Dynamics of Formation of a Helix–Turn–Helix Structure in a Membrane‐Active Peptide: A Time‐Resolved Spectroscopic Study

Trichogin GA IV (nOct-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-IleLol, in which nOct is n-octanoyl and Lol is leucinol) is an antimicrobial peptide member of the lipopeptaibol family, a unique group of membrane-active compounds of fungal origin, characterized by a high content of the nonproteinogenic C-disubstituted glycine Aib (a-aminoisobutyric acid). Owing to the gem-dimethyl substitution on the C atom, Aib exhibits a strong propensity to induce 310/a-helical conformations in peptides, and b-turns as well. X-ray diffraction studies on trichogin GA IV show that the first few residues at the N terminus adopt a right-handed 310-helix, while the rest of the peptide chain forms a distorted, right-handed a-helix. A NMR investigation indicates that this mixed 310/a-helical structure is basically maintained in solution, although the C-terminal region appears to be in equilibrium with disordered states. The hypothesis that the flexible -Gly5-Gly6motif gives rise to a hinge point between the two helical segments has also been advanced. Therefore, trichogin GA IV could represent an interesting model for examining an elementary step in protein folding, that is, the coalescence of a pair of helical segments to form a helix–turn–helix motif. Knowledge of the kinetics of formation of such locally structured domains would be useful for discriminating between different folding mechanisms. To this goal, we applied time-resolved optical spectroscopy techniques, from the nanosecond to the microsecond timescale, on a new analogue of trichogin GA IV with the sequence Fmoc-Aib-Gly-Leu-Aib-Gly-Gly-Leu-TOAC-Gly-Ile-Leu-OMe, denoted F0T8. F0T8 differs from trichogin GA IV by the substitution of i) the n-octanoyl lipid chain at the N terminus by Fmoc (fluoren-9-ylmethyloxycarbonyl), a well-known fluorescent group, ii) -Aib8by TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid), and iii) the 1,2-aminoalcohol Lol moiety group at the C terminus by Leu-OMe. Permeabilization experiments on phospholipid bilayers (Supporting Information) proved that these substitutions enhance the membrane activity of the peptide investigated. Fmoc populates singlet and triplet states with high yields, while TOAC, a stable nitroxidebased radical species, exhibits a strong quenching activity on the excited states of aromatic molecules. We have recently shown that for Fmoc···TOAC interchromophoric distances larger than 9 A, long-range dipolar interactions determine the photophysical relaxation of the interacting pair (Fçrster energy transfer), while for distances shorter than 4–5 A an electron exchange (Dexter energy transfer) mechanism largely predominates. Photophysical-quenching efficiencies were determined with respect to the reference compound Fmoc-Aib-Gly-Leu-Aib-GlyGly-Leu-Aib-Gly-Ile-Leu-OMe (denoted as F0), in which the TOAC quencher is missing. To characterize the conformational heterogeneity and the dynamical behaviour of F0T8, we carried out a time-resolved spectroscopic investigation in three solvents: i) methanol, a polar medium exhibiting a relatively low viscosity and a low competition for hydrogen bond formation; ii) glycerol (Glc) /methanol (8:2, v/v), a mixed solvent showing strong temperature-dependent viscosity that allowed us to sample the peptide dynamics over a broad range of conditions; iii) sodium dodecyl sulfate (SDS) micelles, a well-known membrane-mimicking environment that reproduces the encapsulation effect of an organized medium on the conformational heterogeneity of F0T8. The circular dichroism (CD) spectra of F0T8 in the three solvents are reported in Figure 1.