title: Quantitative analysis of tropomyosin linear polymerization equilibrium as a function of ionic strength.
abstract: Tropomyosin is a coiled-coil protein that polymerizes by head-to-tail interactions in an ionic strength-dependent manner. We produced a recombinant full-length chicken alpha-tropomyosin containing a 5-hydroxytryptophan residue at position 269 (formerly an alanine), 15 residues from the C terminus, and show that its fluorescence intensity specifically reports tropomyosin head-to-tail interactions. We used this property to quantitatively study the monomer-polymer equilibrium in tropomyosin and to calculate the equilibrium constant of the head-to-tail interaction as a function of ionic strength. Our results show that the affinity constant changes by almost 2 orders of magnitude over an ionic strength range of 50 mm (between I = 0.045 and 0.095). We were also able to calculate the average polymer length as a function of concentration and ionic strength, which is an important parameter in the interpretation of binding isotherms of tropomyosin with other thin filament proteins such as actin and troponin.
authors.affiliation: Departamento de Bioquimica, Instituto de Quimica, Universidade de São Paulo CP 26.077, CEP 05599-970 São Paulo, SP, Brazil.
authors.forename: Aurea D
authors.initials: AD
authors.lastname: Sousa
authors.forename: Chuck S
authors.initials: CS
authors.lastname: Farah
mesh_terms: D006916:5-Hydroxytryptophan / Q000737:chemistry; D001483:Base Sequence; D001704:Biopolymers / Q000737:chemistry*; D002942:Circular Dichroism; D017931:DNA Primers; D005456:Fluorescent Dyes; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000737:chemistry; D014335:Tropomyosin / Q000737:chemistry*
chemical_list: D001704:Biopolymers; D017931:DNA Primers; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D014335:Tropomyosin; D006916:5-Hydroxytryptophan