Site-specific labelling of proteins with a rigid lanthanide-binding tag.
This paper describes a generic method for the site-specific attachment of lanthanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide-binding peptide tag to the single cysteine residue present in the N-terminal DNA-binding domain of the Escherichia coli arginine repressor. Complexes with Y(3+), Tb(3+), Dy(3+), Ho(3+), Er(3+), Tm(3+) and Yb(3+) ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide-binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisotropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long-range structure information in the determination of 3D protein structures by NMR spectroscopy.
Su
Xun-Cheng
XC
Research School of Chemistry, Australian National University Canberra, ACT 0200, Australia.
Huber
Thomas
T
Dixon
Nicholas E
NE
Otting
Gottfried
G
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004220:Disulfides; D028581:Lanthanoid Series Elements; D010455:Peptides; D011506:Proteins; D003545:Cysteine