Utilzing Forster Resonance Energy Transfer (FRET) and Photocrosslinking to Visualize Conformational Changes of Alpha-Synuclein

Abstract

Intrinsically disordered proteins (IDPs) make up an expanding category of proteins defined by their conformational heterogeneity. Of these, α-Synuclein (αS), like many other IDPs, has been recognized for its ability to self associate to produce cytotoxic oligomeric and fibrillar species, which have been shown to play a causative role in Parkinson's Disease (PD). Despite the recognized role of αS in PD, there has yet to emerge consensus models of the disordered ensemble or fibrillar species of this protein. Although these structural studies have been dominated by the use of NMR, we focus on using techniques such as Förster Resonance Energy Transfer (FRET) and photocrosslinking, which can be applied in both in vitro and in cell-based experiments. As a proof of concept, we have first directed our attention towards visualizing the compaction of αS induced by trimethylamine-N-oxide (TMAO). After generating a library of analogs consisting of two probe pairs, sensitive to two different distance ranges, ensemble FRET measurements were performed to collect a set of intramolecular distances. These distances were utilized as constraints within Monte Carlo simulations to generate models of the TMAO-compacted ensemble. Furthermore, in separate experiments, we demonstrate that photocrosslinking can be effectively used to identify the binding sites of small molecules to the surface of αS fibril. Lastly, we highlight the use of these techniques within αS fibrils and demonstrate the use of these data in refining already existing fibril models.