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Endocannabinoid peptides, or "pepcans," are endogenous ligands of the CB1 cannabinoid receptor. Depending on their length, they display diverse activity: For instance, the nona-peptide Pepcan-9, also known as hemopressin, is a powerful inhibitor of CB1, whereas the longer variant Pepcan-12, which extends by only three amino acid residues at the N-terminus, acts on both CB1 and CB2 as an allosteric modulator, although with diverse effects. Despite active research on their pharmacological applications, very little is known about structure-activity relationships of pepcans. Different structures have been proposed for the nona-peptide, which has also been reported to form fibrillar aggregates. This might have affected the outcome and reproducibility of bioactivity studies. In an attempt of elucidating the determinants of both biological activity and aggregation propensity of Pepcan-9 and Pepcan-12, we have performed their structure characterization in solvent systems characterized by different polarity and pH. We have found that, while disordered in aqueous environment, both peptides display helical structure in less polar environment, mimicking the proteic receptor milieu. In the case of Pepcan-9, this structure is fully consistent with the observed modulation of the CB1. For Pepcan-12, whose allosteric binding site is still unknown, the presented structure is compatible with the binding at one of the previously proposed allosteric sites on CB1. These findings open the way to structure-driven design of selective peptide modulators of CB1.

Natural sweet proteins may be used as sugar replacer in simple liquid food systems but their applicability in more complex matrices has not been investigated yet. Gelling agent nature and texture characteristics as well as type and distribution of a stimulus in a gel could affect taste perception through inhibition or enhancement of tastants migration to the receptors. The mechanical, nonoral texture and time-intensity sweetness characteristics of sweet proteins MNEI and super sweet Y65R mutant, aspartame and saccharin added at a concentration iso-sweet to 40 g/L of sucrose in three agar gel concentrations (1%, 1.5%, and 2%) were evaluated. The results have shown that agar concentration and agar sweetener interaction particularly affect mechanical fracture stress and non oral hardness of the sweetened gels. Time intensity results illustrated that unlike in solution, the intensity of sweet taste in a gelled system over time decreases. Indeed, the behavior of the sweet proteins differed greatly in the gelled system compared to when they are in solution

3D domain swapping (3D‐DS) is a complex protein aggregation process for which no unique mechanism exists. We report an analysis of 3D‐DS in bovine seminal ribonuclease, a homodimeric protein whose subunits are linked by two disulfide bridges, based on NMR and biochemical studies. The presence of the covalent bonds between the subunits stabilizes the unswapped dimer, and allows distinct evaluation of the structural and dynamic effects of the swapping with respect to the dimerization process. In comparison with the monomeric subunit, which, in solution has a compact structure without any propensity for local unfolding, both swapped and unswapped dimers show increased flexibility. NMR analysis, together with urea denaturation and hydrogen–deuterium exchange data, indicates that the two dimers have increased conformational fluctuations. Furthermore, we found that the rate‐limiting step of both the swapping and unswapping pathways is the detachment of the N‐terminal helices from the monomers. These results suggest a new general mechanism in which a dimeric intermediate could facilitate 3D‐DS in globular proteins. Structured digital abstract http://www.uniprot.org/uniprot/P00669 and http://www.uniprot.org/uniprot/P00669 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0077 (http://www.ebi.ac.uk/intact/interaction/EBI-8870415), BS‐RNase is a unique dimeric protein, because it undergoes 3D domain swapping and exists as dimer also in the unswapped state. Our structural and dynamical comparative analysis among the swapped and unswapped isomers indicates that dimerization prepares the swapping, influencing the protein dynamics and conformational fluctuations. We suggest that this mechanism could be of more general occurrence.

Additional file 4: Table S1. Composition of the G-M17 medium.

