Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger.

Several variants of glucoamylase 1 (GA1) from Aspergillus niger were created in which the highly O-glycosylated peptide (aa 468--508) connecting the (alpha/alpha)(6)-barrel catalytic domain and the starch binding domain was substituted at the gene level by equivalent segments of glucoamylases from Hormoconis resinae, Humicola grisea, and Rhizopus oryzae encoding 5, 19, and 36 amino acid residues. Variants were constructed in which the H. resinae linker was elongated by proline-rich sequences as this linker itself apparently was too short to allow formation of the corresponding protein variant. Size and isoelectric point of GA1 variants reflected differences in linker length, posttranslational modification, and net charge. While calculated polypeptide chain molecular masses for wild-type GA1, a nonnatural proline-rich linker variant, H. grisea, and R. oryzae linker variants were 65,784, 63,777, 63,912, and 65,614 Da, respectively, MALDI-TOF-MS gave values of 82,042, 73,800, 73,413, and 90,793 Da, respectively, where the latter value could partly be explained by an N-glycosylation site introduced near the linker C-terminus. The k(cat) and K(m) for hydrolysis of maltooligodextrins and soluble starch, and the rate of hydrolysis of barley starch granules were essentially the same for the variants as for wild-type GA1. beta-Cyclodextrin, acarbose, and two heterobidentate inhibitors were found by isothermal titration calorimetry to bind to the catalytic and starch binding domains of the linker variants, indicating that the function of the active site and the starch binding site was maintained. The stability of GA1 linker variants toward GdnHCl and heat, however, was reduced compared to wild-type.

Mutational analysis of affinity and selectivity of kringle-tetranectin interaction. Grafting novel kringle affinity ontp the trtranectin lectin scaffold.

C-type lectin-like domains are found in many proteins, where they mediate binding to a wide diversity of compounds, including carbohydrates, lipids, and proteins. The binding of a C-type lectin-like domain to a ligand is often influenced by calcium. Recently, we have identified a site in the C-type lectin-like domain of tetranectin, involving Lys-148, Glu-150, and Asp-165, which mediates calcium-sensitive binding to plasminogen kringle 4. Here, we investigate the effect of conservative substitutions of these and a neighboring amino acid residue. Substitution of Thr-149 in tetranectin with a tyrosine residue considerably increases the affinity for plasminogen kringle 4, and, in addition, confers affinity for plasminogen kringle 2. As shown by isothermal titration calorimetry analysis, this new interaction is stronger than the binding of wild-type tetranectin to plasminogen kringle 4. This study provides further insight into molecular determinants of importance for binding selectivity and affinity of C-type lectin kringle interactions.

Tetranectin-binding site on plasminogen kringle 4 involves the lysine-binding pocket and at least one additional amino acid residue.

Kringle domains are found in a number of proteins where they govern protein-protein interactions. These interactions are often sensitive to lysine and lysine analogues, and the kringle-lysine interaction has been used as a model system for investigating kringle-protein interactions. In this study, we analyze the interaction of wild-type and six single-residue mutants of recombinant plasminogen kringle 4 expressed in Escherichia coli with the recombinant C-type lectin domain of tetranectin and trans-aminomethyl-cyclohexanoic acid (t-AMCHA) using isothermal titration calorimetry. We find that all amino acid residues of plasminogen kringle 4 found to be involved in t-AMCHA binding are also involved in binding tetranectin. Notably, one amino acid residue of plasminogen kringle 4, Arg 32, not involved in binding t-AMCHA, is critical for binding tetranectin. We also find that Asp 57 and Asp 55 of plasminogen kringle 4, which both were found to interact with the low molecular weight ligand with an almost identical geometry in the crystal of the complex, are not of equal functional importance in t-AMCHA binding. Mutating Asp 57 to an Asn totally eliminates binding, whereas the Asp 55 to Asn, like the Arg 71 to Gln mutation, was found only to decrease affinity.

Exact analysis of competition ligand binding by displacement isothermal titration calorimetry.

