Structure, Self-Assembly, and Phase Behavior of Neuroactive N-Acyl GABAs: Doxorubicin Encapsulation in NPGABA/DPPC Liposomes and Release Studies.

N-Acylated amino acids and neurotransmitters in mammals exert significant biological effects on the nervous system, immune responses, and vasculature. N-Acyl derivatives of γ-aminobutyric acid (N-acyl GABA), which belong to both classes mentioned above, are prominent among them. In this work, a homologous series of N-acyl GABAs bearing saturated N-acyl chains (C8-C18) have been synthesized and characterized with respect to self-assembly, thermotropic phase behavior, and supramolecular organization. Differential scanning calorimetric studies revealed that the transition enthalpies and entropies of N-acyl GABAs are linearly dependent on the acyl chain length. The crystal structure of N-tridecanoyl GABA showed that the molecules are packed in bilayers with the acyl chains aligned parallel to the bilayer normal and that the carboxyl groups from opposite layers associate to form dimeric structures involving strong O-H···O hydrogen bonds. In addition, N-H···O and C-H···O hydrogen bonds between amide moieties of adjacent molecules within each layer stabilize the molecular packing. Powder X-ray diffraction studies showed odd-even alternation in the d spacings, suggesting that the odd chain and even chain compounds pack differently. Equimolar mixtures of N-palmitoyl GABA and dipalmitoylphosphatidylcholine (DPPC) were found to form stable unilamellar vesicles with diameters of ∼300-340 nm, which could encapsulate doxorubicin, an anticancer drug, with higher efficiency and better release characteristics than DPPC liposomes at physiologically relevant pH. These liposomes exhibit faster release of doxorubicin at acidic pH (<7.0), indicating their potential utility as drug carriers in cancer chemotherapy.

Effect of temperature and pH on the structure and stability of tumor-specific lectin jacalin and insights into the location of its tryptophan residues: CD, DSC and fluorescence studies.

Jacalin, the jackfruit seed lectin, exhibits high specificity for the tumor-specific T-antigen and is used in various biomedical and biotechnological applications. Here, we report biophysical studies on the thermal unfolding of jacalin and the effect of pH and temperature on its secondary structure. Differential scanning calorimetric (DSC) studies revealed that native jacalin unfolds at ∼60 °C and that carbohydrate binding stabilizes the protein structure. Circular dichroism spectroscopic studies indicated that the secondary structure of jacalin remains mostly unaffected over pH 2.0-9.0, whereas considerable changes were observed in the tertiary structure. DSC experiments demonstrated that jacalin exhibits two overlapping transitions between pH 2 and 5, which could be attributed to dissociation of the tetrameric protein into subunits and their unfolding. Interestingly, only one transition between pH 6 and 9 was observed, suggesting that the subunit dissociation and unfolding occur simultaneously. While quenching of the protein intrinsic fluorescence by acrylamide increased significantly upon carbohydrate binding, quenching by succinimide is essentially unaffected. We attribute this difference to increased exposure of Trp-123 in the α-chain as it is involved in carbohydrate binding. Both acrylamide and succinimide gave biphasic Stern-Volmer plots, consistent with differential accessibility of the two tryptophan residues of jacalin to them.

Contrasting effects of molecular crowding on the membrane-perturbing and chaperone-like activities of major bovine seminal plasma protein, PDC-109.

Crowded environments inside cells and biological fluids greatly affect protein stability and activity. PDC-109, a polydisperse oligomeric protein of the bovine seminal plasma selectively binds choline phospholipids on the sperm cell surface and causes membrane destabilization and lipid efflux, leading to acrosome reaction. PDC-109 also exhibits chaperone-like activity (CLA) and protects client proteins against various kinds of stress, such as high temperature and low pH. In the present work, we have investigated the effect of molecular crowding on these two different activities of PDC-109 employing Dextran 70 (D70) - a widely used polymeric dextran - as the crowding agent. The results obtained show that presence of D70 markedly increases membrane destabilization by PDC-109. Isothermal titration calorimetric studies revealed that under crowded condition the binding affinity of PDC-109 for choline phospholipids increases approximately 3-fold, which could in turn facilitate membrane destabilization. In contrast, under identical conditions, its CLA was reduced significantly. The decreased CLA could be correlated to reduced surface hydrophobicity, which was due to stabilization of the protein oligomers. These results establish that molecular crowding exhibits contrasting effects on two different functional activities of PDC-109 and highlight the importance of microenvironment of proteins in modulating their functional activities.