MNEI has been proved to be a high-potency sweetener that could be used in the beverage industry to satisfy consumer demands for low-caloric products. Until now its sweetness profile was studied only in liquid samples. So the objectives of this study were to characterize the sweetness intensity profile of MNEI protein in simple gelled systems, and to characterize the dominance sensation of more complex gelled systems in comparison with other sweeteners. First, the sweetness intensity of MNEI and of three commercial sweeteners (aspartame, saccharin, and sucrose) was determined in aqueous solutions and in gel, at different agar concentrations, by the time-Intensity method. Then, gels at 1.5% agar concentration, containing flavoring, coloring, and acidifying agent were evaluated using the temporal dominance of sensation method to study the interaction between sweetness and other sensory attributes. Differences regarding sweeteners in solutions and gels were reported showing a decrease of sweetness intensity perception when sweeteners were added in a gelled matrix, especially for MNEI. Additionally, MNEI seemed to be the sweetener most affected by all evaluation conditions (coloring and acidifying agents) in the TDS method. The results are an important scientific contribution to the knowledge of MNEI sensory profile in gelled and complex matrices that could increase its range of food applications. Practical applications: The use of both time intensity and temporal dominance of sensation methods in order to evaluate the temporal sweetness profile of MNEI-based gelled system represented a new approach in the study of sweet proteins. Indeed, the use of the TDS represented an innovative approach to study the effect of color on sweetness and flavor perception. According to our results, MNEI gels did not perform well comparing to the other sweeteners in semisolid matrix. However, further studies should be carried out in order to better understand the interaction between MNEI protein and the gelling as well as the coloring agents. Regarding the application in semisolid foods, it could be suggested the use of new technologies like encapsulation in order to protect the protein from experimental conditions and prevent its interaction with the sweet taste receptor.

International audience

International audience; Wir beschreiben die sensitive Messung von alpha-Protonen in voll protonierten Proteinen durch Festkörper-NMR mit Rotation um den magischen Winkel (MAS) von 100-111 kHz. Die exzellente Auflösung in der Cα-Hα-Ebene wird für 5 Proteine gezeigt, darunter Mikrokristalle, ein sedimentierter Komplex, ein Kapsid und Amyloidfibrillen. Ausgehend davon wurde ein Satz 3D-Spektren für die sequenzspezifische Zuordnung der Rückgrat- und der aliphatischen Seitenketten-Resonanzen entwickelt. Dabei wurden nur 500 μg Probe verwendet. Diese Entwicklungen beschleunigen strukturelle Untersuchungen von biomolekularen Komplexen, die nur in Submilligramm-Mengen verfügbar sind, ohne die Notwendigkeit einer Deuterierung.; We demonstrate sensitive detection of alpha protons of fully protonated proteins by solid-state NMR with 100-111 kHz magic-angle spinning (MAS). The excellent resolution in the Cα-Hα plane is demonstrated for 5 proteins, including microcrystals, a sedimented complex, a capsid and amyloid fibrils. A set of 3D spectra based on a Cα-Hα detection block was developed and applied for the sequence-specific backbone and aliphatic side-chain resonance assignment using only 500 μg of sample. These developments accelerate structural studies of biomolecular assemblies available in submilligram quantities without the need of protein deuteration.

Le proteine dolci costituiscono un'accattivante alternativa ad alcuni dei più tradizionali dolcificanti, come il saccarosio, perché rispondono all???esigenza dei consumatori sempre più orientati verso prodotti con un basso contenuto calorico. Il presente lavoro è incentrato sulla caratterizzazione sensoriale di una proteina dolcificante modificata, MNEI, ottenuta attraverso espressione ricombinante in cellule di E. coli con rese elevate. In particolare sono state valutate le soglie di percezione (SP) e di riconoscimento (SR) della proteina sottoposta a diverse condizioni sperimentali, con l???obiettivo di validarne un suo impiego in campo alimentare, nel settore delle bevande. Gli obiettivi specifici sono stati lo studio dell???effetto della concentrazione di sali, dei valori di pH, di storia termica e di temperatura d???assaggio, sulle SP e SR. Lo studio delle SP e SR della proteina MNEI è stato effettuato utilizzando soluzioni modello, composte da acqua, proteina e in taluni casi da diverse quantità di acido citrico. Per la valutazione delle soglie della proteina sono stati utilizzati due metodi sensoriali già ampiamente applicati: ???Metodo della Differenza da un Riferimento??? e ???Metodo dei Limiti???. È stato inoltre usato un metodo modificato, che introduceva la richiesta di informazioni di tipo qualitativo nelle stesse sedute del test della ???Differenza da un Riferimento???. I risultati sperimentali hanno evidenziato un effetto significativo della concentrazione di sali sulla SP: più l???acqua è ricca in sali, più si alza la soglia di percezione della proteina. Anche la temperatura di assaggio delle soluzioni ha un effetto significativo sulla SP: a 10°C la SP è più alta che a 20°C. Né le variazioni di pH né la storia termica influenzano le soglie della MNEI, confermando che la proteina è stabile sia alle alte temperature che a bassi valori di pH. La richiesta di informazioni anche qualitative agli assaggiatori durante i test di valutazione della soglia non migliora la validità dei risultati ottenuti.

