title: 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.
authors.affiliation: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Collaborative Innovation Center of Chemistry for Life Sciences, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, 430071, Hubei, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Wen-Xue
authors.initials: WX
authors.lastname: Jiang
authors.affiliation: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Collaborative Innovation Center of Chemistry for Life Sciences, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, 430071, Hubei, China.
authors.forename: Xin-Hua
authors.initials: XH
authors.lastname: Gu
authors.affiliation: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Collaborative Innovation Center of Chemistry for Life Sciences, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, 430071, Hubei, China. dongxu@wipm.ac.cn.
authors.forename: Xu
authors.initials: X
authors.lastname: Dong
authors.affiliation: CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Collaborative Innovation Center of Chemistry for Life Sciences, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, 430071, Hubei, China. tanglab@wipm.ac.cn.|University of Chinese Academy of Sciences, Beijing, 100049, China. tanglab@wipm.ac.cn.
authors.forename: Chun
authors.identifier: http://orcid.org/0000-0001-6477-6500
authors.initials: C
authors.lastname: Tang
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001386:Azides / Q000737:chemistry; D001426:Bacterial Proteins; D061565:Cycloaddition Reaction / Q000379:methods; D006801:Humans; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D004369:Pentetic Acid / Q000031:analogs & derivatives; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D013113:Spin Labels; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D000480:Alkynes; D000596:Amino Acids; D001386:Azides; D001426:Bacterial Proteins; D028581:Lanthanoid Series Elements; D011506:Proteins; D013113:Spin Labels; D025801:Ubiquitin; C060068:diethylenetriaminetetraacetic acid; D004369:Pentetic Acid; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System
keywords: Azide-alkyne cycloaddition; Protein structure; Pseudo-contact shift; Transient protein complex; Unnatural amino acid