Isolated α-turns in peptides: a selected literature survey.

The results of classifying into various types the 68 examples of isolated α-turns in the X-ray diffraction crystal structures of peptides documented in the literature are presented and discussed in this review article. α-Turns characterized by the trans disposition of all ω torsion angles are common for the backbone linear peptides investigated. In contrast, the cis arrangement of the N-terminal (ωi + 1 ) torsion angle, among those generated by the three residues internal to the α-turn, is a peculiar feature of 65% of the cyclic peptides. Among linear and cyclic peptides featuring the all-trans disposition of the ω torsion angles, only one third of the α-turns display φ,ψ values not too far from those characterizing regular α-helices. In general, our findings, taken together, suggest that a significant conformational diversity is compatible with the formation of an intramolecularly H-bonded C13 -member pseudocycle (α-turn) in linear and cyclic peptides.

Peptide Self-Assembled Nanostructures: From Models to Therapeutic Peptides.

Self-assembly is the most suitable approach to obtaining peptide-based materials on the nano- and mesoscopic scales. Applications span from peptide drugs for personalized therapy to light harvesting and electron conductive media for solar energy production and bioelectronics, respectively. In this study, we will discuss the self-assembly of selected model and bioactive peptides, in particular reviewing our recent work on the formation of peptide architectures of nano- and mesoscopic size in solution and on solid substrates. The hierarchical and cooperative characters of peptide self-assembly will be highlighted, focusing on the structural and dynamical properties of the peptide building blocks and on the nature of the intermolecular interactions driving the aggregation phenomena in a given environment. These results will pave the way for the understanding of the still-debated mechanism of action of an antimicrobial peptide (trichogin GA IV) and the pharmacokinetic properties of a peptide drug (semaglutide) currently in use for the therapy of type-II diabetes.

Is Cys(MTSL) the Best α-Amino Acid Residue to Electron Spin Labeling of Synthetically Accessible Peptide Molecules with Nitroxides?

Electron paramagnetic resonance spectroscopy, particularly its pulse technique double electron-electron resonance (DEER) (also termed PELDOR), is rapidly becoming an extremely useful tool for the experimental determination of side chain-to-side chain distances between free radicals in molecules fundamental for life, such as polypeptides. Among appropriate probes, the most popular are undoubtedly nitroxide electron spin labels. In this context, suitable biosynthetically derived, helical regions of proteins, along with synthetic peptides with amphiphilic properties and antibacterial activities, are the most extensively investigated compounds. A strict requirement for a precise distance measurement has been identified in a minimal dynamic flexibility of the two nitroxide-bearing α-amino acid side chains. To this end, in this study, we have experimentally compared in detail the side-chain mobility properties of the two currently most widely utilized residues, namely, Cys(MTSL) and 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). In particular, two double-labeled, chemically synthesized 20-mer peptide molecules have been adopted as appropriate templates for our investigation on the determination of the model intramolecular separations. These double-Cys(MTSL) and double-TOAC compounds are both analogues of the almost completely rigid backbone peptide ruler which we have envisaged and 3D structurally analyzed as our original, unlabeled compound. Here, we have clearly found that the TOAC side-chain labels are largely more 3D structurally restricted than the MTSL labels. From this result, we conclude that the TOAC residue offers more precise information than the Cys(MTSL) residue on the side chain-to-side chain distance distribution in synthetically accessible peptide molecules.

Cα-Methyl-l-valine: A Preferential Choice over α-Aminoisobutyric Acid for Designing Right-Handed α-Helical Scaffolds.

In synthetic peptides containing Gly and coded α-amino acids, one of the most common practices to enhance their helical extent is to incorporate a large number of l-Ala residues along with noncoded, strongly foldameric α-aminoisobutyric acid (Aib) units. Earlier studies have established that Aib-based peptides, with propensity for both the 310- and α-helices, have a tendency to form ordered three-dimensional structure that is much stronger than that exhibited by their l-Ala rich counterparts. However, the achiral nature of Aib induces an inherent, equal preference for the right- and left-handed helical conformations as found in Aib homopeptide stretches. This property poses challenges in the analysis of a model peptide helical conformation based on chirospectroscopic techniques like electronic circular dichroism (ECD), a very important tool for assigning secondary structures. To overcome such ambiguity, we have synthesized and investigated a thermally stable 14-mer peptide in which each of the Aib residues of our previously designed and reported analogue ABGY (where B stands for Aib) is replaced by Cα-methyl-l-valine (L-AMV). Analysis of the results described here from complementary ECD and 1H nuclear magnetic resonance spectroscopic techniques in a variety of environments firmly establishes that the L-AMV-containing peptide exhibits a significantly stronger preference compared to that of its Aib parent in terms of conferring α-helical character. Furthermore, being a chiral α-amino acid, L-AMV shows an intrinsic, extremely strong bias for a quite specific (right-handed) screw sense. These findings emphasize the relevance of L-AMV as a more appropriate unit for the design of right-handed α-helical peptide models that may be utilized as conformationally constrained scaffolds.

