The effects of biological crowders on fibrillization, structure, diffusion, and conformational dynamics of α-synuclein

alpha-synuclein is an intrinsically disordered protein (IDP) whose aggregation in presynaptic neuronal cells is a pathological hallmark of Lewy body formation and Parkinson's disease. This aggregation process is likely affected by the crowded macromolecular cellular environment. In this study, alpha-synuclein was studied in the presence of both a synthetic crowder, Ficoll70, and a biological crowder composed of lysed cells that better mimics the biocomplexity of the cellular environment. N-15-H-1 HSQC NMR results show similar alpha-synuclein chemical shifts in non-crowded and all crowded conditions implying that it remains similarly unstructured in all conditions. Nevertheless, both HSQC NMR and fluorescence measurements indicate that, only in the cell lysate, alpha-synuclein forms aggregates over a timescale of 48 h. N-15-edited diffusion measurements indicate that all crowders slow down the alpha-synuclein's diffusivity. Interestingly, at high concentrations, alpha-synuclein diffuses faster in cell lysate than in Ficoll70, possibly due to additional soft (e.g., electrostatic or hydrophobic) interactions. N-15-edited relaxation measurements show that some residues are more mobile in cell lysate than in Ficoll70; the rates that are most different are predominantly in hydrophobic residues. We thus examined cell lysates with reduced hydrophobicity and found slower dynamics (higher relaxation rates) in several alpha-synuclein residues. Taken together, these experiments suggest that while cell lysate does not substantially affect alpha-synuclein structure (HSQC spectra), it does affect chain dynamics and translational diffusion, and strongly affects aggregation over a timescale of days, in a manner that is different from either no crowder or an artificial crowder: soft hydrophobic interactions are implicated.