Mutational analysis of the structural organization of polyglutamine aggregates.

The formation of amyloid-like aggregates by expanded polyglutamine (polyGln) sequences is suspected to play a critical role in the neuropathology of Huntington's disease and other expanded CAG-repeat diseases. To probe the folding of the polyGln sequence in the aggregate, we replaced Gln-Gln pairs at different sequence intervals with Pro-Gly pairs, elements that are compatible with beta-turn formation and incompatible with beta-extended chain. We find that PGQ9 and PGQ10, peptides consisting of four Q9 or Q10 elements interspersed with PG elements, undergo spontaneous aggregation as efficiently as a Q45 sequence, whereas the corresponding PGQ7 and PGQ8 peptides aggregate much less readily. Furthermore, a PDGQ9 sequence containing d-prolines aggregates more efficiently than the peptide with l-prolines, consistent with beta-turn formation in aggregate structure. Introduction of one additional Pro residue in the center of a Q9 element within PGQ9 completely blocks the peptide's ability to aggregate. This strongly suggests that the Q9 elements are required to be in extended chain for efficient aggregation to occur. We determined the critical nucleus for aggregation nucleation of the PGQ9 peptide to be one, a result identical to that for unbroken polyGln sequences. The PGQN peptide aggregates are structurally quite similar to Q45 aggregates, as judged by heterologous seeding aggregation kinetics, recognition by an anti-polyGln aggregate antibody, and electron microscopy. The results suggest that polyGln aggregate structure consists of alternating elements of extended chain and turn. In the future it should be possible to conduct detailed and interpretable mutational studies in the PGQ9 background.