Approaches to the assignment of 19 F resonances from 3-fluorophenylalanine labeled calmodulin using solution state NMR

Traditional single site replacement mutations (in this case, phenylalanine to tyrosine) were compared with methods which exclusively employ 15 N and 19 F-edited two- and three-dimensional NMR experiments for purposes of assigning 19 F 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 1 H- 15 N HSQC spectra and calcium binding assays in the presence and absence of 3-FPhe. The 19 F 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 19 F NMR spectra with significant chemical shift perturbations of the remaining resonances, and provided only a single definitive assignment. Although 1 H- 19 F heteronucleclear NOEs proved weak, 19 F-edited 1 H- 1 H NOESY connectivities were relatively easy to establish by making use of the 3 J FH coupling between the fluorine nucleus and the adjacent fluorophenylalanine δ proton. 19 F-edited NOESY connectivities between the δ protons and α and β nuclei in addition to 15 N-edited 1 H, 1 H NOESY crosspeaks proved sufficient to assign 4 of 8 19 F 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 19 F NMR resonances. Our studies suggest that 19 F-edited NOESY NMR spectra are generally adequate for complete assignment without the need to resort to mutational analysis.