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 T1 and T2 relaxation measurements, 19F T2 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 60â??76% 19F enrichment, without any detectable perturbations from labeling.
Content Type Journal Article
DOI 10.1007/s10858-010-9443-7
Authors
Julianne L. Kitevski-LeBlanc, Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd., North Mississauga, ON L5L 1C6, Canada
Ferenc Evanics, Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd., North Mississauga, ON L5L 1C6, Canada
R. Scott Prosser, Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd., North Mississauga, ON L5L 1C6, Canada
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Optimizing 19F NMR protein spectroscopy by fractional biosynthetic labeling
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