Alpha-synuclein structures from fluorescence energy-transfer kinetics: implications for the role of the protein in Parkinson's disease

Abstract

Parkinson's disease is associated with the deposition and accumulation of alpha-synuclein fibrils in the brain. A30P and A53T mutations have been linked to the early-onset familial disease state. Time-resolved tryptophan fluorescence energy-transfer measurements have been used to probe the structures of pseudo-wild-type and mutant (A30P) alpha-synucleins at physiological pH (7.4), in acidic pH (4.4) solutions, and in the presence of SDS micelles, a membrane mimic. Fluorescent donor-energy acceptor (DA) distance distributions for six different tryptophan/3-nitro-tyrosine pairs reveal the presence of compact, intermediate, and extended conformations of the protein. CD spectra indicate that the protein develops substantial helical structure in the presence of SDS micelles. DA distributions show that micelles induce compaction in the N-terminal region and expansion of the acidic C terminus. In acidic solutions, there is an increased population of collapsed structures in the C-terminal region. Energy-transfer measurements demonstrate that the average DA distances for the W4-Y19 and Y19-W39 pairs are longer in one of the two disease-related mutants (A30P).

Fig. 1.

Primary amino acid sequence of human α-syn. The seven imperfect repeats are underlined. Two point mutations linked to early-onset PD (A30P and A53T) are colored gray. Red, acidic; blue, basic; green, aromatic. Residues in boldface have been either mutated (W, F, and Y) or chemically modified [Y(NO₂)] for FET studies.

Fig. 2.

Trp-to-Y(NO₂) distance distributions for freely jointed polymer chain models of α-syns under a variety of solution conditions. Green, 40 mM SDS in 20 mM NaP_i buffer, pH 7.4; blue, 20 mM NaP_i buffer, pH 7.4; red, 20 mM NaOAc buffer, pH 4.4.

Fig. 3.

Distributions of Trp-to-Y(NO₂) distances in α-syns extracted from FET kinetics. Results are shown for W4–Y(NO₂)19 (A), Y(NO₂)19–W39 (B), W4–Y(NO₂)39 (C), Y(NO₂)74–W94 (D), W94–Y(NO₂)136 (E), and W4–Y(NO₂)136 (F) under a variety of solution conditions. Green, 40 mM SDS in 20 mM NaP_i buffer, pH 7.4; blue, 20 mM NaP_i buffer, pH 7.4; red, 20 mM NaOAc buffer, pH 4.4. Center-to-center distances calculated for an ideal helix are shown as black lines in A–D.

Fig. 4.

Difference distance distributions of disease-related A30P α-syns extracted from FET kinetics under a variety of solution conditions. Results are shown for W4/Y(NO₂)19/A30P and Y(NO₂)19/A30P/W39. Green, 40 mM SDS in 20 mM NaP_i buffer, pH 7.4; blue, 20 mM NaP_i buffer, pH 7.4; red, 20 mM NaOAc buffer, pH 4.4.