Primary structure determination and physicochemical characterization of DSP-3, a phosphatidylcholine binding glycoprotein of donkey seminal plasma.

Major proteins of the seminal plasma in a variety of mammals such as bovine PDC-109, equine HSP-1/2, and donkey DSP-1 contain fibronectin type-II (FnII) domains and are referred to as FnII family proteins. To further our understanding on these proteins, we carried out detailed studies on DSP-3, another FnII protein of donkey seminal plasma. High-resolution mass-spectrometric studies revealed that DSP-3 contains 106 amino acid residues and is heterogeneously glycosylated with multiple acetylations on the glycans. Interestingly, high homology was observed between DSP-1 and HSP-1 (118 identical residues) than between DSP-1 and DSP-3 (72 identical residues). Circular dichroism (CD) spectroscopic and differential scanning calorimetric (DSC) studies showed that DSP-3 unfolds at ~45 °C and binding of phosphorylcholine (PrC) - the head group moiety of choline phospholipids - increases the thermal stability. Analysis of DSC data suggested that unlike PDC-109 and DSP-1, which exist as mixtures of polydisperse oligomers, DSP-3 most likely exists as a monomer. Ligand binding studies monitoring changes in protein intrinsic fluorescence indicated that DSP-3 binds lyso-phosphatidylcholine (Ka = 1.08 × 105 M-1) with ~80-fold higher affinity than PrC (Ka = 1.39 × 103 M-1). Binding of DSP-3 to erythrocytes leads to membrane perturbation, suggesting that its binding to sperm plasma membrane could be physiologically significant.

Subunit association, and thermal and chemical unfolding of Cucurbitaceae phloem exudate lectins. A review.

Detailed characterization of protein (un)folding intermediates is crucial for understanding the (un)folding pathway, aggregation, stability and their functional properties. In recent years, stress-inducible lectins are being investigated with much interest. In plants phloem proteins PP1 and PP2 are major components of the phloem fluid. While PP1 is a structural protein, PP2 exhibits lectin activity, and was proposed to play key roles in wound sealing, anti-pathogenic activity, and transportation of various molecules including RNA within the plant. Cucurbitaceae fruits contain high concentrations of PP2 lectins, which recognize chitooligosaccharides with high specificity. Although the presence of PP2 lectins in the phloem exudate of Cucurbitaceae species was documented over 40 years ago, so far only a few proteins from this family have been purified and characterized in detail. This review summarizes the results of biophysical studies aimed at investigating the oligomeric status of these lectins, their thermal stability, structural perturbations caused by changes in pH and addition of chaotropic agents and characterization of intermediates observed in the unfolding process. The implications of these results in the functional roles played by PP2 type lectins in their native environment are discussed. Finally, perspectives for future biophysical research on these proteins are given.

Macromolecular Crowding Significantly Affects the Conformational Features and Carbohydrate Binding Properties of CIA17, a PP2-Type Lectin from Coccinia indica.

