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TFE however is not representative of a native membrane environment, and the study then progressed to exploring the assembly of E5 homo-dimers in SDS detergent micelles which provide a more membrane-like environment and have been shown previously to maintain the formation of the E5 TM domain

homodimer (Oates, Hicks et al., 2008). Since it has been shown for other TM domains, including for Ii and MHC in this study, that the oligomeric state can be modulated by the detergent concentration, the affect of SDS concentration on the NMR spectra of E5 was explored and 15N-1H HSQC spectra of E5TM1 were

acquired at varying SDS concentrations.

Figure 6.7. Overlay of15N-1H HSQC spectra for E5 TM peptides in TFE and SDS

Comparison of15N-1H HSQC spectra of (a) E5TM1 and (b) E5TM2 peptides acquired in TFE

(grey) and SDS (black) showing the same pattern of distribution of the resonances and thus facilitating the assignment of the resonance observed for spectra acquired of E5 peptides in SDS.

Assignment of resonances for E5 peptides acquired in SDS is difficult due to the increased signal broadening introduced by the slower tumbling of the larger peptide-micelle complex. To aid assignment, the spectra acquired in SDS were compared to those acquired in TFE. Comparison of Figure 6.7 to Figure 6.3, the relative position of the resonances does not significantly change upon solubilization of the E5 TM peptides in SDS micelles, thus the assignments made for resonances acquired in TFE can aid those in SDS. Notably, as expected the peak widths are greatly increased for resonances from peptides solubilised in SDS micelles.

For spectra acquired in SDS, as shown in Figure 6.8a, 2D contour plots revealed the presence of two sets of three resonances with each doublet separated by

fractions of a ppm. Comparison of 1D projections through the HSQC spectrum (Figure 6.8b) revealed the relative intensities of these two sets were dependent upon the concentration of SDS with one diminishing as the concentration was increased with concomitant increase in the second set, as shown in Figure 6.8b. This is indicative of the presence of two species that are in slow exchange on the NMR timescale. It has been shown for other TM peptides that the oligomeric state of the protein can be modulated by the concentration of detergent with shifts to lower oligomeric states as the detergent concentration is increased, so it is likely therefore that these two sets of resonances represent dimeric and monomeric species of E5. Therefore, the set of resonances observed at low detergent concentration were assigned to the dimeric state of E5TM1. This type of splitting

pattern has been observed previously in other NMR studies of TM helix-helix interactions and also attributed to the assembly of monomers into oligomers (Gratkowski, Dai et al., 2002; Wu, Shih et al., 2007).

Interestingly, Leu21 displays a multiplet signal at low micelle concentration in contrast to the broad singlet observed for the other two resonances. It is possible that this is due to the close packing of the helices in the dimer interface restricting the motion of the Leu side chain causing it to adopt multiple rotameric forms. Rotamers of Leu side chains at closely packed dimer interfaces have been observed previously for closely packed dimers of transmembrane domains (MacKenzie, Prestegard et al., 1996). It is conceivable that this could lead to small changes in the backbone conformation and hence small changes in the amide 1H chemical shift. Consistent with this interpretation is the observation that the multiplet collapses to just a single peak as the detergent is increased and E5 becomes more monomeric, which presumably releases the Leu side chain from its restricted motion at the interface.

Figure 6.8.15N-1H HSQC spectra of E5TM1in SDS detergent

(a) Stacked 2D contour plots from15N-1H HSQC spectra of E5TM1as a function of SDS detergent

concentration. (b) 1D projection of plane from 2D spectra showing the resonance doubling and change in relative intensity with detergent concentration. Residue assignments are indicated at the top and the set of peaks attributed to monomer (M) and dimer (D) are indicated.

Figure 6.9.15N-1H HSQC spectra of E5TM2in SDS detergent

Contour plot of two-dimensional 15N-1H HSQC spectra of E5TM2 solubilised in 40 mM SDS

detergent. (b) 1D projections of plane from 2D spectra showing the resonance doubling and change in relative intensity with the peptide:SDS micelle ratio.

It has been suggested that such a change could be due to non-specific interactions (Wu, Shih et al., 2007). To determine if this was a specific interaction or merely an artefact of low micelle concentration we designed a further peptide, E5TM2

which possessed 15N-labelled residues at three positions in the expected interface and at three positions distal to the interface. For residues residing at the interface of the E5 dimer we would expect to observe chemical shift changes upon association of the peptides due to their altered chemical environment. However, as shown in Figure 6.9a, all the resonances observed in the15N-1H HSQC spectra of E5TM2 also exhibit the same resonance splitting. Furthermore, as shown in Figure

6.9b, the relative intensities of the two sets of resonances are modulated by increasing detergent concentration in a similar fashion to E5TM1, and were

therefore assigned to monomer and dimer forms as indicated.

It has been reported that the average difference in backbone amide chemical shifts in a15N-1H HSQC can be used to identify interfacial residues in oligomers since theoretically resonances from interfacial residues should undergo a more significant shift than those of other residues in the helix due to the significantly altered chemical environment (Wu, Shih et al., 2007). The average 15N 1H backbone chemical shift difference for the assigned resonances from E5TM1 and E5TM2 were calculated as described in Section 2.20, according to the method in Wu et al (Wu, Shih et al., 2007). As shown in Figure 6.10, the greatest average differences were observed for those residues in the helix expected to be at the interface namely, Ala14, Leu21, Leu24, and Phe28.

Figure 6.10. Average backbone1H and15N amide chemical shift differences

Average backbone1H and15N amide chemical shift differences for all assigned resonances from

15_N-1_{H HSQC spectra of E5}

TMpeptides in SDS. Δδ was calculated as described in Section 2.20.

These data show that the oligomeric state of TM domains in detergent micelles can be monitored by changes in chemical shift using15N-1H HSQC spectra and that the shift in equilibrium between monomeric and oligomeric species is highly dependent on the detergent micelle concentration. Our data suggest that the E5

TM domain forms dimers in SDS micelles and moreover provides atomic level information about the structure of the E5 dimer consistent with previous published results.

6.1.4 Helical content of E5 is unaffected by peptide: micelle