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Mechanical Manipulation of Single Titin Molecules with Laser Tweezers

  • Miklós S. Z. Kellermayer
  • Steven Smith
  • Carlos Bustamante
  • Henk L. Granzier
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 481)

Abstract

Titin (also known as connectin) is a giant filamentous polypeptide of multi-domain construction spanning between the Z- and M-lines of the vertebrate muscle sarcomere. The molecule is significant in maintaining sarcomeric structural integrity and generating passive muscle force via its elastic properties. Here we summarize our efforts to characterize titin’s elastic properties by manipulating single molecules with force-measuring laser tweezers. The titin molecule can be described as an entropic spring in which domain unfolding occurs at high forces during stretch and refolding at low forces during release. Statistical analysis of a large number (>500) of stretch-release experiments and comparison of experimental data with the predictions of the wormlike chain theory permit the estimation of unfolded titin’s mean persistence length as 16.86 Å (±0.11 SD). The slow rates of unfolding and refolding compared with the rates of stretch and release, respectively, result in a state of non-equilibrium and the display of force hysteresis. Folding kinetics as the source of non-equilibrium is directly demonstrated here by the abolishment of force hysteresis in the presence of chemical denaturant. Experimental observations were well simulated by superimposing a simple domain folding kinetics model on the wormlike chain behavior of titin and considering the characteristics of the compliant laser trap. The original video presentation of this paper may be viewed on the web at http://www.pote.hu/ mm/prezentacio/mkpres/mkpres. htm.URL

Keywords

Persistence Length Partial Release Contour Length Thick Filament Passive Force 
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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References

