Philippe Lagant

Force-field and vibrational spectra of oligosaccharides with different glycosidic linkages—Part I. Trehalose dihydrate, sophorose monohydrate and laminaribiose

Abstract The vibrational spectra of the disaccharides, trehalose dihydrate, sophorose monohydrate and laminaribiose, have been recorded in the crystalline state in the 4000-100 cm−1 spectral region for the IR spectra and in the 4000-20 cm−1 spectral range for the Raman spectra. These three disaccharides exhibit the same monosaccharide composition (i.e. glucose residue), but differ in the position and configuration of the glycosidic linkage (α, 1-1; β, 1-2 and β, 1-3 for trehalose, sophorose and laminaribiose, respectively). Most of these spectra have not yet been reported, particularly in the low frequency range. They constitute the basis of theoretical calculations of normal modes of vibration. Normal coordinate analysis has been made in the crystalline state using a modified Urey-Bradley-Shimanouchi intramolecular potential energy combined with a specific intermolecular potential energy function. The force field parameters are transformed from initial works on both anomers of glucose. The vibrational assignments of the observed bands are made on the basis of the potential energy distributions. It appears that the greatest part of the vibrational modes is very highly coupled vibrations. The calculated vibrational frequencies agree very well with the observed frequencies in the whole spectra, particularly in the “fingerprint” regions and in the low frequency range. The bands observed at 733, 773 and 755 cm−1 for trehalose dihydrate, sophorose monohydrate and laminaribiose, respectively, are calculated at 728, 772 and 755 cm−1 and are due to bending modes of heavy atoms involved in the corresponding glycosidic linkage C1ue5f8O1ue5f8C′x. Moreover, some known characteristic structural regions may be divided into different parts that have a specific significance. Ale standard deviation between calculated and observed frequencies below 1500 cm−1 leads to values of 3.0, 3.7 and 4.2 cm−1 for the three disaccharides, respectively.

Force field and vibrational spectra of oligosaccharides with different glycosidic linkages—Part II. Maltose monohydrate, cellobiose and gentiobiose

Abstract Complete Raman and IR spectra of maltose monohydrate, cellobiose and gentiobiose have been recorded in the crystalline state. These three disaccharides present the same monosaccharide composition of the glucose molecule and the remaining studied position (1–4 and 1–6) of the glycosidic linkage. Moreover, maltose and cellobiose present the different configurations of the glycosidic linkage α, 1–4 and β, 1–4, respectively. These data will constitute the support for theoretical calculations of normal modes of vibration. The assignments of the calculated bands of vibration will be made on the basis of the potential energy distributions using a modified Urey—Bradley—Shimanouchi intramolecular potential energy combined with a specific intermolecular potential energy function. The calculations show that using a correct initial force field, it is possible to reproduce correctly the density of observed vibrational states for large molecules such as disaccharides. The standard deviation between calculated and observed frequencies, below 1500 cm −1 , leads to values of 4.7, 4.2 and 4.6 cm − for maltose monohydrate, cellobiose and gentiobiose, respectively. Our previous investigations on trehalose dihydrate, sophorose monohydrate and laminaribiose are confirmed in this study and complete the previous assignments for the whole set of disaccharides.

Function and Molecular Modeling of the Interaction between Human Interleukin 6 and Its HNK-1 Oligosaccharide Ligands

Interleukin 6 (IL-6) is endowed with a lectin activity for oligosaccharide ligands possessing the HNK-1 epitope (3-sulfated glucuronic acid) found on some mammalian glycoproteinN-glycans (Cebo, C., Dambrouck, T., Maes, E., Laden, C., Strecker, G., Michalski, J. C., and Zanetta, J. P. (2001)J. Biol. Chem. 276, 5685–5691). Using high affinity oligosaccharide ligands, it is demonstrated that this lectin activity is responsible for the early dephosphorylation of tyrosine residues found on specific proteins induced by interleukin 6 in human resting lymphocytes. The gp130 glycoprotein, the signal-transducing molecule of the IL-6 pathway, is itself a molecule possessing the HNK-1 epitope. This indicates that IL-6 is a bi-functional molecule able to extracellularly associate its α-receptor with the gp130 surface complex. Computational modeling indicates that the lower energy conformers of the high affinity ligands of IL-6 have a common structure. Docking experiments of these conformers suggest that the carbohydrate recognition domain of IL-6 is localized in the domain previously identified as site 3 of IL-6 (Somers, W., Stahl, M., and Seehra, J. S. (1997) EMBO J. 16, 989–997), already known to be involved in interactions with gp130.

The SPASIBA force field as an essential tool for studying the structure and dynamics of saccharides

The SPASIBA force field has been applied to the determination of the structure and dynamical properties of various disaccharides. It has been shown that the experimental properties (structure, dipole moment, conformational relative energies) are satisfactorily predicted. The anomeric and exo-anomeric effects are confidently reproduced without specific terms for the alpha and beta anomers and the type of glycosidic linkages.

