title: DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra.
abstract: Structural studies of proteins and protein-ligand complexes by nuclear magnetic resonance (NMR) spectroscopy can be greatly enhanced by site-specific attachment of lanthanide ions to create paramagnetic centers. In particular, pseudocontact shifts (PCS) generated by paramagnetic lanthanides contain important and unique long-range structure information. Here, we present a high-affinity lanthanide binding tag that can be attached to single cysteine residues of proteins. The new tag has many advantageous features that are not available in this combination from previously published tags: (i) it binds lanthanide ions very tightly, minimizing the generation of nonspecific effects, (ii) it produces PCSs with high reliability as its bulkiness prevents complete motional averaging of PCSs, (iii) it can be attached to single cysteine residues, alleviating the need of detailed prior knowledge of the 3D structure of the target protein, and (iv) it does not display conformational exchange phenomena that would increase the number of signals in the NMR spectrum. The performance of the tag is demonstrated with the N-terminal domain of the E. coli arginine repressor and the A28C mutant of human ubiquitin.
authors.affiliation: Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Parkville, VIC 3052, Australia.
authors.forename: Bim
authors.initials: B
authors.lastname: Graham
authors.forename: Choy Theng
authors.initials: CT
authors.lastname: Loh
authors.forename: James David
authors.initials: JD
authors.lastname: Swarbrick
authors.forename: Phuc
authors.initials: P
authors.lastname: Ung
authors.forename: James
authors.initials: J
authors.lastname: Shin
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Yagi
authors.forename: Xinying
authors.initials: X
authors.lastname: Jia
authors.forename: Sandeep
authors.initials: S
authors.lastname: Chhabra
authors.forename: Nicholas
authors.initials: N
authors.lastname: Barlow
authors.forename: Guido
authors.initials: G
authors.lastname: Pintacuda
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
mesh_terms: D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006801:Humans; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012097:Repressor Proteins / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics
chemical_list: C490177:ArgR protein, E coli; D029968:Escherichia coli Proteins; D006573:Heterocyclic Compounds, 1-Ring; D028581:Lanthanoid Series Elements; D011506:Proteins; D012097:Repressor Proteins; D025801:Ubiquitin; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D003545:Cysteine