Molecular design of N-linked tetravalent glycosides bearing N-acetylglucosamine,N,N′-diacetylchitobiose and N-acetyllactosamine: Analysis of cross-linking activities with WGA and ECA lectins

Two types of nonspacer- and spacer-N-linked tetravalent glycosides bearing N-acetylglucosamine (GlcNAc), N,N′-diacetylchitobiose [(GlcNAc)₂] and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. The interactions of wheat germ (Triticum vulgaris) agglutinin (WGA) and coral tree (Erythrina cristagalli) agglutinin (ECA) with a series of tetravalent glycosides and related compounds were studied using a hemagglutination inhibition assay, a precipitation assay, double-diffusion test, and an optical biosensor based on surface plasmon resonance (SPR). The tetravalent glycosides were found to be capable of binding and precipitating the lectins as tetravalent ligands. Strong interactions with WGA, due to a combination of multivalency effects and spacer effects, were observed for tetravalent glycosides bearing flexible tandem GlcNAc. The chelate effect leads to large rate enhancement for the tetravalent system with favorable orientation of ligands. Our simple strategy produced multivalent glycosides with strong cross-linking activity for lectin as a specific coagulant.     

SPR and ITC determination of the kinetics and the thermodynamics of bivalent versus monovalent sugar ligand–lectin interactions

A kinetic study of the interaction of bivalent and monovalent sugar ligands with a lectin was undertaken with the aid of surface plasmon resonance (SPR) method. The study involved a series of bivalent α-d-mannopyranoside containing sugar ligands, with systematic variation in the distance between the sugar ligands. The detailed kinetic studies showed that bivalent ligands underwent a faster association (k _on) and a slower dissociation (k _off) of the ligand–lectin complexes, in comparison to the monovalent ligand–lectin complexes. The kinetic constants were complemented further by assessing the thermodynamic parameters with the aid of isothermal titration calorimetry (ITC). The initiation of cross-linking of ligand–lectin interactions emerge from the early stages of the complexation. The dynamic light scattering (DLS) and the transmission electron microscopy (TEM) techniques allowed judging the sizes and morphologies of the complex in the solution and solid states, respectively.     

Development of covalent antagonists for β1- and β2-adrenergic receptors

The selective covalent tethering of ligands to a specific GPCR binding site has attracted considerable interest in structural biology, molecular pharmacology and drug design. We recently reported on a covalently binding noradrenaline analog (FAUC37) facilitating crystallization of the β₂-adrenergic receptor (β₂AR^H2.64C) in an active state. We herein present the stereospecific synthesis of covalently binding disulfide ligands based on the pharmacophores of adrenergic β₁- and β₂ receptor antagonists. Radioligand depletion experiments revealed that the disulfide-functionalized ligands were able to rapidly form a covalent bond with a specific cysteine residue of the receptor mutants β₁AR^I2.64C and β₂AR^H2.64C. The propranolol derivative (S)-1a induced nearly complete irreversible blockage of the β₂AR^H2.64C within 30 min incubation. The CGP20712A-based ligand (S)-4 showed efficient covalent blocking of the β₂AR^H2.64C at very low concentrations. The analog (S)-5a revealed extraordinary covalent cross-linking at the β₁AR^I2.64C and β₂AR^H2.64C mutant while retaining a 41-fold selectivity for the β₁AR wild type over β₂AR. These compounds may serve as valuable molecular tools for studying β₁/β₂ subtype selectivity or investigations on GPCR trafficking and dimerization.     

Binding thermodynamics of phosphorylated inhibitors to triosephosphate isomerase and the contribution of electrostatic interactions

