Exploring Peptide Conformation through CD and FTIR Spectroscopy: Effects of Glycine Exclusion in Opioid-Based Chimeric Peptides

In our earlier work, we reported the designing and synthesis of three synthetic chimeric analogs of YFa (YGGFMKKKFMRFamide), [Ser5]YFa (MOD-1), and [O-Glu-Ser5]YFa (MOD-3), along with [O-Gal-Ser5]YFa (MOD-5). These peptides demonstrated a tendency to adopt a helical conformation in the presence of the membrane-mimicking solvent trifluoroethanol (TFE). However, in comparison to the parent chimeric peptide MOD-1, a noticeable decrease in helicity was observed in the glycosylated analogs MOD-3, MOD-5, and [O-Gal-Ser5]YFa, indicating that glycosylation alters their structural stability. Building on these findings, we have now synthesized three new conformationally constrained analogs: YGFMKKKFMRFamide (MOD-2), YGFSKKKFMRFamide (MOD-4), and YGFS(O-Glu)KKKFMRFamide (MOD-6), derived from YFa, (MOD-1), and [Ser4]YFa (MOD-3) , respectively, has been designed and synthesized to scrutinize the resulting conformational changes in the chimeric peptides due to the removal of glycine from the active opioid sequence of chimeric peptides. These modifications were introduced to further investigate the resulting conformational changes in these chimeric peptides. We employed both Fourier Transform Infrared (FTIR) and Circular Dichroism (CD) spectroscopic techniques to comprehensively study their conformational stability and flexibility under different conditions, including variations in pH, TFE concentration, and the presence of perturbants. FTIR spectroscopy revealed that these analogs, both in solid and solution phases, predominantly exhibit vibrational characteristics associated with 3-10 helix and/or β-turn (type III) conformations, which are largely stabilized by 1←3 intramolecular hydrogen bonding. CD analysis also showed that the chimeric peptides changed from random coil to α-helical structure with the removal of glycine residues, reflecting an important impact of glycine on peptide folding. pH-dependent analysis also revealed that all of the analogs retained their helical structure under pH values ranging from 6 to 9, reflecting structural stability under physiological conditions. Additionally, experiments on the conformational tendencies in different concentrations of the helicogenic solvent TFE revealed that the increasing concentration of TFE favored higher structural orderliness, which was then disrupted when perturbants such as urea were added. Such knowledge sheds important light on the function of glycosylation and conformational constraint in regulating the structural dynamics of chimeric peptides, setting the stage for the rational design of peptide-based therapeutics with greater bioavailability and stability for neurological use.