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Protein–carbohydrate interactions play a major role in several cellular and biological processes. Elucidating the factors influencing the binding affinity of protein–carbohydrate complexes and predicting their free energy of binding provide deep insights for understanding the recognition mechanism. In this work, we have collected the experimental binding affinity data for a set of 389 protein–carbohydrate complexes and derived several structure-based features such as contact potentials, interaction energy, number of binding residues and contacts between different types of atoms. Our analysis on the relationship between binding affinity and structural features revealed that the important factors depend on the type of the complex based on number of carbohydrate and protein chains. Specifically, binding site residues, accessible surface area, interactions between various atoms and energy contributions are important to understand the binding affinity. Further, we have developed multiple regression equations for predicting the binding affinity of protein–carbohydrate complexes belonging to six categories of protein–carbohydrate complexes. Our method showed an average correlation and mean absolute error of 0.731 and 1.149 kcal/mol, respectively, between experimental and predicted binding affinities on a jackknife test. We have developed a web server PCA-Pred, Protein–Carbohydrate Affinity Predictor, for predicting the binding affinity of protein–carbohydrate complexes. The web server is freely accessible at https://web.iitm.ac.in/bioinfo2/pcapred/. The web server is implemented using HTML and Python and supports recent versions of major browsers such as Chrome, Firefox, IE10 and Opera.

Background Protein-carbohydrate interactions play vital roles in several biological processes in living organisms. The comparative analysis of binding site residues along with stabilizing residues in protein-carbohydrate complexes provides ample insights to understand the structure, function and recognition mechanism. Objective The main objective of this study is to identify and analyze the residues, which are involved in both folding and binding of the protein-carbohydrate complexes. Methods We have identified the stabilizing residues using the knowledge of hydrophobicity, longrange interactions and conservation, as well as binding site residues using a distance cutoff of 3.5A between any heavy atoms in protein and ligand. Residues, which are common in stabilizing and binding, are termed as key residues. These key resides are analyzed with various sequence and structure based parameters such as frequency of occurrence, surrounding hydrophobicity, longrange order and conservation score. Results In this work, we have identified 2.45% binding site residues in a non-redundant dataset of 1130 complexes using distance-based criteria and 7.07% stabilizing residues using the concepts of hydrophobicity, long-range interactions and conservation of residues. Further, 5.9% of binding and 2.04% of stabilizing residues are common to each other, which are termed as key residues. The key residues have been analysed based on protein classes, carbohydrate types, gene ontology functional classifications, amino acid preference and structure-based parameters. We found that all-β, α+β and α/β have more key residues than other protein classes and most of the KRs are present in β-strands, which shows their importance in stability and binding of complexes. On the ligand side, Lsaccharide has the highest number of key residues and it has a high percentage of KRs in SRs and BRs than other carbohydrate types. Further, polar and charged residues have a high tendency to serve as key residues. Classifications based on gene ontology terms revealed that Lys is preferred in all the three groups: molecular functions, biological processes and cellular components. Key residues have 6 to 9 contacts within the protein and make only one contact with the carbohydrate ligand. These contacts are dominant to form polar-nonpolar contacts followed by the contacts between charged atoms. Further, the influence of sequence and structural parameters such as surrounding hydrophobicity, solvent accessibility, secondary structure, long-range order and conservation score has been discussed. Conclusion The results obtained in the present work provide deep insights for understanding the interplay between stability and binding in protein-carbohydrate complexes.

The aim of the study is histochemical identification of the components of protein-carbohydrate complex of wheat grains with selenium sources during germination. We formulated a hypothesis about the stimulating effect of 4.4-di [3 (5-methyldipirazolyl] selenid (DMDPS) on the biosynthesis of substances with a bactericidal effect, which include mucoid substances and glycoproteids in the composition of the components of the protein-carbohydrate complex. We confirm the hypothesis on histochemical identification of the components of the protein-carbohydrate complex of wheat grain during germination with sources of selenium. To identify acid mucopolysaccharides, transverse and longitudinal sections of the grain were stained with Alcian blue according to Steedman. Schiff-iodic acid revealed neutral glycoproteins and was clarified in xyllol. The sections were then placed into the balm and viewed in a Zeiss Axioscop 40 FX light microscope using a Levenhuk C 510 NG digital camera. To detect methionine, sections were stained with Schiff’s reagent; to identify cysteine (SH-groups), sections were treated with a 0.05% solution of 2.2-dioxy-6.6-dinaphthyl disulfide. The obtained images were analyzed using Photoshop CS 5 tools. Positive effect of wheat germination with DMDPA on the content of both neutral glycoproteids and mucopolysaccharides, with a direct correlation relationship, and on the content of methionine and cysteine was proved.

The 3D structure of a complex formed by the acidic fibroblast growth factor (FGF-1) and a specifically designed synthetic heparin hexasaccharide has been determined by NMR spectroscopy. This hexasaccharide can substitute natural heparins in FGF-1 mitogenesis assays, in spite of not inducing any apparent dimerization of the growth factor. The use of this well defined synthetic heparin analogue has allowed us to perform a detailed NMR structural analysis of the heparin-FGF interaction, overcoming the limitations of NMR to deal with the high molecular mass and heterogeneity of the FGF-1 oligomers formed in the presence of natural heparin fragments. Our results confirm that glycosaminoglycans induced FGF-1 dimerization either in a cis or trans disposition with respect to the heparin chain is not an absolute requirement for biological activity.

Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranose) is a non-reducing diglucoside found in various organisms that serves as a carbohydrate reserve and as an agent that protects against a variety of physical and chemical stresses. Deinococcus radiodurans possesses an alternative biosynthesis pathway for the synthesis of trehalose from maltooligosaccharides. This reaction is mediated by two enzymes: maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase). Here, we present the 1.1A resolution crystal structure of MTHase. It consists of three major domains: two beta-sheet domains and a conserved glycosidase (beta/alpha)8 barrel catalytic domain. Three subdomains consisting of short insertions were identified within the catalytic domain. Subsequently, structures of MTHase in complex with maltose and trehalose were obtained at 1.2 A and 1.5 A resolution, respectively. These structures reveal the importance of the three inserted subdomains in providing the key residues required for substrate recognition. Trehalose is recognised specifically in the +1 and +2 binding subsites by an extensive hydrogen-bonding network and a strong hydrophobic stacking interaction in between two aromatic residues. Moreover, upon binding to maltose, which mimics the substrate sugar chain, a major concerted conformational change traps the sugar chain in the active site. The presence of magnesium in the active site of the MTHase-maltose complex suggests that MTHase activity may be regulated by divalent cations.

The structure of a pectin-bound complex of rhamnogalacturonase was modeled to identify the amino acid residues involved in catalysis and substrate binding. The "hairy" region of pectin, represented by six repeating stretches of (1-->4)-D-galacturonate-(1-->2)-L-rhamnose dimer, was flexibly docked into the putative binding site of rhamnogalacturonase from Aspergillus aculeatus whose X-ray structure is known. A search of the complex configurational space was performed using AutoDock for the dimeric and tetrameric sugar units in which the -1 galacturonate residue has various ring conformations. Then the plausible AutoDock solutions were manually extended to the dodecameric pectin models. Subsequently, the resulting complex models were subjected to solvated molecular dynamics using AMBER. In the best model, the substrate has an extended pseudo-threefold helix with the -1 ring in a 4H3 half-chair that approaches the transition state conformation. The catalytic machinery is clearly defined: Asp197 is a general acid and the activated water bound between Asp177 and Glu198 is a nucleophile. The active site is similar, with a small yet significant difference, to that of polygalacturonase that degrades the pectic "smooth" region of linear homopolymer of D-(1-->4)-linked galacturonic acid. Rhamnogalacturonase has ten binding subsites ranging from -3 to +7, while polygalacturonase has eight subsites from -5 to +3. The model suggests that the eight amino acids including three arginine and three lysine residues, all of which are invariantly conserved in the rhamnogalacturonase family of proteins, are important in substrate binding. The present study may aid in designing mutational studies to characterize rhamnogalacturonase.

We present an automated docking protocol specifically optimized to predict the structure and affinity of a protein-carbohydrate complex. A scoring function was developed based on a training set of 30 protein-carbohydrate complexes of known structure and affinity. Combinations of several models for hydrogen bonding, torsional entropy loss, and solvation were tested for their ability to fit the training set data, and the best model was used with AutoDock. The electrostatic empirical coefficient is larger than in a previously obtained model using a training set comprised of various types of protein-ligand complexes, indicating that electrostatic interactions play a more important role in determining the affinity between a carbohydrate and a protein. The differences in the relative weighting of the empirical coefficients in the model yields predicted free energies for the training set with a standard error of 1.403 kcal/mol. The new scoring function was tested on 17 Aspergillus niger glucoamylase inhibitors for which binding energies had been determined experimentally. Free energies of complex formation were predicted with a residual standard error of 1.101 kcal/mol. The new scoring function therefore provides a robust method for predicting free energies of formation and optimal conformations of carbohydrate-protein complexes.

The mutant M 46 was selected from the wild strain F of Rhodospirillum rubrum after treatment with the mutagen 1-methyl-3-nitro-1-nitrosoguanidine. M 46 is unable to grow photosynthetically. It synthesizes a nonfunctional bacteriochlorophyll in small quantities under semiaerobic conditions. The characteristic bacteriochlorophyll in vivo absorption band at 880 nm is shifted to 872 nm, and the usual absorption band at 807 nm is completely absent. The mutant does not produce unsaturated carotenoids. Under semiaerobic conditions in the dark a bacteriopheophytin-protein-carbohydrate-containing complex is excreted. This process correlated with the bacteriochlorophyll synthesis but starts later. The complex was precipitated by ammonium suflate, purified by gel filtration and density gradient centrifugation. In electron micrographs of the negatively stained complex long filamentous aggregates of the macromolecules are to be seen. The filaments have a diameter of 40 ± 5 A . The sedimentation constant is 22.4 S; the diffusion constant is 1.24 F. The molecular weight was calculated to be 1.5·10 6 . The complex consists of 21.5% bacteriopheophytin, 61.0% protein (the amino acid composition is given), 10.0% glucose and 7.5% glucosamine. The complex was split into subunits by treatment with sodium dodecylsulfate together with urea. The molecular weight of the subunit is lower than 100 000. We assume that the pigment complex may be the well known P 800, which apparently cannot be incorporated into the photosynthetic unit.

The effect of divalent cations on the turbidity and pH of suspensions of isolated cell envelopes of a marine pseudomonad has been examined. As the concentration of Mg2+, Ca2+, or Mn2+ was increased the turbidity and pH of the suspensions increased. A concentration of 0.010 M of divalent cations produced the same turbidity in a suspension of cell envelopes as 1.0 M NaCl. The envelopes released soluble non-dialyzable material (Ndf) when suspended in 0.01 M NaCl but not when suspended in 0.001 M solutions of the divalent cations. Whole cells of the marine pseudomonad released an Ndf when suspended in 0.5 M sucrose. The Ndf from both cells and envelopes contained lipid, protein, and carbohydrate. Analytical ultracentrifugation of the Ndf from both cells and envelopes revealed the presence of only one major component with a sedimentation coefficient of 9.7 ± 0.07. Electrophoresis of Ndf gave rise to a component which stained for carbohydrate and another which stained for protein. Both moved to the anode.