Structural Analysis of Nonapeptides Derived from Elastin.

Elastin-derived peptides are released from the extracellular matrix remodeling by numerous proteases and seem to regulate many biological processes, notably cancer progression. The canonical elastin peptide is VGVAPG, which harbors the XGXXPG consensus pattern, allowing interaction with the elastin receptor complex located at the surface of cells. Besides these elastokines, another class of peptides has been identified. This group of bioactive elastin peptides presents the XGXPGXGXG consensus sequence, but the reason for their bioactivity remains unexplained. To better understand their nature and structure-function relationships, herein we searched the current databases for this nonapeptide motif and observed that the XGXPGXGXG elastin peptides define a specific group of tandemly repeated patterns. Further, we focused on four tandemly repeated human elastin nonapeptides, i.e., AGIPGLGVG, VGVPGLGVG, AGVPGLGVG, and AGVPGFGAG. These peptides were analyzed by means of optical spectroscopies and molecular dynamics. Ultraviolet-circular dichroism and Raman spectra are consistent with a mixture of ß-turn, ß-strand, and random-chain secondary elements in aqueous media. Quantitative analysis of their conformations suggested that turns corresponded to half of the total population of structural elements, whereas the remaining half were equally distributed between ß-strand and unordered chains. These distributions were confirmed by molecular dynamics simulations. Altogether, our data suggest that these highly dynamic peptides harbor a type II ß-turn located in their central part. We hypothesize that this structural element could explain their specific bioactivity.

Lipid Unsaturation Properties Govern the Sensitivity of Membranes to Photoinduced Oxidative Stress.

Unsaturated lipid oxidation is a fundamental process involved in different aspects of cellular bioenergetics; dysregulation of lipid oxidation is often associated with cell aging and death. To study how lipid oxidation affects membrane biophysics, we used a chlorin photosensitizer to oxidize vesicles of various lipid compositions and degrees of unsaturation in a controlled manner. We observed different shape transitions that can be interpreted as an increase in the area of the targeted membrane followed by a decrease. These area modifications induced by the chemical modification of the membrane upon oxidation were followed in situ by Raman tweezers microspectroscopy. We found that the membrane area increase corresponds to the lipids' peroxidation and is initiated by the delocalization of the targeted double bonds in the tails of the lipids. The subsequent decrease of membrane area can be explained by the formation of cleaved secondary products. As a result of these area changes, we observe vesicle permeabilization after a time lag that is characterized in relation with the level of unsaturation. The evolution of photosensitized vesicle radius was measured and yields an estimation of the mechanical changes of the membrane over oxidation time. The membrane is both weakened and permeabilized by the oxidation. Interestingly, the effect of unsaturation level on the dynamics of vesicles undergoing photooxidation is not trivial and thus carefully discussed. Our findings shed light on the fundamental dynamic mechanisms underlying the oxidation of lipid membranes and highlight the role of unsaturations on their physical and chemical properties.

Structural changes and picosecond to second dynamics of cytochrome c in interaction with nitric oxide in ferrous and ferric redox states.

Apart from its role in electron transfer, mitochondrial cytochrome c also plays a role in apoptosis and is subject to nitrosylation. The cleavage of the Fe-Met80 bond plays a role in several processes including the release of Cyt c from mitochondria or increase of its peroxidase activity. Nitrosylation of Cyt c precludes the reformation of the disrupted Fe-Met80 bond and was shown to occur during apoptosis. These physiological properties are associated with a conformational change of the heme center of Cyt c. Here, we demonstrate that NO binding induces pronounced heme conformational changes in the six-coordinate Cyt c-NO complex. Equilibrium and time-resolved Raman data reveal that the heme structural conformation depends both on the nature of the distal iron ligand (NO or Met80) and on the Fe2+ or Fe3+ heme redox state. Upon nitrosylation, the heme ruffling distortion is greatly enhanced for ferrous Cyt c. Contrastingly, the initial strong heme distortion in native ferric Cyt c almost disappears after NO binding. We measured the heme coordination dynamics in the picosecond to second time range and identified Met80 and NO rebinding phases using time-resolved Raman and absorption spectroscopies. Dissociation of NO instantly produces 5-coordinate heme with a domed structure which continues to rearrange within 15 ps, while the initial ruffling distortion disappears. The rates of Cyt c-NO complex formation measured by transient absorption are kon = 1.81 × 106 M-1 s-1 for ferric Cyt c and 83 M-1 s-1 for ferrous Cyt c. After NO dissociation and exit from the heme pocket, the rebinding of Met80 to the heme iron takes place 6 orders of magnitude more slowly (3-5 µs) than Met80 rebinding in the absence of NO (5 ps). Altogether, these data reveal the structural and dynamic properties of Cyt c in interaction with nitric oxide relevant for the molecular mechanism of apoptosis.

