Titin is a striated muscle-specific giant protein (M(r) approximately 3,000,000) that consists predominantly of two classes of approximately 100 amino acid motifs, class I and class II, that repeat along the molecule. Titin is found inside the sarcomere, in close proximity to both actin and myosin filaments. Several biochemical studies have found that titin interacts with myosin and actin. In the present work we investigated 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 sliding on top of a lawn of myosin molecules. We used genetically expressed titin fragments containing either a single class I motif (Ti I), a single 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 either myosin, heavy meromyosin, or myosin subfragment-1. In contrast, the linked fragment (Ti I-II) strongly inhibited actin sliding. Ti I-II-induced inhibition was observed with full-length myosin, heavy meromyosin, and myosin subfragment-1. The degree of inhibition was largest with myosin subfragment-1, intermediate with heavy meromyosin, and smallest with myosin. In vitro binding assays and electrophoretic analyses revealed that the inhibition is most likely caused by interaction between the actin filament and the titin I-II fragment. The physiological relevance of the novel finding of motility inhibition by titin fragments is discussed.
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