THE EFFECT OF GENETICALLY EXPRESSED CARDIAC TITIN FRAGMENTS ON IN-VITRO ACTIN MOTILITY

Titin is a striated muscle-specific giant protein (M(r) similar to 3,0 00,000) that consists predominantly of two classes of similar to 100 a mino acid motifs, Glass I and class II, that repeat along the molecule . Titin is found inside the sarcomere, in close proximity to both acti n and myosin filaments. Several biochemical studies have found that ti tin interacts with myosin and actin. In the present work we investigat ed whether this biochemical interaction is functionally significant by studying the effect of titin on actomyosin interaction in an in vitro motility assay where fluorescently labeled actin filaments are slidin g on top of a lawn of myosin molecules. We used genetically expressed titin fragments containing either a single class I motif (Ti I), a sin gle class II motif (Ti II), or the two motifs linked together (Ti I-II ). Neither Ti I nor Ti II alone affected actin-filament sliding on eit her myosin, heavy meromyosin, or myosin subfragment-1. In contrast, th e linked fragment (Ti I-II) strongly inhibited actin sliding. Ti I-II- induced inhibition was observed with full-length myosin, heavy meromyo sin, and myosin subfragment-1. The degree of inhibition was largest wi th myosin subfragment-1, intermediate with heavy meromyosin, and small est with myosin. In vitro binding assays acid electrophoretic analyses revealed that the inhibition is most likely caused by interaction bet ween the actin filament and the titin I-II fragment. The physiological relevance of the novel finding of motility inhibition by titin fragme nts is discussed.