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

Abstract

α-synuclein is an intrinsically disordered protein (IDP) whose spontaneous 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 cellular environment. In this study, α-synuclein was studied in the presence of a synthetic crowder, Ficoll70, and biological crowders composed of lysed cells that better mimic the biocomplexity of the cellular environment. ¹⁵N-1H HSQC NMR results from freshly prepared samples show similar α-synuclein chemical shifts in non-crowded and all crowded conditions implying that its structure remains disordered in all conditions. Nevertheless, both HSQC NMR and fluorescence measurements indicate that, only in the cell lysate, α-synuclein forms aggregates at timescales of 48 hours. ¹⁵N-edited diffusion measurements indicated that all crowders slow down the IDP diffusivity; however, at high concentrations, α-synuclein diffuses faster in cell lysate than in Ficoll70, possibly due to additional soft (e.g. electrostatic or hydrophobic) interactions. ¹⁵N-edited relaxation measurements show that some residues are more mobile in cell lysate than in Ficoll70; the most strongly different rates are predominantly in hydrophobic residues. I thus examined cell lysates with reduced hydrophobicity and found higher relaxation rates (slower dynamics) in several α-synuclein residues. Taken together, these experiments suggest that while cell lysate does not substantially affect α-synuclein structure (HSQC spectra), it does affect chain dynamics (transverse relaxation rates) and translational motion (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.