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/).

We compared thermally induced denaturation and aggregation of two isoforms of the isolated myosin head (myosin subfragment 1, S1) containing different “essential” (or “alkali”) light chains, A1 or A2. We applied differential scanning calorimetry (DSC) to investigate the domain structure of these two S1 isoforms. For this purpose, a special calorimetric approach was developed to analyze the DSC profiles of irreversibly denaturing multidomain proteins. Using this approach, we revealed two calorimetric domains in the S1 molecule, the more thermostable domain denaturing in two steps. Comparing the DSC data with temperature dependences of intrinsic fluorescence parameters and S1 ATPase inactivation, we have identified these two calorimetric domains as motor domain and regulatory domain of the myosin head, the motor domain being more thermostable. Some difference between the two S1 isoforms was only revealed by DSC in thermal denaturation of the regulatory domain. We also applied dynamic light scattering (DLS) to analyze the aggregation of S1 isoforms induced by their thermal denaturation. We have found no appreciable difference between these S1 isoforms in their aggregation properties under ionic strength conditions close to those in the muscle fiber (in the presence of 100 mM KCl). Under these conditions kinetics of this process was independent of protein concentration, and the aggregation rate was limited by irreversible denaturation of the S1 motor domain.

Cyclic association-dissociation of myosin and actin, coupled with myosin-catalyzed ATP hydrolysis, is the most essential event in muscle contraction. The globular head of myosin, called subfragment 1 (S1), is responsible for the force generation during contraction. It consists of a globular motor (or catalytic) domain that contains the sites responsible for the ATP hydrolysis and actin binding, and a neck region with a long

S1 prepared by chymotryptic digestion of skeletal muscle myosin lacks the regulatory light chain but does contain the essential light chain, also called alkali light chain [

Another interesting property of the

Previous studies showed a dramatic difference between two S1 isoforms in their heat-induced aggregation measured at low ionic strength: S1(A1) aggregated at much lower temperature than S1(A2) [

In the present work, we applied differential scanning calorimetry (DSC) for a detailed comparative analysis of thermal denaturation of two S1 isoforms, S1(A1) and S1(A2), and, in particular, to investigate the domain structure of these S1 isoforms. For this purpose, a new calorimetric approach was specially developed to analyze the DSC profiles of irreversibly denaturing multidomain proteins (see Section 2). Using this approach, we have revealed two calorimetric domains in the S1 molecule, the more thermostable domain denaturing in two steps. We also applied special approaches to identify these calorimetric domains,

In this section a new method of the analysis of thermograms for multidomain proteins undergoing irreversible denaturation is proposed. Thermograms for such proteins represent the sum of separate peaks corresponding to individual calorimetric domains (since calorimetric domain is a part of protein molecule that denatures cooperatively and independently from the other parts). Up to date, there has not been proposed any general analytical solution for the problem of the decomposition of thermograms into individual domains with the simultaneous determination of the kinetic parameters for these domains.

For the sake of simplicity it is assumed that denaturation of each individual calorimetric domain is described by the one-stage model of irreversible denaturation:
_{a} is the experimental energy of activation, ^{*} is the temperature at which ^{−1} (the dimension of ^{−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:

Kurganov _{a}

The thermogram
_{i}_{i}_{ann} = _{i}_{i}_{i}_{i}_{ann} gives us the _{i}_{i}_{i}_{ann}) · _{ann}], where _{i}_{ann}) is the rate constant for denaturation of _{ann} calculated from the Arrhenius _{i}_{ann} and _{ann} and parametrically depending on _{a,i}

If the calorimetric experiment with preliminary incubation (annealing) of the protein preparation has been carried out (_{i, N}

If domains are numbered in order of increasing thermostability, the following condition holds at _{1,} _{j}_{2,} _{j}_{N, j}_{a} and ^{*} for (_{i}_{–1,} _{j}_{i}_{–1,} _{j}_{i}_{,} _{j}^{st}, 2^{nd}, . . . , (_{1,} _{j}_{2,} _{j}_{i}_{–2,} _{j}

