The Relaxation Properties of Myofibrils Are Compromised by Amino Acids that Stabilize α-Tropomyosin

We investigated the functional impact of α-tropomyosin (Tm) substituted with one (D137L) or two (D137L/G126R) stabilizing amino acid substitutions on the mechanical behavior of rabbit psoas skeletal myofibrils by replacing endogenous Tm and troponin (Tn) with recombinant Tm mutants and purified skeletal Tn. Force recordings from myofibrils (15°C) at saturating [Ca(2+)] showed that Tm-stabilizing substitutions did not significantly affect the maximal isometric tension and the rates of force activation (k(ACT)) and redevelopment (k(TR)). However, a clear effect was observed on force relaxation: myofibrils with D137L/G126R or D137L Tm showed prolonged durations of the slow phase of relaxation and decreased rates of the fast phase. Both Tm-stabilizing substitutions strongly decreased the slack sarcomere length (SL) at submaximal activating [Ca(2+)] and increased the steepness of the SL-passive tension relation. These effects were reversed by addition of 10 mM 2,3-butanedione 2-monoxime. Myofibrils also showed an apparent increase in Ca(2+) sensitivity. Measurements of myofibrillar ATPase activity in the absence of Ca(2+) showed a significant increase in the presence of these Tms, indicating that single and double stabilizing substitutions compromise the full inhibition of contraction in the relaxed state. These data can be understood with the three-state (blocked-closed-open) theory of muscle regulation, according to which the mutations increase the contribution of the active open state in the absence of Ca(2+) (M(−)). Force measurements on myofibrils substituted with C-terminal truncated TnI showed similar compromised relaxation effects, indicating the importance of TnI-Tm interactions in maintaining the blocked state. It appears that reducing the flexibility of native Tm coiled-coil structure decreases the optimum interactions of the central part of Tm with the C-terminal region of TnI. This results in a shift away from the blocked state, allowing myosin binding and activity in the absence of Ca(2+). This work provides a basis for understanding the effects of disease-producing mutations in muscle proteins

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

The difference between ADP-beryllium fluoride and ADP-aluminium fluoride complexes of the spin-labeled myosin subfragment 1

AbstractElectron paramagnetic resonance (EPR) spectroscopy was used for investigation of the structure of spin-labeled myosin subfragment 1 (S1) containing ADP and phosphate analogues, such as orthovanadate, aluminium fluoride (AlF4), and beryllium fluoride (BeFx). It has been shown that the local conformational changes in the region of Cys-707, induced by formation of the S1-ADP-BeFx complex, differ from those of S1 containing ADP-AlF4 or other phosphate analogues but are similar to the changes which occur in the presence of ADP or ATPγS. It is suggested that S1-ADP-AlF4 and S1-ADP-BeFx complexes represent structural analogues of different transition states of the ATPase cycle, namely the intermediate states S1**-ADP-Pi and S1*-ATP, respectively

Charge changes in loop 2 affect the thermal unfolding of the myosin motor domain bound to F-actin

The thermal unfolding of Dictyostelium discoideum myosin head fragments with alterations in the actin-binding surface loop 2 was studied by differential scanning calorimetry. Lengthening of loop 2 without concomitant charge changes led to decreases in the transition temperature of not more than 1.8 C. Insertions with multiple positive or negative charges had a stronger destabilizing effect and led to reductions in the thermal transition temperature of up to 3.7 C. In the presence of nucleotide, most mutants displayed similar or higher transition temperatures than M765. Only constructs M765(11/+6) and M765(20/+12) with long positively charged inserts showed transition temperatures that were more than 2 C below the values measured for M765 in the presence of ADP, ADP-Vi, and ADP-BeF3. Interaction with F-actin in the presence of ADP shifted the thermal transition of M765 by 6 C, from 49.1 to 55.1 C. The actin-induced increase in thermal stability varied between 1.2 and 9.1 C and showed a strong correlation with the mutant constructs affinity for actin. Our results show that length and charge changes in loop 2 do not significantly affect nucleotide-induced structural changes in the myosin motor domain, but they affect structural changes that occur when the motor domain is strongly bound to actin and affect the coupling between the actin- and nucleotide-binding sites

Structural and Functional Peculiarities of Cytoplasmic Tropomyosin Isoforms, the Products of TPM1 and TPM4 Genes

Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are α-helical coiled-coil protein dimers forming a continuous head-to-tail polymer along the actin filament. Human cells produce a large number of Tpm isoforms that are thought to play a significant role in determining actin cytoskeletal functions. Even though the role of these Tpm isoforms in different non-muscle cells is more or less studied in many laboratories, little is known about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five cytoplasmic Tpm isoforms (Tpm1.5, Tpm 1.6, Tpm1.7, Tpm1.12, and Tpm 4.2), which are the products of two different genes, TPM1 and TPM4, and also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results demonstrate that structural and functional properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. The revealed differences can be important in further studies to explain why various Tpm isoforms interact uniquely with actin filaments, thus playing an important role in the organization and dynamics of the cytoskeleton