Marta De Zotti

Alamethicin Topology in Phospholipid Membranes by Oriented Solid-state NMR and EPR Spectroscopies : a Comparison

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.

Trichogin GA IV: an antibacterial and protease-resistant peptide

The antibacterial and hemolytic activities of the amphiphilic helical, membrane‐active, lipopeptaibol trichogin GA IV and its [Leu11‐OMe] analogue were compared to those of the partially helical or non‐helical 8‐meric or 4‐meric, C‐terminal short sequences, respectively. The study on trichogin GA IV was extended to several methicillin‐resistant Staphylococcus aureus strains. Using a large set of enzymes, we also evaluated the resistance to proteolysis of all of the four peptides. Copyright

Left-handed helical preference in an achiral peptide chain is induced by an l-amino acid in an N-terminal type II β-turn

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.

Structure of Self-Aggregated Alamethicin in ePC Membranes Detected by Pulsed Electron-Electron Double Resonance and Electron Spin Echo Envelope Modulation Spectroscopies

PELDOR spectroscopy was exploited to study the self-assembled super-structure of the [Glu(OMe)(7,18,19)]alamethicin molecules in vesicular membranes at peptide to lipid molar ratios in the range of 1:70-1:200. The peptide molecules were site-specifically labeled with TOAC electron spins. From the magnetic dipole-dipole interaction between the nitroxides of the monolabeled constituents and the PELDOR decay patterns measured at 77 K, intermolecular-distance distribution functions were obtained and the number of aggregated molecules (n approximately 4) was estimated. The distance distribution functions exhibit a similar maximum at 2.3 nm. In contrast to Alm16, for Alm1 and Alm8 additional maxima were recorded at 3.2 and approximately 5.2 nm. From ESEEM experiments and based on the membrane polarity profiles, the penetration depths of the different spin-labeled positions into the membrane were qualitatively estimated. It was found that the water accessibility of the spin-labels follows the order TOAC-1 > TOAC-8 approximately TOAC-16. The geometric data obtained are discussed in terms of a penknife molecular model. At least two peptide chains are aligned parallel and eight ester groups of the polar Glu(OMe)(18,19) residues are suggested to stabilize the self-aggregate superstructure.

Handedness preference and switching of peptide helices. Part I: Helices based on protein amino acids.

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. Copyright

Synthesis, preferred conformation, protease stability, and membrane activity of heptaibin, a medium‐length peptaibiotic

The medium‐length peptaibiotics are characterized by a primary structure of 14–16 amino acid residues. Despite the interesting antibiotic and antifungal properties exhibited by these membrane‐active peptides, their exact mechanism of action is still unknown. Here, we present our results on heptaibin, a 14‐amino acid peptaibiotic found to exhibit antimicrobial activity against Staphylococcus aureus. We carried out the very challenging synthesis of heptaibin on solid phase and a detailed conformational analysis in solution. The peptaibiotic is folded in a mixed 310‐/α‐helix conformation which exhibits a remarkable amphiphilic character. We also find that it is highly stable toward degradation by proteolytic enzymes and nonhemolytic. Finally, fluorescence leakage experiments using small unilamellar vesicles of three different compositions revealed that heptaibin, although uncharged, is a selective compound for permeabilization of model membranes mimicking the overall negatively charged surface of Gram‐positive bacteria. This latter finding is in agreement with the originally published antimicrobial activity data. Copyright

PELDOR conformational analysis of bis-labeled alamethicin aggregated in phospholipid vesicles.

Alamethicin (Alm) is a linear peptide antibiotic of great interest for its capability to form self-assembled ion channels in lipid membranes. Here, the pulsed electron-electron double resonance technique was used to obtain unique conformational information on the aggregated peptide in the lipid membrane-bound state. Since a specific helical conformation implies a given length to the peptide molecule, a distance r was measured at the nanometer scale via the electron dipole-dipole interaction between two 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin labels synthetically incorporated at positions 1 and 16 of this 19-mer peptide. Two data sets were collected (at 77 K): (i) from aggregates of Alm in hydrated egg-yolk phosphocholine (ePC) vesicles (at peptide-to-lipid ratios of 1:200 and 1:75) and (ii) from nonaggregated Alm in pure (nonhydrated) ePC and in solvents of different polarity. The intramolecular distance between the two labels obtained in this manner is in excellent agreement with that calculated on the basis of an almost fully developed alpha-helical conformation for this peptide and is found to be independent of the molecular aggregated state and the environment polarity as well.

Alamethicin in bicelles: orientation, aggregation, and bilayer modification as a function of peptide concentration.

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.

Concerning Selectivity in the Oxidation of Peptides by Dioxiranes. Further Insight into the Effect of Carbamate Protecting Groups

With use of methyl(trifluoromethyl)dioxirane (TFDO), the oxidation of some tripeptide esters protected at the N-terminus with carbamate or amide groups could be achieved efficiently under mild conditions with no loss of configuration at the chiral centers. Expanding on preliminary investigations, it is found that, while peptides protected with amide groups (PG = Ac-, Tfa-, Piv-) undergo exclusive hydroxylation at the side chain, their analogues bearing a carbamate group (PG = Cbz-, Moc-, Boc-, TcBoc-) give competitive and/or concurrent hydroxylation at the terminal N-H moiety. Valuable nitro derivatives are also formed as a result of oxidative deprotection of the carbamate group with excess dioxirane. A rationale is proposed to explain the dependence of the selectivity upon the nature of the protecting group.

Mimicking Nature: A Novel Peptide-based Bio-inspired Approach for Solar Energy Conversion

A bioinspired approach is applied to photoelectric conversion devices. A 3(10)-helical hexapeptide bearing a pyrene unit is immobilized on a gold-covered TiO2 surface. The device is integrated for the first time in a dye-sensitized solar cell, exhibiting stability after several measurements. The approach could have promising applications in the field of optoelectronics.