Effects of Two Familial Hypertrophic Cardiomyopathy Mutations in α-Tropomyosin, Asp175Asn and Glu180Gly, on the Thermal Unfolding of Actin-Bound Tropomyosin

Abstract

Differential scanning calorimetry was used to investigate the thermal unfolding of native alpha-tropomyosin (Tm), wild-type alpha-Tm expressed in Escherichia coli and the wild-type alpha-Tm carrying either of two missense mutations associated with familial hypertrophic cardiomyopathy, D175N or E180G. Recombinant alpha-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 approximately 42 and 51 degrees C. In the presence of phalloidin-stabilized F-actin, a new cooperative transition appears at 46-47 degrees 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 alpha-Tm shows a melting profile and temperature dependence of dissociation from F-actin similar to those for wild-type alpha-Tm. The actin-induced stabilization of E180G alpha-Tm is significantly less than for wild-type alpha-Tm and D175N alpha-Tm, and this property could contribute to the more severe myopathy phenotype reported for this mutation.

Conserved Asp-137 Is Important for both Structure and Regulatory Functions of Cardiac α-Tropomyosin (α-TM) in a Novel Transgenic Mouse Model Expressing α-TM-D137L

“…Previously, the dilated cardiomyopathy-linked E54K mutation was also reported to increase the temperature stability of ␣-TM (45). In contrast, increased flexibility appears to be a characteristic of hypertrophic cardiomyopathy-linked TM mutations (E180G, D175N, K70T, and A63V) (17,46,47). These findings indicate a possible association between TM flexibility and the complex mechanisms leading to cardiac disorders.…”

“…The calorimetric traces were corrected for instrumental background by subtracting a scan with buffer in both cells. The DSC data were analyzed using Origin version 7.0 (MicroCal) as described (17). The heat capacity (C p ) versus temperature curve was analyzed to determine the transition temperature (T m ) and the calorimetric enthalpy of transition (⌬H cal ).…”

Conserved Noncanonical Residue Gly-126 Confers Instability to the Middle Part of the Tropomyosin Molecule

“…The heat capacity curves obtained for Tm WT and its mutants were subjected to deconvolution analysis as was described previously (21,22), and the results show that each DSC profile can be decomposed into two separate thermal transitions (calorimetric domains) (Fig. 4).…”

“…The Maxchelator program was used to calculate the free Ca 2ϩ concentration. DSC Measurements-DSC experiments on recombinant ␣-Tm WT, Tm G126A, and Tm G126R were performed as described earlier for ␣-Tm and its various mutants (21,22). All Tm species were fully reduced before experiments by heating in the presence of ␤-mercaptoethanol with following preheating of the samples in the calorimeter cell.…”

“…Light Scattering-Thermally induced dissociation of Tm⅐F-actin complexes was detected by changes in light scattering at 90°, and the dissociation curves were analyzed as described earlier (21). Light-scattering measurements were performed under the same conditions and at the same heating rate as the DSC experiments.…”