期刊
JOURNAL OF BIOMOLECULAR NMR
卷 48, 期 2, 页码 113-121出版社
SPRINGER
DOI: 10.1007/s10858-010-9443-7
关键词
F-19 NMR; Calmodulin; Fractional labeling; Biosynthetic labeling; 3-fluorophenylalanine
资金
- Natural Sciences and Engineering Research Council of Canada (NSERC) [261980]
- CIHR
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, F-19 and H-1-N-15 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 F-19 and H-1-N-15 HSQC spectra,N-15 T-1 and T-2 relaxation measurements, F-19 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% F-19 labeled samples, F-19 and H-1-N-15 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 N-15 dynamics. In this study, reasonable signal to noise can be achieved between 60-76% F-19 enrichment, without any detectable perturbations from labeling.
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