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Related Experiment Videos

Mechanical unfolding intermediates in titin modules.

P E Marszalek1, H Lu, H Li

  • 1Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905, USA.

Nature
|November 26, 1999
PubMed
Summary

Titin

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Biochemistry

Background:

  • Titin provides passive muscle elasticity through its modular structure.
  • Previous studies suggest titin domains unfold in an all-or-none manner under force.
  • Naturally occurring titin's heterogeneity complicates single-molecule studies.

Purpose of the Study:

  • To investigate the mechanical properties of engineered titin proteins.
  • To understand the initial response of titin immunoglobulin domains to stretching forces.
  • To identify and characterize the 'unfolding intermediate' in titin elasticity.

Main Methods:

  • Engineered single proteins with multiple copies of human cardiac titin immunoglobulin domains.
  • Utilized atomic force microscopy (AFM) for single-molecule elongation experiments.
  • Performed steered molecular dynamics (SMD) simulations to model hydrogen bond rupture.
  • Employed site-directed mutagenesis to disrupt specific hydrogen bonds.

Main Results:

  • Observed an abrupt ~7 Å extension per domain before full unfolding.
  • Identified a reversible 'unfolding intermediate' state.
  • SMD simulations indicated hydrogen bond rupture near the N-terminus causes ~6 Å extension.
  • Site-directed mutagenesis abolished the unfolding intermediate.
  • The intermediate contributes ~15% to the domain's slack length.

Conclusions:

  • An 'unfolding intermediate' is a significant, previously unrecognized contributor to titin elasticity.
  • This intermediate involves reversible hydrogen bond disruption within titin domains.
  • Understanding this intermediate refines models of muscle passive mechanics.

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