Creation of Aggregation-Defective α-Synuclein Variants by Engineering the Sequence Connecting β-Strand-Forming Domains

The aggregation of a-synuclein (aS), which is implicated in the pathology of Parkinson's disease, produces fibrils in which layers of parallel, in-register beta-sheetloop beta-sheets are formed. The effects of sequence variation in the loop-forming region (referred to as the linker region) on aS aggregation have yet to be systematically studied. In the study described here, we created and characterized aS variants containing mutations in the linker regions. Our results indicate that although the physicochemical properties of the linker region, evaluated based on an intrinsic property of a single amino acid, still play a significant role in aggregation, additional factors can also determine aggregation of aS linker mutants. Our analyses suggest that these factors include a pairwise potential for parallel in-register beta-sheet formation. A linker variant displaying significantly reduced self-aggregation interfered with aS aggregation by inhibiting the conversion of aS soluble species to aS insoluble fibrils. We anticipate that linker mutations could serve as a novel method of creating aS variants that are aggregationdefective and/or inhibit aS aggregation.