Titin-Thin Filament Interaction and Potential Role in Muscle Function
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Titin (connectin) is a giant polypeptide that forms a single-molecule filamental structure extending from the M-line to the Z-line in the sarcomere of striated muscle. The primary structure of titin consists mainly of repeats of two types of ~100-amino acid modules (fibronectin type III and immunoglobulin-like motifs, respectively) and a Pro rich segment named the PEVK domain. The I-band region of titin shows an elasticity important to the passive properties of the myofibril. To investigate the biological function of titin, we cloned cDNA segments encoding single or linked structural modules of titin into expression vectors to produce non-fusion titin fragments in E. coli. High level expression of titin fragments was achieved and effective purification procedures were developed. We also developed specific monoclonal antibodies against the titin fragments and solid-phase protein-binding assays to investigate the interaction of the titin structural modules and other sarcomeric proteins. The results show that the immunoglobin-like module that are enriched in the I-band titin binds to F-actin. In contrast to the rigid association of A-band titin with the thick filament, the relatively weak titin-actin binding suggests that the I-band titin may reversibly interact with the thin filament during muscle contraction. This hypothesis is supported by an epitope similarity between the actin-binding site of caldesmon and the immunoglobulin-like module of titin, which suggests analogous functions of caldesmon and titin in organizing the contractile proteins. Together with data from mechanical studies demonstrating that the titin-actin interaction may contribute to the passive property of cardiac muscle in a Ca2+-dependent manner, we speculate that the Ca2+-mediated thin filament regulation may coordinate the function of titin during muscle contraction and relaxation.
KeywordsThin Filament Sarcomeric Protein Passive Property Recombinant Expression Plasmid PEVK Domain
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