Tropomyosins containing 5-hydroxytryptophan: probes specific for interactions in thin filament

Author(s)

Aurea Denise de Sousa Soller Ferreira

• Document type: Doctoral Thesis
• Press: São Paulo. , gráficos, ilustrações, tabelas.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2002-03-08

Examining board members

Chuck Shaker Farah; Sergio Teixeira Ferreira; Shirley Schreier; Martha Meriwether Sorenson; Sérgio Verjovski Almeida

• Advisor: Shaker Chuck Farah
• Field of knowledge: Biological Sciences - Biochemistry
• Indexed in: Banco de Dados Bibliográficos da USP-DEDALUS
• Location: Universidade de São Paulo. Biblioteca do Conjunto das Químicas; T/574.19245; S725t

Abstract

Tropomyosin (Tm) is a coiled-coil protein that polymerizes by head-totail interactions in an ionic-strength-dependent manner. In skeletal muscle,Tm interacts with troponin (Tn) to modulate muscle contraction via its Ca2+-dependent repositioning on the actin filament. Since residues 258-275 present a striated muscle-specific pattern atypical for a coiled-coil structure, we produced a total of 18 polymerizable (ASTm) and non-polymerizable (nfTm) versions of recombinant Tm with tryptophan analogues probes incorporated at specific positions near their C-termini. We found three mutants whose fluorescence are specifically sensitive to Tn-binding (position 263), actin-binding (261) or Tm polymerization (269). We used these mutants to: i) quantitatively investigate the ASTm monomer - polymer equilibrium and its dependence on ionic strength and determine a minimum number effective charges involved in stabilizing the head-to-tai/ interaction, ii) demonstrate that amino acid residue 263 of Tm interacts with residues 77-157 of TnT and that neighboring amino acid sequences along the primary structure of TnT modulate this interaction, iii) quantitatively analyze the binding of the ASTm mutants to Tn, in the presence and in the absence of actin and Ca2+; and iv) qualitatively investigate the influence of the head-to-tail overlap in Tm binding to actin and/or Tn. (AU)