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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Gary S. Shaw

Abstract

Failure to repair injured sarcolemmal membranes leads to muscular dystrophy, a degenerative disorder that results in increasing weakness and gradual wasting of skeletal muscles. Mutations in the gene encoding dysferlin are causative for limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy (MM) forms of the disease. Dysferlin is a Ca2+-sensitive membrane repair protein involved in trafficking of proteins and vesicles around injured membranes in skeletal muscle cells. It is a cytosolic-facing, membrane bound protein composed of seven intermittently spaced C2 domains (C2A-C2G). Dysferlin activity is mediated by the Ca2+-dependent actions of the C2 domains. The main goals of this thesis were to characterize the structure, dynamics and Ca2+-binding mechanisms of the C2 domains, and assess the impact of pathogenic substitutions on the C2 domains.

First, the dynamics of the C2A domain in both Ca2+-free and Ca2+-bound state was comprehensively probed using NMR spectroscopy, which revealed a remarkable flexibility change in the loop region upon Ca2+ binding. The Ca2+-binding properties of the C2A domain was studied on the basis of the crystal structure of the Ca2+-bound C2A, which determined the stoichiometry, binding sites and affinities. Further, mutagenesis study revealed the important role of the electrostatic potential contributed by non-Ca2+-coordinating residues, which provides novel insights into the mechanism of Ca2+ binding to the dysferlin C2A domain as a link for membrane repair.

To understand the consequences of pathogenic mutations, three substituted C2A proteins were generated and analyzed. It was demonstrated that there is dramatic decrease in stability resulted from the substitutions. The unfolding or improper folding of the substituted C2A domain is predicted to be responsible for impaired dysferlin function in the membrane repair process, and consequently the wasting of skeletal muscles in muscular dystrophy patients.

Finally, proteins encompassing the C2B and C2C domains of dysferlin were designed and generated using a combination of computational and experimental methods. The precise domain boundaries of the C2B and C2C domains were determined, which will provide useful information for the further characterization of dysferlin structure.

Summary for Lay Audience

The cell membrane separates the interior of cells from the outside environment and helps maintain the well-being of cells. However, when cells are subject to physical tearing (which happens frequently), the membrane can be damaged, leading to the damage of the equilibrium of cell. This is why a membrane repair system mediated by proteins is required for cell survival. Proteins are biological molecules that can perform an array of functions within living organisms. Each protein has its specific three-dimensional structure, and the function is directly related to the structure.

One protein that regulates cell membrane repair is dysferlin. Abnormalities of dysferlin caused by gene mutations lead to muscle diseases with detrimental consequences. In cells, dysferlin functions with the help of calcium ions. Calcium ions play a vital role in the physiological processes of organisms and cells, usually by selectively and reversibly binding to partner proteins. This binding causes changes of the protein structure, thereby activating the protein function. Thus, knowing when and how calcium ions bind to dysferlin is important to the understanding of its functions, and the mechanisms of inherited muscle diseases.

In this thesis, the 3-D structure of a calcium-binding region in dysferlin was determined by biophysical techniques. Not only the calcium-binding sites within this region were clearly observed at atomic level, how calcium changes the dysferlin structure was also elucidated. It was discovered that this structural change is directly related to the function of dysferlin in membrane repair. Furthermore, we were able to artificially generate the abnormal forms of dysferlin protein when gene mutation occurs. We found that some properties of dysferlin were altered. This helps the analysis for the cause of function loss in membrane repair, that subsequently leads to muscle diseases. Finally, an additional region of dysferlin was explored, which may have a distinct role in regulating the function of dysferlin.

This work represents an important step forward in fully explaining the mechanisms of membrane repair by dysferlin. The structural and biochemical study here will have a significant impact on the understanding of related diseases and the development of drug therapeutics in the future.

Available for download on Wednesday, March 17, 2021

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