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Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction

Nature volume 400, pages 425–430 (1999)Cite this article

Abstract

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.

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Figure 1: Bifunctional rhodamine labelling of RLC.
Figure 2: Polarization ratios P ∥, x P ⊥ and yP ⊥ from single muscle fibres containing each of the four BR–RLCs.
Figure 3: Conformation of the myosin head (catalytic domain, red; heavy-chain residues 707–843, blue; regulatory and essential light chains, magenta and yellow, respectively) bound in rigor to the actin filament (brown, grey, green).
Figure 4: Mechanical and RLC-region orientation transients.
Figure 5: Tilt and twist of the RLC region.

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Acknowledgements

This work was supported by the UK MRC, Wellcome Trust, NIH, MDA and AHA. We thank S. Howell for mass spectrometric analyses, M. Hirshberg and I. Dobbie for help with molecular graphics, and V. R. N. Munasinghe for technical support with BR-I2.

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Authors and Affiliations

  1. National Institute for Medical Research , The Ridgeway, Mill Hill, London, NW7 1AA, UK

    J. E. T. Corrie, B. D. Brandmeier, R. E. Ferguson &  D. R. Trentham

  2. MRC Laboratory of Molecular Biology , Hills Road, Cambridge, CB2 2QH, UK

    J. Kendrick-Jones

  3. Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, 19104-6083, Pennsylvania, USA

    S. C. Hopkins, U. A. van der Heide & Y. E. Goldman

  4. Randall Institute, King's College London, 2629 Drury Lane, London, WC2B 5RL, UK

    C. Sabido-David, R. E. Dale, S. Criddle & M. Irving

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  1. J. E. T. Corrie
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Correspondence to M. Irving.

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Corrie, J., Brandmeier, B., Ferguson, R. et al. Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction. Nature 400, 425–430 (1999). https://doi.org/10.1038/22704

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