Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Reverse engineering of the giant muscle protein titin


Through the study of single molecules it has become possible to explain the function of many of the complex molecular assemblies found in cells1,2,3,4,5. The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ6,7,8,9,10 and at the level of single molecules11,12,13,14. These studies suggested that titin is not a simple entropic spring but has a complex structure-dependent elasticity. Here we use protein engineering and single-molecule atomic force microscopy15 to examine the mechanical components that form the elastic region of human cardiac titin16,17. We show that when these mechanical elements are combined, they explain the macroscopic behaviour of titin in intact muscle6. Our studies show the functional reconstitution of a protein from the sum of its parts.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The proximal and distal tandem Ig regions of cardiac titin have different mechanical properties.
Figure 2: Single-molecule AFM measurements of the mechanical properties of the N2B and PEVK regions of titin.
Figure 3: Single-molecule data explain the extensibility of the individual titin segments measured in situ.
Figure 4: Single-molecule data predict the force–extension curve of cardiac muscle.

Similar content being viewed by others


  1. Sigworth, F. J. & Neher, E. Single Na+ channel currents observed in cultured rat muscle cells. Nature 287, 447–449 (1980)

    Article  ADS  CAS  Google Scholar 

  2. Bustamante, C., Smith, S. B., Liphardt, J. & Smith, D. Single-molecule studies of DNA mechanics. Curr. Opin. Struct. Biol. 10, 279–285 (2000)

    Article  CAS  Google Scholar 

  3. Smith, D. E. et al. The bacteriophage φ29 portal motor can package DNA against a large internal force. Nature 413, 748–752 (2001)

    Article  ADS  CAS  Google Scholar 

  4. Finer, J. T., Simmons, R. M. & Spudich, J. A. Single myosin molecule mechanics: piconewton forces and nanometre steps. Nature 368, 113–119 (1994)

    Article  ADS  CAS  Google Scholar 

  5. Lu, H. & Schulten, K. Steered molecular dynamics simulations of force-induced protein domain unfolding. Proteins Struct. Funct. Genet. 35, 453–463 (1999)

    Article  Google Scholar 

  6. Linke, W. A. et al. I-band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure. J. Cell Biol. 146, 631–644 (1999)

    Article  CAS  Google Scholar 

  7. Maruyama, K. Connectin/titin, giant elastic protein of muscle. FASEB J. 11, 341–345 (1997)

    Article  CAS  Google Scholar 

  8. Wang, K. Titin/connectin and nebulin: giant protein rulers of muscle structure and function. Adv. Biophys. 33, 123–134 (1996)

    Article  CAS  Google Scholar 

  9. Gregorio, C. C., Granzier, H., Sorimachi, H. & Labeit, S. Muscle assembly: a titanic achievement? Curr. Opin. Cell Biol. 11, 18–25 (1999)

    Article  CAS  Google Scholar 

  10. Trinick, J. & Tskhovrebova, L. Titin: a molecular control freak. Trends Cell Biol. 9, 377–380 (1999)

    Article  CAS  Google Scholar 

  11. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M. & Gaub, H. E. Reversible unfolding of individual immunoglobin domains by AFM. Science 276, 1109–1112 (1997)

    Article  CAS  Google Scholar 

  12. Kellermayer, M., Smith, S., Granzier, H. & Bustamante, C. Folding-unfolding transitions in single titin molecules characterized with laser tweezers. Science 276, 1112–1116 (1997)

    Article  CAS  Google Scholar 

  13. Tskhovrebova, L., Trinick, J., Sleep, J. A. & Simmons, R. M. Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature 387, 308–312 (1997)

    Article  ADS  CAS  Google Scholar 

  14. Tskhovrebova, L. & Trinick, J. Direct visualization of extensibility in isolated titin molecules. J. Mol. Biol. 265, 100–106 (1997)

