Mechanical unfolding intermediates in titin modules

Nature. 1999 Nov 4;402(6757):100-3. doi: 10.1038/47083.


The modular protein titin, which is responsible for the passive elasticity of muscle, is subjected to stretching forces. Previous work on the experimental elongation of single titin molecules has suggested that force causes consecutive unfolding of each domain in an all-or-none fashion. To avoid problems associated with the heterogeneity of the modular, naturally occurring titin, we engineered single proteins to have multiple copies of single immunoglobulin domains of human cardiac titin. Here we report the elongation of these molecules using the atomic force microscope. We find an abrupt extension of each domain by approximately 7 A before the first unfolding event. This fast initial extension before a full unfolding event produces a reversible 'unfolding intermediate' Steered molecular dynamics simulations show that the rupture of a pair of hydrogen bonds near the amino terminus of the protein domain causes an extension of about 6 A, which is in good agreement with our observations. Disruption of these hydrogen bonds by site-directed mutagenesis eliminates the unfolding intermediate. The unfolding intermediate extends titin domains by approximately 15% of their slack length, and is therefore likely to be an important previously unrecognized component of titin elasticity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Biomechanical Phenomena
  • Computer Simulation
  • Connectin
  • Humans
  • Hydrogen Bonding
  • Microscopy, Atomic Force
  • Models, Molecular
  • Muscle Proteins / genetics
  • Muscle Proteins / metabolism*
  • Myocardium / chemistry
  • Protein Folding*
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism


  • Connectin
  • Muscle Proteins
  • Recombinant Proteins
  • TTN protein, human
  • Protein Kinases

Associated data

  • PDB/1TIT