Invited Speaker 23rd International Society of Magnetic Resonance Conference 2023

Visualizing Proteins in Mammalian Cells by 19F NMR Spectroscopy (#18)

Angela Gronenborn 1 2 3 , Wenkai Zhu 2 4 , Manas Seal 5 6 , Veronica Frydman 3 6 , Xun-Cheng Su 6 , Daniella Goldfarb 6 7
  1. University of Pittsburgh, Pittsburgh, PA, United States
  2. Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA
  3. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA
  4. Department of Structural Biology, University of Pittsburgh, Pittsburgh
  5. Pittsburgh Center for HIV Protein Interactions, Pittsburgh, PA, USA
  6. Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, Israel
  7. State Key Labratory of Elemento-Organic Chemistry, College of Chemistry, Nankkai University, Tianjin, China

In-cell NMR spectroscopy is a powerful tool to investigate the behavior of biologically important molecules in physiologically relevant environments. We studied proteins in mammalian cells and showed that interactions with cellular components frequently broaden resonances in 1H-15N HSQC spectra beyond detection. This contrasts findings from 19F spectroscopy, where resonances for selectively fluorinated proteins are readily observed. Importantly, for in-cell NMR studies, 19F is an ideal reporter since it is absent from all naturally occurring biological macromolecules, and fluorine can be readily incorporated into proteins biosynthetically via natural or non-natural amino acids. We present 19F in-cell NMR results for proteins introduced into mammalian cells by electroporation. Our data demonstrate that fluorine signals are quickly and efficiently detected in one-dimensional (1D) NMR in-cell spectra, even for proteins whose 1H-15N HSQC spectra are devoid of all but very few resonances due to interactions with other components in the cellular environment. In addition, we show that 19F paramagnetic relaxation enhancements (19F PREs) can provide valuable distance information for structure characterization in physiological contexts. The in-cell 19F PRE-derived distances are in good agreement with in-cell 19F ENDOR measurements on the same paramagnetic proteins, thus providing an effective means to obtain accurate distances in the cellular milieu.

  1. Zhu, W.; Guseman, A. J.; Bhinderwala, F.; Lu, M.; Su, X.-C.; Gronenborn, A. M., Visualizing Proteins in Mammalian Cells by 19F NMR Spectroscopy. Angew. Chem. Int. Ed. 2022, 61 (23), e202201097.
  2. Seal, M., Zhu, W., Dalaloyan, A., Feintuch, A., Bogdanov, A., Frydman, V., Su, X.-C., Gronenborn, A. M., Goldfarb, D., GdIII-19F Distance Measurements for Proteins in Cells by Electron-Nuclear Double Resonance. Angew. Chem. Int. Ed. 2023, 62, e202218780;