Ensemble Approach for NMR Structure Refinement against 1H Paramagnetic Relaxation Enhancement Data Arising from a Flexible Paramagnetic Group Attached to a Macromolecule

Junji Iwahara, Charles D. Schwieters, G. Marius Clore

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255 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)5879-5896
Number of pages18
JournalJournal of the American Chemical Society
Issue number18
StatePublished - May 12 2004
Externally publishedYes

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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