Probing the E/K Peptide Coiled-Coil Assembly by Double Electron-Electron Resonance and Circular Dichroism.

Double electron-electron resonance (DEER, also known as PELDOR) and circular dichroism (CD) spectroscopies were explored for the purpose of studying the specificity of the conformation of peptides induced by their assembly into a self-recognizing system. The E and K peptides are known to form a coiled-coil heterodimer. Two paramagnetic TOAC α-amino acid residues were incorporated into each of the peptides (denoted as K** and E**), and a three-dimensional structural investigation in the presence or absence of their unlabeled counterparts E and K was performed. The TOAC spin-labels, replacing two Ala residues in each compound, are covalently and quasi-rigidly connected to the peptide backbone. They are known not to disturb the native structure, so that any conformational change can easily be monitored and assigned. DEER spectroscopy enables the measurement of the intramolecular electron spin-spin distance distribution between the two TOAC labels, within a length range of 1.5-8 nm. This method allows the individual conformational changes for the K**, K**/E, E**, and E**/K molecules to be investigated in glassy frozen solutions. Our data reveal that the conformations of the E** and K** peptides are strongly influenced by the presence of their counterparts. The results are discussed with those from CD spectroscopy and with reference to the already reported nuclear magnetic resonance data. We conclude that the combined DEER/TOAC approach allows us to obtain accurate and reliable information about the conformation of the peptides before and after their assembly into coiled-coil heterodimers. Applications of this induced fit method to other two-component, but more complex, systems, like a receptor and antagonists, a receptor and a hormone, and an enzyme and a ligand, are discussed.

Effect on the Conformation of a Terminally Blocked, (E) ß,γ-Unsaturated δ-Amino Acid Residue Induced by Carbon Methylation.

Peptides are well-known to play a fundamental therapeutic role and to represent building blocks for numerous useful biomaterials. Stabilizing their active 3D-structure by appropriate modifications remains, however, a challenge. In this study, we have expanded the available literature information on the conformational propensities of a promising backbone change of a terminally blocked δ-amino acid residue, a dipeptide mimic, by replacing its central amide moiety with an (E) Cß═Cγ alkene unit. Specifically, we have examined by DFT calculations, X-ray diffraction in the crystalline state, and FT-IR absorption/NMR spectroscopies in solution the extended vs folded preferences of analogues of this prototype system either unmodified or possessing single or multiple methyl group substituents on each of its four -CH2-CH═CH-CH2- main-chain carbon atoms. The theoretical and experimental results obtained clearly point to the conclusion that increasing the number of adequately positioned methylations will enhance the preference of the original sequence to fold, thus opening interesting perspectives in the design of conformationally constrained peptidomimetics.

Electron spin echo detection of stochastic molecular librations: Non-cooperative motions on solid surface.

In frozen biological media and molecular glasses only restricted motions exist; because of the weakness and disorder of intermolecular bonds these motions may have stochastic nature. Electron spin echo (ESE) spectroscopy of spin-labeled molecules allows detecting their restricted stochastic rotations (stochastic molecular librations). As in molecular disordered media motions may be highly cooperative, it would be desirable to investigate their spectroscopic manifestation also in the systems where cooperative effects would be certainly ruled out. In this work, ESE of spin-labeled molecules adsorbed on inorganic SiO2 surface was investigated in a wide temperature range. The rate of motion-induced spin relaxation was found to become measurable above 130 K, increasing with temperature and attaining then a saturating behavior with a well-defined maximum near 250 K. For two types of molecules differing remarkably in their size and polarity (a small highly-polar nitroxide radical and a large spin-labeled peptide), quite similar results were obtained. This saturating behavior was quantitatively reproduced in simulations within a simple model of jump between two close orientations. Comparison with experiment allowed estimate that at 250 K the correlation time of the motion τc is of the order of several tens of nanoseconds and the angle α between two orientations is around 0.02 rad. As the found saturating behavior is a property of individual motions, for any other molecular system an excess of the spin relaxation rate above the maximum found here for adsorbed molecules may be ascribed to cooperative motions. Comparison with literature data on molecular systems of different origin has shown that effects of cooperativity indeed are present and, moreover, may be very essential.