Evidently, in the part of thermogram corresponding to high temperatures it is possible to select a region where all domains except for the most thermostable one are already completely denatured. In this part of the thermogram
_{a}^{*} for the most thermostable domain to be estimated. With a knowledge of parameters _{a}^{*}, we can calculate _{N}_{N}_{N}_{N}_{N}

Based on these considerations one can propose the following recursive method for the estimation of parameters of calorimetric domains for multidomain protein. After obtaining of
_{a, N}_{N}

However, when determining parameters of

When the corresponding annealing procedures have been carried out and all thermograms have been obtained, the multidimensional non-linear optimization in the parameter space may be used for final calculation of parameters _{a}^{*} and Δ

The method is based on annealing of 1st, 2nd, . . . , (_{a}^{*} for two domains are non-identical, the conditions of annealing may be selected at which _{i}_{–1} ≈ 0 and _{i}_{i}_{−1} and _{i}_{i}_{−1} _{i}^{*} 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 ^{*} values the minimum reasonable difference between parameters _{a}

Taking into account the above-mentioned requirements to the annealing conditions, we can propose the following method for the selection of _{ann} and _{ann}. For definiteness we fix the ratio _{i}_{−1}/_{i}_{i}_{−1} and _{i}_{ann} is a curve _{ann} = _{ann}) established by the conditions:

Let us consider how the above-described principles of DSC data analysis can be applied to S1(A1) thermal denaturation in the presence of 100 mM KCl. _{a}^{*} and Δ_{trans}_{ann} and _{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.

It was proposed from earlier DSC studies that the total heat sorption curve of S1 is composed of three independent calorimetric domains [

We have proposed somewhat more complicated model for the S1 thermal denaturation, which presumes two calorimetric domains in the S1 molecule. According to this model, the less thermostable domain (designated as domain I) undergoes one-stage irreversible denaturation, whereas thermal denaturation of more thermostable domain (calorimetric domain II) is described by two-stage scheme:
_{1} and _{2} obey the Arrhenius law (_{1}, Δ_{2} > 0). Correspondingly, the thermogram of such a domain can be described as follows
_{N}_{I}_{0}. Obviously, before annealing,
_{1} and _{2} from the Arrhenius 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.

To find the parameters of the S1 calorimetric domains according to the above described two-domain model for the S1 thermal denaturation, we have used multidimensional nonlinear optimization in the space of these parameters. The following input data were simultaneously used for this analysis: the thermograms of native S1(A1) and S1(A2) preparations, the thermograms of these preparations subjected to two different annealing procedures (_{a}^{*}, ^{−1}).

Similar experiments were performed with S1(A1) under low ionic strength conditions, as well as with S1(A2) independently of ionic strength. ^{*} of the least thermostable calorimetric domain (transition 1 in

In order to confirm the difference between S1(A1) and S1(A2) in the ^{*} parameter of domain I, we have performed additional optimization procedure, as follows. All calorimetric parameters (except only ^{*} of domain I) were allowed to change and domains’ I ^{*} 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 ^{*} values equality are quite different from curves 1. Thus, we can conclude that this assumption is erroneous, and therefore the difference in domains’ I ^{*} 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.

To identify the S1 calorimetric domains (

_{320} and _{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.

For a more detailed analysis of the changes in the fluorescent properties of S1 isoforms induced by their thermal denaturation, we applied the method of parametric plots [

It should be noted that detailed quantitative analysis of parametric dependences is rather difficult, mainly because of small number of experimental points in the region of transition processes. An increase of the number of points decreases the data accumulation time thus raising the noise. Another problem is imperfect correspondence between temperature scales of the instruments used, calorimeter and fluorimeter, which cannot be fully eliminated even by the calibration procedure. Extremely high sensitivity of non-linear models to even small errors can make the results of fluorescence data optimization incomparable with those obtained from the DSC data analysis. Therefore, to compare more precisely the temperature-induced changes in fluorescent properties of S1 isoforms with thermal denaturation of their calorimetric domains, we analyzed the temperature dependences of normalized parameter _{min} and _{max} represent minimal and maximal values of the parameter

