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As discussed in the previous section, the difficulty of measuring NH RDCs in Otting medium emphasises the requirement of measuring multiple types of RDC in phage. These RDCs can be measured for other couplings in the peptide plane.

2.2.6.1 Approaches for measurement of H-C’ and N-C’ RDCs on the ribosome

Measurement of H-C’ and N-C’ RDCs has the advantage that these two RDCs can be measured simultaneously in a 2D N-H correlation spectrum with relatively high sensitivity. However, the sizes of these two couplings are relatively small compared to N-H RDCs, being ∼3-fold and ∼8-fold smaller, respectively.

The simplest method to measure these two couplings is to remove the 13_{C’ decoupling} pulse from the indirect evolution time in a 15_{N HSQC or TROSY sequence. As well as} generating a splitting in the indirect 15_{N dimension, the resulting doublet components are} also displaced in the acquisition proton dimension. This is due to the natural E.COSY effect, which arises because there is no change in the C’ spin-state during the back-transfer of

magnetisation from 15_{N to} 1_{H. The N-C’ and H-C’ splittings are then measured from the} displacement of the doublet components in the 15_{N and proton dimensions, respectively.} However, this requires spectra of very high resolution to separate the E.COSY components. This problem can be avoided by recording the data in an IPAP fashion. The disadvantage of the IPAP approach is that the long constant-time period required for the IPAP element (1/2JN-C’∼30 ms) causes sensitivity losses.

To evaluate if the precision of measurement will be sufficient relative to predicted range of RDC values to allow meaningful interpretation of the data, IPAP-E.COSY(C’)-15_N- HSQC-TROSY spectra were measured on a 10-µM 13_C, 15_{N labelled ribosome sample.} Splittings were extracted from combined spectra representing a total acquisition time of 40 hours (spectra are shown in Figure A.7 on page 217).

Peak positions, signal-to-noise ratios and linewidths were analysed for 75 peaks. The calculated RMS uncertainties are 1.9 Hz and 0.9 Hz for JH-C’ and JN-C’, respectively. These uncertainties should be compared to the predicted range of RDCs. Assuming DNH_a would be the same as previously obtained for ribosomes aligned in phage (20 Hz), the magnitudes of the expected couplings would fall in the range 0-12 Hz and 0-5 Hz for H-C’ and N-C’, respectively. Assuming that the aligned splittings can be measured with the same precision as the isotropic splittings would yield estimated uncertainties of 2.7 Hz and 1.2 Hz for DH-C’ and DN-C’, respectively. Therefore, the predicted uncertainties are ∼20 % of the maximum expected coupling for both the H-C’ (2.7/12=0.22) and N-C’ (1.2/5=0.24) coupling types. This level of fractional uncertainty corresponds to a Q factor of 0.45-0.5, indicating that while in isolation the utility of these couplings would be limited, they could still be useful when combined with the N-H couplings.

However, in practice, the proton linewidth for the aligned sample will be significantly broader than for the isotropic sample due to the proton-proton dipolar couplings, and hence the uncertainties in the H-C’ RDCs may be too large for this coupling to be useful.

In conclusion, it may be possible to measure the N-C’ couplings at sufficient precision to provide useful information, but the H-C’ couplings will be less precise due to broad

linewidths in the aligned sample and are unlikely to be useful.

2.2.6.2 Approach to measure C’-Cα_RDCs

The final type of coupling considered in this work was the C’-Cα_{couplings. The magnitude} of this coupling is one-fifth of the N-H coupling, which is in between the magnitude of the H-C’ and N-C’ couplings. The measurement of the C’-Cα coupling via the 13_{C HSQC} spectrum is associated with the same difficulties as described for Hα-Cαcouplings, including peak overlap and poor resolution due to the limited13C acquisition time. The latter problem is even more important for measuring C’-Cα couplings, since the size of the RDCs is 10 times smaller than those for the Hα-Cα coupling (hence requiring narrower linewidths to allow measurement with sufficient precision).

An alternative approach is to measure the splittings in a 3D fashion, using an HNCO- type sequence. The HNCO-type experiment is approximately three times more sensitive than the HN(CO)CA sequence tested during the attempt to measure Hα-Cα splittings, and hence might be feasible for use with ribosome samples. The advantage is that a longer acquisition time can be used on C’ than Cα, since the C’ acquisition time is not limited by evolution of other carbon-carbon couplings.

To evaluate the sensitivity and resulting uncertainties of this approach, a 3D in-phase spectrum using an IPAP-BEST-HNCO sequence was measured for 12 hours on a 20-µM isolated L7/L12 sample. The 2D H-C’ projection of the 3D in-phase HNCO spectrum is shown in Figure 2.35, left panel. Peaks representing 80 % of the residues were picked for the signal-to-noise ratio and linewidth analysis. The average signal-to-noise ratio is 10 with an average linewidth of 27 Hz. Extending the total acquisition time to 72 hours would improve the signal-to-noise ratio to 25, which would then yield an estimated peak position uncertainty of 0.5×(27/25) = 0.5 Hz. Assuming a similar signal-to-noise ratio could be obtained for the ribosome sample leads to a predicted uncertainty in the measured RDCs of ∼1 Hz. Again assuming an alignment strength corresponding to a DNH

a of 20 Hz, the predicted maximum RDC for the C’-Cα coupling would be 8 Hz. Therefore, the estimated

7.5 8.0 8.5 9.0 9.5 174 176 178 180 182 7.5 8.0 8.5 9.0 9.5 174 176 178 180 182 δC δ_H ppm ppm ppm δC δ_H ppm

Figure 2.35: Left panel: 2D H-C’ projection of the 3D in-phase HNCO spectrum of isolated L7/L12. Spectrum recorded on 20 µM isolated L7/L12 (fraction 2) and contoured with 5× RMS noise. Right panel: 2D H-C’ IPAP-BEST-HNCO spectrum of ribosome-bound L7/L12 (10 µM 70S ribosomes) with 3× RMS noise. Both spectra were recorded for 12 hours in Tico buffer at 25 °C.

uncertainty is approximately 12 % of the maximum expected RDC, which corresponds to Q factor of 0.25-0.3, which is sufficiently small to be useful.

On the basis of the above analysis, an attempt was made to measure the C’-Cα splittings for a phage-aligned ribosome sample using the IPAP-BEST-HNCO sequence. The experiment was acquired as a 2D H-C’ spectrum to maximise the sensitivity. The measured spectrum for 12 hours total acquisition time is shown in Figure 2.35, right panel. Only a few peaks were observed, which correspond to peaks from the linker region and other intense unassigned peaks in the15N HSQC spectrum of the ribosome.

In conclusion, this approach appears not to be feasible due to the low sensitivity of the spectrum measured on the ribosome. The difference in sensitivity between the isotropic spectrum of the isolated L7/L12 and the phage-aligned spectrum of ribosome-bound L7/L12 is the result of both the increased relaxation rates of ribosome-bound L7/L12 and the decrease in the signal-to-noise ratio due to the increased proton linewidths in the aligned sample (unresolved proton-proton couplings). The triple-resonance experiments are more sensitive to small differences in relaxation rates than simple 2D experiments due to the long delays and multiple transfer steps in the pulse sequence of the triple-resonance experiments.