Correction: An EPR study of ampullosporin A, a medium-length peptaibiotic, in bicelles and vesicles.

Correction for 'An EPR study of ampullosporin A, a medium-length peptaibiotic, in bicelles and vesicles' by Marco Bortolus et al., Phys. Chem. Chem. Phys., 2016, 18, 749-760.

Isolated α-turn and incipient γ-helix.

The unique abilities of homo-oligo-adamantyl peptides to adopt α- and γ-turn conformations are demonstrated by X-ray diffraction, and NMR and FT-IR absorption spectroscopies. Assembled by an Ugi multiple component reaction strategy, N α-formyl-adamantyl tripeptide iso-propyl and tert-butyl amides are respectively found to adopt an isolated α-turn and an incipient γ-helix conformation by X-ray diffraction crystallography. The shortest example of a single α-turn with ideal geometry is observed in the crystalline state. In solution both peptides predominantly assume γ-helical structures.

Trichogin GA IV Alignment and Oligomerization in Phospholipid Bilayers.

Trichogin GA IV is a short peptaibol with antimicrobial activity. This uncharged, but amphipathic, sequence is aligned at the membrane interface and undergoes a transition to an aggregated state that inserts more deeply into the membrane, an assembly that predominates at a peptide-to-lipid ratio (P/L) of 1:20. In this work, the natural trichogin sequence was prepared and reconstituted into oriented lipid bilayers. The 15 N NMR chemical shift is indicative of a well-defined alignment of the peptide parallel to the membrane surface at P/Ls of 1:120 and 1:20. When the P/L is increased to 1:8, an additional peptide topology is observed that is indicative of a heterogeneous orientation, with helix alignments ranging from around the magic angle to perfectly in-plane. The topological preference of the trichogin helix for an orientation parallel to the membrane surface was confirmed by attenuated total reflection FTIR spectroscopy. Furthermore, 19 F CODEX experiments were performed on a trichogin sequence with 19 F-Phe at position 10. The CODEX decay is in agreement with a tetrameric complex, in which the 19 F sites are about 9-9.5 Šapart. Thus, a model emerges in which the monomeric peptide aligns along the membrane surface. When the peptide concentration increases, first dimeric and then tetrameric assemblies form, made up from helices oriented predominantly parallel to the membrane surface. The formation of these aggregates correlates with the release of vesicle contents including relatively large molecules.

Heterochiral Ala/(αMe)Aze sequential oligopeptides: Synthesis and conformational study.

α-Amino acid residues with a ϕ,ψ constrained conformation are known to significantly bias the peptide backbone 3D structure. An intriguing member of this class of compounds is (αMe)Aze, characterized by an Nα -alkylated four-membered ring and Cα -methylation. We have already reported that (S)-(αMe)Aze, when followed by (S)-Ala in the homochiral dipeptide sequential motif -(S)-(αMe)Aze-(S)-Ala-, tends to generate the unprecedented γ-bend ribbon conformation, as formation of a regular, fully intramolecularly H-bonded γ-helix is precluded, due to the occurrence of a tertiary amide bond every two residues. In this work, we have expanded this study to the preparation and 3D structural analysis of the heterochiral (S)-Ala/(R)-(αMe)Aze sequential peptides from dimer to hexamer. Our conformational results show that members of this series may fold in type-II ß-turns or in γ-turns depending on the experimental conditions.

Controlling the Formation of Peptide Films: Fully Developed Helical Peptides are Required to Obtain a Homogenous Coating over a Large Area.

The influence of conformational dynamics on the self-assembly process of a conformationally constrained analogue of the natural antimicrobial peptide Trichogin GA IV was analysed by spectroscopic methods, microscopy imaging at nanometre resolution, and molecular dynamics simulations. The formation of peptide films at the air/water interface and their deposition on a graphite or a mica substrate were investigated. A combination of experimental evidence with molecular dynamics simulation was used to demonstrate that only the fully developed helical structure of the analogue promotes formation of ordered aggregates that nucleate the growth of micrometric rods, which give rise to homogenous coating over wide regions of the hydrophilic mica. This work proves the influence of helix flexibility on peptide self-organization and orientation on surfaces, key steps in the design of bioinspired organic/inorganic hybrid materials.

Peptide antibiotic trichogin in model membranes: Self-association and capture of fatty acids.

