Nath S.,Laboratory of Blomolecular Dynamics |
Meuvis J.,Laboratory of Blomolecular Dynamics |
Hendrix J.,Laboratory of Blomolecular Dynamics |
Carl S.A.,Catholic University of Leuven |
Engelborghs Y.,Laboratory of Blomolecular Dynamics
Biophysical Journal | Year: 2010
The kinetics of aggregation of a-synuclein are usually studied by turbidity or Thio-T fluorescence. Here we follow the disappearance of monomers and the formation of early oligomers using fluorescence correlation spectroscopy. Alexa488labeled A140C-synuclein was used as a fluorescent probe in trace amounts in the presence of excess unlabeled a-synuclein. Repeated short measurements produce a distribution of diffusion coefficients. Initially, a sharp peak is obtained corresponding to monomers, followed by a distinct transient population and the gradual formation of broader-sized distributions of higher oligomers. The kinetics of aggregation can be followed by the decreasing number of fast-diffusing species. Both the disappearance of fast-diffusing species and the appearance of turbidity can be fitted to the Finke-Watzky equation, but the apparent rate constants obtained are different. This reflects the fact that the disappearance of fast species occurs largely during the lag phase of turbidity development, due to the limited sensitivity of turbidity to the early aggregation process. The nucleation of the early oligomers is concentration-dependent and accompanied by a conformational change that precedes /5-structure formation, and can be visualized using fluorescence resonance energy transfer between the donor-labeled N-terminus and the acceptor-labeled cysteine in the mutant A140C. © 2010 by the Biophysical Society.