From bulk to plasmonic nanoparticle surfaces: the behavior of two potent therapeutic peptides, octreotide and pasireotide.

Octreotide and pasireotide are two cyclic somatostatin analogues with an important clinical use in the treatment and diagnosis of neuroendocrine tumors. Herein, by the combined use of several techniques (UV-visible absorption, fluorescence, circular dichroism, ζ-potential, transmission electron microscopy, Raman scattering, surface-enhanced Raman scattering, and quantum mechanical calculations) we have followed the structural dynamics of these analogues in the bulk, as well as their binding sites on plasmonic (gold and silver) colloids. In contrast to the previously derived conclusions, the two peptides seem to possess completely different conformational features. Octreotide, a cyclic octapeptide, is formed by a moderately flexible type-II'ß-turn maintained by a deformable disulfide linkage. Pasireotide, in which the cyclic character is made possible by peptide bonds, manifests a rigid backbone formed by two oppositely placed tight turns of different types, i.e.γ-turn and type-I ß-turn. Owing to their cationic character, both analogues induce aggregation of negatively charged gold and silver colloids. Nevertheless, despite their notable structural differences, both peptides bind onto gold nanoparticles through their unique d-Trp residue. In contrast, their binding to silver colloids seems to be of electrostatic nature, as formed through monodentate or bidentate ionic pairs.

Electronic acceleration of atomic motions and disordering in bismuth.

The development of X-ray and electron diffraction methods with ultrahigh time resolution has made it possible to map directly, at the atomic level, structural changes in solids induced by laser excitation. This has resulted in unprecedented insights into the lattice dynamics of solids undergoing phase transitions. In aluminium, for example, femtosecond optical excitation hardly affects the potential energy surface of the lattice; instead, melting of the material is governed by the transfer of thermal energy between the excited electrons and the initially cold lattice. In semiconductors, in contrast, exciting approximately 10 per cent of the valence electrons results in non-thermal lattice collapse owing to the antibonding character of the conduction band. These different material responses raise the intriguing question of how Peierls-distorted systems such as bismuth will respond to strong excitations. The evolution of the atomic configuration of bismuth upon excitation of its A(1g) lattice mode, which involves damped oscillations of atoms along the direction of the Peierls distortion of the crystal, has been probed, but the actual melting of the material has not yet been investigated. Here we present a femtosecond electron diffraction study of the structural changes in crystalline bismuth as it undergoes laser-induced melting. We find that the dynamics of the phase transition depend strongly on the excitation intensity, with melting occurring within 190 fs (that is, within half a period of the unperturbed A(1g) lattice mode) at the highest excitation. We attribute the surprising speed of the melting process to laser-induced changes in the potential energy surface of the lattice, which result in strong acceleration of the atoms along the longitudinal direction of the lattice and efficient coupling of this motion to an unstable transverse vibrational mode. That is, the atomic motions in crystalline bismuth can be electronically accelerated so that the solid-to-liquid phase transition occurs on a sub-vibrational timescale.

Low concentration structural dynamics of lanreotide and somatostatin-14.

Lanreotide, a synthetic cyclic octapeptide, analogue of the peptide hormone somatostatin-14 (SST-14), is routinely used as a long-acting medication in the management of neuroendocrine tumors. Despite its therapeutic importance, low concentration structural data is still lacking for lanreotide. In fact, the major part of the previous structural investigations were focused on the remarkable aggregation properties of this peptide, appearing at high concentrations (>5 mM). Here, we have applied three optical spectroscopic techniques, i.e. fluorescence, circular dichroism and Raman scattering, for analyzing the structural dynamics at the concentrations below 5 mM, where lanreotide exists either in a monomer state or at the first stages of aggregation. The obtained data from lanreotide were discussed through their comparison with those collected from SST-14, leading us to the following conclusions: (i) The central D-Trp residue, forming with its adjacent Lys the main receptor interacting part of lanreotide, keeps a constant high rotational freedom whatever the environment (water, water/methanol, methanol). (ii) A solvent-dependent tight ß-turn, belonging to the type-II' family, is revealed in lanreotide. (iii) Raman data analyzed by band decomposition in the amide (I and III) regions allowed estimation of different secondary structural elements within the millimolar range. Interestingly, the applied protocol shows a perfect agreement between the structural features provided by the amide I and amide III Raman markers.

