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

Abstract

We investigated the functional impact of a-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 2þ ] 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 2þ ] 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 2þ sensitivity. Measurements of myofibrillar ATPase activity in the absence of Ca 2þ 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 . This work provides a basis for understanding the effects of disease-producing mutations in muscle proteins.

Precise Binding of Tropomyosin on Actin Involves Sequence-Dependent Variance in Coiled-Coil Twisting

“…d-position aspartate 137 in tropomyosin breaks this rule. Surrounding g-position glycine (G126) and a-position methionine residues (M127, M141) also are unusual and may wedge open the surrounding core marginally (21)(22)(23)(24)(25)(26)(27)(28). Strikingly, the four-residue quartet is strictly conserved in vertebrate muscle tropomyosins.…”

“…It is sometimes proposed that the D137-induced ''core gap'' in tropomyosin's hydrophobic stripe provides molecular flexibility (21,22,(24)(25)(26)(27)(28), generally construed as flexural flexibility, required for normal tropomyosin regulatory translations across the actin filament. Consistent with this notion, the region surrounding D137 is susceptible to proteolytic digestion presumably related to transient coiled-coil separation (21).…”

“…If correct, it therefore follows that stretches of coiled coils neighboring these residues might provide tropomyosin with the flexibility needed to associate with successive actin subunits along thin filaments if an ''induced-fitting'' mechanism is involved in actin-binding of tropomyosin (9,29). This notion is supported by circumstantial evidence that heat-induced local instability of actin-free native tropomyosin occurs at a lower temperature when compared with D137L tropomyosin constructs expressed in Escherichia coli that contain a more canonical leucine at position 137 (21,(25)(26)(27)(28). Alternatively, residues 137 or 218 may simply hardwire local geometry of tropomyosin to facilitate shape complementarity to F-actin or improve the precision of F-actin-tropomyosin interactions but without having significant impact on local enhanced flexibility (10,23).…”

Functional role of the core gap in the middle part of tropomyosin

“…RMSD stabilized after 5 ns of the 25 20 ns-long trajectories. To compare the similarities and difference between the effects of these mutations and the stabilizing ones in the same part of the Tpm molecule, we also performed a MD simulation of the D137L/C190A and G126R/D137L/C190A Tpm constructs whose properties were studied previously [20][21][22][23]30,31]. The results of the MD simulation are shown in Videos S1-S5.…”

“…These stabilizing substitutions also significantly changed the functional properties of Tpm both in studies on isolated proteins or regulated actin filaments in solution (i.e. thin filaments reconstituted from F‐actin, Tpm and Tn) and in experiments on reconstituted myofibrils or on a transgenic mouse model expressing cardiac Tpm with the D137L mutation . In particular, the stabilizing substitutions D137L, G126R, and D137L/G126R enhanced maximal sliding velocity of regulated actin filaments in the in vitro motility assay at high Ca 2+ concentrations and increased Ca 2+ sensitivity of the filament sliding along a surface covered by fast skeletal muscle myosin .…”