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A mechanical unfolding intermediate in an actin-crosslinking protein

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Abstract

Many F-actin crosslinking proteins consist of two actin-binding domains separated by a rod domain that can vary considerably in length and structure. In this study, we used single-molecule force spectroscopy to investigate the mechanics of the immunoglobulin (Ig) rod domains of filamin from Dictyostelium discoideum (ddFLN). We find that one of the six Ig domains unfolds at lower forces than do those of all other domains and exhibits a stable unfolding intermediate on its mechanical unfolding pathway. Amino acid inserts into various loops of this domain lead to contour length changes in the single-molecule unfolding pattern. These changes allowed us to map the stable core of ∼60 amino acids that constitutes the unfolding intermediate. Fast refolding in combination with low unfolding forces suggest a potential in vivo role for this domain as a mechanically extensible element within the ddFLN rod.

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Figure 1: Single-molecule mechanics of ddFLN.
Figure 2: Single-domain force spectroscopy.
Figure 3: Characterization of the unfolding intermediate structure in single-molecule experiments using loop mutations.
Figure 4: Series of three unfolding (black) and refolding (red) cycles of a single ddFLN1-5 rod.

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Acknowledgements

We thank H. Gaub and E. Paci for helpful discussions and R. Müller for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft to A.A.N. and the Sonderforschungsbereich 413 to M.R. and M.S.

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Authors and Affiliations

  1. Lehrstuhl für Angewandte Physik, Ludwig-Maximilians-Universität München, Amalienstrasse 54, München, 80799, Germany

    Ingo Schwaiger, Angelika Kardinal & Matthias Rief

  2. Institut für Biochemie I, Med. Fakultät der Universität zu Köln, Joseph-Stelzmann-Strasse 52, Köln, 50931, Germany

    Angelika A Noegel

  3. Adolf-Butenandt-Institut Zellbiologie, Ludwig-Maximilians-Universität München, Schillerstrasse 42, München, 80336, Germany

    Michael Schleicher

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  1. Ingo Schwaiger
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  2. Angelika Kardinal
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  4. Angelika A Noegel
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  5. Matthias Rief
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Correspondence to Matthias Rief.

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Schwaiger, I., Kardinal, A., Schleicher, M. et al. A mechanical unfolding intermediate in an actin-crosslinking protein. Nat Struct Mol Biol 11, 81–85 (2004). https://doi.org/10.1038/nsmb705

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