Electrostatic interactions have a central role in some biological processes, such as recognition of charged ligands by proteins. We characterized the binding energetics of yeast triosephosphate isomerase (TIM) with phosphorylated inhibitors 2-phosphoglycollate (2PG) and phosphoglycolohydroxamate (PGH). We determined the thermodynamic parameters of the binding process (K_b, ΔG_b, ΔH_b, ΔS_b and ΔC_p) with different concentrations of NaCl, using fluorimetric and calorimetric titrations in the conventional mode of ITC and a novel method, multithermal titration calorimetry (MTC), which enabled us to measure ΔC_p in a single experiment. We ruled out specific interactions of Na⁺ and Cl^- with the native enzyme and did not detect significant linked protonation effects upon the binding of inhibitors. Increasing ionic strength (I) caused K_b, ΔG_b and ΔH_b to become less favorable, while ΔS_b became less unfavorable. From the variation of K_b with I, we determined the electrostatic contribution of TIM−2PG and TIM−PGH to ΔG_b at I = 0.06 M and 25 °C to be 36% and 26%, respectively. The greater affinity of PGH for TIM is due to a more favorable ΔH_b compared to 2PG (by 19-24 kJ mol^-1 at 25 °C). This difference is compatible with PGH establishing up to five more hydrogen bonds with TIM. Both binding ΔC_ps were negative, and less negative with increasing ionic strength. ΔC_ps at I = 0.06 M were much more negative than predicted by surface area models. Water molecules trapped in the interface when ligands bind to protein could explain the highly negative ΔCps. Thermodynamic binding functions for TIM−2PG changed more with ionic strength than those for TIM−PGH. This greater dependence is consistent with linked, but compensated, protonation equilibriums yielding the dianionic species of 2PG that binds to TIM, process that is not required for PGH.     

Interactions of aromatic mannosyl disulfide derivatives with Concanavalin A: synthesis, thermodynamic and NMR spectroscopy studies

α-d-Mannopyranosyl units were attached to an aromatic scaffold through disulfide linkages to obtain mono- to trivalent glycosylated ligands for lectin binding studies. Isothermal titration calorimetric (ITC) measurements indicated that binding affinities of these derivatives to Concanavalin A (Con A) were comparable to or slightly higher than that of methyl α-d-mannopyranoside (K_a values in the range of 10⁴ M⁻¹). The stoichiometries of the lectin-ligand complexes were in agreement with the formal valencies (1–3) of the respective ligands indicating cross-linking in interactions with the di- and trivalent derivatives. Multivalency effects could not, however, be observed with the latter. These ligands were shown to bind to the carbohydrate binding site of Con A using saturation transfer difference (STD) NMR competition experiments.     

Thermodynamic Profiling of HIV RREIIB RNA-Zinc Finger Interactions

The interactions between the HIV Rev-responsive element (RRE) RNA and the HIV regulatory protein Rev, are crucial for the HIV life-cycle. Earlier, we showed that single C₂H₂ zinc fingers (znfs) have the same binding site as the Rev peptide and exhibit nanomolar affinity. In this study, the specific role of amino acid side chains and molecular processes involved with complex formation were investigated by perturbation of the binding energetics via changes in temperature, pH, buffers, and salt concentrations, as well as znf and RNA mutations, by isothermal titration calorimetry. Interestingly, despite the large cationic charge on the znfs, the number of interactions with the RNA phosphate backbone was lower than intuitively expected. The presence of binding induced protonation was established by ITC and localized by NMR to a histidine on the znf β-sheet. The ΔC_p of znf-RNA binding was observed to be substantially negative and could not be accounted for by conventional solvent-accessible surface area models. An alternative model, based on the extent of hydrogen bond changes as a result of differences in ligand-induced water displacement at the binding site, provided reasonable explanation of the trends in ΔC_p, as well as ΔH and ΔS. Our studies show that incorporation of favorable interactions at the solvent-excluded binding interface can be used to alleviate the unfavorable enthalpic penalties of displacing water molecules from the hydrated RNA surface.     

Shape readout of AT‐rich DNA by carbohydrates

Gene expression can be altered by small molecules that target DNA; sequence as well as shape selectivities are both extremely important for DNA recognition by intercalating and groove‐binding ligands. We have characterized a carbohydrate scaffold (1) exhibiting DNA “shape readout” properties. Thermodynamic studies with 1 and model duplex DNAs demonstrate the molecule's high affinity and selectivity towards B* form (continuous AT‐rich) DNA. Isothermal titration calorimetry (ITC), circular dichroism (CD) titration, ultraviolet (UV) thermal denaturation, and Differential Scanning Calorimetry were used to characterize the binding of 1 with a B* form AT‐rich DNA duplex d[5′‐G₂A₆T₆C₂‐3′]. The binding constant was determined using ITC at various temperatures, salt concentrations, and pH. ITC titrations were fit using a two‐binding site model. The first binding event was shown to have a 1:1 binding stoichiometry and was predominantly entropy‐driven with a binding constant of approximately 10⁸ M^?1. ITC‐derived binding enthalpies were used to obtain the binding‐induced change in heat capacity (ΔC_p) of ?225 ± 19 cal/mol·K. The ionic strength dependence of the binding constant indicated a significant electrolytic contribution in ligand:DNA binding, with approximately four to five ion pairs involved in binding. Ligand 1 displayed a significantly higher affinity towards AT‐tract DNA over sequences containing GC inserts, and binding experiments revealed the order of binding affinity for 1 with DNA duplexes: contiguous B* form AT‐rich DNA (d[5′‐G₂A₆T₆C₂‐3′]) >B form alternate AT‐rich DNA (d[5′‐G₂(AT)₆C_2‐3′]) > A form GC‐rich DNA (d[5′‐A₂G₆C₆T₂‐3′]), demonstrating the preference of ligand 1 for B* form DNA. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 720–732, 2014.     

