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

Kremneva, Elena, Boussouf, Sabrina, Nikolaeva, Olga, Maytum, Robin, Geeves, Michael A., 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. (doi:10.1529/biophysj.104.048793) (The full text of this publication is not currently available from this repository. You may be able to access a copy if URLs are provided) (KAR id:13201)

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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
DOI/Identification number: 10.1529/biophysj.104.048793
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 > Sciences > School of Biosciences
Depositing User: Michael Geeves
Date Deposited: 29 May 2009 06:05 UTC
Last Modified: 23 Jan 2020 04:02 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/13201 (The current URI for this page, for reference purposes)
Geeves, Michael A.: https://orcid.org/0000-0002-9364-8898
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