Purification and characterization of polyglutamine-containing apomyoglobin mutants as models for polyglutamine disease.

摘要

Expanded CAG diseases are a group of nine inherited and progressive neurodegenerative disorders. In affected individuals, specific proteins have an unusually long polyglutamine (polyQ) stretch, with a strong inverse correlation between polyQ length and age of onset of symptoms. Disease-associated proteins are unique to each disorder and share no other known sequence or structural homologies; all aggregate into intracellular inclusions that are generally believed to be pathological. The expanded polyQ domain appears to be directly linked to a toxic gain of function.;Many strategies have been used to study polyQ-related aggregation in vitro including studying polyQ peptides, the disease-implicated proteins, and model proteins with inserted, non-native polyQ sequences. In this study, the last strategy was employed and polyQ sequences were engineered into the model host protein apomyoglobin. In this study, a method is presented by which a library of polyQ codon lengths above and below the critical length were rapidly and economically generated in the flexible CD loop region of apomyoglobin. Mutants were also synthesized with N-terminal polyQ sequences. A robust purification process for polyQ-containing apomyoglobin mutants is presented. Using denaturing buffer conditions and multiple chromatography steps, polyQ lengths up to Q46 were purified with greater than 90% purity and in sufficient yields for most biophysical assays. Importantly, purified protein finished product was also monomeric.;Circular dichroism and fluorescence spectroscopy were used to assay secondary and tertiary structure of the mutants. Aggregation kinetics were studied by assessing monomer loss and sedimentation while aggregate morphology was studied by transmission electron microscopy. Overall, the mutants' structural and aggregation characteristics correlated more significantly with polyQ sequence position than with polyQ length. A glycine-serine insert in the CD loop was also produced as a control for structural perturbation due to sequence insertion. This mutant caused structural perturbations and aggregation analogous to an equal length polyQ insert.;The research presented in this dissertation broadly impacts the field of polyQ disease research. In vivo, certain polyQ sequence positions may promote aggregation more than others explaining why many polyQ-containing proteins cause no disease. Overall, these data challenge the theory that only expanded polyQ is sufficient for disease.