Calorimetric and spectroscopic studies of aminoglycoside binding to AT-rich DNA triple helices

Calorimetric and fluorescence techniques were used to characterize the binding of aminoglycosides-neomycin, paromomycin, and ribostamycin, with 5′-dA₁₂-x-dT₁₂-x-dT₁₂-3′ intramolecular DNA triplex (x = hexaethylene glycol) and poly(dA)·2poly(dT) triplex. Our results demonstrate the following features: (1) UV thermal analysis reveals that the T_m for triplex decreases with increasing pH value in the presence of neomycin, while the T_m for the duplex remains unchanged. (2) The binding affinity of neomycin decreases with increased pH, although there is an increase in observed binding enthalpy. (3) ITC studies conducted in two buffers (sodium cacodylate and MOPS) yield the number of protonated drug amino groups (Δn) as 0.29 and 0.40 for neomycin and paromomycin interaction with 5′-dA₁₂-x-dT₁₂-x-dT₁₂-3′, respectively. (4) The specific heat capacity change (ΔC_p) determined by ITC studies is negative, with more negative values at lower salt concentrations. From 100 mM to 250 mM KCl, the ΔC_p ranges from −402 to −60 cal/(mol K) for neomycin. At pH 5.5, a more positive ΔC_p is observed, with a value of −98 cal/(mol K) at 100 mM KCl. ΔC_p is not significantly affected by ionic strength. (5) Salt dependence studies reveal that there are at least three amino groups of neomycin participating in the electrostatic interactions with the triplex. (6) FID studies using thiazole orange were used to derive the AC₅₀ (aminoglycoside concentration needed to displace 50% of the dye from the triplex) values. Neomycin shows a seven fold higher affinity than paromomycin and eleven fold higher affinity than ribostamycin at pH 6.8. (7) Modeling studies, consistent with UV and ITC results, show the importance of an additional positive charge in triplex recognition by neomycin. The modeling and thermodynamic studies indicate that neomycin binding to the DNA triplex depends upon significant contributions from charge as well as shape complementarity of the drug to the DNA triplex Watson–Hoogsteen groove.     

Application of isothermal titration calorimetry in bioinorganic chemistry

The thermodynamics of metals ions binding to proteins and other biological molecules can be measured with isothermal titration calorimetry (ITC), which quantifies the binding enthalpy (ΔH°) and generates a binding isotherm. A fit of the isotherm provides the binding constant (K), thereby allowing the free energy (ΔG°) and ultimately the entropy (ΔS°) of binding to be determined. The temperature dependence of ΔH° can then provide the change in heat capacity (ΔC _p°) upon binding. However, ITC measurements of metal binding can be compromised by undesired reactions (e.g., precipitation, hydrolysis, and redox), and generally involve competing equilibria with the buffer and protons, which contribute to the experimental values (K _ITC, ΔH _ITC). Guidelines and factors that need to be considered for ITC measurements involving metal ions are outlined. A general analysis of the experimental ITC values that accounts for the contributions of metal–buffer speciation and proton competition and provides condition-independent thermodynamic values (K, ΔH°) for metal binding is developed and validated.     

A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline,Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate

4-O-β-Di-N-acetylchitobiosyl moranoline (2) and 4-O-β-tri-N-acetylchitotriosyl moranoline (3) were produced by lysozyme-mediated transglycosylation from the substrates tetra-N-acetylchitotetraose, (GlcNAc)₄, and moranoline, and the binding modes of 2 and 3 to hen egg white lysozyme (HEWL) was examined by inhibition kinetics, isothermal titration calorimetry (ITC), and x-ray crystallography. Compounds 2 and 3 specifically bound to HEWL, acting as competitive inhibitors with K_i values of 2.01 × 10⁻⁵ and 1.84 × 10⁻⁶ m, respectively. From ITC analysis, the binding of 3 was found to be driven by favorable enthalpy change (ΔH_r°), which is similar to those obtained for 2 and (GlcNAc)₄. However, the entropy loss (−TΔS_r°) for the binding of 3 was smaller than those of 2 and (GlcNAc)₄. Thus the binding of 3 was found to be more favorable than those of the others. Judging from the K_d value of 3 (760 nm), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. X-ray crystal structure of HEWL in a complex with 3 showed that compound 3 binds to subsites −4 to −1 and the moranoline moiety adopts an undistorted ⁴C₁ chair conformation almost overlapping with the −1 sugar covalently bound to Asp-52 of HEWL (Vocadlo, Davies, G. J., Laine, R., and Withers, S. G. (2001) Nature 412, 835–838). From these results, we concluded that compound 3 serves as a transition-state analogue for lysozyme providing additional evidence supporting the covalent glycosyl-enzyme intermediate in the catalytic reaction.     

