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...

Artificial templates can control out-of-equilibrium self-assembly in an enzyme-mediated dynamic system of cyclodextrins, even allowing access to products not selected in Nature.