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

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

Scientific, institutional and personal rivalries among Soviet geographers in the late Stalin era

Scientific, institutional and personal rivalries between three key centres of geographical research and scholarship (the Academy of Sciences Institute of Geography and the Faculties of Geography at Moscow and Leningrad State Universities) are surveyed for the period from 1945 to the early 1950s. It is argued that the debates and rivalries between members of the three institutions appear to have been motivated by a variety of scientific, ideological, institutional and personal factors, but that genuine scientific disagreements were at least as important as political and ideological factors in influencing the course of the debates and in determining their final outcome

CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin Subfragment 1

<div><p>Myosin head (myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF₄^- and S1-ADP-BeF_x that mimic S1 ATPase intermediate states S1**-ADP-P_i and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1), we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF₄^- and S1-ADP-BeF_x significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle.</p></div

Temperature dependence of the excess molar heat capacity (C_p) obtained by DSC for S1 in the complexes S1-ADP-BeF_x (A) and S1-ADP-AlF₄^- (B), and their decomposition into individual thermal transitions (calorimetric domains 1 and 2).

<p>Decomposition of the DSC curves (solid lines) into individual thermal transitions (dotted lines) was performed as described earlier for the nucleotide-free S1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137517#pone.0137517.ref028" target="_blank">28</a>]. The curves shown by dashed lines represent the results of the fitting of the DSC data in a framework of the two-domain model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137517#pone.0137517.ref028" target="_blank">28</a>]. The values of calorimetric enthalpy (Δ<i>H</i>_cal) for each thermal transition are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137517#pone.0137517.t001" target="_blank">Table 1</a>.</p

Temperature-induced changes in fluorescence measured on the nucleotide-free S1 for labeled recombinant LC1 mutants associated with the S1 regulatory domain (red circles) and for the tryptophan fluorescence of S1 (blue circles).

<p>The labeled Cys residues in the LC1 were Cys-99 (A), Cys-142 (B), Cys-127 (C), and Cys-160 (D). The changes in fluorescence were measured by the changes of values of normalized parameters <i>L</i> and <i>A</i> for the AEDANS-labeled LC1 and tryptophan residues, respectively, calculated as described in Materials and methods. Red and blue solid lines represent the results of the data analysis according to a sigmoidal function (Boltzmann) for LC1 and tryptophan, respectively.</p

Representative temperature-induced changes in fluorescence measured on the S1 ternary complexes S1-ADP-BeF_x (A–C) and S1-ADP-AlF₄ (D–F) for the labeled recombinant LC1 mutants associated with the S1 regulatory domain (red circles) and for the tryptophan fluorescence of S1 (blue circles).

<p>The AEDANS-labeled Cys residues in the LC1 were Cys-99 (A,D), Cys-127 (B), Cys-142 (F), Cys-160 (C), as well as Cys-180 of the wild-type LC1 (E). The changes in the fluorescence were measured by changes of the values of normalized parameters <i>L</i> and <i>A</i> for the labeled LC1 and tryptophan residues, respectively, which were calculated as described in Materials and methods. Red and blue solid lines represent the results of the data analysis according to a sigmoidal function (Boltzmann) for LC1 and tryptophan, respectively.</p

Temperature dependences obtained in the presence or absence of HspB5 for S1 aggregation (A) and for fluorescence of labels specifically attached to Cys-707 in the S1 motor domain (B) or to Cys-180 of the wild-type LC1 in the S1 regulatory domain (C).

<p>(<b>A</b>) S1 aggregation was measured by an increase in turbidity (apparent optical density at 350 nm) of the solution in the absence (blue curve) or in the presence (red curve) of HspB5. Green curve represents the optical density of HspB5 in the absence of S1. (<b>B,C</b>) The changes in the fluorescence were measured by changes of the values of normalized parameter <i>L</i> for fluorescent label (IAEDANS) attached to the motor (B) or regulatory (C) domain of the S1 molecule. The changes of parameter <i>L</i> in the absence or in the presence of HspB5 are shown by blue and red circles, respectively. Correspondingly, blue and red solid lines represent the results of the data analysis according to a sigmoidal function (Boltzmann). Protein concentration was 0.15 mg/mL for both S1 and HspB5. Heating rate was 1°C/min.</p