UNFOLDING DOMAINS OF RECOMBINANT FUSION ALPHA-ALPHA-TROPOMYOSIN

The thermal unfolding of the coiled-coil alpha-helix of recombinant alphaalpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35-degrees-C involving the middle of the molecule; (2) a major transition at 46-degrees-C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56-degrees-C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70-degrees-C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56-degrees-C or 70-degrees-C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a crosslink to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46-degrees-C from the stabilized N-terminal unfolding domain at 56-degrees-C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.