Ripening process in exocarps of scarlet eggplant (Solanum aethiopicum) and banana (Musa spp.) investigated by Raman spectroscopy.

In this work, we used Raman spectroscopy to identify compounds present at different maturation stages of the exocarp of scarlet eggplant and two banana cultivars, 'prata' and 'nanica'. Raman spectral analyses of both fruits showed bands attributed to phenolic acids, flavonoids, carotenoids, and fatty acids. During the scarlet eggplant's maturation process, Raman spectral profile changes are mainly observed in the carotenoid content rather than flavonoids. Furthermore, it is suggested that naringenin chalcone together with ß-carotene determines the orange-red color of the ripe stage. Variations in chemical composition among the maturation stages of bananas were observed predominantly in 'prata' when compared to 'nanica'. In contrast to scarlet eggplant changes in the spectral profile were more evident in the content of the flavonoid/phenolic acids. The in situ analysis was demonstrated to be useful as a guide in selecting bioactive compounds on demand from low-cost horticultural waste.

The Diversity of Linear Conjugated Polyenes and Colours in Nature: Raman Spectroscopy as a Diagnostic Tool.

This review is centered on the linear conjugated polyenes, which encompasses chromatic biomolecules, such as carotenoids, polyunsaturated aldehydes and polyolefinic fatty acids. The linear extension of the conjugated double bonds in these molecules is the main feature that determines the spectroscopic properties as light-absorbing. These classes of compounds are responsible for the yellow, orange, red and purple colors which are observed in their parent flora and fauna in nature. Raman spectroscopy has been used as analytical tool for the characterization of these molecules, mainly due to the strong light scattering produced by the delocalized pi electrons in the carbon chain. In addition, conjugated polyenes are one of the main target molecular species for astrobiology, and we also present a brief discussion of the use of Raman spectroscopy as one of the main analytical tools for the detection of polyenes extra-terrestrially.

Rapid assessment of chemical compounds from Phyllogorgia dilatata using Raman spectroscopy

Abstract The gorgonian Phyllogorgia dilatata is endemic to the Brazilian coast which is listed as threatened with extinction. This species is known to produce sterols, mono- to tetra-terpenes, conjugated polyenals and peptides. The main objective of this study is to present an alternative method for identification of different classes of compounds based upon a Raman mapping technique using FT-Raman spectroscopy. The Raman analysis performed directly on the tissues (in situ) revealed the occurrence of peridinin, diadinoxanthin, conjugated polyenal and linoleic acid, that were also confirmed by Raman analysis of partitioned crude extracts. We have demonstrated that the technique has potential for use in guiding chromatographic separations and in providing information with respect to the early stages of a tissue necrosis through “purpling”. It may become a valuable non-destructive technique for monitoring the accumulation or production of metabolites during a biological interaction.

Raman spectroscopy as a tool in differentiating conjugated polyenes from synthetic and natural sources.

This work presents the Raman spectroscopic characterization of synthetic analogs of natural conjugated polyenals found in octocorals, focusing the unequivocal identification of the chemical species present in these systems. The synthetic material was produced by the autocondensation reaction of crotonaldehyde, generating a demethylated conjugated polyene containing 11 carbon-carbon double bonds, with just a methyl group on the end of the carbon chain. The resonance Raman spectra of such pigment has shown the existence of enhanced modes assigned to ν1(CC) and ν2(CC) modes of the main chain. For the resonance Raman spectra of natural pigments from octocorals collected in the Brazilian coast, besides the previously cited bands, it could be also observed the presence of the ν4(CCH3), related to the vibrational mode who describes the vibration of the methyl group of the central carbon chain of carotenoids. Other interesting point is the observation of overtones and combination bands, which for carotenoids involves the presence of the ν4 mode, whereas for the synthetic polyene this band, besides be seen at a slightly different wavenumber position, does not appear as an enhanced mode and also as a combination, such as for the natural carotenoids. Theoretical molecular orbital analysis of polyenal-11 and lycopene has shown the structural differences which are also responsible for the resonance Raman data, based on the appearance of the (CH3) vibrational mode in the resonant transition only for lycopene. At last, the Raman band at ca. 1010 cm(-1), assigned to the (CH3) vibrational mode, can be used for attributing the presence of each one of the conjugated polyenes: the resonance Raman spectrum containing the band at ca. 1010 cm(-1) refers to the carotenoid (in this case lycopene), and the absence of such band in resonance conditions refers to the polyenal (in this case the polyenal-11).

