Quantitative analysis of tropomyosin linear polymerization equilibrium as a function of ionic strength.
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.
Sousa
Aurea D
AD
Departamento de Bioquimica, Instituto de Quimica, Universidade de São Paulo CP 26.077, CEP 05599-970 São Paulo, SP, Brazil.
Farah
Chuck S
CS
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*
D001704:Biopolymers; D017931:DNA Primers; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D014335:Tropomyosin; D006916:5-Hydroxytryptophan