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

Elena Kremneva ^*, Sabrina Boussouf , Olga Nikolaeva , Robin Maytum , Michael A. Geeves  and Dmitrii I. Levitsky ^* 

^* A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow 119071, Russia; A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia; and Department of Biosciences, University of Kent at Canterbury, Canterbury, Kent CT2 7NJ, United Kingdom

Correspondence: Address reprint requests to Michael A. Geeves, Dept. of Biosciences, University of Kent at Canterbury, Canterbury, Kent CT2 7NJ, UK. Tel: 44-1227-827597; Fax: 44-1227-763912; E-mail: m.a.geeves{at}kent.ac.uk.

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

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