Roopa Kenoth

Fluorescence and absorption spectroscopic studies on the interaction of porphyrins with snake gourd (Trichosanthes anguina) seed lectin.

The interaction of several free-base porphyrins and their corresponding copper(II) and zinc(II) derivatives with the galactose-specific lectin from snake gourd (Trichosanthes anguina) seeds has been investigated by absorption and fluorescence spectroscopic techniques. The lectin dimer contains two apparently equivalent binding sites for the porphyrins. Association constants obtained for the interaction of various porphyrins with the lectin are in the range 1.7 x 10(4)-6.2 x 10(5) M(-1), with the metalloporphyrins being seen to have higher affinity for the lectin compared with the free-base analogues. Both positively charged and negatively charged porphyrins bind to snake gourd seed lectin (SGSL) with comparable affinities, suggesting that binding occurs primarily via hydrophobic interactions. Further, binding of porphyrins is found to be largely unaffected by the presence of the sugar ligand, lactose, indicating that the binding sites for the carbohydrate and porphyrin are different. This study thus suggests that the lectin may serve as a receptor for some endogenous non-carbohydrate, hydrophobic ligand in vivo, in addition to the saccharide ligands. It also opens up the possibility of employing the T. anguina lectin in applications such as photodynamic therapy, which involve the use of porphyrins.

Functional Equality in the Absence of Structural Similarity AN ADDED DIMENSION TO MOLECULAR MIMICRY

The crystal structure ofmeso-tetrasulfonatophenylporphyrin complexed with concanavalin A (ConA) was determined at 1.9 Å resolution. Comparison of this structure with that of ConA bound to methyl α-d-mannopyranoside provided direct structural evidence of molecular mimicry in the context of ligand receptor binding. The sulfonatophenyl group of meso-tetrasulfonatophenylporphyrin occupies the same binding site on ConA as that of methyl α-d-mannopyranoside, a natural ligand. A pair of stacked porphyrin molecules stabilizes the crystal structure by end-to-end cross-linking with ConA resulting in a network similar to that observed upon agglutination of cells by lectins. The porphyrin binds to ConA predominantly through hydrogen bonds and water-mediated interactions. The sandwiched water molecules in the complex play a cementing role, facilitating favorable binding of porphyrin. Seven of the eight hydrogen bonds observed between methyl α-d-mannopyranoside and ConA are mimicked by the sulfonatophenyl group of porphyrin after incorporating two water molecules. Thus, the similarity in chemical interactions was manifested in terms of functional mimicry despite the obvious structural dissimilarity between the sugar and the porphyrin.

Steady-state and time-resolved fluorescence studies on Trichosanthes cucumerina seed lectin.

Steady-state and time-resolved fluorescence spectroscopic studies have been carried out on Trichosanthes cucumerina seed lectin (TCSL). The fluorescence emission maximum of TCSL in the native state as well as in the presence of 0.1 M lactose is centered around 331 nm, which shifts to 347 nm upon denaturation with 8 M urea, indicating that all the tryptophan residues of this protein in the native state are in a predominantly hydrophobic environment. The exposure and accessibility of the tryptophan residues of TCSL and the effect of ligand binding on them were probed by quenching studies employing two neutral quenchers (acrylamide and succinimide), an anionic quencher (I(-)) and a cationic quencher (Cs(+)). Quenching was highest with acrylamide and succinimide with the latter, which is bulkier, yielding slightly lower quenching values, whereas the extent of quenching obtained with the ionic quenchers, I(-) and Cs(+) was significantly lower. The presence of 0.1 M lactose led to a slight increase in the quenching with acrylamide and iodide, whereas quenching with succinimide and cesium ion was not significantly affected. When TCSL was denatured with 8 M urea, both acrylamide and succinimide yielded upward-curving Stern-Volmer plots, indicating that the quenching mechanism involves both dynamic and static components. Quenching data obtained with I(-) and Cs(+) on the urea-denatured protein suggest that charged residues could be present in close proximity to some of the Trp residues. The Stern-Volmer plots with Cs(+) yielded biphasic quenching profiles, indicating that the Trp residues in TCSL fall into at least two groups that differ considerably in their accessibility and/or environment. In time-resolved fluorescence experiments, the decay curves could be best fit to biexponential patterns, with lifetimes of 1.78 and 4.75 ns for the native protein and 2.15 and 5.14 ns in the presence of 0.1 M lactose.

