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

Abstract

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-BeFx that mimic S1 ATPase intermediate states S1**-ADP-Pi 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-BeFx 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.

Chimeric 14-3-3 proteins for unraveling interactions with intrinsically disordered partners

“…To further characterize the conformation change between unbound and bound states of the CH1 chimera, we used differential scanning fluorimetry 42 . We compared CH1 chimera with the unphosphorylated control and the core 14-3-3σ∆C dimer (Fig.…”

“…Before the experiment, the samples were equilibrated for 10 min at the initial temperature (10 °C). The ratio of I 320 (T)/ I 365 (T) normalized from 0 to 100% represented the dependence of completeness of thermal transition, of an unfolded fraction, on temperature and was used to estimate half-transition temperatures 42 . When possible, the single wavelength was used to build analogous transition curves 53 .…”

Chimeric 14-3-3 proteins for unravelling interactions with intrinsically disordered partners

“…To further characterize CH1 chimera, we applied fluorimetry and followed changes in the intensity of intrinsic tryptophan fluorescence of CH1 or the 14-3-3σ∆C dimer at two wavelengths (320 and 365 nm), transformed into temperature dependencies of the unfolded protein fraction ( Fig. 2B) [42], as well as changes in light scattering during the heating of the samples at a constant rate ( Fig. 2C).…”

“…Peaks I and II of the CH1 profile are marked. B -Intrinsic tryptophan fluorescence spectra of CH1 (green) or 14-3-3σ∆C (blue) samples (1.5 µM) excited at 297 nm (slits width 5 nm) (insert) and heating of 14-3-3σ∆C (1.5 µM) and CH1 (0.7-7.7 µM) samples from 10 to 80 o C at a constant rate of 1 ºC/min (direction is shown by arrow) analyzed by plotting fraction unfolded against temperature [42]. C -Aggregation curves for the samples presented on panel B as temperature dependencies of light scattering accompanying aggregation.…”

“…Before the experiment the samples were equilibrated for 30 min at the initial temperature (10 ºC). The ratio of I 320 (T)/I 365 (T) normalized from 0 to 100% represented the dependence of completeness of thermal transition, of unfolded fraction, on temperature and was used to estimate half-transition temperatures [42]. When possible, single wavelength was used to build analogous transition curves [56].…”