Effects of two stabilizing substitutions, D137L and G126R, in the middle part of α-tropomyosin on the domain structure of its molecule

Abstract

We applied differential scanning calorimetry (DSC) to investigate the effects of substitutions D137L and G126R (i.e. replacement of conserved non-canonical residues Asp137 and Gly126 by canonical ones) in the middle part of tropomyosin (Tm), as well as the combined one, D137L/G126R, on the thermal unfolding of Tm. Special approaches (e.g. combination of DSC with measurements of temperature dependences of pyrene excimer fluorescence) were applied to assign separate thermal transitions (calorimetric domains) on the DSC profiles to the certain parts of Tm molecule. The results show that substitutions D137L and G126R (and, especially, the combined one, D137L/G126R) may stabilize not only the middle region of Tm, but also the other parts of its molecule including N- and C-terminal parts. These results suggest that the stabilization of the Tm middle part can be transmitted along the coiled-coil length displaying long-range effects.

Keywords: Coiled-coil stabilization; Differential scanning calorimetry; Domain structure; Pyrene excimer fluorescence; Thermal unfolding; Tropomyosin.