Figure 1. Determination of interacting surfaces using cross‐saturation. The left image shows the pulse sequence used in the cross‐saturation NMR experiment described. The middle image shows the cartoon representation of the protein–RNA complex the experiment was recorded on (the Nova‐1 KH3–RNA complex). The right image shows the – correlation spectrum recorded from a control experiment where a region well outside the chemical shift range of both the protein and the RNA was irradiated (left) and the difference spectrum after subtracting an interleaved spectrum with irradiation centered on the RNA anomeric protons chemical shift (6.0 ppm). Resonances showing a strong cross‐saturation effect are boxed and the corresponding residues are in pink in the middle panel's cartoon representation of the protein.

Figure 2. Determination of dissociation constant for the KSRP KH3 : UAGUAU interaction – workflow. The left panel shows an overlay of the – correlation spectra recorded during the titration of KSRP KH3 with UAGUAU RNA. The blow‐up highlights the progress of a peak during the titration from red (free protein) to blue (1 : 8 protein/RNA). In the middle panel the average chemical shift change is plotted against the RNA/protein ratio. The fitted binding isotherm is in red. The displayed equation is used to obtain the dissociation constant on the right using nonlinear regression analysis and assuming a one‐site binding model.

Figure 3. Deviation from ideality in the intermediate‐exchange regime. Simulation of chemical shift changes observed during the titration of an RNA‐binding domain plotted against the RNA/protein ratios. The protein–RNA complex has a K _d of 1 µM. The three curves represent the changes in three protein resonances that show a 10 (circle), 20 (square) and 50 (star) Hz difference between the free and fully bound position. Courtesy of Dr. Thomas A. Frenkiel.

Figure 4. Determination of interacting surfaces using PRE – workflow. The top left panel shows a representation of the secondary structure of the RNA molecule. The labelled U, displayed at the bottom is attached to the first U of the tetraloop. The top right and bottom right panels display the – correlation spectra of the oxidized and reduced states of the RNA/protein complex, respectively. The bottom left panel shows the mapping of the residue showing a significant PRE on the protein structure.

Figure 5. SIA – workflow. The top left panel displays the 16 RNA pools used in the KH3 SIA assay. The top right panel shows a section of the – correlation spectra of KH3. Each of the four images shows the superimposition of the spectra recorded during a titration at protein/RNA ratios of 1 : 0, 1 : 1 and 1 : 4 (red, green, and blue respectively). The histogram in the bottom right panel records the data from these four titrations. Here the weighted chemical shift change is plotted against the residue number and the four values from the four titrations are represented in different colors. Finally, the bottom left panel displays the SIA score in a graphic format.