Effects of two familial hypertrophic cardiomyopathy mutations in α-tropomyosin, Asp175Asn and Glut180Gly, on the thermal unfolding of actin-bound tropomyosin

Differential scanning calorimetry was used to investigate the thermal unfolding of native α-tropomyosin (Tm), wild-type α-Tm expressed in Escherichia coli and the wild-type α-Tm carrying either of two missense mutations associated with familial hypertrophic cardiomyopathy, D175N or E180G. Recombinant α-Tm was expressed with an N-terminal Ala-Ser extension to substitute for the essential N-terminal acetylation of the native Tm. Native and Ala-Ser-Tm were indistinguishable in our assays. In the absence of F-actin, the thermal unfolding of Tm was reversible and the heat sorption curve of Tm with Cys-190 reduced was decomposed into two separate calorimetric domains with maxima at ∼42 and 51°C. In the presence of phalloidin-stabilized F-actin, a new cooperative transition appears at 46-47°C and completely disappears after the irreversible denaturation of F-actin. A good correlation was found to exist between the maximum of this peak and the temperature of half-maximal dissociation of the F-actin/Tm complex as determined by light scattering experiments. We conclude that Tm thermal denaturation only occurs upon its dissociation from F-actin. In the presence of F-actin, D175N α-Tm shows a melting profile and temperature dependence of dissociation from F-actin similar to those for wild-type α-Tm. The actin-induced stabilization of E180G α-Tm is significantly less than for wild-type α-Tm and D175N α-Tm, and this property could contribute to the more severe myopathy phenotype reported for this mutation.