Conjugated polyenes as chemical probes of life signature: use of Raman spectroscopy to differentiate polyenic pigments.

Polyenes, which are represented by carotenes, carotenoids and conjugated polyenals, are some of the most important targets for astrobiology, because they can provide strong evidence of the presence of organic compounds in the most extreme environments, such as on Mars. Raman spectroscopy has been used as the main analytical tool in the identification of such compounds, for the greatest variety of living species, from microorganisms to animals and plants. However, using only the position of the characteristic Raman bands can lead to errors in tentatively identifying chemicals. In this work, we present a series of observations that can provide a more complete and robust way to analyse the Raman spectrum of a polyenal, in which the position, the intensity, the use of various laser lines for excitation, and the combination of more than one pigment can be considered in the complete analysis.

Raman spectroscopic study of antioxidant pigments from cup corals Tubastraea spp.

Chemical investigation of nonindigenous Tubastraea coccinea and T. tagusensis by Raman spectroscopy resulted in the identification of carotenoids and indolic alkaloids. Comparison of Raman data obtained for the in situ and crude extracts has shown the potential of the technique for characterizing samples which are metabolic fingerprints, by means of band analysis. Raman bands at ca. 1520, 1160, and 1005 cm(-1) assigned to ν1(C═C), ν2(C-C), and ρ3(C-CH3) modes were attributed to astaxanthin, and the band at 1665 cm(-1) could be assigned to the ν(C-N), ν(C-O), and ν(C-C) coupled mode of the iminoimidazolinone from aplysinopsin. The antioxidant activity of the crude extracts has also been demonstrated, suggesting a possible role of these classes of compounds in the studied corals.

Structure of the Ebola fusion peptide in a membrane-mimetic environment and the interaction with lipid rafts.

The fusion peptide EBO(16) (GAAIGLAWIPYFGPAA) comprises the fusion domain of an internal sequence located in the envelope fusion glycoprotein (GP2) of the Ebola virus. This region interacts with the cellular membrane of the host and leads to membrane fusion. To gain insight into the mechanism of the peptide-membrane interaction and fusion, insertion of the peptide was modeled by experiments in which the tryptophan fluorescence and (1)H NMR were monitored in the presence of sodium dodecyl sulfate micelles or in the presence of detergent-resistant membrane fractions. In the presence of SDS micelles, EBO(16) undergoes a random coil-helix transition, showing a tendency to self-associate. The three-dimensional structure displays a 3(10)-helix in the central part of molecule, similar to the fusion peptides of many known membrane fusion proteins. Our results also reveal that EBO(16) can interact with detergent-resistant membrane fractions and strongly suggest that Trp-8 and Phe-12 are important for structure maintenance within the membrane bilayer. Replacement of tryptophan 8 with alanine (W8A) resulted in dramatic loss of helical structure, proving the importance of the aromatic ring in stabilizing the helix. Molecular dynamics studies of the interaction between the peptide and the target membrane also corroborated the crucial participation of these aromatic residues. The aromatic-aromatic interaction may provide a mechanism for the free energy coupling between random coil-helical transition and membrane anchoring. Our data shed light on the structural "domains" of fusion peptides and provide a clue for the development of a drug that might block the early steps of viral infection.

Structure of a membrane-binding domain from a non-enveloped animal virus: insights into the mechanism of membrane permeability and cellular entry.

