A Mutagenesis-Free Approach to Assignment of ¹⁹F 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.

Recent advances in the application of solution NMR spectroscopy to multi-span integral membrane proteins

“…PCS-induced shifts in resonance positions are sometimes so large that resonance assignments originally made using diamagnetic samples cannot be applied to the PCS-affected spectra necessitating extensive resonance reassignment. When utilizing a probe for inducing PCS, one avenue for simplifying spectra is to incorporate 19 F sites, either by expressing the protein in cell growth media containing fluorinated amino acids or by chemically modifying free Cys-SH positions using thiol-reactive fluorinated reagents [241, 244, 246, 248-252]. The sparse 19 F NMR spectra are then recorded under both paramagnetic and diamagnetic conditions.…”

Fluorine-19 NMR of integral membrane proteins illustrated with studies of GPCRs

“…Heteronuclear and homonuclear two- and three-dimensional experiments with 19 F have been applied also with soluble proteins, but few applications have been described with membrane proteins, probably mainly because of limited sensitivity [20 • ,25,26,27,28,29]. …”

“…Auxotrophic bacterial strains are available, and fluorinated aromatic amino acid analogs, such as m-monofluorotyrosine, 4-, 5- and 6- monofluorotryptophan, and o-, m- and p-monofluorophenylalanine are commercial reagents, which makes the labeling process quite efficient [37]. 13 C- and 15 N-enriched fluorinated amino acid analogs have been used to reduce spectral overlap in 19 F-detected multidimensional heteronuclear correlation NMR experiments, and to obtain NMR-based assignments of the 19 F signals [28,29,38]. Detailed biosynthetic labeling protocols can be found in earlier reviews [3,37].…”

19F-Modified Proteins and 19F-Containing Ligands as Tools in Solution NMR Studies of Protein Interactions

“…Unfortunately, this method can fail, if the substitutions result in structural perturbations that alter the resonance frequencies of the remaining resonances. Two alternative approaches have been proposed and applied: (i) mutagenesis of a residue near the residue to be assigned (Drake, Bourret, Luck, Simon, & Falke, 1993) and (ii) NMR-based assignments that combine side-chain correlation experiments with traditional backbone assignments (Kitevski-Leblanc et al, 2009). The latter strategy relies on the availability of the protein structure or prior backbone 1 H, 13 C, 15 N resonance assignments.…”