Outlook

The characteristic features associated with α-helical structure in peptides are present in the CD spectra for the peptoids, despite the lack of amide hydrogen prohibiting the

3. Experimental Secondary Structural Characterisation

formation of inter-backbone hydrogen bond stabilised structures in peptoids. The spectra we collected also resemble the spectra of pure N spe peptoid helices and variations thereof published in the literature [5, 31, 36, 63]. It therefore seems likely that the inclusion of N spe residues in the primary sequences that we have studied is responsible for inducing their helical secondary structure. This is clearly demonstrated in the case of substituting N spe for its opposite handed counterpart N rpe, or the similar but achiral N phe. The former inverts the CD spectrum and the latter results in a the total loss of CD signal. Additionally, within the repeat motif sequences the total percentage of chiral N spe residues varies and the CD spectra increase in intensity as the overall proportion of N spe increases, indicating that the sequences become more helical. These results demonstrate the power of the choice of chiral monomer in controlling the overall secondary structure, including its handedness.

While the increase in helical character with increasing proportion of N spe was perhaps predictable, less so was the influence of the structure of the charged side chain group. Peptoids with N ae side chains appear to be less helical than their identical sequence counterparts with the longer N Lys side chains. This was observed to be the case, without exception, for every sequence explored within this chapter. The physical reasons for this cannot be inferred from CD spectra alone, however it could result from the increased length and therefore flexibility of N Lys relative to N ae allowing sequences to rearrange, minimising electrostatic repulsions and allowing the peptoid backbone to adopt a folded conformation at lesser cost.

In addition, across the library as a whole, the N Lys sequences were considerably more varied in spectral intensity and shape than their N ae counterparts. This indicates that the former side chain is a better choice for the development of combinatorial libraries with conformational diversity than the latter. It would be interesting to investigate the structure of additional peptoids with the same primary sequences as those studied here but with charged side chains of varying length. The peptoid analogue of ornithine would be a good starting point for such an investigation as it is intermediate in length between N ae and N Lys but otherwise structurally analogous. Further investigations into the ef- fect of altering the length of the charged side chain group in these sequences may also provide insight into the self-association which is observed in (N aeN speN spe)3 but not (N LysN speN spe)3.

The conformational diversity within the scrambled sequence spectra is very difficult to correlate to any particular property of the the primary sequences. It seems likely that the positioning of both the charged and the aromatic residues affect the overall secondary structure and therefore it is difficult to de-convolute the effect of each of these on any individual spectrum. Further investigation into a new library of sequences with systematic substitution of residues in particular positions may help rationalise our results here in the

3. Experimental Secondary Structural Characterisation

future. Nevertheless, we have clearly demonstrated that sequences that do not contain a standard repeating motif of residues do still adopt stable helical conformations in aqueous solution. These structures are thermally robust over a wide range of temperatures, as are the repeat motif sequences. This is a strong indication that the helices formed in peptoids are stabilised by non-covalent interactions.

The spectral intensity for the peptoids is increased in octanol relative to PBS, with the intensity of the second minima greatly enhanced relative to the first. This indicates that the peptoids adopt a distinctly different structural conformations in the two solvent conditions, evident in the change in overall spectral shape. This provides the basis for the work in the following chapters, where we investigate these structural differences compu- tationally and then use them to examine the nature of interactions between peptoids and model biological membranes, via CD spectroscopy.

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4.0

Computational Structure Prediction

In this chapter we use molecular dynamics (MD) to study peptoid secondary structure. The results presented here concern the adaptation of the General Amber Force Field for peptoid simulations and subsequent investigation of the structural preferences of the repeat motif sequences in water and octanol. Results are discussed in the context of the experimental data presented previously and the literature on peptoid simulation, which is reviewed at the beginning of this chapter.

4.1

Introduction and Review of Literature

Application of computational techniques to the study of peptoids could be extremely use- ful in understanding relationships between primary sequence and secondary structure. In particular, such methods can reveal molecular level details which are inaccessible by ex- perimental techniques. Since the first attempts at computational peptoid studies, these have been enhancing our understanding of the peptoid folding and stabilisation process by enabling researchers to examine in detail the conformational rearrangements of pep- toids in various conditions. A review of the published efforts to elucidate precise peptoid secondary structural characteristics is presented in the following sections. This concerns the application of quantum chemical methods to predict the properties of small model systems and subsequently attempts to accurately capture peptoid behaviour through the use of MD with atomistic force fields.

4.1.1

Strategies for Peptoid Simulation

The computational approaches to peptoid structure prediction have largely followed the same routes as those applied to protein folding. Unlike protein and peptide simulation however, the field of peptoid simulation is still relatively new and there is considerable scope for development [1]. Ab initio methods, which are too computationally expensive to apply to large systems, have been carried out on peptoid monomers and fragments [2–4]. The data obtained from these calculations has been utilised in building atomistic models and coarse grained models in turn [1]. Due to the highly involved and time-consuming