Year: 2025
Type: article
Abstract: Aggregates of non-globular proteins are associated to several degenerative disorders, e.g. α-synuclein and tau involved in Parkinson’s and Alzheimer’s diseases. Do these proteins suffer progressive changes in conformations and interactions in pathologic situations? In-cell NMR provides atomic-scale information in live cells but, until now, only at ~283 K in the case of unfolded proteins. Here, we report new labeling and acquisition methods enabling in-cell NMR at 310 K to study these proteins at micromolar concentrations, i.e. native cellular abundances. We used stable human cell lines expressing α-synuclein or tau upon induction in a culture medium supplemented with 13 C-labeled amino acids, or precursors thereof. Acquiring 13 Cα- 13 CO spectra permitted an early residue-resolved analysis of α-synuclein and tau at 310 K and <10 μM in HEK cells at 700 MHz. We detected disordered conformations and identical patterns of cellular interactions for α-synuclein wild-type and two mutants (F4A, A30P). Only the disordered N-terminus of tau was observable, even upon microtubule dismantling by colchicin. Our approach offers an excellent scalability -in signal and resolution-up to 1.2 GHz. 13 C-labeling and 13 C-detected NMR in live human cells are thus viable techniques for in-cell structural biology.
DOI: 10.1021/jacs.5c15061
Institutions: Centre National de la Recherche Scientifique, Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Université Paris-Saclay, Institut de Biologie Intégrative de la Cellule, Université Paris Cité, Cibles Thérapeutiques et conception de médicaments, CEA Paris-Saclay - Etablissement de Saclay, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement, CEA Paris-Saclay, Médicaments et Technologies pour la Santé, MetaboHUB
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
Awards: G1052072530, Regulation of the DNA-damage response by phosphorylation clusters in the p53 signaling network, G1669488153, G2247417377, G2303391026, G2360965829, G4180087395, G502948375, G5296751580, G654663749, G686757768, G7237471465, G7240651115, DNA damage signaling studied by in-cell NMR: Structural and functional regulation of Mdm2 through its phosphorylation status., Infrastructure Française pour la Biologie Structurale Intégrée
OpenAlex ID: W4416998287