Early aggregation steps in alpha-synuclein as measured by FCS and FRET: evidence for a contagious conformational change.

Abstract:

:The kinetics of aggregation of alpha-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. Alexa488-labeled A140C-synuclein was used as a fluorescent probe in trace amounts in the presence of excess unlabeled alpha-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 beta-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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Nath S,Meuvis J,Hendrix J,Carl SA,Engelborghs Y

doi

10.1016/j.bpj.2009.12.4290

subject

Has Abstract

pub_date

2010-04-07 00:00:00

pages

1302-11

issue

7

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(09)06139-6

journal_volume

98

pub_type

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