Identification of a Unique “Stability Control Region” that Controls Protein Stability of Tropomyosin: A Two-stranded α-Helical Coiled-coil

Abstract

Nine recombinant chicken skeletal α-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 α-helical coiled-coils but are significantly less stable (Tm between 26–28.5 °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 (Tm 40–43 °C) for a ΔTm of 16–18 °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.