Use this URL to cite or link to this record in EThOS:
Title: Tropomyosin heterodimers in cardiac muscle regulation
Author: Kalyva, Athanasia
ISNI:       0000 0004 2677 8777
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2009
Availability of Full Text:
Access from EThOS:
Access from Institution:
The binding of myosin to actin in the skeletal and heart muscles can be regulated by tropomyosin (Tm), troponin (Tn) and Cat+. Two Tm isoforms are expressed in striated muscles, namely the skeletal a and ß isoforms, which are combined in vivo to form the as homodimers and the aß heterodimers. It is known that, in contrast to the skeletal muscles, the level of the aß heterodimer in a healthy heart is low. In the hearts of smaller animals, the as homodimer is predominantly present. Overexpression of the ß isoform in transgenic mice, resulted in the formation of the aß heterodimer in heart and was lethal with the heart exhibiting several pathological abnormalities. It is not known how the skeletal aß heterodimer regulates heart contraction and why it can be associated with cardiomyopathies. This question has not been addressed to date because purification of the aß heterodimer was problematic. The major achievement of the project presented in this thesis was the development of a method to successfully assemble and purify in vitro the skeletal Tm heterodimer. The aß heterodimers were formed by thermally-induced chain dissociation of bacterially expressed as and ßß homodimers followed by chain exchange and dimer recombination. The ßß homodimers used were tagged at their N-termini (with His or Strep affinity tags). The presence of the small affinity tag prevented the preferential recombination of the tagged-ß monomers into the ßß homodimer and allowed the formation of the target aß heterodimer. This novel method was also applied for the in vitro formation of other types of heterodimers that were carries of a cardiomyopathy mutation, namely the aD175N or the aE180G mutations. It is presented here that heterodimers carrying aß subunit are less thermally stable than homodimers or heterodimers composed by a subunits. Also the heterodimers had reduced affinity for actin when compared to the homodimers. However all heterodimers with the skeletal Tn could efficiently regulate the binding of myosin to actin. The in vitro formation and the purification of the aß heterodimer, now allows the further characterisation of the molecule and will lead in a better understanding of the regulation of heart muscle contraction.
Supervisor: Geeves, Michael A. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Q Science