Generation of pseudocontact shifts in proteins with lanthanides using small “clickable” nitrilotriacetic acid and iminodiacetic acid tags
Date
2015
Authors
Loh, Choy-Theng
Graham, Bim
Abdelkader, Elwy
Tuck, Kellie L.
Otting, Gottfried
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Publisher
Wiley-VCH Verlag
Abstract
Pseudocontact shifts (PCS) induced by paramagnetic lanthanide ions provide unique long-range structural information in nuclear magnetic resonance (NMR) spectra, but the site-specific attachment of lanthanide tags to proteins remains a challenge. Here we incorporated p-azido-phenylalanine (AzF) site-specifically into the proteins ubiquitin and GB1, and ligated the AzF residue with alkyne derivatives of small nitrilotriacetic acid and iminodiacetic acid tags using the Cu(I) -catalysed "click" reaction. These tags form lanthanide complexes with no or only a small net charge and produced sizeable PCSs with paramagnetic lanthanide ions in all mutants tested. The PCSs were readily fitted by single magnetic susceptibility anisotropy tensors. Protein precipitation during the click reaction was greatly alleviated by the presence of 150 mM NaCl.
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Keywords
NMR spectroscopy, click chemistry, lanthanide-binding tags, p-azido-L-phenylalanine, pseudocontact shifts
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Source
Chemistry - A European Journal
Type
Journal article
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Open Access
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