The effect of macromolecular crowding on the conformational features and carbohydrate binding properties of CIA17, a PP2-type lectin, was investigated employing polymeric dextrans D6, D40, and D70 (Mr ∼ 6, 40, and 70 kDa, respectively) as crowders. While the secondary structure of CIA17 was significantly affected by D6, with a considerable decrease in the number of ß-sheets and ß-turns with a corresponding increase in the number of unordered structures, relatively smaller changes were induced by D40 and D70. However, differential scanning calorimetry (DSC) studies revealed that the thermal stability of the protein remains unchanged in the presence of crowders. While the larger dextrans, D70 and D40, induced modest quenching (∼10%) of the protein fluorescence by a static pathway, the smaller D6 induced a higher degree of quenching (37%), which involved both static and collisional quenching processes. The results of fluorescence correlation spectroscopy measurements together with DSC studies suggested that CIA17 forms larger oligomers in the presence of D40 and D70 but D6 prevents the formation of higher-order oligomers. The association constant for the CIA17-chitooligosaccharide interaction increased by ∼30% and 160% in the presence of D40 and D70, respectively, but decreased by ∼30% in the presence of D6. The higher binding affinity can be attributed to the excluded volume effect, i.e., an increased effective concentration of the protein in the presence of D40 and D70, whereas D6, being smaller, possibly penetrates into the protein interior, disrupting the water structure around the protein and also inducing conformational changes, resulting in weaker binding. These observations demonstrate that molecular crowding significantly affects the carbohydrate binding characteristics of lectins, which can modulate their physiological function.

Base-triggerable lauryl sarcosinate-dodecyl sulfate catanionic liposomes: structure, biophysical characterization, and drug entrapment/release studies.

Equimolar mixtures of oppositely charged single-chain amphiphiles form a variety of phases, including vesicles. Such catanionic mixed lipid systems show high stability and exhibit versatile physicochemical properties. In the present study we have investigated the aggregation behaviour of lauryl sarcosinate hydrochloride (LS·HCl) in aqueous dispersion as well as its interaction with the anionic surfactant sodium dodecyl sulfate (SDS). The CMC of LS·HCl was estimated to be ∼5 mM by isothermal titration calorimetry (ITC) and fluorescence spectroscopy using pyrene as the fluorescent probe. Turbidimetric and ITC studies on the interaction of LS·HCl with SDS demonstrated that the two surfactants form an equimolar catanionic complex. The crystal structure of the lauryl sarcosinate-dodecyl sulfate (LS-DS) complex revealed that the complex is stabilized by classical N-H⋯O as well as C-H⋯O hydrogen bonds, besides the electrostatic attraction between LS (cation) and DS (anion) and dispersion interactions between the hydrocarbon chains. Differential scanning calorimetry studies revealed that the phase transition of the equimolar LS-DS complex is significantly reduced compared to the analogous LG-DS and LA-DS complexes in the fully hydrated state. Dynamic light scattering, atomic force microscopy and transmission electron microscopy studies demonstrated that the LS-DS catanionic complex forms stable medium-sized vesicles (diameter of ∼300-500 nm). In vitro studies with 5-fluorouracil and rhodamine 6G showed efficient entrapment and release of these two anti-cancer drugs in the physiologically relevant pH range of 6.0-8.0, but with contrasting pH dependences. These observations indicate that LS-DS catanionic vesicles may find application in designing drug delivery systems.

Cucurbitaceae phloem exudate lectins: Purification, molecular characterization and carbohydrate binding characteristics.

Much of the plant lectin research was focused on these proteins from seeds, whereas lectins from other plant tissues have been less investigated. Although presence of lectins in the phloem exudate of Cucurbitaceae species was reported over 40 years ago, only a few proteins from this family have been purified and characterized with respect to ligand binding properties, primary and secondary structures, while no 3D structure of a member of this family is known so far. Unlike lectins from other plant families and sources (e.g., seeds and tubers), which exhibit specificity towards different carbohydrate structures, all the Cucurbitaceae phloem exudate lectins characterized so far have been shown to recognize only chitooligosaccharides or glycans containing chitooligosaccharides. Interestingly, some of these proteins also bind various types of RNAs, suggesting that they may also play a role in the transport of RNA information molecules in the phloem. The present review gives an overview of the current knowledge of Cucurbitaceae phloem exudate lectins with regard to their purification, determination of primary and secondary structures, elucidation of thermodynamics and kinetics of carbohydrate binding and computational modeling to get information on their 3D structures. Finally, future perspectives of research on this important class of proteins are considered.

Effect of Sorbitol on Alpha-Crystallin Structure and Function.

