The Use of Lanthanides for Solution Structure Determination of Biomolecules by NMR: Evaluation of the Methodology with EDTA Derivatives as Model Systems

The methodology of using lanthanide ions to obtain structures of biological macromolecules in liquid solution from proton NMR measurements was critically examined with indole-EDTA and benzyl-EDTA as model systems. Both of these molecules form tight monodentate complexes with lanthanide ions. Shifts of proton resonance frequencies induced by the binding of Pr³⁺, Nd³⁺, Eu³⁺, and Yb³⁺(LIS) were measured at 5-deg intervals from 5 to 60 °C at a frequency of 300 MHz. Enhancements of proton spin-lattice relaxation rates, T₁^-1 and line widths due to the ion binding (LIR) were measured at 30 °C at frequencies of 200, 300, and 470 MHz. La³⁺ and Lu³⁺ served as diamagnetic references. Structures of these molecules determined from molecular mechanics calculations formed a basis for analysis of the NMR data. Those structures along with 2D-COSY and proton decoupling experiments were combined to assign the proton resonances which were all in slow exchange. A method of analysis of the LIS utilizing linear least-squares fitting of the data to expressions involving elements of the magnetic susceptibility tensors of the ions is given. Theoretical values for the magnetic properties of the ions were not used to separate the contact (through bond) and pseudocontact (through space) contributions to the LIS. The contact shifts were included as fitting parameters. The temperature dependence of the LIS was of no use in making assignments or in separating the contact and pseudocontact shifts; it was only useful for unraveling overlapping resonances. The results showed differences in the orientation of the principal axes of the magnetic susceptibility tensors of the ions within the same molecule as well as deviations of the orientations from the “near”-symmetry directions of the molecules. Furthermore, the magnetic susceptibility tensors deviated greatly from axial symmetry with asymmetry parameters ranging from 0.09 to 0.97. For Yb³⁺, LIS analysis was not feasible because unresolved scalar couplings precluded the making of complete assignments. In general. the LIR yielded reasonable values for the ratios of distances of the protons from the ion. In order to determine detailed molecular structure from these kinds of measurements the coordinates of at least five paramagnetic nuclei in the molecule must be known from other sources. LIR and LIS values, the latter obtained with at least two different ions, are essential to establish distance and orientation, respectively.