-violet fluorescence of actin. Determination of native actin content in actin preparations. FEBS Lett
**1976**, 62, 4–6. [Google Scholar] - Turoverov, KK; Kuznetsova, IM. Intrinsic fluorescence of actin. J. Fluoresc
**2003**, 13, 41–57. [Google Scholar] - Staiano, M; Scognamiglio, V; Rossi, M; D’Auria, S; Stepanenko, OV; Kuznetsova, IM; Turoverov, KK. Unfolding and refolding of the glutamine-binding protein from Escherichia coli and its complex with glutamine induced by guanidine hydrochloride. Biochemistry
**2005**, 44, 5625–5633. [Google Scholar] - Panusz, HT; Graczyk, G; Wilmanska, D; Skarzynski, J. Analysis of orthophosphate-pyrophosphate mixtures resulting from weak pyrophosphatase activities. Anal. Biochem
**1970**, 35, 494–504. [Google Scholar] - Markov, DI; Nikolaeva, OP; Levitsky, DI. Effects of myosin “essential” light chain A1 on aggregation properties of the myosin head. Acta Naturae
**2010**, 2(2(5)), 77–81. [Google Scholar]

**Figure 1.**Temperature dependence of ${C}_{\text{p}}^{\text{ex}}$ for S1(A1) in the presence of 100 mM KCl. Checking of the validity of the three-domain model. (

**A**) DSC profile for the original protein preparation (black) and the simulated curve for the most thermostable calorimetric domain (red); (

**B**) DSC profile for the same S1(A1) preparation obtained after annealing at 46 °C for 15 min (black) and its fitting in the framework of the one-stage irreversible model (green).

**Figure 2.**Analysis of the DSC data obtained for S1(A1) in the presence of 100 mM KCl in the framework of the two-domain model. Black curves correspond to the experimental data. (

**A**) DSC profile for the original preparation; (

**B**and

**C**) DSC-profiles for the same preparation after annealing at 46 °C for 15 min and at 42 °C for 20 min, respectively. The results of the fitting are represented by the colored curves. Cyan corresponds to the full DSC profile. Red, green, and blue curves correspond to transitions 1, 2, and 3, respectively; (

**D**) DSC profile for the S1(A1) preparation after annealing at 44 °C for 17 min. The colored curves were simulated using parameters given in Table 1.

**Figure 3.**Analysis of the DSC data obtained for S1 isoforms in the framework of the two-domain model. (

**A**) S1(A2) in the presence of 100 mM KCl; (

**B**,

**C**) S1(A1) and S1(A2) at low ionic strength, in the absence of KCl (20 mM Hepes, pH 7.3). Black curves correspond to the experimental data. The results of the fitting are represented by the colored curves. Cyan corresponds to the full DSC profile. Red, green, and blue curves correspond to transitions 1, 2, and 3, respectively. The colored curves were simulated using parameters given in Table 1.

**Figure 4.**Comparison of the experimental DSC profiles (black curves) obtained after control annealing procedure (17 min at 44 °C) for S1(A1) (

**A**) and S1(A2) (

**B**) with simulated curves (red and blue). See the text for more details.

**Figure 5.**Three-dimensional structure of chymotryptic S1. Tryptophan residues are colored by red, nucleotide associated with ATPase site is colored by yellow.

**Figure 6.**Normalized spectra of intrinsic tryptophan fluorescence of S1(A1) (solid line curves) and S1(A2) (dashed line curves) measured before (red curves) and after (blue curves) the heating of S1 isoforms up to 70 °C performed in the presence of 100 mM KCl with heating rate of 1 °C/min.

**Figure 7.**Parametric dependence of intensity of fluorescence at 365 nm on fluorescence intensity at 320 nm, characterizing the temperature-induced denaturation of S1(A1) (

**A**) and S1(A2) (

**B**). The variable parameter is the temperature. Fluorescence characteristics of native and completely denatured Trp-containing regions of S1 are represented by green and red lines, respectively. Values of fluorescence intensities are expressed in relative units. Both parametric curves were obtained from the heating of both S1 isoforms with constant rate of 1 °C/min in presence of 100 mM KCl.

**Figure 8.**Temperature-induced changes of the S1(A1) (

**A**) and S1(A2) (

**B**) intrinsic fluorescence measured by the changes of the value of normalized parameter A (black circles) in comparison with fraction of conversion for calorimetric transitions 1, 2, and 3 obtained from DSC experiments (colored curves). Normalized parameter A was calculated according to Equation 25. Fraction of conversion of transition 2 was calculated as 1 – x

_{N}; fraction of conversion of transition 3 was calculated as x

_{D}, where x

_{N}and x

_{D}are fractions of the states N and D of calorimetric domain II according to scheme (19).

**Figure 9.**Typical kinetics of heat-induced inactivation of S1 K

^{+}-EDTA-ATPase obtained at different temperatures for S1(A2) preparation in presence of 100 mM KCl. Protein concentration was 0.5 mg/mL. The experimental data are shown by colored points, and solid curves represent the results of their mono-exponential decay fitting.

