Optimizing 19F NMR protein spectroscopy by fractional biosynthetic labeling

Abstract

In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species, where resolution is dramatically improved over that achieved by complete enrichment. In NMR studies of calmodulin, a 148 residue calcium binding protein, ¹⁹F and ¹H-¹⁵N HSQC spectra reveal a significant extent of line broadening and the appearance of minor conformers in the presence of complete (>95%) 3-fluorophenylalanine labeling. The effects of varying levels of enrichment of 3-fluorophenylalanine (i.e. between 3 and >95%) were further studied by ¹⁹F and ¹H-¹⁵N HSQC spectra, ¹⁵N T(1) and T(2) relaxation measurements, ¹⁹F T(2) relaxation, translational diffusion and heat denaturation experiments via circular dichroism. Our results show that while several properties, including translational diffusion and thermal stability show little variation between non-fluorinated and >95% ¹⁹F labeled samples, ¹⁹F and ¹H-¹⁵N HSQC spectra show significant improvements in line widths and resolution at or below 76% enrichment. Moreover, high levels of fluorination (>80%) appear to increase protein disorder as evidenced by backbone ¹⁵N dynamics. In this study, reasonable signal to noise can be achieved between 60-76% ¹⁹F enrichment, without any detectable perturbations from labeling.

Aromatic 19F-13C TROSY: a background-free approach to probe biomolecular structure, function, and dynamics

“…If incorporation of a given 19 F-labelled amino acid is found to be detrimental, fractional labelling can be used to dial down the percentage of incorporation. This strategy was recently applied to improve the stability and spectral quality of 3-fluoro-phenylalanine labelled calmodulin 32 .…”

What does fluorine do to a protein? Thermodynamic, and highly-resolved structural insights into fluorine-labelled variants of the cold shock protein

“…In this context, it has been shown that extensively fluorinated amino acids can be particularly effective in increasing protein stability 28 due to the increase in buried hydrophobic surface area as identified in structures solved by X-ray crystallography 29 . Kitevski-LeBlanc and co-workers have shown that 19 F enrichment in fluoro-phenyalanine labelled calmodulin results in an increasing protein disorder which can be diminished by a decreased level of fluorination 30 . Other work has shown that the structural integrity of a small single domain protein is conserved when one fluoro-phenylalanine is incorporated 31 and that fluoro-tryptophan labelling of various sites in fluoroacetate dehalogenase does not modify its three-dimensional structural characteristics compared to the wild type 22 .…”

Selective Radical Trifluoromethylation of Native Residues in Proteins

“… 58 In particular, we showed through fractional CF 3 -“labeling” 19 F-NMR spectra can be obtained directly with no significant line broadening. 59 Although Trp residues are rare, their frequent role and in proximity to protein–ligand/protein interactions and presence at hydrophobic interfaces makes them useful probes in PrOF assays. 60 − 63 We speculate too that the reactivity observed here with typically inaccessible sites reflects both the lack of bulk (no ‘linker’/ “zero size”) and prior observations that biphasic/interfacial regions enhance small molecule radical reactions.…”