Mammalian In-cell NMR reveals the intracellular conformation, dynamics and post-translational modifications of the protein alpha-synuclein

PHILIPP SELENKO; DANIELLA GOLDFARB; SILVIA VERZINI; JONAS KOSTEN; BEATA BEKEI; FRANCOIS-XAVIER THEILLET; ANDREA MARTORANA; BINOLFI, ANDRES; MARCEL STUIVER; HONOR M. ROSE; ANTONIO LIMATOLA

Angra dos Reis

Congreso; 16th NMR user Meeting; 2017

Proteins in their native environments do not exist as isolated, unperturbed entities. They can be post-translationally modified or be part of large macromolecular complexes, and depending on their sub-cellular localization they are exposed to different degrees of macromolecular crowding, varied pH values and/or transient interactions with other proteins, metabolites, nucleic acids or lipidic membranes.1 All of these modulate the structure and function of proteins and to understand protein behavior in detail one needs to look at them in their native environments. 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 intrinsically disordered protein alpha-synuclein (aSyn) in mammalian cells. Using electroporation to efficiently deliver isotopically enriched aSyn into neuronal and non-neuronal cells, we delineate its intracellular behavior by in-cell NMR spectroscopy.2 In addition, by using oxidatively modified aSyn molecules we studied endogenous enzymatic redox repair pathways and identified permanently modified aSyn sites that might accumulate and exert neuronal toxicity under stress conditions.3 Our results contribute to the understanding of the native conformations of aSyn and lay the ground to further perform high-resolution investigations of proteins in their physiological environments.References.1.Theillet F-X., Binolfi A., Frembgen-Kesner T., Hingorani K., Sarkar M., Kyne C., Li C., Crowley P., Gierasch L., Pielak G., Elcock A. H., Gershenson A. and Selenko P. Chem. Rev. 114 (2014) 6661.2.Theillet F-X., Binolfi A., Bekei B., Martorana A., Rose H. M., Stuiver M., Verzini S., Lorenz D., van Rossum M., Goldfarb D., Selenko P. Nature 530 (2016) 45.3.Binolfi A., Limatola A., Verzini S., Kosten J., Theillet F-X., Rose H. M., Bekei B., Stuiver M., van Rossum M., Selenko P. Nat. Commun. 7 (2016) 10251.