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.

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.

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.

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.

Intramolecular backbone···backbone hydrogen bonds in polypeptide conformations. The other way around: ɛ-turn.

In this study, we performed a detailed literature survey of the ɛ-turn in peptides and proteins. This three-dimensional structural feature is characterized by an eleven-membered pseudo-cycle closed by an intramolecular backbone…backbone H-bond. Interestingly, in this motif the direction of the N-H…O = C H-bond runs opposite to that of the much more popular and extensively investigated α-, ß-, and γ-turns. We did not authenticate unequivocally the ɛ-turn main-chain reversal topology in any linear short peptide. However, it is frequently observed in small cyclic peptides formed by four, five, and six amino acid residues with stringent geometric requirements. Rather surprisingly, ɛ-turns do occur in proteins, although to a relatively moderate extent, as an isolated feature or in the turn segment of hairpin motifs based on two antiparallel, pleated ß-strands. Moreover, the ɛ-turn may also host not only the seven-membered, intramolecularly H-bonded, pseudo-cycle termed γ-turn, either of the classic or inverse type, but also one (or even two) cis peptide bond(s) or a ß-bulge conformation. Based on their ϕ, ψ backbone torsion angles, we were able to classify the protein ɛ-turns in six different families. Conformational energy computations using the DFT methodology were also performed on the ɛ-turns adopted by the amino acid triplet -Gly-Gly-Gly- (Gly is the most commonly found residue at each of the three positions in our analysis of proteins). Again, in this computational study, six families of turns were identified, but only some of them resemble rather closely those extracted from our investigation on proteins.

En route towards the peptide γ-helix: X-ray diffraction analyses and conformational energy calculations of Adm-rich short peptides.

We performed the solution-phase synthesis of a set of model peptides, including homo-oligomers, based on the 2-aminoadamantane-2-carboxylic acid (Adm) residue, an extremely bulky, highly lipophilic, tricyclic, achiral, Cα -tetrasubstituted α-amino acid. In particular, for the difficult peptide coupling reaction between two Adm residues, we took advantage of the Meldal's α-azidoacyl chloride approach. Most of the synthesized Adm peptides were characterized by single-crystal X-ray diffraction analyses. The results indicate a significant propensity for the Adm residue to adopt γ-turn and γ-turn-like conformations. Interestingly, we found that a -CO-(Adm)2 -NH- sequence is folded in the crystal state into a regular, incipient γ-helix, at variance with the behavior of all of the homo-dipeptides from Cα -tetrasubstituted α-amino acids already investigated, which tend to adopt either the ß-turn or the fully extended conformation. Our density functional theory conformational energy calculations on the terminally blocked homo-peptides (n = 2-8) fully confirmed the crystal-state data, strongly supporting the view that this rigid Cα -tetrasubstituted α-amino acid residue is largely the most effective building block for γ-helix induction, although to a limited length (anti-cooperative effect). Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Endothioxopeptides: A conformational overview.

Although thionamides would have been first prepared two centuries ago and their chemical and spectroscopic properties extensively investigated, only much more recently (since about 1985) a well deserved but still insufficient attention has been paid to their endothioxopeptide subfamily which nonetheless currently represents a rapidly emerging area of great scientific interest in the broader field of foldameric compounds based on biologically relevant building blocks. After two brief sections offering information on the unfortunately still limited number of endothioxopeptides discovered from natural sources but also on the impressive advancements registered in the last few years in their synthetic methods, this review article outlines the results of a detailed literature survey on the ongoing great, but not systematic, progress related to the conformational consequences generated by incorporating one (or more) thionamide group(s) into a polypeptide chain. Finally, a short discussion of the growing, but still in its infancy, class of the endoselenoxopeptide congeners is also presented.

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

Ampullosporin A is a medium-length (14-amino acid long) hydrophobic peptide of the peptaibol family. In this work, electron paramagnetic resonance and circular dichroism spectroscopies were applied to study the interaction of synthetic ampullosporin A and three spin-labeled analogs with small unilamellar vesicles and bicelles. Zwitterionic vesicles were used to investigate the conformation and the penetration depth of the peptide at room temperature. Bicelles were employed in combination with EPR spectroscopy to study the order, dynamics, orientation, aggregation and the 3D-structure of the peptide at near physiological temperature. In the membrane, the peptide adopts a helical structure that changes in nature depending on the thickness of the membrane-mimetic system, from mostly α-helical in vesicles to a more elongated helix in bicelles, suggesting an increase in the 310-helical content. The orientation assumed by the peptide also shows a dependence on the membrane-mimetic system: in bicelles, ampullosporin A has a transmembrane orientation at a peptide-to-lipid (P : L) ratio of 1 : 100 and higher, while in vesicles it undergoes a transition from a parallel to a transmembrane orientation as a function of the P : L ratio. In bicelles, the peptide was found to be monomeric at a P : L ratio of 1 : 25 and lower. Overall, the comparison of the results obtained in the two membrane-mimetic systems showed that ampullosporin A has a rather flexible structure that readily adapts to the bilayer thickness.