The possible use of ribonucleases in the treatment of diseases, including cancer, has received a considerable interest in the last years. The necessary requirements that convert a ribonuclease into a cytotoxin for malignant tumour cells include binding to cell membrane, translocation into the cytosol and RNA degradation. Among the mammalian ribonucleases, only bovine seminal ribonuclease (BS-RNase) fulfils all these requirements (1). We have designed a BS_RNase variant with an enforced cluster of positive charges at the N-termini surface to improve its cytotoxic potential according to literature data indicating that the protein approaches the membrane with a preferred orientation (2). We found that the G38K-BS-RNase variant displays an increased citotoxicity for tumour cells and we hypothesized the higher cytotoxicity to be connected to its enhanced interaction with membranes. A detailed investigation of the membrane interaction mechanism of BS-RNase and G38K-BS-RNase, using a combination of biophysical techniques, including Surface Plasmon Resonance and Electron Spin Resonance, supports this idea, and indicates for both of them a common two-step mechanism, based on an initial binding to the bilayer surface followed by a shallow penetration in the lipid core. Protein binding , driven by electrostatic interactions, effectively perturbs lipid packing and dynamics. Our results, while ruling out the hypothesis that the protein crosses the membrane interior, support the hypothesis of a BS-RNase entry mediated by an endocytosis mechanism. Overall, these studies shed light on the mechanism of ribonuclease membrane binding and perturbation, and provide a rational basis to design proteins with higher antitumor potential.

BS-RNase, a homodimeric protein displaying selective cytotoxicity towards tumor cells, is isolated as a mixture of two isoforms, MxM and M=M, with and without exchange of the monomers’ N-termini. In the cytosolic reducing environment, MxM is converted into a non-covalent dimer (termed NCD), in which the monomers remain intertwined through their N-terminal ends. The quaternary structure renders the reduced protein resistant to the ribonuclease inhibitor (RI), a protein that binds most ribonucleases with very high affinity. On the other hand, upon selective reduction M=M is converted in two monomers, which are readily bound and inactivated by RI. Based on these considerations, it has been proposed that the cytotoxic activity of BS-RNase relies on the 3D structure and stability of its NCD derivative. Here, we report a comparison of the thermodynamic and chemical stability of the NCD form of BS-RNase with that of the monomeric derivative, together with an investigation of the thermal dissociation mechanism revealing the presence of a dimeric intermediate. In addition, we report that the substitution of Arg80 with Ser decreases significantly the cytotoxic activity of BS-RNase and the stability of the NCD form with respect to the parent protein, but does not affect the ribonucleolytic activity nor the dissociation mechanism. The data show the importance of Arg80 for the cytotoxicity of BS-RNase and also support the hypothesis that the reduced derivative of BS-RNase is responsible for its cytotoxic activity.

Bovine seminal RNase (BS RNase) is the only naturally dimeric member of the pancreatic-type RNase superfamily. The enzyme is a mixture of two isoforms: 1) M=M, dimeric through two antiparallel disulfides occurring between the two subunits; 2) MxM (70% of the total), characterized by the swapping of the N-termini besides the mentioned disulfides1. When dissolved in 40% acetic acid and subjected to lyophilisation, BS RNase forms a mixture of oligomeric aggregates2, as does RNase A, the proto-type of the super-family3. However, while the oligomers of the pancreatic variant have been extensively characterized3-5, the multimers of BS RNase6 are presently less known. To deepen their characterization we induced BS RNase self-association by using the same conditions used with RNase A, i.e. lyophilisation of enzyme solutions at low pH, or thermally-induced aggregation of concentrated protein solutions in various media. The multimers obtained were analyzed by SEC, cation-exchange chromatography, cross-linking, electrophoresis, proteolysis, and enzymatic assays. The main results obtained are: - BS-RNase forms several 3D domain-swapped conformers, in particular at least two tetramers; one is probably a totally N-terminal-swapped isoform4, while the other contains a C-terminal swapping. - The C-swapped tetramer is less stable than the N-swapped one. - BS RNase multimers larger than tetramers appear to be a mixture of various isoforms, similarly to what occurs with RNase A3. These multimers seem to contain, again, a C-terminus swapping. - The validity of the results reported have been strengthened by the studies performed with a K113N BS RNase mutant.

