A chitotetrose specific lectin from Luffa acutangula: physico-chemical properties and the assignment of orientation of sugars in the lectin binding site

A chitooligosaccharide specific lectin (Luffa acutangula agglutinin) has been purified from the exudate of ridge gourd fruits by affinity chromatography on soybean agglutinin-glycopeptides coupled to Sepharose-6B. The affinity purified lectin was found homogeneous by polyacrylamide gel electrophoresis, in sodium dodecyl sulphate-polyacrylamide gels, by gel filtration on Sephadex G-100 and by sedimentation velocity experiments. The relative molecular weight of this lectin is determined to be 48,000±1,000 by gel chromatography and sedimentation equilibrium experiments. The sedimentation coefficient (S20, w) was obtained to be 4.06 S. The Stokes' radius of the protein was found to be 2.9 nm by gel filtration. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis the lectin gave a molecular weight of 24,000 in the presence as well as absence of 2-mercaptoethanol. The subunits in this dimeric lectin are therefore held by non-covalent interactions alone. The lectin is not a glycoprotein and circular dichroism spectral studies indicate that this lectin has 31% α-helix and no β-sheet. The lectin is found to bind specifically to chitooligosaccharides and the affinity of the lectin increases with increasing oligosaccharide chain length as monitored by near ultra-violet-circular dichroism and intrinsic fluorescence titration. The values of ΔG, ΔH and ΔS for the binding process showed a pronounced dependence on the size of the oligosaccharide. The values for both ΔH and ΔS show a significant increase with increase in the oligosaccharide chain length showing that the binding of higher oligomers is progressively more favoured thermodynamically than chitobiose itself. The thermodynamic data is consistent with an extended binding site in the lectin which accommodates a tetrasaccharide. Based on the thermodynamic data, blue shifts and fluorescence enhancement, spatial orientation of chitooligosaccharides in the combining site of the lectin is assigned

Elucidation of the combining site of Coccinia indica agglutinin (CIA) by thermodynamic analyses of its ligand binding

Analyses of the ligand size dependence of the fluorescence spectra of CIA together with the thermodynamic parameters for the lectin reveal that its combining site spans the tetrasaccharide chitotetraose. Moreover the fourth sugar residue of chitoligosaccharide is proximal to a highly fluorescent tryptophan

A23187-Channel behaviour: fluorescence study

Pyranine entrapped soylipid liposomes have been used as a model system to study the proton transport across membrane in the presence of A23187, a carboxylic ionophore specific for electroneutral exchange of divalent cations. An apparent rate constant (kapp) for transport of protons has been determined from the rate of change of fluorescence intensity of pyranine by stopped flow rapid kinetics in the presence of proton gradient The variation of kapp has been studied as a function of ionophore concentration and the results have been compared with gramicidin-a well known channel former under the similar experimental conditions. The rates thus obtained showed that A23187 is not only a simple carrier but also shows channel behaviour at high concentration of ionophore

Carbohydrate specificity and salt-bridge mediated conformational change in acidic winged bean agglutinin

Structures of two crystal forms of the dimeric acidic winged bean agglutinin (WBAII) complexed with methyl-α-D-galactose have been determined at 3.0 Å and 3.3 Å resolution. The subunit structure and dimerisation of the lectin are similar to those of the basic lectin from winged bean (WBAI) and the lectin from Erythrina corallodendron (EcorL). The conformation of a loop and its orientation with respect to the rest of the molecule in WBAII are, however, different from those in all the other legume lectins of known structure. This difference appears to have been caused by the formation of two strategically placed salt bridges in the former. Modelling based on the crystal structures provides a rationale for the specificity of the lectin, which is very different from that of WBAI, for the H-antigenic determinant responsible for O blood group reactivity. It also leads to a qualitative explanation for the thermodynamic data on sugar-binding to the lectin, with special emphasis on the role of a tyrosyl residue in the variable loop in the sugar-binding region in generating the carbohydrate specificity of WBAII

Crystallographic identification of an ordered C-terminal domain and a second nucleotide-binding site in RecA: new insights into allostery

RecA protein is a crucial and central component of the homologous recombination and DNA repair machinery. Despite numerous studies on the protein, several issues concerning its action, including the allosteric regulation mechanism have remained unclear. Here we report, for the first time, a crystal structure of a complex of Mycobacterium smegmatis RecA (MsRecA) with dATP, which exhibits a fully ordered C-terminal domain, with a second dATP molecule bound to it. ATP binding is an essential step for all activities of RecA, since it triggers the formation of active nucleoprotein filaments. In the crystal filament, dATP at the first site communicates with a dATP of the second site of an adjacent subunit, through conserved residues, suggesting a new route for allosteric regulation. In addition, subtle but definite changes observed in the orientation of the nucleotide at the first site and in the positions of the segment preceding loop L2 as well as in the segment 102–105 situated between the 2 nt, all appear to be concerted and suggestive of a biological role for the second bound nucleotide

Thermodynamics of target peptide recognition by calmodulin and a calmodulin analogue: implications for the role of the central linker

The thermodynamics of interaction of two model peptides melittin and mastoparan with bovine brain calmodulin (CAM) and a smaller CAM analogue, a calcium binding protein from Entamoeba histolytica (CaBP) in 10 mM MOPS buffer (pH 7.0) was examined using isothermal titration calorimetry (ITC). These data show that CAM binds to both the peptides and the enthalpy of binding is endothermic for melittin and exothermic for mastoparan at 25°C. CaBP binds to the longer peptide melittin, but does not bind to mastoparan, the binding enthalpy being endothermic in nature. Concurrently, we also observe a larger increase in α-helicity upon the binding of melittin to CAM when compared to CaBP. The role of hydrophobic interactions in the binding process has also been examined using 8-anilino-1-naphthalene-sulphonic acid (ANS) binding monitored by ITC. These results have been employed to rationalize the energetic consequences of the binding reaction

Concurrence of Danish Dementia and Cataract: Insights from the Interactions of Dementia Associated Peptides with Eye Lens α-Crystallin

Familial Danish Dementia (FDD) is an autosomal disease, which is distinguished by gradual loss of vision, deafness, progressive ataxia and dementia. Cataract is the first manifestation of the disease. In this article, we demonstrate a specific correlation between the poisoning of the chaperone activity of the rat eye lens α-crystallins, loss of lens transparency in organ culture by the pathogenic form of the Danish dementia peptide, i.e. the reduced Danish dementia peptide (redADan peptide), by a combination of ex vivo, in vitro, biophysical and biochemical techniques. The interaction of redADan peptide and lens crystallins are very specific when compared with another chaperone, HSP-70, underscoring the specificity of the pathogenic form of Danish dementia peptide, redADan, for the early onset of cataract in this disease