title: A mutagenesis-free approach to assignment of (19)F NMR resonances in biosynthetically labeled proteins.
abstract: Solution NMR studies of protein structure and dynamics using fluorinated amino acid probes are a valuable addition to the repertoire of existing (13)C, (15)N, and (1)H experiments. Despite the numerous advantages of the (19)F nucleus in NMR, protein studies are complicated by the dependence of resonance assignments on site-directed mutagenesis methods which are laborious and often problematic. Here we report an NMR-based route to the assignment of fluorine resonances in (13)C,(15)N-3-fluoro-l-tyrosine labeled calmodulin. The assignment begins with the correlation of the fluorine nucleus to the delta proton in the novel (13)C,(15)N-enriched probe which is achieved using a CT-HCCF-COSY experiment. Connection to the backbone is made through two additional solution NMR experiments, namely the (H(beta))C(beta)(C(gamma)C(delta))H(delta) and HNCACB. Assignments are completed using either previously published backbone chemical shift data or obtained experimentally provided uniform (13)C,(15)N labeling procedures are employed during protein expression. Additional benefits of the (13)C,(15)N-3-fluoro-l-tyrosine probe include the reduction of spectral overlap through ((13)C(19)F) CT-HSQCs, as well as the ability to monitor side chain dynamics using (19)F T(1), T(2), and the (13)C-(19)F NOE.
authors.affiliation: Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada.
authors.forename: Julianne L
authors.initials: JL
authors.lastname: Kitevski-LeBlanc
authors.forename: M Sameer
authors.initials: MS
authors.lastname: Al-Abdul-Wahid
authors.forename: R Scott
authors.initials: RS
authors.lastname: Prosser
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000737:chemistry*; D005461:Fluorine; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D012996:Solutions; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry; D014982:Xenopus laevis
chemical_list: D002147:Calmodulin; D012996:Solutions; C001245:3-fluorotyrosine; D005461:Fluorine; D014443:Tyrosine