The stability of the folded structure of a protein molecule is generally limited to a narrow range of environmental parameters. Depending on temperature, pH and types of co-solutes and on the evolutionary history of the protein itself, the tertiary structure may become less tightly bound and undergo to a more or less extensive unfolding that can lead to either unspecific aggregation or ordered adducts with other molecules or even to oligomeric aggregates endorsed with new functions. In the same way, specific sequence modification in hot points of the protein may lead to an “open” monomeric species which can interact in a complementary fashion with a similarly opened molecule (domain swapping) to produce a thermodynamic stable or kinetically trapped transient dimeric form (swapped dimer)[1]. The dimer is stabilized by the pre-existing surfaces of the swapped domains, whereas the flexible loops connecting these domains give rise to a somewhat ‘destructured quaternary assembly’ of the chains that strongly increases the potentiality for higher aggregation [2]. Bovine pancreatic ribonuclease (RNase A) is able to swap both the C-terminal - strand (residues 116-124) and the N-terminal -helix (residues 1-13), thus forming two different dimers [3-4]. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein forms the C-terminal swapped dimer [4]. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. Surprisingly, the mutation not only affects the swapping of the C-terminal b-strand but also that of the N-terminal helix and the stability of the two swapped dimers. These results, which are discussed on the bases of the X-ray structures of the monomeric and C-terminal swapped dimeric forms of P114A and on the available biochemical and structural data on RNase A dimers, reveal a cross-talk between the chain termini in the swapping process of RNase A.

Pancreatic ribonucleases, thanks to their ability to degrade RNA, represent potential antitumor therapeutics. It has been suggested that RNases have to reach the cytosol and to avoid the binding of the ribonuclease inhibitor (RI) in order to exert their cytotoxic activity1. Among antitumor RNases, bovine seminal ribonuclease (BS-RNase) is an interesting case: it is a covalent homodimer with two interchain antiparallel disulphide bridges between Cys31 and Cys322-3, which in the reducing cellular compartment of cytosol convert into a non covalent dimeric form (NCD) stabilized by the swapping of N-terminal helices4. It has been suggested that NCD-BS avoids inhibition by RI because it adopts a compact quaternary structure that interferes with binding [4]. Structural studies have indicated a key role in determining this dimeric assembly for specific amino acid residues: Pro19 and Leu285. Using the homology mutation approach, few structural features of BS-RNase have been introduced in bovine pancreatic ribonuclease (RNase A), which does not show antitumor action. The introduction of Leu28, Cys31 and Cys32 (LCC) and that of the same residues plus Pro19 (PLCC) or the introduction of the hinge peptide sequence (residues 16-22) of BS-RNase and the two cysteines (GNPSCC) in the sequence of RNase A produced dimeric variants which do exhibit a cytotoxic activity, though at a much lower level than BS-RNase6-7. Structural determination of NCD forms of these mutants has allowed to rationalyze their biological activities: all the dimers have open quaternary structures, which can be easily sequestered by RI. The comparison of these structures with those previously reported for other RNase swapped dimers has suggested that an additional important role in determining the compact quaternary structure of NCD-BS can be played also by Gly167. Therefore, the GPLCC mutant of RNase A (with substitution S16G, A19P, Q28L, R31C and S32C) has been produced and found to exert a cytotoxic activity on SVT2 cells comparable to that of seminal enzyme. In order to verify whether NCDGPLCC has a quaternary assembly similar to NCD-BS, its structure has been determined from two different crystal forms obtained from similar crystallization conditions. Surprisingly, NCD-GPLCC shows two different quaternary structures, both of them distinct with respect to NCD-BS. As predicted7, the introduction of Gly16 determines a hinge peptide conformation similar to that observed in NCD-BS. However, in each structure this occurs only for one of the two hinge peptides, and the resulting quaternary assembly is still compatible with the RI binding. Altogether these data represent an encouraging step toward a rational design of a dimeric form of RNase A endowed with a strong cytotoxic activity and point up the necessity of a deeper characterization of its complex with RI, in terms of binding constants and residual ribonuclease activity.

Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to better understand the molecular bases of their action, and also to design targeted structural/functional modifications. Peamaclein is a recently identified 7 kDa peach allergen that has been associated with severe allergic reactions in sensitive subjects. This protein represents the first component of a new allergen family, which has no 3D structure available yet. Here, we report the first experimental data on the 3D-structure of Peamaclein. Almost 75% of the backbone resonances, including two helical stretches in the N-terminal region, and four out of six cysteine pairs have been assigned by 2D-NMR using a natural protein sample. Simulated gastrointestinal digestion experiments have highlighted that Peamaclein is even more resistant to digestion than the peach major allergen Pru p 3. Only the heat-denatured protein becomes sensitive to intestinal proteases. Similar to Pru p 3, Peamaclein keeps its native 3D-structure up to 90 degrees C, but it becomes unfolded at temperatures of 100-120 degrees C. Heat denaturation affects the immunological properties of both peach allergens, which lose at least partially their IgE-binding epitopes. In conclusion, the data collected in this study provide a first set of information on the molecular properties of Peamaclein. Future studies could lead to the possible use of the denatured form of this protein as a vaccine, and of the inclusion of cooked peach in the diet of subjects allergic to Peamaclein. (C) 2014 Wiley Periodicals, Inc.

E' stato realizzato un sito web di divulgazione della Chimica: http://www.whatischemistry.unina.it/ Il sito vuole rispondere alle esigenze di divulgazione scientifica della nostra società e al problema dell'immagine della chimica presso il pubblico. Il sito si caratterizza per la correttezza scientifica, la semplicità del linguaggio, il legame con l'esperienza quotidiana. Sono trattati alcuni concetti fondamentali e diversi argomenti applicativi. E' sottolineata l'importanza della chimica per le scienze della vita. Sono discussi i rischi della chimica e i possibili rimedi. L'opera non ha pretese didattiche ma cerca di sollecitare la curiosità e l'interesse, è destinata a un pubblico internazionale ed è interamente in italiano e in inglese.

What is Chemistry is a website fot the general public about Chemistry. It is found at URL http://www.whatischemistry.unina.it/ It tries to address the negative image of Chemistry often transmitted by mass media. It is not a full learning tool, it just tries to stimulate interest. All chemical contents are correct and aligned to the present state of knowledge. The language is extremely plain and the contents should be understood by people lacking any background in Science.

Bovine seminal ribonuclease (BS-RNase) is a covalent homodimeric enzyme homologous to pancreatic ribonuclease (RNase A), endowed with a number of special biological functions. It is isolated as an equilibrium mixture of swapped (MxM) and unswapped (M=M) dimers. The interchanged N termini are hinged on the main bodies through the peptide 16-22, which changes conformation in the two isomers. At variance with other proteins, domain swapping in BS-RNase involves two dimers having a similar and highly constrained quaternary association, mainly dictated by two interchain disulfide bonds. This provides the opportunity to study the intrinsic ability to swap as a function of the hinge sequence, without additional effects arising from dissociation or quaternary structure modifications. Two variants, having Pro19 or the whole sequence of the hinge replaced by the corresponding residues of RNase A, show equilibrium and kinetic parameters of the swapping similar to those of the parent protein. In comparison, the x-ray structures of MxM indicate, within a substantial constancy of the quaternary association, a greater mobility of the hinge residues. The relative insensitivity of the swapping tendency to the substitutions in the hinge region, and in particular to the replacement of Pro19 by Ala, contrasts with the results obtained for other swapped proteins and can be rationalized in terms of the unique features of the seminal enzyme. Moreover, the results indirectly lend credit to the hypothesis that the major role of Pro19 resides in directing the assembly of the non-covalent dimer, the species produced by selective reduction of the interchain disulfides and considered responsible for the special biological functions of BS-RNase.

The effects of selected mutations at the O-interface on the equilibrium between the MxM and M=M forma of bovine seminal ribonuclease have been studied.

The relative insesitivity of the swapping tendency of bovine seminal ribonuclease to the substitutions in the hinge peptide has been rationalized in terms of the unique features of this enzyme.