In this article, we review the relevant results obtained during almost 60 years of research on a specific aspect of stereochemistry, namely handedness preference and switches between right-handed and left-handed helical peptide structures generated by protein amino acids or appropriately designed, side-chain modified analogs. In particular, we present and discuss here experimental and theoretical data on three categories of those screw-sense issues: (i) right-handed/left-handed α-helix transitions underwent by peptides rich in Asp, specific Asp β-esters, and Asn; (ii) comparison of the preferred conformations adopted by helical host-guest peptide series, each characterized by an amino acid residue (e.g. Ile or its diastereomer aIle) endowed with two chiral centers in its chemical structure; and (iii) right-handed (type I)/left-handed (type II) poly-(Pro)n helix transitions monitored for peptides rich in Pro itself or its analogs with a pyrrolidine ring substitution, particularly at the biologically important position 4. The unique modular and chiral properties of peptides, combined with their relatively easy synthesis, the chance to shape them into the desired conformation, and the enormous chemical diversity of their coded and non-coded α-amino acid building blocks, offer a huge opportunity to structural chemists for applications to bioscience and nanoscience problems.

Trichogin GA IV (GAIV) is an antimicrobial peptide of the peptaibol family, like the extensively studied alamethicin (Alm). GAIV acts by perturbing membrane permeability. Previous data have shown that pore formation is related to GAIV aggregation and insertion in the hydrophobic core of the membrane. This behavior is similar to that of Alm and in agreement with a barrel-stave mechanism, in which transmembrane oriented peptides aggregate to form a channel. However, while the 19-amino acid long Alm has a length comparable to the membrane thickness, GAIV comprises only 10 amino acids, and its helix is about half the normal bilayer thickness. Here, we report the results of neutron reflectivity measurements, showing that GAIV inserts in the hydrophobic region of the membrane, causing a significant thinning of the bilayer. Molecular dynamics simulations of GAIV/membrane systems were also performed. For these studies we developed a novel approach for constructing the initial configuration, by embedding the short peptide in the hydrophobic core of the bilayer. These calculations indicated that in the transmembrane orientation GAIV interacts strongly with the polar phospholipid headgroups, drawing them towards its N- and C-termini, inducing membrane thinning and becoming able to span the bilayer. Finally, vesicle leakage experiments demonstrated that GAIV activity is significantly higher with thinner membranes, becoming similar to that of Alm when the bilayer thickness is comparable to its size. Overall, these data indicate that a barrel-stave mechanism of pore formation might be possible for GAIV and for similarly short peptaibols despite their relatively small size.

Trichogin GA IV, isolated from the fungus Trichoderma longibrachiatum, is the prototype of lipopeptaibols, the sub-class of short-length peptaibiotics exhibiting membrane-modifying properties. This peptaibol is predominantly folded in a mixed 3(10)-/α- helical conformation with a clear, albeit modest, amphiphilic character, which is likely to be responsible for its capability to perturb bacterial membranes and to induce cell death. In previous papers, we reported on the interesting biological properties of trichogin GA IV, namely its good activity against Gram positive bacteria, in particular methicillin-resistant S. aureus strains, its stability towards proteolytic degradation, and its low hemolytic activity. Aiming at broadening the antimicrobial activity spectrum by increasing the peptide helical amphiphilicity, in this work we synthesized, by solution and solid-phase methodologies, purified and fully characterized a set of trichogin GA IV analogs in which the four Gly residues at positions 2, 5, 6, 9, lying in the poorly hydrophilic face of the helical structure, are substituted by one (position 2, 5, 6 or 9), two (positions 5 and 6), three (positions 2, 5, and 9), and four (positions 2, 5, 6, and 9) Lys residues. The conformational preferences of the Lys-containing analogs were assessed by FT-IR absorption, CD and 2D-NMR techniques in aqueous, organic, and membrane-mimetic environments. Interestingly, it turns out that the presence of charged residues induces a transition of the helical conformation adopted by the peptaibols (from 3(10)- to α-helix) as a function of pH in a reversible process. The role played in the analogs by the markedly increased amphiphilicity was further tested by fluorescence leakage experiments in model membranes, protease resistance, antibacterial and antifungal activities, cytotoxicity, and hemolysis. Taken together, our biological results provide evidence that some of the least substituted among these analogs are good candidates for the development of new membrane-active antimicrobial agents.

