title: 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).
abstract: 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), residues 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 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 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) 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 Å 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 wild type 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 rodlike molecule.
authors.affiliation: From the Program in Structural Biology and Biophysics, Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, Colorado 80045.
authors.forename: J Paul
authors.initials: JP
authors.lastname: Kirwan
authors.forename: Robert S
authors.initials: RS
authors.lastname: Hodges
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D057927:Hydrophobic and Hydrophilic Interactions; D020125:Mutation, Missense; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C490773:Tpm1 protein, rat; D014335:Tropomyosin
keywords: Circular Dichroism (CD); Contractile Protein; Differential Scanning Calorimetry (DSC); Protein Stability; Recombinant Protein Expression; Stability Control Region; Stability Signal Transmission; Tropomyosin; Tropomyosin N-domain 1–131; Two-stranded Coiled-coils