Abstract Traditional single site replacement mutations (in this case, phenylalanine to tyrosine) were compared with methods which exclusively employ 15N and 19F-edited two- and three-dimensional NMR experiments for purposes of assigning 19F NMR resonances from calmodulin (CaM), biosynthetically labeled with 3-fluorophenylalanine (3-FPhe). The global substitution of 3-FPhe for native phenylalanine was tolerated in CaM as evidenced by a comparison of 1H-15N HSQC spectra and calcium binding assays in the presence and absence of 3-FPhe. The 19F NMR spectrum reveals six resolved resonances, one of which integrates to three 3-FPhe species, making for a total of eight fluorophenylalanines. Single phenylalanine to tyrosine mutants of five phenylalanine positions resulted in 19F NMR spectra with significant chemical shift perturbations of the remaining resonances, and provided only a single definitive assignment. Although 1H-19F heteronucleclear NOEs proved weak, 19F-edited 1H-1H NOESY connectivities were relatively easy to establish by making use of the 3JFH coupling between the fluorine nucleus and the adjacent fluorophenylalanine δ proton. 19F-edited NOESY connectivities between the δ protons and α and β nuclei in addition to 15N-edited 1H, 1H NOESY crosspeaks proved sufficient to assign 4 of 8 19F resonances. Controlled cleavage of the protein into two fragments using trypsin, and a repetition of the above 2D and 3D techniques resulted in unambiguous assignments of all 8 19F NMR resonances. Our studies suggest that 19F-edited NOESY NMR spectra are generally adequate for complete assignment without the need to resort to mutational analysis.
- Content Type Journal Article
- DOI 10.1007/s10858-010-9415-y
- Authors
- Julianne L. Kitevski-LeBlanc, University of Toronto Department of Chemistry UTM, 3359 Mississauga Rd. North Mississauga ON L5L 1C6 Canada
- Ferenc Evanics, University of Toronto Department of Chemistry UTM, 3359 Mississauga Rd. North Mississauga ON L5L 1C6 Canada
- R. Scott Prosser, University of Toronto Department of Chemistry UTM, 3359 Mississauga Rd. North Mississauga ON L5L 1C6 Canada
Source: Journal of Biomolecular NMR