Localising nuclear spins by pseudocontact shifts from a single tagging site

Abstract

Abstract. Ligating a protein at a specific site with a tag molecule containing a paramagnetic metal ion provides a versatile way of generating pseudocontact shifts (PCS) in nuclear magnetic resonance (NMR) spectra. PCSs can be observed for nuclear spins far from the tagging site and PCSs generated from multiple tagging sites have been shown to enable highly accurate structure determinations. The present work investigates the situation, where only the local structure of a protein region or bound ligand is to be determined rather than the structure of the entire molecular system. In this case, the need for gathering structural information from tags deployed at multiple sites may be queried. Our study presents a computational simulation of the structural information available from samples produced with single tags attached at up to six different sites, up to six different tags attached to a single site, as well as scenarios in-between. The results indicate that the number of tags is much more important than the number of tagging sites. This has important practical implications, as it is much easier to identify a single site that is suitable for tagging than multiple ones. In an initial experimental demonstration with the ubiquitin mutant S57C, PCSs generated with four different tags at a single site are shown to accurately pinpoint the location of amide protons in different segments of the protein.

Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine

“…Chemical assembly of lanthanoid tags on cysteine residues has been proposed previously using pnictogens as mediator between the thiol groups of cysteine and tag, but the pnictogen system depends on a carefully constructed di-cysteine motif, where both cysteine residues need to present their thiol groups in a suitable geometry, as well as assistance by other amino acid side chains of the protein to immobilise the lanthanoid ion sufficiently to enable PCS measurements (Nitsche et al, 2017). The approach of the present work is much more straightforward and general, and it enables the construction of a series of different tags that produce different χ tensors as required for high-resolution structure analysis of specific sites of interest in a protein (Orton et al, 2022).…”

“…Regarding PCSs, the present work established a new set of tags that are inexpensive, small and relatively rigid, suitable for installation on a highly solvent-exposed cysteine thiol group and capable of delivering χ tensor fits with good quality factors. Furthermore, constructing the tags with cysteine or penicillamine generated different PCSs and χ tensors of different orientations, as required for site-specific structure analysis by PCSs (Orton et al, 2022). The titrations with paramagnetic lanthanoid ions tended to result in broader NMR signals of the paramagnetic species before the complete disappearance of the signals of the free protein.…”

“…The present study shows that significantly different χtensor orientations can be obtained by transforming the DCP tags with 1,2-aminothiols of different chirality and with different chemical groups. DCP-tag assembly thus establishes a highly attractive concept for producing the multiple different tensor orientations required to determine local protein structure by multiple PCSs (Orton et al, 2022) or analyse protein motions by RDCs (Tolman et al, 2001;Peti et al, 2002;Vögeli et al, 2008). Although FDCP is currently not yet commercially available, its synthesis is straightforward and inexpensive.…”

“…Furthermore, the z axes of the tensors obtained with the different tags diverted significantly, with the closest alignment (observed between the DCP-(D-Cys) 2 and DCP-(L-pen) 2 tags) featuring an angle of 11 • between the respective z axes. The family of tags of the present work thus presents a good basis for generating largely independent χ tensors, as required for obtaining structural in-formation from multiple tags attached to a single site (Orton et al, 2022).…”