Alpha-actinin is an actin cross linking protein found in all eucaryotic cells. Alpha-actinin is the smallest member of the spectrin-dystrophin-alpha-actinin family of proteins. In the Z-disk of skeletal muscle alpha-actinin cross links antiparallel actin filaments to form the I-band. In smooth muscle alpha-actinin is located in adhesion plaques and dense bodies. In tissue cultured cells, alpha-actinin is located in focal adhesions and stress fibers, structural analogs of adhesion plaques and dense bodies. Molecular defects in alpha-actinin are thought to cause some forms of the muscle disease nemaline myopathy, while defects in the closely related molecule dystrophin cause the disease Duchenne muscular dystrophy. There is very little 3-D structural information available for these molecules. The work of this project is the first step in the long-range goal of investigating the complex molecular interactions that occur in Z-disks, adhesion plaques and focal adhesions and other sites where actin filaments are used to form muscle and cytoskeletal structures. Dr. Taylor and colleagues have recently shown that 2-D crystals of smooth muscle alpha-actinin suitable for electron crystallography can be formed on positively charged lipid monolayers. This is the first reported crystallization of alpha-actinin thereby facilitating a 3-D structure determination. It is proposed to exploit this advance and use these crystals to determine the structure of alpha-actinin according to the following specific aims: (1) Produce a 3-D image of the smooth muscle alpha-actinin to verify the suggested alignment of protein domains. (2) produce a 3-D image of the complex between smooth muscle alpha actinin and actin to determine the orientation when bound to actin. (3) Crystallize Acanthamoeba alpha-actinin and obtain an image that reveals the alignment of protein domains. (4) Examine the orientation of actin filaments cross linked with Acanthamoeba alpha-actinin. (5) Determine conditions that will both produce crystals of one or more alpha-actinin isoforms of a size suitable for an atomic structure analysis and preserve their crystalline order in the electron microscope.
| Taylor, Dianne W; Kelly, Deborah F; Cheng, Anchi et al. (2007) On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy. J Struct Biol 160:305-12 |
| Erickson, H P; Taylor, D W; Taylor, K A et al. (1996) Bacterial cell division protein FtsZ assembles into protofilament sheets and minirings, structural homologs of tubulin polymers. Proc Natl Acad Sci U S A 93:519-23 |