Phenylalanine 35 and tyrosine 82 are involved in the uptake and release of ligand by porcine odorant-binding protein

Structural and molecular dynamics studies have pointed out the role of aromatic residues in the uptake of ligand by porcine odorant-binding protein (pOBP). The shift of Tyr82 from its position during the opening of the binding cavity has been shown, and was supposed to participate in the entrance of the ligand. Several Phe residues in the vicinity of Tyr82 could also participate in the binding process. To clarify their involvement, we performed molecular dynamics studies to simulate the dissociation of undecanal, a ligand previously co-crystallized with pOBP. The results confirmed the key-role of Tyr82 and pointed out the participation of Phe35 in controlling the reorientation of undecanal towards the exit. To bring experimental support to both published (binding) and present simulations (dissociation), we have mutated these two residues and over expressed the wild type pOBP, the two single mutants and the double mutant in the yeast Pichia pastoris. As fluorescence spectroscopy implies the uptake of the fluorescent probe and release in displacement experiments, we monitored the binding ability of the four proteins for 1-aminoanthracene (1-AMA). The experimental results indicated that both residues are involved in the uptake of ligand as the three mutated proteins were unable to bind 1-AMA, contrary to the wild type recombinant pOBP that bound 1-AMA with the expected affinity.

Binding specificity of recombinant odorant-binding protein isoforms is driven by phosphorylation.

Native porcine odorant-binding protein (OBP) bears eleven sites of phosphorylation, which are not always occupied in the molecular population, suggesting that different isoforms could co-exist in animal tissues. As phosphorylation is a dynamic process resulting in temporary conformational changes that regulate the function of target proteins, we investigated the possibility that OBP isoforms could display different binding affinities to biologically relevant ligands. The availability of recombinant proteins is of particular interest for the study of protein/ligand structure-function relationships, but prokaryotic expression systems do not perform eukaryotic post-translational modifications. To investigate the role of phosphorylation in the binding capacities of OBP isoforms, we produced recombinant porcine OBP in two eukaryotic systems, the yeast, Pichia pastoris, and the mammalian CHO cell line. Isoforms were separated by anion exchange HPLC, and their phosphorylation sites were mapped by MALDI-TOF mass spectrometry and compared to those of the native protein. Binding experiments with ligands of biological relevance in the pig, Sus scrofa, were performed by fluorescence spectroscopy on two isoforms of recombinant OBP expressed in the yeast. The two isoforms, differing only by their phosphorylation pattern, displayed different binding properties, suggesting that binding specificity is driven by phosphorylation.

Development of the force field parameters for phosphoimidazole and phosphohistidine

Phosphorylation of histidine‐containing proteins is a key step in the mechanism of many phosphate transfer enzymes (kinases, phosphatases) and is the first stage in a wide variety of signal transduction cascades in bacteria, yeast, higher plants, and mammals. Studies of structural and dynamical aspects of such enzymes in the phosphorylated intermediate states are important for understanding the intimate molecular mechanisms of their functioning. Such information may be obtained via molecular dynamics and/or docking simulations, but in this case appropriate force field parameters for phosphohistidine should be explicitly defined. In the present article we describe development of the GROMOS96 force field parameters for phosphoimidazole molecule—a realistic model of the phosphohistidine side chain. The parameterization is based on the results of ab initio quantum chemical calculations with subsequent refinement and testing using molecular mechanics and molecular dynamics simulations. The set of force constants and equilibrium geometry is employed to derive force field for the phosphohistidine moiety. Resulting parameters and topology are incorporated into the molecular modeling package GROMACS and used in molecular dynamics simulations of a phosphohistidine‐containing protein in explicit solvent.

Dynamical analysis of low-frequency depolarized Raman spectra of a neat monodisperse hyperbranched polymer between 333 and 123 K: Poly(propylene imine) DAB-dendr-(CN)64

Abstract VH-polarized Raman spectra of the title system between 4 – 250 cm−1 and at 333, 300, 273, 243, 173, 150, and 123 K show varying dynamical aspects depending on temperature and frequency range. At 123 K and between 10 – 50 cm−1 the system shows phonon motions, with a crossover at frequency νC ∼ 25 cm−1 from a Debye to a fraction regime of spectral dimension d ∼ 1.1 and a correlation length which decreases with increasing temperatures. This implies that the open branching pattern of the molecules assumes, on the average, a more compact configuration with rising kinetic energies. Above 123 K, increasing temperatures cause increasing excess intensity from intramolecular relaxation mechanisms involving the motions of the molecules terminal (CH2)2CN groups, with zero-frequency rates extrapolating to a critical temperature of TC ∼ 213 K, a value which is close to the thermodynamic glass transition point TG = 233 K of the system.

A density functional derived vibrational force field for dopamine

Dopamine (figure 1) is one of the most important neurotraasmitters of the nervous system in invertebrates and vertebrates species. This molecule is related to several neurobiological dysfunctions like Parkinson disease Ab inition methods are particularly well suited to investigate the physical properties (geometry, vibrational frequencies, force constants...). The density functional theory (DFT) method was employed here for dopamine. The optimized structure and harmonic vibrational normal modes were obtained from the gaussian 94 using the 6-31G(df, p)(5d, 7f) basis set.