The antimicrobial action of peptides in bacterial membranes is commonly related to their mode of self-assembling which results in pore formation. To optimize peptide antibiotic use for therapeutic purposes, a study on the concentration dependence of self-assembling process is thus desirable. In this work, we investigate this dependence for peptaibol trichogin GA IV (Tric) in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane in the range of peptide concentrations between 0.5 and 3.3 mol%. Pulsed double electron-electron resonance (PELDOR) applied on spin-labeled peptide analogs highlights the onset of peptide dimerization above a critical peptide concentration value, namely ~ 2 mol%. Electron spin echo (ESE) envelope modulation (ESEEM) for D2O-hydrated bilayers shows that dimerization is accompanied by peptide re-orientation towards a trans-membrane disposition. For spin-labeled stearic acids (5-DSA) in POPC bilayers, the study of ESE decays and ESEEM in the presence of a deuterated peptide analog indicates that above the critical peptide concentration the 5-DSA molecules are attracted by peptide molecules, forming nanoclusters. As the 5-DSA molecules represent a model for the behavior of fatty acids participating in bacterial membrane homeostasis, such capturing action by Tric may represent an additional mechanism of its antibiotic activity.

A novel peptide conformation: the γ-bend ribbon.

Unlike the extensively investigated relationship between the peptide ß-bend ribbon and its prototypical 310-helix conformation, the corresponding relationship between the narrower γ-bend ribbon and its regular γ-helix counterpart still remains to be studied, as the latter 3D-structures have not yet been experimentally authenticated. In this paper, we describe the results of the first characterization, both in the crystal state and in solution, of the γ-bend ribbon conformation using X-ray diffraction and FT-IR absorption, electronic CD and 2D-NMR spectroscopies applied to an appropriate set of synthetic, homo-chiral, sequential dipeptide oligomers based on (S)-Ala and the known γ-bend inducer, Cα-tetrasubstituted, N-alkylated α-amino acid residue (S)-Cα-methyl-azetidine-carboxylic acid.

Tuning the Morphology of Nanostructured Peptide Films by the Introduction of a Secondary Structure Conformational Constraint: A Case Study of Hierarchical Self-Assembly.

Peptide self-assembly is ubiquitous in nature. It governs the organization of proteins, controlling their folding kinetics and preserving their structural stability and bioactivity. In this connection, model oligopeptides may give important insights into the molecular mechanisms and elementary forces driving the formation of supramolecular structures. In this contribution, we show that a single residue substitution, that is, Aib (α-aminoisobutyric acid) in place of Ala at position 4 of an -(l-Ala)5-homo-oligomer, strongly alters the aggregation process. In particular, this process is initiated by the formation of small peptide clusters that promote aggregation on the nanometer scale and, through a hierarchical self-assembly, lead to mesoscopic structures of micrometric dimensions. Furthermore, we show that the use of the well-established Langmuir-Blodgett technique represents an effective strategy for coating extended areas of inorganic substrates by densely packed peptide layers, thus paving the way for application of peptide films as templates for biomineralization, biocompatible coating of surfaces, and scaffolds for tissue engineering.

Alamethicin self-assembling in lipid membranes: concentration dependence from pulsed EPR of spin labels.

The antimicrobial action of the peptide antibiotic alamethicin (Alm) is commonly related to peptide self-assembling resulting in the formation of voltage-dependent channels in bacterial membranes, which induces ion permeation. To obtain a deeper insight into the mechanism of channel formation, it is useful to know the dependence of self-assembling on peptide concentration. With this aim, we studied Alm F50/5 spin-labeled analogs in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane, for peptide-to-lipid (P/L) ratios varying between 1/1500 and 1/100. Pulsed electron-electron double resonance (PELDOR) spectroscopy reveals that even at the lowest concentration investigated, the Alm molecules assemble into dimers. Moreover, under these conditions, electron spin echo envelope modulation (ESEEM) spectroscopy of D2O-hydrated membranes shows an abrupt change from the in-plane to the trans-membrane orientation of the peptide. Therefore, we hypothesize that dimer formation and peptide reorientation are concurrent processes and represent the initial step of peptide self-assembling. By increasing peptide concentration, higher oligomers are formed. A simple kinetic model of equilibrium among monomers, dimers, and pentamers allows for satisfactorily describing the experimental PELDOR data. The inter-label distances in the oligomers obtained from PELDOR experiments become better resolved with increasing P/L ratio, thus suggesting that the supramolecular organization of the higher-order oligomers becomes more defined.

Conformational properties, membrane interaction, and antibacterial activity of the peptaibiotic chalciporin A: Multitechnique spectroscopic and biophysical investigations on the natural compound and labeled analogs.