A rigorous method for the least-squares nonlinear regression analysis of displacement isothermal titration calorimetric data is presented. The method can fit the binding isotherm of a ligand which is competitively inhibited in its binding by another bound ligand to a molecule with n identical and independent binding sites. There are no other assumptions for the method and no approximations. Analysis of previously published data of the strong binding of acarbose to glucoamylase is presented as an example. The regression equations have been programmed for the Origin software supplied with the widely used titration calorimeters from Microcal, Inc., and an Origin Function Definition File with instructions is freely available from the author upon e-mail request.

Glucoamylase: structure/function relationships, and protein engineering.

Glucoamylases are inverting exo-acting starch hydrolases releasing beta-glucose from the non-reducing ends of starch and related substrates. The majority of glucoamylases are multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain by an O-glycosylated linker region. Three-dimensional structures have been determined of free and inhibitor complexed glucoamylases from Aspergillus awamori var. X100, Aspergillus niger, and Saccharomycopsis fibuligera. The catalytic domain folds as a twisted (alpha/alpha)(6)-barrel with a central funnel-shaped active site, while the starch-binding domain folds as an antiparallel beta-barrel and has two binding sites for starch or beta-cyclodextrin. Certain glucoamylases are widely applied industrially in the manufacture of glucose and fructose syrups. For more than a decade mutational investigations of glucoamylase have addressed fundamental structure/function relationships in the binding and catalytic mechanisms. In parallel, issues of relevance for application have been pursued using protein engineering to improve the industrial properties. The present review focuses on recent findings on the catalytic site, mechanism of action, substrate recognition, the linker region, the multidomain architecture, the engineering of specificity and stability, and roles of individual substrate binding subsites.

Thermodynamics of reversible and irreversible unfolding and domain interactions of glucoamylase from Aspergillus niger studied by differential scanning and isothermal titration calorimetry.

The stability of three forms of glucoamylase from Aspergillus niger has been investigated by differential scanning and isothermal titration calorimetry: Glucoamylase 1 (GA1), which consists of a catalytic domain and a starch-binding domain (SBD) connected by a heavily O-glycosylated linker region; glucoamylase 2 (GA2), which lacks SBD; and a proteolytically cleaved glucoamylase (GACD), which contains the catalytic domain and part of the linker region. The structures of the catalytic domain with part of the linker region and of SBD are known from crystallography and NMR, respectively, but the precise spatial arrangement of the two domains in GA1 is unknown. To investigate the stability of the three glucoamylase forms, we unfolded the enzymes thermally by differential scanning calorimetry (DSC). Aggregation occurs upon heating GA1 and GA2 at pH values between 2.5 and 5.0, whereas no aggregation is observed at higher pH (5.5-7.5). At all pH values, the catalytic domain of GA1 and GA2 unfolds irreversibly, while SBD unfolds reversibly in the pH range 5. 5-7.5 where aggregation does not occur. The unfolding of the catalytic domain of all glucoamylase forms seems to follow an irreversible one-step mechanism with no observable reversible intermediates on the experimental time scale. SBD of GA1 unfolds reversibly, and the ratio between the van't Hoff and calorimetric enthalpies is 1.4 +/- 0.1. Assignment of peaks of the DSC profile to the domains at pH 7.5 is achieved by using two different ligands: Acarbose, a very strong inhibitor that binds exclusively to the catalytic domain, and beta-cyclodextrin, a small starch analogue of which 2 molecules bind solely to the two binding sites present in SBD. Differences are seen in the unfolding processes of GA1 and GA2 since the former unfolds with one peak at all pH values, while the calorimetric trace of the latter can be resolved into more peaks depending on pH and the chemical composition of the buffers. In general, peaks corresponding to unfolding of GA2 are more complex than the peaks of GA1 and GACD. Some part of GA2 unfolds before the rest of the molecule which may correspond to the linker region or a particular early unfolding part of the catalytic domain. This leads to the conclusion that the structure of the GA2 molecule has a larger cooperative unfolding unit and is less stable than the structures of GA1 and GACD and that the C-terminal part of the linker region has a destabilizing effect on the catalytic domain.