The relationship between the tyrosine residue 850-830 cm-1 Raman doublet intensity ratio and the aromatic side chain χ1 torsion angle.

Tyrosine (Tyr) residue in a peptide chain is characterized by the presence of seven Raman markers, referred to as Yi (i = 1, …, 7), distributed over the middle wavenumber spectral region. Particularly, the changes observed in the relative intensity of Y5 and Y6 markers, appearing as a side by side doublet at ca. 850-830 cm-1, has received a great attention. Primarily assigned to a Fermi-resonance effect between phenol ring planar and nonplanar modes, former density functional theory calculations led us to affiliate the Y5-Y6 doublet to two distinct fundamental modes. Furthermore, despite the previous assumptions, it was evidenced that the reversal of the doublet intensity ratio cannot be solely explained by hydrogen bonding on the phenol hydroxyl group involved in Tyr. Herein, upon analyzing the observed and theoretical data collected from the cationic species of the tripeptide Gly-Tyr-Gly, the crucial effect of the aromatic side chain orientation, especially that of the χ1 torsion angle defined around the CαCß bond, on the Tyr doublet intensity ratio has been evidenced.

Relationships between conformational and vibrational features of tryptophan characteristic Raman markers.

Tryptophan (Trp) residue provides characteristic vibrational markers to the middle wavenumber spectral region of the Raman spectra recorded from peptides and proteins. In this report, we were particularly interested in eight Trp Raman markers, referred to as Wi (i = 1,…,8). All responsible for pronounced Raman lines, these markers originate from indole moiety, a bicyclic conjugated segment involved in the Trp structure. Numerous investigations have previously attempted to relate the variations observed in the spectral features of these markers to the environmental changes of Trp residues. To emphasize the most important points we can mention (i) the variations in the Raman profile of W4 (∼1360 cm-1) and W5 (∼1340 cm-1), frequently observed as a doublet with variable intensity ratio. These two markers were thought to result from a Fermi-resonance effect between certain planar and nonplanar modes; (ii) the changes observed in the wavenumbers and relative intensities of W4, W7 (∼880 cm-1) and W8 (∼760 cm-1) were supposed to be related to the accessibility of Trp to surrounding water molecules; and (iii) the wavenumber fluctuations of W3 (∼1550 cm-1), taken as a Trp side chain orientational marker. However, some ambiguities still exist regarding the interpretation of these markers, needing further clarification. Herein, upon a joint experimental and theoretical analysis based on a multiconformational approach, attention was paid to the relationships between structural and vibrational features of three indole-containing compounds with increasing structural complexity, i.e., skatole (3-methylindole), tryptophan, and tripeptide Gly-Trp-Gly. This study clearly shows that the existing assignments given to certain Trp Raman markers should be reconsidered, especially those based on the Fermi-resonance origin of W4-W5 (∼1360-1340 cm-1) doublet, as well as the purely environmental dependence of W7 and W8 markers.

Identification of extracellular vesicles from their Raman spectra via self-supervised learning.

Extracellular vesicles (EVs) released from cells attract interest for their possible role in health and diseases. The detection and characterization of EVs is challenging due to the lack of specialized methodologies. Raman spectroscopy, however, has been suggested as a novel approach for biochemical analysis of EVs. To extract information from the spectra, a novel deep learning architecture is explored as a versatile variant of autoencoders. The proposed architecture considers the frequency range separately from the intensity of the spectra. This enables the model to adapt to the frequency range, rather than requiring that all spectra be pre-processed to the same frequency range as it was trained on. It is demonstrated that the proposed architecture accepts Raman spectra of EVs and lipoproteins from 13 biological sources and from two laboratories. High reconstruction accuracy is maintained despite large variances in frequency range and noise level. It is also shown that the architecture is able to cluster the biological nanoparticles by their Raman spectra and differentiate them by their origin without pre-processing of the spectra or supervision during learning. The model performs label-free differentiation, including separating EVs from activated vs. non-activated blood platelets and EVs/lipoproteins from prostate cancer patients versus non-cancer controls. The differentiation is evaluated by creating a neural network classifier that observes the features extracted by the model to classify the spectra according to their sample origin. The classification reveals a test sensitivity of 92.2 % and selectivity of 92.3 % over 769 measurements from two labs that have different measurement configurations.