In this work, an extensive set of spectroscopic and biophysical techniques (including FT-IR absorption, CD, 2D-NMR, fluorescence, and CW/PELDOR EPR) was used to study the conformational preferences, membrane interaction, and bioactivity properties of the naturally occurring synthetic 14-mer peptaibiotic chalciporin A, characterized by a relatively low (≈20%), uncommon proportion of the strongly helicogenic Aib residue. In addition to the unlabeled peptide, we gained in-depth information from the study of two labeled analogs, characterized by one or two residues of the helicogenic, nitroxyl radical-containing TOAC. All three compounds were prepared using the SPPS methodology, which was carefully modified in the course of the syntheses of TOAC-labeled analogs in view of the poorly reactive α-amino function of this very bulky residue and the specific requirements of its free-radical side chain. Despite its potentially high flexibility, our results point to a predominant, partly amphiphilic, α-helical conformation for this peptaibiotic. Therefore, not surprisingly, we found an effective membrane affinity and a remarkable penetration propensity. However, chalciporin A exhibits a selectivity in its antibacterial activity not in agreement with that typical of the other members of this peptide class.

Synthesis of Intrinsically Blue-Colored bis-Nitronyl Nitroxide Peptidomimetic Templates and Their Conformational Preferences as Revealed by a Combined Spectroscopic Analysis.

The intrinsically blue-colored Ullman imidazolinyl nitronyl nitroxide (NN) mono-radicals have found various applications, in particular as spin probes and organic magnetic materials. Here, we present the solution-phase synthesis, extensive characterization, and conformational analysis of the first peptidomimetics with two pendant, chiral nitronyl nitroxide free radical units. Two (R)-Aic(NN) residues, where Aic(NN) is 2-amino-5-nitronylnitroxide-indan-2-carboxylic acid, have been inserted at positions i and i+3 of the pentapeptide Boc-(R)-Aic(NN)-(Ala)2-(R)-Aic(NN)-Ala-OMe and the hexapeptide Boc-[Ala-(R)-Aic(NN)-Ala]2-OMe as well. The two compounds were obtained in good yields and high purities. Thanks to a combination of several spectroscopic techniques (IR absorption, NMR, VCD, and EPR) we gained clear evidence that both compounds adopt a right-handed 310-helical conformation with both nitronyl nitroxide pendants positioned on the same side of the helix. This peptidomimetic/free radical system is a potentially excellent template for the preparation of a set of appropriate analogs with exciting applications in the area of host-guest organic chemistry, or to spectroscopically evaluate in-depth the intramolecular exchange interactions in this type of probe.

Tuning morphological architectures generated through living supramolecular assembly of a helical foldamer end-capped with two complementary nucleobases.

Two appropriately functionalized nucleobases, thymine and adenine, have been covalently linked at the N- and C-termini, respectively, of two α-aminoisobutyric acid-rich helical peptide foldamers, aiming at driving self-assembly through complementary recognition. A crystal-state analysis (by X-ray diffraction) on the shorter, achiral foldamer 1 unambiguously shows that adeninethymine base pairing, through Watson-Crick intermolecular H-bonding, does take place between either end of each peptide molecule. In the crystals, π-stacking between base pairs is also observed. Evidence for time-dependent foldameroldamer associations for the longer, chiral foldamer 2 in solution is provided by circular dichroism measurements. The self-assembly of foldamer 2, through living supramolecular polymerization, eventually leads to the formation of twisted fibers. Such a supramolecular organization can be affected by addition of either pristine adenine or thymine, that acts as a "terminator" by selectively matching a pairing nucleobase at one end of the foldamer. The co-assembly of foldamer 2 with a porphyrin-derivatized thymine, under appropriate experimental conditions, leads to the formation of vesicles which, in turn, can be converted to the fiber morphology by changing the environmental polarity. Conversely, dendrimeric, star polymer-like microstructures are generated when the supramolecular assembly of foldamer 2 is seeded by adenine-capped gold nanoparticles.

Integrated Computational Approach to the Electron Paramagnetic Resonance Characterization of Rigid 310-Helical Peptides with TOAC Nitroxide Spin Labels.

We address the interpretation, via an integrated computational approach, of the experimental continuous-wave electron paramagnetic resonance (cw-EPR) spectra of a complete set of conformationally highly restricted, stable 310-helical peptides from hexa- to nonamers, each bis-labeled with nitroxide radical-containing TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid) residues. The usefulness of TOAC for this type of analysis has been shown already to be due to its cyclic piperidine side chain, which is rigidly connected to the peptide backbone α-carbon. The TOAC α-amino acids are separated by two, three, four, and five intervening residues. This set of compounds has allowed us to modulate both the radical···radical distance and the relative orientation parameters. To further validate our conclusion, a comparative analysis has been carried out on three singly TOAC-labeled peptides of similar main-chain length.