Myosin-induced Movement Of Tropomyosin Isoforms On Actin Filament

Abstract

Tropomyosins (TMs) are a family of proteins which regulate actin interactions with myosins and other actin-binding proteins. TMs flexibility is thought to underlie the mechanism of TM-dependent regulation. In this work involvement of C-terminal sequence of TM in myosin-induced shift of TM during filament activation was analyzed. Recombinant αTM variants with C-terminus encoded by exon 9a (TM1b9a) or 9d (TM5a) were used. Both isoforms belong to short class of TMs with N-terminus encoded by exon 1b. To assess the degree of TM flexibility we measured FRET between AEDANS, which was attached to each TM isoform either in Cys153 or in Cys28, and DABMI bound to actin in Cys374. TM's Cys153 is a native residue located in central region of TM, Cys28 was introduced in N-terminal region using recombinant DNA methods. When bound to actin alone both TM isoforms show moderate flexibility with TM-actin distances ranging between 38.5 and 44.3 A. Myosin S1 induced >10 A movement of N-terminal region of TM5a, whereas it's central region was moved by about 4 A. More uniform movement of the central and N-terminal regions was observed for TM1b9a. We conclude that TM's C-terminal sequence determines flexibility of the molecule. Supported by The Wellcome Trust.