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N labelled RRM1 was titrated with the RNA substrate EDEN7 (UGUUUGU). Significant chemical shift perturbations (CSPs) were seen for several residues, confirming that RRM1 does bind to the EDEN7 sequence. Most residues were in fast exchange, and so their assignments could simply be tracked from point to point throughout the titration. In Figure 4.14 are shown overlaid spectra of the unbound protein and the protein bound to RNA. Highlighted are the peaks for some of the residues which show the greatest perturbation throughout the titration.

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Figure 4.14: Overlaid 15N TROSY spectra for the titration of RRM1 with the RNA substrate EDEN7 (UGUUUGU) The spectrum of the unbound protein is shown in blue, and the fully bound protein in red. Intermediate titration points are shown as a spectrum. Insets are expanded views of some of the most affected peaks. Most of the affected residues are in fast exchange, such as Phe19, Gly21, Cys61, Cys62 and Leu85. There are some residues that are in intermediate exchange such as Gln22, for which the peak from the free form is highlighted, but the bound peak is lost due to broadening. There are also residues in slow exchange such as Gln93, where both the peak is visible in both the free and fully bound forms, but not the intervening titration points. This prevents the peak being tracked through the titration, which can present problems in determining the assignment of the bound form.

While most residues are in fast exchange there are a few, such as Gln22 which are in intermediate exchange, and so broaden out and are lost in the early stages of the titration. Additional peaks also grow in during the later stages of the titration from the bound forms of residues in slow exchange, but the assignment of these new peaks is in some cases ambiguous due to overlap. It can be stated these residues are definitely involved in binding the RNA, but the chemical shift perturbation cannot be quantified unless the corresponding peak from the bound form can be located. In cases of slow exchange where bound assignments are unclear a minimum CSP can be stated based on the closest unassigned peak in the bound spectrum. This is not possible in some intermediate exchange situations as

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the peak from the bound form may be too broad to observe. Residues in more dynamic regions of the protein are more likely to fall into this category as they tended to give weak, broad peaks even in the spectrum of the unbound protein.

The nitrogen chemical shift has a greater dispersion than the proton chemical shift. When combining the changes in the proton and nitrogen chemical shifts into a single chemical shift perturbation value (CSP), they must therefore be weighted so that changes in the proton dimension are not obscured. CSP values were calculated throughout using the formula below.

CSP N H

Where N is the difference in chemical shift in the nitrogen dimension between

the saturation point and the zero point of the titration and H is the corresponding difference in the proton dimension. The CSP values for each residue can then be plotted against residue number to show those regions of the protein that are involved in binding the RNA.

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Figure 4.15: CSP values for each residue of RRM1 on titration with EDEN7. Residues which are in slow exchange, for which the values are therefore minimum CSPs rather than exact values, are highlighted in red. Gaps are due to prolines, unassigned residues and peaks for which CSPs could not be accurately calculated due to signal overlap.

When RRM1 binds to the EDEN7 RNA substrate, the most perturbed residues are concentrated in the 18 - 23, 59 - 63 and 93 - 101 regions. While these regions are far apart in the protein chain they actually form adjacent strands of the -sheet in the folded protein, and so represent a single coherent RNA binding patch. This binding patch can be more easily visualised by mapping the CSP values for each residue onto the structure of the protein. In this case the CSPs were mapped onto the relevant part of the CELF1 RRM1 and RRM2 structure produced by Jun et al, which is available in the PDB (ID: 2DHS). This map of the binding surface is shown in Figure 4.16.

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Figure 4.16: CSP maps on the structure of RRM1, produced by truncation of Jun et al. NMR

structure of the first 187 residues of CELF1 from the PDB. RRM2 has been removed by deletion of residues 102 187. The CSP values have been mapped onto the structure in red: the brighter the red colour, the greater the magnitude of the CSP. Residues which are definitely affected but do not have quantified CSP values due to intermediate or slow exchange preventing the peaks from the bound form being located are shown in yellow. Residues for which no data can be obtained (i.e. prolines and unassigned residues) are shown in black. This image was produced using the molecular graphics package Molmol, as were all subsequent CSP maps.

