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Hidden complexity in the mechanical properties of titin

Nature volume 422, pages 446–449 (2003)Cite this article

Abstract

Individual molecules of the giant protein titin span the A-bands and I-bands that make up striated muscle. The I-band region of titin is responsible for passive elasticity in such muscle1,2,3,4, and contains tandem arrays of immunoglobulin domains. One such domain (I27) has been investigated extensively, using dynamic force spectroscopy and simulation5,6,7,8,9,10,11,12. However, the relevance of these studies to the behaviour of the protein under physiological conditions was not established. Force studies reveal a lengthening of I27 without complete unfolding, forming a stable intermediate that has been suggested to be an important component of titin elasticity6. To develop a more complete picture of the forced unfolding pathway, we use mutant titins—certain mutations allow the role of the partly unfolded intermediate to be investigated in more depth. Here we show that, under physiological forces, the partly unfolded intermediate does not contribute to mechanical strength. We also propose a unified forced unfolding model of all I27 analogues studied, and conclude that I27 can withstand higher forces in muscle than was predicted previously.

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Figure 1: Ribbon diagram of I27, showing the β-strands labelled A–G and the location of the mutations described.
Figure 2: The dynamical transition N to I in the unfolding free-energy diagram of I27.
Figure 3: Unfolding kinetics of WT I27 and mutants.

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References

  1. Furst, D. O., Osborn, M., Nave, R. & Weber, K. The organization of titin filaments in the half-sarcomere revealed by monoclonal-antibodies in immunoelectron microscopy—A map of 10 nonrepetitive epitopes starting at the Z-line extends close to the M-line. J. Cell Biol. 106, 1563–1572 (1988)

    Article  CAS  Google Scholar 

  2. Labeit, S., Gautel, M., Lakey, A. & Trinick, J. Towards a molecular understanding of titin. EMBO J. 11, 1711–1716 (1992)

    Article  CAS  Google Scholar 

  3. 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 

  4. Linke, W. A., Stockmeier, M. R., Ivemeyer, M., Hosser, H. & Mundel, P. Characterizing titin's I-band Ig domain region as an entropic spring. J. Cell Sci. 111, 1567–1574 (1998)

    CAS  PubMed  Google Scholar 

  5. 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 

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

    Article  ADS  CAS  Google Scholar 

  7. Li, H. B., Carrion-Vazquez, M., Oberhauser, A. F., Marszalek, P. E. & Fernandez, J. M. Point mutations alter the mechanical stability of immunoglobulin modules. Nature Struct. Biol. 7, 1117–1120 (2000)

    Article  CAS  Google Scholar 

  8. Brockwell, D. J. et al. The effect of core destabilization on the mechanical resistance of I27. Biophys. J. 83, 458–472 (2002)

    Article  ADS  CAS  Google Scholar 

  9. Fowler, S. B. et al. Mechanical unfolding of a titin Ig domain: Structure of unfolding intermediate revealed by combining AFM, molecular dynamics simulations, NMR and protein engineering. J. Mol. Biol. 322, 841–849 (2002)

    Article  CAS  Google Scholar 

  10. Li, H. B. et al. Reverse engineering of the giant muscle protein titin. Nature 418, 998–1002 (2002)

    Article  ADS  CAS  Google Scholar 

  11. Lu, H., Isralewitz, B., Krammer, A., Vogel, V. & Schulten, K. Unfolding of titin immunoglobulin domains by steered molecular dynamics simulation. Biophys. J. 75, 662–671 (1998)

    Article  ADS  CAS  Google Scholar 

  12. Lu, H. & Schulten, K. The key event in force-induced unfolding of titin's immunoglobulin domains. Biophys. J. 79, 51–65 (2000)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  14. Trinick, J. Cytoskeleton—Titin as a scaffold and spring. Curr. Biol. 6, 258–260 (1996)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Ma, K., Kan, L. S. & Wang, K. Polyproline II helix is a key structural motif of the elastic PEVK segment of titin. Biochemistry 40, 3427–3438 (2001)

