Kremneva, E. and Boussouf, S. and Nikolaeva, O. and Maytum, R. and Geeves, M.A. and Levitsky, D.I. (2004) Effects of two familial hypertrophic cardiomyopathy mutations in alpha-tropomyosin, Asp175Asn and Glu180Gly, on the thermal unfolding of actin-bound tropomyosin. Biophys J, 87 (6). pp. 3922-33. ISSN 0006-3495 .
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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.
|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|
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences|
|Depositing User:||Michael Geeves|
|Date Deposited:||29 May 2009 06:05|
|Last Modified:||29 May 2009 06:05|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/13201 (The current URI for this page, for reference purposes)|
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