Utility of 1H NMR chemical shifts in determining RNA structure and dynamics

Abstract

The development of methods for predicting NMR chemical shifts with high accuracy and speed is increasingly allowing use of these abundant, readily accessible measurements in determining the structure and dynamics of proteins. For nucleic acids, however, despite the availability of semiempirical methods for predicting (1)H chemical shifts, their use in determining the structure and dynamics has not yet been examined. Here, we show that (1)H chemical shifts offer powerful restraints for RNA structure determination, allowing discrimination of native structure from non-native states to within 2-4 Å, and <3 Å when highly flexible residues are ignored. Theoretical simulations shows that although (1)H chemical shifts can provide valuable information for constructing RNA dynamic ensembles, large uncertainties in the chemical shift predictions and inherent degeneracies lead to higher uncertainties as compared to residual dipolar couplings.

Figure 1

Measured vs. predicted chemical shift in RNA database. Root mean square difference (CS^RMSD) between experimental and (A) NUCHEMICS and (B) SHIFTS predicted chemical shifts prior to (red) and post (blue) minimization of input structure. In cases where multiple conformers exist we report the results for the structure that gives the lowest CS^RMSD after minimization.

Figure 2

Resolving structure using ¹H chemical shifts. (A) Correlation between SHIFTS (red) and NUCHEMICS (black) CS^RMSD and structure^RMSD for the A-form duplex, UUCG tetraloop, hTR internal loop, and pseudoknot. CS^RMSD and structure^RMSD are calculated over conformational pool consisting of native-like and unfolded conformers (see text). Plot where made by binning data along structure^RMSD. Bin widths were 0.50 Å. Averages and error were calculated over data in each bin. (B) Correlation plots between measured and predicted SHIFTS and NUCHEMICS chemical shifts for the A-form duplex, UUCG tetraloop, internal loop and pseudoknot. In each case, the chemical shifts shown are those calculated from conformer with lowest CS^RMSD, which is indicated on each plot. (C) Structural overlay of the average NMR structure with conformers with the lowest SHIFT and NUCHEMICS unweighted (left) and weighted (right) CS^RMSD. The NMR, SHIFTS, and NUCHEMICS structures are shown in blue, red and black, respectively. The structure^RMSD between the structures is indicated below each.