Abstract
Intracellular macromolecular crowding can lead to increased aggregation of proteins, especially those that lack a natively folded conformation. Crowding may also be mimicked by the addition of polymers like polyethylene glycol (PEG) in vitro. α-Synuclein is an intrinsically disordered protein that exhibits increased aggregation and amyloid fibril formation in a crowded environment. Two hypotheses have been proposed to explain this observation. One is the excluded volume effect positing that reduced water activity in a crowded environment leads to increased effective protein concentration, promoting aggregation. An alternate explanation is that increased crowding facilitates conversion to a non-native form increasing the rate of aggregation. In this work, we have segregated these two hypotheses to investigate which one is operating. We show that mere increase in concentration of α-synuclein is not enough to induce aggregation and consequent fibrillation. In vitro, we find a complex relationship between PEG concentrations and aggregation, in which smaller PEGs delay fibrillation; while, larger ones promote fibril nucleation. In turn, while PEG600 did not increase the rate of aggregation, PEG1000 did and PEG4000 and PEG12000 slowed it but led to a higher overall fibril burden in the latter to cases. In cells, PEG4000 reduces the aggregation of α-synuclein but in a way specific to the cellular environment/due to cellular factors. The aggregation of the similarly sized, globular lysozyme does not increase in vitro when at the same concentrations with either PEG8000 or PEG12000. Thus, natively disordered α-synuclein undergoes a conformational transition in specific types of crowded environment, forming an aggregation-prone conformer.
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Acknowledgements
pRSETB-α-SYN was received as a gift from Prof. Roberto Cappai, University of Melbourne, Australia. p426GAL A53Tα-SYN-GFP was a gift from late Prof. Susan Lindquist, Massachusetts Institute of Technology, USA. Partial financial support from Science and Engineering Research Board is acknowledged. SJ is grateful to the Council for Scientific and Industrial Research for the award of senior research fellowship.
Funding
Partial financial support from Science and Engineering Research Board (CRG/2018/000458) is acknowledged. SJ is grateful to the Council for Scientific and Industrial Research for the award of senior research fellowship.
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Biswas, S., Bhadra, A., Lakhera, S. et al. Molecular crowding accelerates aggregation of α-synuclein by altering its folding pathway. Eur Biophys J 50, 59–67 (2021). https://doi.org/10.1007/s00249-020-01486-1
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DOI: https://doi.org/10.1007/s00249-020-01486-1