Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins

Abstract

Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclenbased paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide−alkyne cycloaddition to a genetically encoded p-azido-L-phenylalanine residue with a tether that proved sufficiently short and rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies.

Information content of long-range NMR data for the characterization of conformational heterogeneity

“…However, the presence of an anisotropic magnetic susceptibility also gives rise to pseudocontact shifts (PCS) (Kurland and McGarvey, 1970), which are reporters on the positions of the nuclei in the principal axis frame of the magnetic susceptibility tensor centered on the paramagnetic site, and therefore contain information about the structure/shape of a molecule. The use of paramagnetism-induced restraints (Gochin and Roder, 1995a; Gochin and Roder, 1995b; Banci et al, 1996; Banci et al, 1998; Bertini et al, 2001a; Gaponenko et al, 2004; Bertini et al, 2005; Diaz-Moreno et al, 2005; Jensen et al, 2006; Bertini et al, 2008; Schmitz et al, 2012; Yagi et al, 2013b) is becoming increasingly popular because of the introduction of lanthanide binding tags (Barthelmes et al, 2011; Wöhnert et al, 2003; Rodriguez-Castañeda et al, 2006; Su et al, 2006; John and Otting, 2007; Pintacuda et al, 2007; Zhuang et al, 2008; Su et al, 2008b; Su et al, 2008a; Keizers et al, 2008; Häussinger et al, 2009; Su and Otting, 2010; Hass et al, 2010; Man et al, 2010; Das Gupta et al, 2011; Saio et al, 2011; Swarbrick et al, 2011b; Swarbrick et al, 2011a; Bertini et al, 2012a; Liu et al, 2012; Kobashigawa et al, 2012; Cerofolini et al, 2013; Yagi et al, 2013a; Gempf et al, 2013; Loh et al, 2013), that extend the range of applications from paramagnetic metalloproteins (Banci et al, 1996; Banci et al, 1997) (or proteins in which the naturally occurring metal can be replaced by a paramagnetic one (Allegrozzi et al, 2000; Bertini et al, 2001a; Bertini et al, 2001c; Bertini et al, 2001b; Bertini et al, 2003; Bertini et al, 2004b; Balayssac et al, 2008; Bertini et al, 2010a; Luchinat et al, 2012b)) to, in principle, any protein.…”

FANTEN: a new web-based interface for the analysis of magnetic anisotropy-induced NMR data

“…The advantages offered by paramagnetic restraints are more and more exploited thanks to the development of paramagnetic tags that can be attached to diamagnetic proteins, so that PCSs and RDCs can be easily measured also for molecules without a natural metal binding site (Wöhnert et al 2003;Rodriguez-Castañeda et al 2006;Su et al 2006;Su et al 2008a;Keizers et al 2008;Su et al 2008b;Zhuang et al 2008;Häussinger et al 2009;Su and Otting 2010;Hass et al 2010;Man et al 2010;Das Gupta et al 2011;Liu et al 2012;Cerofolini et al 2013;Yagi et al 2013a;Gempf et al 2013;Kobashigawa et al 2012;Saio et al 2011;Watanabe et al 2010;Loh et al 2013;Swarbrick et al 2011aSwarbrick et al , 2011b.…”

Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA)

“…Large conformational spaces for the paramagnetic center lead to observed PRE measurements whose properties may be influenced by the flexibility of the probe itself. Rigid paramagnetic probes have been introduced into proteins to counter this problem (Liu et al 2014c; Loh et al 2013), but the location of anchoring cysteine residues in the protein limit their placement and their often-bulky structure can also affect native protein structure. The incorporation of unnatural amino acids (UAAs) addresses both the issues of bulk and local dynamics and allows for homogeneous protein samples.…”

“…Of the more than 100 unnatural amino acids incorporated successfully, a small fraction is useful for NMR. These include isotopically labeled p-methoxy-phenylalanine and its fluorinated analogs (Cellitti et al 2008; Deiters et al 2005), photocaged unnatural amino acids for site-specific labeling (Jones et al 2010), metal-chelating unnatural amino acids (Lee et al 2009; Loh et al 2013; Nguyen et al 2011), and an amino acid that ligates with a lanthanide tag (Liu and Schultz 2010). Incorporation of isotopically labeled amino acids at specific residues have helped identify ligand binding sites and conformational changes of large proteins (Deiters et al 2005).…”

“…Previous lanthanide tags could only be non-covalently bound to the N- or C- termini of a protein, resulting in flexibility of the tag and poor measurements. While the lanthanide-p-azido-L-phenylalanine (AzF) fusion allowed for placement of the tag independent of the location of other residues in the protein, there are limitations that hinder its widespread application (Loh et al 2013). The lanthanide tag is also limited by the need for known structural data for tether optimization.…”