title: Engineering [Ln(DPA)3] 3- binding sites in proteins: a widely applicable method for tagging proteins with lanthanide ions.
abstract: Paramagnetic relaxation enhancements from unpaired electrons observed in nuclear magnetic resonance (NMR) spectra present powerful long-range distance restraints. The most frequently used paramagnetic tags, however, are tethered to the protein via disulfide bonds, requiring proteins with single cysteine residues for covalent attachment. Here we present a straightforward strategy to tag proteins site-specifically with paramagnetic lanthanides without a tether and independent of cysteine residues. It relies on preferential binding of the complex between three dipicolinic acid molecules (DPA) and a lanthanide ion (Ln(3+)), [Ln(DPA)(3)](3-), to a pair of positively charged amino acids whose charges are not compensated by negatively charged residues nearby. This situation rarely occurs in wild-type proteins, allowing the creation of specific binding sites simply by introduction of positively charged residues that are positioned far from glutamate or aspartate residues. The concept is demonstrated with the hnRNPLL RRM1 domain. In addition, we show that histidine- and arginine-tags present binding sites for [Ln(DPA)(3)](3-).
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia.
authors.forename: Xinying
authors.initials: X
authors.lastname: Jia
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Yagi
authors.forename: Xun-Cheng
authors.initials: XC
authors.lastname: Su
authors.forename: Mitchell
authors.initials: M
authors.lastname: Stanton-Cook
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
mesh_terms: D000818:Animals; D001665:Binding Sites; D029968:Escherichia coli Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D007553:Isotope Labeling / Q000379:methods*; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010848:Picolinic Acids / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D028581:Lanthanoid Series Elements; D010848:Picolinic Acids; D011506:Proteins; C004860:dipicolinic acid