In-Cell Residue-Resolved NMR of Micromolar α-Synuclein and Tau at 310 K

Aggregates of nonglobular proteins are associated with several degenerative disorders, e.g., α-synuclein and tau involved in Parkinson's and Alzheimer's diseases. Do these proteins undergo progressive changes in their 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 ¹³C-labeled amino acids, or precursors thereof. Acquiring ¹³Cα-¹³CO 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 patterns of extended cellular interactions for α-synuclein wild-type and two mutants (F4A, A30P), which suggests the appearance of a subpopulation binding to lipid membrane at 310 K. Only the disordered N-terminus of tau was observable, even upon microtubule dismantling by colchicine. This shows that supplementary binding partners interfere with tau in cells. Our approach offers an excellent scalability, in signal, and resolution, up to 1.2 GHz. ¹³C-labeling and ¹³C-detected NMR spectroscopy in live human cells are thus viable techniques for in-cell structural biology.