Abstract
The study of intrinsically disordered proteins (IDPs) by NMR often suffers from highly overlapped resonances that prevent unambiguous chemical‐shift assignments, and data analysis that relies on well‐separated resonances. We present a covalent paramagnetic lanthanide‐binding tag (LBT) for increasing the chemical‐shift dispersion and facilitating the chemical‐shift assignment of challenging, repeat‐containing IDPs. Linkage of the DOTA‐based LBT to a cysteine residue induces pseudo‐contact shifts (PCS) for resonances more than 20 residues from the spin‐labeling site. This leads to increased chemical‐shift dispersion and decreased signal overlap, thereby greatly facilitating chemical‐shift assignment. This approach is applicable to IDPs of varying sizes and complexity, and is particularly helpful for repeat‐containing IDPs and low‐complexity regions. This results in improved efficiency for IDP analysis and binding studies.
Highlights
Introduction of the YbM8 tag is a general approach that can alleviate the problem of chemical-shift degeneracy in intrinsically disordered proteins (IDPs), and in repeat regions in particular
The study of intrinsically disordered proteins (IDPs) by NMR often suffers from highly overlapped resonances that prevent unambiguous chemical-shift assignments, and data analysis that relies on well-separated resonances
We present a covalent paramagnetic lanthanide-binding tag (LBT) for increasing the chemical-shift dispersion and facilitating the chemical-shift assignment of challenging, repeat-containing IDPs
Summary
Introduction of the YbM8 tag is a general approach that can alleviate the problem of chemical-shift degeneracy in IDPs, and in repeat regions in particular. We present a covalent paramagnetic lanthanide-binding tag (LBT) for increasing the chemical-shift dispersion and facilitating the chemical-shift assignment of challenging, repeat-containing IDPs. Linkage of the DOTA-based LBT to a cysteine residue induces pseudo-contact shifts (PCS) for resonances more than 20 residues from the spin-labeling site. NMR spectroscopy is a well-suited method for studying the residual structure, dynamics, and interactions of IDPs with atomic resolution under near-native conditions.[4] The key step for these studies is the assignment of NMR resonances, which is challenging for IDPs owing to poor chemical-shift dispersion, which results in severe spectral overlap.
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