Pseudocontact Shifts in Biomolecular NMR Spectroscopy

Abstract

Paramagnetic centers in biomolecules, such as specific metal ions that are bound to a protein, affect the nuclei in their surrounding in various ways. One of these effects is the pseudocontact shift (PCS), which leads to strong chemical shift perturbations of nuclear spins, with a remarkably long range of 50 Å and beyond. The PCS in solution NMR is an effect originating from the anisotropic part of the dipole–dipole interaction between the magnetic momentum of unpaired electrons and nuclear spins. The PCS contains spatial information that can be exploited in multiple ways to characterize structure, function, and dynamics of biomacromolecules. It can be used to refine structures, magnify effects of dynamics, help resonance assignments, allows for an intermolecular positioning system, and gives structural information in sensitivity-limited situations where all other methods fail. Here, we review applications of the PCS in biomolecular solution NMR spectroscopy, starting from early works on natural metalloproteins, following the development of non-natural tags to chelate and attach lanthanoid ions to any biomolecular target to advanced applications on large biomolecular complexes and inside living cells. We thus hope to not only highlight past applications but also shed light on the tremendous potential the PCS has in structural biology.

Localising nuclear spins by pseudocontact shifts from a single tagging site

“…The ability to work with a single site for tagging can thus be of critical importance, aside from the convenience of working with a single protein construct rather than multiple mutants. In a fortuitous development, an increasing number of different lanthanoid tags have recently been published that are designed for attachment to cysteine residues and deliver different χ tensor orientations (Miao et al, 2022;Müntener et al, 2022). The present work shows that different enantiomers of the same tag can produce quite different χ tensor orientations.…”

“…Most of the tags designed for the generation of PCSs in proteins are based on complexes of lanthanoid ions that can be attached to single cysteine residues. Many variants of such single-arm tags have been produced with the aim of generating large PCSs, avoiding multiple tag conformations, producing stable bonds with cysteines, and keeping structural perturbations of the tagged proteins to a minimum (Nitsche and Otting, 2017;Joss and Häussinger, 2019;Su and Chen, 2019;Miao et al, 2022;Müntener et al, 2022). As shown in the present work, the availability of a variety of tags turns out to be of great benefit for structure determinations of specific sites of interest, as they allow the generation of χ tensors of different relative orientations with a single tagging site.…”

“…Pseudocontact shifts (PCSs), which are generated by paramagnetic metal ions with fast-relaxing electron spins, provide outstanding restraints for the structure refinement of biological macromolecules (Luchinat et al, 2018). To make full use of PCS restraints, a large number of metal tags have been developed in recent years with the express purpose to install a paramagnetic centre on proteins, measure PCSs, and gain structural information (Liu et al, 2014;Nitsche and Otting, 2017;Su and Chen, 2019;Joss and Häussinger, 2019;Saio and Ishimori, 2020;Miao et al, 2022;Müntener et al, 2022). With restraints from multiple tagging sites, PCSs of backbone amide protons have been shown to be sufficient for 3D structure determinations of proteins (Schmitz et al, 2012;Yagi et al, 2013;Pilla et al, 2016Pilla et al, , 2017Cucuzza et al, 2021).…”

Localising nuclear spins by pseudocontact shifts from a single tagging site

“…The ability to work with a single site for tagging can thus be of criticial importance, aside from the convenience of working with a single protein construct rather than multiple mutants. In a fortuitous development, an increasing number of different lanthanoid tags have recently been published that are designed for attachment to cysteine residues and deliver different ∆χ-tensor orientations (Miao et al, 2022;Müntener et al, 2022). The present work shows that different enantiomers of the same tag can produce quite different ∆χ-tensor orientations.…”

“…Most of the tags designed for the generation of PCSs in proteins are based on complexes of lanthanoid ions that can be attached to single cysteine residues. Many variants of such single-arm tags have been produced with the aim to generate large PCSs, avoid multiple tag conformations, produce stable bonds with cysteines and keep structural perturbations of the tagged proteins to a minimum (Nitsche et al, 2017;Joss et al, 2019;Su et al, 2019;Miao et al, 2022;Müntener et al, 2022). As shown in the present work, the availability of a variety of tags turns out to be of great benefit for structure determinations of specific sites of interest, as they allow generating ∆χ tensors of different relative orientations with a single tagging site.…”