Facile synthesis of cyclopeptide-centered multivalent glycoclusters with ‘click chemistry’ and molecular recognition study by surface plasmon resonance

A facile synthesis of cyclopeptide-centered multivalent glycoclusters using Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes, so called ‘click chemistry’, has been developed. The affinities of mannose-specific protein Concanavalin A (Con A) toward two synthetic glycoclusters respectively bearing divalent or tetravalent mannoses were investigated by surface plasmon resonance (SPR). It is founded that the tetravalent glycocluster has 3.0-fold increase in binding affinity relative to the divalent glycoluster (valency-corrected values), which indicates the potential of this system in investigating carbohydrate–protein interactions.     

Photoswitchable cluster glycosides as tools to probe carbohydrate-protein interactions: synthesis and lectin-binding studies of azobenzene containing multivalent sugar ligands

Synthetic cluster glycosides have often been used to unravel mechanisms of carbohydrate-protein interactions. Although synthetic cluster glycosides are constituted on scaffolds to achieve high avidities in lectin binding, there have been no known attempts to modulate the orientations of the sugar clusters with the aid of a functional scaffold onto which the sugar units are linked. Herein, we describe synthesis, physical, and lectin-binding studies of a series of alpha-D-mannopyranoside and beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranoside glycoclusters that are attached to a photoswitchable azobenzenoid core. These glycoclusters were synthesized by the amidation of amine-tethered glycopyranosides with azobenzene carbonyl chlorides. From kinetic studies, the cis forms of the azobenzene-glycopyranoside derivative were found to be more stable in aqueous solutions than in organic solvents. Molecular modeling studies were performed to estimate the relative geometries of the photoswitchable glycoclusters in the trans- and cis-isomeric forms. Isothermal titration calorimetry (ITC) was employed to assess the binding of these glycoclusters to lectins peanut agglutinin (PNA) and concanavalin A (Con A). Although binding affinities were enhanced several orders higher as the valency of the sugar was increased, a biphasic-binding profile in ITC plots was observed during few glycoclusters lectin-binding processes. The biphasic-binding profile indicates a "cooperativity" in the binding process. An important outcome of this study is that in addition to inherent clustering of the sugar units as a molecular feature, an induced clustering emanates because of the isomerization of the trans form of the azobenzene scaffold to the cis-isomeric form.     

Stabilization of Invertase at the Cell Wall-Site of Cycad Pollens by Complex-Formation with Pectic Substances

A series of dansyl-labeled glycosides with di-, tetra-, and hexasaccharides carrying the terminal N-acetyllactosamine (LacNAc) sequence were synthesized as acceptor substrates for α2,6- and α2,3-sialyltransferases. As an alternative design, dansyl-labeled LacNAc glycoside carrying a long-spacer linked glycan was engineered by replacement of the LacNAc or lactose units with an alkyl chain. In addition, we designed a dansyl-labeled bi-antennary LacNAc glycoside as an N-linked oligosaccharide mimetic, such as asialo-α₁-acid glycoprotein. The kinetic parameters for the transfer reaction of synthesized dansyl-labeled glycosides by sialyltransferases were determined by the fluorescent HPLC method. The catalytic efficiencies (V _max/K _m) of acceptor substrates carrying the terminal LacNAc sequence with various length glycans in the array for α2,6- and α2,3-sialyltransferases decreased in a glycan length-dependent manner. Furthermore, of the acceptor substrates tested, dansyl-labeled bi-antennary LacNAc glycoside displayed the most favorable K _m value for α2,6- and α2,3-sialyltransferases.     

