title: Lanthanide tags for site-specific ligation to an unnatural amino acid and generation of pseudocontact shifts in proteins.
abstract: Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclen-based paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide-alkyne cycloaddition to a genetically encoded p-azido-l-phenylalanine residue with a tether that proved sufficiently short and rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies.
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
authors.forename: Choy Theng
authors.initials: CT
authors.lastname: Loh
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ozawa
authors.forename: Kellie L
authors.initials: KL
authors.lastname: Tuck
authors.forename: Nicholas
authors.initials: N
authors.lastname: Barlow
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
authors.forename: Bim
authors.initials: B
authors.lastname: Graham
mesh_terms: D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D002384:Catalysis; D057930:Click Chemistry; D003300:Copper / Q000737:chemistry; D000080027:Cyclams; D061565:Cycloaddition Reaction; D004926:Escherichia coli / Q000235:genetics; D006571:Heterocyclic Compounds / Q000737:chemistry; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000235:genetics; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics
chemical_list: D000480:Alkynes; D001386:Azides; D000080027:Cyclams; D006571:Heterocyclic Compounds; C471213:His-His-His-His-His-His; D028581:Lanthanoid Series Elements; D009842:Oligopeptides; D011506:Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D010649:Phenylalanine; D006639:Histidine; D003300:Copper; C038072:cyclen