The gamma(1)-peptide is a 21-residue lipid-binding domain from the non-enveloped Flock House virus (FHV). Unlike enveloped viruses, the entry of non-enveloped viruses into cells is believed to occur without membrane fusion. In this study, we performed NMR experiments to establish the solution structure of a membrane-binding peptide from a small non-enveloped icosahedral virus. The three-dimensional structure of the FHV gamma(1)-domain was determined at pH 6.5 and 4.0 in a hydrophobic environment. The secondary and tertiary structures were evaluated in the context of the capacity of the peptide for permeabilizing membrane vesicles of different lipid composition, as measured by fluorescence assays. At both pH values, the peptide has a kinked structure, similar to the fusion domain from the enveloped viruses. The secondary structure was similar in three different hydrophobic environments as follows: water/trifluoroethanol, SDS, and membrane vesicles of different compositions. The ability of the peptide to induce vesicle leakage was highly dependent on the membrane composition. Although the gamma-peptide shares some structural properties to fusion domains of enveloped viruses, it did not induce membrane fusion. Our results suggest that small protein components such as the gamma-peptide in nodaviruses (such as FHV) and VP4 in picornaviruses have a crucial role in conducting nucleic acids through cellular membranes and that their structures resemble the fusion domains of membrane proteins from enveloped viruses.

Reversible aggregation plays a crucial role on the folding landscape of p53 core domain.

The role of tumor suppressor protein p53 in cell cycle control depends on its flexible and partially unstructured conformation, which makes it crucial to understand its folding landscape. Here we report an intermediate structure of the core domain of the tumor suppressor protein p53 (p53C) during equilibrium and kinetic folding/unfolding transitions induced by guanidinium chloride. This partially folded structure was undetectable when investigated by intrinsic fluorescence. Indeed, the fluorescence data showed a simple two-state transition. On the other hand, analysis of far ultraviolet circular dichroism in 1.0 M guanidinium chloride demonstrated a high content of secondary structure, and the use of an extrinsic fluorescent probe, 4,4'-dianilino-1,1' binaphthyl-5,5'-disulfonic acid, indicated an increase in exposure of the hydrophobic core at 1 M guanidinium chloride. This partially folded conformation of p53C was plagued by aggregation, as suggested by one-dimensional NMR and demonstrated by light-scattering and gel-filtration chromatography. Dissociation by high pressure of these aggregates reveals the reversibility of the process and that the aggregates have water-excluded cavities. Kinetic measurements show that the intermediate formed in a parallel reaction between unfolded and folded structures and that it is under fine energetic control. They are not only crucial to the folding pathway of p53C but may explain as well the vulnerability of p53C to undergo departure of the native to an inactive state, which makes the cell susceptible to malignant transformation.

Conversion of wild-type p53 core domain into a conformation that mimics a hot-spot mutant.

The wild-type p53 protein can be driven into a conformation corresponding to that adopted by structural mutant forms by heterodimerization with a mutant subunit. To seek partially folded states of the wild-type p53 core domain (p53C) we used high hydrostatic pressure (HP) and subzero temperatures. Aggregation of the protein was observed in parallel with its pressure denaturation at 25 and 37 degrees C. However, when HP experiments were performed at 4 degrees C, the extent of denaturation and aggregation was significantly less pronounced. On the other hand, subzero temperatures under pressure led to cold denaturation and yielded a non-aggregated, alternative conformation of p53C. Nuclear magnetic resonance (1H15N-NMR) data showed that the alternative p53C conformation resembled that of the hot-spot oncogenic mutant R248Q. This alternative state was as susceptible to denaturation and aggregation as the mutant R248Q when subjected to HP at 25 degrees C. Together these data demonstrate that wild-type p53C adopts an alternative conformation with a mutant-like stability, consistent with the dominant-negative effect caused by many mutants. This alternative conformation is likely related to inactive forms that appear in vivo, usually driven by interaction with mutant proteins. Therefore, it can be a valuable target in the search for ways to interfere with protein misfolding and hence to prevent tumor development.