In producing this map, residues with CSP values of less than 0.1 are considered to undergo no significant change on binding, and are shown in grey. Residues for which no data is available, specifically the prolines and the unassigned residues are shown in black. CSP values are displayed as a colour gradient, with bright red indicating the most affected residues, and dark red indicating lower CSPs. Residues without quantifiable CSPs due to slow exchange preventing location of the peak for the bound form are shown in yellow. As slow exchange is associated with a large difference in chemical shifts between the free and bound forms these residues are presumably the most disrupted on RNA binding, and would show the largest CSPs if exact values could be calculated.

From this CSP map, it can be seen that the RNA binding site is across the face of

104 some significantly perturbed residues in the loops at the lower edge of the sheet

as shown in Figure 4.16, such as Gln22. The most affected residues, such as Cys61 and Gly21 lie in the classic RNP regions in -sheets 1 and 3. The conserved aromatic residues in the RNP regions (Phe19 and Phe63) are also affected. This is consistent with them forming stacking interactions with the RNA bases, as is commonly seen for RRMs. Some residues near the C-terminus (e.g. Asp98) appear to be involved in RNA binding despite being beyond the folded region of RRM1. It was not clear whether this was an artefact caused by the position at which the protein was truncated, or if some residues beyond the C- terminus of the structured domain are in fact involved in binding.

It was noted that this titration seemed to reach saturation rather earlier than would be expected for simple 1:1 binding, based on the point at which the 15N TROSY spectrum ceased to vary significantly between titration points. Very few changes in chemical shift were seen after a 0.6: 1 ratio of RNA to protein was reached. Examples of the titration curves for some of the most affected residues, such as Gly21 and Cys61 are shown in Figure 4.17.

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Figure 4.17: Chemical shift perturbations at each titration point for a selection of the most affected residues for which CSPs can be calculated throughout the titration. The protein concentration was 500 µM. All reach an apparent plateau before an RNA concentration of 500 µM is reached.

Since the protein concentration in this experiment was 500 µM, in a 1:1 model the titration would not be expected to reach saturation before this concentration of RNA was reached. A possible explanation for this was that the complex being formed did not have a simple 1:1 stoichiometry. If RRM1 is recognising a UGU site then the EDEN7 RNA used (UGUUUGU) potentially had two identical binding sites. This would permit a 2:1 protein to RNA complex to form, resulting in all of the protein being in the bound form after a 0.5:1 ratio of RNA to protein was reached.

The titration was repeated with the EDEN3 (UGU) RNA substrate. The protein concentration for this titration, and all further titrations with RRM1, was reduced to 400 µM.

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Figure 4.18: Above is a histogram of chemical shift perturbations for the titration of RRM1 with UGU. The assignment for the bound peak of Gln22 was unclear, so a minimum CSP estimate to the closest unassigned peak has been included for this residue. Below is the CSP map for the titration of RRM1 with EDEN3 (the trinucleotide UGU). The protein concentration was 400 µM, and the titration was conducted at 298 K.

The UGU RNA substrate was still bound by RRM1, with almost all residues in fast exchange in the NMR titration. Only Gln22 still appeared to be in slow exchange, and so only has a minimum CSP value. The same residues in the RNP regions are disrupted, including the aromatic residues Phe19 and Phe63, which can therefore be assumed to be still stacking with two of the three RNA bases. The only residues which appeared unaffected in the UGU titration, but showed

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significant CSPs in the EDEN7 case are Asp98, Glu100 and some of the weakly affected residues such as Ile45 in 2.

Significant CSPs show that RRM1 is still binding to this shorter RNA substrate, and the set of affected residues appears to be very similar. The UGU substrate was therefore not only binding to RRM1, but occupying the whole of the binding site across the -sheet as effectively as the longer EDEN7 substrate. From this it was concluded that UGU sites are the key component of the EDEN motif, and are sufficient for binding of RRM1. It also supported the possibility of two RRM1 protein molecules binding to EDEN7 (UGUUUGU) with one protein at each UGU site.

The titration curves for some of the most affected residues suggest titration had not quite reached saturation point at a 1:1 ratio of RNA to protein (see Figure 4.19). Again this is consistent with the UGU substrate forming a 1:1 complex while EDEN7 forms a 2:1 complex with RRM1.

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Figure 4.19: Titration curves for a selection of residues in the titration of the RNA sequence UGU into 400 µM RRM1. The curves for Gly21 and Cys61 definitely do not reach a maximum until an RNA concentration of 400µM. This is less clear for the other residues shown.