Ensemble Approach for NMR Structure Refinement against1H Paramagnetic Relaxation Enhancement Data Arising from a Flexible Paramagnetic Group Attached to a Macromolecule

Abstract

Paramagnetic relaxation enhancement (PRE) measurements on ¹H nuclei have the potential to play an important role in NMR structure determination of macromolecules by providing unique long-range (10−35 Å) distance information. Recent methodological advances for covalently attaching paramagnetic groups at specific sites on both proteins and nucleic acids have permitted the application of the PRE to various biological macromolecules. However, because artificially introduced paramagnetic groups are exposed to solvent and linked to the macromolecule by several freely rotatable bonds, they are intrinsically flexible. This renders conventional back-calculation of the ¹H-PRE using a single-point representation inaccurate, thereby severely limiting the utility of the ¹H-PRE as a tool for structure refinement. To circumvent these limitations, we have developed a theoretical framework and computational strategy with which to accurately back-calculate ¹H-PREs arising from flexible paramagnetic groups attached to macromolecules. In this scheme, the ¹H-PRE is calculated using a modified Solomon−Bloembergen equation incorporating a “model-free” formalism, based on a multiple-structure representation of the paramagnetic group in simulated annealing calculations. The ensemble approach for ¹H-PRE back-calculation was examined using several SRY/DNA complexes incorporating dT-EDTA−Mn²⁺ at three distinct sites in the DNA, permitting a large data set comprising 435 experimental backbone and side-chain ¹H-PREs to be obtained in a straightforward manner from 2D through-bond correlation experiments. Calculations employing complete cross-validation demonstrate that the ensemble representation provides a means to accurately utilize backbone and side-chain ¹H-PRE data arising from a flexible paramagnetic group in structure refinement. The results of ¹H-PRE based refinement, in conjunction with previously obtained NMR restraints, indicate that significant gains in accuracy can be readily obtained. This is particularly significant in the case of macromolecular complexes where intermolecular translational restraints derived from nuclear Overhauser enhancement data may be limited.