Monitoring the Interactions of a Ternary Complex Using NMR Spectroscopy: The Case of Sugars, Polyphenols, and Proteins

Gaining insight into intermolecular interactions between multiple species is possible at an atomic level by looking at different parameters using different NMR techniques. In the specific case of the astringency sensation, in which at least three molecular species are involved, different NMR techniques combined with dynamic light scattering and molecular modeling contribute to decipher the role of each component in the interaction mode and to assess the thermodynamic parameters governing this complex interaction. The binding process between a saliva peptide, a polyphenol, and polysaccharides was monitored by following ¹H chemical shift variations, changes in NMR peak areas, and size of the formed complex. These NMR experiments deliver a complete picture of the association pathway, assessed by dynamic light scattering and molecular dynamics simulations: all of the data collected converge toward a comprehensive mode of interaction in which sugars indirectly play a role in astringency by sequestering part of the polyphenols, reducing their effective concentration to bind saliva proteins

The Colloidal State of Tannins Impacts the Nature of Their Interaction with Proteins: The Case of Salivary Proline-Rich Protein/Procyanidins Binding

While the definition of tannins has been historically associated with its propensity to bind proteins in a nonspecific way, it is now admitted that specific interaction also occurs. The case of the astringency perception is a good example to illustrate this phenomenon: astringency is commonly described as a tactile sensation induced by the precipitation of a complex composed of proline-rich proteins present in the human saliva and tannins present in beverages such as tea or red wines. In the present work, the interactions between a human saliva protein segment and three different procyanidins (B1, B3, and C2) were investigated at the atomic level by NMR and molecular dynamics. The data provided evidence for (i) an increase in affinity compared to shortest human saliva peptides, which is accounted for by protein “wraping around” the tannin, (ii) a specificity in the interaction below tannin critical micelle concentration (CMC) of ca. 10 mM, with an affinity scale such that C2 > B1 > B3, and (iii) a nonspecific binding above tannin CMC that conducts irremediably to the precipitation of the tannins/protein complex. Such physicochemical findings describe in accurate terms saliva protein–tannin interactions and provide support for a more subtle description by oenologists of wine astringency perception in the mouth

Molecular Dynamics Analysis of a Novel β3 Pro189Ser Mutation in a Patient with Glanzmann Thrombasthenia Differentially Affecting αIIbβ3 and αvβ3 Expression

<div><p>Mutations in <i>ITGA2B</i> and <i>ITGB3</i> cause Glanzmann thrombasthenia, an inherited bleeding disorder in which platelets fail to aggregate when stimulated. Whereas an absence of expression or qualitative defects of αIIbβ3 mainly affect platelets and megakaryocytes, αvβ3 has a widespread tissue distribution. Little is known of how amino acid substitutions of β3 comparatively affect the expression and structure of both integrins. We now report computer modelling including molecular dynamics simulations of extracellular head domains of αIIbβ3 and αvβ3 to determine the role of a novel β3 Pro189Ser (P163S in the mature protein) substitution that abrogates αIIbβ3 expression in platelets while allowing synthesis of αvβ3. Transfection of wild-type and mutated integrins in CHO cells confirmed that only αvβ3 surface expression was maintained. Modeling initially confirmed that replacement of αIIb by αv in the dimer results in a significant decrease in surface contacts at the subunit interface. For αIIbβ3, the presence of β3S163 specifically displaces an α-helix starting at position 259 and interacting with β3R261 while there is a moderate 11% increase in intra-subunit H-bonds and a very weak decrease in the global H-bond network. In contrast, for αvβ3, S163 has different effects with β3R261 coming deeper into the propeller with a 43% increase in intra-subunit H-bonds but with little effect on the global H-bond network. Compared to the WT integrins, the P163S mutation induces a small increase in the inter-subunit fluctuations for αIIbβ3 but a more rigid structure for αvβ3. Overall, this mutation stabilizes αvβ3 despite preventing αIIbβ3 expression.</p></div

Effect of the β3P163 substitution on the backbone flexibility of β3 within the integrin complex.

