Earliest events in α-synuclein fibrillation probed with the fluorescence of intrinsic tyrosines

• Intrinsic fluorescence of α-synuclein reports the earliest events of the aggregation.
• In the sub-7 ms after acidification, a conformational change occurs in the protein.
• 4–10 s after acidification, the protein starts to aggregate.

The fluorescence of the four tyrosines of α-synuclein (Syn) was used for probing the earliest events preceding the fibrillation of Syn, during the onset of the so-called lag-time of fibrillation. Steady-state fluorescence experiments revealed an increase in the fluorescence intensity (FI) for Syn solutions at pH values 3 and 2, in comparison with pH 7, and fluorescence decays indicated that the FI increase did not result from suppression of excited-state proton transfer from the tyrosines to aspartates and glutamates, exposure of tyrosines to more hydrophobic environments, or reduction of homo-energy transfer. Instead, the FI increase was due to changes in the population of the tyrosine rotamers at low pH values.Stopped-flow experiments (pH-jumps) showed that the FI enhancement involves two processes: a fast (sub-7 ms) intramolecular (concentration-independent) process, which we assign to the protein collapse at low pH, and a slower intermolecular (concentration-dependent) process of protein dimerization/oligomerization, starting at 4–10 s after acidification.To the best of our knowledge, this is the first work on the experimental detection of these earliest processes in the fibrillation of Syn.

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