Baboolal, TG, Sakamoto, T, Forgacs, E et al. (8 more authors) (2009) The SAH domain extends the functional length of the myosin lever. Proceedings of the National Academy of Sciences of USA, 106 (52). pp. 22193-22198. ISSN 1091-6490
Abstract
Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding IQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to two IQ motifs (Myo5–2IQ). Electron microscopy of this chimera (Myo5–2IQ-SAH) showed the SAH domain was straight and 17 nm long as predicted, restoring the truncated lever to the length of wild-type (Myo5–6IQ). The power stroke (of 21.5 nm) measured in the optical trap was slightly less than that for Myo5–6IQ but much greater than for Myo5–2IQ. Myo5–2IQ-SAH moved processively along actin at physiological ATP concentrations with similar stride and run lengths to Myo5–6IQ in in-vitro single molecule assays. In comparison,Myo5–2IQ is not processive under these conditions. Solution biochemical experiments indicated that the rear head did not mechanically gate the rate of ADP release from the lead head, unlike Myo5–6IQ. These data show that the SAH domain can form part of a functional lever in myosins, although its mechanical stiffness might be lower. More generally, we conclude that SAH domains can act as stiff structural extensions in aqueous solution and this structural role may be important in other proteins.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2009, National Academy of Sciences. This is an author produced version of a paper published in Proceedings of the National Academy of Sciences of USA. Uploaded in accordance with the publisher's self-archiving policy. In order to comply with the publisher requirements the University does not require the author to sign a non-exclusive licence for this paper. |
Keywords: | ATPase; electron microscopy; optical trap; single alpha helix |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Medicine (Leeds) > Institute of Rheumatology & Musculoskeletal Medicine (LIRMM) (Leeds) > Experimental Musculoskeletal Medicine (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 30 Jul 2014 10:29 |
Last Modified: | 21 Jan 2018 10:06 |
Published Version: | http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=... |
Status: | Published |
Publisher: | National Academy of Sciences |
Refereed: | Yes |
Identification Number: | 10.1073/pnas.0909851106 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:79932 |