Thermodynamics of oligosaccharide binding to a monoclonal antibody specific for a Salmonella O-antigen point to hydrophobic interactions in the binding site.

The thermodynamic characteristics of oligosaccharide binding to an antibody binding site that is dominated by aromatic amino acids suggest that the hydrophobic effect contributes substantially to complex formation as well as hydrogen bonding and van der Waals interactions. A detailed titration microcalorimetric study on the temperature dependence of the binding of a trisaccharide, representing the epitope of a Salmonella O-antigen, showed that its maximum binding to the monoclonal antibody Se155-4 occurs just below room temperature and both enthalpy and entropy changes are strongly dependent on temperature in a mutually compensating manner. The heat capacity change also shows an unusually strong temperature dependence being large and negative above room temperature and positive below. van't Hoff analysis of the temperature dependence of the binding constant yielded a biphasic curve with two apparent intrinsic enthalpy estimations (approximately -100 kJ mol-1 above 18 degrees C and approximately +100 kJ mol-1 below), each very different from the calorimetrically determined enthalpies (ranging from about -60 kJ mol-1 to -20 kJ mol-1). This was interpreted as being due to large enthalpy contributions from concomitant reactions, most notably changes in solvation. Linear plots, -delta H0 versus -T delta S0, observed for temperature-dependent measurements mirror the behavior seen for a series of functional group replacements, suggesting that the molecular and physical origin of these phenomena are closely related and linked to the role of water in complex formation. The thermodynamic results are compared to the mode of binding determined from a 2.05-A resolution structure of the Fab-oligosaccharide complex, and with literature data for the heat capacities of sugars in aqueous solution and for the thermodynamics of carbohydrate binding to transport proteins and lectins

Bacterial expression and secretion of various single-chain Fv genes encoding proteins specific for a Salmonella serotype B O-antigen.

Active single-chain Fv molecules encoded by synthetic genes have been expressed and secreted to the periplasm of Escherichia coli using the ompA secretory signal. Four different constructs were developed to investigate the effects of peptide linker design and VL-VH orientation on expression, secretion, and binding to a Salmonella O-polysaccharide antigen. Peptide linker sequences derived from the elbow regions of the Fab molecule were used alone or in combination with the flexible (GGGGS)2 sequence. VL and VH domain order in the single chain molecules had a profound effect on the level of secretion but hardly influenced total expression levels, which were approximately 50 mg/liter, chiefly in the form of inclusion bodies. With VL in the NH2-terminal position, the amount of secreted product obtained was 2.4 mg/liter, but when VH occupied this position the yield was less than 5% of this value. Enzyme immunoassays of the four products showed domain order and linker sequence affected antigen binding by less than an order of magnitude. Attempts to express active Fv from dicistronic DNA were unsuccessful, but active Fv was obtained from single-chain Fv by enzymic cleavage at a site in the elbow linker peptide. The thermodynamic binding parameters of intact and cleaved single-chain Fvs determined by titration microcalorimetry were similar to those of bacterially produced Fab and mouse IgG

A 1-acetamido derivative of 6-epi-valienamine: an inhibitor of a diverse group of β-N-acetylglucosaminidases

The synthesis of an analogue of 6-epi-valienamine bearing an acetamido group and its characterisation as an inhibitor of β-N-acetylglucosaminidases are described. The compound is a good inhibitor of both human O-GlcNAcase and human β-hexosaminidase, as well as two bacterial β-N-acetylglucosaminidases. A 3-D structure of the complex of Bacteroides thetaiotaomicron BtGH84 with the inhibitor shows the unsaturated ring is surprisingly distorted away from its favoured solution phase conformation and reveals potential for improved inhibitor potency

Intrinsic Thermodynamics of Protein-Ligand Binding by Isothermal Titration Calorimetry as Aid to Drug Design

Isothermal titration calorimetry (ITC) is one of the main techniques to determine specific interactions between molecules dissolved in aqueous solution. This technique is commonly used in drug development programs when low-molecular-weight molecules are sought that bind tightly and specifically to a protein (disease target) molecule. The method allows a complete thermodynamic characterization of an interaction, i.e., ITC enables direct determination of the model-independent observed interaction change in enthalpy (ΔH) and a model-dependent observed interaction affinity (change in Gibbs free energy, ΔG) in a single experiment. The product of temperature and change in entropy (TΔS) can be obtained by the subtraction of ΔG from ΔH, and the change in heat capacity (ΔC p ) can be determined as a slope of the temperature dependence of the binding ΔH. Despite the apparent value of ITC in characterization of interactions, it is often forgotten that many protein-ligand binding reactions are linked to protonation-deprotonation reactions or various conformational changes. In such cases, it is important to determine the linked-reaction contributions and obtain the intrinsic values of the changes in Gibbs energy (affinity), enthalpy, and entropy. These energy values can then be used in various SAR-type structure-thermodynamics and combined with structure-kinetics correlations in drug design, when searching for small molecules that would bind the protein target molecule. This manuscript provides a detailed protocol on how to determine the intrinsic values of protein-ligand binding thermodynamics by ITC

Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay – Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII

The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90αN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 ºC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90αN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding

Modulation of Wnt/β-catenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer

Using a screen for Wnt/β-catenin inhibitors, a family of 8-hydroxyquinolone derivatives with in vivo anti-cancer properties was identified. Analysis of microarray data for the lead compound N-((8-hydroxy-7-quinolinyl) (4-methylphenyl)methyl)benzamide (HQBA) using the Connectivity Map database suggested that it is an iron chelator that mimics the hypoxic response. HQBA chelates Fe2+ with a dissociation constant of ∼10−19 , with much weaker binding to Fe3+ and other transition metals. HQBA inhibited proliferation of multiple cell lines in culture, and blocked the progression of established spontaneous cancers in two distinct genetically engineered mouse models of mammary cancer, MMTV-Wnt1 and MMTV-PyMT mice, without overt toxicity. HQBA may inhibit an iron-dependent factor that regulates cell-type-specific β-catenin-driven transcription. It inhibits cancer cell proliferation independently of its effect on β-catenin signaling, as it works equally well in MMTV-PyMT tumors and diverse β-catenin-independent cell lines. HQBA is a promising specific intracellular Fe2+ chelator with activity against spontaneous mouse mammary cancers

The anti-sigma factor RsrA responds to oxidative stress by reburying its hydrophobic core

Redox-regulated effector systems that counteract oxidative stress are essential for all forms of life. Here we uncover a new paradigm for sensing oxidative stress centred on the hydrophobic core of a sensor protein. RsrA is an archetypal zinc-binding anti-sigma factor that responds to disulfide stress in the cytoplasm of Actinobacteria. We show that RsrA utilizes its hydrophobic core to bind the sigma factor σ R preventing its association with RNA polymerase, and that zinc plays a central role in maintaining this high-affinity complex. Oxidation of RsrA is limited by the rate of zinc release, which weakens the RsrA-σ R complex by accelerating its dissociation. The subsequent trigger disulfide, formed between specific combinations of RsrA's three zinc-binding cysteines, precipitates structural collapse to a compact state where all σ R-binding residues are sequestered back into its hydrophobic core, releasing σ R to activate transcription of anti-oxidant genes