Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins

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• 作者：Choy Theng Loh ; Kiyoshi Ozawa ; Kellie L. Tuck ; Nicholas Barlow ; Thomas Huber ; Gottfried Otting ; Bim Graham
• 刊名：Bioconjugate Chemistry
• 出版年：2013
• 出版时间：February 20, 2013
• 年：2013
• 卷：24
• 期：2
• 页码：260-268
• 全文大小：390K
• 年卷期：v.24,no.2(February 20, 2013)
• ISSN：1520-4812

文摘

Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclen-based paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide鈥揳lkyne cycloaddition to a genetically encoded p-azido-l-phenylalanine residue with a tether that proved sufficiently short and rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies.