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Electron microscope studies of myosin VI

Reference	BB/C506699/1
Principal Investigator / Supervisor	Professor John Trinick
Co-Investigators / Co-Supervisors	Dr William Nicholson, Mr M Walker
Institution	University of Leeds
Department	Institute of Membrane & Systems Biology
Funding type	Research
Value (£)	378,467
Status	Completed
Type	Research Grant
Start date	01/09/2004
End date	31/12/2007
Duration	40 months

Abstract

This project concerns the structure and properties of class 1 myosins, which are found from amoebe to humans. The project is a collaboration between the Leeds Molecular Contractility Group, which specialises in electron microscopy of motor proteins, and Dr Lynne Coluccio (Boston) who is the leading authority on myosin 1. The structure of myosin 1 molecules will be examined in preparations provided by Dr Coluccio, both purified from liver and expressed in baculovirus. Molecules will be studied free and attached to actin filaments, using both negatively stained and unstained, frozen-hydrated specimens. Frozen specimens will use a new F20 FEG microscope with 4k CCD camera recently installed in Leeds. Single particle image processing using SPIDER and IMAGIC software will produce 2-D image averages of stained molecules and 3-D reconstructions of frozen molecules attached to actin. The Leeds group has already had considerable success in studying structural changes responsible for movement in myosins II, V and dynein. Preliminary data have already provided detailed image averages of myosin 1. During the project, the structure of myosin 1 molecules will be studied with a variety of adenine nucleotides bound: ATP, ADP, ADP.Pi non-hydrolysable analogues (ATP-gamma S, AMP-PNP, ADP.vanadate, etc.) and no nucleotide. 3-D reconstructions of molecules attached to actin will use a new method that treats windowed filament segments as the particles for single particle image processing. Results will be compared with existing EM, optical trap and crystal structure data from myosin I and other classes. The binding of calmodulin light chains under carefully controlled calcium concentrations will be studied as a means to understanding myosin 1 regulation. The structures of molecules with attached cargo proteins (identified and provided by Dr Coluccio) will be examined.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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