The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine.

Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon resonance and isothermal calorimetry binding analyses using single-residue and deletion mutant tetranectin derivatives produced in Escherichia coli showed that the kringle 4 binding site resides in the carbohydrate recognition domain and includes residues of the putative carbohydrate binding site. Furthermore, the binding analysis revealed that the interaction is sensitive to calcium in addition to lysine.

Thermodynamics of binding of heterobidentate ligands consisting of spacer-connected acarbose and beta-cyclodextrin to the catalytic and starch-binding domains of glucoamylase from Aspergillus niger shows that the catalytic and starch-binding sites are in close proximity in space.

The binding to glucoamylase 1 from Aspergillus niger (GA1) of a series of four synthetic heterobidentate ligands of acarbose and beta-cyclodextrin (beta-CD) linked together has been studied by isothermal titration calorimetry. GA1 consists of a catalytic and a starch-binding domain (SBD) connected by a heavily O-glycosylated linker region. Acarbose is a strong inhibitor of glucoamylase and binds exclusively in the catalytic site, while the cyclic starch mimic beta-CD binds exclusively to the two sites of SBD. No spacer or spacer arms of 14, 36, and 73 A in their extended conformations connect acarbose and beta-CD. These compounds were used as probes for bidentate ligand binding to both domains in order to estimate the distance between the catalytic site and the SBD binding site in solution. DeltaH of binding of the four heterobidentate ligands is within experimental uncertainty equal to the sum of DeltaH of binding of free acarbose and beta-CD, indicating ligand binding to both domains. However, the binding constants are 4-5 orders of magnitude smaller than for the binding of acarbose (K approximately 10(12) M-1), increasing with spacer length from 2 x 10(7) M-1 for no spacer to 1 x 10(8) M-1 for the 73 A spacer. Subsequent titrations with beta-CD of the glucoamylase-bidentate ligand complexes revealed that only one of the two binding sites of SBD was vacant. Further titrations with acarbose to these mixtures showed complete displacement of the acarbose moiety of the bidentate ligands from the catalytic sites. These experiments show that the bidentate ligands bind to both the catalytic domain and SBD. The weakening of the bidentate ligand binding compared to acarbose is a purely entropic effect point to steric hindrance between SBD and the beta-CD moiety. To test this, titrations of glucoamylase 2, a form containing the catalytic domain and the linker region but lacking SBD, with the bidentate ligands were carried out. The results were indistinguishable from the binding of free acarbose. Thus, the reduced affinity of the bidentate ligands observed with GA1 stems from interactions due to SBD. The results show that the catalytic and starch-binding sites are in close proximity in solution and thus indicate considerable flexibility of the linker region.

Thermodynamics of ligand binding to acyl-coenzyme A binding protein studied by titration calorimetry.

Ligand binding to recombinant bovine acyl-CoA binding protein (ACBP) was examined using isothermal microcalorimetry. Microcalorimetric measurements confirm that the binding affinity of acyl-CoA esters for ACBP is strongly dependent on the length of the acyl chain with a clear preference for acyl-CoA esters containing more than eight carbon atoms and that the 3'-phosphate of the ribose accounts for almost half of the binding energy. Binding of acyl-CoA esters, with increasing chain length, to ACBP was clearly enthalpically driven with a slightly unfavorable entropic contribution. Accessible surface areas derived from the measured enthalpies were compared to those calculated from sets of three-dimensional solution structures and showed reasonable correlation, confirming the enthalphically driven binding. Binding of dodecanoyl-CoA to ACBP was studied at various temperatures and was characterized by a weak temperature dependence on delta G zero and a strong enthalpy-entropy compensation. This was a direct consequence of a large heat capacity delta Cp caused by the presence of strong hydrophobic interactions. Furthermore, the binding of dodecanoyl-CoA was studied at various pH values and ionic strengths. The data presented here state that ACBP binds long-chain acyl-CoA esters with very high affinity and suggest that ACBP acts as a housekeeping protein with no pronounced built-in specificity.

