A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues
The HaloTag-TEV cassette allows bright fluorescent detection of the tagged protein in tissues, as shown here for the giant protein titin in skeletal muscle. The cassette also enables probing protein mechanics in tissues and ex vivo using single-molecule force spectroscopy.
Rivas-Pardo JA, Li Y, Martonfalvi Z, Tapia-Rojo R, Unger A, Fernandez-Trasancos A, Herrero-Galan E, Velazquez-Carreras D, Fernandez JM, Linke WA, Alegre-Cebollada J.
Nat Commun 2020 Apr; 11: 2060.
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.
PubMed: 32345978. Doi: 10.1038/s41467-020-15465-9. OPEN Free PMC