<p>RMSF values are calculated for each residue within WT β3 (A) and P163S β3 (B) in complex with either αIIb (heavy line) or αv (faint line). Red arrows indicate the position of the mutation. The largest changes are seen approximately 100 amino acids forward from the mutation (dotted box).</p

Chemical Details on Nucleolipid Supramolecular Architecture: Molecular Modeling and Physicochemical Studies

Nucleolipids are currently under investigation as vectors for oligonucleotides (ON) delivery thanks to their supramolecular organization properties and their ability to develop specific interactions (i.e., stacking and potential Watson and Crick hydrogen bonds) for lipoplexes formation. To investigate the factors that govern the interaction events at a molecular level and optimize nucleolipid chemical structures, physicochemical experiments (tensiometry, AFM, BAM, and ellipsometry) combined with molecular dynamics simulation were performed on a series of zwitterionic nucleolipids (PUPC, DPUPC, PAPC) featuring a phosphocholine chain (PC). After construction and initial equilibration, simulations of pure nucleolipid bilayers were run for 100 ns at constant temperature and pressure, and their properties were compared to experimental data and to natural dipalmitoylphosphatidylcholine (DPPC) bilayers. Nucleolipid-based membranes are significantly more ordered and compact than DPPC bilayers mainly due to the presence of many intermolecular interactions between nucleoside polar heads. The hydrophilic phosphocholine moieties connected to the 5′ hydroxyls are located above the bilayers, penalizing nucleic bases accessibility for further interactions with ON. Hence, a neutral nucleolipid (PUOH) without hydrophilic phosphocholine was inserted in the membranes. Simulations and experimental analysis of nucleolipid membranes in interaction with a single strand RNA structure indicate that PUOH interacts with ON in the subphase. This study demonstrates that molecular modeling can be used to determine the interactions between oligonucleotide and nucleolipids

Maximum intraspecific distance and distance to the nearest neighbor of the 157 species with two or more sequences delimited under the Initial Morphological Assessment (A) and Morphological Reassessment (B) approaches.

<p>Maximum intraspecific distance and distance to the nearest neighbor of the 157 species with two or more sequences delimited under the Initial Morphological Assessment (A) and Morphological Reassessment (B) approaches.</p

Timeline plots of the β3 secondary structure.

<p>Illustrated are the WT form (A, C) and the P163S mutated form (B, D) either associated with αIIb (A, B) or αv (C, D) subunits. Shown are molecular dynamics time and primary sequence: time (60 ns) is on the horizontal axis and primary sequence is on the vertical axis. The following color code is used for the secondary structure: dark green = turn, yellow = β-sheet, pink = α-helix, blue = 3–10 helix, red = pi-helix, white = random. Position 163 is indicated by red arrows and the region concerned by the major changes is framed with a dotted box.</p

Recursive and Initial partitions delimited with the Automatic Barcode Gap Discovery approach (ABGD) used to uncover Tiger Moths diversity along an elevational gradient in southern Brazilian Atlantic Forest.

<p>Recursive and Initial partitions delimited with the Automatic Barcode Gap Discovery approach (ABGD) used to uncover Tiger Moths diversity along an elevational gradient in southern Brazilian Atlantic Forest.</p

Static modeling showing the positioning of β3P163.

<p>Panel (A) represents computer-drawn ribbon diagrams of the WT αIIb and β3 headpiece complex and panel (B) the corresponding structure for WT αv and β3 subunits. Interacting surfaces are colored in blue for αIIb or αv, and in red for β3. Amino acids forming a H-bond with their counterpart in the other subunit are represented as sticks. H-bonds are shown as dotted lines. Interactions modified by the mutation are highlighted in boxes A.1 and B.1. The mutated proline is colored in yellow. Models were obtained using the PyMol Molecular Graphics System, version 1.3, Schrödinger, LLC and 3fcs and 1u8c pdb files for the crystal structure of αIIb in complex with β3 and αv in complex with β3 in bent conformations.</p

Results of the Morphological Reassessment (MRA) to assess incongruences between species delimited according to the IMA and BIN systems.

<p>Results of the Morphological Reassessment (MRA) to assess incongruences between species delimited according to the IMA and BIN systems.</p