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 delta proton. (19)F-edited NOESY connectivities between the delta protons and alpha and beta 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.
Kitevski-Leblanc
Julianne L
JL
Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd. North, Mississauga, ON, L5L 1C6, Canada.
Evanics
Ferenc
F
Scott Prosser
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D005461:Fluorine / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D029867:Xenopus Proteins / Q000737:chemistry / Q000378:metabolism
D002147:Calmodulin; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D012996:Solutions; D029867:Xenopus Proteins; D005461:Fluorine; D014443:Tyrosine; C027218:3-fluorophenylalanine; D010649:Phenylalanine; D014357:Trypsin
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