LAS CONSECUENCIAS DEL DARSE CUENTA

Peptoids reported in the literature to form stable helices generally contain a regular pattern of helix inducing residues in the primary sequence. The repeat motif peptoid CD spectra demonstrate that the proportion of N spe in the primary sequence affects the secondary structure but the positioning of these residues has also been reported to be influential in sequences containing a mixture of chiral and non-chiral residues [32]. In addition, the positioning of charged residues such as lysine and histidine has been shown to influence the overall helical character of peptide sequences [58] so the distribution of N ae and N Lys may be of consequence in the peptoid sequences. To investigate this we collected CD spectra for the scrambled sequence peptoids, with both charged side chain variants, in PBS at 20°C. We have subdivided the scrambled sequences into two groups based on the number of adjacent N spe residues in each sequence. The spectra for the first subset of sequences, which contain a maximum of two adjacent N spe, are shown in Figure 3.19a. The spectra for the second subset of sequences, each of which contain up to 4 adjacent N spe are shown in Figure 3.19b.

Each of the scrambled peptoids has a spectrum that indicates helical secondary struc- ture. The positions and intensities of the spectral minima are listed in Table 3.4. Trends observed in the CD spectra of the repeat motif peptoids were also observed in the scram- bled sequences. There is very little variation in the positions of the minima across the scrambled sequences with both charged side chains. However, there is considerable varia- tion in spectral intensity. Contrary to expectation, the repeat motif variants (S1 and S8)

3. Experimental Secondary Structural Characterisation

Fig. 3.19: CD spectra of scrambled sequences peptoids in 0.01 M PBS. (a) Shows the spectra for sequences 1, 6 and 7 and (b) shows the spectra of sequences 2, 3, 4 and 5. The N ae side chain sequences are shown as solid lines and the N ae side chain sequences as dashed lines in the same colours. Peptoid concentration is 25 µM in each case.

were not the most helical of the library by the measure of the spectral intensity at λ2 or the ratio of the minima, R2/1. Substituting N Lys for N ae results in a more helical pep- toid for every sequence variation. This was particularly clear in the case of the sequences with up to 2 adjacent N spe residues (Figure 3.19a) where all of the N Lys sequences have a considerably greater signal intensity that any of the N ae sequences. There is a less obvious distinction between the N Lys and N ae variants of the sequences containing up to 4 adjacent N spe residues, although in each case the N Lys variant does have a greater overall helicity, as measured by the intensity of λ2. There was not a clear variation in spectral intensity between the two subgroups, indicating that increasing the number of adjacent N spe residues without regard to the specific positioning along the chain does not moderate the overall helical content or conformation where measured by spectral inten- sity. This is contrary to the findings of Shin et al. who propose adjacent N spe residues synergistically enhance the helical character of sequences in acetonitrile [32]. However, the Shin et al. study only considered heptamers with 27% N spe (2 residues) whereas the scrambled sequences discussed here have 67% N spe and therefore the synergistic effect of the adjacent residues may be diminished in sequences with a higher overall percentage of helix inducing residues.

There is some variation in the ratio of the two minima and hence the spectral shape of the scrambled sequences. R2/1 for the scrambled sequence are depicted in Figure 3.20a where the magenta bars represent the N ae variants and the teal bars the N Lys variants.

3. Experimental Secondary Structural Characterisation

Side Chain Sequence λmin1 (nm) λmin2 (nm) θM RE,min1 θM RE,min2

N ae S1 202.8 218.7 -25877 -27998 S2 203.0 218.3 -24816 -27683 S3 203.3 219.1 -22942 -29395 S4 203.7 218.6 -24113 -26674 S5 203.1 218.8 -25664 -28854 S6 203.7 218.0 -24494 -25430 S7 203.5 217.6 -28151 -29191 N Lys S8 202.3 218.7 -32320 -36100 S9 202.1 218.9 -35647 -40175 S10 202.8 219.4 -22382 -32026 S11 202.8 218.9 -26140 -28245 S12 202.8 219.2 -24329 -29015 S13 202.8 218.2 -37006 -36236 S14 202.9 218.2 -35648 -34742

Tab. 3.4: Wavelengths of the 2 minima and corresponding MRE values (in units of deg cm2dmol−1) for scrambled sequences 1-14. In the graphic representations of the sequences yellow cir- cles represent N spe residues, pink circles represent N ae residues and teal circles represent N Lys residues.

