Next, we assessed if the effects observed on peptide potency upon constraining could be correlated with changes in peptide conformation (Fig. ). The control N-Myc73-94 and N-Myc73-94-E86C/S90C variants showed similar CD spectra, characterized by the presence of a minima in mean residue ellipticity (MRE) at a wavelength of 200 nm, slightly shifted to 205 nm for N-Myc73-94-E86C/S90C-ox (Fig. and). Qualitatively, the variants exhibited only limited differences in helicity, with all adopting predominantly random coil conformations in solution (estimated % helicity ~ 17 to 21; Table ). In contrast, N-Myc73-94-N85C/G89C-mal exhibited a different CD spectrum, indicating that the mal constraint in this position destabilizes the random coil conformation of the peptide and biases it in favor of another defined conformation (Fig. ).