Spectral features of d3'-EBS1*·IBS1*

In order to solve the solution structure of the full d3'- EBS1*·IBS1* construct (Figure 51), IBS1* (numbered 59-65) was added in a 1:1 ratio to d3'-EBS1*. First NMR measurements resulted in very well resolved spectra. Even without the help of partially deuterated samples the sequential walk could be completely assigned. Figure 52A shows the sequential walk region and its assignment of d3'- EBS1*·IBS1*. It can be easily seen that even with the addition of seven nucleotides the resolution of the spectra is much better than in the absence of IBS1*. This is due to the

formation of a second helix and this includes the stabilization of the loop leading to a well structured part, which is confirmed by the very good dispersion of the resonances throughout the spectrum. With 2D [1H,1H]-NOESY spectra that were acquired at different temperatures and mixing times (see Materials and Methods) all H1', H2, H5, H6, and H8 protons could be assigned (Appendix 13). The H2' sugar protons were assigned by using a deuterated sample. The H3' resonances were then assigned with the help of 2D [1H,1H]-NOESY spectra with 60 ms and 120 ms mixing time. Subsequently, the assignment of most other sugar protons became possible (H4', H5', and H5''; Appendix 13).

Figure 50 Comparison of (A) the secondary structures of d3'-EBS1* (left) together with the section of the

hairpin including EBS1 of the group II intron from Oceanobacillus iheyensis(249) (right) and (B) of the solution

structure of d3'-EBS1* (left) with the crystal structure of the corresponding section (right). The nucleotides in the loop are coloured in red and the d3'-stem nucleotides in blue. EBS1 is indicated by a red arrow. Numbering of the nucleotides corresponds to the one in the full ribozymes.

Figure 51 Secondary structure of

d3'-EBS1*·IBS1*. The colouring scheme corresponds to Figure 30.

To confirm the sequential walk of IBS1*, a phase sensitive ge-2D w1,w2 13C,15N-

filtered/edited-NOESY experiment using a watergate H2O suppression was recorded.(325-328)

In this spectrum only protons that are bound to 14N or 12C are detected, but they have to interact through H-bonds with a 15N,13C labeled substrate. The X-filter is based on the evolution of J(XH) for 1H-13C systems whereas 1H-12C remains unaltered during a spin echo

period. Thus, at the end of this period, 1H-12C is on the y-axis whereas 1H-13C is on the x-axis. A 90° proton pulse applied from the x or y axis rotates only one of these two magnetizations to the z-axis, whereas the other component remains in the transverse plane. This experiment is widely used to study protein-ligand complexes where the protein is 15N,13C labeled and the ligand is at natural abundance.(325-328) In our case, the hairpin d3'-EBS1* is 15N,13C labeled and IBS1* is at natural abundance, thus only NOEs of IBS1* are detected. Figure 52B shows the double filtered-NOESY spectrum and Figure 52C the corresponding TOCSY spectrum. The sequential walk of IBS1* is identical with the one in the NOESY of the completely unlabeled d3'-EBS1*·IBS1*, thus confirming the assignment. The TOCSY spectrum shows the four H5-H6 crosspeaks of the pyrimidines of IBS1*.

In Appendix 42 the double X half-filtered-NOESY-HSQC with 15N as third dimension of

Figure 52 (A) 2D [1H,1H]-NOESY spectrum of the sequential walk region of d3'-EBS1*·IBS1* (100% D2O, pD = 6.83, 110 mM KCl, 10 µM EDTA) recorded at 700 MHz and 298 K. The solid line shows the sequential walk through the helical region, black broken lines the one of the loop including EBS1*, and red dotted lines the

one of IBS1. (B) ge-2D w1,w213C,15N-filtered-NOESY spectrum of d3'-EBS1*·IBS1* (90% H2O/10% D2O, pH =

6.45, 50 mM KCl, 10 µM EDTA) to detect the unlabeled IBS1* protons that are bound to labeled d3'-EBS1*.

The sequential walk through IBS1* confirms the one in the 2D [1H,1H]-NOSEY spectrum of the unlabeled RNA.

(C) Double filtered TOCSY spectrum of d3'-EBS1*·IBS1* (90% H2O/10% D2O, pH = 6.45, 50 mM KCl, 10 µM

Results and Discussion 71 d3'-EBS1*·IBS1* is shown. Only resonances from

the 15N,13C-labeled d3'-EBS1* to unlabeled IBS1* are visible. Resonances to H2O from labeled d3'-

EBS1* are also observed.

