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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Raman tweezers microspectroscopy of circa 100 nm extracellular vesicles.

The technique of Raman tweezers microspectroscopy (RTM) for the global biomolecular content characterization of a single extracellular vesicle (EV) or a small number of EVs or other nanoscale bioparticles in an aqueous dispersion in the difficult-to-access size range of near 100 nm is described in detail. The particularities and potential of RTM are demonstrated using the examples of DOPC liposomes, exosomes from human urine and rat hepatocytes, and a mixed sample of the transfection reagent FuGENE in diluted DNA solution. The approach of biomolecular component analysis for the estimation of the main biomolecular contributions (proteins, lipids, nucleic acids, carotenoids, etc.) is proposed and discussed. Direct Raman evidence for strong intra-sample biomolecular heterogeneity of individual optically trapped EVs, due to variable contributions from nucleic acids and carotenoids in some preparations, is reported. On the basis of the results obtained, we are making an attempt to convince the scientific community that RTM is a promising method of single-EV research; to our knowledge, it is the only technique available at the moment that provides unique information about the global biomolecular composition of a single vesicle or a small number of vesicles, thus being capable of unravelling the high diversity of EV subpopulations, which is one of the most significant urgent challenges to overcome. Possible RTM applications include, among others, searching for DNA biomarkers, cancer diagnosis, and discrimination between different subpopulations of EVs, lipid bodies, protein aggregates and viruses.

Phosphorescence Kinetics of Singlet Oxygen Produced by Photosensitization in Spherical Nanoparticles. Part II. The Case of Hypericin-Loaded Low-Density Lipoprotein Particles.

The phosphorescence kinetics of singlet oxygen produced by photosensitized hypericin (Hyp) molecules inside low-density lipoprotein (LDL) particles was studied experimentally and by means of numerical and analytical modeling. The phosphorescence signal was measured after short laser pulse irradiation of aqueous Hyp/LDL solutions. The Hyp triplet state lifetime determined by a laser flash-photolysis measurement was 5.3 × 10-6 s. The numerical and the analytical model described in part I of the present work (DOI: 10.1021/acs.jpcb.8b00658) were used to analyze the observed phosphorescence kinetics of singlet oxygen. It was shown that singlet oxygen diffuses out of LDL particles on a time scale shorter than 0.1 µs. The total (integrated) concentration of singlet oxygen inside LDL is more than an order of magnitude smaller than the total singlet oxygen concentration in the solvent. The time course of singlet oxygen concentrations inside and outside the particles was calculated using simplified representations of the LDL internal structure. The experimental phosphorescence data were fitted by a linear combination of these concentrations using the emission factor E (the ratio of the radiative singlet oxygen depopulation rate constants inside and outside LDL) as a fitting parameter. The emission factor was determined to be E = 6.7 ± 2.5. Control measurements were carried out by adding sodium azide, a strong singlet oxygen quencher, to the solution.

Phosphorescence Kinetics of Singlet Oxygen Produced by Photosensitization in Spherical Nanoparticles. Part I. Theory.

The singlet oxygen produced by energy transfer between an excited photosensitizer (pts) and ground-state oxygen molecules plays a key role in photodynamic therapy. Different nanocarrier systems are extensively studied to promote targeted pts delivery in a host body. The phosphorescence kinetics of the singlet oxygen produced by the short laser pulse photosensitization of pts inside nanoparticles is influenced by singlet oxygen diffusion from the particles to the surrounding medium. Two theoretical models are presented in this work: a more complex numerical one and a simple analytical one. Both the models predict the time course of singlet oxygen concentration inside and outside of the spherical particles following short-pulse excitation of pts. On the basis of the comparison of the numerical and analytical results, a semiempirical analytical formula is derived to calculate the characteristic diffusion time of singlet oxygen from the nanoparticles to the surrounding solvent. The phosphorescence intensity of singlet oxygen produced in pts-loaded nanocarrier systems can be calculated as a linear combination of the two concentrations (inside and outside the particles), taking the different phosphorescence emission rate constants into account.

