Effects of two familial hypertrophic cardiomyopathy mutations in alpha-tropomyosin, Asp175Asn and Glu180Gly, on the thermal unfolding of actin-bound tropomyosin

Kremneva, Elena and Boussouf, Sabrina and Nikolaeva, Olga and Maytum, Robin and Geeves, Michael A. and Levitsky, Dmitrii I. (2004) Effects of two familial hypertrophic cardiomyopathy mutations in alpha-tropomyosin, Asp175Asn and Glu180Gly, on the thermal unfolding of actin-bound tropomyosin. Biophysical Journal, 87 (6). pp. 3922-33. ISSN 0006-3495 . (The full text of this publication is not available from this repository)

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Official URL
http://dx.doi.org/10.1529/biophysj.104.048793

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.

Item Type: Article
Additional information: 0006-3495 (Print) Journal Article
Uncontrolled keywords: Actins/*chemistry Amino Acid Substitution Binding Sites Cardiomyopathy, Hypertrophic, Familial/*genetics/*metabolism Mutagenesis, Site-Directed Mutation Protein Binding Protein Denaturation Protein Folding Research Support, Non-U.S. Gov't Structure-Activity Relationship Temperature Tropomyosin/*chemistry/genetics
Subjects: Q Science
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences
Depositing User: Michael Geeves
Date Deposited: 29 May 2009 06:05
Last Modified: 16 Apr 2014 10:58
Resource URI: http://kar.kent.ac.uk/id/eprint/13201 (The current URI for this page, for reference purposes)
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