Secondary Structural Response to Temperature

Thermally induced unfolding is often observed in peptides via changes in CD spectral shape over a range of temperatures. In the simplest case the structural transition observed is from a folded state at low temperatures to an unfolded state at higher temperatures. The midpoint of the transition represents the melting temperature of the peptoid. The temperature dependent CD spectra for a given peptide (or peptoid) can be used to cal- culate thermodynamic properties including the free energy of unfolding. The melt curves for peptides that show cooperative thermal unfolding are characteristically sigmoidal in shape [61].

Peptoids have been widely reported to exhibit remarkable thermal stability, main- taining their folded conformations at much higher temperatures than many antimicrobial peptides [2,36,62]. We investigated the thermal unfolding of the repeat motif and scram- bled sequence peptoids in PBS by measuring ellipticity at the wavelength of the second spectral minimum (λ2) as a function of temperature, between 20 °C and 90 °C. Addition- ally, full spectra were collected at 10°C intervals to give an indication of how the spectral shape varied with temperature.

The melt curves for the repeat motif peptoids are shown in Figure 3.25a. The change in intensity of λ2between 20°C and 90 °C is small, providing very little evidence of thermally induced unfolding of any of the sequences. Each peptoid shows a slight decrease in signal intensity at λ2 with increasing temperature. The total loss of spectral intensity was in the region of 10-15% for each sequence over the temperature range studied.

During the course of the peptoid melts there was very little change in spectral shape, which can be seen in the spectra collected at 10 °C intervals. The melt spectra for RM3 are shown in Figure 3.25b as an example of this behaviour and are generally representative of the behaviour of all the sequences. From the full spectra it is evident that the positions of the minima gradually shift to longer wavelengths over the course of the melt. This shift is exaggerated in the case of λ1 as the loss of spectral resolution occurs at longer wavelengths as temperature increases. For this reason the intensity of λ2 was chosen over λ1 for monitoring.

The two-state analysis often applied to peptides could not be applied to this data due to the very slow rate of change in the CD spectra over the measured temperature range. Though each peptoid is clearly in a folded state at 20°C there is no convergence of the spectra over certain temperature ranges (within those studied). It is possible that lower temperatures could induce further folding and therefore we do not know what the spectra of the maximally folded and minimally folded states would look like and hence

3. Experimental Secondary Structural Characterisation

Fig. 3.25: (a) Melt curves for peptoids RM1-6 in 0.01 M PBS. Peptoid concentration is 25 µM in each case. (b) Full spectra for RM3 in PBS collected every 10 °C over the course of a melt, from 20 °C (black line) to 90 °C (purple line). On completion of the melt the sample temperature was returned to 20°C and an addition spectrum collected which is shown as the black dashed line.

cannot meaningfully fit a two state model two the data. Given the many reports in the literature asserting that peptoid secondary structures are extremely robust, particularly in response to temperature, the melt curves obtained for the repeat motif sequences were not unexpected. Given however, that the sequences reported in the literature all contain a regular repeating motif assumed to be important in stabilising the secondary structure, it is interesting to consider how the scrambling of sequences affects this and whether disruption to the regular patterning of N spe residues is influential.

The melt curves for the scrambled sequences in our peptoid library are shown in Figure 3.26a and 3.26b with the library again divided into two by the number of adjacent N spe residues in each sequence. Like the repeat motifs, the scrambled sequences showed very little change in spectral shape and intensity over the range of temperatures studied. The

3. Experimental Secondary Structural Characterisation

Fig. 3.26: (a) Melt curves for the scrambled sequences with a maximum of 2 adjacent N spe residues in 0.01 M PBS. (b) Melt curves for the scrambled sequences with a maximum of 4 adjacent N spe residues in 0.01 M PBS. Peptoid concentration is 25 µM in each case. (c) Melt curves for every scrambled sequence with data normalised to the mean residue ellipticity at 20°C. The sequences with N ae charged side chains are shown in magenta and the sequences with N Lys charged side chains are shown in teal with sequences 1 and 8 (the repeat motif 2 sequences) shown as solid lines and the scrambled variants shown in dotted and dashed lines.

melt curves normalised to the spectral intensity at 20 °C are shown in Figure 3.26c. The sequences with the N ae charged side chain are shown in pink and the N Lys charged side chain shown in teal. Peptoids S1 and S8 (which are the repeat motif sequences RM3 anf RM4) are shown as solid lines while the scrambled versions are dotted and dashed. The normalised melt curves reveal that, overall, the sequences with the N ae charged side chains show a greater loss of spectral intensity over the course of the melts than the sequences with the N Lys charged side chains. The exception to this is S1, the repeat motif N ae sequence. The repeat motif N Lys sequence also shows the smallest overall loss of intensity of any of the sequences, indicating that for all the sequences containing 4 cationic and 8 aromatic residues, the two containing the repeat motif were the least sensitive to temperature change within the range investigated.

It seems intuitive that RM3 and RM4 exhibit the least thermal unfolding behaviour of the scrambled sequences. These have the most regular distribution of charge along the backbone and therefore also the most regular distribution of hydrophobic residues. The sequences with a maximum of 4 adjacent N spe residues showed a greater loss of intensity

3. Experimental Secondary Structural Characterisation

at λ2 over the course of the melt than those with a maximum of 2 adjacent N spe residues. The sequences with N ae side chains and a maximum of 2 adjacent N spe residues showed a 17-20% loss of intensity where their N Lys counterparts had a 10-15% loss of intensity. The sequences with N ae side chains a maximum of 4 adjacent N spe residues showed a 19-23% loss of intensity and their N Lys counterparts had a 17-19% loss of intensity. It therefore appears that a regular distribution of charge along the peptoid stabilises the sequences to a small extent at increasing temperatures.