Expression and comparison of tropomyosin isoform actin-binding properties and their resolution within the thin-filament proteome

A thesis submitted to the University of Bedfordshire in fulfilment of the requirements for the degree of Doctor of Philosophy ; Tropomyosins(Tm) are a group of proteins that regulate the actin filaments in both muscle and non-muscle cells. In mammalian cells four Tm species are found: α-Tm (fast) encoded by α-Tm /TPM1 gene, β-Tm, encoded by β-Tm/ TPM2 gene, α-Tm (slow) encoded by γTm gene/ TPM3 and δ-Tm encoded by δTm / TPM4gene. Mutations in Tm are linked to many cardiac and skeletal diseases like hypertrophic cardiac myopathy (TPM1 and TPM2), familial cardiac myopathy (TPM1) and skeletal diseases like nemaline myopathy (TPM2 and TPM3) along with other sarcomere proteins. The hypothesis on which this study is based is, the isoform composition in both muscle and non-muscle cells adapts in response to disease and physiological changes. A significant part of that adaptation is changes in the thin filament protein isoforms expressed and the post translational modifications of these proteins. In this study Tpm3.12st isoform of γTm and other striated muscle tropomyosin isoforms (Tpm1 and Tpm2) and a non-muscle Tmp4 were characterised using a variety of techniques. The aim was to enhance our understanding of the role of tropomyosin interactions in regards to its efficiency of actin binding capacity as well as its effect on actin polymerisation. Human tropomyosin 3 (Tpm3.12st) was expressed in E. coli to produce recombinant protein with three N-terminal sequence variants (Met, MM and (M)ASM). The proteins were characterised for their binding affinity with actin as this isoform has not been well characterised so far. Its properties are compared with other striated muscle tropomyosin Tpm1.1st and Tpm2.2st and non-muscle Tpm4.1cy. The proteins were purified through ion exchange chromatography and the purity was checked by using SDS-PAGE and UV spectrometry. The molecular weights of the recombinant proteins produced were confirmed by mass spectrometry. Cosedimentation assays were performed for their actin binding affinity ...