Location trumps length: polyglutamine-mediated changes in folding and aggregation of a host protein.

Expanded CAG diseases are progressive neurodegenerative disorders in which specific proteins have an unusually long polyglutamine stretch. Although these proteins share no other sequence or structural homologies, they all aggregate into intracellular inclusions that are believed to be pathological. We sought to determine what impact the position and number of glutamines have on the structure and aggregation of the host protein, apomyoglobin. Variable-length polyQ tracts were inserted either into the loop between the C- and D-helices (Q(n)CD) or at the N-terminus (Q(n)NT). The Q(n)CD mutants lost some α-helix and gained unordered and/or ß-sheet in a length-dependent manner. These mutants were partially unfolded and rapidly assembled into soluble chain-like oligomers. In sharp contrast, the Q(n)NT mutants largely retained wild-type tertiary structure but associated into long, fibrillar aggregates. Control proteins with glycine-serine repeats (GS(8)CD and GS(8)NT) were produced. GS(8)CD exhibited similar structural perturbations and aggregation characteristics to an analogously sized Q(16)CD, indicating that the observed effects are independent of amino acid composition. In contrast to Q(16)NT, GS(8)NT did not form fibrillar aggregates. Thus, soluble oligomers are produced through structural perturbation and do not require polyQ, whereas classic fibrils arise from specific polyQ intermolecular interactions in the absence of misfolding.

A strategy for generating polyglutamine 'length libraries' in model host proteins.

Huntington's disease is one of nine known neurodegenerative diseases in which a disease-specific protein contains an unusually long polyglutamine (polyQ) stretch. The proteins associated with each disease are unrelated in sequence, size, structure, function or location of the mutation. In all cases, there is an apparent critical number of glutamines below which individuals do not develop disease. Expansion of the polyQ domain is closely associated with misfolding and aggregation of the protein. It is not yet well understood how the length of the polyQ tract, and its location within a given protein, is related to misfolding and to disease. In this work we developed a strategy for generating length libraries of polyQ-containing proteins, with the polyQ inserted at an arbitrary location. This strategy facilitates systematic, detailed study of the relationship among polyQ length, context and misfolding.