Neither charged side chain consistently results in a greater value of R2/1 than the other in the equivalent sequence. In 3 out of the 7 sequences the N ae variant has a higher value of R2/1, while in the other 4 R2/1is greater for the N Lys variant. Overall R2/1 varies to a greater extent with the sequence pattern than the structure of the charged side chain, as was also the case for the repeat motif sequences. Sequence 3 in particular stands out as it has a particularly high value of R_2/1 relative to the rest of the scrambled and repeat motif sequences. This sequence is unique among the library for having a string of 4 adjacent N spe residues close in the C-terminal region of the sequence, though it does not have one in the C-terminal position itself. In addition, it is the only sequence with 2 sets of 4 adjacent N spe. Potentially this high density of stabilising aromatic residues could help to stabilise the otherwise flexible C-terminal end of the sequence, resulting in the enhanced helical character of the CD spectrum relative to the others in the library. Alternatively this higher ratio could indicate that this sequence is not monomeric but instead forms helical bundles, though there is no other evidence that this is the case.

The scrambled sequences with N Lys residues vary more in both R_2/1 and overall intensity than their N ae counterparts. This indicates that the positioning of N Lys residues in different places among the N spe causes a greater disruption to the helical structure (or on the other hand promotes folding to a greater extent) than the equivalent positioning of N ae. The standard deviation in MRE at λ2 is approximately 1335 deg cm2 dmol−1 for the N ae sequences and approximately 4275 deg cm2 dmol−1 for the N Lys sequences. The standard deviation in R2/1 was 0.08 for the N ae sequences and 0.16 for the N Lys

3. Experimental Secondary Structural Characterisation

Fig. 3.20: (a) Ratio of mean residue ellipticity at λ2 to mean residue ellipticity at λ1 (R2/1) for

the scrambled sequence library with data for N ae side chains shown in magenta and N Lys side chains in teal. (b) MRE at λ2for each scrambled sequence with N ae variants

shown in magenta and N Lys in teal.

sequences.

It is possible that the increased length of the N Lys side chain relative to N ae allows the peptoids containing the former greater flexibility to minimise electrostatic repulsions between the charged groups and this in turn alters the backbone conformation. The N ae side chains, with less flexibility to rearrange, could induce strain in the peptoid backbone as the entropic penalty associated with rearranging the side chains to minimise electrostatic repulsions would be higher.

The particular positioning of the charged residues may also affect the scrambled se- quence spectra. Rules relating to charges in end positions stabilising helices in peptides may be transferable to peptoids as they do not concern the hydrogen bonding network but rather the helix macrodipole. The C=O dipoles in the peptoid helix described by Armand et al. [5] are almost parallel to the helical axis. This results in a helix macrodipole, similar but opposite in direction to macrodipoles observed in peptide α-helices, since the C=O point towards the N-terminus in the peptoid and the C-terminus in the peptide. The knowledge that charged amino acids near the C-terminus stabilise helices in peptides led Armand et al. to predict that charged residues near the N-terminus may stabilise peptoid helices. It could be argued that this is what we observe in the scrambled sequences as 1, 3, 4 and 5, which all have a charged residue at the N terminus, appear to have more intensely helical CD spectra than the others in the library.

Overall it is very difficult to de-convolute the effects that different factors may have on the CD spectra for the scrambled sequences. Switching the charged side chain in otherwise equivalent sequences has the most distinctive effect on the helicity, with the longer N Lys resulting in more helical sequences that the shorter N ae. The effect of the particular

3. Experimental Secondary Structural Characterisation

placement of residues is more difficult to identify and quantify as the positioning of both the aromatic N spe residues and the charged residues could influence the peptoid secondary structure. In addition the total helical content could be crudely quantified by either the MRE at λ2 or R2/1 but different scrambled sequences appear more helical than others depending on which of these is chosen.