Extracellular vesicles from activated platelets possess a phospholipid-rich biomolecular profile and enhance prothrombinase activity.

BACKGROUND: Extracellular vesicles (EVs), in particular those derived from activated platelets, are associated with a risk of future venous thromboembolism. OBJECTIVES: To study the biomolecular profile and function characteristics of EVs from control (unstimulated) and activated platelets. METHODS: Biomolecular profiling of single or very few (1-4) platelet-EVs (control/stimulated) was performed by Raman tweezers microspectroscopy. The effects of such EVs on the coagulation system were comprehensively studied. RESULTS: Raman tweezers microspectroscopy of platelet-EVs followed by biomolecular component analysis revealed for the first time 3 subsets of EVs: (i) protein rich, (ii) protein/lipid rich, and (iii) lipid rich. EVs from control platelets presented a heterogeneous biomolecular profile, with protein-rich EVs being the main subset (58.7% ± 3.5%). Notably, the protein-rich subset may contain a minor contribution from other extracellular particles, including protein aggregates. In contrast, EVs from activated platelets were more homogeneous, dominated by the protein/lipid-rich subset (>85%), and enriched in phospholipids. Functionally, EVs from activated platelets increased thrombin generation by 52.4% and shortened plasma coagulation time by 34.6% ± 10.0% compared with 18.6% ± 13.9% mediated by EVs from control platelets (P = .015). The increased procoagulant activity was predominantly mediated by phosphatidylserine. Detailed investigation showed that EVs from activated platelets increased the activity of the prothrombinase complex (factor Va:FXa:FII) by more than 6-fold. CONCLUSION: Our study reports a novel quantitative biomolecular characterization of platelet-EVs possessing a homogenous and phospholipid-enriched profile in response to platelet activation. Such characteristics are accompanied with an increased phosphatidylserine-dependent procoagulant activity. Further investigation of a possible role of platelet-EVs in the pathogenesis of venous thromboembolism is warranted.

Picosecond primary structural transition of the heme is retarded after nitric oxide binding to heme proteins.

We investigated the ultrafast structural transitions of the heme induced by nitric oxide (NO) binding for several heme proteins by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We probed the heme iron motion by the evolution of the iron-histidine Raman band intensity after NO photolysis. Unexpectedly, we found that the heme response and iron motion do not follow the kinetics of NO rebinding. Whereas NO dissociation induces quasi-instantaneous iron motion and heme doming (<0.6 ps), the reverse process results in a much slower picosecond movement of the iron toward the planar heme configuration after NO binding. The time constant for this primary domed-to-planar heme transition varies among proteins (approximately 30 ps for myoglobin and its H64V mutant, approximately 15 ps for hemoglobin, approximately 7 ps for dehaloperoxidase, and approximately 6 ps for cytochrome c) and depends upon constraints exerted by the protein structure on the heme cofactor. This observed phenomenon constitutes the primary structural transition in heme proteins induced by NO binding.

The H-NOX protein structure adapts to different mechanisms in sensors interacting with nitric oxide.

Some classes of bacteria within phyla possess protein sensors identified as homologous to the heme domain of soluble guanylate cyclase, the mammalian NO-receptor. Named H-NOX domain (Heme-Nitric Oxide or OXygen-binding), their heme binds nitric oxide (NO) and O2 for some of them. The signaling pathways where these proteins act as NO or O2 sensors appear various and are fully established for only some species. Here, we investigated the reactivity of H-NOX from bacterial species toward NO with a mechanistic point of view using time-resolved spectroscopy. The present data show that H-NOXs modulate the dynamics of NO as a function of temperature, but in different ranges, changing its affinity by changing the probability of NO rebinding after dissociation in the picosecond time scale. This fundamental mechanism provides a means to adapt the heme structural response to the environment. In one particular H-NOX sensor the heme distortion induced by NO binding is relaxed in an ultrafast manner (∼15 ps) after NO dissociation, contrarily to other H-NOX proteins, providing another sensing mechanism through the H-NOX domain. Overall, our study links molecular dynamics with functional mechanism and adaptation.

