Journal of Muscle Research & Cell Motility

, Volume 23, Issue 5–6, pp 513–521 | Cite as

Unfolding of titin domains studied by molecular dynamics simulations

  • Mu Gao
  • Hui Lu
  • Klaus Schulten


Titin, a ∼1 μm long protein found in striated muscle myofibrils, possesses unique elastic properties. The extensible behavior of titin has been demonstrated in atomic force microscopy and optical tweezer experiments to involve the reversible unfolding of individual immunoglobulin-like (Ig) domains. We have used steered molecular dynamics (SMD), a novel computer simulation method, to investigate the mechanical response of single titin Ig domains upon stress. Simulations of stretching Ig domains I1 and I27 have been performed in a solvent of explicit water molecules. The SMD approach provides a detailed structural and dynamic description of how Ig domains react to external forces. Validation of SMD results includes both qualitative and quantitative agreement with AFM recordings. Furthermore, combining SMD with single molecule experimental data leads to a comprehensive understanding of Ig domains' mechanical properties. A set of backbone hydrogen bonds that link the domains' terminal β-strands play a key role in the mechanical resistance to external forces. Slight differences in architecture permit a mechanical unfolding intermediate for I27, but not for I1. Refolding simulations of I27 demonstrate a locking mechanism.


Steer Molecular Dynamic Atomic Force Microscopy Experiment Steer Molecular Dynamic Simulation Titin Domain Single Molecule Atomic Force Microscopy 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. Best RB, Li B, Steward A, Daggett V and Clarke J (2001) Can nonmechanical proteins withstand force? stretching barnase by atomic force microscopy and molecular dynamics simulation. Biophys J 81: 2344-2356.PubMedGoogle Scholar
  2. Brünger AT (1992) X-PLOR, Version 3.1: A System for X-ray Crystallography and NMR. The Howard Hughes Medical Institute and Department of Molecular Biophysics and Biochemistry, Yale University.Google Scholar
  3. Carrion-Vazquez M, Oberhauser A, Fowler S, Marszalek P, Broedel S, Clarke J and Fernandez J (1999) Mechanical and chemical unfolding of a single protein: a comparison. Proc Natl Acad Sci USA 96: 3694-3699.PubMedCrossRefGoogle Scholar
  4. Carrion-Vazquez M, Oberhauser AF, Fisher TE, Marszalek PE, Li H and Fernandez JM (2000) Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering. Prog Biophys Mol Biol 74: 63-91.PubMedCrossRefGoogle Scholar
  5. Craig D, Krammer A, Schulten K and Vogel V (2001) Comparison of the early stages of forced unfolding of fibronectin type III modules. Proc Natl Acad Sci USA 98: 5590-5595.PubMedCrossRefGoogle Scholar
  6. Erickson H (1994) Reversible unfolding of fibronectin type III and immunoglobulin domains provides the structural basis for stretch and elasticity of titin and fibronectin. Proc Natl Acad Sci USA 91: 10114-10118.PubMedCrossRefGoogle Scholar
  7. Erickson H (1997) Stretching single protein modules: titin is a weird spring. Science 276: 1090-1093.PubMedCrossRefGoogle Scholar
  8. Evans E and Ritchie K (1999) Strength of a weak bond connecting flexible polymer chains. Biophys J 76: 2439-2447.PubMedGoogle Scholar
  9. Freiburg A, Trombitas K, Hell W, Cazorla O, Fougerousse F, Centner T, Kolmerer B, Witt C, Beckmann J, Gregorio C, Granzier H and Labeit S (2000) 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.PubMedGoogle Scholar
  10. Gao M, Lu H and Schulten K (2001) Simulated refolding of stretched titin immunoglobulin domains. Biophys J 81: 2268-2277.PubMedGoogle Scholar
  11. Gao M, Craig D, Vogel V and Schulten K (2002a) Identifying unfolding intermediates of FN-III 10 by steered molecular dynamics. J Mol Biol 323: 939-950.PubMedCrossRefGoogle Scholar
  12. Gao M, Wilmanns M and Schulten K (2002b) Steered molecular dynamics studies of titin I1 domain unfolding. Biophys J, 83: 3435-3445.PubMedGoogle Scholar
  13. Granzier H and Labeit S (2002) Cardiac titin: an adjustable multifunctional spring. J Physiol 541: 335-342.PubMedCrossRefGoogle Scholar
  14. Grubmüller H, Heymann B and Tavan P (1996) Ligand binding and molecular mechanics calculation of the Streptavidin-Biotin Rupture Force. Science 271: 997-999.PubMedGoogle Scholar
  15. Improta S, Politou A and Pastore A (1996) Immunoglobulin-like modules from titin I-band: extensible components of muscle elasticity. Structure 4: 323-337.PubMedCrossRefGoogle Scholar
