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Mechanical unfolding intermediates in titin modules

Nature volume 402, pages 100–103 (1999)Cite this article

Abstract

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 fashion1,2,3,4,5,6. 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 titin7. Here we report the elongation of these molecules using the atomic force microscope. We find an abrupt extension of each domain by ∼7 Å before the first unfolding event. This fast initial extension before a full unfolding event produces a reversible ‘unfolding intermediate’. Steered molecular dynamics8,9 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 Å, 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 ∼15% of their slack length, and is therefore likely to be an important previously unrecognized component of titin elasticity.

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Figure 1: Force peaks corresponding to the sequential unfolding of immunoglobulin-like domains of human cardiac titin, showing large hump-like deviations from the WLC model of entropic elasticity (arrows).
Figure 2: The size of the hump-like deviation depends linearly on the number of folded modules.
Figure 3: Steered molecular dynamics simulations of I27 extensibility under constant force.
Figure 4: Removal of the deviation from the WLC model by introducing a point mutation in the I27 polyprotein (I279-K6P).

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Acknowledgements

We thank M. Gautel for a gift of the I27-134/pET8c plasmid, and T. Fisher for comments on the manuscript. This work was supported by NIH and NSF grants to J.M.F., P.E.M. A.F.O. and K.S., and by the Roy J. Carver Charitable Trust to K.S.

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Authors and Affiliations

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

    Piotr E. Marszalek, Hongbin Li, Mariano Carrion-Vazquez, Andres F. Oberhauser & Julio M. Fernandez

  2. Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, 61801, Illinois, USA

    Hui Lu & Klaus Schulten

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  1. Piotr E. Marszalek
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Correspondence to Julio M. Fernandez.

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Marszalek, P., Lu, H., Li, H. et al. Mechanical unfolding intermediates in titin modules. Nature 402, 100–103 (1999). https://doi.org/10.1038/47083

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