Loss of eye lens transparency due to cataract is the leading cause of blindness all over the world. While aggregation of lens crystallins is the most common endpoint in various types of cataracts, chaperone-like activity (CLA) of α-crystallin preventing protein aggregation is considered to be important for maintaining the eye lens transparency. Osmotic stress due to increased accumulation of sorbitol under hyperglycemic conditions is believed to be one of the mechanisms for diabetic cataract. In addition, compromised CLA of α-crystallin in diabetic cataract has been reported. However, the effect of sorbitol on the structure and function of α-crystallin has not been elucidated yet. Hence, in the present exploratory study, we described the effect of varying concentrations of sorbitol on the structure and function of α-crystallin. Alpha-crystallin purified from the rat lens was incubated with varying concentrations of sorbitol in the dark under sterile conditions for up to 5 days. At the end of incubation, structural properties and CLA were evaluated by spectroscopic methods. Interestingly, different concentrations of sorbitol showed contrasting results: at lower concentrations (5 and 50 mM) there was a decrease in CLA and subtle alterations in secondary and tertiary structure but not at higher concentrations (500 mM). Though, these results shed a light on the effect of sorbitol on α-crystallin structure-function, further studies are required to understand the mechanism of the observed effects and their implication to cataractogenesis.

Deciphering the thermotolerance of chitinase O from Chitiniphilus shinanonensis by in vitro and in silico studies.

Biochemical and biophysical studies revealed that chitinase O from Chitiniphilus shinanonensis (CsChiO) exhibits considerable thermotolerance, possibly due to the formation of a stable structural conformation. CsChiO is an exochitinase with a temperature optimum of 70 °C. The secondary structures of CsChiO and its catalytic domain (Cat-CsChiO) are only marginally affected upon heating up to 90 °C, as revealed by circular dichroism (CD) spectroscopy. Differential scanning calorimetric (DSC) studies revealed that CsChiO exhibits two endothermic transitions at ca. 51 °C (Tm1) and 59 °C (Tm2), whereas Cat-CsChiO shows a single endothermic transition at 52 °C. Together, the CD and DSC analyses suggested that the catalytic domain of CsChiO undergoes a thermotropic transition at ~52 °C from native state to another stable structural conformation. Results from molecular dynamic simulations corroborated that Cat-CsChiO adopts a stable structural conformation above 50 °C by partial unfolding. Thermotolerant CsChiO would be useful for the conversion of chitin, which is highly abundant, to biologically active COS. This study unveiled the adaptability of enzymes/proteins in nature to perform biological functions at elevated temperatures.

Low-pH Molten Globule-Like Form of CIA17, a Chitooligosaccharide-Specific Lectin from the Phloem Exudate of Coccinia indica, Retains Carbohydrate-Binding Ability.

pH-induced changes in the conformation, structural dynamics, and carbohydrate-binding activity of Coccinia indica agglutinin (CIA17), a PP2-type lectin, were investigated employing biophysical approaches. The secondary structure of CIA17 remains nearly unaltered over a wide pH range (2.0-8.5), while the tertiary structure of the protein exhibits considerable changes. A decrease in the fluorescence intensity and excited-state lifetime at low pH indicated perturbation in the local conformation (near Trp residues) of CIA17, which was further supported by enhancement in the Trp accessibility toward charged quenchers under acidic conditions. Fluorescence correlation spectroscopic studies indicated that at pH 2.0, CIA17 exists as a monomer over the concentration range of 10-200 nM and forms dimers at higher concentrations (KD ∼ 387 nM) but could not form higher oligomers even at ∼150-fold higher concentrations, unlike under native conditions at pH 7.4. Thermal unfolding of the low pH intermediate involves two distinct steps: dissociation of a dimer to a monomer, followed by the unfolding of the monomer. These results strongly suggest that the acid-induced unfolding pathway of CIA17 involves the formation of a monomeric molten globule-like intermediate, which retains appreciable carbohydrate-binding ability. These observations are of great physiological significance since the PP2 proteins are involved in plant defense responses.

Purification, molecular characterization and ligand binding properties of the major donkey seminal plasma protein DSP-1.

Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the protein is heterogeneously glycosylated and also contains multiple acetylations, occurring in the attached glycans. Structural and thermal stability studies on DSP-1 employing CD spectroscopy and differential scanning calorimetry showed that the protein unfolds at ~43 °C and binding to phosphorylcholine (PrC) - the head group moiety of choline phospholipids - increases its thermal stability. Intrinsic fluorescence titrations revealed that DSP-1 recognizes lyso-phosphatidylcholine with over 100-fold higher affinity than PrC. Further, interaction of DSP-1 with erythrocytes, a model cell membrane, revealed that DSP-1 binding is mediated by a specific interaction with choline phospholipids and results in membrane perturbation, suggesting that binding of this protein to sperm plasma membrane could be physiologically significant.

Packing polymorphism, odd-even alternation and thermotropic phase transitions in N-,O-diacylethanolamines with varying N-acyl chains. A combined experimental and computational study.

N-,O-Diacylethanolamines (DAEs) are derived by simple esterification of bioactive N-acylethanolamines, which are present in plant and animal tissues. In this study, two homologous series of DAEs, namely N-acyl (n = 8-15), O-palmitoylethanolamines (Nn-O16s) and N-acyl (n = 8-14), O-pentadecanoylethanolamines (Nn-O15s) were synthesized and characterized with respect to thermotropic phase transitions, crystal structures and intermolecular interactions. In addition, computational studies were performed to get a molecular level insight into the role of different factors in selective polymorphism in Nn-O16s and Nn-O15s. Differential scanning calorimetric studies revealed that dry Nn-O16s exhibit odd-even alternation in their calorimetric properties, which is absent in Nn-O15s. The 3-dimensional structures of three Nn-O16s (n = 12-14) and two Nn-O15s (n = 12, 14) have been determined by single-crystal X-ray diffraction. Analysis of the molecular packing in these crystals showed the presence of two packing polymorphs (α and ß) in the crystal lattice of Nn-O16s, whereas only the ß polymorph was observed in the Nn-O15s. Further, intermolecular hydrogen bonding interactions (N-H⋯O and C-H⋯O) and dispersion interactions among acyl chains have been found to stabilize the molecular packing observed in the crystal lattice. Molecular dynamics simulations show that the ß polymorph is slightly energetically preferred over the α polymorph in all the systems due to favorable packing of terminal methyl groups at the interlayers. These findings are relevant for understanding the interactions of the DAEs with membrane lipids and proteins.

DSC and FCS Studies Reveal the Mechanism of Thermal and Chemical Unfolding of CIA17, a Polydisperse Oligomeric Protein from Coccinia Indica.

The mechanism of thermal and chemical unfolding of Coccinia indica agglutinin (CIA17), a chitooligosacharide-specific phloem exudate lectin, was investigated by biophysical approaches. DSC studies revealed that the unfolding thermogram of CIA17 consists of three components (Tm ∼ 98, 106, and 109 °C), which could be attributed to the dissociation of protein oligomers into constituent dimers, dissociation of the dimers into monomers, and unfolding of the monomers. Intrinsic fluorescence studies on the chemical denaturation by guanidinium thiocyanate and guanidinium chloride indicated the presence of two distinct steps in the unfolding pathway, which could be assigned to dissociation of the dimeric protein into monomers and unfolding of the monomers. Results of fluorescence correlation spectroscopic studies could be interpreted in terms of the following model: CIA17 forms oligomeric structures in a concentration dependent manner, with the protein existing as a monomer below 1 nM concentration but associating to form dimers at higher concentrations (KD ≈ 2.9 nM). The dimers associate to yield tetramers with a KD of ∼50 µM, which further associate to form higher oligomers with further increase in concentration. These results are consistent with the proposed role of CIA17 as a key player in the defense response of the plant against microbes and insects.

Structure, thermotropic phase behavior and membrane interaction of N-acyl-ß-alaninols. Homologs of stress-combating N-acylethanolamines.