Thermodynamics of inhibitor binding to mutant forms of glucoamylase from Aspergillus niger determined by isothermal titration calorimetry.

We have investigated the binding of mutant forms of glucoamylase from Aspergillus niger to the inhibitors 1-deoxynojirimycin and acarbose. The mutants studied comprise a group of single amino acid replacements in conserved regions near the active site of the enzyme. For each mutant we have determined both the affinities for the two inhibitors and the thermodynamic state functions for binding using titration microcalorimetry. We find that acarbose binds to all the mutants with a wide range of binding constants (10(4) < Ka < 10(13) M-1). In contrast, 1-deoxynojirimycin shows either binding at near wild-type affinity (Ka approximately equal to 10(4) M-1) or no detectable binding. The changes in the affinities of the mutant enzymes are rationalized in terms of the known three-dimensional structure of the wild-type enzyme with subsites 1, 2, and 3 being important for acarbose binding while only subsite 1 is critical for 1-deoxynojirimycin binding. In most of the mutants studied the magnitudes of the enthalpies and the entropies of binding of the mutant enzymes differed from those of the wild-type enzyme with the mutant enzymes having a relatively large portion of their binding energy composed of enthalpy and a relatively small proportion composed of entropy. The pattern of changes in the enthalpy and entropy is hypothesized to be due to changes in the structural complementarity of the binding pocket and the inhibitor.

Evidence for the extended helical nature of polysaccharide epitopes. The 2.8 A resolution structure and thermodynamics of ligand binding of an antigen binding fragment specific for alpha-(2-->8)-polysialic acid.

The antigen binding fragment from an IgG2a kappa murine monoclonal antibody with specificity for alpha-(2-->8)-linked sialic acid polymers has been prepared and crystallized in the absence of hapten. Crystals were grown by vapor diffusion equilibrium with 16-18% polyethylene glycol 4000 solutions. The structure was solved by molecular replacement methods and refined to a conventional R factor of 0.164 for data to 2.8 A. The binding site is observed to display a shape and distribution of charges that is complementary to that of the predicted conformation of the oligosaccharide epitope. A thermodynamic description of ligand binding has been compiled for oligosaccharides ranging in length from 9 to 41 residues, and the data for the largest ligand has been used in a novel way to estimate the size of the antigen binding site. A model of antigen binding is presented that satisfies this thermodynamic data, as well as a previously reported requirement of conformational specificity of the oligosaccharide. X-ray crystallographic and thermodynamic evidence are consistent with a binding site that accommodates at least eight sialic acid residues.

Thermodynamics of ligand binding to the starch-binding domain of glucoamylase from Aspergillus niger.

The thermodynamics of ligand binding to the starch-binding domain (SBD) of glucoamylase from Aspergillus niger has been studied using titration calorimetry. The ligand binding was studied both with the SBD fragment as well as glucoamylase G1 which contains both a catalytic domain and SBD. The ligands were beta-cyclodextrin and three thiopanose analogues [panose = alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glcp] each including an alpha-(1-->6) thioglycosidic linkage at the non-reducing end. beta-Cyclodextrin binds more strongly than the thiopanose analogues and these have a slightly increasing binding constant with chain length. The reactions are enthalpy-driven with unfavourable contributions from entropy and the variations in enthalpy and entropy compensate each other linearly. SBD was shown to have two binding sites that appear to bind identically and independently in the isolated binding domain, whereas they interact with each other in a negatively cooperative fashion when the catalytic domain of glucoamylase is present (glucoamylase G1). In glucoamylase G1 one site of SBD has an increased binding constant compared to the SBD fragment, whereas the other has the same association constant. The change in binding constant and induced cooperativity were not due to interactions with the catalytic binding site, since binding of beta-cyclodextrin was the same both when the catalytic site was occupied by the strong inhibitor acarbose and when the catalytic site was free.