For further identification of the calorimetric domains of S1 isoforms we studied the temperature-induced inactivation of their ATPase. _{a}^{*} obtained from the DSC studies (

The irreversible thermal denaturation of S1 is accompanied by its aggregation [

Unlike thermal denaturation, aggregation of proteins is always a reaction of interaction between two (or more than two) proteins molecules. Hence, at least one of its stages should be the second-order (or higher order) reaction. In other words, one can expect that, in general, aggregation of proteins should depend on their concentration. Nevertheless, our preliminary DLS results obtained in the presence of 100 mM KCl with mixed (not separated into isoforms) S1 preparation have demonstrated that under these conditions the heat-induced aggregation of S1, as measured by an increase in the hydrodynamic radius (_{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. _{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 _{h}_{0}), and _{2R} is the time interval over which the hydrodynamic radius of the protein aggregates increases twofold. The parameter _{2R} characterizes the rate of aggregation [_{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 (_{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 _{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 _{2R} value on temperature. _{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 _{h} growth are well described by _{2R} values at different temperatures. The temperature dependences of the _{2R} values for both S1 isoforms are presented on _{2R} values coincide with the _{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.

We also calculated the parameters _{a}^{*} of the Arrhenius equation (_{h} growth process of S1 aggregates. The _{a}^{*} 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

For better understanding of the delay in the aggregation process (_{0} value), we also investigated the temperature dependences of _{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 _{h} values of 3000 nm (data not shown). _{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 _{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 (

A unique feature of the S1 thermal denaturation is a “blue shift” of its intrinsic fluorescence spectrum (_{h} fast growth (black points and curve in

Based on the data obtained, we can propose the following mechanism of S1 thermal aggregation. The first stage of this process is nucleation, when several partially or fully denatured S1 molecules stick together and form the primary aggregates (so-called start aggregates [_{0} in the

The above proposed mechanism of S1 thermal aggregation disagrees with earlier proposed scheme of heat-induced aggregation of proteins [

It should be noted that the earlier proposed model of heat-induced aggregation of proteins based on the interaction between start aggregates [

S1 from rabbit skeletal myosin was prepared by digestion of myosin filaments with TLCK-treated ^{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 [

DSC experiments were performed on a DASM-4M differential scanning microcalorimeter (Institute for Biological Instrumentation, Pushchino, Russia) as described earlier [_{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 [

The thermogram for a protein undergoing one-stage reversible as well as one-stage irreversible denaturation may be described by the following equation

In the case of the multidomain protein or protein with several thermal transitions the thermogram is an algebraic sum of the equations of the type 28:
_{p, i}

As a null approximation the approach which is close to that described by Filimonov _{p, i}_{i}_{p, i}_{i}_{i}_{i}

Substitute _{p} value, the inaccuracy introduced in the

On this basis, the estimation of the chemical base line and its subtraction may be carried out as follows. We select four points on the experimental dependence of heat absorption _{p}(_{1}) corresponds to the beginning of the linear part of the thermogram before the peak of excess heat absorption, the second point (_{2}) corresponds to the beginning of the peak and simultaneously the end of the linear part before the peak, the third point (_{3}) corresponds to the end of the peak and the beginning of the linear part after the peak and the fourth point (_{4}) corresponds to the end of the linear part after the peak. The linear part before the peak is taken as
_{CBL}(_{1} to _{2}. In the interval from _{3} to _{4} the chemical base line is taken equal to
_{2} to _{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 Δ_{1} to _{4} is calculated by the formula:

The calculated excess heat capacity is used for estimation of Δ

The proposed method of the estimation of the chemical base line is close to the algorithm elaborated by Filimonov _{p, i}

Fluorescence studies were performed on a Cary Eclipse spectrofluorimeter (Varian) equipped with a Peltier-controlled cell holder and thermoprobes. Intrinsic tryptophan fluorescence was measured at protein concentration of 0.05 mg/mL in 20 mM Hepes-KOH (pH 7.3) containing 1 mM MgCl_{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

The thermally induced inactivation of S1 isoforms’ ATPase was measured after heating the protein (0.5 mg/mL) at constant temperature in the medium containing 20 mM Hepes (pH 7.3), 1 mM MgCl_{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 [_{4} to final concentration of 2.5%.

