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Abstract 19F NMR spectroscopy is an attractive and growing area of research with broad applications in biochemistry, chemical biology, medicinal chemistry, and materials science. We have explored fast magic angle spinning (MAS)19F solid‐state NMR spectroscopy in assemblies of HIV‐1 capsid protein. Tryptophan residues with fluorine substitution at the 5‐position of the indole ring were used as the reporters. The19F chemical shifts for the five tryptophan residues are distinct, reflecting differences in their local environment. Spin‐diffusion and radio‐frequency‐driven‐recoupling experiments were performed at MAS frequencies of 35 kHz and 40–60 kHz, respectively. Fast MAS frequencies of 40–60 kHz are essential for consistently establishing19F–19F correlations, yielding interatomic distances of the order of 20 Å. Our results demonstrate the potential of fast MAS19F NMR spectroscopy for structural analysis in large biological assemblies.
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High‐Efficiency Trifluoromethyl‐Methionine Incorporation into Cyclophilin A by Cell‐Free Synthesis for 19 F NMR Studies
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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- Award ID(s):
- 2116534
- PAR ID:
- 10558479
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- Volume:
- 64
- Issue:
- 7
- ISSN:
- 1433-7851
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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