Generation of Pseudocontact Shifts in Proteins with Lanthanides Using Small “Clickable” Nitrilotriacetic Acid and Iminodiacetic Acid Tags |
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| Authors: | Dr Choy‐Theng Loh Dr Bim Graham Elwy H Abdelkader Dr Kellie L Tuck Prof Gottfried Otting |
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| Institution: | 1. Research School of Chemistry, Australian National University, Canberra ACT 2601 (Australia);2. Monash Institute of Pharmaceutical Sciences, Monash University, Clayton VIC 3800 (Australia);3. School of Chemistry, Monash University, Clayton VIC 3800 (Australia) |
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| 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 CuI‐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: | click chemistry lanthanide‐binding tags NMR spectroscopy p‐azido‐L‐phenylalanine pseudocontact shifts |
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