The chemical shift pattern of the adenosine H2 protons of A10, A16 and A20 in the absence of IBS1* resonates more downfield as the usual ones observed for AU base pairs (see Section 2.2.5, Appendix 35). Upon addition of IBS1* the chemical shift of A10H2 is hardly affected showing that A10 is still neither fully buried within the helix nor flipped out. In comparison the chemical shifts of A16H2 as well as A20H2

experience an upfield shift (Appendix 35). A16H2 shifts about 0.5 ppm upfield being now in the area where H2 resonances appear in AU base pairs. Since A16 basepairs with U62 this additionally shows that IBS1* binds to EBS1*. The resonance of the H2 of A20 shifts only about 0.2 ppm to smaller ppm values. A20 is not involved in a base pair but this intermediate shift shows that it is stuck in between two neighbouring base pairs.

Very weak correlations for U12H6- G13H1', G13H8-H1', G13H8-G14H1' as well as for G14H8-H1' already indicate an unusual conformation in this region. NOEs were found from the unpaired nucleotides in the loop A10, U11, U12, and A20 to C59 of IBS1* and are listed

Table 10 Unusual NOEs observed in the 2D

[1H,1H]-NOESY from C59 to A10, U11, U12

and A20 (see also Appendix 17). NOE from: to:

A10H1' C59H5, C59H6 A10H2' C59H5 A10H2 C59H1', C59H2', C59H5, and C59H6 U11H1' C59H1', C59H5, and C59H6 U11H5 C59H5 U11H6 C59H5 and C59H6 U12H1' C59H1', C59H5, C59H6 and C59H5 U12H5 C59H5 U12H6 C59H5 A20H2 C59H1'

Figure 53 Two sections of the 2D [1H,1H]-TOCSY of d3'- EBS1*·IBS1*. The upper panel shows the H1'-H2' crosspeaks that indicate a 2'-endo sugar conformation. In d3'- EBS1*·IBS1* such a sugar pucker is found at the helix end (black), and in the unpaired nucleotides A10, U11 and U12 (yellow). The H5-H6 crosspeaks of d3'-EBS1* are shown in the lower panel. These crosspeaks help to assign the

in Table 10. All these NOEs show that C59 is embedded within the loop pointing into the direction of the three unpaired nucleotides A10, U11, and U12 (see also Section 2.2.9 and Figure 58).

The sugar pucker of the individual residues was investigated by a 2D [1H,1H]-TOCSY experiment acquired at 298 K (Figure 53). Very strong H1'- H2' correlations were found for A10, U11, and U12. These nucleotides are besides A20 the only ones, which are not incorporated in a base pair. A H1'- H2' crosspeak is not found for A20, suggesting that this nucleotide is indeed stuck in between the flanking nucleotides and therefore connecting EBS1* to the stem in an A-form like manner (see Section 2.2.9). G1, C29 and C65 at the helical ends show a weak, but still visible H1'-H2'

correlation. For C59 located at the 5'-end of IBS1* no H1'-H2' crosspeak is found, what one might have expected. As already mentioned C59 is pointing towards the middle of the loop thus being trapped. Figure 53 shows another region in the TOCSY spectrum that comprises H5-H6 correlations. 18 H5-H6 resonances of d3'-EBS1*·IBS1* were clearly observed, which confirm the assignments in 2D [1H,1H]-NOESY spectra.

Exchangeable protons were assigned by 2D [1H,1H]-NOESY spectra in 90% H2O/10%

D2O at 278 K and 293 K with a mixing time of 150 ms and watergate H2O suppression

(Figure 54). The assignment was supported by a 2D [1H,15N]-HSQC, which only shows the NH-resonances of the 15N,13C-labeled part, i.e. the d3'-EBS1* hairpin. The imino region reveals two strong crosspeaks originating from G21-U9 and G14-U64. In addition to the eight Watson-Crick base pairs of the stem, which were already observed in d3'-EBS1*, six additional Watson-Crick base pairs were found in d3'-EBS1*·IBS1*. This shows that IBS1* is

Figure 54 Assignment of imino resonances in d3'-

EBS1*·IBS1*. The imino section of the 2D [1H,1H]-NOESY

spectra acquired in 90% H2O/10% D2O at 278 K is shown in the

lower panel. In the upper panel the corresponding section of the

Results and Discussion 73 completely bound to EBS1*. To confirm the base pairing pattern, a 2D JNN HNN-COSY(118)

was recorded with the d3'-EBS1* hairpin 15N,13C-labeled and IBS1* at natural abundance (Appendix 15). This spectrum revealed for the stem the same base pairs like in d3'-EBS1*. G14N1-H1, U18N3-H3 and G19N1-H1 resonances were observed showing that these nucleotides are not solvent exposed anymore like it was the case in the absence of IBS1*. A missing correlation for G13 in the 2D JNN HNN-COSY as well as in the 2D [1H,15N]-HSQC

experiment suggests that this imino proton is partly solvent exposed. This suggestion is confirmed by the solution structure, which shows that G13 is pointing outwards (see Section 2.2.9). In addition, the missing crosspeak can derive from a fraying end of the EBS1*·IBS1* duplex as is well known for other RNA as well as DNA structures.(329-336)