A terminally protected dipeptide: from crystal structure and self-assembly, through co-assembly with carbon-based materials, to a ternary catalyst for reduction chemistry in water.

A terminally protected, hydrophobic dipeptide Boc-L-Cys(Me)-L-Leu-OMe (1) was synthesized and its 3D-structure was determined by single crystal X-ray diffraction analysis. This peptide is able to hierarchically self-assemble in a variety of superstructures, including hollow rods, ranging from the nano- to the macroscale, and organogels. In addition, 1 is able to drive fullerene (C60) or multiwalled carbon nanotubes (MWCNTs) in an organogel by co-assembling with them. A hybrid 1-C60­MWCNT organogel was prepared and converted (through a high vacuum-drying process) into a robust, high-volume, water insoluble, solid material where C60 is well dispersed over the entire superstructure. This ternary material was successfully tested as a catalyst for: (i) the reduction reaction of water-soluble azo compounds mediated by NaBH4 and UV-light with an overall performance remarkably better than that provided by C60 alone, and (ii) the NaBH4-mediated reduction of benzoic acid to benzyl alcohol. Our results suggest that the self-assembly properties of 1 might be related to the occurrence in its single crystal structure of a sixfold screw axis, a feature shared by most of the linear peptides known so far to give rise to nanotubes.

Peptide flatlandia: a new-concept peptide for positioning of electroactive probes in proximity to a metal surface.

A helical hexapeptide was designed to link in a rigid parallel orientation to a gold surface. The peptide sequence of the newly synthesized compound is characterized by the presence of two 4-amino-1,2-dithiolane-4-carboxylic acid (Adt) residues (positions 1 and 4) to promote a bidentate interaction with the gold surface, two L-Ala residues (positions 2 and 5) and two-aminoisobutyric acid (Aib) residues (positions 3 and 6) to favor a high population of the 310-helix conformation. Furthermore, a ferrocenoyl (Fc) probe was inserted at the N-terminus to investigate the electronic conduction properties of the peptide. X-Ray photoelectron spectroscopy and scanning tunneling microscopy techniques were used to characterize the binding of the peptide to the gold surface and the morphology of the peptide layer, respectively. Several electrochemical (cyclic voltammetry, chronoamperometry, square wave voltammetry) techniques were applied to analyze the electrochemical activity of the Fc probe, along with the influence of the peptide 3D-structure and the peptide layer morphology on electron transfer processes.

Peptide δ-Turn: Literature Survey and Recent Progress.

Among the various types of α-peptide folding motifs, δ-turn, which requires a central cis-amide disposition, has been one of the least extensively investigated. In particular, this main-chain reversal topology has been studied in-depth neither in linear/cyclic peptides nor in proteins. This Minireview article assembles and critically analyzes relevant data from a literature survey on the δ-turn conformation in those compounds. Unpublished results from recent conformational energy calculations and a preliminary solution-state analysis on a small model peptide, currently ongoing in our laboratories, are also briefly outlined.

The fluorescence and infrared absorption probe para-cyanophenylalanine: Effect of labeling on the behavior of different membrane-interacting peptides.

Total syntheses and complete characterizations of singly substituted PheCN -based analogs of alamethicin AlaP, which is active on model and natural membranes, and the TM peptide, which inserts in a transmembrane orientation in lipid bilayers, are reported. The syntheses of the AlaP analogs were performed in solution, while those of TM and its analogs were carried out by solid phase. Using the cyanophenyl fluorescence and infrared (IR) absorption probe, an in-depth investigation of the self-association, membrane-interacting, permeabilizing, and orientation properties of these peptides were conducted. The aromatic residue incorporated induces only a negligible modification to the properties of the parent peptides. The PheCN IR absorption band was located between 2228 and 2230 cm(-1) for all peptides, irrespective of the position of labeling. By contrast, as the width of this band varied significantly with the depth of probe insertion in the bilayer, it could represent a good marker of the PheCN position in phospholipid membranes.

4-Cyano-α-methyl-l-phenylalanine as a spectroscopic marker for the investigation of peptaibiotic-membrane interactions.

Two analogs of the ten-amino acid residue, membrane-active lipopeptaibiotic trichogin GA IV, mono-labeled with 4-cyano-α-methyl-L-phenylalanine, a potentially useful fluorescence and IR absorption probe of the local microenvironment, were synthesized by the solid-phase methodology and conformationally characterized. The single modification was incorporated either at the N-terminus (position 1) or near the C-terminus (position 8) of the peptide main chain. In both cases, the replaced amino acid was the equally helicogenic α-aminoisobutyric acid (Aib) residue. We performed a solution conformational analysis by use of FT-IR absorption, CD, and 2D-NMR spectroscopies. The results indicate that both labeled analogs essentially maintain the overall helical propensity of the naturally occurring lipopeptaibiotic. Peptide-membrane interactions were assessed by fluorescence and ATR-IR absorption techniques. Analogies and differences between the two peptides were highlighted. Taken together, our data confirm literature results that some of the spectroscopic parameters of the 4-cyanobenzyl chromophore are sensitive markers of the local microenvironment.