Dynamical Behavior of Somatostatin-14 and Its Cyclic Analogues as Analyzed in Bulk and on Plasmonic Silver Nanoparticles.

Primarily known as the inhibitor of growth hormone release, the role of somatostatin in many other inhibiting activities upon binding to its five G-protein-coupled receptors has been elucidated. Because of the short half-life of somatostatin, a number of synthetic analogues were elaborated for this peptide hormone. Herein, after recalling the main somatostatin therapeutic interests, we present the dynamical behavior of somatostatin-14 and its two currently used synthetic cyclic analogues, octreotide and pasireotide. Physical techniques, such as fluorescence, UV-visible absorption, circular dichroism, Raman spectroscopy, surface-enhanced Raman spectroscopy, and transmission electron microscopy, were jointly used in order to get information on the solution structural features, as well as on the anchoring sites of the three peptides on silver colloids. While somatostatin-14 adopts a rather unordered chain within the submillimolar concentration range, its cyclic analogues were revealed to be ordered, i.e., stabilized either in a type-II' ß-turn (octreotide) or in a face-to-face γ-turn/type-I ß-turn (pasireotide) structure. Nevertheless, a progressive structuring trend was observed in somatostatin-14 upon increasing concentration to the millimolar range. Because of their cationic character, the three peptides have revealed their capability to bind onto negatively charged silver nanoparticles. The high affinity of the peptides toward metallic particles seems to be extremely promising for the elaboration of somatostatin-based functionalized plasmonic nanoparticles that can be used in diagnosis, drug delivery, and therapy.

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.

Raman scattering-based multiconformational analysis for probing the structural differences between acetylcholine and acetylthiocholine.

Acetylcholine is the first discovered neurotransmitter that has received a great attention regarding its capability of binding to several cellular targets. The chemical composition of acetylcholine, including a positively charged trimethylammonium and a carbonyl group, as well as its conformational flexibility was pointed out as the key factors in the stabilization of its interactions. Here, the possibilities offered by a Raman scattering-based multiconformatioal analysis to access the most stable conformers of acetylcholine, is discussed. To control the validity of this protocol, acetylcholine and one of its closely structured analogues, acetylthiocholine, were simultaneously analyzed. Solution Raman spectra revealed distinct and well resolved strong markers for each molecule. Density functional theory calculations were consistent with the fact that the energy order of the low energy conformers is considerably affected by the acyloxy oxygen→sulfur atom substitution. Raman spectra were calculated on the basis of the thermal average of the spectra arising from the low energy conformers. It has been evidenced that the carbonyl and trimethylammonium groups are the most favorable hydration sites in aqueous environment. Taking into account the large gap between the carbonyl bond-stretch and aliphatic bending bands, Raman spectra also allowed separation of the HOH bending vibrations arising from the bound and bulk water molecules.

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.

Protonation-deprotonation and structural dynamics of antidiabetic drug metformin.

Since the late 1950s, metformin is the worldwide first-line pharmacologic treatment for type 2 diabetes. Beyond the fact that the mode of action of this drug has always been very difficult to elucidate, little is known about its physicochemical properties in aqueous solution. Herein, we focus on the protonation-deprotonation features of metformin by using jointly Raman scattering and theoretical calculations. Vibrational markers evidence the fact that within a wide pH interval extended at either side of the physiological one, i.e. ∼7 ± 4, metformin is mainly monoprotonated. Although the biprotonated form appears as major population at very low pH values (<1.5), Raman markers of neutral species do not dominate even at very high pH values (>13), presumably because of the extreme basicity of metformin as described by recent NMR measurements. Density functional theory calculations using both explicit and implicit hydration models, have led to presume a possible coexistence of two possible monoprotonated forms in aqueous environment. In conclusion, the biophysical features of this molecule and the amount used in clinical practice might certainly explain the pleiotropic actions toward several targets where metformin could be a permanent cationic partner, a proton donor/acceptor, as well as a good candidate for stabilizing the so-called π→π interactions.