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Abstract Fluorine‐19 NMR spectroscopy has emerged as a powerful tool for studying protein structure, dynamics, and interactions. Of particular interest is the exploitation of trifluoromethyl (tfm) groups, given their high sensitivity and superior transverse relaxation properties, compared to single fluorine atoms. However, biosynthetic incorporation of tfm‐bearing amino acids remains challenging due to cytotoxicity and incompatibility with natural tRNA synthetases. Here, we report on overcoming this challenge using cell‐free synthesis, incorporating trifluoromethyl‐methionine (tfmM) into the protein Cyclophilin A (CypA) with remarkably high efficiency, impossible via biosynthetic means. Importantly, we demonstrate that tfmM CypA binds a native substrate, the N‐terminal domain of HIV‐1 capsid protein (HIV‐1 CA‐NTD), and retains peptidyl prolylcis/transisomerase activity. It also binds the peptide inhibitor Cyclosporine A (CsA) with the same affinity as non‐labeled, wild‐type CypA. Furthermore, we show that19F isotope shifts and19F solvent paramagnetic relaxation enhancements (PREs) provide valuable structural information on surface exposure. Taken together, our study illustrates that tfmM can be readily incorporated into proteins at very high levels by cell‐free synthesis without disturbing protein structure and function, significantly expanding the scope of19F NMR spectroscopy for studying protein structure and dynamics.
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Deciphering the Mechanistic Basis for Perfluoroalkyl‐Protein Interactions
Abstract Although rarely used in nature, fluorine has emerged as an important elemental ingredient in the design of proteins with altered folding, stability, oligomerization propensities, and bioactivity. Adding to the molecular modification toolbox, here we report the ability of privileged perfluorinated amphiphiles to noncovalently decorate proteins to alter their conformational plasticity and potentiate their dispersion into fluorous phases. Employing a complementary suite of biophysical, in‐silico and in‐vitro approaches, we establish structure‐activity relationships defining these phenomena and investigate their impact on protein structural dynamics and intracellular trafficking. Notably, we show that the lead compound, perfluorononanoic acid, is 106times more potent in inducing non‐native protein secondary structure in select proteins than is the well‐known helix inducer trifluoroethanol, and also significantly enhances the cellular uptake of complexed proteins. These findings could advance the rational design of fluorinated proteins, inform on potential modes of toxicity for perfluoroalkyl substances, and guide the development of fluorine‐modified biologics with desirable functional properties for drug discovery and delivery applications.
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- Award ID(s):
- 1845053
- PAR ID:
- 10419113
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ChemBioChem
- Volume:
- 24
- Issue:
- 13
- ISSN:
- 1439-4227
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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