    Article  CAS  Google Scholar 

  17. Linke, W. A. & Granzier, H. A spring tale: New facts on titin elasticity. Biophys. J. 75, 2613–2614 (1998)

    Article  ADS  CAS  Google Scholar 

  18. Minajeva, A., Kulke, M., Fernandez, J. M. & Linke, W. A. Unfolding of titin domains explains the viscoelastic behavior of skeletal myofibrils. Biophys. J. 80, 1442–1451 (2001)

    Article  CAS  Google Scholar 

  19. Tskhovrebova, L. & Trinick, J. Flexibility and extensibility in the titin molecule: Analysis of electron microscope data. J. Mol. Biol. 310, 755–771 (2001)

    Article  CAS  Google Scholar 

  20. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. Gautel, M., Lehtonen, E. & Pietruschka, F. Assembly of the cardiac I-band region of titin/connectin: Expression of the cardiac-specific regions and their structural relation to the elastic segments. J. Muscle Res. Cell Motil. 17, 449–461 (1996)

    Article  CAS  Google Scholar 

  24. Fowler, S. B. & Clarke, J. Mapping the folding pathway of an immunoglobulin domain: Structural detail from phi value analysis and movement of the transition state. Structure 9, 355–366 (2001)

    Article  CAS  Google Scholar 

  25. Best, R. B., Fowler, S. B., Toca-Herrera, J. L. & Clarke, J. A simple method for probing the mechanical unfolding pathway of proteins in detail. Proc. Natl Acad. Sci. USA 99, 12143–12148 (2002)

    Article  ADS  CAS  Google Scholar 

  26. Steward, A., Toca-Herrera, J. L. & Clarke, J. Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy. Protein Sci. 11, 2179–2183 (2002)

    Article  CAS  Google Scholar 

  27. 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 

  28. Evans, E. Probing the relation between force—lifetime—and chemistry in single molecular bonds. Annu. Rev. Biophys. Biomol. Struct. 30, 105–128 (2001)

    Article  CAS  Google Scholar 

  29. Press, W. H., Teukolsky, S. A., Vetterling, W. T. & Flannery, B. P. Numerical Recipes in C: The Art of Scientific Computing (Cambridge Univ. Press, Cambridge, UK, 1992)

    MATH  Google Scholar 

  30. Improta, S., Politou, A. S. & Pastore, A. Immunoglobulin-like modules from titin I-band: Extensible components of muscle elasticity. Structure 4, 323–337 (1996)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

P.M.W. is an EPSRC Advanced Research Fellow, and J.C. is a Wellcome Trust Senior Research Fellow. Funding was provided by the EPSRC, the MRC and the Wellcome Trust. S.B.F. was supported by the EPSRC and Newnham College, and R.B.B. by the Cambridge Commonwealth Trust.

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Author notes

  1. José Luis Toca-Herrera

    Present address: Center for Ultrastructure Research, Universität für Bodenkultur Wien, Gregor Mendel Strasse 33, A-1180, Vienna

Authors and Affiliations

  1. Laboratory of Biophysics and Surface Analysis, School of Pharmaceutical Sciences, University of Nottingham, Nottingham, NG7 2RD, UK

    Philip M. Williams

  2. Cambridge University Chemical Laboratory, MRC Centre for Protein Engineering, Lensfield Road, CB2 1EW, Cambridge, UK

    Susan B. Fowler, Robert B. Best, José Luis Toca-Herrera, Kathryn A. Scott, Annette Steward & Jane Clarke

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Correspondence to Jane Clarke.

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Supplementary information

41586_2003_BFnature01517_MOESM1_ESM.pdf

Supplementary Information: Extends Methods section of manuscript. Describes data analysis in detail. Discusses alternative analysis method. (PDF 150 kb)

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Williams, P., Fowler, S., Best, R. et al. Hidden complexity in the mechanical properties of titin. Nature 422, 446–449 (2003). https://doi.org/10.1038/nature01517

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