Galectins bind to the multivalent glycoprotein asialofetuin with enhanced affinities and a gradient of decreasing binding constants

Our previous isothermal titration microcalorimetry (ITC) studies of the binding of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) showed negative binding cooperativity that was due to the carbohydrate ligands and not the proteins [Dam, T. K., et al. (2002) Biochemistry 41, 1351-1358]. The negative cooperativity was associated with the decreasing functional valence of the carbohydrates upon progressive binding of their epitopes. The present study also shows negative cooperativity in the ITC binding data of asialofetuin (ASF), a glycoprotein that possesses nine LacNAc epitopes, to galectin-1, -2, -3, -4, -5, and -7, and truncated, monomer versions of galectin-3 and -5, which are members of a family of animal lectins. Although the observed K(a) values for binding of ASF to the galectins and two truncated forms are only 50-80-fold greater than that of LacNAc, analysis of the data in terms of the relationship between the observed macroscopic free energy of binding and the decreasing microscopic free energies of binding of the epitopes shows that the first LacNAc epitope of ASF binds with approximately 6000-fold higher affinity than the last epitope. Thus, the microscopic binding constants of the galectins for the first epitope(s) of ASF are in the nanomolar range, with a gradient of decreasing binding constants of the remaining epitopes. The results indicate that the above galectins bind with fractional, high affinities to multivalent glycoproteins such as ASF, independent of the quaternary structures of the galectins. These findings have important implications for the binding of galectins to multivalent carbohydrate receptors.     

Cysteine Cross-linking Defines the Extracellular Gate for the Leishmania donovani Nucleoside Transporter 1.1 (LdNT1.1)

Equilibrative nucleoside transporters are a unique family of proteins that enable uptake of nucleosides/nucleobases into a wide range of eukaryotes and internalize a myriad of drugs used in the treatment of cancer, heart disease, AIDs, and parasitic infections. In previous work we generated a structural model for such a transporter, the LdNT1.1 nucleoside permease from the parasitic protozoan Leishmania donovani, using ab initio computation. The model suggested that aromatic residues present in transmembrane helices 1, 2, and 7 interact to form an extracellular gate that closes the permeation pathway in the inward-open conformation. Mutation of residues Phe-48_TM1 and Trp-75_TM2 abrogated transport activity, consistent with such prediction. In this study cysteine mutagenesis and oxidative cross-linking were combined to analyze proximity relationships of helices 1, 2, and 7 in LdNT1.1. Disulfide bond formation between introduced paired cysteines at the interface of such helices (A61C_TM1/F74C_TM2, A61C_TM1/G350C_TM7, and F74C_TM2/G350C_TM7) was analyzed by transport measurement and gel mobility shifts upon oxidation with Cu (II)-(1,10-phenanthroline)₃. In all cases cross-linking inhibited transport. However, if LdNT1.1 ligands were included during cross-linking, inhibition of transport was reduced, suggesting that ligands moved the three gating helices apart. Moreover, all paired cysteine mutants exhibited a mobility shift upon oxidation, corroborating the formation of a disulfide bond. These data support the notion that helices 1, 2, and 7 constitute the extracellular gate of LdNT1.1, thus further validating the computational model and the previously demonstrated importance of F48_TM1 and Trp-75_TM2 in tethering together helices that are part of the gate.     

Evidence for dimer/tetramer equilibrium in Trypanosoma brucei 6-phosphogluconate dehydrogenase

6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer–tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in ΔC_p, with NADP binding ΔC_p closer to what is expected of crystallographic structures, while NADPH ΔC_p is three times larger. The estimated dimer–tetramer association constant is 1.5 · 10⁶ M^? 1, and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme.     

Synthesis and structures of aryl-substituted tetramethylcyclopentadienyl dinuclear metal carbonyl complexes

A series of 14 aryl-substituted tetramethylcyclopentadienyl dinuclear metal carbonyl complexes have been synthesized by treating the corresponding ligands (C₅Me₄C₆H₄X-4) (X = H, Me, Cl, OMe) with Ru₃(CO)₁₂, Fe(CO)₅, or Mo(CO)₃(MeCN)₃, respectively in refluxing xylene. It showed that the electronic effects of the substituents had influence on the molecular structures and reactions of the complexes, especially for the ruthenium and molybdenum complexes. In the reactions of aryl-substituted tetramethylcyclopentadiene with Mo(CO)₃(MeCN)₃, the electron-withdrawing effect of the substituent in the para position of benzene ring is favorable to produce the Mo-Mo triple bonded complexes, but the electron-donor effect of the substituent in the para position of benzene ring is favorable to produce the Mo-Mo single bonded complexes. In a given condition, the Mo-Mo single bonded complex could be transformed into the corresponding Mo-Mo triple bonded complex. The structures of nine complexes were determined by single crystal X-ray diffraction.     

