Spectroscopic Probes of the Muscle Cytoskeleton Our long-term goal is to define the molecular structure and dynamics that determine the functions of dystrophin (Dys) and utrophin (Utr) in striated muscle, in order to provide a much needed foundation for the understanding of the roles of these proteins in muscle function and disease, such as Duchenne (DMD) and Becker (BMD) muscular dystrophies. To accomplish this, conventional methods of structural biology (microscopy, crystallography) are not sufficient, so we are carrying out the first applications of site-directed spectroscopic probes (phosphorescence, fluorescence, and electron paramagnetic magnetic resonance [EPR]) to these proteins. The focus of the current proposal is to elucidate the structural dynamics of functional interactions of actin with Dys and Utr. We hypothesize that the pathophysiology of DMD and BMD arises in part from the failure of the ablated or mutated Dys to interact appropriately with actin, reducing the resilience of the muscle cytoskeleton (costamere), as revealed by direct spectroscopic detection of structural dynamics. We propose that the structure and dynamics of these complexes are important for understanding the pathophysiology of DMD and BMD, and their possible reversal by gene or protein therapy, using Dys or Utr or smaller constructs. We will use probes on Dys, Utr, and actin, to ask, How do Dys and Utr affect the structural dynamics of actin? What segments of these proteins are crucial for these effects? How do structures of Dys and Utr in solution, free and bound to actin, compare with each other? with proposed therapeutic constructs being tested in mdx mice? with those obtained previously by xray or EM? How does actin affect the structural dynamics of Utr and Dys? How are these results affected by Dys mutations that cause DMD or BMD? How do the answers to these questions differ when the g isoform of actin is used? These questions will be addressed using time-resolved phosphorescence anisotropy to detect rotational dynamics, fluorescence and EPR to map protein structures and interactions, and computational simulation to integrate these results with those of crystallography and EM. This project is likely to have a major impact on the understanding of the muscle cytoskeleton, with particular relevance to muscular dystrophy, because the project is unique and timely. Our proposal is the first thorough structural investigation of the actin-dystrophin and actin-utrophin system. This is possible because of an innovative collaboration between two laboratories - the Thomas laboratory, which leads the world in spectroscopic probes of muscle proteins, and the Ervasti laboratory, which leads the world in the expression and purification of the relevant proteins, and their physiological testing n mouse models. This project is timely, because recent work points to the functional importance of these interactions in disease and therapy. The findings of the proposed research will provide structure-function guidelines for future therapeutic development.
The goal of this project is to provide direct molecular insight into the proteins of the muscle cytoskeleton, which plays a major role in muscular dystrophy and other diseases. We will use an innovative approach involving molecular probes, molecular biology, and structural biology. This work is designed to provide crucial information needed for the development of molecular therapies.
|Avery, Adam W; Fealey, Michael E; Wang, Fengbin et al. (2017) Structural basis for high-affinity actin binding revealed by a ?-III-spectrin SCA5 missense mutation. Nat Commun 8:1350|
|Avery, Adam W; Thomas, David D; Hays, Thomas S (2017) ?-III-spectrin spinocerebellar ataxia type 5 mutation reveals a dominant cytoskeletal mechanism that underlies dendritic arborization. Proc Natl Acad Sci U S A 114:E9376-E9385|
|Avery, Adam W; Crain, Jonathan; Thomas, David D et al. (2016) A human ?-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding. Sci Rep 6:21375|
|McCaffrey, Jesse E; James, Zachary M; Thomas, David D (2015) Optimization of bicelle lipid composition and temperature for EPR spectroscopy of aligned membranes. J Magn Reson 250:71-5|
|McCarthy, Megan R; Thompson, Andrew R; Nitu, Florentin et al. (2015) Impact of methionine oxidation on calmodulin structural dynamics. Biochem Biophys Res Commun 456:567-72|
|Petersen, Karl J; Peterson, Kurt C; Muretta, Joseph M et al. (2014) Fluorescence lifetime plate reader: resolution and precision meet high-throughput. Rev Sci Instrum 85:113101|
|Moen, Rebecca J; Klein, Jennifer C; Thomas, David D (2014) Electron paramagnetic resonance resolves effects of oxidative stress on muscle proteins. Exerc Sport Sci Rev 42:30-6|
|Prochniewicz, Ewa; Guhathakurta, Piyali; Thomas, David D (2013) The structural dynamics of actin during active interaction with myosin depends on the isoform of the essential light chain. Biochemistry 52:1622-30|
|Moen, Rebecca J; Thomas, David D; Klein, Jennifer C (2013) Conformationally trapping the actin-binding cleft of myosin with a bifunctional spin label. J Biol Chem 288:3016-24|
|Colson, Brett A; Rybakova, Inna N; Prochniewicz, Ewa et al. (2012) Cardiac myosin binding protein-C restricts intrafilament torsional dynamics of actin in a phosphorylation-dependent manner. Proc Natl Acad Sci U S A 109:20437-42|