A triple-emission fluorescent probe reveals distinctive amyloid fibrillar polymorphism of wild-type alpha-synuclein and its familial Parkinson's disease mutants.
Intracytoplasmic neuronal deposits containing amyloid fibrils of the 140-amino acid presynaptic protein alpha-synuclein (AS) are the hallmark of Parkinson's (PD) disease and related neurodegenerative disorders. Three point mutations (A53T, A30P, and E46K) are linked to early onset PD. Compared to the wild-type (WT) protein, the mutants aggregate faster in vitro, but their fibrillar products are quite similar. Using the extrinsic multiple-emission probe 4'-(diethylamino)-3-hydroxyflavone (FE), we demonstrate unique and distinct spectroscopic signatures for the amyloid fibrils formed by the WT and mutant AS, presumably indicative of subtle differences in supramolecular structure. The two well-separated emission bands of the FE probe originate from a proton transfer reaction in the excited state. The ratiometric response constitutes a sensitive, tunable reporter of microenvironmental properties such as polarity and hydrogen bonding. The very distinctive fluorescence spectra of the FE probe bound to the four AS variants reflect different tautomeric equilibria in the excited state and the existence of at least two different binding sites in the fibrils for the dye. Deconvolution of the two-dimensional excitation-emission spectra leads to estimations of different local dielectric constants and extents of hydration characteristic of the proteins. The sensitivity of such a simple external probe to conformational alterations induced by point mutations is unprecedented and provides new insight into key phenomena related to amyloid fibrils: plasticity, polymorphism, propagation of structural features, and structure-function relationships underlying toxicity.
Celej
M Soledad
MS
Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Caarls
Wouter
W
Demchenko
Alexander P
AP
Jovin
Thomas M
TM
D000682:Amyloid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015415:Biomarkers / Q000737:chemistry / Q000378:metabolism; D005419:Flavonoids / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D018625:Microscopy, Atomic Force; D010300:Parkinson Disease / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011110:Polymorphism, Genetic; D013050:Spectrometry, Fluorescence; D051844:alpha-Synuclein / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure
C090856:4'-diethylamino-3-hydroxyflavone; D000682:Amyloid; D015415:Biomarkers; D005419:Flavonoids; D005456:Fluorescent Dyes; D051844:alpha-Synuclein