  1. Bell GI. Models for the specific adhesion of cells to cells. Science 1978;200:618–627.PubMedCrossRefGoogle Scholar
  2. Bustamante CJ, Marko JF, Siggia ED, Smith SB. Entropic elasticity of λ-phage DNA. Science 1994;265:1599–1600.PubMedCrossRefGoogle Scholar
  3. Erickson, HP. 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 1994;91:10114–8.PubMedCrossRefGoogle Scholar
  4. Evans E, Ritchie K. Dynamic strength of molecular adhesion bonds. Biophys J 1997;72:1541–1555.PubMedCrossRefGoogle Scholar
  5. Fürst DO, Osborn M, Nave R, Weber K. The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map often nonrepetitive epitopes starting at the Z line extends close to the M line. J Cell Biol 1988;106:1563–72.PubMedCrossRefGoogle Scholar
  6. Granzier HLM, Akster HA, ter Keurs HED. Effect of thin filament length on the force-sarcomere length relation of skeletal muscle. Am J Physiol1991;260:C1060–C1070.PubMedGoogle Scholar
  7. Granzier HLM, Irving T. Passive tension in cardiac muscle: the contribution of collagen, titin, microtubules and intermediate filaments. Biophys J 1995;68:1027–1044.PubMedCrossRefGoogle Scholar
  8. Gregorio CC, Granzier H, Sorimachi H, Labeit S. Muscle assembly: a titanic achievement? Curr Opin Cell Biol 1999;11:18–25 (Review).PubMedCrossRefGoogle Scholar
  9. Higuchi H, Nakauchi Y, Maruyama K, Fujime S. Characterization of beta-connectin (titin 2) from striated muscle by dynamic light scattering. Biophys J 1993;65:1906–15.PubMedCrossRefGoogle Scholar
  10. Higuchi H, Suzuki T, Kimura S, Yoshioka T, Maruyama K, Umazume Y. Localization and elasticity of connectin (titin) filaments in skinned frog muscle fibres subjected to partial depolymerization of thick filaments. J Muscle Res Cell Motil 1992;13:285–94.PubMedCrossRefGoogle Scholar
  11. Horowits R, Kempner ES, Bisher ME, Podolsky RJ. A physiological role for titin and nebulin in skeletal muscle. Nature 1986;323:160–4.PubMedCrossRefGoogle Scholar
  12. Horowits R, Podolsky RJ. The positional stability of thick filaments in activated skeletal muscle depends on sarcomere length: evidence for the role of titin filaments. J Cell Biol 1987;105:2217–23.PubMedCrossRefGoogle Scholar
  13. Itoh Y, Suzuki T, Kimura S, Ohashi K, Higuchi H, Sawada H, et al., Extensible and less-extensible domains of connectin filaments in stretched vertebrate skeletal muscle sarcomeres as detected by immunofluorescence and immunoelectron microscopy using monoclonal antibodies. J Biochem 1988;104:504–508.PubMedGoogle Scholar
  14. Kellermayer MSZ, Granzier HLM. Calcium-dependent inhibition of in vitro thin-filament motility by native titin. FEBS Lett 1996;380:281–286.PubMedCrossRefGoogle Scholar
  15. Kellermayer MSZ, Granzier HLM. Elastic properties of single titin molecules made visible through fluorescent F-actin binding. Biochem Biophys Res Commun 1996b;221:491–497.PubMedCrossRefGoogle Scholar
  16. Kellermayer MSZ, Smith SB, Granzier HL, Bustamante C. Folding-unfolding transitions in single titin molecules characterized with laser tweezers. Science 1997;276:1112–1116.PubMedCrossRefGoogle Scholar
  17. Kellermayer MSZ, Smith SB, Bustamante C, Granzier HL. Complete unfolding of the titin molecule under external force. J Struct Biol 1998;122:197–205.PubMedCrossRefGoogle Scholar
  18. Kratky O, Porod G. Rec Trav Chim 1949;68:1106.CrossRefGoogle Scholar
  19. Kuhn W, Grün F. Kolloid Z 1942;101:248.CrossRefGoogle Scholar
  20. Labeit S, Kolmerer B. Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science 1995;270:293–296.PubMedCrossRefGoogle Scholar
  21. Marko JF, Siggia ED. Stretching DNA. Macromolecules 1995;28:8759–8770.CrossRefGoogle Scholar
  22. Maruyama K. Connectin/titin, giant elastic protein of muscle. FASEB J 1997;11:341–345.PubMedGoogle Scholar
  23. Rief M, Fernandez JM, Gaub HE. Elastically coupled two-level systems as a model for biopolymer extensibility. Phys Rev Lett 1998a;81:4764–4767.CrossRefGoogle Scholar
  24. Rief M, Gautel M, Oesterhelt F, Fernandez JM, Gaub HE. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 1997;276:1109–1112.PubMedCrossRefGoogle Scholar
  25. Rief M, Gautel M, Schemmel A, Gaub H. The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy. Biophys J.1998b;75:3008–14.PubMedCrossRefGoogle Scholar
  26. Smith SB, Cui Y, Bustamante C. Overstretching B-DNA: The elastic response of individual double-stranded and single-stranded DNA molecules. Science 1996;271:795–799.PubMedCrossRefGoogle Scholar
  27. Smith SB, Finzi L, Bustamante C. Direct mechanical measurements of elasticity of single DNA molecules by using magnetic beads. Science 1992;258:1122–1126.PubMedCrossRefGoogle Scholar
  28. Svoboda K, Block S. Biological applications of optical forces. Annu Rev Biophys Biomol Struct 1994;23:247–285.PubMedCrossRefGoogle Scholar
  29. Trinick J. Elastic filaments and giant proteins in muscle. Curr Opinion Cell Biol 1991;3:112–118.PubMedCrossRefGoogle Scholar
  30. Trinick J. Cytoskeleton: Titin as a scaffold and spring. Current Biology 1996;6:258–260.PubMedCrossRefGoogle Scholar
  31. Trombitás K, Jin J-P, and Granzier HL. The mechanically active domain of titin in cardiac muscle. Circ Res 1995;77:856–61.PubMedCrossRefGoogle Scholar
  32. Trombitás K, Pollack GH. Elastic properties of the titin filament in the Z-line region of vertebrate striated muscle. J Muscle Res Cell Motil 1993;14:416–22.PubMedCrossRefGoogle Scholar
  33. Tskhovrebova L, Trinick J, Sleep JA, Simmons RM. Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature 1997;387:308–312.PubMedCrossRefGoogle Scholar
  34. Wang K. Titin/connectin and nebulin: giant protein rulers of muscle structure and function. Adv Biophys 1996;33:123–134.PubMedCrossRefGoogle Scholar
  35. Whiting A, Wardale J, Trinick J. Does titin regulate the length of muscle thick filaments? J Mol Biol 1989;205:263–8.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2000

Authors and Affiliations

  • Miklós S. Z. Kellermayer
    • 1
  • Steven Smith
    • 2
  • Carlos Bustamante
    • 2
  • Henk L. Granzier
    • 3
  1. 1.Dept. BiophysicsPécs University Medical SchoolPécsHungary
  2. 2.Dept. PhysicsUniversity of CaliforniaBerkeleyUSA
  3. 3.Dept. VCAPPWashington State UniversityPullmanUSA

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