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Determining Stable Single Alpha Helical (SAH) Domain Properties by Circular Dichroism and Atomic Force Microscopy

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Molecular Motors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1805))

Abstract

Stable, single α-helical (SAH) domains exist in a number of unconventional myosin isoforms, as well as other proteins. These domains are formed from sequences rich in charged residues (Arg, Lys, and Glu), they can be hundreds of residues long, and in isolation they can tolerate significant changes in pH and salt concentration without loss in helicity. Here we describe methods for the preparation and purification of SAH domains and SAH domain-containing constructs, using the myosin 10 SAH domain as an example. We go on to describe the use of circular dichroism spectroscopy and force spectroscopy with the atomic force microscope for the elucidation of structural and mechanical properties of these unusual helical species.

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Acknowledgments

This work was supported by Biotechnology and Biological Sciences Research Council grants BB/I007423/1 and BB/M009114/1.

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Author notes

  1. Matthew Batchelor and Marcin Wolny contributed equally to this work.

Authors and Affiliations

  1. Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK

    Matthew Batchelor, Marcin Wolny, Marta Kurzawa, Peter J. Knight & Michelle Peckham

  2. Astbury Centre for Structural Molecular Biology and School of Physics and Astronomy, University of Leeds, Leeds, LS2 9JT, UK

    Lorna Dougan

Authors
  1. Matthew Batchelor
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  2. Marcin Wolny
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  3. Marta Kurzawa
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  4. Lorna Dougan
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  5. Peter J. Knight
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  6. Michelle Peckham
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Corresponding author

Correspondence to Michelle Peckham .

Editor information

Editors and Affiliations

  1. National Museum of Natural History, CNRS UMR7196 / INSERM U1154, Paris, France

    Christophe Lavelle

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Batchelor, M., Wolny, M., Kurzawa, M., Dougan, L., Knight, P.J., Peckham, M. (2018). Determining Stable Single Alpha Helical (SAH) Domain Properties by Circular Dichroism and Atomic Force Microscopy. In: Lavelle, C. (eds) Molecular Motors. Methods in Molecular Biology, vol 1805. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8556-2_10

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  • DOI: https://doi.org/10.1007/978-1-4939-8556-2_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8554-8

  • Online ISBN: 978-1-4939-8556-2

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