caa a22 4500 445325860 CHVBK 20180317142720.0 cr unu---uuuuu 170323e20110801xx s 000 0 eng 10.1007/s10858-011-9529-x doi (NATIONALLICENCE)springer-10.1007/s10858-011-9529-x Engineering [Ln(DPA)3]3− binding sites in proteins: a widely applicable method for tagging proteins with lanthanide ions [Elektronische Daten] [Xinying Jia, Hiromasa Yagi, Xun-Cheng Su, Mitchell Stanton-Cook, Thomas Huber, Gottfried Otting] 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 (Ln3+), [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−. Springer Science+Business Media B.V., 2011 Lanthanide tag nationallicence NMR spectroscopy nationallicence Paramagnetic relaxation enhancements nationallicence RRM domain nationallicence [Ln(DPA)3]3− nationallicence Jia Xinying Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut Yagi Hiromasa Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut Su Xun-Cheng Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut Stanton-Cook Mitchell Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut Huber Thomas Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut Otting Gottfried Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut Journal of Biomolecular NMR Springer Netherlands 50/4(2011-08-01), 411-420 0925-2738 50:4<411 2011 50 10858 https://doi.org/10.1007/s10858-011-9529-x text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9529-x text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Jia Xinying Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut NATIONALLICENCE 700 1- Yagi Hiromasa Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut NATIONALLICENCE 700 1- Su Xun-Cheng Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut NATIONALLICENCE 700 1- Stanton-Cook Mitchell Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut NATIONALLICENCE 700 1- Huber Thomas Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut NATIONALLICENCE 700 1- Otting Gottfried Research School of Chemistry, Australian National University, 0200, Canberra, ACT, Australia aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 50/4(2011-08-01), 411-420 0925-2738 50:4<411 2011 50 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer