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Flexibility in the heads of myosin 5

Revill, Derek John (2013) Flexibility in the heads of myosin 5. PhD thesis, University of Leeds.

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Class 5 myosins are motor proteins involved in the transport and localisation of diverse cargoes in eukaryotic cells. Molecules have two heads and a tail and can move along actin filaments, by making step-like movements driven by MgATP hydrolysis. Each head comprises an actin-binding motor and a lever, an extended α-helix carrying six light chains bound to IQ motifs. The lever is an essential mechanical element in myosin 5 function, but an understanding of its mechanical properties and how these derive from its substructure is lacking. To address this, the first aim of this study was to look for and characterise flexibility within the heads of myosin 5. A second aim was to test the effect of altering the IQ motif spacing on the properties and behaviour of the molecule. The structure and flexibility of the head region of mammalian myosin 5a was studied using single-particle image processing of images from negative stain electron microscopy (EM). Image averaging revealed features of the motor and lever and provided evidence of independent rotational freedom of the motor about the lever axis, and thermally-driven flexing at the motor-lever junction. A stiffness of 32-51 pN·nm·rad-2 for the flexing was inferred, which represents a significant compliance in the unattached head. Variation in light chain orientations at each IQ motif suggested torsional flexibility. Lever bending analysis, by decomposition of average lever shapes into Fourier modes, yielded a persistence length of 50.9 ± 18.6 nm. All these results indicate that, under EM conditions, the unattached head is more flexible than has previously been measured for the actinbound complex in optical trap experiments. Lever mutants with altered IQ motif spacings were constructed and initial characterisations by negative stain EM, MgATPase assay and single-molecule fluorescence microscopy were made. For the latter, two calmodulin point mutants were made, for site-specific labelling and attachment to the lever as a fluorescent probe. One lever mutant, with closerspaced IQ motifs, resembled wild-type and had similar actin-activated MgATPase activity. However, its motile properties remain undetermined. Another mutant, with wider-spaced IQ motifs did not bind calmodulin and it had no actin-activated MgATPase. The molecule appeared misfolded but retained residual actin-binding properties. These contrasting results highlight the importance of the lever’s structure in the function of the molecule.

Item Type: Thesis (PhD)
ISBN: 978-0-85731-370-6
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Identification Number/EthosID: uk.bl.ethos.589342
Depositing User: Repository Administrator
Date Deposited: 24 Jan 2014 09:26
Last Modified: 25 Nov 2015 13:41
URI: http://etheses.whiterose.ac.uk/id/eprint/5066

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