The goal of this Training Plan is to prepare me for a career as an independent investigator in biomedical research, with a focus on muscle aging and degeneration. With my advisor, I have devised a plan that combines my interest in clinical science with a rigorous training in basic research techniques in biochemistry and biophysics. This proposal focuses on the interactions of actin with other two other filamentous muscle proteins, dystrophin and utrophin. While this project is directly related to a specific inherited disease of muscle degeneration, Duchenne muscular dystrophy, this training will prepare me for a wide range of future studies on muscle degeneration:
AIM 1. Effects of dystrophin, and its engineered constructs, on actin structural dynamics. Dystrophin and selected deletion fragments, previously shown to bind actin, will be expressed and purified from insect cells using the baculovirus expression system, following procedures documented in our collaborator's previous papers. Actin will be labeled at selected sites with fluorescent and phosphorescent dyes, following procedures documented in our previous papers. Then laser-excited time-resolved phosphorescence anisotropy (TPA) and fluorescence will be used to determine the effects of the dystrophin constructs on actin structural dynamics. These experiments will reveal (a) which sites on the surface of actin interact with dystrophin and (b) how dystrophin affects the global and internal flexibility of the actin filament.
AIM 2. Effects of utrophin, and its engineered constructs, on actin structural dynamics. Experiments will be analogous to those in Aim 1, except that site-directed utrophin constructs will be used.
AIM 3. Effects of disease-causing dystrophin mutations on the interaction between dystrophin and actin. Mutations in dystrophin that are known to cause Duchenne or Becker muscular dystrophy will be introduced into the most interesting constructs generated in AIM 1, to determine the effects on actin structural dynamics. For those mutations that change actin structural dynamics without abolishing binding, utrophin constructs (from AIM 2) will be added to ask whether they """"""""rescue"""""""" the actin;i.e., restore the structural dynamics of actin to the state observed with wild-type dystrophin or utrophin. These studies are closely coordinated with a more applied project, expressing utrophin and dystrophin constructs for therapeutic purposes. Information from my research will aid in the strategic development of these therapeutic constructs. The biochemical and biophysical approaches I am learning in this project will prepare me well for a career that combines structural biophysics with analysis of muscle degeneration.
|Lin, Ava Yun; Prochniewicz, Ewa; Henderson, Davin M et al. (2012) Impacts of dystrophin and utrophin domains on actin structural dynamics: implications for therapeutic design. J Mol Biol 420:87-98|
|Henderson, Davin M; Lin, Ava Yun; Thomas, David D et al. (2012) The carboxy-terminal third of dystrophin enhances actin binding activity. J Mol Biol 416:414-24|
|Lin, Ava Y; Prochniewicz, Ewa; James, Zachary M et al. (2011) Large-scale opening of utrophin's tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism. Proc Natl Acad Sci U S A 108:12729-33|