Handedness preference and switching of peptide helices. Part II: Helices based on noncoded α-amino acids.

In this second part of our review article on the preferred screw sense and interconversion of peptide helices, we discuss the most significant computational and experimental data published on helices formed by the most extensively investigated categories of noncoded α-amino acids. They are as follows: (i) N-alkylated Gly residues (peptoids), (ii) C(α) -alkylated α-amino acids, (iii) C(α,ß) -sp(2) configurated α-amino acids, and (iv) combinations of residues of types (ii) and (iii). With confidence, the large body of interesting papers examined and classified in this editorial effort will stimulate the development of helical peptides in many diverse areas of biosciences and nanosciences.

Helical screw-sense preferences of peptides based on chiral, Cα-tetrasubstituted α-amino acids.

The preferred helical screw senses of chiral α-amino acids with a C(α)-tetrasubstituted α-carbon atom, as determined in the crystal state by X-ray diffraction analyses on derivatives and peptides, are reviewed. This survey covers C(α)-methylated and C(α)-ethylated α-amino acids, as well as α-amino acids cyclized on the α-carbon, including those characterized by the combination of lack of chirality at the α-carbon with either side-chain or axial chirality. Although, in general, chiral C(α)-tetrasubstituted α-amino acids show a less pronounced bias toward a single helical screw sense than their proteinogenic (C(α)-trisubstituted) counterparts, our analysis highlights significant differences in terms of magnitude and direction of such a bias among the various sub-families of residues, and between individual amino acids within each sub-family as well. The experimental findings can be rationalized, at least in part, on the basis of steric considerations.

Peptides on the surface. PELDOR data for spin-labeled alamethicin F50/5 analogues on organic sorbent.

The PELDOR technique was used to obtain the spectra of distances between spin labels for mono and double TOAC substituted analogues of [Glu(OMe)(7,18,19)] alamethicin F50/5 (Alm') peptaibiotic on the surface of the organic sorbent Oasis HLB and in ethanol solution at 77 K. For the double-labeled Alm', the free radical probes are at positions 1 and 16 (Alm'1,16). The intra- and intermolecular contributions to the PELDOR time traces were separated, with regard to the fractality of the system studied. We established that on HLB the labeled Alm' molecules are prone to aggregation. The distance spectra for Alm'1,16 show that, in both adsorbed state and in ethanol solution, the peptaibiotic is predominantly folded in the α-helix conformation. We assign the asymmetry of the distance spectrum in both cases to the occurrence of an admixture of more elongated α/3(10)-helical conformers. The portion of these conformers is higher for the peptide adsorbed on HLB. We speculate that both the broadening of the basic spectrum line at r(max) = 2.0 nm and the increase in the contribution of elongated conformers might be associated with the spread of the peptaibiotic adsorption sites on HLB as compared with the more uniform Alm'1,16 trap structure in frozen ethanol solution. The aggregates of mono-labeled Alm'1 and Alm'16 also studied. The intermolecular distance spectrum for Alm'1 on HLB is shifted toward longer distances as compared with those of Alm'16. This result suggests that in the aggregates Alm' molecules are preferentially oriented with their C-terminal regions in the vicinity.

Photoinduced electron transfer through peptide-based self-assembled monolayers chemisorbed on gold electrodes: directing the flow-in and flow-out of electrons through peptide helices.

Photoinduced electron transfer (PET) experiments have been carried out on peptide self-assembled monolayers (SAM) chemisorbed on a gold substrate. The oligopeptide building block was exclusively formed by C(α)-tetrasubstituted α-aminoisobutyric residues to attain a helical conformation despite the shortness of the peptide chain. Furthermore, it was functionalized at the C-terminus by a pyrene choromophore to enhance the UV photon capture cross-section of the compound and by a lipoic group at the N-terminus for linking to gold substrates. Electron transfer across the peptide SAM has been studied by photocurrent generation experiments in an electrochemical cell employing a gold substrate modified by chemisorption of a peptide SAM as a working electrode and by steady-state and time-resolved fluorescence experiments in solution and on a gold-coated glass. The results show that the electronic flow through the peptide bridge is strongly asymmetric; i.e., PET from the C-terminus to gold is highly favored with respect to PET in the opposite direction. This effect arises from the polarity of the Au-S linkage (Au(δ+)-S(δ-), junction effect) and from the electrostatic field generated by the peptide helix.

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 ß-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.