Stabilizing the Central Part of Tropomyosin Increases the Bending Stiffness of the Thin Filament

Abstract

A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.

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

“… who also simulated the D137L construct with MD. Although these authors calculated the persistence length of the whole Tpm molecule bound to actin while we estimated it only for an isolated middle segment of unbound Tpm and therefore it was undiminished by such interaction, both calculations show an increase in stiffness caused by the D137L mutation consistent with experimental data . Moore et al .…”

“…However, our single molecule experiments have not shown changes in the unitary force of myosin interaction with reconstructed thin filaments containing these Tpm constructs, which nevertheless strongly affected the cooperativity of the interaction in the in vitro motility assay . We therefore concluded that increase in force in the motility assay induced by these substitutions is caused by an involvement of additional myosin heads in the interaction due to global stabilization of the Tpm structure and global increase in stiffness of the Tpm strand . The M127A/I130A or M141A/Q144A substitutions increased Tpm stiffness only locally probably due to a closure of the interhelical gap (Table and Fig.…”

“…All these Tpm mutants significantly enhanced the maximum sliding velocity of thin filaments at saturating Ca 2+ concentrations and similarly increased the Ca 2+ sensitivity of the velocity. According to explanations given in the previous work, the effects of the stabilizing substitutions D137L and G126R, and especially their combination, D137L/G126R, on the Ca 2+ ‐dependent sliding of reconstituted thin filaments in the in vitro motility assay can be accounted for by stabilization of the filaments expressed in an increase in their bending stiffness as well as by an influence of these substitutions on the interactions between Tpm and certain sites of the myosin head .…”

“…However, the mechanism underlying the effects of these substitutions on the Ca 2+ ‐regulated actin–myosin interaction is not completely identical to that for the stabilizing substitutions D137L/G126R. The replacement of the noncanonical residues Asp137 and Gly126 with the canonical Leu and Arg, respectively, not only decreased flexibility of the Tpm molecule (Fig. A and Table ), but also enhanced sliding velocity of the regulated thin filaments in the in vitro motility assay .…”

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

“…Replacement of these residues with canonical ones, D137L and G126R (9,10), was shown in solution studies to significantly increase coiled-coil stability and decrease molecular mobility or flexibility (9,(11)(12)(13). A significant decrease in the flexibility of reconstructed thin filaments with the combined D137L/G126R substitution was also shown by means of the two-bead optical trap technique (14).…”