Conserved Noncanonical Residue Gly-126 Confers Instability to the Middle Part of the Tropomyosin Molecule

Abstract

Tropomyosin (Tm) is a two-stranded ␣-helical coiled-coil protein with a well established role in regulation of actin cytoskeleton and muscle contraction. It is believed that many Tm functions are enabled by its flexibility whose nature has not been completely understood. We hypothesized that the well conserved non-canonical residue Gly-126 causes local destabilization of Tm. To test this, we substituted Gly-126 in skeletal muscle ␣-Tm either with an Ala residue, which should stabilize the Tm ␣-helix, or with an Arg residue, which is expected to stabilize both ␣-helix and coiled-coil structure of Tm. We have shown that both mutations dramatically reduce the rate of Tm proteolysis by trypsin at Asp-133. Differential scanning calorimetry was used for detailed investigation of thermal unfolding of the Tm mutants, both free in solution and bound to F-actin. It was shown that a significant part of wild type Tm unfolds in a non-cooperative manner at low temperature, and both mutations confer cooperativity to this part of the Tm molecule. The size of the flexible middle part of Tm is estimated to be 60 -70 amino acid residues, about a quarter of the Tm molecule. Thus, our results show that flexibility is unevenly distributed in the Tm molecule and achieves the highest extent in its middle part. We conclude that the highly conserved Gly-126, acting in concert with the previously identified non-canonical Asp-137, destabilizes the middle part of Tm, resulting in a more flexible region that is important for Tm function.

Conserved Asp-137 Is Important for both Structure and Regulatory Functions of Cardiac α-Tropomyosin (α-TM) in a Novel Transgenic Mouse Model Expressing α-TM-D137L

“…It was previously reported that flexibility of the middle region of TM, where residue 137 lies, is not crucial for actin binding and that D137L substitution does not alter the actin binding properties of TM (11,12). However, a direct interaction between the C-terminal mobile domain of cTnI and residue 146 of TM was previously proposed by Mudalige et al (56).…”

“…As determined with DSC, the ␣-TM-D137L variant has higher thermostability than WT, suggesting decreased flexibility. In the description of the ␣-TM and ␣-TM-D137L variant, we have used the term "flexibility" as has been common in previous papers (8,9,11,12). However, it should be recognized that in our case describing a protein as flexible is an interpretation of determinations of stability that may have different interpretations.…”

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

“…Among Tm noncanonical amino acids, recent studies have examined two highly conserved residues in the middle part of the aaTm molecule: D137 (a charged residue in a d hydrophobic position) and G126 (a small, neutral residue in a g charged position). 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).…”

“…Interestingly, Ca 2þ regulation dynamics in the intact thin filament are also affected, as shown by measurements of thin-filament-induced activation of S1 ATPase activity in the presence of D137L or G126R aaTm (9,10), and by the maximal sliding velocity of regulated actin filaments in in vitro motility assays (15,16). All of these studies reported a clear functional change in the presence of one or both stabilizing substitutions and a generalized increase in apparent Ca 2þ sensitivity.…”

“…Thus, loss of this interaction with the Tm mutants appears to be the cause of the relaxation impairment. Our previous studies showed that these mutations increase the stability or dynamic rigidity of Tm in solution (9,10,12). It is possible that this loss of conformational mobility contributes to the loss of a specific interaction between Tm and a region in the C-terminus of TnI.…”