In-cell residue-resolved NMR of micromolar α-synuclein and tau at 310K

Aggregates of non-globular proteins are associated to several degenerative disorders, e.g. -synuclein and tau involved in Parkinsons and Alzheimers 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 -synuclein or tau upon induction in a culture medium supplemented with 13C-labeled amino acids, or precursors thereof. Acquiring 13C-13CO spectra permitted an early residue-resolved analysis of -synuclein and tau at 310 K and <10 M in HEK cells at 700 MHz. We detected disordered conformations and identical patterns of cellular interactions for -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. 13C-labeling and 13C-detected NMR in live human cells are thus viable techniques for in-cell structural biology.