Comparison Of The Conformational Stabilities Of Striated Alpha-Tropomyosins Adapted To Different Temperature Regimes

The conformational stabilities of alpha-striated tropomyosins from warm and cold blooded sources have been compared using circular dichroism (CD) and differential scanning calorimetry. Alpha-tropomyosins from rabbit and shark share 95% sequence identity. There are three replacements in the core: residues Thr179Ala, Ser190Cys and Ser211Ala. At low temperature (pH 7) the two tropomyosins are equivalent in molar ellipticity at 222nm, however, shark tropomyosin unfolds over a wider temperature range than the mammalian counterpart. The dependence of this ellipticity on temperature (0.1M salt, pH 7, 2mg/mL protein + dithiothreitol in the case of rabbit tropomyosin) is characterized by the following transitions: shark, ∼33 (main, ∼two-thirds of total change in signal) and ∼54°C and rabbit, ∼41 (main) and ∼49°C. At ∼10mg/mL, the ΔTm for shark tropomyosin becomes larger on account of an upward shift in the minor transition, otherwise the results of calorimetry are in agreement with those of CD. Analysis of fragments CN1A (residues 11-128, Tm ∼59°C) and CN1B (residues 142 - 281, Tms ∼20 and 35°C) by far-UV CD and intact protein by near-UV CD (Tm ∼ 32°C), shows that the most stable section of shark tropomyosin is located in the amino-terminal half of the molecule, possibly the first ∼100 amino acids. The enhanced flexibility (lower stability) of the remainder of the molecule coincides with the presence of a row of destabilising ‘core’ amino acids between residues 179 - 221. A final point is that conformational stability is insensitive to the presence of a covalently bound phosphate group, consistent with the structural disordering of the corresponding region of each tropomyosin.