Crystallization of Ataxin-3

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DESCRIPTION (provided by applicant): The polyglutamine diseases are incurable neurodegenerative disorders affecting hundreds of thousands of people worldwide; they are caused by the expansion of normally occurring polyglutamine tracts in proteins. Patients suffer a steady deterioration in motor and cognitive abilities, followed by death. The Aim of this research is to investigate the structural basis of polyglutamine disease, focusing specifically on the ataxin-3 protein. The lengths of polyglutamine tracts in proteins vary within the normal population, but never exceed 35- 40 consecutive glutamines in healthy individuals. Mutations that expand the number of these residues to greater than 40 cause disease. Proteins with expanded polyglutamine regions misfold and aggregate into amyloid fibrils; the aggregation and fibril formation are inextricably linked with cellular dysfunction and death. Expanded glutamine repeats disrupt native protein structure and favor the formation of misfolded states, which can assemble into toxic aggregates. An understanding of this aberrant folding process requires a thorough structural knowledge of both the native and pathological states of the protein. Advances have been made in the characterization of fibril architecture, but no three-dimensional structure is currently available for any polyglutamine protein. This lack of structural information drastically hinders progress toward understanding the molecular basis of polyglutamine disease and developing rational therapies. Ataxin-3 is the causative agent of Machado-Joseph disease, the most common polyglutamine disease. The P. I.'s laboratory is developing ataxin-3 as a model system for studying the misfolding and aggregation of polyglutamine proteins. The structural aspects of this work require crystals of ataxin-3 suitable for X-ray diffraction studies. We therefore have developed a systematic and comprehensive approach to obtaining crystals of full-length and truncated forms of ataxin-3. Three Specific Aims are proposed, namely the identification and crystallization of isolated domains of ataxin-3, the use of rational mutagenesis to improve our ability to grow diffraction-quality crystals, and the use of antibody fragments to prepare Fab-ataxin-3 cocrystals. The molecular understanding of ataxin-3 that will be obtained from this work is a prerequisite for any rational design of therapeutics that will interfere with the protein's aberrant folding and aggregation.