Detection of Distinct Domains in Stretched Titin experiments where urea was omitted from the buffer solution. Considering that on Thiazole Orange web average only 22 globular domains became unfolded per titin molecule upon receding-meniscus action, the systematic unfolding in the kinase makes it one of the mechanically weakest titin domains with canonical structure and supports its role as a discrete mechanosensor. The weak positive correlation between the width of the first gap and the length of the corresponding titin molecule suggests that the unfolded N-terminal region of the kinase domain responds to the overall extension of titin, which may have implications on the diffusional access to the ATPbinding pocket. Whether titin kinase might function as a continuous mechanosensor rather than a discrete one only, needs much further investigation. Altogether the MedChemExpress PTH 1-34 topographical distance mapping employed here may allow us to allocate further unfolded domains downstream of the kinase and explore the presence of any spatial pattern in the force-driven structural changes in titin. AFM. Periodically arranged globular structures along the molecule’s contour were identified as individual folded domains, and topographical gaps were correlated with unfolded and extended domains. Based on topographical distance mapping we hypothesize that the gap nearest the M-line end corresponds to the Nterminal part of the kinase domain, raising further implications of this 25837696 domain in sarcomeric mechanosensing. Acknowledgments The authors gratefully acknowledge the assistance of Agnes Iszlai and Katalin Naftz with the preparation of titin, and that of Dorina Koszegi with processing image data. Author Contributions Conceived and designed the experiments: MK ZM. Performed the experiments: MK ZM. Analyzed the data: MK ZM. Contributed reagents/materials/analysis tools: MK ZM. Wrote the paper: MK ZM. Conclusions Titin molecules overstretched by receding meniscus and captured on mica surface were visualized here with high-resolution References 1. Wang K Titin/connectin and nebulin: giant protein rulers of muscle structure and function. Advances In Biophysics 33: 123134. 2. Maruyama K Connectin/titin, giant elastic protein of muscle. FASEB J 11: 341345. 3. Funatsu T, Higuchi H, Ishiwata S Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin. Journal of Cell Biology 110: 5362. 4. Gregorio CC, Granzier H, Sorimachi H, Labeit S Muscle assembly: a titanic achievement Curr Opin Cell Biol 11: 1825. 5. Labeit S, Kolmerer B Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science 270: 293296. 6. Horowits R, Kempner ES, Bisher ME, Podolsky RJ A physiological role for titin and nebulin in skeletal muscle. Nature 323: 160164. doi:10.1038/ 323160a0. 7. Granzier HL, Irving TC Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments. Biophysj 68: 1027 1044. doi:10.1016/S0006-349580278-X. 8. Linke WA, Ivemeyer M, Olivieri N, Kolmerer B, Ru egg JC, et al. Towards a molecular understanding of the elasticity of titin. J Mol Biol 261: 62 71. 9. Kellermayer MS, Smith SB, Granzier HL, Bustamante C Foldingunfolding transitions in single titin molecules characterized with laser tweezers. Science 276: 11121116. 10. Tskhovrebova L, Trinick J, Sleep JA, Simmons RM Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature 387: 308 312. doi:10.1038/387308a0. 11.Detection of Distinct Domains in Stretched Titin experiments where urea was omitted from the buffer solution. Considering that on average only 22 globular domains became unfolded per titin molecule upon receding-meniscus action, the systematic unfolding in the kinase makes it one of the mechanically weakest titin domains with canonical structure and supports its role as a discrete mechanosensor. The weak positive correlation between the width of the first gap and the length of the corresponding titin molecule suggests that the unfolded N-terminal region of the kinase domain responds to the overall extension of titin, which may have implications on the diffusional access to the ATPbinding pocket. Whether titin kinase might function as a continuous mechanosensor rather than a discrete one only, needs much further investigation. Altogether the topographical distance mapping employed here may allow us to allocate further unfolded domains downstream of the kinase and explore the presence of any spatial pattern in the force-driven structural changes in titin. AFM. Periodically arranged globular structures along the molecule’s contour were identified as individual folded domains, and topographical gaps were correlated with unfolded and extended domains. Based on topographical distance mapping we hypothesize that the gap nearest the M-line end corresponds to the Nterminal part of the kinase domain, raising further implications of this 25837696 domain in sarcomeric mechanosensing. Acknowledgments The authors gratefully acknowledge the assistance of Agnes Iszlai and Katalin Naftz with the preparation of titin, and that of Dorina Koszegi with processing image data. Author Contributions Conceived and designed the experiments: MK ZM. Performed the experiments: MK ZM. Analyzed the data: MK ZM. Contributed reagents/materials/analysis tools: MK ZM. Wrote the paper: MK ZM. Conclusions Titin molecules overstretched by receding meniscus and captured on mica surface were visualized here with high-resolution References 1. Wang K Titin/connectin and nebulin: giant protein rulers of muscle structure and function. Advances In Biophysics 33: 123134. 2. Maruyama K Connectin/titin, giant elastic protein of muscle. FASEB J 11: 341345. 3. Funatsu T, Higuchi H, Ishiwata S Elastic filaments in skeletal muscle revealed by selective removal of thin filaments with plasma gelsolin. Journal of Cell Biology 110: 5362. 4. Gregorio CC, Granzier H, Sorimachi H, Labeit S Muscle assembly: a titanic achievement Curr Opin Cell Biol 11: 1825. 5. Labeit S, Kolmerer B Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science 270: 293296. 6. Horowits R, Kempner ES, Bisher ME, Podolsky RJ A physiological role for titin and nebulin in skeletal muscle. Nature 323: 160164. doi:10.1038/ 323160a0. 7. Granzier HL, Irving TC Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments. Biophysj 68: 1027 1044. doi:10.1016/S0006-349580278-X. 8. Linke WA, Ivemeyer M, Olivieri N, Kolmerer B, Ru egg JC, et al. Towards a molecular understanding of the elasticity of titin. J Mol Biol 261: 62 71. 9. Kellermayer MS, Smith SB, Granzier HL, Bustamante C Foldingunfolding transitions in single titin molecules characterized with laser tweezers. Science 276: 11121116. 10. Tskhovrebova L, Trinick J, Sleep JA, Simmons RM Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature 387: 308 312. doi:10.1038/387308a0. 11.