title: A systematic study of labelling an α-helix in a protein with a lanthanide using IDA-SH or NTA-SH tags.
abstract: The previously published IDA-SH and NTA-SH tags are small synthetic lanthanide-binding tags derived from cysteine, which afford site-specific lanthanide labelling by disulfide-bond formation with a cysteine residue of the target protein. Following attachment to a single cysteine in an α-helix, sizeable pseudocontact shifts (PCS) can be observed, if the lanthanide is immobilized by additional coordination to a negatively charged amino-acid side chain that is located in a neighboring turn of the helix. To identify the best labelling strategy for PCS measurements, we performed a systematic study, where IDA-SH or NTA-SH tags were ligated to a cysteine residue in position i of an α-helix, and aspartate or glutamate residues were placed in the positions i - 4 or i + 4. The largest anisotropy components of the magnetic susceptibility tensor were observed for an NTA-SH tag in position i with a glutamate residue in position i - 4. While the NTA-SH tag produced sizeable PCSs regardless of the presence of nearby carboxyl groups of the protein, the IDA-SH tag generated a good lanthanide binding site only if an aspartate was placed in position i + 4. The findings provide a firm basis for the design of site-directed mutants that are suitable for the reliable generation of PCSs in proteins with paramagnetic lanthanides.
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 0200, Australia.
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Yagi
authors.forename: Ansis
authors.initials: A
authors.lastname: Maleckis
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
mesh_terms: D016880:Anisotropy; D003545:Cysteine / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: C110909:Erp29 protein, rat; D006360:Heat-Shock Proteins; D028581:Lanthanoid Series Elements; D011506:Proteins; D003545:Cysteine