Abstract
Aggregation of neuronal protein a-synuclein leads to the formation of amyloid fibrils, which are associated with the development of Parkinson's disease. The mechanism of a-synuclein pathology is not fully understood and is a subject of active research in the field.
To tackle this problem, the fusions of fluorescent proteins to a-synuclein C-terminus are often used in cellular and animal studies. The effects induced by such a-synuclein sequence extension on a-synuclein aggregation propensity are, however, not systematically examined despite the evidence that the negatively charged C-terminus plays a critical role in the regulation of a-synuclein aggregation.
In this work, we investigated how the charge and length variations of the C-terminus affect the aggregation propensity of a-synuclein. To address these questions, we prepared mutants of a-synuclein carrying additional moieties of different charge and length at the protein C-terminus.
We determined the rates of two different aggregation stages (primary nucleation and elongation) based on a thioflavin T kinetic assay. We observed that all mutants bearing neutrally charged moieties of different length fibrilized slower than wild-type a-synuclein.
The primary nucleation and elongation rates strongly decreased with increase of the C-terminal extension length. Meanwhile, charge variation of the C-terminus significantly changed the rate of a-synuclein nucleation, but did not markedly affect the rate of fibril elongation.
Our data demonstrate that both the charge and length of the C-terminus play an important role at the stage of initial fibril formation, but the stage of fibril elongation is affected mainly by the length of C-terminal extension. In addition, our results suggest that there are at least two steps of incorporation of a-synuclein monomers into the amyloid fibril: namely, the initial monomer binding to the fibril end (charge-dependent, relatively fast), and the subsequent conformational change of the protein