Identification and characterization of a multispecific monoclonal antibody G2 against chicken prion protein

We previously generated a monoclonal antibody (mAb), G2, by immunizing mice with Residues 174–247 of the chicken prion protein (ChPrP^C). In this study, we found that G2 possessed an extremely unusual characteristic for a mAb; in particular, it could react with at least three proteins other than ChPrP^C, the original antigenic protein. We immunoscreened a complementary DNA library from chicken brain DNA and found three proteins (SEPT3, ATP6V1C1, and C6H10orf76) that reacts with G2. There were no regions of amino acid sequence similarity between ChPrP^C and SEPT3, ATP6V1C1, or C6H10orf76. We selected ATP6V1C1 as a representative of the three proteins and identified the epitope within ATP6V1C1 that reacts with G2. The amino acid sequence of the G2 epitope within ATP6V1C1 (Pep8) was not related to the G2 epitope within ChPrP^C (Pep18mer). However, enzyme-linked immunosorbent assay, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) experiments indicated that these two peptides have similar binding affinity for G2. The apparent K_D values of Pep18mer and Pep8 obtained from SPR experiments were 2.9 × 10⁻⁸ and 1.6 × 10⁻⁸ M, respectively. Antibody inhibition test using each peptide indicated that the binding sites of the two different peptides overlapped each other. We observed that these two peptides substantially differed in several binding characteristics. Based on the SPR experiments, the association and dissociation rate constants of Pep18mer were higher than those of Pep8. A clear difference was also observed in ITC experiments. These differences may be explained by G2 adopting different binding conformations and undergoing different binding pathways.     

Cooperative binding ensures the obligatory melibiose/Na+ cotransport in MelB

MelB catalyzes the obligatory cotransport of melibiose with Na⁺, Li⁺, or H⁺. Crystal structure determination of the Salmonella typhimurium MelB (MelB_St) has revealed a typical major facilitator superfamily (MFS) fold at a periplasmic open conformation. Cooperative binding of Na⁺ and melibiose has been previously established. To determine why cotranslocation of sugar solute and cation is obligatory, we analyzed each binding in the thermodynamic cycle using three independent methods, including the determination of melting temperature by circular dichroism spectroscopy, heat capacity change (ΔC_p), and regulatory phosphotransferase EIIA^Glc binding with isothermal titration calorimetry (ITC). We found that MelB_St thermostability is increased by either substrate (Na⁺ or melibiose) and observed a cooperative effect of both substrates. ITC measurements showed that either binary formation yields a positive sign in the ΔC_p, suggesting MelB_St hydration and a likely widening of the periplasmic cavity. Conversely, formation of a ternary complex yields negative values in ΔCp, suggesting MelB_St dehydration and cavity closure. Lastly, we observed that EIIA^Glc, which has been suggested to trap MelB_St at an outward-open state, readily binds to the MelB_St apo state at an affinity similar to MelB_St/Na⁺. However, it has a suboptimal binding to the ternary state, implying that MelB_St in the ternary complex may be conformationally distant from the EIIA^Glc-preferred outward-facing conformation. Our results consistently support the notion that binding of one substrate (Na⁺ or melibiose) favors MelB_St at open states, whereas the cooperative binding of both substrates triggers the alternating-access process, thus suggesting this conformational regulation could ensure the obligatory cotransport.     

Synthetic assembly of novel avidin-biotin-GlcNAc (ABG) complex as an attractive bio-probe and its interaction with wheat germ agglutinin (WGA)

A tetravalent GlcNAc pendant glycocluster was constructed with terminal biotin through C₆ linker. To acquire the multivalent carbohydrate-protein interactions, we synthesized a glycopolymer of tetrameric structure using N-acetyl-d-glucosamine (GlcNAc) as the target carbohydrate by the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as coupling reagent, followed by biotin-avidin complexation leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). The dynamic light scattering (DLS) system was implied for size detection and to check the binding affinity of GlcNAc conjugate with a WGA lectin we use fluorometric assay by means of specific excitation of tryptophan at λ_ex 295 nm and it was found to be very high K_a ∼ 1.39 × 10⁷ M⁻¹ in case of ABG complex as compared to GlcNAc only K_a ∼ 1.01 × 10⁴ M⁻¹ with the phenomenon proven to be due to glycocluster effect.