    Article  CAS  Google Scholar 

  15. Fisher, T. E., Marszalek, P. E. & Fernandez, J. M. Stretching single molecules into novel conformations using the atomic force microscope. Nature Struct. Biol. 7, 719–724 (2000)

    Article  CAS  Google Scholar 

  16. Freiburg, A. et al. Series of exon-skipping events in the elastic spring region of titin as the structural basis for myofibrillar elastic diversity. Circ. Res. 86, 1114–1121 (2000)

    Article  CAS  Google Scholar 

  17. Labeit, S. & Kolmerer, B. Titins, giant proteins in charge of muscle ultrastructure and elasticity. Science 270, 293–296 (1995)

    Article  ADS  CAS  Google Scholar 

  18. Carrion-Vazquez, M. et al. Mechanical and chemical unfolding of a single protein: a comparison. Proc. Natl Acad. Sci. USA 96, 3694–3699 (1999)

    Article  ADS  CAS  Google Scholar 

  19. Li, H. B., Oberhauser, A. F., Fowler, S. B., Clarke, J. & Fernandez, J. M. Atomic force microscopy reveals the mechanical design of a modular protein. Proc. Natl Acad. Sci. USA 97, 6527–6531 (2000)

    Article  ADS  CAS  Google Scholar 

  20. Li, H. B. et al. Multiple conformations of PEVK proteins detected by single-molecule techniques. Proc. Natl Acad. Sci. USA 98, 10682–10686 (2001)

    Article  ADS  CAS  Google Scholar 

  21. Best, R. B., Li, B., Steward, A., Daggett, V. & Clarke, J. Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation. Biophys. J. 81, 2344–2356 (2001)

    Article  CAS  Google Scholar 

  22. Marko, J. F. & Siggia, E. D. Stretching DNA. Macromolecules 28, 8759–8770 (1995)

    Article  ADS  CAS  Google Scholar 

  23. Bell, G. I. Models for the specific adhesion of cells to cells. Science 200, 618–627 (1978)

    Article  ADS  CAS  Google Scholar 

  24. Marszalek, P. E. et al. Mechanical unfolding intermediates in titin modules. Nature 402, 100–103 (1999)

    Article  ADS  CAS  Google Scholar 

  25. Trombitas, K., Freiburg, A., Centner, T., Labeit, S. & Granzier, H. Molecular dissection of N2B cardiac titin's extensibility. Biophys. J. 77, 3189–3196 (1999)

    Article  CAS  Google Scholar 

  26. Bueche, F. Physical Properties of Polymers 37 (Interscience, New York, 1962)

    Google Scholar 

  27. Linke, W. A. et al. Towards a molecular understanding of the elasticity of titin. J. Mol. Biol. 261, 62–71 (1996)

    Article  CAS  Google Scholar 

  28. Higuchi, H., Nakauchi, Y., Maruyama, K. & Fujime, S. Characterization of beta-connectin (titin 2) from striated muscle by dynamic light scattering. Biophys. J. 65, 1906–1915 (1993)

    Article  ADS  CAS  Google Scholar 

  29. Allen, D. G. & Kentish, J. C. The cellular basis of the length-tension relation in cardiac muscle. J. Mol. Cell Cardiol. 17, 821–840 (1985)

    Article  CAS  Google Scholar 

  30. Scott, K. A., Steward, A., Fowler, S. B. & Clarke, J. Titin; a multidomain protein that behaves as the sum of its parts. J. Mol. Biol. 315, 819–829 (2002)

    Article  CAS  Google Scholar 

Download references


We thank H. Erickson for the electron microscope pictures of I27 polyproteins. W.A.L. thanks the German Research Foundation for a Heisenberg fellowship. This work was supported by the National Institutes of Health (J.M.F.)

Author information

Authors and Affiliations


Corresponding author

Correspondence to Julio M. Fernandez.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, H., Linke, W., Oberhauser, A. et al. Reverse engineering of the giant muscle protein titin. Nature 418, 998–1002 (2002).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing