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Structural changes and their relationship to mechanical work during muscle contraction - real-time studies of a protein conformational change

Objective



Research objectives and content The aim of this project is to understand how protein motion within the regulatory domain of the myosin molecule contributes to muscle contraction. Prior work by Dr Trentham's research group and their collaborators has established that the C-terminal domain of the regulatory light chain tilts and rotates during cross-bridge movement. This project aims to map the movement of the N-terminal domain. In X-ray diffraction analysis both domains are wrapped around a long a-helix of the myosin heavy chain. The part of the a-helix around which the C-terminal domain is wrapped comprises a long unbroken helix extending from the catalytic (ie ATPase and actin binding) domain of myosin, but the a-helix then undergoes a sharp break followed by a further helix creating a 'hook'. The N-terminal domain is wrapped around the hook region, hence understanding how the N-terminal domain moves will determine the nature of the hook movement. Modelling suggests that rotation in the C- terminal domain may, by virtue of the hook linkage, translate into productive sliding of the myosin backbone with respect to actin filaments giving rise to muscle contraction: this model will be tested as outlined in Research Plan (pages 11-14). Experimental and theoretical content will be predominantly based on the following; fluorescence and fluorescence polarisation to monitor dipole orientation and motion during contraction; mutagenesis and expression of regulatory light chains (RLC); labeling and purification of RLC labelled with a bifunctional rhodamine (to provide the oriented dipole); equilibrium binding studies of labelled RLC with myosin heavy chain; functional (enzymatic and physiological) studies of isolated myosin and fibres, each reconstituted with labelled RLC, to ensure viable preparations; NMR studies of RLC and heavy chain peptides for solution structures; muscle physiology. Training content (objective. benefit and expected impact) My overall training objective is that I will learn how to study protein conformational change in real-time and the biophysics of muscle contraction. I will be part of a well-structured PhD programme (see Host Institution pages 15-16). It will be of long term advantage to my career to be fluent in scientific English (literature, seminar presentation). I will be trained in a wide range of biophysical, physiological and biochemical techniques, especially in spectroscopy - these are specified in my Research Plan (pages 11-14). The benefit to me is outlined in the above training objective. I also look forward to applying my Physics background to interpret the spectroscopic and mechanical data I will acquire in the context of major biological problems. The impact of this study is that I hope it will provide important new insights into the mechanism of muscle contraction and define in real-time the motion of a protein conformational change.

Coordinator

MRC National Institute for Medical Research
Address
The Ridgeway Mill Hill
NW7 1AA London
United Kingdom