Identification of a unique "stability control region" that controls protein stability of tropomyosin: A two-stranded alpha-helical coiled-coil.

Nine recombinant chicken skeletal alpha-tropomyosin proteins were prepared, eight C-terminal deletion constructs and the full length protein (1-81, 1-92, 1-99, 1-105, 1-110, 1-119, 1-131, 1-260 and 1-284) and characterized by circular dichroism spectroscopy and analytical ultracentrifugation. We identified for the first time, a stability control region between residues 97 and 118. Fragments of tropomyosin lacking this region (1-81, 1-92, and 1-99) still fold into two-stranded alpha-helical coiled-coils but are significantly less stable (T(m) between 26-28.5 degrees C) than longer fragments containing this region (1-119, 1-131, 1-260 and 1-284) which show a large increase in their thermal midpoints (T(m) 40-43 degrees C) for a DeltaT(m) of 16-18 degrees C between 1-99 and 1-119. We further investigated two additional fragments that ended between residues 99 and 119, that is fragments 1-105 and 1-110. These fragments were more stable than 1-99 and less stable than 1-119, and showed that there were three separate sites that synergistically contribute to the large jump in protein stability (electrostatic clusters 97-104 and 112-118, and a hydrophobic interaction from Leu 110). All the residues involved in these stabilizing interactions are located outside the hydrophobic core a and d positions that have been shown to be the major contributor to coiled-coil stability. Our results show clearly that protein stability is more complex than previously thought and unique sites can synergistically control protein stability over long distances.