Characterizing the magnetic susceptibility tensor of lanthanide-containing polymethylated-DOTA complexes

Abstract

Lanthanide complexes based on the DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) cage are commonly used as phase contrast agents in magnetic resonance imaging, but can also be utilized in structural NMR applications due to their ability to induce either paramagnetic relaxation enhancement or a pseudocontact shift (PCS) depending on the choice of the lanthanide. The size and sign of the PCS for any given atom is determined by its coordinates relative to the metal center, and the characteristics of the lanthanide's magnetic susceptibility tensor. Using a polymethylated DOTA tag (Ln-M8-SPy) conjugated to ubiquitin, we calculated the position of the metal center and characterized the susceptibility tensor for a number of lanthanides (dysprosium, thulium, and ytterbium) under a range of pH and temperature conditions. We found that there was a difference in temperature sensitivity for each of the complexes studied, which depended on the size of the lanthanide ion as well as the isomeric state of the cage. Using ¹⁷O-NMR, we confirmed that the temperature sensitivity of the compounds was enhanced by the presence of an apically bound water molecule. Since amide-containing lanthanide complexes are known to be pH sensitive and can be used as probes of physiological pH, we also investigated the effect of pH on the Ln-M8-SPy susceptibility tensor, but we found that the changes in this pH range (5.0-7.4) were not significant.

Keywords: DOTA; Lanthanide; Magnetic susceptibility tensor; PCS; Pseudocontact shift.

Fig. 1

PCS temperature dependence for Dy-M8-Ub^S57C. a Ln-M8-SPy, a lanthanide-containing tag that induces PCS, is present in two NMR-observable forms; twisted square antiprism (TSAP) and square antiprism (SAP). The two structures interconvert on a slow time scale and are related via rotation of the carboxylate and amide pendant arms. The two isomers are displayed in (b), using the Pr-M8-SPy structure calculated in (Opina et al. 2016), in sticks (PyMol) with carbon (grey), nitrogen (blue), oxygen (red), praseodymium (green), polar hydrogens (white), and sulfur (yellow). Non-polar hydrogens are not displayed. c A small section of the ¹H/¹⁵N−HSQC spectrum for Lu- and Dy-M8-Ub^S57C showing the PCS for residue I36 at a range of temperatures. At 288 K, Lu-M8-Ub^S57C, the diamagnetic version of the complex, is shown in dark red, and the paramagnetic version, Dy-M8-Ub^S57C is shown in purple. Two peaks are observed for Dy-M8-Ub^S57C since each of the two isomers induces different PCS (TSAP, left, minor isomer; SAP, right, major isomer). The PCS is measured as the difference in chemical shift (ppm) between the paramagnetic and diamagnetic peaks. As the temperature increases to 298 K, the PCS value decreases for both isomers (dark orange, Lu- and dark blue, Dy-M8-Ub^S57C). This trend is continued at 308 K (light orange, Lu- and cyan, Dy-M8-Ub^S57C). Proton PCS (ppm) of Dy-M8-Ub^S57C are shown on a per-residue basis at three temperatures (288 K, purple; 298 K dark blue; 308 K cyan) for d the SAP isomer and e the TSAP isomer. Note that the PCS values of the SAP isomer are more sensitive to temperature

Fig. 2

Temperature dependence of the lanthanide metal center position for Dy-M8-Ub^S57C. a Observed vs calculated PCS for the SAP isomer at 288 K (cyan), 298 K (dark blue), and 308 K (purple) and the TSAP isomer at 288 K (yellow), 298 K (orange), and 308 K (red). Minimization of the metal center position using PCS values as restraints was carried out using Xplor-NIH for the TSAP and SAP isomers simultaneously. The lowest energy structure was used to back-calculate expected values. b Minimized metal center positions for each of the isomers are shown in spheres at the three temperatures [color system as in (a)], with ubiquitin shown in green cartoon. c Isosurfaces of SAP and TSAP isomer-induced PCS values where the blue and red lobes signify +5 ppm and −5 ppm, respectively, for Dy-M8-Ub^S57C at pH 7.4, 298 K

Fig. 3

PCS pH dependence for Dy-M8-Ub^S57C. Zoom of the ¹H/¹⁵N-HSQC spectrum for a Lu- and b Dy-M8-Ub^S57C showing the PCS for residue K6 at pH 5.0 and 6.2. In (b), a large difference is observed between the peak position of the Dy-M8-Ub^S57C at pH 5.0 and 6.2, induced by the SAP isomer, which could be mistaken as entirely caused by paramagnetism. Since there is also a large change in the peak position of the diamagnetic version a in this pH range, this is an entirely diamagnetic effect, and underlines the importance of using a reference compound in calculating PCS. Proton PCS values (ppm) of Dy-M8-Ub^S57C are shown on a per-residue basis at three pH values (pH 5.0, purple; pH 6.2, dark blue; pH 7.4, cyan) for c the SAP isomer and d the TSAP isomer

Fig. 4

pH dependence of the lanthanide metal center position for Dy-M8-Ub^S57C. a Observed vs calculated PCS for the SAP isomer at pH 5.0 (cyan), pH 6.2 (dark blue), and pH 7.4 (purple) and the TSAP isomer at pH 5.0 (yellow), pH 6.2 (orange), and pH 7.4 (red). Minimization of the metal center position using PCS values as restraints was carried out using Xplor-NIH for the TSAP and SAP isomers simultaneously. The lowest energy structure was used to back-calculate expected values. b Minimized metal center positions for each of the isomers are shown in spheres at the three pH values [color system as in (a)], with ubiquitin shown in green cartoon. c Stick representations of the TSAP (red) and SAP (cyan) isomers of the Dy-M8 tag are shown, with the corresponding metal centers displayed using spheres. It should be noted that although the metal positions are calculated using PCS restraints and can therefore be considered accurate, other atoms in the tag are not calculated using experimental restraints and should therefore not be treated as reliable, but are simply shown as an estimate of their approximate distance from the ubiquitin surface

Fig. 5

Temperature and pH dependence of the axial (χ_a) and rhombic (χ_r) components of the magnetic susceptibility tensor for Dy-, Tm-, and Yb-M8-Ub^S57C. Mean and standard deviation are calculated as described in Table 1. The SAP isomer is shown in red and the TSAP isomer is in black, throughout. a–c The axial (χ_a) component of the susceptibility tensors for the SAP and TSAP isomers of Dy-M8-Ub^S57C and the TSAP isomer of Tm-M8-Ub^S57C decrease in magnitude with increased temperature, with the strongest effect for the SAP isomer of Dy-M8-Ub^S57C. No change is observed for the TSAP isomer of Yb-M8-Ub^S57C. d–f The rhombic (χ_r) component of the susceptibility tensors for the SAP and TSAP isomers of Dy-M8-Ub^S57C, and the TSAP isomer of Tm- and Yb-M8-Ub^S57C as a function of temperature. g–i The axial (χ_a) component of the susceptibility tensors as a function of pH. j– I The rhombic (χ_r) component of the susceptibility tensors as a function of pH