Thermodynamics of inhibitor binding to the catalytic site of glucoamylase from Aspergillus niger determined by displacement titration calorimetry.

The binding of different inhibitors to glucoamylase G2 from Aspergillus niger and its temperature and pH dependencies have been studied by titration calorimetry. The enzyme binds the inhibitors 1-deoxynojirimycin and the pseudo-tetrasaccharide acarbose with association constants of 3 x 10(4) and 9 x 10(11) M-1, respectively, at 27 degrees C. The binding free energy for both ligands is remarkably temperature-invariant in the interval from 9 to 54 degrees C as the result of large compensating changes in enthalpy and entropy. Acarbose and 1-deoxynojirimycin bound with slightly different free energy-pH profiles, with optima at 5.5 and 5.5-7.0, respectively. Variations in delta H degrees and T delta S degrees as a function of pH were substantially larger than variations in delta G degrees in a partly compensatory manner. Two titratable groups at or near subsite 1 of the catalytic site were found to change their pKa slightly upon binding. The hydrogenated forms of acarbose, D-gluco- and L-ido-dihydroacarbose, bind with greatly reduced association constants of 3 x 10(7) and 2 x 10(5) M-1, respectively, and the pseudo-disaccharide methyl acarviosinide, lacking the two glucose units at the reducing end compared to acarbose, has a binding constant of 8 x 10(6) M-1; these values all result from losses in both enthalpy and entropy compared to acarbose. Three thio analogues of the substrate maltose, methyl alpha- and beta-4-thiomaltoside and methyl alpha-4,5'-dithiomaltoside, bind with affinities from 3 x 10(3) to 6 x 10(4) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular recognition of a Salmonella trisaccharide epitope by monoclonal antibody Se155-4.

The binding site of monoclonal antibody Se155-4, which has been the object of successful crystallographic and antibody-engineering studies, is shown by solid-phase immunoassays to be complementary to a branched trisaccharide, alpha-D-Galp(1-->2) [alpha-D-Abep(1-->3)]-alpha-D-Manp(1, rather than to the tetrasaccharide repeating unit alpha-D-Galp(1-->2) [alpha-D-Abep(1-->3)]-alpha-D-Manp(1-->4) alpha-L-Rhap(1- of the bacterial antigen. Specificity for the 3,6-dideoxy-D-xylo-hexose (3,6-dideoxy-D-galactose) epitope present in Salmonella paratyphi B O-antigens was ensured by screening hybridoma experiments with glycoconjugates derived from synthetic oligosaccharides. Detailed epitope mapping of the molecular recognition by modified and monodeoxy oligosaccharide derivatives showed that complementary surfaces and three antibody-saccharide hydrogen bonds are essential for full binding activity. Both hydroxyl groups of the 3,6-dideoxy-D-galactose residue were obligatory for binding and consistent with the directional nature of their involvement in carbohydrate-protein hydrogen bonds; related tetrasaccharides built from the isomeric 3,6-dideoxyhexoses, 3,6-dideoxy-D-glucose, paratose, and 3,6-dideoxy-D-mannose, tyvelose were not bound by the antibody. Titration microcalorimetry measurements were consistent with the hydrogen-bonding map inferred from the crystal structure and suggest that the displacement of water molecules from the binding site accounts for the favorable entropy that accompanies binding of the native trisaccharide determinant. The protein sequences determined for the antibody VL and VH domains reveal somatic mutation of the VL germ line gene, implying that this antibody-binding site results from a mature antibody response.

Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: role of the heavy-chain CDR3 residues.