Dynamics of mitochondrial membranes under photo-oxidative stress with high spatiotemporal resolution.

In our study, we harnessed an original Enhanced Speed Structured Illumination Microscopy (Fast-SIM) imaging setup to explore the dynamics of mitochondrial and inner membrane ultrastructure under specific photo-oxidation stress induced by Chlorin-e6 and light irradiation. Notably, our Fast-SIM system allowed us to observe and quantify a distinct remodeling and shortening of the mitochondrial structure after 60-80 s of irradiation. These changes were accompanied by fusion events of adjacent inner membrane cristae and global swelling of the organelle. Preceding these alterations, a larger sequence was characterized by heightened dynamics within the mitochondrial network, featuring events such as mitochondrial fission, rapid formation of tubular prolongations, and fluctuations in cristae structure. Our findings provide compelling evidence that, among enhanced-resolution microscopy techniques, Fast-SIM emerges as the most suitable approach for non-invasive dynamic studies of mitochondrial structure in living cells. For the first time, this approach allows quantitative and qualitative characterization of successive steps in the photo-induced oxidation process with sufficient spatial and temporal resolution.

Side chain flexibility and protonation states of sulfur atom containing amino acids.

We present a set of new data allowing elucidation of the energetic, conformational and vibrational features of cysteine (Cys) and methionine (Met), i.e. two natural amino acids (AAs) containing a sulfur atom in their side chains. Special attention has been paid to cysteine, for which vibrational features were analysed in a wide pH range (6-to-12), where its backbone can switch from a zwitterionic to an anionic form, and its side chain SH group can be deprotonated. Through a detailed discussion on the relative acidity of the three protonation sites of this AA, as well as on the vibrational markers arising from zwitterionic and anionic backbones, we could assign the spectra recorded at pH 6, 9.2 and 12 to three species, referred to as Cys(0), Cys(1-)(a) and Cys(2-), where the superscripts designate their global net charges. To bring clarification to the structural and vibrational features, quantum mechanical calculations based on the Density Functional Theory (DFT) were carried out, allowing (i) a quasi exhaustive energetic and side chain conformational analysis through 804 clusters of explicitly hydrated AAs; (ii) simulation of the observed aqueous solution vibrational spectra of Cys(0), Cys(-2) and Met by means of the theoretical data obtained from their conformationally distinct lowest energy clusters.

Energy maps, side chain conformational flexibility, and vibrational features of polar amino acids L-serine and L-threonine in aqueous environment.

A comprehensive description of the energetic, conformational, and vibrational features of the two amino acids (AAs) with polar side chains, i.e., serine and threonine, in aqueous environment, is provided. To adequately analyze the side chain conformational flexibility of these amino acids, we resorted to quantum mechanical calculations with the use of density functional theory, which allowed the determination of the energetic features of these AAs through 236 clusters. Each cluster contains a zwitterionic AA surrounded by seven explicit water molecules. The obtained data could evidence the effect of the side chain conformational angle (χ(1) and χ(2)) as well as the location of water molecules on the energy landscapes of both AAs. Four of the lowest energy clusters of each AA, which give rise to distinct side chain conformations, were selected in order to reproduce the FT-IR and Raman spectra recorded in aqueous solutions and to assign the vibrational modes responsible of the main observed bands.

Multiconformational analysis of tripeptides upon consideration of implicit and explicit hydration effects.

During the last two decades, numerous observed data obtained by various physical techniques, also supported by molecular modeling approaches, have highlighted the structuring features of tripeptides, as well as their aggregation properties. Herein, we focus on the structural dynamics of four trimers, i.e., Gly-Gly-Gly, Gly-Ala-Gly, Ala-Ala-Ala and Ala-Phe-Ala, in an aqueous environment. Density functional theory calculations (DFT) were carried out to assess the stability of four types of secondary structures, i.e., ß-strand, polyproline-II (pP-II), α-helix and γ-turn, of which the formation had been described in these tripeptides. Both implicit and explicit hydration effects were analyzed on the conformational and energetic features of trimers. It has been shown that the use of M062X functional (versus B3LYP) improve the stability of intramolecular H-bonds, especially in inverse γ-turn structures, as well as the energetic and conformational equilibrium in all tripeptides. Explicit hydration reflected by the presence of five water molecules around the backbone polar sites (NH3+, N-H, CO and NH2) considerably changes the conformational landscapes of the trimers. Characteristic intramolecular and intermolecular interactions evidenced by the calculations, were emphasized.

Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles.

Extracellular vesicles (EVs) are heterogeneous lipid containers with a complex molecular cargo comprising several populations with unique roles in biological processes. These vesicles are closely associated with specific physiological features, which makes them invaluable in the detection and monitoring of various diseases. EVs play a key role in pathophysiological processes by actively triggering genetic or metabolic responses. However, the heterogeneity of their structure and composition hinders their application in medical diagnosis and therapies. This diversity makes it difficult to establish their exact physiological roles, and the functions and composition of different EV (sub)populations. Ensemble averaging approaches currently employed for EV characterization, such as western blotting or 'omics' technologies, tend to obscure rather than reveal these heterogeneities. Recent developments in single-vesicle analysis have made it possible to overcome these limitations and have facilitated the development of practical clinical applications. In this review, we discuss the benefits and challenges inherent to the current methods for the analysis of single vesicles and review the contribution of these approaches to the understanding of EV biology. We describe the contributions of these recent technological advances to the characterization and phenotyping of EVs, examination of the role of EVs in cell-to-cell communication pathways and the identification and validation of EVs as disease biomarkers. Finally, we discuss the potential of innovative single-vesicle imaging and analysis methodologies using microfluidic devices, which promise to deliver rapid and effective basic and practical applications for minimally invasive prognosis systems.

Characterization of a multicore fiber image guide for nonlinear endoscopic imaging using two-photon fluorescence and second-harmonic generation.

Multiphoton microscopy (MPM) has the capacity to record second-harmonic generation (SHG) and endogenous two-photon excitation fluorescence (2PEF) signals emitted from biological tissues. The development of fiber-based miniaturized endomicroscopes delivering pulses in the femtosecond range will allow the transfer of MPM to clinical endoscopy. We present real-time SHG and 2PEF ex vivo images using an endomicroscope, which totally complies with clinical endoscopy regulations. This system is based on the proximal scanning of a commercial multicore image guide (IG). For understanding the inhomogeneities of the recorded images, we quantitatively characterize the IG at the single-core level during nonlinear excitation. The obtained results suggest that these inhomogeneities originate from the variable core geometries that, therefore, exhibit variable nonlinear and dispersive properties. Finally, we propose a method based on modulation of dispersion precompensation to address the image inhomogeneity issue and, as a proof of concept, we demonstrate its capability to improve the nonlinear image quality.

Disorder-to-Order Markers of a Cyclic Hexapeptide Inspired from the Binding Site of Fertilin ß Involved in Fertilization Process.

Synthetic peptides mimicking the binding site of fertilin ß to its receptor, integrin α6ß1, were shown to inhibit sperm-egg fusion when added to in vitro media. In contrast, the synthetic cyclic hexapeptide, cyclo(Cys1-Ser2-Phe3-Glu4-Glu5-Cys6), named as cFEE, proved to stimulate gamete fusion. Owing to its biological specificity, this hexapeptide could help improve the in vitro fertilization pregnancy rate in human. In an attempt to establish the structure-activity relationship of cFEE, its structural dynamics was herein analyzed by means of ultraviolet circular dichroism (UV-CD) and Raman scattering. The low concentration CD profile in water, containing mainly a deep minimum at ∼202 nm, is consistent with a rather unordered chain. However, an ordering trend of the peptide loop has been observed in a less polar solvent such as methanol, where the UV-CD signal shape is formed by a double negative marker at ∼202/215 nm, indicating the presence of a type-II' ß-turn. Raman spectra recorded in aqueous samples upon a 100-fold concentration increase, still showed an important population (∼30%) of the disordered structure. The structural flexibility of the disulfide bridge was confirmed by the Raman markers arising from the Cys1-Cys6 disulfide bond-stretch motions. Density functional theory calculations highlighted the formation of the type-II' ß-turn on the four central residues of cFEE (i.e., -Ser2-Phe3-Glu4-Glu5-) either with a left- or with a right-handed disulfide. The structure with a left-handed S-S bond, however, appears to be more stable.