  16. Isralewitz B, Gao M and Schulten K (2001) Steered molecular dynamics and mechanical functions of proteins. Curr Op Struct Biol 11: 224-230.CrossRefGoogle Scholar
  17. Isralewitz B, Izrailev S and Schulten K (1997) Binding pathway of retinal to bacterio-opsin: a prediction by molecular dynamics simulations. Biophys J 73: 2972-2979.PubMedGoogle Scholar
  18. Izrailev S, Stepaniants S, Balsera M, Oono Y and Schulten K (1997) Molecular dynamics study of unbinding of the Avidin-Biotin complex. Biophys J 72: 1568-1581.PubMedGoogle Scholar
  19. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW and Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79: 926-935.CrossRefGoogle Scholar
  20. Kalé L, Skeel R, Bhandarkar M, Brunner R, Gursoy A, Krawetz N, Phillips J, Shinozaki A, Varadarajan K and Schulten K (1999) NAMD2: greater scalability for parallel molecular dynamics. J Comp Phys 151: 283-312.CrossRefGoogle Scholar
  21. Kellermayer M, Smith S, Granzier H and Bustamante C (1997) Folding-unfolding transition in single titin modules characterized with laser tweezers. Science 276: 1112-1116.PubMedCrossRefGoogle Scholar
  22. Klimov DK and Thirumalai D (1999) Stretching single-domain proteins: phase diagram and kinetics of force-induced unfolding. Proc Natl Acad Sci USA 96: 1306-1315.CrossRefGoogle Scholar
  23. Klimov DK and Thirumalai D (2000) Native topology determines force-induced unfolding pathways in globular proteins. Proc Natl Acad Sci USA 97: 7254-7259.PubMedCrossRefGoogle Scholar
  24. Krammer A, Craig D, Thomas WE, Schulten K and Vogel V (2002) A structural model for force regulated integrin binding to fibronectin's RGD-synergy site. Matrix Biology 21: 139-147.PubMedCrossRefGoogle Scholar
  25. Krammer A, Lu H, Isralewitz B, Schulten K and Vogel V (1999) Forced unfolding of the fibronectin type III module reveals a tensile molecular recognition switch. Proc Natl Acad Sci USA 96: 1351-1356.PubMedCrossRefGoogle Scholar
  26. Labeit S and Kolmerer B (1995) Titins, giant proteins in charge of muscle ultrastructure and elasticity. Science 270: 293-296.PubMedGoogle Scholar
  27. Li H, Linke W, Oberhauser AF, Carrion-Vazquez M, Kerkvliet JG, Lu H, Marszalek PE and Fernandez JM (2002) Reverse engineering of the giant muscle protein titin. Nature 418: 998-1002.PubMedCrossRefGoogle Scholar
  28. Li H, Mariano CV, Oberhauser AF, Marszalek PE and Fernandez JM (2001a) Point mutations alter the mechanical stability of immunoglobulin modules. Nature Struct Biol 7: 1117-1120.Google Scholar
  29. Li H, Oberhauser AF, Redick SD, Carrion-Vazquez M, Erikson H and Fernandez JM (2001b) Multiple conformations of PEVK proteins detected by single-molecule techniques. Proc Natl Acad Sci USA 98: 10682-10686.PubMedCrossRefGoogle Scholar
  30. Linke WA (2000) Stretching molecular springs: elasticity of titin filaments in vertebrate striated muscle. Histol Histopathol 15: 799-811.PubMedGoogle Scholar
  31. Linke WA, Ivemeyer M, Mundel P, Stockmeier MR and Kolmerer B (1998) Nature of PEVK-titin elasticity in skeletal muscle. Proc Natl Acad Sci USA 95: 8052-8057.PubMedCrossRefGoogle Scholar
  32. LuH, Isralewitz B, Krammer A, Vogel V and Schulten K (1998) Unfolding of titin immunoglobulin domains by steered molecular dynamics simulation. Biophys J 75: 662-671.Google Scholar
  33. Lu H and Schulten K (1999a) Steered molecular dynamics simulation of conformational changes of immunoglobulin domain I27 interpret atomic force microscopy observations. Chem Phys 247: 141-153.CrossRefGoogle Scholar
  34. Lu H and Schulten K (1999b) Steered molecular dynamics simulations of force-induced protein domain unfolding. Proteins Struct Func Gen 35: 453-463.CrossRefGoogle Scholar
  35. Lu H and Schulten K (2000) The key event in force-induced unfolding of titin's immunoglobulin domains. Biophys J 79: 51-65.PubMedGoogle Scholar
  36. Ma K, Kan L and Wang K (2001) Polyproline II helix is a key structural motif of the elastic PEVK segment of titin. Biochemistry 40: 3427-3438.PubMedCrossRefGoogle Scholar
  37. MacKerell Jr. AD, Bashford D, Bellott M, Dunbrack Jr. RL, Evanseck J, Field MJ, Fischer S, Gao J, Guo H, Ha S, Joseph D, Kuchnir L, Kuczera K, Lau FTK, Mattos C, Michnick S, Ngo T, Nguyen DT, Prodhom B, Reiher IWE, Roux B, Schlenkrich M, Smith J, Stote R, Straub J, Watanabe M, Wiorkiewicz-Kuczera J, Yin D and Karplus M (1998) All-hydrogen empirical potential for molecular modeling and dynamics studies of proteins using the CHARMM22 force field. J Phys Chem B 102: 3586-3616.CrossRefGoogle Scholar