ß-Alaninol and its derivatives were reported to exhibit interesting biological and pharmacological activities and showed potential application in formulating drug delivery vehicles. In the present study, we report the synthesis and characterization of N-acyl-ß-alaninols (NABAOHs) bearing saturated acyl chains (n = 8-20) with respect to thermotropic phase behavior, supramolecular organization and interaction with diacylphosphatidylcholine, a major membrane lipid. Results obtained from DSC and powder XRD studies revealed that the transition temperatures (Tt), transition enthalpies (ΔHt), transition entropies (ΔSt) and d-spacings of NABAOHs show odd-even alteration. A linear dependence was observed in the values of ΔHt and ΔSt on the acyl chain length, independently for even and odd acyl chains in both dry and hydrated states; further, the even chainlength molecules exhibited higher values than the odd chainlength series. The crystals structures of N-lauroyl-ß-alaninol and N-palmitoyl-ß-alaninol, solved in monoclinic system in the P21/c space group, show that the NABAOHs adopt a tilted bilayer structure. A number of NH⋯O, O-H⋯O, and C-H⋯O hydrogen bonds between the hydroxyl and amide moieties of the head groups of NABAOH molecules belonging to adjacent and opposite layers stabilize the overall supramolecular organization of the self-assembled bilayer system. DSC studies on the interaction of N-myristoyl-ß-alaninol (NMBAOH) with dimyristoylphosphatidylcholine (DMPC) indicate that these two lipids mix well up to 45 mol% NMBAOH, whereas phase separation was observed at higher contents of NMBAOH. Transmission electron microscopic studies reveal that mixtures containing 20-50 mol% NMBAOH form stable ULVs of 90-150 nm diameter, suitable for use in drug delivery applications.

New Class of Chitosanase from Bacillus amyloliquefaciens for the Generation of Chitooligosaccharides.

Chitooligosaccharides (COS) generated from either chitin (chitin oligosaccharides) or chitosan (chitosan oligosaccharides) have a wide range of applications in agriculture, medicine, and other fields. Here, we report the characterization of a chitosanase from Bacillus amyloliquefaciens (BamCsn) and the importance of a tryptophan (Trp), W204, for BamCsn activity. BamCsn hydrolyzed the chitosan polymer by an endo mode. It also hydrolyzed chitin oligosaccharides and interestingly exhibited transglycosylation activity on chitotetraose and chitopentaose. Mutation of W204, a nonconserved amino acid in chitosanases, to W204A abolished the hydrolytic activity of BamCsn, with a change in the structure that resulted in a decreased affinity for the substrate and impaired the catalytic ability. Phylogenetic analysis revealed that BamCsn could belong to a new class of chitosanases that showed unique properties like transglycosylation, cleavage of chitin oligosaccharides, and the presence of W204 residues, which is important for activity. Chitosanases belonging to the BamCsn class showed a high potential to generate COS from chitinous substrates.

Purification, biochemical/biophysical characterization and chitooligosaccharide binding to BGL24, a new PP2-type phloem exudate lectin from bottle gourd (Lagenaria siceraria).

Phloem Protein 2 (PP2), highly abundant in the sieve elements of plants, plays a significant role in wound sealing and anti-pathogenic responses. In this study, we report the purification and characterization of a new PP2-type lectin, BGL24 from the phloem exudate of bottle gourd (Lagenaria siceraria). BGL24 is a homodimer with a subunit mass of ~24 kDa and exhibits high specificity for chitooligosaccharides. The isoelectric point of BGL24 was estimated from zeta potential measurements as 5.95. Partial amino acid sequence obtained by mass spectrometric studies indicated that BGL24 exhibits extensive homology with other PP2-type phloem exudate lectins. CD spectroscopic measurements revealed that the lectin contains predominantly ß-sheets, with low α-helical content. CD spectroscopic and DSC studies showed that BGL24 exhibits high thermal stability with an unfolding temperature of ~82 °C, and that its secondary structure is essentially unaltered between pH 3.0 and 8.0. Fluorescence titrations employing 4-methylumbelliferyl-ß-D-N,N',N″-triacetylchitotrioside as an indicator ligand revealed that the association constants for BGL24-chitooligosaccharide interaction increase considerably when the ligand size is increased from chitotriose to chitotetraose, whereas only marginal increase was observed for chitopentaose and chitohexaose. BGL24 exhibited moderate cytotoxicity against MDA-MB-231 breast cancer cells, whereas its effect on normal splenocytes was marginal.

