Significance: A key question in structural biology is how protein properties mapped out under simplified conditions in vitro transfer to the complex environment in live cells. The answer, it appears, varies. Defying predictions from steric crowding effects, experimental data have shown that cells in some cases stabilize and in other cases destabilize the native protein structures. In this study, we reconcile these seemingly conflicting results by showing that the in-cell effect on protein thermodynamics is sequence specific: The outcome depends both on the individual target protein and on its detailed host-cell environment.

Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson’s disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson’s disease protein alpha-synuclein (α-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched α-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal α-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of α-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered α-Syn in cells.