A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues
Open Access
- 28 April 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature Communications
- Vol. 11 (1), 1-13
- https://doi.org/10.1038/s41467-020-15465-9
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.This publication has 79 references indexed in Scilit:
- Truncations of Titin Causing Dilated CardiomyopathyThe New England Journal of Medicine, 2012
- Genetic Variation in Titin in Arrhythmogenic Right Ventricular Cardiomyopathy–Overlap SyndromesCirculation, 2011
- Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteinsNature, 2011
- Titin visualization in real time reveals an unexpected level of mobility within and between sarcomeresThe Journal of cell biology, 2011
- Tuning Passive Mechanics through Differential Splicing of Titin during Skeletal Muscle DevelopmentBiophysical Journal, 2009
- PKC Phosphorylation of Titin’s PEVK ElementCirculation Research, 2009
- A combined approach to improving large-scale production of tobacco etch virus proteaseProtein Expression and Purification, 2007
- The Mechanical Fingerprint of a Parallel Polyprotein DimerBiophysical Journal, 2007
- Contour Length and Refolding Rate of a Small Protein Controlled by Engineered Disulfide BondsBiophysical Journal, 2007
- Entropic Elasticity of λ-Phage DNAScience, 1994