28 April 2020

A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues.

DOI : 10.1038/s41467-020-15465-9

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Andrés Rivas-Pardo, PhD.

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.

Participating Center Researchers

Mechanical Biology Laboratory

PhD in Sciences, Universidad de Chile, Chile.


LEER MÁS
Edificio2

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