Site‐Specific Labelling of Proteins with a Rigid Lanthanide‐Binding Tag

Su, Xun Cheng; Huber, Thomas; Dixon, Nicholas E.; Otting, Gottfried

doi:10.1002/cbic.200600142

Cited by 89 publications

(110 citation statements)

References 25 publications

Supporting

Mentioning

109

Contrasting

Order By: Relevance

“…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.…”

Section: Introductionmentioning

confidence: 98%

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

et al. 2014

View full text Add to dashboard Cite

Pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) arising from the presence of paramagnetic metal ions in proteins as well as RDCs due to partial orientation induced by external orienting media are nowadays routinely measured as a part of the NMR characterization of biologically relevant systems. PCSs and RDCs are becoming more and more popular as restraints (1) to determine and/or refine protein structures in solution, (2) to monitor the extent of conformational heterogeneity in systems composed of rigid domains which can reorient with respect to one another, and (3) to obtain structural information in protein-protein complexes. The use of both PCSs and RDCs proceeds through the determination of the anisotropy tensors which are at the origin of these NMR observables. A new user-friendly web tool, called FANTEN (Finding ANisotropy TENsors), has been developed for the determination of the anisotropy tensors related to PCSs and RDCs and has been made freely available through the WeNMR ( http://fanten-enmr.cerm.unifi.it:8080 ) gateway. The program has many new features not available in other existing programs, among which the possibility of a joint analysis of several sets of PCS and RDC data and the possibility to perform rigid body minimizations.

show abstract

Section: Introductionmentioning

confidence: 98%

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

et al. 2014

View full text Add to dashboard Cite

show abstract

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

View full text Add to dashboard Cite

Long-range NMR data, namely residual dipolar couplings (RDCs) from external alignment and paramagnetic data, are becoming increasingly popular for the characterization of conformational heterogeneity of multidomain biomacromolecules and protein complexes. The question addressed here is how much information is contained in these averaged data. We have analyzed and compared the information content of conformationally averaged RDCs caused by steric alignment and of both RDCs and pseudocontact shifts caused by paramagnetic alignment, and found that, despite the substantial differences, they contain a similar amount of information. Furthermore, using several synthetic tests we find that both sets of data are equally good towards recovering the major state(s) in conformational distributions.

show abstract

“…9 Lanthanides bind dipicolinic acid (DPA) with nanomolar affinity for the first DPA ligand and decreasing affinities for additional DPA ligands up to the [Ln(DPA) 3 ] 3-complex. 12 A protein derivatized with one molecule of 1 thus leaves the free coordination sites on any metal ion bound to 1 available for binding to additional ligands.…”

mentioning

confidence: 99%

A Dipicolinic Acid Tag for Rigid Lanthanide Tagging of Proteins and Paramagnetic NMR Spectroscopy

et al. 2008

Self Cite

View full text Add to dashboard Cite

Site-specific labeling of a protein with a lanthanide ion (Ln 3+ ) provides access to a wealth of paramagnetic effects that contain long-range structural information and can be measured by nuclear magnetic resonance (NMR) spectroscopy. 1 In particular, pseudocontact shifts (PCS) induced by lanthanides can be used to determine the structure of protein-protein and protein-ligand complexes rapidly from a minimum amount of NMR data 2 and the paramagnetically induced weak alignment of the protein in the magnetic field leads to residual dipolar couplings (RDCs) which contain detailed information about the structure and dynamics of proteins.3 Correspondingly, many efforts have been directed toward the development of tags for site-specific attachment of lanthanides to proteins, including fusions with lanthanide-binding peptides 4 and chemical derivatization of cysteine residues with synthetic peptides and lanthanide-chelating reagents. 5-10Several features must be considered in the design of suitable lanthanide tags. (i) Rigidity of the attachment, as PCS and RDCs are greatly reduced when the lanthanide tag reorientates with respect to the protein. 6,10 In addition, variation in the metal ion position prevents interpretation of the PCS by a single magnetic susceptibility anisotropy (∆ ) tensor. (ii) Enantiomeric purity, as metal ions can act as chiral centers and different enantiomers of the metalchelate would lead to diastereomeric protein-tag constructs, therefore doubling the number of NMR peaks. 5,7,11 (iii) Proximity of the metal ion to the protein, as accurate determination of the ∆ tensor requires adequate sampling of the space around the metal ion.10 (iv) Ease of use. For example, lanthanide tags that are anchored to the protein via two disulfide bonds immobilize the tag well with respect to the protein. This requires, however, two cysteine residues with thiol groups positioned at the correct distance to react with a single tag molecule.8 Here we describe a simple lanthanide tag and strategy for immobilization of a lanthanide close to the protein using a single cysteine residue.4-Mercaptomethyl-dipicolinic acid (4MMDPA, 1) coordinates metal ions in a nonchiral fashion and can be readily attached to a cysteine thiol group via a disulfide bridge using established dithionitrobenzoate chemistry.9 Lanthanides bind dipicolinic acid (DPA) with nanomolar affinity for the first DPA ligand and decreasing affinities for additional DPA ligands up to the [Ln(DPA) 3 ] 3-complex. 12 A protein derivatized with one molecule of 1 thus leaves the free coordination sites on any metal ion bound to 1 available for binding to additional ligands. In the case of lanthanides, carboxyl groups of the protein are particularly suitable ligands that can act as additional anchors for tethering the lanthanide ion to the protein. Simultaneous coordination of the lanthanide ion by 1 and one or several protein carboxyl groups provides an avenue for creating high-affinity lanthanide binding sites that immobilize the lanthanide ion with respect to t...

show abstract

Site‐Specific Labelling of Proteins with a Rigid Lanthanide‐Binding Tag

Cited by 89 publications

References 25 publications

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

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

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

A Dipicolinic Acid Tag for Rigid Lanthanide Tagging of Proteins and Paramagnetic NMR Spectroscopy

Contact Info

Product

Resources

About