The carbohydrate-binding site in Fab fragments of an antibody specific for Salmonella serogroup B O-polysaccharide has been probed by site-directed mutagenesis using an Escherichia coli expression system. Of the six hypervariable loops, the CDR3 of the heavy chain was selected for exhaustive study because of its significant contribution to binding-site topography. A total of 90 mutants were produced and screened by an affinity electrophoresis/Western blotting method. Those of particular interest were further characterized by enzyme immunoassay, and on this basis seven of the mutant Fabs were selected for thermodynamic characterization by titration microcalorimetry. With regard to residues that hydrogen bond to ligand through backbone interactions, Gly102H could not be substituted, while several side chains could be introduced at Gly100H and Tyr103H with relatively little effect on antigen binding. There was, however, a preference for nonpolar side chains at position 103H. Substitution of His101H with carboxylate and amide side chains gave mutants with binding affinities approaching that of the wild type; complete side-chain removal by mutation to Gly was tolerated with a 10-fold reduction in binding constant. Analysis of binding by titration microcalorimetry revealed some dramatic thermodynamic changes hidden by the similarity of the binding constants. Similar effects were observed with residue changes in an Arg-Asp salt-bridge at the base of the loop. These results indicate that alterations to higher affinity anti-carbohydrate antibodies are characterized by an enthalpy-entropy compensation factor which allows for fundamental changes in the nature of the binding interactions but impedes engineering for increases in affinity.

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.

Hydrolysis of substrate analogues catalysed by beta-D-glucosidase from Aspergillus niger. Part II: Deoxy and deoxyhalo derivatives of cellobiose.

The hydrolysis of sixteen mainly deoxy and deoxyhalo derivatives of celloboise catalysed by beta-D-glucosidase from Aspergillus niger has been studied by means of 1H NMR spectroscopy and progress-curve enzyme kinetics in both single-substrate and competition experiments. In the non-reducing ring of cellobiose it was found that the hydroxy groups at positions 2', 3', and 4' are essential for the enzymatic hydrolysis. The primary hydroxy group on 6' in this ring is, although important for the hydrolysis, not essential. The analogues modified at positions 3' and 4' and the 6'-bromo-6'-deoxy derivative were not inhibitors, whereas the 2'-deoxy derivative inhibited the enzymatic hydrolysis of methyl beta-cellobioside to some extent. Of the analogues modified in the reducing ring, some were hydrolysed faster (e.g. the deoxy compounds) and some slower than methyl beta-cellobioside in single-substrate experiments, but all derivatives were hydrolysed at a lower rate than this reference substrate in direct competition and displayed relatively weak inhibitory effects. The results are interpreted qualitatively with respect to changes in the free binding energies of the substrates and catalytic transition states based on the Michaelis-Menten mechanism, and some mechanistic implications of these findings are discussed.

Sensitive titration microcalorimetric study of the binding of Salmonella O-antigenic oligosaccharides by a monoclonal antibody.

The binding of several oligosaccharide haptens by a monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-antigen was studied by titration microcalorimetry. In the software developed by Wiseman et al. [Wiseman, T., Williston, S. & Brandts, J.F. (1989) Anal. Biochem. 17, 131-137] the number of binding sites/macromolecule is one of the optional regression parameters in the non-linear least-squares analysis of the calorimetric data. Instead, an approach was adopted in which the concentration of binding sites was treated as a regression parameter, obviating the requirement for precise values of antibody absorption coefficients and minimizing effects due to partially inactive antibody preparations. Furthermore, performing the least-squares analysis in two steps, first using a differential heat mode and then an integral heat mode, was shown to yield the most accurate results. The technique gave accurate results using not more than 1-2 mumol ligand and less than 7 mg antibody. Haptens 2-5 were oligomers of the O-antigenic repeating unit varying in chain length by 2-5 repeating units and a trisaccharide glycoside 1, which filled the binding site. The latter hapten exhibited a favourable entropy contribution to binding (delta Go = -31 kJ.mol-1; delta Ho = -21 kJ.mol-1 and -T delta So -10 kJ.mol-1), while all four oligomers 2-5 showed a constant binding energy delta Go = -33 kJ.mol-1, composed of increasingly stronger enthalpy forces compensated by an increasingly unfavourable entropy contribution. These observations are compared with results from enzyme immunoassays and a high-resolution crystal structure for the dodecasaccharide 3 bound to the Fab derived from Se155-4.