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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.

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.

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.

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.