  38. Marszalek PE, Lu H, Li H, Carrion-Vazquez M, Oberhauser AF, Schulten K and Fernandez JM (1999) Mechanical unfolding intermediates in titin modules. Nature 402: 100-103.PubMedCrossRefGoogle Scholar
  39. Maruyama K (1997) Connectin/titin, a giant elastic protein of muscle. FASEB J 11: 341-345.PubMedGoogle Scholar
  40. Mayans O, Wuerges J, Canela S, Gautel M and Wilmanns M (2001) Structural evidence for a possible role of reversible disulphide bridge formation in the elasticity of the muscle protein titin. Structure 9: 331-340.PubMedCrossRefGoogle Scholar
  41. Minajeva A, Kulke M, Fernandez JM and Linke WA (2001) Unfolding of titin domains explains the viscoelastic behavior of skeletal myofibrils. Biophys J 80: 1442-1451.PubMedGoogle Scholar
  42. Oberhauser A, Badilla-Fernandez C, Carrion-Vazquez M and Fernandez J (2002) The mechanical hierarchies of fibronectin observed with single molecule AFM. J Mol Biol 319: 433-447.PubMedCrossRefGoogle Scholar
  43. Oberhauser AF, Marszalek PE, Erickson H and Fernandez J (1998) The molecular elasticity of tenascin, an extracellular matrix protein. Nature 393: 181-185.PubMedCrossRefGoogle Scholar
  44. Paci E and Karplus M (1999) Forced unfolding of fibronectin type 3 modules: an analysis by biased molecular dynamics simulations. J Mol Biol 288: 441-459.PubMedCrossRefGoogle Scholar
  45. Paci E and Karplus M (2000) Unfolding proteins by external forces and temperature: the importance of topology and energetics. Proc Natl Acad Sci USA 97: 6521-6526.PubMedCrossRefGoogle Scholar
  46. Politou AS, Thomas D and Pastore A (1995) The folding and the stability of titin immunoglobulin-like modules, with implications for mechanism of elasticity. Biophys J 69: 2601-2610.PubMedGoogle Scholar
  47. Rief M, Gautel M, Oesterhelt F, Fernandez JM and Gaub HE (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276: 1109-1112.PubMedCrossRefGoogle Scholar
  48. Rief M, Gautel M, Schemmel A and Gaub H (1998) The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by AFM. Biophys J 75: 3008-3014.PubMedGoogle Scholar
  49. Rief M, Pascual J, Saraste M and Gaub H (1999) Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles. J Mol Biol 286: 553-561.PubMedCrossRefGoogle Scholar
  50. Rohs R, Etchebest C and Lavery R (1999) Unraveling proteins: a molecular mechanics study. Biophys J 76: 2760-2768.PubMedCrossRefGoogle Scholar
  51. Schulten K, Schulten Z and Szabo A (1980) Reactions governed by a binomial redistribution process. The ehrenfest urn problem. Physica 100A: 599-614.Google Scholar
  52. Schulten K, Schulten Z and Szabo A (1981) Dynamics of reactions involving diffusive barrier crossing. J Chem Phys 74: 4426-4432.CrossRefGoogle Scholar
  53. Socci N, Onuchic J and Wolynes P (1999) Stretching lattice models of protein folding. Proc Natl Acad Sci USA 96: 2031-2035.PubMedCrossRefGoogle Scholar
  54. Soteriou A, Clarke A, Martin S and Trinick J (1993) Titin folding energy and elasticity. Proc R Soc Lond B (Biol. Sci.) 254: 83-86.Google Scholar
  55. Szabo A, Schulten K and Schulten Z (1980) First passage time approach to diffusion controlled reactions. J Chem Phys 72: 4350-4357.CrossRefGoogle Scholar
  56. Trombitas K, Greaser M, Labeit S, Jin J, Kellermayer M, Helmes M and Granzier H (1998) Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments. J Cell Biol 140: 853-859.PubMedCrossRefGoogle Scholar
  57. Tskhovrebova L and Trinick J (2002) Role of titin in vertebrate striated muscle. Proc R Soc Lond B (Biol. Sci.) 357: 199-206.Google Scholar
  58. Tskhovrebova L, Trinick J, Sleep J and Simmons R (1997) Elasticity and unfolding of single molecules of the giant protein titin. Nature 387: 308-312.PubMedCrossRefGoogle Scholar
  59. Wang K (1996) Titin/connectin and nebulin: giant protein ruler of muscle structure and function. Adv Biophys 33: 123-134.PubMedCrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Mu Gao
    • 1
  • Hui Lu
    • 2
  • Klaus Schulten
    • 1
  1. 1.Department of Physics and Beckman InstituteUniversity of Illinois at Urbana-ChampaignUSA
  2. 2.Department of BioengineeringUniversity of Illinois at ChicagoUSA

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