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 (97-118) [Molecular Biophysics]

December 20th, 2013 by Kirwan, J. P., Hodges, R. S.

Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR) 97-118 in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by L110, while 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 α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm1-131 proteins, including the native sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry (DSC). The single mutation L110A destabilizes the entire Tm1-131 molecule, showing that the effect of this mutation is transmitted 165Å 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 ~9°C more stable than native and one that is ~16°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 rod-like molecule.
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