IN-CELL NMR AS A TOOL FOR IN SITU STRUCTURAL BIOLOGY

BINOLFI, ANDRES

Congreso; Reunión Conjunta de Sociedades de BioCiencias 2017; 2017

The structure and function of proteins depend on multiple cellular factors such as localization, post-translational modifications and interactions with biological partners.However, most protein structural studies are typically performed on isolated samples, under conditions that differ substantially from in vivo environments of live cells. The question arises whether protein features observed in vitro correlate with their intracellular behaviors? In this regard, in-cell NMR spectroscopy represents a powerful method to directly study protein structures in intact cells. Here, we present high-resolution in-cell NMR results on the structural and dynamic properties of the human amyloid protein alpha-synuclein (aSyn) in different mammalian cell types, the aggregation of which correlates with the onset of Parkinson´s disease (PD). Using electroporationto efficiently deliver isotopically enriched aSyn into neuronal and non-neuronal cells, we delineate its intracellular behavior by in-cell NMR spectroscopy.1 Additionally, we study the cellular repair of oxidation-damaged aSyn by endogenous enzymes and identify C-terminal modifications sites as remaining permanently damaged, which may lead to neuronal toxicity under oxidative stress conditions.These results allow us to better understand the native conformations of aSyn under physiological cell conditions, further enabling future in situ investigations of intracellular aSyn aggregation in PD and other relatedneurodegenerative disease processes.References:1. Theillet, F. X., Binolfi, A. and Bekei B., et al. Structuraldisorder of monomeric alpha-synuclein persists in mammaliancells. Nature, 530, 45-50, (2016).2. Binolfi, A. et al. Intracellular repair of oxidation-damagedalpha-synuclein fails to target C-terminal modificationsites. Nat. Commun. 7, 10251, (2016).