Conformational stability of human skeletal tropomyosins modified by site-directed mutagenesis

We have used human ß-tropomyosin produced in Escherichiacoli and deletion mutants obtained by site-directed mutagenesisto analyse the conformational stability of this molecule undervarious experimental conditions. Protein engineering has allowedus to answer some questions raised by stability analysis ofthe wild-type tropomyosin. The complex pattern of denaturationis due neither to heterogeneity of the preparation nor to head-to-tailinteractions. The N- and C-termini are not of importance forthe thermal stability of the molecule. On the contrary, deletionof the 31 C-terminus amino acids leads to a dramatic decreaseof the stability observed in guanidinium chloride. This loweringis interpreted as the participation of one more guanidiniumchloride ions to the denaturation equilibrium. Analysis of thestability in presence of organic solvents reveals that acetonitrileand methanol induce opposite effects. Investigation of theseeffects by three methods (CD, fluorescence and electrophoresisthat measure respectively the content in alpha-helix, the contactbetween the two strands and the strands exchange) leads to theconclusion that strand separation can precede the denaturationof the alpha-helix.