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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.