Optimizing ¹⁹F NMR protein spectroscopy by fractional biosynthetic labeling.
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.
Kitevski-LeBlanc
Julianne L
JL
Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd., North Mississauga, ON L5L1C6, Canada.
Evanics
Ferenc
F
Scott Prosser
R
R
D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D017611:Fluorine Compounds / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D056004:Molecular Dynamics Simulation; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D055550:Protein Stability; D029867:Xenopus Proteins / Q000737:chemistry / Q000378:metabolism
D002147:Calmodulin; D017611:Fluorine Compounds; D009587:Nitrogen Isotopes; D029867:Xenopus Proteins; C027218:3-fluorophenylalanine; D010649:Phenylalanine