Journal
CHEMISTRY-A EUROPEAN JOURNAL
Volume 27, Issue 65, Pages 16145-16152Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.202102495
Keywords
lanthanoid binding tags; NMR spectroscopy; paramagnetic effects; protein modifications; protein structures
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Funding
- National Natural Science Foundation of China [21991081, 21273121, 21473095]
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This study demonstrates two rigid, reactive, and stable paramagnetic tags for protein modification, which exhibit high reactivity and rigidity for high-resolution NMR measurements of biological macromolecules and their complexes.
Site specific installation of a paramagnetic ion with magnetic anisotropy in a biomolecule generates valuable structural restraints, such as pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs). These paramagnetic effects can be used to characterize the structures, interactions and dynamics of biological macromolecules and their complexes. Two single-armed DOTA-like tags, BrPSPy-DO3M(S)A-Ln and BrPSPy-6M-DO3M(S)A-Ln, each containing a thiol-specific reacting group, that is, a phenylsulfonyl pyridine moiety, are demonstrated as rigid, reactive and stable paramagnetic tags for protein modification by formation of a reducing resistant thioether bond between the protein and the tag. The two tags present high reactivity with the solvent exposed thiol group in aqueous solution at room temperature. The introduction of Br at the meta-position in pyridine enhances the reactivity of 4-phenylsulfonyl pyridine towards the solvent exposed thiol group in a protein, whereas the ortho-methyl group in pyridine increases the rigidity of the tag in the protein conjugates. The high performance of these two tags has been demonstrated in different cysteine mutants of ubiquitin and GB1. The high reactivity and rigidity of these two tags can be added in the toolbox of paramagnetic tags suitable for the high-resolution NMR measurements of biological macromolecules and their complexes.
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