Parkinson’s disease (PD) is associated with the protein α-Synuclein (α-syn), as it is part of PDs main pathophysiological phenotype. α-Syn is an intrinsically disordered protein (IDP), undergoing rapid conformational changes. Using single-molecule Förster resonance energy transfer (smFRET), previous labs have shown that α-syn undergoes such changes in hundreds of ns, yet recent works from the Lerner lab indicate that they may also occur at the ms timescale. The Lerner lab developed an analytical framework for discerning µs-ms conformational dynamics to reinspect the previous lab’s results, therefore that same doubly-labeled α-syn had to be reproduced. An α-syn double cysteine mutant was mixed with a 1:3:3 molar ratio of protein, FRET donor, and FRET acceptor dye. Size exclusion chromatography proved the most efficient method to filter out free dye. Fluorescence cross-correlation spectroscopy was then implemented for assessing the fraction of donor-acceptor doubly-labeled α-syn.
FRET occurs when the excitation energy of the donor dye is transferred non-radiatively to the acceptor dye, and only if the donor and acceptor dyes are fluorescently active (exhibiting stoichiometry values 0<S<1). As such, overall photon bursts analyzed included photon detection in the acceptor and donor detection channels caused by donor excitation, as well as acceptor photon detections following acceptor excitation. Bursts displaying signatures of low FRET were selected from the overall burst channel data, then further analyzed to identify within-burst FRET dynamics in ms, using multi-parameter photon-by-photon hidden Markov modelling (mpH²MM; Figure 1.). From these preliminary results, α-syn exhibits ms dynamics and not just ns dynamics, paving the way to proving that IDPs may have states of less and more thermodynamic stability.

<b>Figure 1:</b> Early burst dynamics data, with arrows representing transitions between bursts in ms

Figure 1: Early burst dynamics data, with arrows representing transitions between bursts in ms

Eitan Lerner

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