DLS measurements were performed on a Photocor Complex apparatus (Photocor Instruments Inc., USA) equipped with a temperature controller. The sample was illuminated by a 632.8 nm laser light, and the scattering signal was observed at an angle of 90°. DLS data were accumulated and analyzed with multifunctional real-time correlator Photocor-FC. DynaLS software (Alango, Israel) was used for polydisperse analysis of DLS data. The kinetics of S1 isoforms aggregation was studied by measuring an increase in the mean hydrodynamic radius of the particles (_{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 _{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.

The algorithms for estimation of chemical base line, calculation of

Optimization algorithms (embedded into the Optimization Toolbox) allow to calculate only optimal values of the parameters, but not their confidence intervals. Moreover, because of specific features of the calorimetric experiment, the systematic error exceeds many times the random error. This renders the use of regression statistics for estimating the confidence intervals impossible. For this reason we used the following (rather rough) approach to estimate the error in the values of the calorimetric parameters. We carried out a number of different optimization procedures in the process of calculation of the values of these parameters. In one case, all the experimental
_{a}^{*} 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 ^{*}, 40 kJ/mole for _{a}

The optimal values of the rate constants for the heat-induced S1 ATPase inactivation and the optimal values of the _{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.

The values of the rate constants and half-life times of the S1 calorimetric domains were calculated using Arrhenius equation (_{a} and ^{*} values given in

The optimal values of the parameters of Arrhenius equation (_{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 _{2R} parameters and by the error in incubation temperature that was assumed to be equal to 0.5 °C.

In the present study we have found no appreciable difference between S1(A1) and S1(A2) in their aggregation properties under ionic strength conditions close to those in the muscle fiber (in the presence of 100 mM KCl). This means that under these conditions the presence of additional

Some difference between the two S1 isoforms was only revealed by DSC, independently of the ionic strength conditions, in the thermal denaturation of the regulatory domain. This is, however, quite an expected effect as the alkali light chains are associated with this domain of the S1 molecule. Thus, the interaction of the

A noticeable influence of the A1

We thank Andrey K. Tsaturyan (Institute of Mechanics, Moscow State University) and Sergei Yu. Kleimenov (A.N. Bach Institute of Biochemistry RAS, Moscow) for valuable comments and advices.

This work was supported by the Russian Foundation for Basic Research (grants 09-04-00266-a and 08-04-00666-a), the Program “Molecular and Cell Biology” of the Presidium of the Russian Academy of Sciences and by the grant of the President of Russian Federation (grant MK 2965.2009.4).

Temperature dependence of

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. (

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

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

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

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.

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

Temperature-induced changes of the S1(A1) (_{N}_{D}_{N}_{D}

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.

Temperature dependences of the first-order rate constants calculated for heat-induced inactivation of K^{+}-EDTA-ATPase of S1 isoforms. (

Time course of S1(A1) (_{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.

Time course of S1(A1) (_{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.

Comparison of the DLS data on the thermally induced aggregation of S1(A1) (_{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 _{0.5} values shown by blue and red lines for domains I and II, respectively). The _{0.5} values were calculated from the data presented in _{0.5} values for the first stage of its denaturation are only presented).

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

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

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

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

^{*} |
317.5 |
319.0 |
321.2 |
321.6 |
324.5 |
324.7 |

_{a} |
290 |
265 |
400 |
400 |
340 |
380 |

Δ |
200 |
200 |
1030 |
970 |
500 |
300 |

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

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

^{*} |
319.2 |
321.4 |
323.0 |
323.0 |
325.5 |
325.6 |

_{a} |
230 |
250 |
350 |
400 |
390 |
390 |

Δ |
370 |
380 |
1260 |
720 |
400 |
260 |