Alamethicin, a hydrophobic peptide that is considered a paradigm for membrane channel formation, was uniformly labeled with 15N, reconstituted into oriented phosphatidylcholine bilayers at concentrations of 1 or 5 mol %, and investigated by solid-state NMR spectroscopy as a function of temperature. Whereas the peptide adopts a transmembrane alignment in POPC bilayers at all temperatures investigated, it switches from a transmembrane to an in-plane orientation in DPPC membranes when passing the phase transition temperature. This behavior can be explained by an increase in membrane hydrophobic thickness and the resulting hydrophobic mismatch condition. Having established the membrane topology of alamethicin at temperatures above and below the phase transition, ESEEM EPR was used to investigate the water accessibility of alamethicin synthetic analogues carrying the electron spin label TOAC residue at one of positions 1, 8, or 16. Whereas in the transmembrane alignment the labels at positions 8 and 16 are screened from the water phase, this is only the case for the latter position when adopting an orientation parallel to the surface. By comparing the EPR and solid-state NMR data of membrane-associated alamethicin it becomes obvious that the TOAC spin labels and the cryo-temperatures required for EPR spectroscopy have less of an effect on the alamethicin-POPC interactions when compared to DPPC. Finally, at P/L ratios of 1/100, spectral line broadening due to spin-spin interactions and thereby peptide oligomerization within the membrane were detected for transmembrane alamethicin.

Oligomers of the achiral amino acid Aib adopt helical conformations in which the screw-sense may be controlled by a single N-terminal residue. Using crystallographic and NMR techniques, we show that the left- or right-handed sense of helical induction arises from the nature of the β-turn at the N terminus: the tertiary amino acid L-Val induces a left-handed type II β-turn in both the solid state and in solution, while the corresponding quaternary amino acid L-α-methylvaline induces a right-handed type III β-turn.

Alamethicin is a 19-amino-acid residue hydrophobic peptide of the peptaibol family that has been the object of numerous studies for its ability to produce voltage-dependent ion channels in membranes. In this work, for the first time electron paramagnetic resonance spectroscopy was applied to study the interaction of alamethicin with oriented bicelles. We highlighted the effects of increasing peptide concentrations on both the peptide and the membrane in identical conditions, by adopting a twofold spin labeling approach, placing a nitroxide moiety either on the peptide or on the phospholipids. The employment of bicelles affords additional spectral resolution, thanks to the formation of a macroscopically oriented phase that allows to gain information on alamethicin orientation and dynamics. Moreover, the high viscosity of the bicellar solution permits the investigation of the peptide aggregation properties at physiological temperature. We observed that, at 35°C, alamethicin adopts a transmembrane orientation with the peptide axis forming an average angle of 25° with respect to the bilayer normal. Moreover, alamethicin maintains its dynamics and helical tilt constant at all concentrations studied. On the other hand, by increasing the peptide concentration, the bilayer experiences an exponential decrease of the order parameter, but does not undergo micellization, even at the highest peptide to lipid ratio studied (1:20). Finally, the aggregation of the peptide at physiological temperature shows that the peptide is monomeric at peptide to lipid ratios lower than 1:50, then it aggregates with a rather broad distribution in the number of peptides (from 6 to 8) per oligomer.

Fungal species belonging to the Trichoderma genus are commonly used as biocontrol agents against several crop pathogens. Among their secondary metabolites, peptaibols are helical, antimicrobial peptides, which are structurally stable even under extreme pH and temperature conditions. The promise of peptaibols as agrochemicals is, however, hampered by poor water solubility, which inhibits efficient delivery for practical use in crop protection. Using a versatile synthetic strategy, based on green chemistry procedures, we produced water-soluble analogs of the short-length peptaibol trichogin. Although natural trichogin was inactive against the tested fungal plant pathogens (Botrytis cinerea, Bipolaris sorokiniana, Fusarium graminearum, and Penicillium expansum), three analogs completely inhibited fungal growth at low micromolar concentrations. The most effective peptides significantly reduced disease symptoms by B. cinerea on common bean and grapevine leaves and ripe grape berries without visible phytotoxic effects. An in-depth conformational analysis featuring a 3D-structure–activity relationship study indicated that the relative spatial position of cationic residues is crucial for increasing peptide fungicidal activity.

The lipopeptaibol trichogin GA IV is a natural, non-ribosomally synthesized, antimicrobial peptide remarkably resistant to the action of hydrolytic enzymes. This feature may be connected to the multiple presence in its sequence of the non-coded residue α-aminoisobutyric acid (Aib), which is known to be responsible for the adoption of particularly stable helical structures already at the level of short peptides. To investigate the role of Aib residues on the 3D-structure and bioactivity of trichogin GA IV, we synthesized and fully characterized four analogs where one or two Aib residues are replaced by L-Leu. Our extensive conformational studies (including an X-ray diffraction analysis) and biological assays performed on these analogs showed that the Aib to L-Leu replacements do not affect the resistance to proteolysis, but modulate the bioactivity of trichogin GA IV in a 3D-structure related manner.