Purification and biochemical/biophysical characterization of two hexosaminidases from the fresh water mussel, Lamellidens corrianus.

Two thermostable isoforms of a hexosaminidase were purified to homogeneity from the soluble extract of fresh water mussel Lamellidens corrianus, employing a variety of chromatographic techniques. Hexosaminidase A (HexA) is a heterodimer with subunit masses of ~80 and 55 kDa. Hexosaminidase B (HexB) is a homodimer with a subunit mass of 55-60 kDa. Circular dichroism spectroscopic studies indicated that both HexA and HexB contain ß-sheet as the major secondary structural component with considerably lower content of α-helix. The temperature and pH optima of both the isoforms were found to be 60 °C and 4.0, respectively. The IC50 values for HexA with N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, d-galactosamine, d-glucosamine, methyl α-d-mannopyranoside and d-mannose are 3.7, 72.8, 307, 216, 244 and 128 mM, respectively, whereas the corresponding IC50 values for HexB were estimated as 5.1, 61, 68, 190, 92 and 133 mM, respectively. Kinetic parameters KM and Vmax for HexA and B with p-nitrophenyl N-acetyl-ß-d-glucosaminide are 4 mM, 0.23 µmol·min-1·mL-1 and 2.86 mM, 0.29 µmol·min-1·mL-1, respectively, and with p-nitrophenyl N-acetyl-ß-d-galactosaminide are 4.5 mM, 0.054 µmol·min-1·mL-1 and 1.4 mM, 0.14 µmol·min-1·mL-1, respectively. GalNAc inhibited both isoforms in a non-competitive manner, whereas a mixed mode of inhibition was observed with GlcNAc with both forms.

Conserved core tryptophans of FnII domains are crucial for the membranolytic and chaperone-like activities of bovine seminal plasma protein PDC-109.

The fibronectin type II (FnII) domain, present in diverse vertebrate proteins, plays crucial roles in several fundamental biological processes. PDC-109, the major bovine seminal plasma protein, contains two FnII domains that bind to choline phospholipids on sperm plasma membrane and induce lipid efflux crucial for successful fertilization. PDC-109 also exhibits chaperone-like activity and protects other proteins against various types of stress. Here, we show that a core tryptophan residue is highly conserved across species in the FnII domains. Mutation of conserved tryptophan residues W47, W93, and W106 in the FnII domains of PDC-109 to alanine leads to drastic decrease or complete abolition of membrane-binding and chaperone-like activities. These observations suggest that conserved tryptophans are important for the function of FnII proteins.

Chitooligosaccharide binding to CIA17 (Coccinia indica agglutinin). Thermodynamic characterization and formation of higher order complexes.

CIA17 is a PP2-like, homodimeric lectin made up of 17 kDa subunits present in the phloem exudate of ivy gourd (Coccinia indica). Isothermal titration calorimetric (ITC) studies on the interaction of chitooligosaccharides [(GlcNAc)2-6] showed that the dimeric protein has two sugar binding sites which recognize chitotriose with ~70-fold higher affinity than chitobiose, indicating that the binding site is extended in nature. ITC, atomic force microscopic and non-denaturing gel electrophoresis studies revealed that the high-affinity interaction of CIA17 with chitohexaose (Ka = 1.8 × 107 M-1) promotes the formation of protein oligomers. Computational studies involving homology modeling, molecular docking and molecular dynamics simulations on the binding of chitooligosaccharides to CIA17 showed that the protein binding pocket accommodates up to three GlcNAc residues. Interestingly, docking studies with chitohexaose indicated that its two triose units could interact with binding sites on two protein molecules to yield dimeric complexes of the type CIA17-(GlcNAc)6-CIA17, which can extend in length by the binding of additional chitohexaose and CIA17 molecules. These results suggest that PP2 proteins play a role in plant defense against insect/pathogen attack by directly binding with the higher chain length chitooligosaccharides and forming extended, filamentous structures, which facilitate wound sealing.