A pH-Sensitive, Colorful, Lanthanide-Chelating Paramagnetic NMR Probe

Abstract

Paramagnetic lanthanides ions are broadly used in NMR spectroscopy. The effects of unpaired electrons on NMR spectral parameters provide a powerful tool for the characterization of macromolecular structures and dynamics. Here, a new lanthanide-chelating NMR probe, Caged Lanthanide NMR Probe-7 (CLaNP-7), is presented. It can be attached to protein surfaces via two disulfide bridges, yielding a probe that is rigid relative to the protein backbone. CLaNP-7 extends the application range of available probes. It has a yellow color, which is helpful for sample preparation. Its effects are comparable to those of CLaNP-5, but its charge is two units lower (+1) than that of CLaNP-5 (+3), reducing the change in surface potential after probe attachment. It also has a different magnetic susceptibility tensor, so by using both tags, two sets of structural restraints can be obtained per engineered cysteine pair. Moreover, it was found that the orientation of the magnetic susceptibility tensor is pH dependent (pK(a) ≈ 7) when a histidine residue is located in the neighborhood of the probe attachment site. The results show that the His imidazole group interacts with the CLaNP-7 tag. It is proposed that the histidine residue forms a hydrogen bond to a water/hydroxyl molecule that occupies the ninth coordination position on the lanthanide, thus breaking the two-fold symmetry of the CLaNP tag in a pH-dependent way.

Delicate conformational balance of the redox enzyme cytochrome P450cam

“…, respectively, using structures of both the open and closed states of P450cam. These values are in line with previously published values for CLaNP-7 (34). As expected, the position of the paramagnetic centers and the orientation of the Δχ tensor are also identical within error for both states.…”

“…In the structure of the closed state, the lanthanoid is positioned ∼8 Å from the Cα atoms of the two Cys residues (Fig. S3A), consistent with the results of other studies using CLaNP tags (34,(36)(37)(38). In contrast, in the fit of the PCS data to the structure of the open state, the paramagnetic center is placed far from the expected position, due to the moved G-helix (Fig.…”

“…The pET28a plasmid harboring the cDNA of P450cam C334A was used as a template for site-directed mutagenesis. As described in previous studies, the surface-exposed Cys residue at position 334 on P450cam was substituted to Ala to prevent dimerization (45), as well as interference with CLaNP tagging (34). Sitedirected mutagenesis was carried out using the Quik-Change protocol (Stratagene).…”

“…As a consequence, a nucleus experiences distinct distances to the paramagnetic center and positions in the Δχ tensor frame in the two states, and the ensuing differences in PCSs provide a means of differentiating between the two states. We used this approach by mutating residues E195 and A199 to Cys for attachment of the two-armed caged lanthanoid NMR probe-7 (CLaNP-7) (15,34). This mutation site is at the top of the G-helix, which undergoes a significant conformational change when the protein transitions from the closed state to the open state.…”

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

“…Due to the quadratic mathematical form of these restraints, more than one set of PCSs and RDCs are needed to remove degeneracies in the solutions (Dosset et al 2001;Longinetti et al 2002;Bertini et al 2002;Fragai et al 2013). These multiple sets can be obtained by alternative replacement of the paramagnetic ion with other paramagnetic ions with different magnetic susceptibility anisotropy tensors or by attaching, once at a time, paramagnetic tags in different points (Bertini et al 2001;Pintacuda et al 2006;Otting 2010;Liu et al 2012). Finally, PCSs and RDCs can provide information on interdomain mobility in multidomain proteins or internal mobility in protein-protein complexes, whenever internal consistency of all PCSs and RDCs arising from each paramagnetic metal with its own magnetic susceptibility anisotropy tensor cannot be achieved by assuming a single rigid structure (Gardner et al 2005;Longinetti et al 2006;Bertini et al 2007Bertini et al , 2010Bertini et al , 2012bRusso et al 2013;Ravera et al 2014;Andralojc et al 2014).…”