N-Myristoylethanolamine-cholesterol (1:1) complex: first evidence from differential scanning calorimetry, fast-atom-bombardment mass spectrometry and computational modelling.

The interaction of N‐myristoylethanolamine (NMEA) with cholesterol is investigated by differential scanning calorimetry (DSC), fast‐atom‐bombardment mass spectrometry (FAB‐MS) and computational modelling. Addition of cholesterol to NMEA leads to a new phase transition at 55°C besides the chain‐melting transition of NMEA at 72.5°C. The enthalpy of the new transition increases with cholesterol content up to 50 mol%, but decreases thereafter, vanishing at 80 mol%. The enthalpy of the chain‐melting transition of NMEA decreases with an increase in cholesterol; the transition disappears at 50 mol%. FAB‐MS spectra of mixtures of NMEA and cholesterol provide clear signatures of the formation of {[NMEA+cholesterol]+} {[NMEA+cholesterol+Na]+}. These results are consistent with the formation of a 1:1 complex between NMEA and cholesterol. Molecular modelling studies support this experimental finding and provide a plausible structural model for the complex, which highlights multiple H‐bond interactions between the hydroxy group of cholesterol and the hydroxy and carbonyl groups of NMEA besides appreciable dispersion interaction between the hydrocarbon domains of the two molecules.

Physicochemical and saccharide-binding studies on the galactose-specific seed lectin from Trichosanthes cucumerina.

Physicochemical and saccharide-binding studies have been performed on Trichosanthes cucumerina seed lectin (TCSL). The agglutination activity of TCSL is highest in the pH range 8.0-11.0, whereas below pH 7.0 it decreases quite rapidly, which is consistent with the involvement of imidazole side chains of His residues, which titrate in this pH range, in the sugar-binding activity of the lectin. The lectin activity is unaffected between 0 and 60 degrees C, but a sharp decline occurs at higher temperatures. Isoelectric focusing studies show that TCSL has three isoforms with pI values of 5.3, 6.2, and 7.1, with the isoform of pI 6.2 being the most abundant. Circular dichroism spectroscopic studies reveal that TCSL contains about 28.4% beta-sheet, 10.6% beta-turns, 7% polyproline type 2 structure, with the remainder comprising unordered structure; the alpha-helix content is negligible. Binding of 4-methylumbelliferyl-beta-D-galactopyranoside (MeUmbbetaGal) to TCSL results in a significant increase in the fluorescence intensity of the ligand, and this change has been used to obtain the association constant for the interaction. At 25 degrees C, the association constant, K(a), for the TCSL-MeUmbbetaGal interaction was determined as 6.9 x 10(4)M(-1). Binding of nonfluorescent, inhibitory sugars was studied by monitoring their ability to reverse the fluorescence changes observed when MeUmbbetaGal was titrated with TCSL.

Crystallization and preliminary X-ray studies of snake gourd lectin: homology with type II ribosome-inactivating proteins

The lectin from the seeds of snake gourd (Trichosanthes anguina) has been crystallized in two forms using the hanging-drop method. Both the forms are hexagonal, with the asymmetric unit containing one subunit consisting of two polypeptide chains linked through disulfide bridges. Intensity data from one of the forms were collected at room temperature as well as at low temperature to 3 A resolution. Molecular-replacement studies indicate that the lectin is homologous to type II ribosome-inactivating proteins. Partial refinement confirms this conclusion.

Galactose-Specific Seed Lectins from Cucurbitaceae

Lectins, the carbohydrate binding proteins have been studied extensively in view of their ubiquitous nature and wide-ranging applications. As they were originally found in plant seed extracts, much of the work on them was focused on plant seed lectins, especially those from legume seeds whereas much less attention was paid to the lectins from other plant families. During the last two decades many studies have been reported on lectins from the seeds of Cucurbitaceae species. The main focus of the present review is to provide an overview of the current knowledge on these proteins, especially with regard to their physico-chemical characterization, interaction with carbohydrates and hydrophobic ligands, 3-dimensional structure and similarity to type-II ribosome inactivating proteins. The future outlook of research on these galactose-specific proteins is also briefly considered.