Abstract
Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces interact with titin in the sarcomere, enabling muscle contraction. However, testing these mechanical hypotheses on native titin in its natural environment has remained inaccessible to conventional genetics, biophysics and molecular biology tools. To overcome these limitations, here we demonstrate a genetically engineered knock-in mouse model carrying a HaloTag-TEV insertion in titin. Using our system, we have specifically severed the titin filament by digestion with TEV protease, and found that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. HaloTag-based covalent tethering has enabled directed examination of the dynamics of native titin under physiological forces using recently developed magnetic tweezers. At physiological pulling forces lower than 10 pN, titin domains are readily recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Our results support an active role of titin in muscle contraction in coordination with actomyosin motors.
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