Dr. Kumar Tekwani Movellan - 19F dynamic nuclear polarization: a powerful tool for protein structural biology in mammalian cells

Kumar Tekwani Movellan

University of Delaware

19F dynamic nuclear polarization: a powerful tool for protein structural biology in mammalian cells

Obtaining structural information on proteins in their native environments, the cell, is challenging with the current methodologies. The lack of site specificity and sensitivity has been a major limitation. Here, we introduce 19F dynamic nuclear polarization (DNP) combined with magic-angle spinning (MAS) NMR spectroscopy as a powerful technique to study proteins in mammalian cells. We demonstrate this proof-of-concept on SARS-CoV-2 5F-Trp-NNTD protein, introduced by electroporation into human A2780 cells. Using fast magic angle spinning frequencies (30-40 kHz), we obtained 19F DNP signal enhancements of over 35 for SARS-CoV-2 5F-Trp-NNTD, which translates into a reduction in experiment time by a factor of 103, requiring only minutes to record high signal-to-noise ratio MAS NMR spectra on nanomole quantities of protein. The additional sensitivity enabled acquisition of 2D 19F-19F dipolar correlation spectra which exhibit remarkable 19F resolution. These experiments revealed 19F-19F intramolecular cross peaks between fluorine atoms as far as ~10 Å apart with 19F line widths as narrow as ~2 ppm. This work paves the way for in-cell 19F DNP-enhanced MAS NMR applications and is envisioned to be critical for improved understanding of in vivo mechanisms and enabling characterization of drug-protein interactions in cellular environments.