Abstract
Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10 pN, titin domains are recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.
Highlights
Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues
Homozygous and heterozygous animals for the HaloTagTEV insertion are obtained at Mendelian rates
We did not observe any alteration in the ultrastructure of sarcomeres, their resting sarcomere length or in the passive force generated by muscle fibers from the HaloTag-TEV-titin mice (Supplementary Fig. 3)
Summary
Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. Testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. The magnitude of mechanical work produced by titin folding has been proposed to be similar to that delivered by the ATP-driven activity of myosin motors, a realization that has triggered the hypothesis that titin folding is an important contributor to active muscle contraction[15,16] Hypotheses like this are challenging to test experimentally due to the absence of methods to probe native protein mechanics. Our cassette includes a TEV protease recognition site and a HaloTag domain, which in combination allow protein labeling to assess cellular localization, polypeptide severing to probe mechanical function, and clickchemistry-based covalent anchoring for single-protein mechanical manipulation. We propose that the HaloTag-TEV cassette can be applied to examine the performance of a broad spectrum of proteins with mechanical function
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