**Figure 10.**Temperature dependences of the first-order rate constants calculated for heat-induced inactivation of K

^{+}-EDTA-ATPase of S1 isoforms. (

**A**) Comparison of the inactivation rate constants for S1(A1) (magenta) and S1(A2) (cyan). (

**B**,

**C**) Comparison of the ATPase inactivation rate constants (black lines) for S1(A1) (

**B**) and S1(A2) (

**C**) with the rate constants calculated for thermal denaturation of calorimetric domain I (blue lines) and for the first stage of denaturation of calorimetric domain II (red lines). The rate constants for thermal denaturation of calorimetric domains were calculated using parameters given in Table 1.

**Figure 11.**Time course of S1(A1) (

**A**) and S1(A2) (

**B**) aggregation upon heating at 44 °C at different protein concentrations as measured by growth of mean hydrodynamic radius (R

_{h}). All experiments were performed in presence of 100 mM KCl. Distribution of the hydrodynamic radius by size was monomodal during all the period of observation.

**Figure 12.**Time course of S1(A1) (

**A**) and S1(A2) (

**B**) aggregation upon heating at different temperatures as measured by growth of the mean hydrodynamic radius (R

_{h}). All experiments were performed in presence of 100 mM KCl. Protein concentration was constant and equal to 0.5 mg/mL for both S1 isoforms. Distribution of the hydrodynamic radius was monomodal during all the period of observation.

**Figure 13.**Comparison of the DLS data on the thermally induced aggregation of S1(A1) (

**A**) and S1(A2) (

**B**) (the temperature dependences of the t

_{2R}values shown by black lines) with the DSC data on the thermal denaturation of calorimetric domains I and II (the temperature dependences of the t

_{0.5}values shown by blue and red lines for domains I and II, respectively). The t

_{0.5}values were calculated from the data presented in Table 1 (in the case of domain II, the t

_{0.5}values for the first stage of its denaturation are only presented).

**Figure 14.**Temperature dependences of the R

_{h}growth obtained from the DLS experiments for S1(A1) (

**A**) and S1(A2) (

**B**) (black circles and lines) in comparison with fraction of conversion for calorimetric transitions 1, 2, and 3 obtained from the DSC experiments on these S1 isoforms and calculated as in Figure 8 (colored curves). Heating rate was 1 °C/min in both cases. Proteins concentration in the DLS experiments was 0.5 mg/mL.

**Table 1.**The results of the fitting of the DSC data for S1 preparations after final optimization in the framework of the two-domain model.

High ionic strength conditions (100 mM KCl) | ||||||
---|---|---|---|---|---|---|

Parameter | Domain I (transition 1) | Domain II (transition 2) | Domain II (transition 3) | |||

S1(A1) | S1(A2) | S1(A1) | S1(A2) | S1(A1) | S1(A2) | |

T^{*}, K | 317.5 ± 0.4 | 319.0 ± 0.4 | 321.2 ± 0.4 | 321.6 ± 0.4 | 324.5 ± 0.4 | 324.7 ± 0.4 |

E_{a}, kJ/mole | 290 ± 40 | 265 ± 40 | 400 ± 40 | 400 ± 40 | 340 ± 40 | 380 ± 40 |

ΔH, kJ/mole | 200 ± 20 | 200 ± 20 | 1030 ± 100 | 970 ± 100 | 500 ± 50 | 300 ± 30 |

Low ionic strength conditions (no KCl) | ||||||
---|---|---|---|---|---|---|

Parameter | Domain I (transition 1) | Domain II (transition 2) | Domain II (transition 3) | |||

S1(A1) | S1(A2) | S1(A1) | S1(A2) | S1(A1) | S1(A2) | |

T^{*}, K | 319.2 ± 0.4 | 321.4 ± 0.4 | 323.0 ± 0.4 | 323.0 ± 0.4 | 325.5 ± 0.4 | 325.6 ± 0.4 |

E_{a}, kJ/mole | 230 ± 40 | 250 ± 40 | 350 ± 40 | 400 ± 40 | 390 ± 40 | 390 ± 40 |

ΔH, kJ/mole | 370 ± 40 | 380 ± 40 | 1260 ± 130 | 720 ± 70 | 400 ± 40 | 260 ± 30 |

© 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

## Share and Cite

**MDPI and ACS Style**

Markov, D.I.; Zubov, E.O.; Nikolaeva, O.P.; Kurganov, B.I.; Levitsky, D.I.
Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains. *Int. J. Mol. Sci.* **2010**, *11*, 4194-4226.
https://doi.org/10.3390/ijms11114194

**AMA Style**

Markov DI, Zubov EO, Nikolaeva OP, Kurganov BI, Levitsky DI.
Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains. *International Journal of Molecular Sciences*. 2010; 11(11):4194-4226.
https://doi.org/10.3390/ijms11114194

**Chicago/Turabian Style**

Markov, Denis I., Eugene O. Zubov, Olga P. Nikolaeva, Boris I. Kurganov, and Dmitrii I. Levitsky.
2010. "Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains" *International Journal of Molecular Sciences* 11, no. 11: 4194-4226.
https://doi.org/10.3390/ijms11114194