Figure 3. Substrate binding surface of ligase 10C probed by NMR and alanine scanning.

(a) Mapping of NMR chemical shift perturbations (intensity changes ΔI) for the evolved ligase shows regions affected by formation of the complex with RNA substrate. The most perturbed regions are indicated by thicker lines and darker colors. Residues involved in zinc coordination are labeled. (b) Region of ten single alanine mutants that were tested individually is shown in red and mapped onto one NMR conformer. (c) Ligase activity assay of ligase 10C and alanine mutants by gel shift. The lower band corresponds to unligated ³²P-labeled PPP-substrate, and the upper band corresponds to ligated product. Ligation activity is normalized to the activity of ligase 10C. Percentages shown represent the mean values from two independent experiments. Residues with activity below detection limit are shown in blue. (d) Sequence conservation analysis of region 45–54 for ligase enzymes generated by directed evolution¹⁴. The sequences were analyzed using the web based application WebLogo (V 2.8.2) from a dataset of 49 enzyme sequences. Residues E48, Y50, and H51 (blue) which completely lost activity during alanine scanning (see above) were conserved among all sequences. The two residues with 97% reduced activity of their alanine mutant were either conserved (C53) or had one alternative amino acid (C47). None of the other residues in this region were conserved.