Lanthanoid tagging via an unnatural amino acid for protein structure characterization
Abstract
Lanthanoid pseudo-contact shift (PCS) provides long-range structural information between a paramagnetic tag and protein nuclei. However, for proteins with native cysteines, site-specific attachment may only utilize functional groups orthogonal to sulfhydryl chemistry. Here we report two lanthanoid probes, DTTA-C3-yne and DTTA-C4-yne, which can be conjugated to an unnatural amino acid pAzF in the target protein via azide-alkyne cycloaddition. Demonstrated with ubiquitin and cysteine-containing enzyme EIIB, we show that large PCSs of distinct profiles can be generated for each tag/lanthanoid combination. The DTTA-based lanthanoid tags are associated with large magnetic susceptibility tensors owing to the rigidity of the tags. In particular, introduction of the DTTA-C3 tag affords intermolecular PCSs and enables structural characterization of a transient protein complex between ubiquitin and a UBA domain. Together, we have expanded the repertoire of paramagnetic tags and the applicability of paramagnetic NMR.
Source: Journal of Biomolecular NMR