Incorporation of Orthogonal Fluorine Probes for In-Cell Protein NMR

• Sara Catalina Nieto Rincon, Chemistry, Universidad Nacional de Colombia

• Tatyana Polenova, Chemistry and Biochemistry, University of Delaware

Incorporation of Orthogonal Fluorine Probes for In-Cell Protein NMR

Sara Catalina Nieto Rincón¹, Kumar Tekwani Movellan^2,3, João Pedro de Jesus Pereira^2,3, Tatyana Polenova^2,3

¹ Universidad Nacional de Colombia, Department of Chemistry, Bogotá, Colombia, ² University of Delaware, Department of Chemistry and Biochemistry, Newark DE 19716, United States, ³ Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, 1051 BiomedicalScience Tower 3, 3501 Fifth Avenue, Pittsburgh PA 15261, United States

The elucidation of protein structure and dynamics is essential to understanding the molecular mechanisms underlying biological processes. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique to uncover atomic-level structural and dynamic details of biomolecules from a wide range of conditions, including the cells. Nevertheless, studying proteins in the cellular environment by NMR faces several challenges mainly the specificity, sensitivity, and cell viability (Freedberg., & Selenko., 2014).

¹⁹F protein labeling offers information on biomolecules in solution and solid-state NMR spectroscopy in biological systems (Gronenborn., 2022). It is an alternative to the common labels, ¹³C and ¹⁵N, used for NMR studies, especially for in-cell approaches. ¹⁹F offers high sensitivity (83% of ¹H), it is highly responsive to local changes, and because of the variety of moieties available for protein labeling we can exploit a large range of chemical shift. Moreover, ¹⁹F is naturally absent from cellular systems resulting in a background free probe ideal for in-cell NMR studies (Zhu et al., 2022).

The Polenova lab is aimed to develop protocols to incorporate simultaneously two distinct fluorine moieties into proteins, with the purpose of characterizing protein structural changes inside of the mammalian cells. We tested this methodology using the N-terminal domain of the nucleocapsid N protein from SARS-CoV-2 (N^NTD) comprising 134 residues. We are using a strategy combining distinct fluorine labeling protocols, specifically Trp-indole (Crowley et al., 2012) and genetic code expansion protocols (Sharaf & Gronenborn., 2015), to incorporate ¹⁹F on Trp indole and phenylalanine simultaneously. This will provide unique structural NMR restrains for structural characterization inside mammalian cells. We obtained above 75 % fluorine incorporation at position 7 of Trp14, Trp70, and Trp94, and 100% tfmF at position 72. We assigned the 7F-Trp positions of N^NTD by expressing 7F-Trp labeled N^NTD variants W14F, W70F and W94F, where each one of the three Trp residues was mutated to a Phe. These results pave the way for novel MAS NMR in-cell structural biology applications.

References

[1] Freedberg, D. I., & Selenko, P. (2014). Live cell NMR. Annual Review of Biophysics, 43(1), 171-192.

[2] Gronenborn, A. M. (2022). Small, but powerful and attractive: 19F in biomolecular NMR. Structure, 30(1), 6-14.

[3] Zhu, W., Guseman, A. J., Bhinderwala, F., Lu, M., Su, X. C., & Gronenborn, A. M. (2022). Visualizing proteins in mammalian cells by 19F NMR spectroscopy. Angewandte Chemie International Edition, 61(23), e202201097.

[4] Crowley, P. B., Kyne, C., & Monteith, W. B. (2012). Simple and inexpensive incorporation of 19 F-tryptophan for protein NMR spectroscopy. Chemical communications, 48(86), 10681-10683.

[5] Sharaf, N. G., & Gronenborn, A. M. (2015). 19F-modified proteins and 19F-containing ligands as tools in solution NMR studies of protein interactions. In Methods in enzymology (Vol. 565, pp. 67-95).