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

• Published in: Biophysical journal 2004-12, Vol.87 (6), p.3922-3933
• Main Authors: Kremneva, Elena, Boussouf, Sabrina, Nikolaeva, Olga, Maytum, Robin, Geeves, Michael A., Levitsky, Dmitrii I.
• Format: Article
• Language: English
• Subjects: Actins - chemistry; Amino Acid Substitution; Binding Sites; Cardiomyopathy, Hypertrophic, Familial - genetics; Cardiomyopathy, Hypertrophic, Familial - metabolism; Muscle and Contractility; Mutagenesis, Site-Directed; Mutation; Protein Binding; Protein Denaturation; Protein Folding; Structure-Activity Relationship; Temperature; Tropomyosin - chemistry; Tropomyosin - genetics
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1529/biophysj.104.048793

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.