Transmission of Stability Information through the N-domain of Tropomyosin Is Interrupted by a Stabilizing Mutation (A109L) in the Hydrophobic Core of the Stability Control Region (Residues 97-118)

Background: A stability control region (SCR) (residues 97-118) is conserved in vertebrate tropomyosins and controls overall stability. Results: An A109L mutation interrupts the transmission of stability information along the coiled-coil, inducing two domains. Conclusion: The optimum stability provided by the SCR is critical for transmission of the stability signal. Significance: Learning how stability information is transmitted along tropomyosin is crucial to understanding function and signaling. Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded -helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97-118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual -helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual -helices provides an optimum stability and allows functionally beneficial dynamic motion between the -helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1-131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1-131) molecule, showing that the effect of this mutation is transmitted 165 along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is approximate to 9 degrees C more stable than wild type and one that is approximate to 16 degrees C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.