Lanthanide-binding tags (LBTs) are peptide sequences of up to 20 encoded amino acids that tightly and selectively complex lanthanide ions and can sensitize terbium (Tb3+) luminescence. On the basis of these properties, it was predicted that increasing the number of bound lanthanides would improve the capabilities of these tags. Therefore, using a structurally well-characterized single-LBT sequence as a starting point, a "double-LBT" (dLBT), which concatenates two lanthanide-binding motifs, was designed. Herein we report the generation of dLBT peptides and luminescence and NMR studies on a dLBT-tagged ubiquitin fusion protein. These lanthanide-bound constructs are shown to be improved luminescent tags with avid lanthanide binding and up to 3-fold greater luminescence intensity. NMR experiments were conducted on the ubiquitin construct, wherein bound paramagnetic lanthanides were used as alignment-inducing agents to gain residual dipolar couplings, which are valuable restraints for macromolecular structure determination. Together, these results indicate that dLBTs will be valuable chemical tools for biophysical applications leading to new approaches for studying the structure, function, and dynamics of proteins.
"We have shown how to characterize the conformational space sampled by two -domain proteins in a correct quantitative way, by the calculation of the Maximum Occurrence (MO) parameter, which is the maximum percent of time that the system can spend in a given conformation (Longinetti et al., 2006; Bertini et al., 2007; Bertini et al., 2010; Bertini et al., 2011b). This method relies on the use of pseudocontact shifts (pcs) and self-orientation residual dipolar couplings (rdc), originating from a paramagnetic center either bound in a metal binding site or introduced by covalent tagging (Ikegami et al., 2004; Martin et al., 2007; Keizers et al., 2008; Häussinger et al., 2009; Hass et al., 2010; Su and Otting, 2010; Das Gupta et al., 2011), as well as of small angle x-ray scattering (SAXS) data (Petoukhov and Svergun, 2007; Bertini et al., 2009; Bernadó et al., 2010). "
[Show abstract][Hide abstract] ABSTRACT: The MaxOcc web portal is presented for the characterization of the conformational heterogeneity of two-domain proteins, through the calculation of the Maximum Occurrence that each protein conformation can have in agreement with experimental data. Whatever the real ensemble of conformations sampled by a protein, the weight of any conformation cannot exceed the calculated corresponding Maximum Occurrence value. The present portal allows users to compute these values using any combination of restraints like pseudocontact shifts, paramagnetism-based residual dipolar couplings, paramagnetic relaxation enhancements and small angle X-ray scattering profiles, given the 3D structure of the two domains as input. MaxOcc is embedded within the NMR grid services of the WeNMR project and is available via the WeNMR gateway at http://py-enmr.cerm.unifi.it/access/index/maxocc . It can be used freely upon registration to the grid with a digital certificate.
"For the application of paramagnetic lanthanide probes to non-metalloproteins, a wide variety of lanthanide-anchoring tags has been developed, including lanthanide binding peptide tags (Su et al. 2006, 2008a; Gaponenko et al. 2000; Wohnert et al. 2003; Martin et al. 2007; Ma and Opella 2000; Zhuang et al. 2008) and synthetic lanthanide chelating reagents (Dvoretsky et al. 2002; Haberz et al. 2006; Pintacuda et al. 2004; Prudencio et al. 2004; Rodriguez- Castaneda et al. 2006; Ikegami et al. 2004; Leonov et al. 2005; Gaponenko et al. 2002, 2004; Vlasie et al. 2007; Keizers et al. 2007, 2008; Su et al. 2008b; Swarbrick et al. 2011a, b; Graham et al. 2011). These tags are attached to the target proteins through N-or C-terminal fusion (Gaponenko et al. 2000; Wohnert et al. 2003; Martin et al. 2007; Ma and Opella 2000; Zhuang et al. 2008), insertion into the loop region (Barthelmes et al. 2011) or the formation of a disulfide bond with cysteine residues (Su et al. 2006, 2008a, b; Dvoretsky et al. 2002; Haberz et al. 2006; Pintacuda et al. 2004; Prudencio et al. 2004; Ikegami et al. 2004; Leonov et al. 2005; Gaponenko et al. 2002, 2004; Vlasie et al. 2007; Keizers et al. 2007, 2008; Swarbrick et al. 2011a, b; Graham et al. 2011). However, the mobility of the tag relative to the target protein reduces the anisotropic paramagnetic effect (Bertini et al. 2004, 2007; Su et al. 2008a). "
[Show abstract][Hide abstract] ABSTRACT: Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein–protein and protein–ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.
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The online version of this article (doi:10.1007/s10858-012-9623-8) contains supplementary material, which is available to authorized users.
"For example, lanthanide-binding peptides can be attached, or inserted into the sequence of a protein (Wöhnert et al. 2003; Nitz et al. 2004; Martin et al. 2007; Su et al. 2008a). The introduction of a lanthanide binding peptide into loop regions has been found to have a small effect on the overall structure of the protein (Barthelmes et al. 2011). "
[Show abstract][Hide abstract] ABSTRACT: Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large residual dipolar couplings and pseudocontact shifts that could be measured easily and agreed very well with the protein structure. We show that Cys-Ph-TAHA can be used to label large proteins that are biochemically challenging such as the Lac repressor in a 90 kDa ternary complex with DNA and inducer.
Electronic supplementary material
The online version of this article (doi:10.1007/s10858-011-9560-y) contains supplementary material, which is available to authorized users.
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